S4938F-RyR2 mutation in the carboxyl-terminal was expressed in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 gene-editing technique. Ca2+ signaling and electrophysiological properties of beating cardiomyocytes carrying the mutation were studied using total internal reflection fluorescence microscopy (TIRF) and patch clamp technique. In mutant cells, L-type Ca2+ currents (ICa), measured either by depolarizations to zero mV or repolarizations from +100 mV to –50 mV, and their activated Ca2+ transients were significantly smaller, despite their larger caffeine-triggered Ca2+ release signals compared to wild type (WT) cells, suggesting ICa-induced Ca2+ release (CICR) was compromised. The larger SR Ca2+ content of S4938F-RyR2 cells may underlie the higher frequency of spontaneously occurring Ca2+ sparks and Ca2+ transients and their arrhythmogenic phenotype.Type-2 ryanodine receptor (RyR2) is the major Ca Dyadic + sparks . Sparks  myocyte . The magagnitude . Spatialtraction . Over 15 editing ,8,9,10.  of RyR2 ,14,15,16e (CRDS) ,18,19.2+ binding residues of RyR2 located at the interface between the central and C-terminal domains. Near-atomic structural analysis of RyR has identified the Ca2+-binding sites of RyR2 to be formed essentially by five amino acid residues , and the caffeine binding site to be located parallel to aromatic chain of W4645, forming a hydrophobic interaction with I4926, and possibly with hydrogen bonds of E4194, suggesting possible allosteric interaction between caffeine and calcium binding sites of RyR2 [2+ content. It has been proposed that CPVT1 or ventricular tachycardia (VT)-associated RyR2 mutations similarly increase the sensitivity of the channel to luminal Ca2+, which results in an increased open probability of the channel causing aberrant releases of calcium [2+- or caffeine-binding sites, even though they alter the caffeine responses, suggesting that such regulation is mediated through allosteric modulation of the channel. Ca2+ release deficiency-associated C-terminal domain mutations have been shown to have varied effects on caffeine activation of RyR2 expressed in HEK293 cells [2+- and caffeine-binding sites of RyR2 structure [2+ release [The main regulator of RyR2 is the cytoplasmic calcium, that binds to Ca of RyR2 ,21,22. C calcium . It shou93 cells , which rtructure . Clinica2+ signaling properties of S4938F-RyR2 mutant cardiomyocytes. We found higher frequencies of Ca2+ sparks and spontaneously triggered Ca2+ transients consistent with a larger SR Ca2+ content and lower SR Ca2+ leak but suppressed magnitude of ICa-gated or spontaneously triggered Ca2+ transients.In our study, we introduced S4938F-RyR2 mutation in hiPSCs using CRISPR/Cas9 gene editing and characterized the CaRYR2 gene of wild type hiPSC-CMs, CRISPR/Cas9 gene-editing technique was used, as described in RYR2 sequence with mutations  long  S4938F mutant, the 408 bp fragment was further digested into two fragments, resulting in three fragments of 247 + 161 + 119 bp (HpyCHIV4 restriction enzyme site (#), confirming that mutations were introduced in both gene alleles, i.e., homozygous mutations.To introduce the S4938F mutation in the utations A. After products B. Figurebp) long B. HpyCH4+ 119 bp B. Sequen+ 119 bp C showed 2+ release characteristics of S4938F RyR2 mutant cells were altered compared with WT cells, in one set of experiments we measured the cytosolic Fluo-4 AM Ca2+ transients  and SR Ca2+ release, S4938F cells failed to generate significant SR Ca2+ release signals  was also significantly suppressed in S4938F mutant hiPSC-CMs, both measured alone  and cytosolic Ca2+ rise compared to WT cells (2.74 ± 0.16 ΔF/F0 vs. 1.88 ± 0.24 ΔF/F0)  B. FractiNCX was also significantly larger in mutant compared with WT cells , and the current inactivated significantly slower , respectively , even when normalized to the smaller current density, and failed to trigger SR Ca2+ release compared with WT cells , Quantified data of I2+ sparks were measured in intact S4938F RyR2 mutant and WT cells incubated with Fluo4-AM. Two-dimensional imaging of spontaneously igniting calcium sparks were recorded from different cellular regions of interest (ROI). 2+ sparks recorded from different color-coded cellular regions in WT and S4938F mutant cells. Spontaneous sparks ignited more frequently in S4938F mutants compared with WT hiPSC-CMs (29.0 spark/s in S4938F vs. 15.7 spark/s in WT) (2+ content. The sparks histogram in 2+ sparks (labeled by #) in WT and S4938F mutant cells.Cas in WT) B, consis2+ leak, WT and mutant cells were first bathed in a normal Tyrode solution that supports spontaneous Ca2+ releases and beating and then cells were exposed to a zero Ca2+/Na+ solution containing 1 mM tetracaine, followed by rapid application of 5 mM caffeine to quantify the SR Ca2+ content. SR Ca2+ leak was quantified as the decrease in the diastolic fluorescence level in the zero Ca2+/Na+ solution and then was normalized to the SR Ca2+ content triggered by caffeine. 2+ fluorescence changes in spontaneous beating WT and S4938F cells after application of tetracaine followed by 5 mM caffeine in zero Ca2+/Na+ solution. Both the raw  coupling interval, causing episodic ventricular tachycardia .We chose to study the homozygous variant of this mutation, even though in patients this mutation is expressed as heterozygous variant, to minimize complications arising from the heterotetrametric structure of RyR2. Although this approach does not fully reflect the patient pathology, it does provide more precise data on the structure function implications of the mutation.2+, caffeine and ATP are also located. It is not surprising, therefore, that several human-disease-associated C-terminal and pore mutations are expressed as “loss-of-function” mutations. It is likely, therefore, that S4938F-RyR2 mutation affects the transmission of the activating ligand signal to the C-terminal that regulates the ion channel pore open/close state. Indeed, Ca2+-dependent activation of RyR2 in recombinant protein platforms have shown such reduced activating Ca2+ affinity in the S4938F-RyR2 mutant, consistent with our ICa-induced Ca2+ release results. It remains of interest to investigate whether “affinities” to caffeine or other activating ligands are similarly altered by S4938F mutation. We have already reported that mutations in the calcium binding site that disable CICR also suppress caffeine-triggered responses, which suggests crossover effects between calcium and other ligand binding site functions [The S4938F site is located in the carboxyl-terminal tail of RyR2 that directly lines the helix of the ion channel conducting pore in close proximity to the channel’s activation core where the channel binding sites for Caunctions . It was,2. The culture medium was replaced every 2 days and hiPSCs were passaged with Accutase (Thermo Fisher) every 4–6 days. A 10 μM quantity of Y-27632 ROCK inhibitor (Tocris) was added to the culture media for 24 h after each passage. Cell passages between No. 50 and No. 60 were used for the experiments.The wild-type (K3) line of hiPSC was established previously by Si-Tayeb et al.  and was RYR2 gene of hiPSCs. The guide sequence 5′-TTTTGCAGGAATCTTATGTC-3′ adjacent to the Pam sequence (TGG) was identified in exon105 of the RYR2 gene by CRISPR Design Tool and was cloned into the pSpCas9 (BB)-2A-Puro vector  to guide Cas9 exonuclease (RYR2 gene within the guide sequence. Site-directed mutations were introduced by homology-directed repair of the Cas9-digested site using double-stranded plasmid DNA containing a ~400 bp homologous RYR2 genome sequence with mutations (HpyCH4IV to facilitate a process screening for correctly gene-edited hiPSC clones (HpyCH4IV digestion (HpyCHIV4 restriction enzyme site (#) which was used for screening correct cell clones.CRISPR/Cas9 gene editing technique was used to introduce the S4938F mutation into the nuclease A, which utations A. The hoC clones B,C. The igestion B, and diigestion C. Two seScreening primers for S4938F:F4-5′-AGG CTG GTC TTG AAT GCC TG-3′F6-5′-AAC TCA AGC GAT CCT CCC AC-3′R4-5′-GGA GGA AAC AGC TCT TCC GA-3′.Wild type and S4938F mutant hiPSCs were differentiated into cardiomyocytes in 24-well plates, as described in detail previously ,29. Brie2+ signaling experiments. The cells were plated on Vitronectin- or Matrigel-coated 25 mm glass coverslips and were incubated for three to four days before the experiments.The beating hiPSC-CMs were grown in monolayer cell cultures for a minimum of 5 weeks before they were enzymatically  dissociated into single cardiomyocytes for electrophysiological and CaER-GCaMP6-150 cDNA plasmid  (#86918)2, 1 MgCl2, 10 glucose and 10 HEPES, titrated to pH 7.4 with NaOH. Ca2+ transient and Ca2+ sparks were recorded at 70–80 Hz with a depth of penetration of 150 nm. Image sequences and fluorescence signals were detected and analyzed with LASX Leica software . The fluorescence signals were quantified as the ratio of the changes in whole-cell fluorescence (ΔF) and the baseline fluorescence level (F0). The Ca2+ sparks were exported and analyzed with our custom-developed Con2j software [2+ spark analysis was illustrated and described previously [WT and S4938F mutant hiPSC-CMs were imaged using a total internal reflection fluorescence (TIRF) imaging system. Directly before the experiments, the cells were loaded with either 1 µM Fluo-4 AM or 1 µM Fura-2 AM fluorescent dyes for 20 min at 37 °C in Tyrode’s solution containing (in mM) 137 NaCl, 5.4 KCl, 2 CaClsoftware . The meteviously .2+ leak of the SR was measured in spontaneously contracting cells when normal Tyrode solution was rapidly switched to zero Na+/Ca2+ solution containing 1 mM tetracaine to block RyR2. After tetracaine removal, the SR Ca2+ load was measured by rapid administration of 5 mM caffeine. The SR Ca2+ leak was quantified as the difference between the baseline fluorescence level and tetracaine-induced decrease in the diastolic Ca2+ level.Spontaneously beating WT and mutant hiPSC-CMs were imaged after 25–30 min of incubation of Fluo-4 AM at 37 °C. The Ca+-based internal solution containing (in mM) 100 Cs+-Aspartate, 15 NaCl, 30 TEA-Cl, 5 Mg-ATP, 0.5 MgCl2, 0.2 EGTA, 0.3 CaCl2, 10 HEPES and 0.1 Fura-2 pentasodium salt, at pH 7.2 and an osmolality of 300 Osmls. ICa was activated by a 100 ms depolarizing voltage pulse to 0 mV from a holding potential of –50 mV. The ICa-induced SR Ca2+ release and the corresponding cytosolic Ca2+ rise were simultaneously measured with the current. The amplitude of the ICa current was normalized to the membrane capacitance termed ICa density. Single exponential fits were applied to analyze the inactivation of ICaL with tau time constants of inactivation. The magnitude of the Ca2+ fluorescence changes were quantified as ΔF/F0, where ΔF is the amplitude of the fluorescence transient and F0 is the basal fluorescence intensity. ICa tail current-induced Ca2+ transients were measured by repolarization from 100 mV to -50 mV. The Ca2+ content of the SR was measured by rapid application of 5 mM caffeine for 1 s. The carried charge was estimated by calculating the integral of caffeine activated INCX. All the experiments were performed at 25–30 °C. The data were analyzed with the Clampfit10.7 (Molecular Devices) and Origin 9 (Origin lab) software.WT and S4938F mutant hiPSC-CMs were voltage-clamped in the whole cell configuration mode of patch clamp technique. The membrane voltage was obtained using a Dagan amplifier connected to a Digidata 1320A  analogue–digital converter and pClamp software . Borosilicate patch pipettes with resistances of 2–4 MΩ were pulled using a horizontal pipette puller . The cells were dialyzed with Csp ≤ 0.05 (labeled with one asterisk) and p ≤ 0.01 (labeled with two asterisks on each plot). Statistical analysis was performed using Origin 9 software (Origin lab) and MS Excel software . The data are presented as mean ± SEM.Statistical comparisons were performed using Student’s t-test or one-way ANOVA followed by the Tukey post hoc test. The statistical significance level was set to 2+ content and more frequent Ca2+ sparks underlie the increased arrhythmogenicity of the S4938F-RyR2 mutant. The smaller RyR2 Ca2+ leak and suppressed ICa-gated calcium release are likely to result from the close proximity of the mutation and the pore.A higher SR Ca


