We found that the level of free resting calcium in the cell nucleus is significantly lower compared to the soma, sub-membrane, and dendritic tree regions. Calcium release from the endoplasmic reticulum under the action of several stimuli  occurs in all areas studied. Under depolarization, calcium signals developed faster in the dendrites than in other areas, while their amplitude was significantly lower since larger and slower responses inside the soma. The peak value of the calcium response to the application of 10 mM caffeine, ryanodine receptors (RyRs) agonist, does not differ in the sub-membrane zone, central region, and nucleus but significantly decreases in the dendrites. In the presence of caffeine, the delay of Ca2+ signals between various areas under depolarization significantly declined. Thirty percentage of the peak amplitude of Ca2+ transients at prolonged electric field stimulation corresponded to calcium release from the ER store by RyRs, while short-term stimulation did not depend on them. 20 μM dantrolene, RyRs inhibitor, significantly reduces Ca2+ transient under high K+ levels depolarization of the neuron. RyRs-mediated enhancement of the Ca2+ signal is more pronounced in the central part and nucleus compared to the sub-membrane or dendrites regions of the neuron. In summary, using the ratiometric imaging allowed us to obtain additional information about the involvement of RyRs in the intracellular dynamics of Ca2+ signals induced by depolarization or electrical stimulation train, with an underlying change in Ca2+ concentration in various regions of interest in hippocampal pyramidal neurons.The spatio-temporal properties of calcium signals were studied in cultured pyramidal neurons of the hippocampus using two-dimensional fluorescence microscopy and ratiometric dye Fura-2. Depolarization-induced Ca All chemicals were obtained from Sigma-Aldrich .All experimental procedures were performed following ethical principles of the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes  and were approved by the local Animal Ethics Committee of the Bogomoletz Institute of Physiology . All efforts were made to minimize the number and suffering of animals used. All experiments were performed on cultured hippocampal neurons obtained from newborn Wistar rats. Primary cultures were prepared as previously reported  = 20.7 and F380(max) = 169.4; dK(Fura-2) = 224 nM  for 30 min at 37°C and 20 min for de-esterification of the dye. The values of R Shkryl, . The cov Shkryl, . Ca2+ tr340/F380) was calculated. This is done according to the protocol described by Shkryl  mounted on an Olympus IX71 inverted microscope equipped with an Olympus LUCPlanFFN 20×/0.45 lens and MT10 illumination system that included a filter wheel exchanger (340–380 nm) and 150 W xenon arc burner. For the data collection, we use Cell M software . The acquisition speed was 30 Hz for one wave and 1.3 Hz for ratiometric use. For further data analysis, the ratio of 340–380 nm fluorescence intensity . In recorded images with excitation of 340–380 nm lights, the regions of interests (ROIs) were selected. All experiments were performed at room temperature (22°C–25°C). Data are presented as mean ± SEM.2+ release in different regions of different neurons differ significantly  increases induced by two series of 50 pulses of EFS (used to load the ER store) and followed by the application of 10 mM caffeine for 5 s to induce Ca2+ release from the ER stores by ryanodine receptors (RyRs). An additional EFS and 5-s application of the depolarizing solution were applied when the restoration of calcium signals to the resting level. 2D imaging of Ca2+ signals reveals an asynchronous delayed rise of free Ca2+ concentration in the nuclear compared to the sub-membrane, central or dendritic regions  but had the smallest amplitude during EFS, KCl depolarization, and caffeine application. The amplitude of Ca2+ transient in the dendritic tree of pyramidal-like neurons in response to EFS was 1.18 ± 0.09 , which was significantly smaller compared to sub-membrane—1.62 ± 0.17 , central—1.73 ± 0.18 , and nuclear—1.66 ± 0.17  regions of the cell  respectively to sub-membrane—4.76 ± 0.39 , central—4.98 ± 0.38 , and nuclear—5.06 ± 0.39  regions of studied neurons (2+ release from the ER) were significantly reduced in the dendritic tree region to 1.88 ± 0.08  from 2.31 ± 0.11  in sub-membrane, central—2.43 ± 0.13  and nuclear—2.17 ± 0.12  regions of pyramidal neurons.The value of basal free Cathe cell . In the  neurons . As show2+ transients in the various areas of pyramidal-like neurons recorded at 30 Hz frequencies /dt] representing the underlying Ca2+ flux. The Ca2+ flux in the sub-membrane and central regions appeared with increasing and decreasing steps, representing the EFS-induced calcium transients. The maximum flux in the sub-membrane and central regions was observed 0.2 s after initiation and decreased with time. In the nuclear region of the neuron, the flux was delayed and reached its maximum value 0.6 s after the onset with reduced amplitude compared to other regions. The maximal flux rate was in the dendritic tree region  and reduced in the sub-membrane  and central , and minimal in nuclear aria  of the neuron . 2+]i increases caused by 50 pulses of EFS (3A) and 50 mM KCl solution (3B) normalized to the maximal amplitude of Ca2+ transients in various ROIs of the neuron. When recording in x-y-t mode (limited to 30 Hz), Ca2+ signals were insufficient to represent the initial phase of the Ca2+ signal, which was more evident during KCl-induced transients. However, we find signal delays between different ROIs at 25%, 50%, and 75% of the maximal amplitude. It is in the sub-membrane, and central regions were more pronounced and progressed during the EFS-induced Ca2+ transient and less during KCl depolarization, and this parameter of the difference between the dendritic tree and the sub-membrane region of interest increased up to 1 s. In the central and nuclear regions, the delay was almost independent of the type of stimulus and was around 0.2 s. Statistical data are represented in the summarized diagram in part 3C.To show more detailed information about the inhomogeneity of Ca2+ release from the ER occurs under conditions of the opening of the IP3Rs and RyRs  and in the presence of 10 mM caffeine (red trace). In control, the basal level of free calcium was 1.06 ± 0.03 and 1.26 ± 0.05 in the presence of caffeine. The peak amplitude of Ca2+ transients induced by depolarizing solution significantly decreased from 6.47 ± 0.57 (in the control) to 4.58 ± 0.31 under the application of 10 mM caffeine . Under electrical field stimulation changing the amplitude of the caffeine depend on the duration of the stimulation. The peak amplitude of the Ca2+ signal induced by short  EFS did not change appreciably, 0.54 ± 0.08 compared to 0.51 ± 0.07 (n = 13). However, by a long stimulus , this parameter significantly decreases from 2.72 ± 0.58 to 1.89 ± 0.38  with caffeine.1 ± 0.07 ; n = 13.2+ transients under caffeine, but the Ca2+ response significantly decreased under the depolarization solution or long EFS. Thus, with prolonged stimulation of the neuron, Ca2+ release appeared from the ER by RyRs. As we can see from normalized caffeine-induced Ca2+ transients  than in sub-membrane zone and dendritic tree. 2+]i caused by 5 s application of KCl solution normalized to the maximal amplitude of the signal in various regions of interest of the neuron. The short electrical stimulation  does not alter the amplitude of Caansients , a rise 2+ transients. 2+ signal, recorded from a neuron under control condition (black square line) and in the presence of 20 μM dantrolene (red square line). In control, the basal level of free calcium was 1.23 ± 0.03 and 1.10 ± 0.03 in the presence of dantrolene. The amplitude value of depolarizing solution-induced Ca2+ transients under 20 μM dantrolene significantly decreased from 5.30 ± 0.26 to 2.84 ± 0.13 . The ER load with the presence of dantrolene was significantly decreased to 0.682 ± 0.06 from 1.72 ± 0.14 . It further confirms the involvement of RyRs in Ca2+ signaling in cultured hippocampal pyramidal neurons.Additionally, we used 20 μM dantrolene to block RyRs and record high KCl-induced Cat-test. Also, the difference between these signals in the nucleus  were reported to be empty at rest and to accumulate Ca2+ only after its entry via voltage-gated Ca2+ channels in the plasmalemma  of CA1 pyramidal cells induced Ca2+ release not only from dendritic ryanodine-sensitive Ca2+ stores, which was shown previously  in dendrites , the amplitude of calcium responses was significantly lower during EFS but did not differ during depolarization of solutions with a high KCl, which suggests the role of glutamate receptors in these responses.Spontaneous, localized Ca2+ signal. Caffeine interacts with fluorescent calcium indicator dyes  was not enough to enhance the Ca2+ signal through CICR, but adequate at long-lasting excitation (6 s) produce calcium release from the ER store there 30% of the peak amplitude of Ca2+ transient corresponds to RyRs mediated calcium release.In summary, we demonstrate spatial dynamics of free CaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All experimental procedures were performed following ethical principles of the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes  and were approved by the local Animal Ethics Committee of the Bogomoletz Institute of Physiology .VS carried out the experiment; conceived and designed the analyses; collected the data; performed the analysis; wrote the article.


