Patient-derived xenografts (PDX) are widely used as human cancer models. Previous studies demonstrated clonal discordance between PDX and primary cells. However, in acute myeloid leukemia (AML)-PDX models, the significance of the clonal dynamics occurring in PDX remains unclear. By evaluating changes in the variant allele frequencies (VAF) of somatic mutations in serial samples of paired primary AML and their PDX bone marrow cells, we identify the skewing engraftment of relapsed or refractory (R/R) AML clones in 57% of PDX models generated from multiclonal AML cells at diagnosis, even if R/R clones are minor at <5% of VAF in patients. The event-free survival rate of patients whose AML cells successfully engraft in PDX models is consistently lower than that of patients with engraftment failure. We herein demonstrate that primary AML cells including potentially chemotherapy-resistant clones dominantly engraft in AML-PDX models and they enrich pre-existing treatment-resistant subclones. Clonal dynamics and selection have not been fully understood in patient-derived xenografts (PDX) of acute myeloid leukemia (AML). Here, the authors generate 160 AML-PDX models to track the clonal dynamics of primary and relapsed AML, and find selectively enriched subclones that are associated with resistance to therapy. Leukemia-founding clones acquire additional cooperating mutations, yielding subclones that contribute to leukemic progression and/or relapse5. Previous studies reported that acute leukemia comprises genetically diverse subclones, some of which survive after chemotherapy and contribute to disease recurrence7. Genetic analyses of samples from patients who relapsed after chemotherapies showed that a major selection pressure was imposed on leukemia clones, leading to clonal evolution8. A similar genetic analysis of purified subpopulations and their xenografts from paired diagnosis and relapse samples demonstrated that therapy-resistant cells were already present at diagnosis and evolved at relapse9.Relapse and/or chemotherapy resistance are major obstacles to long-term survival in patients with acute myeloid leukemia (AML)12. More recently, xenograft models using immunodeficient mice enabled the ex vivo maintenance and expansion of primary leukemia cells. Patient-derived xenografts (PDX) are widely used as human cancer models mimicking the phenotypes of primary cancer cells15. However, PDX models do not always recapitulate the genetic features of primary cancer cells and polyclonal engraftment has been observed in various solid cancer PDX models17. Although multiple subclones proliferate in a patient, relapse is based on the selection of existing clones or acquisition of additional mutations during disease progression. Therefore, each genetically polyclonal population from cancer cells transplanted into PDX has a different growth ability in xenografts20. In a previous study on DNA copy number alteration profiling and sequencing in colorectal cancer PDX models, the proliferation, persistence, and chemotherapy tolerance of lentivirally marked lineages varied within each clone, and chemotherapy promoted the dominance of previously minor lineages in PDX21. Furthermore, one of the phenotypes representing the quiescent clones that expanded in serial passages of PDX was shown to be responsible for resistance to chemotherapy. A genomic analysis of 15 breast cancer PDX models revealed that polyclonal engraftment was possible; however, clonal selection in PDX was clearly evident22. In another study that systematically analyzed the landscapes of aneuploidy and conducted a copy number analysis of a large cohort of PDX models across multiple human cancers, the landscapes of PDX also markedly diverged from those of the parental tumors during passaging, and the genomic stability of PDX was associated with their responses to chemotherapy or targeted drugs23. Thus, transplanted tumor cells display a variable potential for proliferation and therapy tolerance in PDX models.Since significant patient-to-patient cell heterogeneity complicates the clarification of a common mechanism that controls the biology of AML, a faithful model reflecting the complex heterogeneity of human AML in vivo will contribute to a more detailed understanding of the molecular pathogenesis. Many groups have developed mouse leukemia models, such as those induced by chemicals, viral infection, or irradiation, and transgenic animals expressing AML-associated proto-oncogenes24. However, in AML-PDX models, the significance of the clonal dynamics that occur in PDX and the role of clonal selection between parental patients and their PDX models remain unclear25.In the field of acute leukemia, a previous study demonstrated that 24 out of 48 paired primary tumor and PDX samples showed various degrees of clonal discordance based on variant allele frequency (VAF) changesIn the present study, by generating 160 AML-PDX models from adult AML patients and tracking the dynamics of somatic mutations in serial samples from primary AML cells and their PDX, we show their clonal architecture and the PDX-specific enrichment of AML clones with predictive power for characterizing clonal selection in parental patients.6 viable cells per mouse. The engraftment of primary samples was confirmed when the BM human CD45+ percentage was higher than 20% and serial transplantation at 1 × 106 cells was successful. Patient characteristics are summarized in Table + cells was not confirmed in samples from the other 55 patients (34%); 28 models died within 365 days  without positive hCD45+ in PB, 18 with negative human CD45+ in PB until day 365, 7 with T-cell engraftment, and 2 failed in serial passaging with hCD45+ >5% in PB, but <20% in BM, at day 365. Engraftment was achieved within a median of 106 days  post-transplantation, and was more frequently observed in mice transplanted with more than 5.0 × 106 cells . Successful engraftment was also associated with the M4-FAB type (P = 0.028), relapse/refractory (R/R) AML (P < 0.001), and a higher risk in the European LeukemiaNet (ELN) classification , but not with patient age (P = 0.621), cytogenetic risks (P = 0.053), graft sources (P = 0.060), or infused blast percentages (P = 0.280).We hypothesized that treatment-resistant clones in primary AML cells may engraft and expand in immunodeficient mice. To compare the features of primary AML cells that successfully engraft and proliferate in PDX to those with engraftment failure, we generated AML-PDX models from 160 AML patients. Mononuclear cells isolated from the fresh bone marrow (BM) or peripheral blood (PB) samples of AML patients were intravenously injected into 6-week-old NOD/Shi-scid, IL-2Rγnull (NOG) mice  at 1–15 × 10FLT3 , NPM1 , IDH1 , and WT1 genes  were more frequently observed in engrafted patients than in those with engraftment failure , ASXL1 , RUNX1 , and DNMT3A  genes were also frequent, but not significantly different. In contrast, AML cells with RUNX1-RUNX1T1  and CBFB-MYH11  showed lower engraftment rates. These results revealed the existence of specific clones that have the ability to engraft and proliferate in AML-PDX models as well as their genetic profiles.To obtain further insights into the genetic landscapes of engrafted primary AML cells, the mutation profiles of AML cells were analyzed. Among cells from 160 AML patients, the targeted sequencing of 54 genes , supporting the existence of clonal selection in PDX models , IDH2 , EZH2 , TP53 , NPM1 , and RUNX1  showed uniformity between primary AML and PDX BM cells , FLT3-TKD , WT1  CEBPA , NRAS , KRAS , and DNMT3A . They were enriched more in PDX than in primary AML cells  and PTPN11  were slightly higher in primary AML cells and decreased when engrafted in PDX models  and 72 days , respectively , relapse (N = 29), and refractory (N = 18) AML, relapsed AML  and refractory AML  provided faster engraftment than newly diagnosed AML , which is consistent with few clonal changes in relapsed AML-PDX models. Refractory AML showed the fastest engraftment, suggesting only slight clonal selectivity in the status of these patients , but not with FAB classifications, cytogenetic abnormalities, or the infused blast burden  PDX models, and further passaged (P3-11) PDX models Fig. . Engraftons Fig. . In contels Fig. .Fig. 4ClTo assess individual differences between NOG mice, we transplanted primary AML cells with the same cell count into duplicated mice and evaluated discrepancies in engrafted clones using the VAF of each variant in five AML-PDX models , with 2-year EFS rates of 20.0%  and 53.5% , respectively. Two-year overall survival (OS) and cumulative incidence of relapse rates were also significantly poorer in PDX-engrafted patients, at 28.8%  vs. 50.9%  and 74.6%  vs. 45.4% , respectively  was analyzed in 76 patients who received cytotoxic chemotherapies and whose primary AML cells at diagnosis were transplanted into PDX Fig. . The medBased on these results, we hypothesized that treatment-resistant clones may be identified by investigating engrafted clones in AML-PDX models generated from patients at diagnosis.To elucidate whether selectively expanded subclones in PDX models reflect clonal evolution in the corresponding primary AML patients, we evaluated serial clonal dynamics in 21 newly diagnosed AML patients by analyzing patient samples at diagnosis and R/R and each corresponding PDX model  and WT1C453Y  and/or IDH1R132H  at the diagnosis of AML-M2  and WT1C453Y . This clonal architecture was consistent with that of the patient at relapse after consolidation therapy with RUNX1R107HWR , WT1C453Y , and FLT3-ITD . On the other hand, PDX generated from this relapsed patient’s sample (Relapse-PDX) did not show further clonal selection, but maintained similar clonality to patient BM samples with RUNX1R107HWR , WT1C453Y , and FLT3-ITD . Patient #266 showed the same pattern of clonal diversity , GATA2R330L , and WT1L378Dfs . In the Diagnosis-PDX model, the clone carrying all of these mutations expanded . Once hematological remission was achieved post-induction chemotherapy, this patient relapsed with similar clonality to Diagnosis-PDX .Patient #28 had subclones carrying -M2 Fig. . The numity Fig. . BM AML NPM1W288Cfs and the 57-bp insertion of FLT3-ITD mutations showed similar VAF between the patient at diagnosis  and Diagnosis-PDX . When the patient relapsed after consolidation chemotherapy, major clones carrying NPM1W288Cfs and the 57-bp insertion of FLT3-ITD mutations persisted with similar VAF .In patient #624 Fig. , clones GATA2A372T  and a 48-bp insertion of FLT3-ITD (20.7%). When engrafted in Diagnosis-PDX, clonality markedly changed with the emergence of a frameshift mutation in WT1A382Rfs  and a single nucleotide variant in KRASG12D  with the regression of clones carrying GATA2A372T  and FLT3-ITD . On the other hand, the relapsed AML cells of the same patient after consolidation chemotherapy harbored clones including GATA2A372T , WT1D464G , and the 48-bp insertion of FLT3-ITD . Relapse-PDX showed the concordant clonality of GATA2A372T , WT1D464G , and FLT3-ITD . In this case, although Diagnosis-PDX did not predict the clonal architecture of AML cells at relapse post-consolidation therapy, results from its relapsed sample indicated that AML cells at relapse were less heterogeneous than those before treatment, consistent with the results shown in Fig. Cases of discordance were noted between Diagnosis-PDX models and paired patient samples at relapse, with the emergence of new clones observed only in Diagnosis-PDX. In patient #606 shown in Fig. FLT3-ITD mutation, clones harboring EZH2D657A , NRASG13D , and FLT3-ITD  were detected when quizartinib was initially administered. During the third course of administration, the patient did not respond to quizartinib and AML cells proliferated. BM samples after the first course (Refractory#1) and third course of consolidation therapy (Refractory#2) showed similar clonality with the VAF of EZH2D657A (48.6 and 51.3%), NRASG13D (51.4 and 46.8%), and FLT3-ITD (41.3 and 35.6%) without newly acquired mutations. However, in the PDX of the BM sample at this time of resistance (Refractory#2-PDX), the clone carrying FLT3D835Y, a resistance mutation to quizartinib, newly expanded