Acute myeloid leukaemia (AML) is characterised by the malignant accumulation of myeloid progenitors with a high recurrence rate after chemotherapy. Blasts (leukaemia cells) exhibit a complete myeloid differentiation hierarchy hiding a wide range of temporal information from initial to mature clones, including genesis, phenotypic transformation, and cell fate decisions, which might contribute to relapse in AML patients.Based on the landscape of AML surface antigens generated by mass cytometry (CyTOF), we combined manifold analysis and principal curve-based trajectory inference algorithm to align myelocytes on a single-linear evolution axis by considering their phenotype continuum that correlated with differentiation order. Backtracking the trajectory from mature clusters located automatically at the terminal, we recurred the molecular dynamics during AML progression and confirmed the evolution stage of single cells. We also designed a ‘dispersive antigens in neighbouring clusters exhibition (DANCE)’ feature selection method to simplify and unify trajectories, which enabled the exploration and comparison of relapse-related traits among 43 paediatric AML bone marrow specimens.The feasibility of the proposed trajectory analysis method was verified with public datasets. After aligning single cells on the pseudotime axis, primitive clones were recognized precisely from AML blasts, and the expression of the inner molecules before and after drug stimulation was accurately plotted on the trajectory. Applying DANCE to 43 clinical samples with different responses for chemotherapy, we selected 12 antigens as a general panel for myeloblast differentiation performance, and obtain trajectories to those patients. For the trajectories with unified molecular dynamics, CD11c overexpression in the primitive stage indicated a good chemotherapy outcome. Moreover, a later initial peak of stemness heterogeneity tended to be associated with a higher risk of relapse compared with complete remission.In this study, pseudotime was generated as a new single-cell feature. Minute differences in temporal traits among samples could be exhibited on a trajectory, thus providing a new strategy for predicting AML relapse and monitoring drug responses over time scale.The online version contains supplementary material available at 10.1186/s12859-023-05561-0. Nineteen patients achieved complete remission (CR) after chemotherapy treatment, seven showed non-remission (NR), one showed partial remission, seven patients relapsed, six died due to complications, and three did not have complete follow-up information. Detailed information is provided in Additional File 5 cells per sample. FCS files created for the mixed barcoded specimens were separated into individual datasets using Debarcoder software (https://www.fluidigm.com/products-services/software), and then each sample was filtered to preserve living myeloid cells according to specific metal signals and biomarkers using Flowjo (version x.0.7) . The workflow for differentiation trajectory analysis was verified using a public dataset with samples SJ03, SJ05, SJ11, and SJ16 reported by Levine et al. . FollowiThe early-stage blast was chosen by selecting cells with a pseudotime value < 0.1; these were then visualized on a CD34-CD38 plot by overlapping raw data cells. Furthermore, five reported hierarchical signal pathway responses, G-CSF → pSTAT3, SCF → pAkt, G-CSF → pSTAT5, Flt3-L → pAkt, and IL-10 → pSTAT3, were respectively visualized with an integrative presentation of temporal expression changes before and after stimulation. In brief, temporal changes of each inner molecule in two situations were fitted by locally weighted regression (LOESS) and integrated into a time-expression coordinate system.high/CD11b+ clone as the mature terminal landmark) was applied as a reference to determine the direction of query one for determining the trajectory direction. The correct direction of the query trajectory generated a lower normalized dissimilarity value.We optimized the trajectory inferred by the proposed pipeline with the myeloid blasts of the 43 clinical samples described above. Following data collection and pre-processing, 8000 cells in each sample were selected at random and 20 surface antigens involved in normal hematopoietic processes were used for differentiation-related feature selection. For each sample, molecules with positive expression  were selected. These data were then subjected to hyperbolic arcsine (arcsinh) transformation and clustered using Phenograph for DANCE selection. We created a distance matrix representing the Euclidean distance between each of the two clusters and selected the three shortest-distance neighbouring clones to every cluster. Thus, each cluster would comprise a four-neighbouring group. In every group, the expression levels of single molecules fluctuating on different scales were calculated according to the variance of an antigen in each group; if the variance was < 0.5 in up to 25% of groups, the surface antigen would be preserved. After counting the occurrence of the DANCE-selected antigens among the 43 patients, we identified eight antigens that were selected in the majority of samples, which were combined with the constant differentiation markers CD45, CD38, CD11b, and CD34 to obtain a final antibody combination, which we refer to as the 12-antigen differentiation panel. The new panel was then used to reconstruct the trajectory for each sample. Next, the CellAlign algorithm  was adopThe 32 samples exhibiting excellent trajectory fitting and with complete follow-up information available after chemotherapy were selected for further misaligned expression analysis  × log ], where Ni is the cell number of cluster i in a given time period and Ntotal is the count of test cells along the complete trajectory. The stemness heterogeneity index of each time component was then plotted as a histogram with temporal order on the horizontal axis and the dynamics were fitted by LOESS regression. After detecting the time point of the appearance of the first peak in each sample, we integrated the peak time location in 14 patients with CR, 7 with NR, and 5 with relapse.To explain the temporal features of specific antigen combinations related to AML relapse, we selected 26 samples with a lower mean dissimilarity value than the others  were determined along with the differentiation ranking of the sample.Raw scRNA-seq datasets in FASTQ format were supplied by Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine derived from seven specimens of adult AML bone marrow, including four patients with relapse and three who achieved CR after initial chemotherapy treatment. Data alignment and pre-processing were performed according to a previous study . After vMolecules involved in the 17-gene stemness score assessment  were app+/CD45high blasts, which are generally regarded as mature myelocytes , complete remission with CD11c+ in the early state , NR (n = 7), and relapse (n = 7). Statistical analysis was used to investigate the expression levels of CD11c in the two stages of the four groups: the mean expression levels were higher in both stages of the CR (CD11c+) group compared with those of the other three groups, with the greatest difference in comparison with the CR (CD11c–) group (p < 0.001); the temporal changes in most NR and relapse patients were significantly (p < 0.05) lower than those in the CR (CD11c+) group in the two states  (p = 0.0033). In contrast to the CR (CD11c+) group, which showed significant improvement in CD11c expression in the two stages compared with that of the CR (CD11c–), the NR group exhibited a higher CD11c level only in the mature stage. Of the six patients with high CD11c levels in the primitive stage, four achieved CR, thus indicating the potential prediction of a good prognosis in AML diagnosis. In addition, most patients with poor clinical outcomes had slightly higher expression levels of CD11c during the primitive stages as compared with those in the CR (CD11c+) group.On the differentiation trajectories constructed using the 12-antigen differentiation panel, the dynamics of other tested molecules  with a lower mean dissimilarity value , suggesting that the time peak could serve as an indicator of relapse at the proteomic level. In addition, there was an earlier trend of peak time point of stemness heterogeneity in the NR samples than in the CR samples (p = 0.056), which might be related to the proliferation dynamics of blood system that would be discussed later, but makes NR group hardly to be distinguished with CR. Together, these results indicated the potential for the stemness heterogeneity temporal distribution to be used for diagnostic prediction. The dynamics of stemness heterogeneity for each sample is shown Additional File Shannon entropy is a concept that is used to generally evaluate system complexity, including the quantification of morphological and molecular diversity in AML via flow cytometry , and hasFinally, we tested the proteome-based stemness heterogeneity dynamics using scRNA-seq of three CR patients and four relapse patients. After pre-processing and cell filtering, the remaining monocytes aligned on the trajectory depended on differentiation-related RNA molecules . Specifip = 0.19); the peak time point occurred after time 3, in contrast to the first peak point at time 1 or 2 in the CR samples  than that of the 17-gene stemness heterogeneity (p = 0.19), which might indicate a better utility of a Shannon entropy-based stemness molecular complexity index.The progression of AML is accompanied by an overall down-regulation of founding genes and formation of mature phenotypes. During this process, changes of stemness along the trajectory implied an alternation between earlier and later molecules. Therefore, we assumed that the first peak of stemness heterogeneity was attributed to the initial molecules and that stemness would gradually reduce over time due to competition with evolution-related molecules. We used the 17-gene stemness score as a benchmark to evaluate the risk for AML relapse. The pseudotime point of the first trough in the 17-gene stemness score was defined as the remaining AML-founding factor capability. We found that four relapse patients approached the time point of this first trough later than the three patients who achieved CR; these patients showed relatively consistent stemness heterogeneity  is sufficient to allocate a correct pseudotime to each cell. This approach provides the capability of optimizing detection channels for other aspects of medical research, such as signal molecule drug responses and the evaluation of prognosis based on stemness.In contrast to earlier analyses, which investigated more branches or nodes and thus led to a more complex differentiation trajectory, we adopted a single-linear trajectory inference algorithm to simplify the pattern of AML differentiation. First, we selected 12 antigens by DANCE to identify the commonality of differentiation patterns among patients and then investigated the intra- and inter-tumour heterogeneity along the trajectory. Although we were able to incorporate the temporal behaviour of molecules as a similar temporal ‘yardstick’ in this study, the commonality of different trajectories needs further improvement to apply this method to a greater number of patients. Here, we conceived a group of artificial references for which biomarkers related to standard differentiation were added to specimens before mass cytometry detection. These references are expected to align more efficiently with cells on a uniform trajectory. In addition, the pipeline for analysis and algorithms require certain modifications if they are to automatically recognize the correct global and local direction of a trajectory.https://cn.string-db.org/) (co-expression score: 0.998). According to the literature, the early expression of CD11c and CD18 form integrin for the binding of C3b complement [The trajectory analysis workflow provides us new perspective of AML heterogeneity, and the temporal pattern might indicate prognosis evaluation potential. CD11c early expression is an indicator of good prognosis, but the research is restricted by the sample size. To find out the mechanism of CD11c during AML development, we assumed that CD11c was normally expressed on the surface of mature blasts. As an adhesion molecule, it plays a role in cell-to-cell interaction and recognition, which might influence the chemotherapy prognosis of AML cells. To test the first hypothesis, we investigated the CD11c-CD18 interaction in the STRING website  to identify indicators to guide the therapeutic strategy in the early stages. Furthermore, since differentiation induction therapy has shown good efficacy for AML treatment (ATRA to M3 AML patients) , this neIn this study, we developed a novel trajectory analysis pipeline that recreates the phenotypic dynamics of AML progression based on a single-cell surface antigen landscape. We then optimized the model by determining a common evolution pattern among patients through the proposed DANCE feature selection strategy. The continuous presentation of AML progression reflects an actual differentiation order of single cells and their expressed molecules. On the hierarchy, we identified that abnormal CD11c expression at the primitive stage of AML differentiation predicts a good chemotherapy outcome. We further proposed stemness heterogeneity as an indicator of relapse risk, suggesting that detection of the initial stemness maintenance might contribute to a more accurate prognosis evaluation. Overall, our proposed temporal analysis strategy provides insight into the heterogeneity of AML and may serve as a reference for clinical diagnosis and prognosis.Additional file 1. Sample information, antibody labelling, intermediate data and supplementary figures.