Background: Atrial fibrillation is the most sustained form of arrhythmia in the human population that leads to important electrophysiological and structural cardiac remodeling as it progresses into a chronic form. Calcium is an established key player of cellular electrophysiology in the heart, yet to date, there is no information that maps calcium signaling across the left atrium. Objective: The aim of this study is to determine whether calcium signaling is homogenous throughout the different regions of the left atrium. This work tests the hypothesis that differences across the healthy left atrium contribute to a unique, region-dependent calcium cycling and participates in the pro-arrhythmic activity during atrial fibrillation. Methods: An animal model relevant to human cardiac function (the sheep) was used to characterize both the electrical activity and the calcium signaling of three distinct left atrium regions  in control conditions and after acetylcholine perfusion (5 μM) to induce acute atrial fibrillation. High-resolution dual calcium-voltage optical mapping on the left atria of sheep was performed to explore the spatiotemporal dynamics of calcium signaling in relation to electrophysiological properties. Results: Action potential duration (at 80% repolarization) was not significantly different in the three regions of interest for the three pacing sites. In contrast, the time to 50% calcium transient decay was significantly different depending on the region paced and recorded. Acetylcholine perfusion and burst pacing-induced atrial fibrillation when pulmonary veins and appendage regions were paced but not when the free wall region was. Dantrolene (a ryanodine receptor blocker) did not reduce atrial fibrillation susceptibility. Conclusion: These data provide the first evidence of heterogenous calcium signaling across the healthy left atrium. Such basal regional differences may be exacerbated during the progression of atrial fibrillation and thus play a crucial role in focal arrhythmia initiation without ryanodine receptor gating modification. Cardiac arrhythmias are major causes of morbidity and mortality , with atAF is initiated by the presence of one or more hyper-excitability sources, usually located within the left atria (LA), that lead to asynchronous and chaotic myocardial contractions. Those ectopic foci can sustain AF as a driver and may trigger reentries  in a vu2+) regulation is a major determinant of cellular electrophysiology in the heart i in the atria due to the technical difficulty of simultaneously recording APs and [Ca2+]i from multiple sites with sufficient spatial resolution and speed. Several dual-optical mapping experiments have been performed in rodents [m and [Ca2+]i was performed only once to study AF in the context of ischemia in sheep i across the surface of the LA from a translationally relevant ovine animal model, and we use this novel approach to characterize Vm and [Ca2+]i in three different regions of the LA. This is crucial because, although PV is the main site for ectopic beats in paroxysmal AF, ~30% arise from other areas of the LA, more specifically the free wall (FW) i signals from the atrial endocardium. LA preparations were loaded with either RH237 or Rhod-2 AM via the left circumflex coronary artery. 2+]i signals in the [Ca2+]i camera. The first series of experiments were designed to verify our experimental approach for simultaneously recording optical Vm and [Ca2+]i signals.Conversely, LA loaded with Rhod-2 exhibited null signals in the voltage camera array. This demonstrates a complete spectrum separation between the Vm and [Ca2+]i simultaneously to check the temporal characteristics between Vm and [Ca2+]i. 2+]i and the rise time of Vm signals was shorter than that for [Ca2+]i. The interval between the peak of the APs and the peak of the Ca2+ transients (Δt) can therefore be measured , stimulation electrodes were positioned at the PV, LAA, or FW. 50 and APD80 . In contrast, when LA preparations were perfused with 10 µM of Ach, APD differed depending on the pacing site. When LAA was stimulated, APD80 was longer in LAA than in PV and FW , and APD50 was longer in LAA than in FW but not in PV . Similarly, when the pacing site was FW, APD80 was significantly longer in LAA than in the FW but not in PV (p = 0.0002). Surprisingly, when the pacing site was PV, APD80 and APD50 were similar between the regions of measurements . The electrodes of stimulation were located at PV, LAA, or FW. 2+ transients recorded from LA preparations stimulated at 1000 ms PCL.Next, we investigated the impact of the site of stimulation on the regional Ca50 was significantly shorter in PV than in FW and LAA . When LAA was the stimulation site, TD50 was significantly shorter in LAA compared to FW, but there was no significant difference with PV . When the stimulation site was FW, TD50 was homogeneous in the three regions . Taken together, these results indicate LAA showed the highest variation in terms of Ca2+ handling properties.In the baseline condition, when PV was stimulated, TD50. The only significant decrease was for FW when the stimulation originated from PV (p = 0.02). However, the difference in TD50 between the three regions was more pronounced during acetylcholine perfusion, especially when PV was the stimulation site.The effects of acetylcholine perfusion were modest on TD2+ homeostasis is non-homogeneous across LA. The heterogeneity of Ca2+ cycling means that if the trigger is localized in PV, the different regions of the LA may react discordantly, making some areas more likely to generate arrhythmias  by supporting re-entry circuits. These results are consistent with numerous studies reporting that PV is an important source of ectopic activity, triggering FA events (see Introduction).These results indicate that Cam-Ca2+ delay (Δt). Simultaneous Vm and Ca2+ activation maps were compared between three regions of pacing in LA, as shown in To get more insights into the potential arrhythmogenicity of these regional differences, we quantified the Vp = 0.006; vs. PV: 36.5 ± 5.1 ms p = 0.54) when FW was stimulated. Acetylcholine significantly shortened the Vm-Ca2+ delay by −55 ± 3% on average . As a result, one can assume that if ectopic activity emanates from the PV, the FW appears to be a zone where additional rotors can emerge. Indeed, a short Vm-Ca2+ delay is a measure of the arrhythmogenic susceptibility of a tissue i during an AF episode.m oscillated with a peak-to-peak amplitude that is ~47% of normal Vm amplitude during the pacing cycle length at 1000 ms. In contrast, [Ca2+]i oscillations were nearly completely abolished. Analysis was therefore restricted to Vm signals.Vm signals to investigate the spatiotemporal organization of atrial fibrillation episodes. We used frequency domain analysis on Vp = 0.47). Likewise, the spatial distribution of the RI showed island-like regions, but on average, no significant differences between LAA and PV regions as pacing sites were observed .There is a tendency for more rotors, but with a shorter lifespan, when AF episodes are triggered from PV E. Howeve2+ leak from the sarcoplasmic reticulum can serve as a substrate for atrial arrhythmia induction and persistence (see Introduction); therefore, we next investigated the effect of dantrolene, a RyR stabilizer, on atrial fibrillation inducibility.Animal model and human atrial cardiac myocyte studies have demonstrated that diastolic Cap = 0.31). This result suggests that RyR, without post-translational remodeling, is not involved in AF induction.Surprisingly, dantrolene does not reduce AF inducibility  Ca2+ cycling in the PV region is different from that in the LAA and FW regions, especially during short action potentials and simulation sites in this area. (2) There is no apparent relation between abnormal Vm-Ca2+ delays and AF susceptibility. (3) Dantrolene, a RyR antagonist, does not prevent AF susceptibility without remodeling.The purpose of this study was to determine whether Cam and [Ca2+]i have been extensively applied in single cardiac cell studies using microelectrodes or patch-clamp techniques and Ca2+ fluorescent indicators at the cellular level. At the tissue level, this challenging approach is less used, especially in the atria. The first data from the ventricle demonstrated that by combining Vm-sensitive and Ca2+ indicator dyes with similar excitation but different emission spectra, cross-talk between Vm and Ca2+i recordings could be avoided i show peak-to-peak oscillation during AF that is ~20% of the amplitude of the normal intracellular Ca2+ transient. This is less than what is observed in ventricular fibrillation using a similar approach  [2+ transient, which is smaller in the atria than in the ventricles [2+ silencing phenomenon observed at the cellular level [2+ maps in the LA suggests a causal link between the regional Ca2+ cycling, DADs, and triggered ectopic activity.A short Vf the LA . This def the LA ,29 and hf the LA . Disappog, ~30%) . It may ntricles  and the ar level . However2+ mishandling as a trigger. In addition, acute episodes of AF, in the absence of stress , might not involve a dysregulation of the RyR. Further work is needed to evaluate Ca2+ cycling at the cellular level, e.g., calcium sparks, including in the presence of dantrolene, but also using techniques such as western blotting and immunocytochemistry.In our study, we showed that dantrolene does not prevent AF episodes in sheep. However, dantrolene did appear to suppress AF induction in a sheep model of left myocardial infarction . This obOne limitation of the current study is that AF was induced acutely in sheep hearts by Ach infusion and rapid pacing, which may not be relevant to persistent or chronic forms of AF in humans. Indeed, the acute model of AF does not incorporate pathophysiological structural remodeling, which could conceivably compound the degree of desynchrony observed across the transmural wall. However, in the clinical electrophysiology laboratory, atrial pacing is often performed to induce paroxysmal AF in patients. Because more than 70% of pAF is thought to be driven by enhanced spontaneous activity originating in the PV, rapid atrial pacing may be most relevant to uncovering increased susceptibility to pAF in humans .2+ handling in different regions of the LA of the sheep, an animal relevant to human pathophysiology. The underlying heterogeneities appeared to enhance Ca2+ cycling, with relevance to atrial fibrillation susceptibility. The novel experimental insights we obtained into fundamental arrhythmogenic mechanisms in AF may facilitate the development of safer and more effective mechanism-based therapeutic strategies.In this study, we evaluated the tissular determinants of intracellular Ca


Atrial fibrillation (AF) is the most persistent arrhythmia today, with its prevalence increasing exponentially with the rising age of the population. Particularly at elevated heart rates, a functional abnormality known as cardiac alternans can occur prior to the onset of lethal arrhythmias. Cardiac alternans are a beat-to-beat oscillation of electrical activity and the force of cardiac muscle contraction. Extensive evidence has demonstrated that microvolt T-wave alternans can predict ventricular fibrillation vulnerability and the risk of sudden cardiac death. The majority of our knowledge of the mechanisms of alternans stems from studies of ventricular electrophysiology, although recent studies offer promising evidence of the potential of atrial alternans in predicting the risk of AF. Exciting preclinical and clinical studies have demonstrated a link between atrial alternans and the onset of atrial tachyarrhythmias. Here, we provide a comprehensive review of the clinical utility of atrial alternans in identifying the risk and guiding treatment of AF. Atrial fibrillation (AF) is the most common cardiac arrhythmia observed today, affecting roughly 33 million people globally . DespiteAF was first shown to be instigated by ectopic triggers located in the atrial sleeve of the pulmonary veins by Haïssaguerre et al. . Radio-fTo improve the likelihood of success, multiple theories for identifying triggers and ectopic activity initiating outside the pulmonary veins and instigating AF have been investigated. It is now widely accepted that the development and maintenance of AF require both arrhythmogenic triggers and a suitable substrate . Variati2+) and contraction, have been suggested to play a significant role in the generation of proarrhythmic substrates and facilitate re-entry phenomena that can ultimately lead to sustained AF i dynamics affect Vm regulation via Ca2+-dependent ion currents and transporters that define the bi-directional coupling of Vm and [Ca2+]i. Yet, there remains an ambiguity over the fundamental mechanism for the origin of alternans, perturbations of Vm, or [Ca2+]i regulation, as studies have validated both assumptions. Therefore, the mechanisms of alternans remain