with VAF of 39.7% in addition to pre-existing EZH2D657A (53.3%), NRASG13D (3.5%), and FLT3-ITD (91.5%). In these cases, AML subclones selectively enriched in PDX models recapitulated clonal selection in patients acquiring treatment resistance.We found that minor treatment-resistant clones with lower VAF (<5%) expanded in PDX. In patient #463 Fig. , who rec23. We consistently found that particular AML clones were selected from heterogeneous primary AML cells and enriched though the serial passaging of their AML-PDX models. These results indicate that AML-PDX models did not completely recapitulate the clonality of patients, and indicate the need for caution regarding the application of PDX.In the present study, we generated 105 serially transplantable AML-PDX models, including a series of PDX models from sequential AML patient samples from diagnosis through to the acquisition of resistance to individual treatments. Tumor evolution and copy number alterations during PDX passaging were reported in a previous study on more than 1000 PDX models across 24 solid cancer typesPTPN11 or ASXL1 mutations decreased when engrafted in PDX, and those of CEBPA and NRAS mutations gradually diminished through the passaging of PDX , TET2 (N = 2), TP53 (N = 1) or WT1 (N = 1) dominantly expanded.We also demonstrated the instability of clonal sizes according to genetic variants in engrafted PDX Fig. . The sizPDX Fig. . On the PDX Fig. . Since aNPM1 and DNMT3A genes was associated with a higher engraftment ability, whereas a single mutation was not26. The co-mutation of the NPM1 and DNMT3A genes consistently showed a higher engraftment ability . In addition, most of the genes also carried FLT3 mutations 28. In the present study, the CD34−/38+ fraction dominantly expanded in most PDX models, while the CD34+/CD38− or CD34+/38+ fraction decreased in engrafted PDX  antibody . NOG mice were not pre-conditioned with irradiation. NOG mice were maintained in a 12-hour dark/light cycle, at a temperature of 23 ± 2 °C and a humidity of 55 ± 10%. All animal procedures were approved by the Institutional Animal Care and Use Committee of Nagoya University (M210583-002) and carried out in accordance with the Regulations on Animal Experiments in Nagoya University.Patient samples were acquired from Nagoya University Hospital, Ichinomiya Municipal Hospital, Japanese Red Cross Nagoya First Hospital, and Komaki City Hospital. The present study was approved by the Institutional Review Board of each participating institution and all patients provided informed consent for banking and molecular analyses in accordance with the Declaration of Helsinki. Mononuclear cells isolated from the fresh BM or PB samples of primary acute leukemia patients were intravenously injected into 6-week-old NOG mice  at 1–15 × 10+ reached >0.5% at 2 time points, followed by a flow cytometry assessment of the PB, spleen, and BM for engrafted human cells with the same antibodies. The engraftment of the primary sample was confirmed when the BM human CD45+ percentage was higher than 20% and serial transplantation at 1 × 106 cells was successful, whereas engraftment failure was confirmed when PB human CD45+ did not increase to 0.5% until 365 days post-transplantation of primary leukemia cells or the serial transplantation of 1 × 106 PDX BM cells, or when the human CD45+ percentage in harvested BM was lower than 20%. In AML, the presence of human CD45+CD3+ cells in harvested BM was also regarded as the engraftment failure of primary leukemia cells. After the confirmation of engraftment by successful serial transplantation, primagraft or secondary passaged (P1-2) BM samples were used in the mutational or flow cytometric analysis.Primary AML cells were transplanted into one NOG mouse in each case, except for five cases in which AML cells were transplanted into duplicated mice  with anti-mouse CD45-APC/Cy7 , anti-human CD45-PerCP/Cy5.5  , anti-human CD45-PE , anti-human CD34-APC , anti-human CD38-PE/Cy7 , and anti-human CD3-APC  antibodies . Flow cytometry data were collected using FACSAria2 and FACSDiva software v8.0.1  and analyzed using FlowJo software v8.8.7 . The gating strategy is detailed in Supplementary Fig. + fraction was sorted from PDX BM when it was less than 90% by magnetic cell separation using MACS MicroBeads  following the manufacturer’s recommendations.Pairs of transplanted primary BM or PB samples and BM samples of their PDX were subjected to a sequencing analysis. Genomic DNA was extracted from unfractionated primary BM or PB samples and PDX BM samples using the QIAamp DNA Blood Mini Kit or QIAamp DNA investigator Kit . The human CD4538. Variant Studio software version 3.0  was used and targeting regions with a sequencing depth of more than 100 were considered to be suitable for analysis. Detected genomic lesions that had either been previously published as recurrent in leukemia from the literature or the public database COSMIC or were predicted to alter protein function on tumor suppressor genes were designated as significant somatic mutations. The internal tandem duplication of the FLT3 gene (FLT3-ITD) was examined by a fragment analysis as previously reported39. The functional domains of FLT3 gene were PCR-amplified with forward primers 5’ end labeled with fluorescent dye using AmpliTaq Gold DNA Polymerase with Gold Buffer and MgCl2  according to the manufacturer’s instructions. The PCR products were analyzed using ABI 3500 Genetic Analyzer and 3500 Series Data Collection Software version 1.0  and the amplicons with a size greater than that of wild type were interpreted as positive for the ITD mutation. The number, area and length of mutant peaks on the electropherogram were analyzed using GeneMapper analysis software version 4.1 . The primer sequences can be found in Supplementary Table The target sequencing of 54 genes, which are frequently identified in the presence of myeloid malignancies, was performed using the TruSight Myeloid Sequencing Panel and Illumina MiSeq sequencer according to the manufacturer’s instructions Germline samples were absent and, thus, not analyzed.BCR-ABL1, Minor BCR-ABL1, PML-RARA, CBFB-MYH11, MLL-AF4, MLL-AF9, MLL-ENL, and MLL-ELL) by reverse transcription PCR using AmpliTaq Gold DNA Polymerase with Gold Buffer and MgCl2  according to the manufacturer’s instructions. Amplification was evaluated by agarose gel electrophoresis40. The primer sequences can be found in Supplementary Table RNA was extracted from unfractionated primary BM or PB samples and PDX BM samples using the QIAamp RNA Blood Mini Kit . Complementary DNA was generated from total RNA using SuperScript II Reverse Transcriptase  according to the manufacturer’s instructions. We examined chimeric gene transcripts  and GraphPad Prism 6 .Demographic factors and disease characteristics were compared using the standard Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Here, we provide an updated overview of the mechanistic roles the sarcolemma, sarcoplasmic/endoplasmic reticulum, and mitochondria play in the abnormal and sustained elevation of 2+ homeostasis as potential therapies for DMD.Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disease caused by the loss of dystrophin. DMD is associated with muscle degeneration, necrosis, inflammation, fatty replacement, and fibrosis, resulting in muscle weakness, respiratory and cardiac failure, and premature death. There is no curative treatment. Investigations on disease-causing mechanisms offer an opportunity to identify new therapeutic targets to treat DMD. An abnormal elevation of the intracellular calcium ( Duchenne muscular dystrophy (DMD) is X-linked and is the most common form of muscle wasting disease. It affects 1 in 3,500 to 5,000 male births . In the absence of dystrophin, these pathways are damaged and contribute to muscle pathology. For details on these pathways and their clinical implications in DMD, refer to a recent review by Allen et al.  to the cytosol /endoplasmic reticulum (ER), a major internal Ca2+ store in striated muscles. These changes result in a chronic accumulation of Ca2+ in the cytoplasm. Thus, the lack of dystrophin results in muscle degeneration and improper regeneration, which may contribute to abnormal Among the various disease-causing mechanisms, changes in intracellular calcium  activity decreases calpain activity and improves muscle function in dystrophin and utrophin double-mutant (mdx:utr−/−) mice  in the sarcolemma, which promotes Ca2+ release from the SR via the ryanodine receptor . This process is known as Ca2+-induced Ca2+ release (CICR)  , Orai, transient receptor potential canonical (TRPC) channel, and RyRs. These proteins are involved in Ca2+ store restoration during the intense work of skeletal muscles  Rios, . The mec2+ released from the SR binds to troponin C, a thin filament sarcomeric protein, and initiates muscle contraction. Muscle relaxation is initiated by Ca2+ removal from the cytoplasm. Approximately 70–90% of Ca2+ is removed by SERCA  and resequestrated into the lumen of the SR. Most of the remaining Ca2+ is extruded out of the cell via sarcolemmal Ca2+ transport proteins. These include the sodium (Na+)–Ca2+ exchanger (NCX) and plasma membrane Ca2+ ATPase. A small amount of Ca2+ is taken up by the mitochondria via the mitochondrial uniporter (MCU). Thus, SR Ca2+ cycling is not only important for muscle contraction and relaxation cycle but also helps to maintain cytosolic Ca2+ levels. For extensive reviews on Ca2+ handling in normal skeletal muscle and heart, refer to the recent reviews , the main ROS producer, saves muscle from force loss following eccentric contraction  and direct interaction with actin microfilaments, intermediate filaments, and microtubules  acts as an intracellular messenger and triggers Ca2+ entry through SOCCs  are Ca2+ store depletion and/or membrane stretch. In dystrophin-deficient muscles, evidence shows Ca2+ enters via eccentric contraction-activated stretch channels  channel may also play a role in increased Ca+ levels are increased via the opening of voltage-gated Na+ channels (Nav1.4)  type I , NCX functions in a reverse mode to remove Na+ from the cell. In doing so, NCX moves Ca2+ into the cell. Interestingly, it has been shown that increased SR Ca2+ release in dystrophic muscle can also trigger the NCX to work in a reverse mode and thereby increase In dystrophic muscle, intracellular Na2+ channel, Cav1.2, determines Ca2+ entry in the plateau phase (phase 2) of the action potential in cardiac myocytes. In mdx cardiac myocytes, Cav1.2 activation is significantly increased –deficient dystrophic hamsters, the administration of diltiazem, the L-type Ca2+ antagonist, reduces muscle Ca2+ content  are gap junction proteins, which are important for many physiological processes including coordinated depolarization of muscle, and ion movement between muscle cells  down-regulation of SERCA expression, (ii) posttranslational modification of SERCA protein, and (iii) differential expression and function of SERCA regulators. In the dystrophic myocardium of mouse models and human patients, SERCA2a levels remain unchanged , sarcolipin (SLN), myoregulin (MLN), and dwarf open reading frame (DWORF) , calreticulin, and sarcalumenin (SLM), play an important role in buffering luminal Ca2+ concentrations and regulating SR Ca2+ uptake and release , CSQ-like proteins (CLPs), histidine-rich Camdx mice, whereas these proteins are at low levels in the diaphragm of mdx mice  have been reported in spared extraocular and intrinsic laryngeal muscles of 2+ dysregulation has been extensively studied in dystrophin-deficient cardiac and skeletal muscle tissues, the implications of dystrophin deficiency in smooth muscles in DMD patients or animal models have not been adequately studied. Studies on the gastric and intestinal smooth muscles of mdx mice have shown impaired muscle relaxation, which has been attributed to impairment in 2+ influx through L-type voltage-dependent channels is responsible for the sustained mechanical tone. A recent study showed that the enhanced stretch-induced mdx mice occurs through the activation of TRPC1, TRPC3, and TRPC6 channels  , whereas Ca2+ extrusion occurs through the mitochondrial Na+-Ca2+-Li+ exchanger (mtNCLX) , a large channel in the inner mitochondrial membrane, also contributes to Ca2+ extrusion  due to decreased expression of proteins involved in the PINK1/PARKIN mitophagy pathway , mitofusin 2 (MFN2), and MFN1 are also involved in the physical interactions between ER/SR and mitochondria  . MAMs ar2+ to be rapidly transferred. This design overcomes the low apparent Ca2+ affinity (Kd ~ 15–20 M) of the MCU , RyR2, SERCA2, CSQ, and PLN, have been found enriched at MAMs, supporting the close correlation between this organellar intersection and Ca2+ regulation  failed to decrease RyR1 Ca2+ leak in mdx muscle fibers. These studies further show that NOX4 is activated in the NOX2 ROS-deficient mdx muscle and contributes to RyR1 leak  knockout mouse model of dilated cardiomyopathy  or pharmacological induction of Hsp72 improves several pathological indices in 2+-dependent mitochondrial dysfunction could be due to the opening of mPTP  appears to be a promising strategy to treat both cardiac and skeletal muscle defects in DMD. Therefore, exploring new strategies to improve SERCA function, developing new AAV vectors with improved tropism for both muscle and heart, and identifying small molecules that activate endogenous SERCA pumps will be useful.The use of large animal models: Many Ca2+-targeted therapies were performed in mouse models of DMD. These findings need to be verified in large animal models before moving to human trials.The effects of dystrophin restoration on Ca2+handling: It is unknown whether partial dystrophin restoration alleviates Ca2+-handling defects in dystrophic muscles. This information is not available and needs to be tested. This will help to design a combination therapy of dystrophin restoration with improved Ca2+ handling.DD is a member of the scientific advisory board for Solid Biosciences and equity holders of Solid Biosciences. The Duan laboratory has received research supports unrelated to this project from Solid Biosciences and Edgewise Therapeutics in the past 3 years. SM, EM, and GB have no conflicts of interest, financial or otherwise. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.All authors discussed, wrote, and commented on the manuscript at all stages.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The evolution of flight imposed new challenges on insects when locating and identifying food sources, mates, or enemies. As an adaptation, flying insects developed a remarkably sensitive olfactory system to detect faint odor traces. This ability is linked to the olfactory receptor class of odorant receptors, which are found in insect olfactory sensory neurons. In a subgroup of these neurons, sensitivity can be further enhanced through a process called sensitization. Extracellular calcium ions, calmodulin, and protein kinase C are known to be key factors in this process. While manipulation of mitochondrial calcium im- and export has been shown to influence odor responses in general, the connection of intracellular calcium stores to sensitization has so far been only speculative. Using two pharmacological approaches, we disrupted mitochondrial calcium management in order to explore its importance to sensitization. Overall, our findings reveal that mitochondrial calcium stores are important players in the complex intracellular signaling pathways required for sensitization.2+. OSNs expressing different groups of ORs show varying optimal odor concentration ranges according to environmental needs. Certain types of OSNs, usually attuned to high odor concentrations, allow for the detection of even low signals through the process of sensitization. By increasing the sensitivity of OSNs upon repetitive subthreshold odor stimulation, Drosophila melanogaster can detect even faint and turbulent odor traces during flight. While the influx of extracellular Ca2+ has been previously shown to be a cue for sensitization, our study investigates the importance of intracellular Ca2+ management. Using an open antenna preparation that allows observation and pharmacological manipulation of OSNs, we performed Ca2+ imaging to determine the role of Ca2+ storage in mitochondria. By disturbing the mitochondrial resting potential and induction of the mitochondrial permeability transition pore (mPTP), we show that effective storage of Ca2+ in the mitochondria is vital for sensitization to occur, and release of Ca2+ from the mitochondria to the cytoplasm promptly abolishes sensitization. Our study shows the importance of cellular Ca2+ management for sensitization in an effort to better understand the underlying mechanics of OSN modulation.Insects detect volatile chemosignals with olfactory sensory neurons (OSNs) that express olfactory receptors. Among them, the most sensitive receptors are the odorant receptors (ORs), which form cation channels passing Ca For insects, the sense of smell plays an important role in the location of food sources, mates, breeding substrates as well as to avoid predators and other dangers . To loca+, K+, and Ca2+ I reporting on OR activity. The flies characterized by the genotype w;UAS-GCaMP6f/Cyo;Orco-Gal4/TM6B were reared under a 12 h light: 12 h dark cycle at 25 °C on conventional agar medium. For experiments, antennae of 4–8 day old females were excised and prepared as described in i i . By ectoparation  that allparation . Stimula2+ filling state of mitochondria under resting conditions. We used CCCP, an uncoupler of mitochondrial oxidative phosphorylation, to release the mitochondrial Ca2+ stores passively. CCCP disrupts ATP synthesis by transporting protons across the mitochondrial inner membrane, interfering with the proton gradient and dissipating the mitochondrial membrane potential, allowing mitochondria to release accumulated Ca2+ and stopping further accumulation [For the first experiment, we tested the Camulation .2+ stores as the application of CCCP over 550 s increased the fluorescence intensity by 19.7 ± 7.9%, which indicates an increase of 7.9%  in the control between the first and second VUAA1 stimulation, after CCCP application there was no statistical difference between the pair of stimulations . Mitochondria import Caesponses ,35. We u+ uptake . Treatme2+ import can rescue the effects of auranofin on OR response [2+ stored in mitochondria before the application of Ru360 that could be released by the mPTP . While the difference in means is not significant, other pathways of sensitization might be upregulated to offset the lack of mitochondrial Ca2+-signaling. Further investigations will be needed to determine the contributions of the different signaling pathways involved in sensitization. Alternatively, mitochondrial stores might be directly involved in the internal signaling responsible for sensitization. Mukunda and colleagues speculated that the signaling pathway involving CaM might be driven by the release of intracellular Caproteins . While R2+ buffering allowing for very sensitive and fast Ca2+ signaling. Previous studies have shown that sensitization might function differently in different parts of the OSNs. Modulation by CaM only appears to be significant in the outer dendritic segments . Mitocho2+ [2+ stores was previously shown to not influence OR responses [Further, the function of mitochondria is also influenced by the endoplasmic reticulum, as they are known to be in close contact and transfer Ca2+ . Althougesponses , a possi


Preoperative serum level of carcinoembryonic antigen (pCEA) is generally recognized as a prognostic factor for colorectal cancer (CRC), but the stage-specific role of pCEA in colorectal cancer remains unclear.  We investigated the prognostic significance of pCEA levels in different tumor stages of nonmetastatic CRC patients. X-tile program was used to generate stage-specific cutoff values of pCEA for all patients and two subpopulations (lymph node-positive or -negative). The prognostic significance of pCEA was assessed using Kaplan–Meier analysis and Cox proportional hazards regression analysis. A nomogram model that combined pCEA score and clinical feature indexes was established and evaluated.  Six hundred and fifteen CRC patients at stage I–III were retrospectively analyzed. All of them received curative tumor resection. The P < 0.01) in the subpopulation of lymph node metastasis (stage III) patients, and the relative survival rates in the pCEA-low (≤4.9 ng/mL), pCEA-medium (4.9–27.2 ng/mL), and pCEA-high (≥27.2 ng/mL) groups were 73.4%, 60.5%, and 24.8%, respectively (P < 0.05). However, no such effect was observed in the lymph node nonmetastasis (stage I and II) subgroup. The established nomogram showed acceptable predictive power of the 5-year OS rate (C-index: 0.612) in lymph node-positive CRC patients, with an area under the curve value of 0.772, as assessed by ROC curve analysis.  Two cutoff values were identified in the study population. At a cutoff value of 4.9 ng/mL, a significantly higher 5-year overall survival (OS) rate (82.16%) was observed in the pCEA-low group (<4.9 ng/mL) compared with 65.52% in the pCEA-high group (≥4.9 ng/mL). Furthermore, at the second cutoff value of 27.2 ng/mL, 5-year OS was found to be only 40.9%. Stratification analysis revealed that preoperative serum level of pCEA was an independent prognostic factor (OR = 1.991,  Pretreatment serum CEA levels had different prognostic significance based on the lymph node metastasis status. Among stage III CRC patients, pCEA was an independent prognostic factor. Five-year OS rates could be predicted according to the individual pCEA level at the different cutoff values. Carcinoembryonic antigen (CEA) is a tumor marker in gastrointestinal cancers, particularly colorectal cancer (CRC). It belongs to a superfamily of glycoproteins expressed on cell membranes that play an important role in cell recognition and adhesion. Since it was first described in 1965 , the bioIt is generally accepted that preoperative CEA (pCEA) is not suitable for screening or diagnostic purposes in CRC . HoweverDespite a consensus being reached on the role of pCEA in CRC, the practical application of pCEA as a routine tool was still obstructed by many uncertainties. One was the definition of an “elevated” pCEA level. Because the elevation of preoperative pCEA was associated with the development of tumor stage, the traditional use of a single pCEA cutoff value for prognostic assessment may not be suitable for all CRC patients at other stages –13. ManySix hundred and fifteen eligible patients who were diagnosed with nonmetastatic resectable CRC during the period January 2010 to December 2013 at Fujian Cancer Hospital were recruited. All patients underwent surgery. Data for preoperative serum CEA level, clinicopathological features, and individual characteristics from each patient were retrospectively retrieved from the patient records within the hospital database.th edition of the TNM classification system [XNXM0 resectable CRC; (2) adenocarcinoma confirmed by histopathological examination; (3) physical fitness suitable for surgery; and (4) without receiving any type of adjunctive therapy. The exclusion criteria included the following: (1) over 90 years old; (2) with preexisting or other concomitant cancers; (3) distant metastatic diseases; (4) noncurative resection; (5) multiple primary malignancies; (6) died within 30 days after surgery.In keeping with the 8n system , the incn = 21); (2) with previous or other concomitant cancers (n = 11); (3) distant metastatic disease (n = 31); (4) noncurative resection (n = 12); (5) multiple primary malignancies (n = 11); (6) mortality within 30 d after surgery (n = 0). The process diagram of this article is as Line 1.All patients were followed up by letter or telephone interview. The last follow-up was conducted in January 2018, and the median follow-up period was 57 months . (1) Over 85 years of age  and X-tile program, which identified the cutoff with the minimum P value from log-rank χ2 statistics for the categorical pCEA in terms of survival. Meaningful factors were calculated using the logistic regression method extracted for further analyses. The overall cumulative probability of survival was calculated using the Kaplan–Meier method, and differences were evaluated using the log-rank test.Statistical analyses