Early embryo implantation is a complex phenomenon characterized by the presence of an implantation-competent blastocyst and a receptive endometrium. Embryo development and endometrial receptivity must be synchronized and an adequate two-way dialogue between them is necessary for maternal recognition and implantation. Proteases have been described as blastocyst-secreted proteins involved in the hatching process and early implantation events. These enzymes stimulate intracellular calcium signaling pathways in endometrial epithelial cells (EEC). However, the exact molecular players underlying protease-induced calcium signaling, the subsequent downstream signaling pathways and the biological impact of its activation remain elusive.To identify gene expression of the receptors and ion channels of interest in human and mouse endometrial epithelial cells, RNA sequencing, RT-qPCR and in situ hybridization experiments were conducted. Calcium microfluorimetric experiments were performed to study their functional expression.3R and the STIM1/Orai1 complex. Finally, in vitro experiments in the presence of a specific PAR2 agonist evoked an upregulation of the ‘Window of implantation’ markers in human endometrial epithelial cells.We showed that trypsin evoked intracellular calcium oscillations in EEC of mouse and human, and identified the protease-activated receptor 2 (PAR2) as the molecular entity initiating protease-induced calcium responses in EEC. In addition, this study unraveled the molecular players involved in the downstream signaling of PAR2 by showing that depletion and re-filling of intracellular calcium stores occurs via PLC, IPThese findings provide new insights into the blastocyst-derived protease signaling and allocate a key role for PAR2 as maternal sensor for signals released by the developing blastocyst.The online version contains supplementary material available at 10.1186/s12958-023-01085-7. Human fertility is generally considered as a straightforward event, but even in optimal conditions human pregnancy is only achieved in approximately 30% of any given menstrual cycle . One of It was shown that pre-implantation blastocysts are capable of secreting several chemical molecules, such as growth factors, hormones and trypsin-like proteases to participate in the hatching process . Recentl2+]I) changes in response to blastocyst derived trypsin-like proteases [2). PGE2 will ultimately activate cAMP-regulated pathways in ESCs and influence the endometrial decidualization process [2+]I, it was suggested that the blastocyst-derived trypsin-like proteases would induce Na+ influx and subsequent activation of voltage-dependent Ca2+ channels (VDCC) [+ entry via ENaC would increase the intracellular Na+ concentration and reverse the sodium calcium exchanger (NCX) thereby providing means for Ca2+ entry [2+]I oscillations in Ishikawa cells [2+ influx mechanisms to regulate trypsin-induced [Ca2+]I oscillations in primary human EEC and the physiological consequences of these [Ca2+]I changes on the implantation process are currently unclear.Several molecular sensors for the detection of embryonic signals have been described in the EEC. Originally, the epithelial sodium channel (ENaC) was proposed as a potential detector of embryonic signals, initiating intracellular calcium  . In late2+ entry . In addiwa cells . Current2+]I influxes in EEC. However, we identified PAR2 as a key sensor in the detection of blastocyst signals and its capability to induce [Ca2+]I oscillations in primary EEC of both mouse and human. Ultimately, we unraveled the molecular players involved in the downstream signaling of PAR2 and present the first evidence for a biological consequence of PAR2-induced [Ca2+]I oscillations in the process of pre-decidualization in human EEC.In this study, we were unable to find evidence for the involvement of the ENaC-VDCC connection nor for the ENaC-NCX link to induce I of control mEEC was monitored using Ca2+ microfluorimetric experiments. The onset of perfusion induced a small laminar flow of 307 µl/min over the cells and mimicked shear stress (SS). Intriguingly, application of SS alone evoked [Ca2+]I oscillations in a significant portion of mEEC , for a time period of minimally 25 min, which resulted in [Ca2+]I oscillations in the majority of mEEC (63 ± 5%)    in the presence of SS, with an oscillation frequency similar to application of trypsin (0.26 ± 0.05 and 0.27 ± 0.02 oscillations/min for trypsin and elastase respectively)  Fig. A and C. 3%) Fig. B, C. In 2+]I oscillations induced by serine proteases in mEEC. Previous reports described a potential role of the epithelial sodium channel (ENaC) and the voltage dependent Ca2+ channel (VDCC) in [Ca2+]I responses to embryo-derived proteases I oscillations in mEEC. Typically, a rapid first [Ca2+]I peak was detected, followed by [Ca2+]I oscillations . Interestingly, application of thapsigargin prior to stimulation of trypsin prevented the cells from responding to trypsin completely  channels as part of the downstream signaling.The involvement of PAR2 in I oscillations specifically in EEC, but not in the underlying ESC of both mouse and human origin. Moreover, PAR2 was found to be involved in the translation of blastocyst-secreted proteases like trypsin into [Ca2+]I oscillations, a signaling cascade which depends on the PLC/IP3/SOCE pathway. In addition, the presence of trypsin in the trophectoderm of (hatching) human blastocysts was further confirmed via immune histological staining techniques. These results reveal a novel role for PAR2 in the epithelium-specific reaction to embryonic signals.The communication and complex interplay between the implanting blastocyst and the maternal endometrium are imperative during the embryo implantation process. The molecular players involved in both embryonic signal generation and maternal signal detection have been extensively studied, but a full comprehension of the mechanisms at play is still lacking. Especially the identification of molecular signals governing embryo-uterine communication, and their function in human endometrial epithelial cells remain incomplete. Recently, serine proteases have been acknowledged as blastocyst-secreted proteins that are able to act on the maternal endometrium and influence the implantation process , 12. How2+]I in EEC induced by proteases I response. We did observe that the number of responding cells was decreased upon amiloride application. However, removal of external Na+ did not alter trypsin-induced [Ca2+]I oscillations in mEEC, excluding the potential contribution of ENaC in the [Ca2+]I influx. Possibly, the effect of amiloride on trypsin responses may reflect direct modulation of GPCRs by amiloride, as has been described in earlier studies, especially for the class A GPCRs to which PAR members belong [2+]I influx induced by trypsin. Another recent study proposed PAR1 and PAR2 as important receptors to regulate the trypsin-induced endometrial [Ca2+]I signaling in Ishikawa cells I oscillations. In addition, it was proposed that Na+ entry via trypsin-activated ENaC would depolarize the cellular membrane and increase the intracellular Na+ concentration to reverse the sodium calcium exchanger (NCX) thereby providing means for Ca2+ entry I was detected upon switching to Na+-free solution, suggesting a minor contribution of the NCX in the [Ca2+]I influx.Previous reports have suggested several molecular mechanisms to explain the increased I oscillations in EEC. First, high gene counts were detected for F2rl1 (PAR2) in EEC of mouse and human. Moreover, in situ hybridization and RT-qPCR experiments further confirmed this molecular presence of F2rl2 (PAR2) in different cellular models of mouse and human. Next, functional expression of PAR2 was shown by the use of a selective PAR2 agonist, 2-fu, which could trigger a comparable [Ca2+]I response in EEC of mouse and human as trypsin. Additionally, application of the PAR2 antagonist, I-191 reversibly inhibited [Ca2+]I oscillations induced by either trypsin or 2-fu. Finally, these findings were confirmed in excised uterine tissue fragments via ex vivo Ca2+ imaging experiments, where trypsin or 2-fu were able to induce [Ca2+]I oscillations. Spontaneous [Ca2+]I oscillations could also be observed in the control condition of bath solution without perfusion, which could be explained by the induction of mechanical stimulation of the tissue during experimental handlings prior to the fluorescent measurements (e.g. cutting and tightening of the uterus). Furthermore, our study unraveled part of the underlying molecular mechanism of trypsin-induced [Ca2+]I influx in EEC. The [Ca2+]I oscillations are mainly caused via Ca2+ release from and re-uptake into intracellular stores, often effectuated via PLC activation, generation of IP3 and inducement of store operated calcium entry (SOCE). However, at the moment it is unclear what the underlying oscillator is and whether these [Ca2+]I oscillations also induce oscillations of the membrane potential. Ca2+ microfluorimetric experiments showed a strong reduction of the number of oscillating EEC when trypsin was coapplied in the presence of specific inhibitors of PLC, IP3R, SERCA and CRAC. A previous study in Ishikawa cells proposed a similar mechanism to sustain the trypsin-induced Ca2+ entry via STIM1/Orai1 store operated Ca2+ channels [2+]I oscillations could potentially be explained by the functional expression of mechanosensitive ion channels in primary endometrial epithelial cells, like PIEZO1 [Strong evidence is provided for the involvement of PAR2 in trypsin-induced [Cachannels . Howevere PIEZO1 . The co-e PIEZO1 . It was 2+]I oscillations was assessed during pre-decidualization in human EEC. Interestingly, EEC derived from human showed a significant upregulation of the WOI markers after incubation with the PAR2 agonist, 2-fu. These results indicate that PAR2-induced [Ca2+]I influx potentially play an important role in the regulation of the decidualization of stromal cells, which is key for successful implantation. At the moment additional research is required to further explore the in vivo role of PAR2 activation in the implantation process. Earlier studies did not show major fertility problems in full body PAR2 knock-out mice at first sight [2+]I responses in Ishikawa cells [2+]I responses were short-lived and organized, whereas [Ca2+]I responses induced with conditioned medium obtained from developmentally incompetent embryos were lasting and chaotic. It was speculated that [Ca2+]I patterns generated by the embryo-derived proteases would convey embryo quality towards the maternal endometrium, allowing for implantation of competent blastocysts only. Our data illustrate the presence of oscillatory Ca2+ signals in EEC induced by embryonic signals released by the outgrowing trophoblast cells. Potentially, PAR2-induced [Ca2+]I oscillations are important for the selection of competent blastocysts, in which a lack of [Ca2+]I oscillations or an overload of [Ca2+]I would influence further steps of the implantation process in human, like stromal decidualization and embryo selection.Next, the physiological impact of PAR2-induced I oscillations upon contact with trypsin, a process dependent on Ca2+ release via the PLC/IP3R/SOCE pathways. Evidence is provided for PAR2 as initiator of [Ca2+]I responses and potential regulator of pre-decidualization events in human. These findings could lead to novel clinical applications to the further optimization of the IVF-protocol to improve IVF success rates. However, additional research is required to further unravel the exact role of PAR2 in the early implantation events in human.Our research focused on the effects of blastocyst-derived serine proteases on maternal EEC, providing novel insights in the molecular players and CaAdditional file 1: Supplementary Fig. 1. Progesterone-induced uterine gland knock-out model. Supplementary Fig. 2. Trypsin induced calcium oscillations in endometrial epithelial cells. Supplementary Fig. 3. Expression of Enac in mEEC. Supplementary Fig. 4. Aprotinin has no effect on the 2-fu induced [Ca2+]I oscillations. Supplementary Fig. 5. Stromal cells do not show [Ca2+]I oscillations after stimulation by trypsin. Supplementary Fig. 6. Ex vivo Ca2+ imaging in excised uterine tissue. Supplementary table 1. overview of used RNAscope probes and RT-PCR assays.Additional file 2.Additional file 3.Additional file 4.Additional file 5.