incompletely understood, especially in atrial tissue.Cardiac alternans, first described by Heinrich Hering in 1908 as a cyclic beat-to-beat alternation in contraction force, intracellular Caial (Vm) . Vm dire+) ion channels and their subunits, such as the ultra-rapid delayed rectifier current (IKur) and the transient outward K+ current, initiate faster atrial repolarization, leading to shorter APDs compared with ventricular myocytes [2+ and K+ dynamics. AF may lead to reduced mRNA expression for subunits of L-type Ca2+ channels, resulting in intracellular Ca2+ overload [+ current. These interactions between the ion channel modifications due to aging or ischemia promote shortening of the atrial APD during AF and facilitate atrial APD maladaptation [In the case of atrial tissue, irreversible damage to the atrial substrate over time due to aging causes severe atrial remodeling that can lead to alterations in P-wave duration and morphology . Old agemyocytes . Howeveroverload , as wellaptation ,14.Other factors like intensifying calcium oscillations due to atrial stretch and dilation, remodeling of connexins, and gap junctions also play a role in APD changes ,18. Thes2+ release slope, which initiated Ca2+-driven alternans. These results highlighted that disturbed Ca2+ homeostasis drives proarrhythmic APD alternans in AF patients and may be significantly influenced by RyR kinetics [2+ transients and APD alternans by altering calcium dynamics and increasing their variability. Their results indicated that AF remodeling enhanced spontaneous single-cell Ca2+ release, prolonged the refractory period of the Ca2+ transient, increased the slope of the Ca2+ transient restitution curve, and eventually predisposed hearts to Ca2+ transient alternans [2+ transient amplitude and SR Ca2+ concentration. They demonstrated that the enhanced SR Ca2+ reuptake was the primary reason that HF-induced atrial electrical remodeling increased susceptibility to atrial alternans [To ascertain how cellular alterations in patients with AF impacted the manifestations of alternans at heart rates close to rest, electrophysiological remodeling associated with chronic AF was investigated using a computational model of human atrial tissue by Chang et al. . Their rkinetics , a phenokinetics ,22. Similternans . Furtherlternans  revealedlternans .The role of atrial alternans in AF pathophysiology has been investigated extensively using preclinical animal models over the past couple of decades . Atrial 2+ handling were responsible for a lower alternans threshold in older animals. It was demonstrated that key changes in calcium dynamics including decreased peak L-type Ca2+ current, increased SR Ca2+ content, decreased Ca2+ transient amplitude, and slowed reuptake of Ca2+ into the SR driven by increased Ca2+ buffering predisposed the aged myocytes to developing atrial alternans [A major risk factor for AF is age and studies conducted in sheep models have shown that aging is associated with a reduction in the threshold for action potential alternans, both in-vivo and in isolated myocytes . The maglternans . Their rlternans .+ levels and the risk of AF [+ agonists might be an applicable approach to prevent the development of Ca2+ transient alternans in rabbits [Another interesting study tested the correlation between serum Ksk of AF . In a 12sk of AF . Support rabbits . SpecifiVarious features of the P-wave duration and morphology, corresponding to atrial depolarization, have been studied to predict the incidence of AF ,31,32 T. ElectriIn a clinical study on patients with chronic and paroxysmal AF, inter-regional differences in the slopes of the restitution curves were observed to be greater in patients with chronic AF than in those with paroxysmal AF or healthy controls . It was Another recent case study reported the presence of pulsus alternans, an alternation in the pulse strength that has been linked to severe HF, instigated by atrial flutter, which ceased on cardioversion . A more 2+ channel blockers, digitalis, and amiodarone. For rhythm control, the main goal is to sustain sinus rhythm and attenuate uncomfortable symptoms to improve the everyday quality of life [The current pharmacological treatments for AF are mainly divided into three categories: rate control, rhythm control, and stroke prevention. The target audience for rate control therapy are patients aged >65 years, as these medications help reduce recurring hospitalizations by aiming to maintain a resting heart rate of <80 bpm . Commonl of life .Direct current cardioversion is an effective therapy for AF patients or AF with rapid ventricular response to restore sinus rhythm, and it is mainly recommended for AF patients who do not respond to pharmacological therapies, combined with HF or hemodynamic instability . In addiPulsed field ablation (PFA) is a promising treatment strategy wherein fast and short-duration non-thermal energy is delivered to cardiomyocytes using electroporation, which damages their structural integrity, leading to isolated death of the target cell. In comparison with other thermal ablation procedures, PFA can be delivered ultra-rapidly in sub-seconds by both percutaneously delivered endocardial and surgically placed epicardial catheters . Thanks Another surgical option is Cox maze IV procedure, which is currently the gold-standard surgical treatment for AF, with a 93% non-recurrence of AF at one year and a 78% freedom from AF at five years . FinallyIn interventional procedures, a novel study investigated the termination of spiral wave arrhythmias using light waves, given its minimal side effects and non-invasiveness . Using oThe search for substrate-driven AF assessment and treatment has been gaining increased research attention over the past decade. Variations in P-wave morphology and duration have been directly linked to the substrate dependent conduction abnormalities observed in AF patients . A novelRecent clinical studies have also demonstrated the potential of cardiac alternans as a biomarker of vagal stimulation treatment efficacy in patients with both atrial and ventricular tachyarrhythmias ,49. CardDespite the promising preliminary results highlighting the utility of cardiac alternans in assessing the risk of AF, further studies are warranted to test its efficacy in substrate mapping for guiding ablation therapy. Extensive evidence demonstrating the predictive capabilities of both T-wave repolarization and P-wave atrial alternans has been reported over the past two decades. Yet, its clinical applicability in guiding treatment strategies is limited, primarily because of the patient-to-patient variability and a non-standardized approach for calculating alternans levels, and hence the impending risk of tachyarrhythmias. Recent data suggest a more patient-specific approach would likely be more effective in guiding treatment strategies. Screening for atrial alternans may provide an indication of the risk of AF and help improve patient management strategies.


Gallus gallus) for upward- versus downward-moving visual stimuli. We found that, without any previous visual experience, chicks consistently exhibited a preference to approach stimuli that move upward, against gravity. A control experiment showed that these preferences are not driven by avoidance of downward stimuli. These results show that newborn animals have a gravity prior that attracts them toward upward movement. Movement against gravity can be used as a cue of animacy to orient early approach responses in the absence of previous visual experience.At the beginning of life, inexperienced animals use evolutionary-given preferences (predispositions) to decide what stimuli to attend and approach. Stimuli that contain cues of animacy, such as face-like stimuli, biological motion and changes in speed, are particularly attractive across vertebrate taxa. A strong cue of animacy is upward movement against terrestrial gravity, because only animate objects consistently move upward. To test whether upward movement is spontaneously considered attractive already at birth, we tested the early preferences of dark-hatched chicks ( In fact, early predispositions to approach/attend to cues of animacy have been found in young and inexperienced infants and other animals at the beginning of life –3. TheseGallus gallus) are spontaneously attracted by movement against gravity. Chicks are born with a mature sensory and motor system and have a strong motivation to approach social partners [a). Using automated tracking [To clarify whether newborn animals can use the upward direction of movement as a cue of animacy, we investigated whether newly hatched, visually inexperienced chicks  hatched in darkness in individual compartments. Each chick was tested once for 20 min (four consecutive 5 min bins) in a rectangular arena virtually divided in a starting area (54 × 60 cm), where the chick was initially located, and two stimulus areas (18 × 60 cm) (a). In the stimulus areas, two different video stimuli were simultaneously played in a loop on computer monitors , with left/right side counterbalanced between subjects. In Exp. 1, a red circle (Ø 3.28 cm) moved vertically upward or downward along the centre, with 9.81 m s−2 acceleration . In Exp. 2, the same stimuli moved at uniform speed (3.57 m s−1) . Exp. 3 used the accelerating upward stimulus from Exp. 1 and the uniform upward stimulus from Exp 2 . In Exp. 4, one stimulus moved downwards at the acceleration of terrestrial gravity on one screen, with the other screen blank . Chicks’ centroid position in the arena was automatically tracked with DeepLabCut [At the end of incubation, chicks (× 60 cm) a. In theepLabCut  and usedQ = 4.40, p = 0.221; Exp. 2: Q = 0.34, p = 0.952; Exp. 3: Q = 2.67, p = 0.446). Thus, in Exp. 1–3, we calculated an overall preference index for each chick as an average of the four bins and tested the preference against chance level with a one-sample Wilcoxon test. The latency to first approach (time elapsed between the start of the test and the first stimulus area entered by each chick) was assessed using a Mann–Whitney U-test. In Exp. 4, the Friedman test detected a significant difference across time bins , so the time bins were assessed individually with a Wilcoxon test. In Exp. 4, only one chick chose the no stimulus option, and for this reason differences in latencies could not be analysed. All tests were two-tailed; significance was set at p ≤ 0.05. Analyses were run in Python, figures were prepared using R.Friedman tests showed no significant difference across the four 5 min time bins in Exp. 1–3 −2) in opposite vertical directions . The first-approach latency was shorter for chicks that moved towards the upward stimulus . These results show that upward-moving stimuli are more attractive than downward-moving stimuli. In Exp. 2, we tested the preference for upward versus downward uniform (constant speed) movement . Chicks preferred the upward-moving stimuli , showing that the mere upward direction of movement (without acceleration) is sufficient to elicit a spontaneous preference. Again, chicks had a shorter first-approach latency for upward-moving stimuli . In Exp. 3, we directly compared the responses to accelerating versus uniformly moving upward stimuli of the same average speed . Chicks showed no preference, spending 48.5% of time (median) at the uniform moving stimulus . The first-approach latency was faster for the uniform upward movement , suggesting that chicks might be attracted to the faster movement of the uniform stimulus at ground level . Overall, our results show that upward accelerating and constant speed movements are attractive for visually inexperienced chicks.In Exp. 1, we tested the preference for upward versus downward movement, presenting chicks with red circles that moved according to terrestrial gravity acceleration . Chicks = 0.014 b. The fi (b)p < 0.001; median at subsequent times = 0, p < 0.001, figure 1h,i). These results show that the downward-moving stimulus was not aversive.Chicks are predisposed to flee away from fast-looming approaching stimuli . To test. 4The ability to discriminate between animate and inanimate objects is adaptive from early life, enabling animals to identify relevant information, find support from conspecifics and avoid threats ,15. Upwa