were conducted using SPSS software version 19.0. The distributions of baseline characteristics were compared using either unpaired R 4.0 software. The concordance index (C-index), receiver operating characteristic (ROC) curve, and internal calibration plot were further used to evaluate predictive performance. P values of <0.05 were considered statistically significant.To evaluate the numerical weight of each factor, particularly the influence of pCEA, for predicting the long-term clinical outcome, a nomogram model integrating pCEA level and the AJCC 8th staging system were developed using Among 615 CRC patients, there were 87 (14.15%) at stage I, 237 (38.54%) at stage II, and 291 (47.32%) at stage III. The 1-, 3-, and 5-year overall survival (OS) rates were 99.35%, 97.56%, and 74.30%, respectively. The distribution of pCEA level ranged from 0.3 to 266.2 ng/mL. Detailed information of recruited patients is summarized in X-tile program. Two cutoff values were detected. At the first cutoff value of 4.9 ng/mL, a significantly higher 5-year OS rate (82.16%) was observed in the CEA-low group (<4.9 ng/mL) as compared with 65.52% in the CEA-high group (≥4.9 ng/mL) (P < 0.05)  .P < 0.05) , CEA-medium (4.9–27.2 ng/mL), and CEA-high (>27.2 ng/mL) groups. The relative 5-year survival rates of these three subgroups were 82.6%, 67.5%, and 40.9%, respectively ( < 0.05) .T4 stage, N2 stage, and CEA level were independent prognostic factors for nonmetastatic CRC  (P > 0.05) , the 5-year survival rates of the three subgroups, pCEA-low (≤4.9 ng/mL), pCEA-medium (4.9–27.2 ng/mL), and pCEA-high (≥27.2 ng/mL), were significantly different at 73.4%, 60.5%, and 24.8%, respectively ( < 0.01) . However > 0.05) .Univariant analysis further confirmed that gender (OR = 1.789), pCEA (OR = 1.875), and T4 category (OR = 2.555) were independent factors for lymph node metastasis patients. Meanwhile, pCEA (OR = 1.610) was independent factor for patients with negative lymph nodes . MultivaT category, and N category to predict 3- and 5-year OS in this subgroup . However, in the subgroup with negative lymph nodes, CEA level was not an independent prognostic factor (P=0.326). This result implied that the prognostic significance of CEA in CRC differs from TNM stage. Although our data suggested that pCEA level might be closely related to lymph node metastasis, consistent with previous research [Previous studies are unclear about the common effect of CEA and lymph node metastasis in predicting prognosis –21. Thisresearch , the undOn this topic, some previous literatures also showed a similar research endpoint –27. Howe1 staging. Moreover, a similar association was observed between pCEA levels higher than 27.2 ng/mL and N2 staging. These results are consistent with clinical observations and further support the idea that pCEA level is closely related to the status of lymph node metastasis. Therefore, through this analysis, it may be possible to initially explain the differences in previous clinical research.Interestingly, as shown by the nomogram model established in this study, we found that when pCEA was in the range of 5.0 to 27.2 ng/mL, its contribution to 5-year OS was equivalent to NDespite the current study proving the significant role of pCEA in CRC prognosis, there are some limitations. First, the inherent bias of patient selection could not be completely avoided, which could partly affect surgical outcomes. Second, the case number of patients with high serum CEA levels was relatively limited in this study, which reduced the statistical power. Third, the data collection was localized in one hospital, which suggests that the current results might not be applicable to different regions of China.In conclusion, we demonstrated that pCEA levels can effectively predict prognosis in CRC patients with positive lymph node metastasis. When pCEA levels are higher than 4.9 ng/mL, they generally indicate worse and unfavorable tumor behavior and poor prognosis.


ARs at inhibitory and an increase in the GluA1, but not GluA2 AMPAR subunit at excitatory synapses. This unbalanced regulation of AMPAR subunits results in the incorporation of Ca2+-permeable GluA2-lacking AMPARs and increased intracellular Ca2+ levels due to a higher Ca2+ influx without affecting its release from the internal stores. Increased neuronal activation upon Nogo-A loss-of-function prompts the phosphorylation of the transcription factor CREB and the expression of c-Fos. These results contribute to the understanding of the molecular mechanisms underlying the regulation of the excitation/inhibition balance and thereby of plasticity in the brain.A tight regulation of the balance between inhibitory and excitatory synaptic transmission is a prerequisite for synaptic plasticity in neuronal networks. In this context, the neurite growth inhibitor membrane protein Nogo-A modulates synaptic plasticity, strength, and neurotransmitter receptor dynamics. However, the molecular mechanisms underlying these actions are unknown. We show that Nogo-A loss-of-function in primary mouse hippocampal cultures by application of a function-blocking antibody leads to higher excitation following a decrease in GABA ARs) present at synapses /(F0 − B0), where F0 and B0 represent the mean grey value of the selected ROIs at resting conditions. In transfected neurons with ER-GCaMP6-150, the calculation makes it possible to determine the resting condition and to distinguish negative and positive amplitudes, reflecting the status of Ca2+ binding. To compensate for the bleaching, the recorded Ca2+ traces were filtered and detected by a MatLab in-house tool. Data were normalized to the first time point before treatment because 2 records were acquired before antibody treatment. With respect to the ER-GCamP5g experiments with CPA, wash-in raw data were plotted. For statistical analysis, a repeated-measures ANOVA with a Fisher’s LSD post hoc test was performed.For analysis, the regions of interest (ROIs) were determined using ImageJ software and analyzed with a MatLab-based self-established protocol. For background correction, an additional ROI was drawn on an area with no fluorescence, representing resting activity. The ROIs were placed on the cell body, and for GCaMP5g experiments, additionally on dendritic spines. To evaluate changes in CaARs; [treatment × time, F = 11.44, p < 0.001, Fisher’s LSD post-hoc test). Comparing the average ratio between mEPSCs and mIPSCs after 10 min of treatment with the control or the Nogo-A function-blocking antibody for each individual neuron showed a net shift in E/I balance toward more excitation and less inhibition (Nogo-A has been shown to confine the recruitment of excitatory postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) at the membrane surface ,22, therARs; ) to streARs;  and clashibition . When nomPSCs × time, F = 11.27, p < 0.001, Fisher’s LSD post-hoc test). Next, the change in amplitude relative to the time point before treatment was plotted in percent for the individual cells over time in a heat map  along individual dendrites of single neurons . The neurons were treated with either Nogo-A function-blocking or control antibodies for 10 min and additionally with different pharmacological blockers for AMPARs , CP-AMPARs , and N-Methyl-D-Aspartate (NMDA) receptors  for 10 more min  = 5.217, p < 0.01, Fisher’s LSD post-hoc test; time, F = 13.98, p < 0.001, Fisher’s LSD post-hoc test; 2+ dynamics  = 13.98, p < 0.001, Fisher’s LSD post-hoc test; 2+ dynamics upon Nogo-A loss-of-function, next only CP-AMPARs were blocked by the selective antagonist Naspm. While the Ca2+ transients of neurons treated with Nogo-A blocking antibodies showed a significant rise in amplitude  = 3.456, p = 0.038, Fisher’s LSD post-hoc test; 2+ amplitude was completely abolished and the fluorescence intensity level was reduced by ∼5% relative to the baseline (Nogo-A: 95% ± 8.58). Under control conditions, the presence of Naspm led to a decrease of the Ca2+ transient amplitude by ∼20% (Ctrl Ab: 79% ± 18.73)  = 5.429, p = 0.024, Fisher’s LSD post-hoc test; 2+ transients was not affected, neither the antibody treatment nor by the application of Naspm  = 5.687, p = 0.006, Fisher’s LSD post-hoc test). In contrast, when the competitive NMDAR antagonist APV was washed-in, the increase in amplitude of Ca2+ transients seen upon Nogo-A blocking antibody treatment was completely abolished  = 8.922, p = 0.001, Fisher’s LSD post-hoc test; Nogo-A signaling has been shown to affect, in addition to GluA1 and GluA2, also the expression of NMDAR subunits in hippocampal neurons . Therefobolished . The flu2+ influx upon Nogo-A loss-of-function in primary hippocampal neurons, presumably due to the absence of GluA2-containing receptors at the synaptic membrane. Together with live-cell labeling of GluA1 and GluA2, this strengthens the hypothesis that Nogo-A regulates CP-AMPAR insertion at synapses.These results suggest an AMPAR and NMDAR-dependent increase in Ca2+ dynamics are crucial for synaptic transmission and affect both inhibitory and excitatory signaling. We recently showed that Nogo-A modulates the motility and synaptic localization of inhibitory postsynaptic GABAAR by regulating Ca2+ dynamics within primary hippocampal neurons [2+ influx in the regulation of post-synaptic neurotransmitter receptors. To take this evidence further and exclude the endoplasmic reticulum (ER) as a possible source of the increased intracellular Ca2+ levels, we investigate whether Nogo-A signaling may affect the release of Ca2+ from the ER. In this respect, primary hippocampal neurons were transfected with the genetically encoded ER-targeted Ca2+ sensor ER-GCaMP6-150 and treated with either a Nogo-A function-blocking or a control antibody. Due to the low affinity properties of ER-GCaMP6-150, Ca2+ dynamics were detected in time-lapse imaging experiments  = 3.131, p = 0.030, Fisher’s LSD post-hoc test; time × treatment, F = 1.326, p = 0.272; 2+ transfer through the ER upon Nogo-A loss-of-function, characterized by increased Ca2+ entry into the intracellular Ca2+ storage sites. To further clarify the impact of the ER in the Ca2+ dynamics downstream of Nogo-A, in some experiments the transfer of Ca2+ from the cytosol into the lumen of the ER was blocked by inhibiting the SERCA ATPase with a 5 min application of Cyclopiazonic acid  resulting in a reversible depletion of ER Ca2+ stores [2+ ions into the ER. Interestingly, the fluorescence intensity curve under control conditions showed a significantly smaller increase than the one of neurons treated with Nogo-A blocking antibodies. While upon Nogo-A neutralization, the fluorescence intensity reached its maximum recovery after 15 min and remained at a plateau of −20% of the baseline, under control conditions the increase was slower reaching −30% of the baseline after 35 min of CPA washout  = 6.382, p < 0.001).Ca neurons . Moreoveeriments A via chaectively B. To mea+ stores . Both th2+ efflux from the ER. This conclusion is supported by the lack of difference in the course of the fluorescence intensity curve during the decreasing phase after depletion of ER Ca2+ stores by application of CPA. This suggests an ER-independent increase in Ca2+ dynamics upon Nogo-A loss-of-function. This hypothesis is strengthened by the significant difference in the replenishment of internal stores after CPA wash-out and the higher speed and amplitude of the positive changes in the fluorescence intensity curve. Thus, upon treatment with the Nogo-A function-blocking antibody, an increase can be observed in the concentration of cytosolic Ca2+, which is recycled through a higher reuptake by the ER.In summary, when comparing the two antibody conditions, no difference could be seen in the Catreatment × time, F = 3.307, p = 0.047, Fisher’s LSD post-hoc test; treatment × current injection, F = 2.587, p = 0.006, Fisher’s LSD post-hoc test; Next, we assessed whether the regulation of synaptic properties upon Nogo-A loss-of-function might influence neuronal excitability. Based on the previous observation that Nogo-A affects synaptic transmission, we additionally examined the effect of Nogo-A loss-of-function on the threshold and the frequency of evoked action potentials (AP). To this aim, the