The mitochondrial unfolded protein response (UPRmt) and mitophagy are two mitochondrial quality control (MQC) systems that work at the molecular and organelle levels, respectively, to maintain mitochondrial homeostasis. Under stress conditions, these two processes are simultaneously activated and compensate for each other when one process is insufficient, indicating mechanistic coordination between the UPRmt and mitophagy that is likely controlled by common upstream signals. This review focuses on the molecular signals regulating this coordination and presents evidence showing that this coordination mechanism is impaired during aging and promoted by exercise. Furthermore, the bidirectional regulation of reactive oxygen species (ROS) and AMPK in modulating this mechanism is discussed. The hierarchical surveillance network of MQC can be targeted by exercise-derived ROS to attenuate aging, which offers a molecular basis for potential therapeutic interventions for sarcopenia. Skeletal muscles are multifunctional organs that exhibit high plasticity and adaptability in response to changes in exercise, nutrition, and stress. The age-related decline in muscle mass, strength, and function, known as sarcopenia, is well-established and has been linked to a variety of negative health outcomes, including loss of mobility, metabolic dysfunction, chronic disease susceptibility, and increased mortality risk ,2. The uRegular physical exercise is an integral component of human life that exerts significant effects on preserving health and preventing muscle-related disorders. The soundness and operational efficiency of skeletal muscle mitochondria are crucial for sustaining physical activity because they are responsible for providing energy through oxidative phosphorylation in skeletal muscle cells. Apart from their well-established role as energy-producing organelles, mitochondria also play a vital role in regulating calcium homeostasis, a critical process for muscle contraction. Additionally, mitochondria are key signal-regulating organelles that produce reactive oxygen species (ROS) that modulate redox signaling and regulate energy-sensitive pathways such as AMPK during exercise. Mitochondria exhibit a high degree of plasticity during exercise by adapting their volume, structure, and function, such as augmenting mitochondrial biogenesis, activating mitophagy, and boosting the capacity for protein import into mitochondria and UPRmt ,7. ThereThe UPRmt and mitophagy are integrated into a hierarchical surveillance network of MQC by counteracting the adverse effects caused by mitochondrial damage . The UPRIn recent years, various signaling pathways have been found to regulate the mitophagy-mediated degradation of mitochondria. Interestingly, these pathways also activate the UPRmt, suggesting a possible coordinated control of these two systems that are responsible for the maintenance of mitochondrial quality. The available evidence demonstrates that under stress conditions, the UPRmt and mitophagy are simultaneously activated and compensate for each other when one system becomes insufficient. The coordination of the UPRmt and mitophagy is probably controlled by common upstream signaling pathways, which are part of a complex network of molecular and organelle systems involved in MQC mechanisms. Because dysfunction of the UPRmt and mitophagy is associated with certain diseases, including sarcopenia, identifying the molecular targets involved in this coordination mechanism may hold therapeutic potential for muscle-related disorders .Based on recent research evidence, the exercise-induced activation of UPRmt and mitophagy has been shown to improve muscle quality in elderly individuals. For instance, exercise has been demonstrated to enhance the muscle mitochondrial quality by improving UPRmt activation, which is associated with improvements in exercise performance, including increased muscle strength, maximum running speed, and running distance in older adults . AdditioThis review provides a comprehensive analysis of the multifaceted biological functions of the UPRmt and mitophagy and places a particular emphasis on their coordination mechanism and how it is impacted by aging and exercise. This paper also explores the bidirectional regulation of ROS induced by exercise or aging and its impact on several signaling pathways that are involved in this coordinated mechanism. Understanding the intricate mechanisms underlying mitochondrial homeostasis may provide valuable insights into the development of therapeutic strategies for the treatment of age-related muscle wasting and for promoting muscle health throughout the human lifespan.MQC is the collective term for mechanisms that maintain the quantity, morphology, and function of mitochondria, which are primarily mediated by the interplay among protein import, UPRmt, mitochondrial fusion and fission, mitophagy, and mitochondrial biogenesis ,15. ThesMitochondrial proteome aberrations, which are induced by stressful stimuli, generate biological signals that are transmitted to the nucleus, where they rapidly trigger extensive transcriptional responses and thereby prevent the accumulation of damaged proteins. This feedback loop is referred to as the UPRmt and plays a vital role in the maintenance of mitochondrial homeostasis and cellular health . In mammMitophagy is a selective autophagy mechanism that eliminates depolarized or superfluous mitochondria via the autophagosome–lysosome pathway in response to different cellular energy states, stresses, and signaling cues . The claThe UPRmt and mitophagy play critical roles in repairing unfolded proteins and eliminating damaged mitochondria, respectively. However, the endogenous UPRmt may be insufficient in repairing mitochondrial damage, resulting in inevitable mitophagy activation under stress conditions, regardless of the UPRmt status . An antaFurthermore, both the UPRmt and mitophagy systems can compensate for each other when one is inadequate. For instance, the knockdown of UPRmt proteins, including HSP60, mtHSP70, and LONP1, induces the activation of mitophagy in various tissues ,43,44,45The FUN14 domain-containing 1 (FUNDC1) receptor plays a crucial role in hypoxia-induced mitophagy at the OMM . Upon deCHCHD2, which is also known as mitochondrial–nuclear retrograde regulator 1, has been suggested as a stress-responsive protein in the IMS that enhances mitochondrial respiration by binding to COX. CHCHD2 is responsive to various cellular stresses, leading to its relocation from mitochondria to the nucleus, where it binds to the promoter of hundreds of genes, similar to ATF5, to boost their transcription . In DW7 + levels. SIRT1 is located in both the nucleus and cytoplasm. Mouchiroud et al. showed that NAD+ precursor supplementation or SIRT1 overexpression in primary mouse hepatocytes induces classical UPRmt activation in a SIRT1-dependent manner, indicating the regulatory role of SIRT1 in the UPRmt [Sirtuins, a highly evolutionarily conserved family of deacetylases, primarily depend on NADhe UPRmt . SIRT1 ihe UPRmt . Thus, ihe UPRmt . Additiohe UPRmt . Thus, SIn contrast to SIRT1, SIRT3 is a highly prominent mitochondrial deacetylase that exerts its effects on all aspects of mitochondrial functions, such as mitochondrial fission, transcription and translation, DNA and RNA processing, lipid and amino acid metabolism, the tricarboxylic acid (TCA) cycle and OXPHOS, and confers protection against oxidative stress . FurtherPhosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) at Ser51, which is a critical component of the ISR, is responsible for regulating translation initiation in response to environmental stress and is vital for organismal resilience. This activation of P-eIF2α reduces global mRNA translation while simultaneously enhancing the expression of stress response genes such as amino acid metabolism and apoptosis pathways. This phenomenon creates an opportunity for the cell to repair itself and facilitate the restoration of protein homeostasis. The four main kinases that activate P-eIF2α are heme-regulated inhibitor (HRI), general control nonderepressible 2 (GCN2), protein kinase R (PKR), and PKR-like endoplasmic reticulum kinase (PERK). HRI and GCN2 are activated by amino acid deficiency, whereas PKR is activated by a viral infection, and PERK is activated by endoplasmic reticulum stress . EmerginThe mitochondrial ORF of the 12S type c (MOTS-c) is a mitochondria-derived peptide (MDP) or a “mitokine” that plays a critical role in metabolic regulation and has the potential for health promotion and disease prevention in biomedical research. A recent study showed that endogenous MOTS-c is secreted from exercising muscles in humans . MoreoveThe hormone fibroblast growth factor 21 (FGF21) is a mitokine that ameliorates metabolic dysfunction, reduces body weight, and confers systemic protection against various diseases . AlthougIn summary, the UPRmt and mitophagy are regulated through shared upstream molecular signaling pathways, which underlie their coordinated action in ensuring mitochondrial homeostasis .Aging muscle is characterized by defective organelles, such as increased levels of mutant mtDNA (ΔmtDNA), higher susceptibility to mitochondrial apoptosis, and increased oxidative stress ,79. ThesThe impact of aging on the UPRmt has become increasingly evident, and research shows that the UPRmt is suppressed during the aging process. In skeletal muscle from aging mice, the levels of UPRmt proteins are reduced, and in muscle biopsy samples from sarcopenic humans, the expression of UPRmt genes is decreased, indicating an insufficient UPRmt response to repair or neutralize oxidative stress-induced damage to mitochondrial proteins ,83. ConvConsiderable evidence supports the notion that the UPRmt promotes the extension of the lifespan and health span across several model organisms. In worms, the epigenetic memory of the UPRmt induced by mitochondrial stress during the developmental stage is crucial for lifespan extension . In thisAging tissues are known to accumulate severely damaged organelles, particularly mitochondria. Mitophagy, the process by which damaged mitochondria are cleared, is essential for the maintenance of MQC. Although decreased mitophagy has been directly linked to age-related declines in cardiac and neural organs, the precise impact of aging on mitophagy in skeletal muscle remains unclear. Conflicting evidence has been reported: some studies indicate an increase in mitophagy, and others suggest a decrease. Possible explanations for these inconsistencies include differences in the measurement methods and the notion that an increase in mitophagy may serve compensatory functions but does not necessarily confer oxidative stress resistance because the number of damaged mitochondria continues to increase with age . Evidenc+ levels in the cytosol and nucleus of aging skeletal muscle [During cellular senescence, dysfunction of the MQC system is mainly caused by oxidative stress and redox imbalance, which leads to a breakdown of the coordination mechanism and contributes to mitochondrial dysfunction in aging organs. Although the effect of aging on FUNDC1 expression in skeletal muscle is unknown, its downregulation during aging has been reported in other organs, such as the myocardium . Additiol muscle . In cardl muscle . Similarl muscle . Interesl muscle . The levl muscle ,107,108.In summary, sarcopenia may be caused by a dysfunctional coordination mechanism that disrupts both the UPRmt and mitophagy, which ultimately compromises organelle homeostasis.Physical activity and exercise have been proposed as effective interventions for improving the quality of aging muscles or delaying the onset of sarcopenia. In particular, aerobic exercise has been shown to partially alleviate