Circular RNAs (circRNAs) are a class of covalently closed single-stranded RNA molecules. Four types of circRNAs have been reported in animal cells, and they have typical characteristics in their biogenesis, nuclear export and degradation. Advances in our understanding of the molecular functions of circRNAs in sponging microRNAs, modulating transcription, regulating RNA-binding proteins, as well as encoding proteins have been made very recently. Dysregulated circRNAs are associated with human diseases such as acute myeloid leukemia (AML). In this review, we focus on the recently described mechanisms, role and clinical significance of circRNAs in AML. Although great progress of circRNAs in AML has been achieved, substantial efforts are still required to explore whether circRNAs exert their biological function by other mechanisms such as regulation of gene transcription or serving as translation template in AML. It is also urgent that researchers study the machineries regulating circRNAs fate, the downstream effectors of circRNAs modulatory networks, and the clinical application of circRNAs in AML. Acute myeloid leukemia (AML) is a hematological malignancy characterized by clonal expansion of myeloid blasts cells with uncontrolled proliferation in the bone marrow and peripheral blood . AML hasvia linking an upstream 5′ splicing site to a downstream 3′ splicing site  and some EcircRNAs were proposed to play important role in transcriptional regulation. EIcircRNAs such as circEIF3J and circPAIP2, facilitate transcription initiation by RNA polymerasemer II (Pol II) at the promoter of host gene through recruiting U1 small nuclear ribonucleoprotein (U1 snRNP) in human cells  Figure . Furthera Pol II  ) and 16 healthy hematopoietic stem and progenitor cell samples (HSPCs) through using ribosomla RNA-depleted RNA sequencing , revealing a poor prognosis. Through comparing differentially expressed circRNAs in bone marrow mononuclear cells between EMI-AML and non-EMI AML patients, they found that seven target genes of 17 circRNAs  revealed a poor prognosis  Figure . Through+) compared with those without FLT3-ITD mutation (FLT3-ITD-). CircMYBL2 suppressed cell apoptosis, increased cell proliferation, and promoted cell-cycle progression in FLT3-ITD+ leukemic cells but not FLT3-ITD- cells. Mechanistically, it increased translation of FLT3 kinase by promoting the PTBP1 binding to FLT3 messenger RNA (AML is characterized by aberrant differentiation and abnormal clonal expansion of myeloid blasts . It has nger RNA  (Figure nger RNA . In addinger RNA . Overexpnger RNA , while cnger RNA . While inger RNA  (Figure nger RNA .via modulating miR-1299 and S100 calcium-binding protein A7A expression (Apoptosis, or programmed cell death, plays a key role in the development and homeostasis of the hematopoietic system . Althougpression . CircRNApression . Furtherpression . In addipression .Drug resistance is one of the key factors that lead to the failure of AML chemotherapy . VariousIn conclusion, circRNAs play important role in regulating cell differentiation, cell cycle progress, proliferation and apoptosis, as well as involve in drug resistance through acting as microRNA sponges or interacting with RNA binding proteins in AML. As discussed in above, circRNAs also can regulate gene transcription and serve as translation template to exert their function. However, whether circRNAs exert their function through regulating gene transcription and serving as translation template in AML need to be further explored.CircRNAs have the potential to be diagostic and prognostic biomarkers, and therapeutic targets because that they are highly stable, cell- and tissue-specific expressed, and their expression levels often associated with clinical and pathological characteristics . Differevia intravenous injection inhibited the promotive effect on tumor growth and metastasis in a mouse hepatocellilar carcinoma model (in vitro and blocked tumor growth in GC-patient-derived xgenograft mouse model (in vivo non-small cell lung cancer xenograft model (Increasing reports in patient-derived xenograft mouse model indicated that the siRNAs specifically targeting oncogenic circRNAs can effectively suppress tumor growth . Meng etma model . Anotherse model . Recentlse model . An intese model . Ping etse model . Anotherse model . Wang etse model . Althougse model . In addise model . This mese model . Ishola ft model . Howeverft model .AML is a malignant tumor characterized by the accumulation and clonal expansion of the immature myeloid hematopoietic cells in the bone marrow, with rising morbidity and mortality . Althougin vivo. However, whether translatable circRNAs could applied to the treatment of AML required to be further investigated. Furthermore, increasing evidence indicates the siRNAs specifically targeting oncogenic circRNAs can effectively suppress tumor growth in patient-derived xenograft mouse model (Recently, Qu et al. reported that circRNA vaccine successfully elicited potent neutralizing antibodies and T cell response by encoding the trimeric receptor-binding domain of SARS-CoV-2 spike protein . Their rse model . Howeverse model . HoweverOur understanding of the metabolism and transport of circRNA within and outside the cell is also lacking. It has been reported that excessive circRNAs are transported out of the cell in exosomes . This isIn summary, although great progress of circRNAs in AML has been achieved, substantial efforts are still needed to find whether circRNAs exert their biological function by other mechainsms such as regulation of gene transcription or serving as translation template in AML. It is also urgent that scientists study the machineries regulating circRNAs fate, the downstream effectors of circRNAs modulatory networks, and the clinical application of circRNAs in AML. Better understanding of these will promote our knowledge of circRNAs in AML biology and the development of circRNAs-based diagnosis, prognosis and therapeutic methods for AML.


PIK3IP1, MXD4 and ZMAT3, were found to be FOXO target genes in our cell models. Some of these genes were recently identified as indirect p53-target genes, and their expression was found to be regulated by RFX7 transcription factor, which was found activated upon pharmacological activation of p53, e.g., by Nutlin-3. However, a clear involvement of RFX7 in our model could not be established, but an interplay between FOXO and RFX7 factors seems evident.We have investigated the transcriptional changes occurring in uveal and cutaneous melanoma cell lines upon depletion of MDMX (aka:MDM4). Computational analyses of the mRNAs/genes affected upon MDMX depletion determined that many were containing a p53-bindingsite, but even more contained a FOX recognition site(s). Since connections between MDM2 and FOXO1 had already been published, we investigated whether indeed a subset of the MDMX-regulated genes are dependent on FOXO1/FOXO3 expression. Indeed, a number of such target genes, i.e., The tumor suppressor protein p53 has an important role in cell-fate determination. In cancer cells, the activity of p53 is frequently repressed by high levels of MDMX and/or MDM2. MDM2 is a ubiquitin ligase whose activity results in ubiquitin- and proteasome-dependent p53 degradation, while MDMX inhibits p53-activated transcription by shielding the p53 transactivation domain. Interestingly, the oncogenic functions of MDMX appear to be more wide-spread than inhibition of p53. The present study aimed to elucidate the MDMX-controlled transcriptome. Therefore, we depleted MDMX with four distinct shRNAs from a high MDMX expressing uveal melanoma cell line and determined the effect on the transcriptome by RNAseq. Biological function analyses indicate the inhibition of the cell cycle regulatory genes and stimulation of cell death activating genes upon MDMX depletion. Although the inhibition of p53 activity clearly contributes to the transcription regulation controlled by MDMX, it appeared that the transcriptional regulation of multiple genes did not only rely on p53 expression. Analysis of gene regulatory networks indicated a role for Forkhead box (FOX) transcription factors. Depletion of FOXO proteins partly prevented the transcriptional changes upon MDMX depletion. Furthermore, depletion of FOXO proteins relatively diminished the growth inhibition upon MDMX knockdown, although the knockdown of the FOXO transcription factors also reduces cell growth. In conclusion, the p53-independent oncogenic functions of MDMX could be partially explained by its regulation of FOXO activity. MDM2 or MDMX in mice resulted in embryonic lethality, which was shown to be p53-dependent . Samples were pooled and sequenced on the HiSeq2500, run type paired-end 2 × 50bp + dual index on v4 reagents and flowcells. Read counts were extracted from the BAM files with FeatureCounts (version 1.5.3). For statistical analysis of the read counts, R (version 3.4.2) was used. Read counts were normalized using quantile normalization in the DeSeq package (Version 1.16.1). Differential expression analysis was performed with DeSeq. Gene regulatory network analysis was performed using iRegulon [MEL202 cells containing four Enriched biological processes (GO) were determined using String-DB version 10.5.CAPNS1 and SRPR, and in some experiments also to RPS11 and TBP. Primer sequences are shown in RNA was isolated using the SV total RNA isolation kit (Promega), after which cDNA was synthesized using the reverse transcriptase reaction mixture as indicated by Promega. qPCR was performed using SYBR green mix  in a C1000 touch Thermal Cycler . In independent experiments the expression of target genes was determined and normalized to at least two housekeeping genes After incubation with drugs as indicated, cells were harvested in Laemmli sample buffer. Equal protein amounts were separated using SDS-PAGE and blotted onto polyvinylidene fluoride transfer membranes . After blocking in TBST  containing 10% milk, membranes were incubated with the proper primary antibodies: USP7 (A300-033) and RFX7 (#A303-062A) from Bethyl Laboratories, Montgomery, TX, USA; MDMX (8C6), MDM2 (3G9) and p21 (CP74) from Millipore, Burlington, MA, USA; p53 (DO1), PUMA (G3) and MDM2 (SMP14), all from Santa Cruz Biotechnology, Dallas, TX, USA; MAD2L1 ; Vinculin  and FOXO3 (75D8); . Bands were visualized using chemoluminescence and visualized by exposure to X-ray film or imaged on a Chemidoc machine .p-values < 0.05 were considered to be significant.Data analysis was performed using GraphPad Prism version 9 for Windows . The bars present mean ± SEM. The data were analyzed using ANOVA or t-test, the MDMX-targeting shRNA expression constructs. Efficiency and kinetics of knockdown were tested by incubating the cells with doxycycline for 24, 48 and 72 h and harvesting both RNA and protein for further analysis. These experiments show that the depletion of MDMX was efficient from 24 h onwards with all shRNA constructs used . Hercts used A. Concomt MAD2L1 B. An incMDMX itself  these changes in transcriptome are mediated, we employed the computational method iRegulon, first described to determine the transcriptional effect(s) of Nutlin-3 treatment . AccordiAnalysis of the downregulated genes clearly indicates enrichment for two known repressive transcription (co)regulators, E2F4 and SIN3A. In total, 61 of the 70 (87%) downregulated genes contained one or both repressive transcription regulators binding sites .KIF23, CENPF, PIK3IP1, PTCHD4; PIK3IP1 because, from the iRegulon analysis, this gene was not identified as a p53-target gene but as a FOX target, which is confirmed in the literature [CIP1 and PUMA and the downregulation of MAD2L1 could be confirmed , also expressing wild-type p53 and high levels of MDMX. Moreover, in this cell line, upregulation of nockdown .In addition, we noticed, again, a slight effect on FOXO1 protein levels upon MDMX knockdown in these 92.1 cells . These rCDKN1A, is strongly induced upon Nutlin-3 treatment  were also repressed upon incubation with Nutlin-3  p53-independent growth arrest. We, therefore, determined to what extent the observed MDMX-induced effects on the transcriptome are p53-dependent. As an initial step, we investigated whether specific activation of p53 by Nutlin-3 in MEL202 cells would affect the expression of the same genes as observed upon MDMX depletion. As expected, we observed that the mRNA level of a classical p53 target gene, reatment . SimilarNutlin-3 . To exclaffected . In the y change . It can epletion . CutaneoCDKN1A, PTCHD4 and CYFIP2 upon MDMX depletion is fully p53-dependent  resulting in the mutation of ASP48 to GLU48 and introducing an alternative reading frame resulting in an early translation stop after amino acid position 50 . As a cohat less C.This result fits with the observation that also in the p53-KO cells the FOXO1 protein levels are slightly increased upon MDMX depletion D.CDKN1A and PTCHD4 is p53-dependently upregulated upon MDMX depletion, but increased expression of PIK3IP1 and MXD4 is p53-independent (PIK3IP1 was identified as a FOX(O) target gene, protein expression of FOXO1 was also investigated from cells treated in the same way. Indeed, upregulation of FOXO1 protein levels in 92.1 cells upon MDMX depletion was found to be p53-independent  [First, MEL202/CR-Ctrl and MEL202/CRp53 cells were transduced with lentiviruses either expressing a doxycycline-inducible control shRNA  or a doxycycline-inducible shRNA targeting . (2018) .Subsequently, these four cell lines were transduced with the above-described lentiviruses containing the doxycycline-inducible i-shMDMX#1 or