percentual change of the evoked AP threshold was recorded for CA3 pyramidal neurons in hippocampal organotypic slice cultures in whole patch-clamp electrophysiological experiments. Already after 10 min of blocking the Nogo-A protein with a function-blocking antibody, the threshold for generating APs was reduced. After 20 min of Nogo-A antibody treatment, there was a significant difference by ∼6%  between the Nogo-A blocking antibody and the control condition  Nogo-A Ab: 154.2% ± 20.6; (grey) Ctrl Ab: 97.9% ± 14.9) after 10 min Nogo-A loss-of function when compared to the controls  = 7.366, p = 0.009, Fisher’s LSD post-hoc test; 2+ transients observed upon Nogo-A neutralization is completely abolished by the washing in of the CP-AMPAR blocker Naspm during the last 10 min of the experiment , as welein CREB ,21. To eng c-fos . C-Fos eng c-fos . Thus, tTaken together, these results show that Nogo-A loss-of-function increases neuronal excitability and activation as well as the activity-induced expression of different plasticity-related molecules such as pCREB and cFos.AR dynamics and suppressing neurons excitability in a Ca2+-dependent manner. Indeed, loss-of-function for Nogo-A results in a shift in the E/I balance toward more excitation due to the insertion of a higher proportion of Ca2+-permeable AMPARs, an increased influx of extracellular Ca2+, and the reduction of GABAARs at synaptic sites. These changes result in a Ca2+-dependent increase in the activation of hippocampal neurons followed by the enhanced downstream expression of activity-induced immediate early genes (IEGs).In this study, we addressed the mechanism by which Nogo-A signaling is involved in stabilizing the excitation/inhibition (E/I) balance in mouse hippocampal neurons. We show here that it does so by restricting AMPAR and GABAARs diffusion along the membrane [A receptors. This and previously published studies indicate the ability of Nogo-A to regulate neurotransmitter receptor dynamics in mouse hippocampal neurons for review see [2+-permeable AMPARs (CP-AMPARs), and is also observed following the induction of a homeostatic response [2+ transients upon Nogo-A loss-of-function, which is prevented by the application of Naspm, a specific antagonist of CP-AMPARs [We recently showed that Nogo-A signaling via its S1PR2 receptor suppresses excitatory and promotes inhibitory synaptic transmission  and thatmembrane  in an opview see . Specifiresponse ,50,51. IP-AMPARs ,52. ImpoP-AMPARs ,53. InteP-AMPARs ,17,21.2+ transients  suppressed GABAAR lateral diffusion [2+ influx through the plasma membrane leads to an increase in their lateral diffusion [2+ signals as well as of inhibitory synaptic strength upon Nogo-A loss-of-function occurs solely via the S1PR2 [2+ transients after blocking Nogo-A depends exclusively on its influx across the plasma membrane. This claim is based on the Ca2+ imaging experiments performed using a genetically encoded ER-targeted Ca2+ indicator [2+ concentration. The replenishment of the internal stores occurring after application of the washout of the sarco/endoplasmic reticulum calcium ATPase (SERCA) blocker CPA is significantly faster and stronger in cells treated with Nogo-A blocking antibodies, supporting the increased level of cytoplasmic Ca2+ under these conditions. In this context, it is known that the cytosolic Ca2+ concentration is kept stable by the SERCA actively pumping Ca2+ into the ER [2+ efflux from the ER. However, it is important to mention that we cannot completely exclude an extracellular Ca2+ influx into the ER by activating store-operated calcium entry  upon its phosphorylation [2+ signals and further supported by the increase in CREB phosphorylation and in the expression level of c-Fos in primary hippocampal neurons. Interestingly, a gain-of-function approach via the application of the Nogo-A-Δ20 peptide has been shown to reduce CREB phosphorylation, thereby inducing growth cone collapse in axons of dorsal root ganglion neurons [The long-lasting enhancement of synaptic strength during plasticity processes requires Ca changes . The exp changes . Moreoverylation . In this neurons . Additio neurons  as well  neurons . The act neurons  and c-Fo neurons  are asso neurons ,13 as we neurons ,21.2+-dependent manner. The ability of Nogo-A to modulate neuronal excitability as well as pCREB and c-Fos levels makes it extremely interesting in the context of research addressing the molecular mechanisms regulating learning and memory processes, and particularly the formation of memory engrams in healthy and pathological conditions, like the implementation of unwanted memories in post-traumatic-stress-disorder.Taken together, the results presented here identify Nogo-A as a main player in the molecular mechanisms regulating the E/I balance and synaptic plasticity and define the downstream signaling involved in this activity. Particularly, we propose that Nogo-A signaling stabilizes the E/I balance by controlling the neurotransmitter receptor dynamics in a Ca


Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection  and establishing chronic infection . Calcium ion (Ca2+) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca2+ signaling pathways in bradyzoites remain largely unexplored. Here, we show that Ca2+ responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened Ca2+ responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca2+ imaging demonstrated lower Ca2+ basal levels, reduced magnitude, and slower Ca2+ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with downregulation of Ca2+-ATPases involved in intracellular Ca2+ storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus Ca2+ rapidly restored their intracellular Ca2+ and ATP stores, leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca2+ signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility. Toxoplasma gondii is an obligate intracellular parasite, capable of infecting nearly all warm-blooded animals and frequently causing human infections (T. gondii (fections . The ing. gondii . Followi. gondii . In resp. gondii .2+) signaling  from microneme to facilitate rupture of parasitophorous vacuole membrane (PVM) followed by egress (3) by phosphoinositide-phospholipase C (PI-PLC) and leading to subsequent release of intracellular Ca2+  channels, and plasma membrane type Ca2+-ATPases (PMCAs) are predicted to be present in T. gondii and likely involved in regulating cytosolic Ca2+ influx and efflux  or bradyzoite cysts, as detected by indirect immunofluorescence assay (IFA) or time-lapse video microscopy. We observed that A23187 induced complete egress of tachyzoites from disrupted PVs while only a few bradyzoites were released from cysts that remained largely intact  cells at alkaline pH (8.2) for 7 days. We treated both tachyzoite cultures and in vitro-differentiated cysts with Cay intact . This rey intact  or bradyy intact . We quanaprinast . To examaprinast . Bradyzoaprinast , as show2+-stimulated microneme secretion. To examine the reason for inefficient egress by bradyzoites, we monitored microneme secretion by quantitative secretion analysis of MIC2 fused with Gaussia luciferase (Gluc). The MIC2-Gluc reporter was randomly integrated into the genome of the BAG1-mCherry strain  fractions collected following stimulation  from 3 days to 7 days after induction. Based on this criterion, ~70% of parasites were mature bradyzoites in cysts that were induced for 7 days  showed delayed Ca2+ increase and lower fold changes in bradyzoites compared with tachyzoites in response to A23187 stimulation  from bradyzoites within ex vivo cysts demonstrated lower and slower changes, consistent with lower Ca2+ levels, compared with extracellular tachyzoites in response to Ca2+ agonists  after stimulation by A23187 and zaprinast showed slower Ca2+ responses in extracellular bradyzoites when compared to tachyzoites  vs. time  mTagBFP2 linked by a P2A split peptide  or SRS9 (bradyzoites)  . These t2+ and energy stores to meet the demands of cell to cell transmission. To test this idea, we released bradyzoites using trypsin treatment and then treated extracellular bradyzoites with EC buffer with or without Ca2+ (1.8 mM) and with or without glucose (5.6 mM) for different times and stimulated the Ca2+ responses using ionomycin. Quantitative analysis of Ca2+ fluorescence changes (F/F0) showed that bradyzoites substantially recovered stored Ca2+ in the presence of exogenous Ca2+ and glucose for 1 hr compared to 10 min  with glucose to support glycolysis vs. the glucose analogue 2-deoxy-D-glucose (2-DOG) to block glycolysis  and glucose (5.6 mM) for different times  to measure the adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) levels in bradyzoites treated with EC buffer containing both Cant times . We obsent times , demonst glucose . The ATP glucose . Bradyzo glucose , indicatT. gondii . We also demonstrate that bradyzoites are refractory to stimulation of microneme secretion using either an intracellular reporter monitoring the release of PLP1 based on the dispersion of FNR-mCherry from the cyst matrix or a MIC2-GLuc reporter detecting secretion from extracellular bradyzoites. To explore the basis for these differences, we utilized a dual fluorescent reporter GCaMP6f BAG1-mCherry to monitor changes of cytosolic Ca2+ levels in bradyzoites. Calcium signaling was significantly dampened in bradyzoites as reflected in delayed Ca2+ spikes and lower magnitude of cytosolic Ca2+ increases in response to Ca2+ agonists. Reduced Ca2+ responses were also confirmed using bradyzoites naturally formed in C2C12 skeletal muscle cells and ex vivo cysts isolated from chronically infected mice, indicating that the dampened responses are not simply a consequence of alkaline pH stress during bradyzoites development in vitro. Additionally, we observed similar dampened responses from extracellular bradyzoites, indicating that decreased responses are not simply due to reduced permeability of intact cysts to agonists. To confirm these results, we also utilized Fluo-8/AM to monitor intracellular Ca2+ stores of bradyzoites and observed similar dampened responses. Finally, since Ca2+-dependent fluorescence responses by GCaMP6f or Fluo-8 are only relative and subject to differences in protein or probe levels, we developed a ratiometric Ca2+ reporter that contains GCaMP6f fused with self-cleavage tag P2A-linked mTagBFP2 under the control of the same promoter. Ratiometric measurements of the GCaMP6f signal compared to the Ca2+ insensitive indicator mTagBFP2 determined that bradyzoites have lower resting Ca2+ levels and quantitatively decreased Ca2+ responses relative to tachyzoites in response to Ca2+ agonists. Collectively, these findings conclusively show that bradyzoites have reduced Ca2+ responses whether developed in vitro or in vivo and using a variety of independent methods to assess both Ca2+ levels and physiological responses.Egress is a crucial step in the lytic cycle of apicomplexan parasites, and this response requires the sequential steps of increase in cytoplasmic Camotility . Our stu2+ homeostasis, including differences in expression of Ca2+ channels and Ca2+ pumps relative to tachyzoites. These differences would impact Ca2+ storage pools, affecting cytosolic Ca2+ and signaling. For example, our findings indicate that bradyzoites show reduced responses to ionomycin and thapsigargin, which release Ca2+ primarily from the ER, and in response to NH4Cl, which releases Ca2+ from acidocalcisomes and likely other acidic stores  from the ER to the mitochondria . For in vitro induction of bradyzoites, parasites were cultured in alkaline medium in ambient CO2 as described previously  were maintained in DMEM supplemented with 20% FBS. C2C12 myoblast differentiation and myotube formation were induced in DMEM containing 2% horse serum (Biochrom) by cultivation at 37°C in 5% CO2 for 5 days. Tachyzoites were inoculated into the differentiated muscle cells and cultured for another 7 days to induce bradyzoite formation, during which time the induction medium was changed every 2 days. For harvesting bradyzoites, infected monolayers were scraped into intracellular (IC) buffer  and released from cells by serially passing through 18G, 20G, and 25G needles, followed