sarcopenia-related issues caused by mitochondrial dysfunction, including apoptosis, mitochondrial oxidative stress, myostatin, and inflammatory cytokines . MoreoveTo date, numerous studies have demonstrated that exercise effectively activates the UPRmt pathway in skeletal muscle. For instance, the Hood research group utilized chronic contractile activity to simulate exercise and observed increased levels of UPRmt-related mRNA and proteins in rat skeletal muscle, and similar results have been observed in murine myocytes ,111. AddIn addition, exercise has been shown to counteract the decline in the UPRmt observed with aging. In mouse gastrocnemius muscle, the mRNA levels of UPRmt-related genes, such as Yme1L1 and CLpP, decrease with aging; however, 4 weeks of aerobic exercise increase the expression of these factors, in line with an increase in mitochondrial content . SimilarAcute exercise has been shown to induce mitophagy in skeletal muscle during the recovery period through AMPK-mediated phosphorylation of ULK1 at Ser555 . This prMitophagy has emerged as a critical mechanism underlying exercise adaptation in chronic diseases and aging. Lifelong exercise training can mitigate age-related decreases in the LC3II and BNIP3 levels in human muscle, which are mediated through the PGC-1α-p53 axis . The oveAlthough the role of FUNDC1 in exercise has not been fully explored, studies have shown that the electrical pulse stimulation of C2C12 cells increases the FUNDC1 levels, elevates the LC3-II/I ratio and reduces the p62 levels, whereas the inhibition of FUNDC1 upstream factors decreases its levels, resulting in reduced colocalization of LC3, FUNDC1, and COXIV, suggesting that FUNDC1 mediates exercise-induced mitophagy . In mousIn summary, moderate-intensity muscle contraction cumulatively enhances both the UPRmt and mitophagy and consequently restores organelle homeostasis during old age through the pivotal molecular signaling pathways that regulate the coordination mechanism.2O2 and NO, which often serve as redox-sensitive signals for regulating protein metabolism and mitochondrial function. Recent evidence suggests that moderate exercise enhances the redox balance by upregulating compensatory antioxidant capability, which is the main mediator of the health effects of exercise. This finding contrasts with antioxidant intervention, which inhibits oxidative stress and induces a low-level redox balance, reducing or abolishing exercise-associated stress and preventing the health-promoting effects of exercise. Our group recently demonstrated that inhibiting mitochondrial ROS generation in mice during exercise using MitoTEMPO, a novel cell-penetrating antioxidant targeting mitochondria, reduces the increases in the c-Jun, HSP60, and CLpP levels in the classical pathway of the UPRmt in skeletal muscle, indicating that exercise-derived ROS stimulate the UPRmt [Mitochondria are considered the primary sources of ROS production during exercise, although some researchers suggest that nonmitochondrial sources, such as NADPH oxidases and xanthine oxidases, may also contribute to this effect . Antioxihe UPRmt . Additiohe UPRmt . ROS are2O2 conditions (approximately 10–100 µM), AMPK is activated by glutaredoxin-mediated S-glutathionylation and phosphorylation [2O2 (approximately 200 µM) results in reductions in the S-glutathionylation, phosphorylation, and protein content of AMPK. This observation is consistent with evidence indicating that excessive exercise-induced oxidative stress decreases AMPK expression and disrupts the mitochondrial structure. These divergent effects of ROS on AMPK activity highlight a bidirectional regulation of AMPK by ROS  activates miR-34a through PI3Kα in human bronchial epithelial cells, and miR-34a binds to the 3’UTR of SIRT1 mRNA, inhibiting SIRT1 expression [+ levels. In vivo studies have demonstrated that a decline in NAD+ levels can impair SIRT3 activity, leading to mitochondrial dysfunction and redox disruption due to the acetylation of key enzymes and transcription factors [2O2 upregulates the P-eIF2α/eIF2α and CHOP levels in C2C12 myoblasts [Acute exercise has been demonstrated to increase AMPK phosphorylation at Thr172 and ULK1 phosphorylation at Ser555 in skeletal muscle . Our pres organs . Furthers organs . Based os organs , indicatpression . The exppression . Consequpression . Phloretpression . Converspression . The act factors ,143. Mor factors . Therefo factors . YME1L1  factors . These f factors . Acute eyoblasts . Under myoblasts . Thus, RRecent research has uncovered the involvement of mitokines, specifically the exercise-induced translocation of N1 hypothalamic neuronal MOTS-c to the nucleus for gene transcription. Interestingly, the regulation of MOTS-c appears to be dependent on ROS produced at moderate levels, as evidenced by the reversal of MOTS-c translocation upon treatment with the antioxidant N-acetylcysteine ,150. Fu Exercise-derived ROS act as molecular signals of exercise adaptation that mediate the coordination of the UPRmt and mitophagy through the AMPK-mediated redox-sensitive pathways discussed above.Despite considerable progress toward understanding the classical regulators of the UPRmt and mitophagy, the underlying crosstalk mechanisms remain incompletely understood. The coordination mechanism mediates the activation of both processes under certain conditions, allowing them to compensate for each other when either process is insufficient and thus enhancing mitochondrial homeostasis and vitality. Multiple regulatory signaling pathways mediate this coordinated response, allowing mitochondria to adapt to physiological stress and maintain muscle function. However, this mechanism is attenuated in aging skeletal muscle, leading to abnormal mitochondrial homeostasis and sarcopenia development. Exercise restores the coordination mechanism and subsequent UPRmt and mitophagy to mitigate mitochondrial dysregulation in aging and sarcopenia. The concept of “ROS hormesis” describes how mild exercise-induced redox-sensitive signaling can result in beneficial adaptations that protect against more severe stresses, such as aging. The reductions in physical activity and cellular function during aging can trigger oxidative stress and sarcopenia. However, moderate and transient ROS production during exercise appears to be beneficial. Aging-induced ROS inhibit AMPK and its downstream signaling pathways and thereby suppress the coordination mechanism. Exercise-derived ROS activate AMPK, stimulating coordination, stabilizing mitochondrial homeostasis in aging, and preventing or reversing sarcopenia. The bidirectional regulation of the coordination mechanism by ROS and AMPK highlights the regulatory networks coordinating MQC . Identif


Sporadic Renal Angiomyolipoma (AML) is commonly managed through transcatheter arterial embolization (TAE) or surgical intervention. In this report, we present a remarkable case of tumor regression from Novick Classification level 2 to level 1 in a 54-year-old male with sporadic renal AML and inferior vena cava (IVC) involvement, treated with the administration of everolimus. Subsequently, laparoscopic nephrectomy without cavectomy was performed. Notably, the patient did not present with tuberous sclerosis complex (TSC). Our findings highlight the potential of everolimus in the treatment of sporadic AML, even in cases unrelated to TSC, offering a viable alternative to invasive therapeutic approaches. While sporadic AML are more common, this tumor can also be associated with tuberous sclerosis complex (TSC). Although often asymptomatic, larger AMLs present a potential risk of rupture, necessitating therapeutic intervention such as transcatheter arterial embolization (TAE) or surgical resection.2A 54-year-old male presented with macrohematuria and was found to have a large right renal tumor measuring 110 mm, accompanied by a tumor thrombus in the right renal vein extending into the IVC (Novick Classification level 2). Physical examination revealed no other abnormalities, and there was no significant family history. Given the associated risk of pulmonary embolization due to necrotic tissues, TAE was deemed inappropriate. Due to the potential risk of surgical resection, we administered everolimus 10mg in expectation of tumor shrinkage. There were no adverse effects observed. After three months of therapy, a significant reduction in tumor diameter to 80mm was observed, along with regression of the IVC thrombus .Fig. 1Co2.1Following the successful preoperative therapy, a laparoscopic nephrectomy and thrombectomy were performed. To ensure safety and simplicity, our surgical approach involved temporary clamping of the proximal IVC above the tumor, the distal IVC at the renal vein, and dissection of the right renal vein. By incising the right renal vein, the tumor was successfully extracted as a cohesive mass with the right kidney. Monitoring with ultrasound sonography confirmed complete tumor removal. The thrombus did not adhere to the vein wall, and no fibrin clot was observed. The operation lasted 5 hours and 28 minutes, with a blood loss of 200ml and no complications. The patient was discharged on the sixth postoperative day and has remained recurrence-free during the 5-year follow-up period.2.2Histopathological examination of the primary tumor confirmed the diagnosis of angiomyolipoma. The tumor displayed abundant fat cells, and some vessels exhibited thickened walls. The tumor margin was negative, and no necrotic tissue was present. Due to the patient's request, genetic testing was conducted. Although there were no significant abnormalities in his genetic test.3This report presents the first evidence of the efficacy of everolimus in the treatment of sporadic renal angiomyolipoma (AML) without tuberous sclerosis complex (TSC) genetic defects, while avoiding the need for cavectomy in cases involving inferior vena cava (IVC) thrombus.First, everolimus showed potential as a treatment for sporadic AML cases unrelated to TSC. Everolimus is guideline-endorsement treatment in patients with TSC-related AML. Several case reports showed the efficacy of everolimus even for a large AML.In contrast, the molecular structure of sporadic AML remains not well-proven. In general, sporadic AML is characterized by mutations in the TSC2 gene alone, leaving uncertainty regarding the effectiveness of mTOR inhibitors.Second, a valuable surgical strategy involves incising the renal vein and removing the tumor without cavectomy in cases of AML with IVC thrombus. While 77 cases of AML with IVC thrombus have been reported, irrespective of TSC association, all cases required cavectomy. Previous reports indicate that AML generally does not adhere to the vessel wall, and fibrin clots are rarely observed.However, caution should be exercised regarding the long-term administration of everolimus in patients with IVC thrombus. Incomplete cytoreduction may result in the peeling off of necrotic tissue, which can embolize the pulmonary vein. The prolonged use of drugs necessitates careful consideration to prevent this outcome.There is potential for everolimus to exhibit good efficacy in treating sporadic AML. Guidelines for sporadic AML remain uncertain, in comparison to invasive therapies such as TAE or surgical interventions, everolimus may be considered a potential therapeutic option. Future discussions should delve into the possibility of many sporadic AML cases exhibiting similar positive responses to everolimus treatment.4Everolimus shows promising potential as a non-invasive treatment option for sporadic AML. Our surgical approach provides a safe and efficient method for managing AMLs with IVC thrombus. Continued investigation and discussions are necessary to expand our understanding and refine the management strategies for sporadic AML.None declared.