i-shMDMX#2 cassette or i-shCtrl.Stable cell lines were treated with doxycycline for 72 h, after which the cells were harvested and protein and RNA extracted. In CDKN1A and MDM2, and the-putative-FOX(O) targets PIK3IP1 and MXD4.Subsequently, the mRNA expression of a number of genes was investigated B, includMDM2, CDKN1A and PTCHD4, which are almost completely lost in the MEL202/CR-p53 cells. Depletion of FOXO proteins does not affect levels of MDM2, but slightly increases CDKN1A and PTCHD4 levels. In addition, depletion of FOXOs reduces the increase in MDM2 and PTCHD4 levels upon MDMX knockdown a little, but does not affect the increase in CDKN1A. We included in our analyses of these cells the ZMAT3 gene, previously described as a p53 target gene [PIK3IP1, MXD4 and ZMAT3 expression in CR-Ctrl/i-shCtrl cells, which is completely prevented in CR-Ctrl/i-shF1F3 cells. Moreover, in CRp53/i-shCtrl cells, MDMX depletion still induces PIK3IP1 and MXD4 expression, but not ZMAT3 expression. The simultaneous depletion of the FOXOs completely prevents the induction of PIK3IP1, MXD4 and ZMAT3. Furthermore, depletion of FOXOs reduces basal levels of these genes, especially of PIK3IP1 and ZMAT3. These results indicate that the increase in mRNA levels of a number of genes upon MDMX knockdown is mainly dependent on FOXO expression, while other(s), i.e., ZMAT3, need both the expression of p53 and FOXO to be regulated by MDMX depletion. The same results were obtained in independent biological experiments.As can be seen, in the MEL202/CR-Ctrl cells the MDMX knockdown results increased mRNA levels of get gene  and accoTo verify these results in another cell line, we again used the 92.1 uveal melanoma cell line.These cells were first transduced with lentiviruses expressing either the doxycycline-inducible control shRNA or the doxycycline-inducible FOXO1/FOXO3 shRNA. Subsequently, these cells were transduced with either i-shCtrl, i-shMDMX#1 or i-shMDMX#2.Knockdown of FOXOs and MDMX was checked by analyzing protein lysates harvested after 72 h of treatment with doxycycline or vehicle .Despite the limited knockdown of MDMX knockdown by i-shMDMX#2, in the i-shF1F3 background, RNA was harvested from duplicate plates and mRNA expression of selected genes was analyzed .MDM2, CDKN1A and PTCHD4, in a partly FOXO-dependent manner. PIK3IP1, MXD4 and ZMAT3 expression are also well induced upon MDMX depletion in 92.1 cells. The increase in PIK3IP1 and ZMAT3 is essentially abrogated upon simultaneous depletion of FOXOs, as seen in MEL202 cells. Surprisingly, in 92.1 cells, the depletion of FOXO proteins results in a slight increase in MXD4 expression, but that expression is not further increased upon simultaneous MDMX depletion, still indicating that the enhanced expression upon MDMX depletion needs the presence of FOXO proteins.Depletion of MDMX increases mRNA expression levels of The i-shMDMX#2 cell line shows hardly any induction of the p53-target genes in the i-shF1F3 background, but that can be explained by the inefficient knockdown of MDMX in this cell line.The same sets of MEL202- and 92.1-derived cell lines were used to investigate whether the growth inhibition upon MDMX knockdown is, indeed, partly p53-independent, as we mentioned before, and whether the regulation of FOXOs is involved in this growth inhibition.Therefore, we determined the relative survival after 5 days of doxycycline treatment of the MEL202-derived cell lines. In First, as shown before , growth Secondly, knockdown of the FOXOs also inhibits the growth of MEL202 cells, again partly p53-dependent , indicating a proliferative function of FOXO proteins in these cells.Simultaneous depletion of MDMX and FOXO proteins reduces the relative survival of the cells more than either single knockdown. However, if the effect of MDMX knockdown is calculated relative to either the i-shCtrl + Doxy or the i-shF1F3 + Doxy, it can be seen that in the FOXO-depleted situation the knockdown of MDMX has less of a growth inhibitory effect B.In the CR-Ctrl background, relative survival is 0.288 and 0.264 for i-shMDMX#1 and i-shMDMX#2, respectively, relative to i-shCtrl/Doxy, and 0.425 and 0.606 for i-shMDMX#1 and i-shMDMX#2, respectively, relative to i-shF1F3/Doxy. In the CR-p53 background, relative survival is 0.524 and 0.428 for i-shMDMX#1 and i-shMDMX#2, respectively, relative to i-shCtrl/Doxy, and 0.651 and 0.635 for i-shMDMX#1 and i-shMDMX#2, respectively, relative to i-shF1F3/Doxy. These results suggest that MDMX stimulates the growth of uveal melanoma cell lines by attenuating both p53 and FOXO activity.The depletion of FOXO proteins has a strong effect on the relative survival of the MEL202 cells after 5 days of doxycycline, especially in the CR-Ctrl cells; therefore, we decided to perform the same experiments again with 3 days of doxycycline treatment C. As expThe depletion of FOXO in CR-Ctrl cells reduces relative survival to 71%; additional knockdown of MDMX reduces this further to 44% and 59% . On the other hand, in i-shCtrl cells MDMX depletion reduces the survival from 92% to 46% and 47%. Again, this means that the effect of MDMX depletion is stronger in i-shCtrl cells than in i-shFOXO cells, as shown in To show that the effects of MDMX knockdown and FOXO knockdown is not cell line dependent, we used again the 92.1 cell lines i-shCtrl and i-shF1F3, containing i-shCtrl, i-shMDMX#1 and i-shMDMX#2. The cells were treated for 72 h with doxycycline and relative survival was determined. In the control cells, the survival upon MDMX knockdown by both shRNAs is about 35%. FOXO knockdown reduces survival to 85%. Compared to MEL202 cells, the 92.1 cells appear a bit more sensitive to MDMX depletion but slightly less sensitive to FOXO depletion. The combined depletion of MDMX and FOXO proteins reduces relative survival to about 52% and 72%, so even less effect than only MDMX depletion . As seenVery similar observations were made in two additional independent biological replicate experiments in these 92.1 cells.These results obtained in both MEL202 and 92.1 uveal melanoma cells suggest that indeed the oncogenic function of MDMX is not only the attenuation of p53 tumor suppressor activity but also by diminution of a tumor suppressor function of FOXO transcription factors.Lastly, during the course of these studies it has been reported that the p53-target gene encoding the transcription factor RFX7 is involved in the p53-dependent upregulation of mRNA levels of genes in which no direct p53-binding site in the promoter region could be found .RFX7 at the mRNA level could be found, indicated that the expression of a number of genes, including PIK3IP1 and MXD4, which we identified as FOX(O) target genes, is indirectly increased upon p53 activation via stimulating RFX7 activity. Furthermore, some other genes relatively high up in our list of upregulated genes upon MDMX depletion, including DDIT4, TP53INP1 and PNRC1, are reported as RFX7-target genes [These studies, although no clear upregulation of et genes . Based oet genes , includiRFX7 mRNA levels upon depletion of MDMX, or in CR-Ctrl nor in CR-p53 cells, but that is not necessarily in conflict with the published results on Nutlin-3 treated cells [RFX7 could be found upon reducing FOXO levels, in a p53-independent manner. The expression of RFX5 mRNA, identified as an RFX7 target gene, is slightly downregulated upon MDMX depletion, in a p53-dependent manner. Depletion of FOXO results in downregulation of RFX5 mRNA levels (40–50%) in a p53-independent manner. These results do suggest some cross-talk between RFX5/RFX7 and FOXO transcription factors.As a first step, the mRNA expression of a number of the above-mentioned genes was investigated on the same samples as shown in ed cells . InteresPNRC1 mRNA levels, which is partly p53-dependent. Interestingly, depletion of FOXO results in a slight increase in PNRC1, which is enhanced by simultaneous MDMX depletion in a partly p53-dependent manner, which could be the result of increased RFX7 expression upon FOXO depletion. Most strikingly is the regulation of PDCD4 mRNA levels; we find no increase at all upon MDMX depletion, but a slight downregulation in a p53-dependent manner. Reducing FOXO levels more strongly downregulates PDCD4 expression levels (50–60%), essentially in a p53-independent manner. These PDCD4 results are difficult to reconcile with the earlier mentioned publication [PDCD4 is an -indirectly-p53-upregulated gene, although we cannot exclude cell line specificity.As published , activatlication , indicatRFX7 mRNA levels are not influenced by either MDMX or FOXO depletion in these cells , the authors showed that in some cases not so much the levels of RFX7 protein were increased upon Nutlin-3a treatment, but they noticed a shift towards a somewhat faster migrating protein species, an effect apparently p53-dependent.We initially analyzed the same samples as were shown in PNRC1 mRNA levels, but is not in line with the downregulation of RFX5 and PDCD4 mRNA levels upon FOXO depletion, suggesting a strong context dependency of the effects of FOXO-depletion and/or RFX7 protein activity.Interestingly, depletion of FOXO increases RFX7 protein levels, partly p53-dependent. This observation could explain the increase in These results strongly suggest an interplay between FOXO transcription factor(s) and RFX7, to some extent orchestrated in a p53-dependent manner in MEL202 cells.The effect on the RFX7 protein levels is consistent in a number of independent experiments in MEL202 cells. In the other uveal melanoma cell line, 92.1, the effect is less clear, but a slightly faster migrating band is appearing just below the main RFX7 band upon MDMX-depletion C.RFX5 and PDCD4. In our cell systems the upregulation of ‘RFX7’ target genes PIK3IP1 and MXD4 upon MDMX depletion is almost completely abrogated upon FOXO knockdown. Together these data could suggest that RFX7 and FOXO cooperate in the regulation of these genes, although our observation that RFX7 protein increases upon FOXO depletion is difficult to reconcile with that model.These experiments show that depletion of MDMX affects the migration of RFX7 protein, but we do not see a consistent upregulation of RFX7 target genes like Our studies presented here make it evident that MDMX is affecting the transcription of genes which are implicated in cell cycle regulation and apoptosis. Further analysis of the downregulated MDMX target genes indicates that E2F4- and SIN3A-mediated transcription repression is involved. To determine whether SIN3A or E2F4 is essential for the repression of these target genes will be essentially impossible; firstly, because it has been shown that in SIN3A knock-out cells E2F target genes are regulated  and, secBased on a large meta-analysis performed by Fisher and colleagues, our results suggest that depletion of MDMX liberates p53, resulting in increased levels of p21 protein which in turn leads to the activation of the DP, RB-like, E2F4 and Muvb (DREAM) complex, resulting in the repression of target genes ,74,75. OCDKN1A, TP53INP1, TSPAN11, EDA2R and CYFIP2 [PIK3IP1, MXD4 and PNRC1 is not, or only partially. All three genes were identified as ‘p53-responsive’ in previous studies, but since a clear p53 binding near the promoter region could not be found, they were not assigned as true p53 target gene status [PIK3IP1 has previously been demonstrated to be a direct FOXO3 target [PIK3IP1 and MXD4 and it could be hypothesized that MDMX could also bind and inhibit the activity of FOXO transcription factors. However, we have tried immunoprecipitations multiple times with both FOXO1 and MDMX antibodies in at least two different cell lines but no convincing interaction could be determined.The list of upregulated genes upon MDMX knockdown contains 66 genes potentially regulated directly by p53 of which multiple genes are among the most commonly found p53-activated genes, namely, d CYFIP2 , indicate status . Also in3 target . We coulWe could conclude from our double-inducible FOXO/MDMX knockdown cell lines that the growth inhibition upon MDMX knockdown is partly dependent on the expression of FOXO proteins. However, FOXO depletion inhibits the growth of our tested UM cell lines, also indicating a proliferative function of these proteins, as has been reported for breast cancer cell lines . AlthougPIK3IP1, MXD4 and PNRC1 [Recently, an important function for the transcription factor RFX7 was reported explaining the transcriptional upregulation of genes upon p53-activation but containing no p53 binding site, including some of the genes found in our study like nd PNRC1 . We notiIn conclusion, our data presented here suggest that high MDMX protein levels stimulate the proliferation of cancer cells by inhibiting the activity of p53 and FOXO transcription factor(s). Targeting MDMX as a treatment for cancer should, therefore, be focused on the depletion of MDMX protein levels and not only on the interference with p53-MDMX interaction.