by centrifugation  for 10 min. The pellet containing cysts was resuspended in IC buffer. Bradyzoites were liberated from cysts by digestion with 0.25 mg/ml trypsin at room temperature for 5 min, followed by centrifugation  for 10 min. The supernatant containing liberated bradyzoites was further centrifuged  for 10 min. The pellet containing purified bradyzoites was resuspended in extracellular (EC) buffer  with (1.8 mM Ca2+) or without CaCl2, as indicated for different assays and in the legends.. gondii  was usedeviously . In brie2) for 7 days followed by scraping into PBS containing 0.1% bovine serum albumin (BSA). Cysts were released from host cells by repeated passage through a 23G needle and collected by centrifugation at 150 × g for 10 min. To purify large-scale bradyzoites used for western blotting, pelleted cysts were resuspended in 1 ml PBS containing 10 µl biotinylated Dolichos biflorus agglutinin (DBA) (Vector Laboratories) and 100 µl Pierce Streptavidin Magnetic Beads (Thermo Fisher) and incubated for 1 hr at 4 °C. The beads and absorbed cysts were collected using a magnetic stand and resuspended in 1 ml PBS containing 0.25 mg/ml trypsin and incubated for 10 min at 16°C. The supernatant containing released bradyzoites was separated from the beads and retained. To remove any residual tachyzoites in the supernatant, 5 µl of mAb DG52 pre-coupled to 100 µl Dynabeads Protein G (Thermo Fisher) was added to the supernatant and incubated for 1 hr at 4 °C. The supernatant was separated from the beads, bradyzoites centrifuged at 600 × g, 4°C for 10 min and kept for different assays. To purify bradyzoites used for investigation of recovery of microneme secretion and high-performance liquid chromatography UV (HPLC-UV) analysis of ATP, ADP, and AMP levels in bradyzoites, PBS was replaced with EC buffer without Ca2+ or glucose during the purification by magnetic beads.Tachyzoites were induced to form bradyzoites at a multiplicity of infection (MOI) of 0.5 by culture at pH 8.2 in RPMI 1640 medium under ambient air  (25 μg/ml) and 6-xanthine (6Xa) (50 μg/ml). Single-cell clones containing randomly integrated transgenes were confirmed by diagnostic PCR and by IFA staining. Primers are listed in The BAG1 promoter and the mCherry open reading frame (ORF) were independently PCR-amplified from pNJ-26 and the EGFP ORF was amplified from pSAG1:CAS9-U6:sgUPRT, respectively. The BAG1 promoter fragment and EGFP ORF or mCherry (ORF) were cloned by NEBuilder HiFi DNA Assembly Cloning Kit  into the vector backbone that was produced by double enzymatic digestion of pTUB1:YFP-mAID-3HA, DHFR-TS:HXGPRT using KpnI and NdeI. ME49 ∆The bradyzoite reporter line BAG1-mCherry was transfected with 20 μg of the previously described pMIC2:GLuc-myc, DHFR-TS plasmid , and selThe bradyzoite reporter line BAG1-EGFP was transfected with 20 μg pTUB1:FNR-mCherry, CAT (provided by the Carruthers lab), and selected with 20 μM chloramphenicol. Single-cell clones containing randomly integrated transgenes were confirmed by diagnostic PCR and IFA staining.The ratiometric reporter strain was generated using targeted insertion with CRISPR/Cas9 using previously described methods  to add t0) vs. time were plotted with GraphPad Prism version 6 .For time-lapse microscopy, extracellular parasites were added to glass-bottom culture dishes (MatTek) or intracellular parasites were grown in host cells attached glass-bottom culture dishes. Alternating phase and fluorescence images  were collected on a Zeiss AxioObserver Z1  equipped with an ORCA-ER digital camera (Hamamatsu Photonics) and a ×20 EC Plan-Neofluar objective (N.A. 0.50), 37°C heating unit, and LED illumination for blue, green, red, and far-red wavelengths. Spinning disc images were acquired with a ×100 oil Plan-Apochromat (N.A. 1.46) objective using illumination from 488 nm and 561 nm solid-state lasers (Zeiss) and Evolve 512 Delta EMCCD cameras (Photometrics) attached to the same Zeiss AxioObserver Z1 microscope. Images were acquired and analyzed using Zen software 2.6 blue edition (Zeiss). Fluorescent intensity changes (F/FParasites grown in HFF monolayers on glass coverslips were fixed in 4% (v/v) formaldehyde in PBS for 10 min, permeabilized by 0.25% (v/v) Triton X-100 in PBS for 20 min, and blocked in 3% BSA in PBS. Monolayers were incubated with different primary antibodies and visualized with secondary antibodies conjugated to Alexa Fluor. Coverslips were sealed onto slides using ProLong Gold Antifade containing DAPI (Thermo Fisher Scientific). Images were captured using a ×63 oil Plan-Apochromat lens (N.A. 1.4) on an Axioskop2 MOT Plus Wide Field Fluorescence Microscope . Scale bars and linear adjustments were made to images using Axiovision LE64 software .Samples were prepared in 5× Laemmli buffer containing 100 mM dithiothreitol, boiled for 5 min, separated on polyacrylamide gels by SDS-PAGE, and transferred to nitrocellulose membrane. Membranes were blocked with 5% nonfat milk and probed with primary antibodies diluted in blocking buffer. Membranes were washed with PBS + 0.1% Tween 20, then incubated with goat IR dye-conjugated secondary antibodies (LI-COR Biosciences) in blocking buffer. Membranes were washed several times before scanning on a LI-COR Odyssey imaging system (LI-COR Biosciences).g for 5 min and washing in EC buffer without Ca2+. Parasites were resuspended in EC buffer without Ca2+ and added directly to glass-bottom culture dishes. After addition of agonists, time-lapse images were recorded and analyzed as described above.Freshly harvested parasites were loaded with 500 nM Fluo-8 AM for 10 min at room temperature, followed by centrifugation at 400 × Infected cells were treated with 2 μM A23187 or 500 μM zaprinast for 15 min at 37°C. Following incubation, samples were stained by IFA using antibodies against SAG1 (mouse), GRA7 (rabbit), FITC-conjugated DBA or BAG1 (rabbit), and followed by secondary antibodies conjugated to Alexa Fluor. Samples were examined by fluorescence microscopy, and the percentages of egressed or released parasites per vacuole or cyst was determined at least for 20 vacuoles or cysts per experiment. The maximum egress distance of parasites from vacuole or cysts was measured from scanned tiff images in ImageJ.hxgprt::Fluc tachyzoites, cultured and harvested as indicated above, were used for gating. Approximately 1 × 106 parasites from each sample (ME49 BAG1-mCherry MIC2-GLuc reporter tachyzoites and ME49 BAG1-mCherry MIC2-GLuc reporter bradyzoites) were sorted on Sony SH800S Cell Sorter directly into 500 µl IC buffer followed by centrifugation. Flow cytometry data were processed using FlowJo version 10 .ME49 BAG1-mCherry MIC2-GLuc reporter bradyzoites were induced for 7 days at pH 8.2, harvested in IC buffer as described above, and passed through a 3 μm polycarbonate membrane filter. ME49 ∆Gaussia Luciferase Glow Assay Kit reagent (Thermo Scientific), and luminescence was detected using a Cytation 3 Cell Imaging Multimode Imager . Buffer control values were subtracted from their corresponding sample values to correct for background.FACS-sorted MIC2-GLuc reporter tachyzoites and bradyzoites, or bradyzoites purified by magnetic beads, were suspended with EC buffer and incubated with different agonists at 37°C for 10 min. ESA was collected by centrifugation and mixed with Pierce hxgprt::Fluc tachyzoites and bradyzoites induced for 7 days at pH 8.2 using RNeasy Mini Kit (QIAGEN) combined with QIAshredder (QIAGEN) followed by DNA Removal using DNA-free DNA Removal Kit (Thermo Fisher) and subsequent reverse transcription using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher). Quantitative real-time PCR was performed on Applied Biosystems QuantStudio 3 Real-Time PCR System (Thermo Fisher) using SYBR Green JumpStart Taq ReadyMix (Sigma) with primers shown in RNA was extracted from ME49 ∆Coverslips were precoated by incubation in 50% FBS diluted in PBS for 1 hr at 37°C followed by rinsing in PBS. Freshly harvested tachyzoites or bradyzoites were resuspended in EC buffer, treated with DMSO , or inhibitors , and then added to precoated glass coverslips and incubated at 37°C for 15 min. Coverslips were fixed in 2.5% formalin in PBS for 10 min, and the surface proteins were detected by IFA as described above using anti-SAG1 and anti-SRS9 antibodies as stage-specific markers for tachyzoites and bradyzoites, respectively. Gliding trails were captured by IFA microscopy as described above, and the frequency of trails was measured from tiff images using ImageJ.2) for 7 days followed by scraping into EC buffer without Ca2+ or glucose and repeated passage through a 23G needle. Intact but extracellular cysts were pellet by centrifugation at 150 × g for 10 min and resuspended in EC buffer without Ca2+ or glucose and incubated for ~2 hr at 4°C. MatTek 25 mm glass-bottom dishes (coverslip dishes) were precoated with 2 ml 50% FBS at 4°C overnight and rinsed twice using PBS prior to use. Purified cysts were added to the precoated coverslip dishes in EC buffer without Ca2+ or glucose but containing 0.25 mg/ml trypsin and incubated for 10 min at 16°C. The medium was removed and 2 ml EC buffer ± 1.8 mM Ca2+ and/or ±5.6 mM glucose was added and incubated for 10 min or 1 hr at 16°C. Prior to imaging, the coverslip dishes were heated to 37°C using a Heating Unit XL S (Zeiss) attached to the Zeiss AxioObserver Z1 . Images were collected under bright-field illumination using a ×40 C-Apochromat water immersion objective (N.A. 1.20) and ORCA-ER digital camera (Hamamatsu Photonics) at 1 s intervals for 5 min per field. The percentage of BAG1-mCherry-positive bradyzoites displaying different types of gliding motility was calculated from six movies per sample. Images were imported into NIH ImageJ with a Cell Counter plug-in for quantification of the types of motility based on visual inspection.BAG1-mCherry parasites were induced to form bradyzoites by culture at pH 8.2 in RPMI 1640 medium under ambient air  was used to resuspend cell pellets and incubated for 5 min at room temperature. Extraction was stopped by addition of 17 µl of neutralization buffer (2 M potassium hydroxide) to the samples followed by mixing. Samples were centrifuged at 14,000 × g for 10 min at 4°C, and the supernatant was transferred to a new tube for HPLC analysis. Analysis was performed using an HPLC system consisting of a SPD-20A UV/VIS detector (Shimadzu) equipped with SIL-20A autosampler (Shimadzu), with a Luna Omega Polar C18 column  and LC-20AD pump (Shimadzu). The protocol was setup as isocratic separation using a mobile phase containing 0.1 M ammonium dihydrogen phosphate , pH 6.0, containing 1% methanol with a flow rate of 0.8 ml/min. Injection volume was 30 µl, and peak detection was monitored at 254 nm. A series of standards containing ATP, ADP, and AMP with different concentrations were used to establish retention times and standard calibration curves by calculating peak area. Samples from two independent biological replicates were analyzed using three technical replicates. The retention time and peak areas were used to calculate the corresponding concentration of each nucleotide from each sample according to the standard curve.A previously described method for extraction of ATP, ADP, and AMP  was adapMice were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International-approved facility at Washington University School of Medicine. All animal studies were conducted in accordance with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals, and protocols were approved by the Institutional Animal Care and Use Committee at the School of Medicine, Washington University in St. Louis.Eight-week-old female CD-1 mice (Charles River) were infected with 200 ME49 BAG1-mCherry GCaMP6f tachyzoites by intraperitoneal injection. After 30 days of infection, animals were sacrificed, the brain removed and homogenized, and the number of brain cyst was determined by DBA staining and microscopy as previously described . Eight-wt-tests, while data that were not normally distributed, or contain too few samples to validate the distribution, were analyzed by Mann–Whitney or Kruskal–Wallis nonparametric tests. *p<0.05, **p<0.01, ***p<0.001.Statistical analyses were performed in Prism (GraphPad). Data that passed normally distribution were analyzed by one-way ANOVA or Student’s  Toxoplasma gondii. However, calcium signaling in these stages is rapidly restored following parasite egress and exposure to extracellular calcium and energy sources. The ability of Toxoplasma bradyzoites to rapidly switch between quiescence and a metabolically active state is likely to be essential for maintaining long-lived chronic infections as well as successful transmission.This study shows that calcium signaling is strongly suppressed in the intracellular cyst-forming bradyzoite stages of  public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Malcolm J McConville as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and Dominique Soldati-Favre as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Christopher J Tonkin (Reviewer #2).Thank you for submitting your article "Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.T. gondii bradyzoite stages. A number of issues were identified which may require additional experimental support and/or explicit discussion in the text.The three reviewers noted the novelty of these findings and the insights they provide into the biology of Essential revisions:1. Provide further validation of the Mic2-Gluc assay for measuring microneme secretion in bradyzoites – in particular it would be important to show that bradyzoites express sufficient M2AP to allow use of Mic2-Gluc as a read-out for secretion or confirm using an alternative assay.2. Provide indication of variability in measurements of calcium levels where currently only the mean of just three cells is provided (or repeat with increased number of cells). This is important in Figure 5 where basal calcium levels are being measured.3. Ensure that several points regarding methodology raised by the reviewers are directly addressed in the text, including the use of different time points when measuring ATP levels and gliding motility in extracellular bradyzoites, and discussion of possible effect of bradyzoite isolation protocol on calcium signalling.4. Include discussion on the role of calcium signalling in mobilization of amylopectin carbohydrate stores and activation of bradyzoites in vivoReviewer #1 (Recommendations for the authors):It remains unclear whether repression of calcium signalling in bradyzoites directs these stages into metabolic quiescence, or conversely, that entry into metabolic quiescence decreases the cellular energy balance with concomitant decrease in calcium uptake, depletion of intracellular stores and signalling. Can the authors draw out arguments for one side or the other?As noted above, incubation of artificially released bradyzoites in calcium plus or minus a carbon source leads to increased motility and ATP levels. However, it is likely that activation of bradyzoites will occur earlier in vivo . During natural egress, bradyzoites will be more dependent on intracellular carbohydrate stores rather than exogenous sugars. Mobilization of internal stores is suggested by the finding that calcium alone is sufficient to activate motility in extracellular bradyzoites. Discussion of this possibility, including reference to previous work showing that calcium dependent kinases regulate amylopectin degradation is warranted.Figure 8F. Can the authors provide information on the basal levels of ATP/ADP/AMP in tachyzoites suspended in the same EC buffer 10min/1hr, to allow comparison with bradyzoite data?Reviewer #2 (Recommendations for the authors):The introduction uses language that is a bit too definitive in places. I suggest to tone down.2+, I'd say the evidence suggests it is likely, but it is far from being definitive.– I would not say that ER is the known storage site of ca– Line 346- I would not say 1hr is a rapid change, especially in context of persistence in the brain or transmission (see below)2+ in the ER cannot lead to Mn secretion – this must be a mistake.There are some spelling/grammatical mistakes; for example, Line 80: Accumulation of Ca2+, motility and microneme secretion impact cyst lysis and re-invasion? Are these parameters enough or is there a transition period where bradyzoites become more motile to? Likewise, for transmission, it seems unlikely that an extracellular bradyzoite could hang around in the gut for 1 hr, to bolster energy and ca2+ needed to actively invade cells. Does this mean that high ca2+ and ATP (similar levels to tachyzoites) are not needed for transmission or do the authors think there is other possibilities? These considerations of the physiological relevance are important to discuss.I think the discussion is too long and goes over a lot of what is already presented in Results section. I recommend reducing this but instead discuss more about how these results impact our understanding of transmission and persistence of a latent infection. For example, how do the lower levels of ca Essential revisions:1. Provide further validation of the Mic2-Gluc assay for measuring microneme secretion in bradyzoites – in particular it would be important to show that bradyzoites express sufficient M2AP to allow use of Mic2-Gluc as a read-out for secretion or confirm using an alternative assay.We performed western blotting  and IFA  to confirm that bradyzoites express MIC2-Gluc and M2AP, albeit at lower levels compared with tachyzoites. Moreover, MIC2-GLuc and M2AP were properly co-localized to the apical end in bradyzoites, ruling out the possibility of mis-localization of the MIC2-GLuc reporter. Based on these results, we believe that MIC2-GLuc provides a reliable read-out for microneme secretion in in vitro differentiated bradyzoites. Additionally, the conclusion that MIC secretion is dampened in bradyzoites is also supported by the studies using the FNR-Cherry reporter in Figure 2E,F,G.2. Provide indication of variability in measurements of calcium levels where currently only the mean of just three cells is provided (or repeat with increased number of cells). This is important in Figure 5 where basal calcium levels are being measured.We have quantified more cells in all figures related to fluorescence measurements. For measurements of single parasites in Figure 5B, 5D, 5E, 6F, 8A, 8B and Figure 7 supplement 1A, we have now quantified 10 parasites for each condition and plotted the data as means ±S.D. For in vitro induced cysts or ex vivo cysts in Figure 2G, 3D, 3E, 4C,4G, 6E, 7B and Figure 4 supplement 1A, we measured 5 cysts or vacuoles per condition. Because these samples contain many parasites within each vacuole or cyst, they represent a greater sample size. The data are also plotted a means ±S.D.3. Ensure that several points regarding methodology raised by the reviewers are directly addressed in the text, including the use of different time points when measuring ATP levels and gliding motility in extracellular bradyzoites, and discussion of possible effect of bradyzoite isolation protocol on calcium signalling.We have conducted several new experiments to more closely match the experimental conditions to: (1) rule out that the differences are due to different time points or treatments , (2) define the extent of bradyzoite differentiation , (3) demonstrate the treatment with trypsin does not affect intracellular calcium , and (4) validate consistent expression levels of GCaMP6f in different stages . We also repeated the gliding motility assays  using bradyzoites purified by the same methods used for ATP analysis to match these two experiments more closely. We found the same results in previous experiments that incubation with exogenous glucose and calcium leads to the recovery of gliding motility by bradyzoites.4. Include discussion on the role of calcium signalling in mobilization of amylopectin carbohydrate stores and activation of bradyzoites in vivoThis is an excellent suggestion and we have included it in the Discussion.Reviewer #1 (Recommendations for the authors):It remains unclear whether repression of calcium signalling in bradyzoites directs these stages into metabolic quiescence, or conversely, that entry into metabolic quiescence decreases the cellular energy balance with concomitant decrease in calcium uptake, depletion of intracellular stores and signalling. Can the authors draw out arguments for one side or the other?This is an interesting point, but the current data do not argue strongly for one possibility over the other. We have added this statement to the discussion to highlight this as an interesting area for further research: "One aspect that is not addressed by our studies is whether altered expression of calcium channels and pumps is responsible for reducing energy levels and hence driving quiescence, or whether the altered calcium pathways are a consequence of initial changes in energy production." As noted above, incubation of artificially released bradyzoites in calcium plus or minus a carbon source leads to increased motility and ATP levels. However, it is likely that activation of bradyzoites will occur earlier in vivo . During natural egress, bradyzoites will be more dependent on intracellular carbohydrate stores rather than exogenous sugars. Mobilization of internal stores is suggested by the finding that calcium alone is sufficient to activate motility in extracellular bradyzoites. Discussion of this possibility, including reference to previous work showing that calcium dependent kinases regulate amylopectin degradation is warranted.This is an excellent suggestion and we have included it in the Discussion.Figure 8F. Can the authors provide information on the basal levels of ATP/ADP/AMP in tachyzoites suspended in the same EC buffer 10min/1hr, to allow comparison with bradyzoite data?Toxoplasma gondii tachyzoites do not require carbon source uptake for ATP maintenance, gliding motility and invasion in the first hour of their extracellular life. Int J Parasitol. 2011 Jul;41(8):835-41. doi: 10.1016/j.ijpara.2011.03.005. Epub 2011 Apr 7. PMID: 21515276.)We thank the reviewer for this suggestion. We measured the ATP/ADP/AMP levels in tachyzoites treated with EC buffer for 10 min vs.1 hr and found that treatment did not significantly affect the ATP and ADP levels in tachyzoites but led to increase in AMP levels. These results indicate that extracellular tachyzoites do not depend on uptake of exogenous glucose for ATP production during the first hr they are extracellular, a result that is consistent with the conclusion in a previous publication :The introduction uses language that is a bit too definitive in places. I suggest to tone down.2+, I'd say the evidence suggests it is likely, but it is far from being definitive.– I would not say that ER is the known storage site of caRevised as suggested.– Line 346- I would not say 1hr is a rapid change, especially in context of persistence in the brain or transmission (see below).Revised as suggested.2+ in the ER cannot lead to Mn secretion – this must be a mistake.There are some spelling/grammatical mistakes; for example, Line 80: Accumulation of caRevised as suggested.I think the discussion is too long and goes over a lot of what is already presented in Results section. I recommend reducing this but instead discuss more about how these results2+, motility and microneme secretion impact cyst lysis and re-invasion? Are these parameters enough or is there a transition period where bradyzoites become more motile to? Likewise, for transmission, it seems unlikely that an extracellular bradyzoite could hang around in the gut for 1 hr, to bolster energy and ca2+ needed to actively invade cells. Does this mean that high ca2+ and ATP (similar levels to tachyzoites) are not needed for transmission or do the authors think there is other possibilities? These considerations of the physiological relevance are important to discuss.impact our understanding of transmission and persistence of a latent infection. For example, how do the lower levels of caWe prefer to keep the summary in the Discussion as it provides a synthesis for the many different experimental results that are presented. However, we agree that it would be beneficial to expand the text to address the issue of how altered calcium signaling in bradyzoites impacts transmission and we have now included this topic in the Discussion.