However, the significance of Ca2+ and Ca2+ channels participating in entosis is unclear. Here, it is shown that intracellular Ca2+ signaling regulates entosis via SEPTIN‐Orai1‐Ca2+/CaM‐MLCK‐actomyosin axis. Intracellular Ca2+ oscillations in entotic cells show spatiotemporal variations during engulfment, mediated by Orai1 Ca2+ channels in plasma membranes. SEPTIN controlled polarized distribution of Orai1 for local MLCK activation, resulting in MLC phosphorylation and actomyosin contraction, leads to internalization of invasive cells. Ca2+ chelators and SEPTIN, Orai1, and MLCK inhibitors suppress entosis. This study identifies potential targets for treating entosis‐associated tumors, showing that Orai1 is an entotic Ca2+ channel that provides essential Ca2+ signaling and sheds light on the molecular mechanism underlying entosis that involves SEPTIN filaments, Orai1, and MLCK.Entosis is a non‐apoptotic cell death process that forms characteristic cell‐in‐cell structures in cancers, killing invading cells. Intracellular Ca 2+ signaling by IP3R coupled Orai1 Ca2+ channels regulates entosis via SEPTIN‐Orai1‐Ca2+/CaM‐MLCK‐actomyosin axis. This provides insights into a therapeutic target for entotic regulators promoting entosis‐mediated tumor development.Entosis is a non‐apoptotic cell death process that forms characteristic cell‐in‐cell structures in cancers, killing invading cells. Intracellular Ca Entosis is triggered by several physiological conditions such as matrix detachment, aberrant mitosis, and glucose deprivation. These various pathways for entosis reflect the characteristics of cancer cells, such as abnormal proliferation, anchorage‐independence, and metabolic stress, suggesting that intrinsic properties of cancer cells or their microenvironments can cause entosis. It is associated with a wide range of human diseases, such as genome instability, developmental disorders, and cancer.Entosis, a non‐apoptotic cell death process, engulfs homotypic living cells.1 Alth Following the formation of AJs, they are engulfed through actin polymerization following AJ, mechanical ring, and actomyosin contraction. Lastly, they appear in cell‐in‐cell (CIC) structures, resulting in inside cells escaping and dividing; although, they typically die via lysosomal degradation.Regardless of the mechanism by which entotic cells initiate, they form adherens junctions (AJs) via Ca2+/E‐cadherins.9 Foll Engulfing cells have lower tension than invading cells and swallow neighboring cells with actin‐dependent engulfment activity. Rac1, a key mediator of the actin cytoskeleton, and KRas, the oncogene regulating Rac1 activity, enhance engulfing by regulating actomyosin contractility.Engulfing  and invading  entotic cells form CIC structures through actin polymerization and myosin contraction. Invading cells have higher stiffness with accumulated actomyosin in the cell cortex, opposite to AJ, and the resulting mechanical tension drives CIC invasion. It is regulated by RhoA, Rho‐associated protein kinase (ROCK), and diaphanous‐related formin 1 (Dia1).14 Eng2+ function as intracellular signaling messengers and are involved in cytoskeleton rearrangement, metastasis, metabolism, cell death, and cancer development. An important mechanism for maintaining intracellular Ca2+ levels is store‐operated Ca2+ entry (SOCE), which is mediated by Orai1 Ca2+ channels in plasma membrane (PM) and stromal interaction molecule (STIM) proteins in the endoplasmic reticulum (ER). STIM senses ER Ca2+ concentrations and accumulates at ER‐PM junctions following ER Ca2+ depletion, activating Orai1 channels in adjacent PM. Ca2+ entry through Orai1 channels is associated with several cell signaling processes in most cells.Cytosolic Caelopment.18 An 2), and proteins scaffolding ER to PM. SEPTINs, filament forming GTPases, bind to inner PM through specific interactions with PIP2 and assemble on membrane domains. They promote stable recruitment of Orai1 by maintaining PIP2 organization in PM. Their loss results in abnormal Orai1 clustering and reduced SOCE.Orai1‐STIM1 assembly and stability, and architecture of ER‐PM junctions, are determined by specific protein and lipid components including SEPTINs, phosphatidyl 4,5‐bisphosphate and is activated by CaM in response to an increase in intracellular Ca2+ levels. Activated MLCK phosphorylates the regulatory myosin light chains of myosin II (MLC2), specifically at serine‐19, facilitating actin‐activated myosin and promoting myosin‐driven contraction.Myosin light chain (MLC) phosphorylation is essential for entosis providing contractile force. It is determined by myosin light chain kinase (MLCK), ROCK, and MLC phosphatase (MLCP). MLCK, the best‐studied factor regulating MLC phosphorylation, is Ca2+ is required for E‐cadherin‐mediated cell junction formation during entosis. However, the role of intracellular Ca2+ signaling in entosis, and the mechanism through which it is modulated, remain poorly understood. Entosis‐associated molecular pathways are Ca2+‐dependent. Orai1, a major Ca2+ route, regulates diverse processes related to entosis. For example, Ca2+ influx through Orai1 controls actin organization and dynamics at the immune synapse in T lymphocytes, membrane blebs in amoeboid cells, and leading‐edge in migrating cells. Orai1‐dependent signaling enhances contractile force by regulating actomyosin reorganization. Furthermore, entosis‐associated proteins, including actin, MLC, Ezrin, Rac1, AMP‐activated protein kinase (AMPK), and vinculin are regulated by Orai1 or Orai1 mediating Ca2+ signals. We hypothesized that Orai1 is a major route for Ca2+ influx during entosis.Extracellular Caphocytes,30 mem2+ channel, whose membrane localization is tightly controlled by SEPTINs, resulting in temporal entotic Ca2+ oscillations that affect MLCK‐actomyosin rearrangement during entosis. We identify the mechanism through Orai1‐mediated Ca2+ signaling regulating Ca2+‐dependent engulfment during entosis and provide insights into a therapeutic target for entotic regulators promoting entosis‐mediated tumor development.Here, we provide evidence that Orai1 is an entotic Ca22.12+ signaling drives intracellular processes and communicates between cells. Intracellular Ca2+ concentration exhibits diverse spatiotemporal dynamics, influencing the versatility of Ca2+‐dependent signaling. Hence, we investigated whether and how Ca2+ signaling, particularly Ca2+ channel‐mediated Ca2+ dynamics, is required for entosis. To determine whether intracellular Ca2+ is necessary for entosis, we first measured the entosis efficiency in MCF7 cells treated with BAPTA‐AM, a cell‐permeable Ca2+ chelator. For entosis quantification, adherent MCF7 cells were trypsinized into single cells and cultured in suspension for 2–6 h under the indicated conditions   Figure . When wem Figure , confirm2+ signaling on entosis progression, we measured intracellular Ca2+ concentrations in “engulfing” and “invading” entotic MCF7 cells expressing mCherry‐GCaMP6s, a genetically encoded Ca2+ indicator. Time‐lapse microscopy imaging was performed for 100 min at 3–5 min intervals in 0.3% low melting agarose as described  entosis proceeding cells in Ca2+ dependent manner  decreased entosis efficiency from 15% at 2 h to 4% at 4 h , an inorganic Ca2+ channel blocker for 3 h and showed a decreased entosis efficiency  channels are best known for major Ca2+ channels. Therefore, MCF7 cells were treated with SOC channel blockers, 2‐APB, SKF96365, and YM58483 for 4 h and showed reduced entosis efficiency from 35% to 20%  than at the distal membranes   and lower local Ca2+ concentrations at the CC  or sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) may affect the Ca2+ influx and entosis efficiency. First, we treated MCF7 cells with the IP3R inhibitor, heparin (400 µg mL−1), and Xestospongin C (4 µm), and measured intracellular Ca2+ concentration and entosis efficiency. The cells with the IP3R inhibitor showed a reduced entosis efficiency  or TG (Thapsigargin), two SERCA inhibitors. Our results showed that DBHQ (25 µm) or TG (1 µm) significantly reduce the efficiency of entosis  MCF7 cells to explore the function of Orai1 Cas Figure  and immud Figure . There w2+ channels positively regulate entosis. To investigate whether Orai1 Ca2+ channels induce local Ca2+ influx in entotic cells during the engulfment stages of entosis, we expressed mCherry‐GCaMP6s‐CAAX in Orai1 WT and KO cells, acquired time‐lapse images at 30 s intervals over 4 h, and aligned the end of engulfment to time 0. WT cells showed PM‐localized Ca2+ oscillations before entotic cells formed CIC structures (engulfment stage: ≈ −60 to 0 min) , which alters the assembly and disassembly of SEPTIN networks. Surprisingly, we found that entosis efficiency in MCF7 cells treated with FCF for 4 h reduced from approximately 35% to 15%  for actomyosin contractility and changes in entotic cell morphology.Finally, we explored the molecular mechanism by which the entotic Orai1 Cat Figure . Myosin m) and peptide‐18 . ML‐9 is a classic MLCK inhibitor and P18 is a specific MLCK inhibitor that mimics the inhibitory domain of MLCK by interacting with the catalytic domain of MLCK. MCF7 cells with inhibitors showed reduced entosis efficiency, demonstrating that MLCK activity and phosphorylation of its downstream protein (MLC) might affect entosis  of engulfing and invading cells maintains the stability of the AJ complex, which occasionally forms a ring‐like structure with F‐actin. Besides, pMLC was colocalized with Orai1 during engulfment and was enriched in ID  inhibitors. MLCK inhibitor (P18) or SOC channel blockers (SKF96365) decreased pMLC intensity in ID  and MLCK (P18) inhibitors. We compared the efficiency of entosis between cells treated with both inhibitors and against each other. The effects of MLCK and SOC channel inhibitor on the reduction of entosis were not significantly different. In addition, reduced entosis efficiency was not further decreased when both inhibitors were used Figure . In addiThese findings suggest that Orai1 channel activates CaM/MLCK and myosin (pMLC) in specific cell regions, facilitating engulfment between entotic cells.32+ channel that induces Ca2+ oscillations during entosis. Orai1 channel is the main pore subunit, but it has two mammalian homologs, Orai2 and Orai3. In addition, Orai1 channel functions as the homomeric or heteromeric channel with other Orai members. Particularly, Orai3 has emerged as a potential fine‐tuner for Ca2+ signaling in a variety of cancer cells, including breast cancer cells MCF7. Furthermore, several TRP channels were found to have a corporative function with Orai1 in cancer physiology; however, their function during entosis remains unknown. Ca2+ signaling mediated by Orai1 is also modulated by several STIM and Orai binding proteins, especially calcium‐dependent proteins such asCaM, SARAF, EF‐hand domain family member B (EFHB), and Cortactin. It is, therefore, necessary to investigate the role of Orai1 interacting proteins in entosis in the future.In our study, we demonstrated that Orai1 is a critical Calls MCF7.42 Fur2+ signaling through IP3R, which leads to Ca2+ oscillations during entosis. Entosis is induced via matrix detachment and formation of AJ, which may activate Orai1‐mediated Ca2+ signaling. It has been reported that E‐cadherin ligation stabilizes and activates epidermal growth