Cyclic ADP-ribose (cADPR), synthesized from NAD+ by ADP-ribosyl cyclase family proteins, such as the mammalian cluster of differentiation 38 (CD38), is important for intracellular Ca2+ mobilization for cell functioning. cADPR induces Ca2+ release from endoplasmic reticulum via the ryanodine receptor intracellular Ca2+ channel complex, in which the FK506-binding protein 12.6 works as a cADPR-binding regulatory protein. Recently, involvements of the CD38-cADPR signal system in several human diseases and animal models have been reported. This review describes the biochemical and molecular biological basis of the CD38-cADPR signal system and the diseases caused by its abnormalities.Calcium (Ca Typically, cells respond to a Ca2+ signal that is generated inside the cell in response to the activation of a wide variety of extracellular signals, including those involved in nutrient, neurotransmitter, hormonal, and sensory signaling. In most cases, the initial Ca2+ signal generated in the cell is a specific increase in cytoplasmic Ca2+ concentrations resulting from the release of Ca2+ from internal Ca2+ stores (mainly the endoplasmic reticulum) or the entry of Ca2+ from the external spaces across the plasma membrane. Both routes involve the movement of Ca2+ through Ca2+ channels that are localized within these cellular membranes. While intracellular Ca2+ release from the endoplasmic reticulum occurs via channels activated by inositol 1,4,5-trisphosphate (IP3), cyclic ADP-ribose (cADPR), or Ca2+ itself (Ca2+-induced Ca2+ release), Ca2+ influx across the plasma membrane is achieved via numerous types of Ca2+ channels, including voltage-gated Ca2+ channels and store-operated Ca2+ channels as well as a variety of ligand-gated cation channels.Changes in cytosolic CaIn this review, I focus specifically on the historical background, physiological significance, biochemical and molecular biological bases, and recent topics in the pathophysiology of the CD38-cADPR signal system in mammalian cells and human diseases.2+ levels govern a multitude of cellular processes. Many extracellular stimuli mediate complex changes in Ca2+ through the production of second messengers -5-}2,7-dichloro-6-hydroxy-3-oxo-3H-xanthen-9-yl)phenoxy]ethoxy}-4-methylphenyl]azanediyl}diacetic acid) fluorescence showed cADPR released Ca2+ )  was foun3C > T]) . In addiMost recently, Takeda et al. reported IH, which is a common feature of SAS patient cells, induced the upregulation of renin in juxtaglomerular cells via the upregulation of Cd38 . The IH-


However, how Ca2+ levels are controlled during this process is barely known. Here, we report that intracellular Ca2+ contents increase in response to many senescence inducers in immortalized human mammary epithelial cells (HMECs) and that expression of calbindin 1 (CALB1), a Ca2+-binding protein, is upregulated in this context, through the Ca2+-dependent calcineurin/NFAT pathway. We further show that overexpression of CALB1 buffers the rise in intracellular Ca2+ levels observed in senescent cells. Finally, we suggest that increased expression of Ca2+-binding proteins calbindins is a frequent mark of senescent cells. This work thus supports that, together with Ca2+channels, Ca2+-binding proteins modulate Ca2+ levels and flux during cellular senescence. This opens potential avenues of research to better understand the role of Ca2+ and of Ca2+-binding proteins in regulating cellular senescence.In response to many stresses, such as oncogene activation or DNA damage, cells can enter cellular senescence, a state of proliferation arrest accompanied by a senescence-associated secretory phenotype (SASP). Cellular senescence plays a key role in many physiopathological contexts, including cancer, aging and aging-associated diseases, therefore, it is critical to understand how senescence is regulated. Calcium ions (Ca CIP1 or p16INK4a/Rb axis, which implement the cell cycle arrest or the NF-κB, Notch, and mTOR pathways which control the SASP, how senescence is regulated is not yet fully understood [Cellular senescence is a cell response to many stresses, such as oncogene activation, oxidative stress, telomere shortening, or DNA damage. This response is mainly characterized by a stable cell proliferation arrest and a senescence-associated secretory phenotype (SASP), consisting in the secretion of multiple factors, including, among others, pro-inflammatory cytokines and chemokines, metalloproteases and growth factors. The SASP can activate the immune system to eliminate senescent cells. Senescence is thus a critical barrier against abnormal cell proliferation and tumor formation. However, over time and upon chronic exposure to stresses, senescent cells accumulate and contribute to tumor progression through their SASP, by promoting stemness, epithelial-mesenchymal transition, and migration. Accumulation of senescent cells also critically promotes aging and aging-associated diseases. Even though some molecular pathways are already well known to regulate senescence, such as the p53/p212+) is a powerful secondary messenger driving multiple cellular processes, ranging from cell proliferation and secretion to cell death. Ca2+ intracellular concentrations and fluxes need to be tightly controlled to achieve a fine-tune regulation of cellular responses. A pivotal role of Ca2+ in cellular senescence has recently emerged in the literature [2+ contents and activation of calcium-dependent signaling pathways, such as the calcineurin/nuclear factor of activated T cells (NFAT) pathway, have been reported in some senescence contexts. These increased contents and activity regulate senescence outcomes [2+ levels evolve and are controlled in response to different senescence inducers, especially in epithelial cells, is barely known. Calcium , which acts as an endoplasmic reticulum (ER) Ca2+ release channel, is shown to promote cellular senescence and organismal aging by fostering Ca2+ transfers from ER to mitochondria, through mitochondria-ER contacts (MERCs) [2+ load by inhibiting ITPR3 activity [2+ signals. Calcium-buffering proteins are calcium-binding proteins (CaBP) that tightly control intracellular free Ca2+ concentrations. Indeed, they sequester free Ca2+ by their EF-hand domains and thus fine-tune Ca2+ signaling [Many proteins such as channels, transporters, or pumps can regulate Ca (MERCs) ,9,10. Deactivity . Beyond ignaling . Whether2+ observed in senescent cells. During cellular senescence in non-epithelial cells, CALB1 is not induced, but CALB2 protein might take over this Ca2+-buffering role.In this study, we discovered that the expression of calbindin 1 , a well-known calcium-binding protein, is upregulated in response to many senescence inducers in immortalized human mammary epithelial cells (HMECs) and that CALB1 buffers the rise of Ca2+ levels has been recently reported in some contexts of cellular senescence, and we and others have demonstrated that Ca2+ transfer from endoplasmic reticulum to mitochondria promotes cellular senescence [2+ level alterations are a classical mark of senescent cells, we measured Ca2+ levels in HMEC in proliferation versus senescence using different types of senescence inducers: oncogenes, the chemotherapeutic agent bleomycin, or the p53 stabilizer nutlin-3. For oncogene-induced senescence (OIS), telomerase-expressing HMEC (HMECT) were infected with retroviral vectors encoding Mek or Raf oncogenes in fusion with the ligand binding domain of estrogen receptor (ER) , inducible with 4-HydroxyTamoxifen (4-OHT). These HMECT expressing inducible oncogenes enter OIS upon oncogene activation by adding 4-OHT [2+ probe Fura2, we assessed Ca2+ contents both in the cytosol and in the intracellular stores . The size of intracellular Ca2+ stores was appreciated as the Ca2+ increase in the cytosolic Ca2+ compartment due to the ionomycin-induced membrane permeabilization and the subsequent organelles’ depletion obtained in the absence of external Ca2+. All the senescence inducers provoked an increase in the resting cytosolic Ca2+ concentration and in the intra-organelle Ca2+ content , but induction of mRNA levels of CALB2 , another well-known calcium-buffering protein  production and contributes to ER stress. We recently showed that during senescence, mitochondrial Ca2+ concentration increases notably due to Ca2+ transfer from the ER promoted by MERCs. Accumulation of Ca2+ in mitochondria induces ROS generation and DNA damage, which lead to cell entry into senescence [2+ in the regulation of SASP has also been reported as the precursor of interleukin 1α (IL1α), a key upstream SASP pro-inflammatory cytokine inducing the expression of other SASP cytokines such as IL6 and IL8, is processed by the calcium-dependent calpain protease [2+ also activates mTOR and NLRP3 [2+ in senescent cells might contribute to many features of senescent cells [Together with other works which reported increased Cacontexts ,9,10,11,tood yet ,9,10,11.nescence ,9,10. A protease . Ca2+ alnd NLRP3 ,27, whicnt cells .2+ seems to result in the up-regulation of calcium-buffering protein, CALB1, in senescent HMECT. Indeed, according to our results, chemical inhibition of the calcium-dependent calcineurin/NFAT pathway largely inhibits the upregulation of CALB1 in senescent HMECT, whereas constitutive expression of NFATc1 transcription factor activates CALB1 expression in HMECT. In this latter condition, mRNA level of overexpressed NAFTc1 is very high compared to the level of induction of CALB1 messenger. This difference could be explained by a low level of transcriptionally active NFAT protein and/or CALB1 promoter saturation and/or limitation of cofactors. Our findings are in accordance with a previous report describing that NFAT pathway upregulates CALB1 in another context [2+ release channel by directly binding to its promoter [2+ signaling at several levels. Up to now, this pathway has been reported either to promote or suppress senescence depending on the cell type and context [2+ signaling during cellular senescence.This rise of Ca context . Importapromoter , ITPR2 bpromoter ,9,10. Th context ,29, and 2+ contribution was not investigated in this study. We showed that CALB1 overexpression in HMECT buffers the rise in intracellular Ca2+ contents in oncogene-induced