Background: Atrial fibrillation (AF) is associated with calcium (Ca2+) handling remodeling and increased spontaneous calcium release events (SCaEs). Nevertheless, its exact mechanism remains unclear, resulting in suboptimal primary and secondary preventative strategies.Methods: We searched the PubMed database for studies that investigated the relationship between SCaEs and AF and/or its risk factors. Meta-analysis was used to examine the Ca2+ mechanisms involved in the primary and secondary AF preventative groups.Results: We included a total of 74 studies, out of the identified 446 publications from inception (1982) until March 31, 2020. Forty-five were primary and 29 were secondary prevention studies for AF. The main Ca2+ release events, calcium transient (standardized mean difference (SMD) = 0.49; I2 = 35%; confidence interval (CI) = 0.33–0.66; p < 0.0001), and spark amplitude  were enhanced in the primary diseased group, while calcium transient frequency was increased in the secondary group. Calcium spark frequency was elevated in both the primary diseased and secondary AF groups. One of the key cardiac currents, the L-type calcium current (ICaL) was significantly downregulated in primary diseased  and secondary AF groups . Furthermore, the sodium–calcium exchanger (INCX) and NCX1 protein expression were significantly enhanced in the primary diseased group, while only NCX1 protein expression was shown to increase in the secondary AF studies. The phosphorylation of the ryanodine receptor at S2808 (pRyR-S2808) was significantly elevated in both the primary and secondary groups. It was increased in the primary diseased and proarrhythmic subgroups  and secondary AF group . Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) expression was elevated in the primary diseased and proarrhythmic drug subgroups but substantially reduced in the secondary paroxysmal AF subgroup.Conclusions: Our study identified that ICaL is reduced in both the primary and secondary diseased groups. Furthermore, pRyR-S2808 and NCX1 protein expression are enhanced. The remodeling leads to elevated Ca2+ functional activities, such as increased frequencies or amplitude of Ca2+ spark and Ca2+ transient. The main difference identified between the primary and secondary diseased groups is SERCA expression, which is elevated in the primary diseased group and substantially reduced in the secondary paroxysmal AF subgroup. We believe our study will add new evidence to AF mechanisms and treatment targets. It ifunction , 8.2+ release events are exclusively investigated in myocardial physiology and pathophysiology, as they hold the key to understanding how cardiomyocyte Ca2+ signaling is regulated by ionic channels and Ca2+ proteins  located on the junctional sarcoplasmic reticulum (SR) to release Ca2+ from its stores into the cytosol  to bind to actin in the sarcomere to initiate cardiac contraction (2+ is mainly recycled back into the SR via the SR Ca2+-ATPase (SERCA2a) pump or extruded across the cell membrane through the cardiac sodium–calcium exchanger (NCX1) , which is responsible for transducing cytosolic Ca2+, and calmodulin, a Ca2+-binding messenger protein that modulates RyR activity and transduces Ca2+ signals to other protein kinases or phosphatases  . The redlaxation , 16. SERtion (2+ . Anotherphatases –19.2+ release events (SCaEs) are observed as spontaneous Ca2+ sparks or arrhythmogenic Ca2+ waves are substantially enhanced  guidelines , Ca2+ transient frequency (CaTF), Ca2+ spark amplitude (CaSpA), and Ca2+ transient amplitude (CaTA)], and Ca2+ load and leak. It also included atrial current densities, such as L-type calcium current (ICaL), sodium–calcium exchanger current (INCX), late sodium current (INa−Late), and potassium current (IK), and protein expressions, such as L-type alpha 1C subunit voltage-dependent calcium current (Cav1.2), NCX1, RyR2, phosphorylated ryanodine receptor 2 (pRyR2), SERCA2a, PLN, phosphorylated phospholamban (pPLN), CAMKII, phosphorylated Ca2+/calmodulin-dependent protein kinase II (pCAMKII), and PKA. We further categorized the above results into two main groups: primary and secondary preventative therapies for AF. Primary prevention was divided into three subclasses: the diseased group (risk factors for AF), the application of proarrhythmogenic agents or antiarrhythmogenic agents. Secondary prevention was classified into either the paroxysmal or chronic AF group. The analysis was conducted using R.The atrial cellular activities were extracted from selected studies. It included SCaEs i, seen as Ca2+ sparks. The properties (frequency and amplitude) of these Ca2+ release events are key determinants to the arrhythmogenicity of the cardiomyocytes. In our study, key calcium-handling remodeling including ionic currents, calcium release events, and protein expressions was summarized for the primary prevention and secondary AF groups in I2 = 0%; CI = 0.30–0.89; p = 0.6601) and proarrhythmic drug subgroup      (The evolution of cardiac Ca 0.0001) . When th 0.0001) . The add 0.0054) . A simil 0.0001)  and redu 0.0002) . In addi 0.0002) .I2 = 96%; CI = −0.14–1.76; p < 0.0001   and permanent AF   were significantly elevated in both the secondary paroxysmal and chronic AF subgroups, with respective SMD = 0.81; < 0.0001 , and SMD< 0.0001 , and hig 0.0386) . Surpris 0.0001) .2+ release is affected by the opening of RyR channels from its stores. In particular, SR Ca2+ leak is a major contributor to cardiac arrhythmia. Ca2+ load remained relatively unchanged in both the primary and secondary subgroups, except when antiarrhythmic drugs were applied in the primary group    (SR Ca 0.0001) . No chanon group , but it  0.7583)  and anta 0.0932) .CaL. ICaL was significantly downregulated in the primary diseased subgroup       , antiarr 0.0001) , and sec 0.0001) . These r 0.2027)  and seco 0.0001) .2+]i for Ca2+ recycling is via the cardiac NCX. INCX was significantly enhanced in both the primary diseased and proarrhythmic subgroups       and redu 0.0001) . Likewis 0.0638)  and sign 0.0001) . As oppo< 0.0001 , and SMD= 0.0638 , respectI2 = 64%; CI = 0.12–1.79; p = 0.074)    and S2814 (pRyR-S2814). S2808 is phosphorylated by PKA, while S2814 is modulated by CAMKII. Only pRyR-S2808 was significantly affected in both primary and secondary groups, while total RyR (tRyR) and pRyR-S2814 showed no significant changes. pRyR-S2808 expression was increased in the primary diseased and proarrhythmic subgroups   and the  0.0001)  but was  0.0315) .2+ recycling is via the SERCA2a pump, in which activity is directly controlled by PLN. Few studies reported on SERCA and PLN expression, resulting in inconsistent and non-significant results. SERCA expression was elevated in the primary diseased and proarrhythmic subgroups but substantially reduced in the secondary paroxysmal AF subgroup. Total PLN (tPLN) remained relatively constant in all primary subgroups but was similarly decreased in paroxysmal AF. The results for phosphorylated PLN at sites serine 16 (pPLN-S16) and threonine 17 (pPLN-T17) in both groups were markedly diverse.Another pathway for CaI2 = 60%; CI = 0.77–2.40; p = 0.0192) , and total CAMKII (t CAMKII) by AF groups  (Na), INa−Late, and IK current densities. However, all data extracted were non-significant and/or inconsistent except for INa−Late. INa−Late was moderately enhanced in diseased      and anti= 0.023) . Phospho 0.2206)  and proa 0.7447)  and sign 0.3408) , with loThe action of various drugs or reagents in the different experimental models were summarized in This preclinical systematic review study analyzed 74 articles identified from 446 searched primary and secondary AF prevention articles. Forty-five publications were classified as primary AF prevention studies and 29 others as secondary prevention. To our knowledge, this is the largest study of this kind to explore the association between modulated calcium homeostasis and release events for primary and secondary prevention of AF. Our principal findings are summarized as follows .2+ channels/proteins/mediators, our study found that ICaL was the most widely studied current in both primary and secondary AF prevention, followed by INCX and RyR2 channels.With regard to the key CaCaL was significantly downregulated in primary and secondary diseased groups, which were largely consistent with our results for Cav1.2 protein expression. Antiarrhythmic drugs in the primary group further reduced ICaL significantly.We showed that INCX was only elevated in the primary diseased group.Furthermore, the NCX1 protein expression was significantly enhanced in both the primary and secondary diseased groups, but IIn addition, our study demonstrated that the key phosphorylation expression for RyR was enhanced at serine 2808 in both the primary diseased and secondary AF groups, and inhibited in the primary antiarrhythmic drug subgroup. On the other hand, the other key RyR phosphorylation expression at serine 2814 showed no significant changes in both the primary and secondary diseased groups.SERCA expression was elevated in the primary diseased and proarrhythmic drug subgroups but substantially reduced in the secondary paroxysmal AF subgroup. tPLN remained relatively constant in all primary subgroups but was decreased in paroxysmal AF.2+ signaling mediator CAMKII was increased in the secondary AF group. With its phosphorylation activity at threonine 286, pCAMII-T286 was significantly raised by proarrhythmic drugs and significantly reduced by antiarrhythmic therapies.Finally, the CaNa−Late and its direct effect on arrhythmia. Our study identified many primary preventative publications that showed that INa−Late was moderately enhanced in the primary diseased and proarrhythmic drug subgroups but significantly antagonized by antiarrhythmic agents, with low heterogeneities across all three subgroups.It is noteworthy that there is a growing surge of interest for the late sodium current I2+ functional activities, i.e., Ca2+ spark, Ca2+ transient, and Ca2+ load/leak. In the primary prevention group, CaSpF, CaTA, and CaSpA were significantly enhanced in the diseased subgroup and decreased by antiarrhythmic drug agents. On the other hand, CaTF and CaSpF were significantly elevated in both the secondary paroxysmal and chronic AF subgroups. Interestingly, we discovered that SR Ca2+ load and Ca2+ leak remained relatively constant in the primary and secondary subgroups, except when SR Ca2+ load was reduced when antiarrhythmic drugs were applied in the primary group. Furthermore, we found that Ca2+ leak was raised by proarrhythmic agents and antagonized by antiarrhythmic agents in the primary group.As a result of atrial remodeling in the ionic channels and protein/signaling expressions in diseased and AF conditions, we observed changed CaCaL and dysfunction of the Ca2+-handling proteins, in particular pRyR at S2808. The enhanced pRyR-S2808 activity may increase the frequency of the Ca2+ spark due to calcium-induced calcium releases. This would eventually lead to a high Ca2+ level in the cytosol and enhanced trigger activities via the forward mode of INCX, which was demonstrated in this review as the elevation of INCX activity. Furthermore, the enhancement of INa−Late was shown to potentially play a more significant role in the generation of arrhythmia in our review and recent studies  can be found in the article/SF and SA undertook data extraction, post-processing, and analysis, as well as drafting the manuscript. MG assisted in assessing the quality of the included studies. JZ guided the project and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.