factor receptor (EGFR) signaling and activation of EGFR may result in the activation of phospholipase C gamma and ER Ca2+ depletion, resulting in Orai1 mediated Ca2+ influx. However, several lines of evidence demonstrate that, despite the small conductance of Orai Ca2+ channels and high Ca2+ selectivity, all Orai isoforms are not non‐redundant for the diversity of Ca2+ signaling/oscillations and all SOCE components are independently involved in the generation of Ca2+ oscillations. Through activation of physical receptors, the diversity of Ca2+ oscillations can be expanded, including activation of Src for cancer invasion and fine‐tuning transcriptional activation of the Ca2+‐dependent transcription factor Nuclear Factor of Activated T‐cell (NFAT). Ca2+ oscillations are influenced by positive and negative feedback effects on the Ca2+ release system, which result in fluctuations in IP3 levels or changes in Ca2+ channel activity in intracellular stores. Moreover, IP3 stimulates the IP3R and triggers Ca2+ release from the ER, leading to Ca2+ influx mediated by Orai1, together with our observations that IP3R inhibitor and SERCA inhibitors reduce the entosis efficiency and Ca2+ level during entosis. Therefore, it is reasonable to conclude that the Ca2+ spikes/oscillations, we observed in entosis, would be IP3‐dependent Ca2+ oscillations (which last tens to hundreds of seconds), which are associated with the functional coupling between ER Ca2+ channels (IP3Rs) and PM Orai Ca2+ channels.SOCE activation is dependent on intra‐ER Casignaling51 and2+ oscillations occur primarily during the early to the mid‐engulfment stage. It is a question inherent in our results that few Ca2+ oscillations were observed in entotic cells following the formation of complete CIC structures  present in many types of cells, including MCF7. MCF7 cells express not only Orai1 but also other Ca2+ channels  which can be involved in Ca2+ signaling during the entosis; however, there are no studies about Ca2+ channel mediated signalings and whether these Ca2+ signalings may or may not be detected levels of oscillations. Thus, further study is needed to expand our understanding on how diverse Ca2+ signalings are involved in entosis.Orai1 KO cells showed a reduced entosis with a dramatic decrease in Cas Figure , implyin2+ ions and signalings are absolutely essential, particularly during the early stages of engulfment. First, in the absence of extracellular Ca2+, Ca2+ oscillations did not appear, and entosis occurred very rarely in WT MCF7 cells  or jetPRIME (PolyPlus) according to the manufacturer's instructions.MCF7 and HEK293T cells were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C and 5% CO5 cells per mL) were cultured in the indicated conditions  for 2–6 h on polyHEMA  coated plates. Cells were pelleted at the indicated times, washed in PBS, and fixed in 4% PFA for 10 min at 25 °C. Fixed samples were washed in PBS and stained with Alexa FluorTM 594 Phalloidin , WGA‐488 , and Hoechst 33342  for 30 min at 25 °C. To confirm that MCF7 cells were completely internalized by entosis, the cells were examined under a laser‐scanning confocal microscope LSM780 . Prior to heparin treatment, cells were permeabilized by saponin (10 µg mL−1).MCF7 cells were cultured for at least three days before the experiment. Monolayer cells were trypsinized to a single‐cell suspension and washed briefly with phosphate buffered saline (PBS). Suspended cells  were counted as one.3 (Gadolinium (III) Chloride) , AnCoA4 , YM58483 , SKF96365 , 2‐APB , Heparin , Xestospongin C , saponin , Forchlorfenuron , ML‐9 , Peptide‐18 , and Thapsigargin .The following Chemicals were used. EGTA , GdCl5 cells per mL) were embedded in growth media containing 0.3% low melting agarose and plated on micro‐insert 4 wells in polymer coverslip bottom dishes  coated with polyHEMA. The dishes were centrifuged at 1300 rpm for 1 min and then incubated at 37 °C for 20–30 min to allow the cells to settle down. Time‐lapse microscopy was performed at 37 °C and 5% CO2 in live‐cell incubation chambers.To track entosis, cells  images were obtained every 3 s to 5 min for the indicated time courses on a LSM780 (Zeiss) confocal microscope with Zeiss Plan‐Apochromat 63×/1.4 Oil objective lens. mCherry and eGFP (GCaMP6s) were simultaneously excited at 594 and 488 nm, respectively. Fluorescence emission was collected at 615–840 nm (mCherry) and 510–570 nm (eGFP). Zen software (Zeiss) was used for image acquisition. Images were analyzed using Zen or ImageJ software.F/F, where F was the baseline level). Entotic cell pairs consisting of engulfing and invading cells were counted as one. Ca2+ levels 3–5× above the standard deviation were recorded as Ca2+ signals. The time point at which a complete CIC structure was created was set to 0. The 60–0 min period, before complete cell internalized, was defined as “engulfment”. The period from 0 to 60 min after generating the CIC structures, was defined as “Cell‐in‐Cell”.Graphs showing low pass filtered normalized GCaMP6s intensity were generated. Raw signals were converted into values relative to the baseline ‐coated cover glass for 10 min. After 10 min, the cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.02–0.1% Triton X‐100 in PBS for 10 min at 25 °C, and then blocked with 3% bovine serum albumin (BSA) in PBS for 30 min. After incubating with the indicated antibody for 2–4 h at 25 °C or overnight at 4 °C, the cells were incubated with a conjugated secondary antibody at 25 °C for 1 h. For staining PM, the cells were incubated with CellBrite Red Cytoplasmic Membrane Dyes  at 25 °C for 10 min. For staining cytosol, the cells were incubated with CellTracker Red Dyes  at 25 °C for 10 min. For staining F‐actin, the cells were incubated with Alexa FluorTM 594 Phalloidin  at 25 °C for 10 min. Last, the nuclei were stained with Hoechst 33342 (Molecular Probes) for 10 min. Confocal fluorescence images were acquired using LSM780 confocal microscope  and LSM980 (Zeiss) with a Zeiss 63× and 100× oil objective lens .HEK293T cells were plated into 12‐well plates. The cells were transfected with lentiviral constructs together with packaging plasmids . Supernatants were collected 48 h post‐transfection. MCF7 cells were infected with 100–500 µL of lentivirus‐containing medium and incubated for 48 h.−1 hygromycin  for 7 days. Cell colonies were isolated after 2–3 weeks. To check for genome editing, the region surrounding the target site of the guide RNA was amplified using PCR (forward primer: 5’‐ATGCATCCGGAGCCCGCCCCGCCCCCGAGC‐3’ and reverse primer: 5’‐CATGGCGAAGCCGGAGAGCAG‐3’). PCR products were subsequently purified via agarose gel extraction and then sequenced. Sequencing of PCR fragments from the Orai1 KO MCF7 cells revealed 1 base‐pair deletion confirming successful Orai1 KO. . Protein expression was confirmed using Western blot analysis. For immunofluorescence assay, CellTracker Red  was used for labeling Orai1 KO cells.MCF7 Orai1 KO cells were made with a CRISPR‐Cas9 system. Guide RNA sequences for human Orai1 (sense 5’‐GATCGGCCAGAGTTACTCCGAGG‐3’ and antisense 5’‐ CCTCGGAGTAACTCTGGCCGATC ‐3’) were inserted into the pRGEN vector and pRGEN‐reporter (ToolGen). MCF7 cells were transfected with pRGEN‐Orai1, pRGEN‐Cas9, and pRGEN‐report using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. After 2 days, transfected cells were selected with 200 µg mLTotal RNA was extracted from the isolated cells using RIboEX (GeneAll) following the manufacturer's protocol. cDNA was synthesized from 1 µg of RNA using oligo (dT) primers and First Strand cDNA Synthesis Kit (TOYOBO). PCR amplification was conducted on the C1000 Touch thermal Cycler (Bio‐Rad) using the following amplification conditions: initial denaturation at 95 °C for 3 min and then 35–45 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min; an additional extension at 72 °C for 10 min, 16 °C hold. PCR products were subsequently purified via agarose gel extraction. h.STIM1 (For 5'‐AAGGCATTACTGGCGCTGAACCATGG‐3' and Rev 5'‐ACGGGAAGAATCCAAATGTGGAGAGC‐3'), h.STIM2 (For 5'‐AACGCTGAAATGCAGCTAGCTATTGC‐3' and Rev 5'‐CGTTCTCGTAAACAAGTTGTCAACTC‐3'), h.Orai2 (For 5'‐GGCCATGGTGGAGGTGCAGCTGGAG‐3' and Rev 5'‐GAGTTCAGGTTGTGGATGTTGCT‐3'), h.Orai3 (For 5'‐TGGGTCAAGTTTGTGCCCATTGG‐3' and Rev 5'‐TGCTGCAGACGCAGAGGACCG‐3'), and GAPDH (For 5'‐CTGAACGGGAAGCTCACTGGCATG‐3' and Rev 5'‐AGGTCCACCACCCTGTTGCTGTAGC‐3')m Tris‐HCL pH 7.4, 150 mm NaCl, 1% Triton X‐100, 0.1% SDS), and lysed for 10 min on ice. Lysates were centrifuged at 12 000 rpm at 4 °C for 10 min. For soluble protein blotting, samples were mixed with 1/4 volume of 4× reducing sample buffer (200 mm Tris‐HCl (pH6.8), 8% SDS, 0.4% bromophenol blue, 40% glycerol, and 20% β‐mercaptoethanol) and boiled at 95 °C for 5 min. Samples were separated using 10% polyacrylamide SDS‐PAGE and transferred onto 0.45 µm pore size PVDF membranes . The membrane was blocked with TBS‐T plus 7% SKIM‐milk and incubated overnight at 4 °C with primary antibodies diluted in TBS‐T plus 3% BSA. Blots were incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies and detected using enhanced chemiluminescence (Pierce). Densitometry analysis was performed using the ImageJ software (NIH).Cells were scraped into ice‐cold cell lysis buffer . Lysates were centrifuged at 12 000 rpm for 10 min and the supernatant was incubated overnight at 4 °C with anti‐Flag M2 agarose beads (Sigma–Aldrich). Lysates and immunoprecipitated samples were run on SDS‐PAGE gels, probed with horseradish peroxidase (HRP)‐conjugated secondary antibodies, and detected by enhanced chemiluminescence (Thermo Fisher Scientific).HEK293T cells were transfected with the indicated constructs for 18 h. Transfected cells were washed three times with PBS and lysed with lysis buffer , SEPT2 , FLAG , and GFP Orai1 , Orai2 , Orai3 , STIM1 , STIM2 , p‐MLC2 (S19) , MLC , GAPDH , m of Fluo‐4 AM (Invitrogen) for 30 min at 37 °C. Ca2+ imaging was performed in 0 or 2 mm Ca2+ Ringer's solution with an IX81 microscope (Olympus) equipped with an Olympus × 40 oil objective lens (NA 1.30) and with a fluorescent arc lamp (LAMDA LS), excitation filter wheel , stage controller , and a CCD camera  at 25 °C. Images were processed with MetaMorph software (Molecular Devices) and analyzed with excel and GraphPad Prism 5. TG  was used for inducing ER Ca2+ depletion.MCF7 cells were loaded with 1 µ2+ were determined as mean variation between the Fluo‐4 fluorescence intensities obtained during the stimulus (F) and the resting state (F0), as follows: F–F0.The changes in intracellular Cat‐test was used to determine pairwise statistical significance. ***p < 0.001; **p < 0.01; and *p < 0.05.All generated data were recorded in Excel and analyzed using GraphPad Prism 5 software. Results are presented as means ± SEM. The student's The authors declare no conflict of interest.Wrote the manuscript, conceived the study, and designed and interpreted experiments: A.R.L. and C.Y.P. Performed all experiments: A.R.L.Supporting InformationClick here for additional data file.Supplemental Movie 1Click here for additional data file.Supplemental Movie 2Click here for additional data file.Supplemental Movie 3Click here for additional data file.Supplemental Movie 4Click here for additional data file.Supplemental Movie 5Click here for additional data file.Supplemental Movie 6Click here for additional data file.