senescence. However, we did not observe in our preliminary experiments any impact of this overexpression on cell proliferation arrest induced by Mek activation (data not shown). CALB1 was reported to protect cells from death by buffering Ca2+ [2+ sensor, interacting in a calcium-dependent manner with other proteins. Notably, after Ca2+ fixation to its EF-hands, CALB1 interacts with and inhibits caspase 3, a key effector caspase in the apoptosis process [2+ concentration triggers cell death, one interesting hypothesis would be that CALB1 expression is upregulated in response to senescence-inducing stresses in order to buffer the rise in intracellular Ca2+ levels and avoid its cytotoxic effects. This negative feedback loop would be established by the calcium-dependent calcineurin/NFAT pathway. The observation of an increased expression of calbindins in response to many senescence-inducing stresses in several cell types suggests that this could be a general mechanism in senescent cells. This idea will need to be explored by investigating the impact of downregulating the expression of calbindins on senescence.The role of CALB1 in senescence remains to be explored. It was recently reported that a high level of CALB1 could inhibit senescence induction by promoting p53 degradation through HDM2 activation in cancer cells , but Ca2ing Ca2+ . Moreove process . This in process . As a toAltogether, by identifying the induction of the expression of calbindins in senescent cells, our work points out a new feature of senescent cells and provides a better understanding of calcium-related processes in cellular senescence. Here, we focused our work on normal senescent cells, it will also be interesting to investigate whether these mechanisms are shared by senescent cancer cells, as some molecular features they express are different . This wo2. (Z)-4-hydroxytamoxifen (4-OHT) (Sigma-Aldrich) was used at 100 nM, bleomycin  at 12 µg/mL, nutlin-3 (Sigma-Aldrich) at 1 µM, H2O2 (Sigma) at 250 µM, TGFβ  at 0.5 ng/mL, KCl (Sigma-Aldrich) at 65 mM and FK-506  at 2 µM.Human mammary epithelial cells  were cultured in a mammary epithelial cell growth medium  supplemented with 1% penicillin/streptomycin . Human fetal lung fibroblasts MRC5 (ATCC), 293 GP retrovirus-producing cells, and 293 T lentivirus-producing cells  were cultured in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum  and 1% penicillin/streptomycin . All the cells were cultured at 37 °C with 5% COHMEC were first infected with pWZL-Blast-Flag-HA-hTERT retroviral vector   according to the manufacturer’s instructions. RNAs were reverse transcribed with First-Strand cDNA Synthesis Kit  following the manufacturer’s recommendations. TaqMan real-time quantitative PCR was then run with PCR mixture containing TaqMan mix (Roche), 200 nM of primers, Universal Probe Library probe , and cDNA template. Reactions were performed in triplicate on a FX96 Thermocycler (Bio-Rad). The comparative Ct (ΔΔCT) method and normalization with GAPDH housekeeping gene were used to calculate the relative amount of mRNA. Sequences of primers are as follows: CALB1:Sens 5′-aagatccgttcggtacagctt-3′, Anti-sens 5′-ctgaaggatctgtgcgagaa-3′; CALB2:Sens 5′-tcatttcctttttgtttttctcg-3′, Anti-sens 5′-gcgatcttcacattttacgaca-3′; NFATc1: Sens 5′-ggtcagttttcgcttccatc-3′, Anti-sens 5′-ccaaggtcattttcgtggag-3′; GAPDH: Sens 5′-agccacatcgctcagacac-3′, Anti-sens 5′-gcccaatacgaccaaatcc-3′.Cells were lysed in 6X Laemmli buffer  with 15% β-mercaptoethanol and boiled for 5 min at 100 °C. After measuring protein concentrations with Bio-Rad Protein Assay kit according to the manufacturer’s recommendations, total cell extracts were separated using 12% acrylamide gel by SDS-PAGE electrophoresis, and proteins were transferred to nitrocellulose membranes . Membranes were blocked for 1 h in Tris buffer saline , with 0.05% Tween-20 (TBS-T) and 5% milk and primary antibodies added and incubated overnight at 4 °C. An anti-CALB1 rabbit polyclonal antibody  and anti-α-tubulin mouse monoclonal antibody  were used. After washes in TBS-T, membranes were incubated for 1 h at room temperature with anti-rabbit  or anti-mouse  HRP-coupled secondary antibody (1/5000 dilution). Peroxidase activity was revealed using an enhanced chemiluminescence Western Blotting detection reagents  and visualized by luminography.Cells plated on Lab-Tek chamber slides (Thermo Fisher) were washed once with PBS and fixed with ice-cold methanol 10 min at −20 °C. Cells were then rinsed once in TBS-T and incubated in TBS-T-20% FBS for 30 min at room temperature. Cells were then incubated overnight at 4 °C with primary antibody against CALB1 . The following day, cells were washed and incubated with anti-rabbit IgG coupled with Alexa Fluor 488  for 1 h at room temperature. Cells were then washed with PBS and counterstained with Hoechst (Sigma-Aldrich) for 10 min at room temperature. Pictures were taken with a Nikon fluorescence microscope (CRCL cellular imaging platform).2+ experiments were achieved as previously described [2+-containing buffer . Live Ca2+ imaging was performed at room temperature and in the absence of external Ca2+ . MetaFluor 6.3  was used for image analysis. After 2 min at resting state, cells were stimulated with 1 µM ionomycin in order to deplete intracellular Ca2+ stores. Results are presented as a ratio of fluorescence intensity of 340/380 nm (F340/F380) as this ratio is strictly related to intracellular free calcium [Cytosolic Caescribed . Briefly calcium .t-test  or Mann–Whitney test  was used unless stated otherwise in the figure legends. p-values are indicated in figures.Graphs show means and SEM obtained with several independent experiments (n indicated in figure legends) or show data extracted from transcriptome analyses as mentioned in figure legends. Statistical analyses were performed with GraphPad Prism 8 software and are specified in figure legends. Before proceeding to any analysis, the normality of the samples was evaluated. Unpaired


Duranta erecta Linn. has a longstanding history for use in folk remedy for several disorders. Its hydroalcoholic extract has been investigated intensely in the treatment of many ailments, but to date very few data are presented to explain the pharmacological use of its oil. In this study, the chemical profiles of the leaf oils extracted from three Egyptian Duranta erecta cultivars, namely ‘Green’, ‘Golden edge’, and ‘Variegata’ are traced using GC-MS analysis. D. erecta ‘Green’ showed predominance of vitamin E (22.7%) and thunbergol (15%) whereas D. erecta ‘Golden edge’ and ‘Variegata’ contained tetratetracontane as a major component in their oils. The highest phenolic and flavonoid contents, displayed as gallic acid and rutin equivalents per gram oil, respectively, were observed in the ‘Golden edge’ and ‘Variegata’ cultivars, which was reflected by their strong DPPH and ABTS scavenging activities as well as the highest reducing power in both CUPRAC and FRAP assays. D. erecta ‘Green’ displayed better metal chelating potential, which may be attributed to its content of vitamin E. All cultivars showed similar enzyme inhibitory profiles. The best inhibition of α-glucosidase and α-amylase was observed by D. erecta ‘Green’. In silico studies of the major constituents docked on the active sites of the target enzymes NADPH oxidase, amylase, glucosidase, butyrylcholinesterase, and tyrosinase revealed high binding scores, which justified the biological activities of the tested oils. Duranta erecta (D. repens) Linn. (Verbenaceae) has long been traditionally used as a folk medicine in countries such as Brazil, Nigeria, India, Philippines, and Bangladesh [D. erecta hydroponics were of potential use for the removal of domestic pathogens from wastewater [n-hexane fraction [ngladesh . The ricstewater . This flstewater , antivirstewater , antimicstewater , urolithstewater , antioxistewater ,7,8,9, astewater ,11, antistewater , anticoastewater , antihypstewater ,15,16, lstewater , and antstewater ,18 activfraction .D. erecta alcoholic extract and its pure isolated compounds on controlling glucose levels. Flavonoid-rich fractions displayed promising α-glucosidase and α-amylase inhibition, therefore reducing the sharp rise of glucose after meals [D. erecta hydroalcoholic extract over thirty days showed significant improvement in their glucose tolerance, with a subsequent reduction in insulin resistance. Network pharmacology supported by in silico studies revealed that there are nearly 36 different metabolites in D. erecta that modulate up to 31 different targets and pathways involved in the pathogenesis of diabetes, with tyrosine phosphatase 1B and phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) being the chiefly regulated target and pathway, respectively [D. erecta enhanced the glucose uptake in yeast where in silico studies revealed that α-onocerin (one of the constituents of the extract) had the highest binding score to glucose transporter-2 protein. Isoprenylated flavonoids in D. erecta alcoholic extract significantly inhibited prolyl endopeptidase, whose levels seem to change in neuropsychiatric disorders such as Alzheimer, mania, and depression [Hyperglycemia induces a sequence of biochemical, phenotypic, and pathological alterations which could increase the risk of cardiovascular disorders and promotes the accumulation of free radicals and advanced glycation end-products leading to hyperglycemia-induced oxidative stress . Insuliner meals ,23. The er meals . Anotherer meals  revealedectively . Patil eectively  showed tpression .Duranta presented the medical efficacy of its alcoholic extract especially as enzyme inhibitors, very few conclusive data were reported on the activity of its oil. Herein, we present the GC-MS metabolomic analysis of the oil obtained by the n-hexane extraction of the leaves of three different cultivars of D. erecta growing in Egypt, viz. ‘Green’, ‘Golden edge’, and ‘Variegata’. The total phenolic and flavonoid content of their extracts are studied with correlation to their in vitro antioxidant activities and their reducing power through DPPH, ABTS·+, CUPRAC, FRAP, metal chelation, and phosphomolybdenum assays. Moreover, their enzymatic inhibition properties are assessed on acetylcholine