Caliciviridae that infects wild and domestic rabbits and hares (lagomorphs). At present, RHDV2 has caused huge economic losses to the commercial rabbit trade and led to a decline in the number of wild lagomorphs worldwide. We performed a necropsy and pathological observations on five dead rabbits on a rabbit farm in Tai’an, China. The results were highly similar to the clinical and pathological changes of typical RHD. RHDV2 strain was isolated and identified by RT-PCR, and partial gene sequencing and genetic evolution analysis were carried out. There were significant differences in genetic characteristics and antigenicity between RHDV2 and classical RHDV strain, and the vaccine prepared with the RHDV strain cannot effectively prevent rabbit infection with RHDV2. Therefore, we evaluated the protective efficacy of a novel rabbit hemorrhagic virus baculovirus vector inactivated vaccine (VP60) in clinical application by animal regression experiment. The result showed that VP60 could effectively induce humoral immunity in rabbits. The vaccine itself had no significant effect on the health status of rabbits. This study suggested that the clinical application of VP60 may provide new ideas for preventing the spread of RHD2.Rabbit hemorrhagic disease (RHD) is known as rabbit plague and hemorrhagic pneumonia. It is an acute, septic, and highly fatal infectious disease caused by the Lagovirus rabbit hemorrhagic disease virus (RHDV) in the family  Rabbit hemorrhagic disease is an acute infectious disease caused by RHDV. The disease could occur all year round. The incubation period is 48–72 h. The incidence and mortality of young and adult rabbits over 3 months are higher up to 90%), which is transmitted through the respiratory tract, digestive tract, skin, and other ways. Systemic septicemia was even found in dead rabbits . The vir0%, whichThe virus of RHD is endemic in most parts of the world, and genetic mutations in RHDV have also occurred. In 2010, a new strain of RHDV was first identified in France named RHDV2 . In PortCurrently, vaccination is the most effective way to prevent and control RHDV2. RHDV2 and the classical RHDV strain G1-G6 have similar typical clinical signs of death in rabbits, but there are significant differences in genetic characteristics and antigenicity ,29. AccoThe nucleotide diversity of the capsid protein (VP60) of RHDV2 differs from classical RHDV by more than 15%. As the main structural protein of RHDV, VP60 could self-assemble into virus-like particles (VLPs) in a baculovirus vector expression system. Therefore, the VP60 gene is an attractive choice for the detection of RHDV by Reverse-Transcription Polymerase Chain Reaction (RT-PCR) amplification and sequencing, as it is transcribed as sub-genomic RNA at high copy number during replication. It has the advantages of easy culture, expression, and post-translational protein modification . AccordiIn this study, we investigated a rabbit farm in Tai’an, Shandong Province. Approximately 1500 rabbits were present on the farm prior to the disease outbreak. Within ten days of the outbreak, 744 rabbits died, resulting in an average mortality rate of 49.6%. The rabbit farm promptly followed the regulations stipulated by the Animal Epidemic Prevention Law of China for the disposal of the affected rabbits during the outbreak. Then, we conducted clinical and pathological examinations, which showed classic clinical signs of rabbit hemorrhagic disease. The VP60 gene of the isolate was sequenced, and the results showed that the isolate was very similar to RHDV2. On this basis, we studied the effectiveness of a new commercialized rabbit plague baculovirus vector (VP60) vaccine against RHDV2. The challenge test and antibody detection were carried out, and the effect of the vaccine itself on rabbits was studied. The aim was to provide practical ideas for the clinical prevention of new rabbit hemorrhagic disease.SteadyPure Virus DNA/RNA Extraction Kit, Evo M-MLV Plus 1st Strand cDNA Synthesis Kit, and ApexHF HS DNA Polymerase CL were purchased from Accurate Biotechnology (Hunan) Co., Ltd. . IgG Ab and IgM Ab ELISA kits were from Shanghai Jianglai Biotechnology Co., Ltd. . Rabbit Viral Haemorrhagic Disease Baculovirus Vector Vaccine and Inactivated (Strain VP60) was obtained by Qilu Animal Health Products Co. Ltd. . Gel Extraction Kit D2500 was purchased from Omega Bio-Tek . The 35-day-old New Zealand white rabbits were provided by Qingdao Kangda Biotechnology Co., Ltd. .New Zealand white rabbits, approximately 4 months of age, from an RHDV2-free commercial facility were assessed for general health. Rabbits were raised at 22 °C temperature and 50% humidity and fed with sterile food and water. The rabbits were randomly divided into a control group, classic vaccine group, new vaccine group, and classic + new vaccine group (30 rabbits in each group). The control group did not do any treatment. Each rabbit in the classic vaccine group and the new vaccine group was injected subcutaneously with 2 mL of the vaccine in the neck, and the classic + new vaccine group was injected subcutaneously with 1 mL of the two vaccines on both sides of the neck. Then, the temperature and weight of each group of rabbits were recorded every day within 7 days after the vaccine injection. At 7, 14, 21, 28, and 35 days after vaccination, blood samples were collected by cardiac puncture, and antibody detection was performed after the serum was obtained. w/v) suspension centrifuged at 4000 rpm/min for 10 min . The clarified supernatant was kept at −20 °C until use. Moreover, 2 mL of supernatant was subcutaneously injected into the neck of each rabbit, and deaths were recorded within 35 days after vaccine injection.RHDV2 was discovered on a rabbit farm in Tai’an, China. The liver tissues  preserved at −80 °C were repeatedly frozen, thawed, and ground three times, then pooled and homogenized in sterile phosphate-buffered saline (PBS) to obtain a 10%  to detect the serum antibody level. The absorbance was measured at 450 nm with a microplate reader. A standard curve was constructed based on the absorbance of the control sample to calculate the serum IgG and IgM content, respectively.During the whole measurement process, the room temperature was not changed more than 3 °C, and noise interference was avoided. The rabbits stopped feeding 2 h before the measurement and were placed in a suitable device until it was completed. The body temperature was measured with an anal thermometer with a precision of ±0.1 °C. The depth and time of inserting the anal thermometer into the anus were the same for each rabbit, the depth was about 1 cm, and the time was not less than one and a half minutes. The temperature was measured every 30 min, and the average of the two-body temperatures was recorded. The body weight was measured using an electronic scale, then recorded the data after the rabbit was quiet.p < 0.05.All data were expressed as mean ± standard deviation (S.D.). GraphPad Prism 8.0 was used to draw the statistical chart. Statistical differences were evaluated by one-way analysis of variance (ANOVA) in SPSS Statistics 25, followed by Duncan’s multiple range test. Statistically significant differences were assumed at Before death, the rabbits showed struggling motion, prostration, dyspnoea, and occasionally bloody discharges from nostrils. In addition, we found that the clinical signs and histopathological changes of dead rabbits were very similar to rabbit hemorrhagic diseases through clinical necropsy and histopathological examination. From We designed primers based on the reported RHDV2 VP60 gene sequence (GenBank: HE819400.1) and used RT-PCR to detect RHDV2 VP60 RNA in liver tissue . We clonp < 0.05). It could be seen that the new rabbit plague vaccine can effectively stimulate the body to produce antibodies and have a protective effect on it.According to the antibody test results, the new rabbit plague vaccine has a good immunization effect, and the antibody concentration in the body continues to increase from day 0 to day 35. As shown in To verify the reliability of the new RHDV2 vaccine, we conducted the RHDV2 challenge experiment . The resBy measuring the body weight, we found that 7 days after immunization, the weight gain of rabbits in the new vaccine group and the classic vaccine group was insignificant compared with that of the control group. Still, the weight gain of the mixed vaccinated rabbits was smaller than that of the control group A. We speSince the 1890s, rabbit hemorrhagic disease has spread all over the world. As a highly infectious and fatal disease, it has caused great damage to the rabbit breeding industry, the ecological environment and the national economy . In 2010In the case of RHDV2 discovered by Toh et al. in Singapore, the liver had a severe acute liver injury, diffuse necrosis and degeneration of liver cells, and many inflammatory cell infiltrations . Hänske In this study, the RHDV2 VP60 gene was obtained from the liver of infected rabbits; then, we performed a phylogenetic analysis of the VP60 gene sequence. Through investigation, we found that the new isolate is in the same branch as the other RHDV2 strains. The nucleotide homology analysis revealed that RHDV2 isolates in China and Europe were closely related, which may indicate rapid growth transcontinental transmission of the virus and support the conclusion that the strain collected from Tai’an, China, in 2020 belongs to the RHDV2 (GI.2) gene group. In addition, viruses often recombination ,42,43. IThere was a strong demand for effective vaccines to protect rabbits against this disease since, in 1984, RHDV1 killed millions of rabbits in commercial husbandries in China ,44. HoweIn this study, we conducted clinical and pathological examinations on a rabbit farm in Tai’an and found the typical clinical signs of rabbit viral hemorrhagic disease. We sequenced the VP60 gene of the isolate and found that the isolate was very similar to the SC2020 gene sequence through the phylogenetic tree, indicating that the cause of this disease was caused by RHDV2, which may have spread over long distances in China. On this basis, we explored the effectiveness of the new rabbit plague baculovirus vector (VP60) vaccine against RHDV2 through challenge experiments and antibody detection and studied the effect of the vaccine itself on rabbits. It was proved that the VP60 vaccine showed a strong protective force against RHDV2 attack 5 weeks after vaccination and could effectively induce humoral immunity in rabbits. When used alone, it is safe and effective for rabbits. Our purpose is to provide practical ideas for the clinical prevention of new rabbit hemorrhagic diseases.


Moreovene auxin .− into cells with two H+, and passive diffusion also delivers auxin into the cells. On one hand, TIR1/AFB receptors perceive auxin to promote cellular H+ influx across the PM through so far undiscovered channels or transporters/antiporters, resulting in rapid apoplast alkalinization. The Ca2+ transient contributes to H+ influx , a Ca2+ channel, to rapidly stimulate PM depolarization  and TRANSMEMBRANE KINASEs (TMKs) are excellent candidates . Remarkaongation . Conversongation . Howeverhibition . Nonether needed . Collectr needed Figure 1Besides apoplast alkalinization, other cellular processes such as cortical microtubule re-orientation  and vacu2+ signaling consists of an array of receptors that perceive extracellular cues and Ca2+ channels that transport Ca2+ into the cells, formulating a specific Ca2+ signature (2+-DEPENDNT PROTEIN KINASEs (CDPKs), and CALCINEURIN B-LIKE PROTEINs (CBLs) have been identified in land plants as Ca2+ sensors. Plant-specific CBL-INTERACTING PROTEIN KINASEs (CIPKs) act as major downstream signaling components of the CBL sensors to orchestrate central CBL-CIPK signaling networks and fine-tune plant adaptive growth in response to developmental and environmental cues  receptor mutant , has also been shown to rapidly (within 2 min) arrest primary root elongation in a similar way to auxin . The appr mutant . Their rr mutant . It is iongation . Unexpec peptide . Notablyimension . Thus, Rimension Figure 1FER plays multiple roles in plant development, including cortical microtubule re-orientation  and intr2+ sensor in FER-mediated pollen tube reception  as a strong candidate to enhance cytoplasmic calcium concentrations . NORTIA eception . Therefomponents  in RALF1mponents Figure 1FER is a Ser/Thr receptor kinase that phosphorylates itself or substrates in a context-specific manner . RALF1 iArabidopsis primary root depends not only on the AFB1 subcellular localization but also on specific AFB1 protein properties  and reversible effects of auxin and RALF1 peptide provide mechanistic insights into rapid primary root elongation regulation. Nevertheless, open questions remain to be unanswered. The key unresolved question is how TIR1/AFB and TMK1 kinase confer primary root rapid reactions, and which downstream players function in non-canonical auxin actions. Phosphoproteomics analysis has revealed that many proteins are rapidly phosphorylated independently of the TIR1/AFB pathway . Only a operties . Furtheroperties . Hence, clc2-1/3-1 mutant, impaired in CME, shows resistance to RALF1 peptide in root growth inhibition (2+) and MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) cascades to exert its rapidness , and the hibition . It is lapidness , but thiapidness . Hence, apidness  are requ+ influx Figure 1All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.