esterase, butyl cholinesterase, tyrosinase, amylase, and glucosidase enzymes. In silico studies are implemented which further supported the bioassays.Since the majority of reports on Duranta erecta, viz. ‘Green’, ‘Golden edge’, and ‘Variegata’ (ca. 100 g each) were collected in February 2022 from a botanical garden in Nasr City, Cairo. Authentication of the plant samples were kindly performed by Taxonomy Specialist Terase Labib, Consultant of Plant Taxonomy at the Ministry of Agriculture, and El-Orman Botanical Garden, Giza, Egypt. The leaves were dried, powdered, and macerated overnight in 100 mL n-hexane. The extract was filtered, and the filtrate was concentrated under vacuum. This process was repeated thrice over three consecutive days (3 × 100 mL) to yield 1.31 mg, 1.12 mg, and 1.09 mg of dark yellow (D. erecta ‘Green’), light yellow (D. erecta ‘Golden edge’), and light yellow (D. erecta ‘Variegata’) oily residues, respectively, which were subjected to GC-MS analysis and biological investigations. Voucher specimens  were deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, Cairo, Egypt.The fresh leaves of three different cultivars of v/v in n-hexane), and oven and injector temperatures adjusted to 50 °C and 280 °C, respectively. Initial column temperature was set at 50 °C then gradually increased at a rate of 5 °C/min until reaching 300 °C, allowing metabolites of different boiling points and molecular weights to get separated at 70 eV EI ionization mode. Identification of oil components was based on comparison of their mass spectra and Kovat’s indices to those of a series of n-alkanes (C8–C30) injected under the same GC condition and to those reported in NIST online mass library, as well as matching with the literature [GC-MS analysis was performed on a Shimadzu QP2010  coupled to a quadrupole mass spectrometer. Separation was done on the low polarity diphenyl dimethyl polysiloxane Rtx-5MS (30 m × 0.25 mm i.d. × 0.25 µm thickness) capillary column  in a split injection mode with a split ratio 1:15. Helium was the carrier gas flowing at a rate of 1.37 mL/min, sample injection volume 1 µL  was used to determine if there were any differences in the tested extracts. The Pearson correlation test was used to examine the relationship between total bioactive components and biological activities. The statistical procedures were performed by using GraphPad 9.0.® software [The X-ray 3D structures of NADPH oxidase, butyrylcholinesterase, tyrosinase, α-amylase, and α-glucosidase were downloaded from the protein data bank using the following IDs: 2cdu, 6esj, 5m8q, 4gqq and 3wy2, respectively. All the docking studies were conducted using MOE 2019software , which wn-hexane from three Egyptian cultivars of D. erecta (D. repens), viz. ‘Green’, ‘Golden edge’, and ‘Variegata’ is presented in D. repens, which although obtained from the same country (Nigeria) and under the same extraction protocol (hydrodistillation), their metabolic profile was different.A detailed chemoprofiling of the oils extracted by p-vinylanisole (11.7%), whereas styrene (52.5%) and palmitic acid (55.7%) were the major constituents of the oils obtained from the fruits and roots, respectively. The stem oil was in contrast rich in alkanes such as tetracosane (22.2%), tricosane (19.3%), pentacosane (18%), and docosane (14.2%). Here, the leaf oils of the three Egyptian D. erecta cultivars showed dissimilar metabolic profiles to a large extent  and the triterpene steroidal alkene 24-norursa-3,12-diene (12.09%). Octadecatrienoic acid ethyl ester (5.4%) and olean-12-en-3-one (5.7%) were present in minor percentages. The leaf oil of D. erecta ‘Golden edge’ was predominantely rich in the unbranched long chain alkane, tetratetracontane, constituting ca. 61.97% of the whole oil followed by vitamin E, which represented 14.5%. Similarly, D. erecta ‘Variegata’ was highly rich in tetratetracontane (75.5%) but, in contrast to the ‘Golden edge’, stigmasterol (5.03%) and squalene (4.96%) were predominant in vitamin E (3.5%) in the oil of their leaves. The structures of the major metabolites are presented in D. erecta were determined by spectrophotometric methods. The results are presented in D. erecta ‘Golden edge’, with 12.07 mg GAE/g and 8.51 mg RE/g, respectively. D. erecta ‘Green’ contained the lowest level of these bioactive compounds, which were ca. 8.51 mg GAE/g and 1.49 mg RE/g, respectively. The content of the total phenolic compounds was clearly dependent on the tested Duranta cultivars. Consistent with our results, the concentration of flavonoids and phenolics were different in the cultivars of a plant species.Phenolic compounds play a key role in the management of many diseases ,38,39,40Duranta oils, although the content of the total bioactive compounds in some of its members have been reported in several publications [To the best of our knowledge, there is no scientific data on ications ,8,41. Reications ,43. TherD. erecta cultivars and the results are presented in Duranta oils. The free radical scavenging potential was assessed using DPPH and ABTS. D. erecta ‘Golden edge’ showed the strongest DPPH scavenging ability (12.93 mg TE/g), whereas D. erecta ‘Variegata’ displayed the best ABTS scavenging capacity (8.98 mg TE/g). The results of DPPH and ABTS revealed that D. erecta ‘Green’ had no ability to scavenge free radicals. The reducing power of the oils, which is their ability to donate an electron to stabilize free radicals, were studied using CUPRAC (ability to reduce Cu2+ to Cu+) and FRAP (ability to reduce Fe3+ to Fe2+) assays. The best reducing power was observed for D. erecta ‘Variegata’ , followed by ‘Golden edge’ and ‘Green’.Currently, natural antioxidants are replaced with synthetic alternatives, which appeared to have adverse effects on humans . In thisp > 0.05).Apparently, the free radical scavenging and reducing abilities of the tested oils are consistent with their levels of total bioactive compounds. This fact was also confirmed by Pearson’s correlation analysis, and strong correlation values (R > 0.7) were determined . ConsistD. erecta ‘Green’ with 16.37 mg EDTAE/g, followed by ‘Golden edge’ (10.71 mg EDTAE/g) and ‘Variegata’ (2.77 mg EDTAE/g). The observed metal chelating ability could be explained by the presence of tocopherol in the tested oil. These findings are in accordance with those in the literature where tocopherol has been reported as a good metal chelator [Duranta, the antioxidant activities of the oils of the three cultivars of D. erecta are moderate to some extent.The chelation of transition metals is an important mechanism and prevents the formation of the most dangerous hydroxyl radical. The best ability for metal chelation was observed in chelator ,48,49. Achelator  have expDuranta erecta cultivars against cholinesterases (AChE and BChE), tyrosinase, amylase, and glucosidase. The results are presented in D. erecta ‘Variegata’. Tyrosinase is the main catalyst in the synthesis of melanin and its inhibition is important to treat hyperpigmentation problems. As presented in p > 0.05).Some diseases, such as Alzheimer’s disease, diabetes mellitus, or obesity, affect millions of people, and their prevalence is increasing every day. With this in mind, new and effective strategies are becoming increasingly popular as a subject of study. Enzymes are critical in managing the diseases mentioned above. The symptoms of the diseases could be controlled by inhibiting important enzymes. Some enzymes, such as acetylcholinesterase, amylase, and lipase, have been identified as targets for certain diseases, such as Alzheimer’s, diabetes, and obesity, respectively. In this regard, several synthetic enzyme inhibitors have been approved in the treatment of diseases, but the majority of which have unpleasant side effects ,51,52,53D. erecta ‘Green’ and ‘Golden edge’ with 0.32 mmol ACAE/g. However, the strongest glucosidase inhibition was observed in D. erecta ‘Green’ (1.31 mmol ACAE) and ‘Variegata’ (1.24 mmol ACAE). It is clear from Duranta is limited [D. erecta oils.Amylase and glucosidase were selected as targets for antidiabetic effects and the best amylase inhibition was recorded by  limited ,23,56 anThe three major compounds, viz. α-tocopherol, thunbergol, and tetratetracontane were docked into the active site vicinity of the five enzymes . As presented in For the NADPH oxidase, the compounds tetratetracontane, α-tocopherol, and thunbergol achieved docking scores of −10.3, −13.9, and −11.4 kcal/mol, respectively. Tetratetracontane interacted with the amino acid residues Cys42, Phe245, and Met33, whereas α-tocopherol interacted with Cys42, Pro298, Phe245, and Asp282 . ThunberFor the BChE enzyme, tetratetracontane, α-tocopherol, and thunbergol achieved docking scores of −11.1, −13.9, and −9.4 kcal/mol, respectively. As depicted from Tetratetracontane, α-tocopherol, and thunbergol achieved docking scores of −7, −7.8, and −7.2 kcal/mol, respectively, for the amylase enzyme. Tetratetracontane interacted with amylase only through Phe406, α-tocopherol interacted at Asp236, Ser245, and Gly285 while thunbergol bounded at Trp280, Gly281, and Gly283 .For tyrosinase, tetratetracontane, α-tocopherol, and thunbergol achieved docking scores of −8.5, −10.3, and −8.7 kcal/mol, respectively. On glucosidase, tetratetracontane, α-tocopherol, and thunbergol achieved docking scores of −12.1, −13.5, and −13.3 kcal/mol, respectively. Duranta erecta is an important herb with several medicinal and industrial applications. Phytochemical and pharmacological studies have previously reported on its hydroalcoholic extract; however, few documented its oil and properties. GC-MS investigations on three different cultivars of D. erecta growing in Egypt showed significant variations in their constituent profiles. Mono- and sesquiterpene hydrocarbons were almost absent in Duranta oil with predominance in fatty esters, acids, alkanes, alkenes, sterols, alcohols, tocopherols, and some phenolics. In vitro antioxidant assays showed that the oils displayed good radical scavenging activities and promising reducing power, which was likewise supported by the in silico data. Therefore, we strongly recommend further investigations on Duranta oils, such as toxicity studies, animal testing, and bioavailability.