We have demonstrated in vivo and in vitro that intracellular Ca2+ increases in the gastric epithelial cells directly adjacent to a damaged cell, and that this Ca2+ rise is essential for the cellular migration that rapidly repairs the epithelium (restitution). While intracellular Ca2+ has been shown to be an important signaling factor during epithelial restitution, the source from which this intracellular Ca2+ originates remains unclear. Using gastric organoids derived from mice transgenic for a genetically encoded Ca2+ indicator, we sought to investigate the potential sources of intracellular Ca2+ mobilization. During confocal imaging, photodamage (PD) was induced to 1–2 gastric organoid epithelial cells and epithelial restitution measured simultaneously with changes in intracellular Ca2+ (measured as FRET/CFP ratio in migrating cells adjacent to the damaged area). Inhibition of voltage‐gated Ca2+ channels  or store‐operated calcium entry  resulted in delayed repair and dampened intracellular Ca2+ response. Furthermore, inhibition of phospholipase C  or inositol trisphosphate receptor  likewise resulted in delayed repair and dampened Ca2+ response. Results suggest both extracellular and intracellular Ca2+ sources are essential for supplying the Ca2+ mobilization that stimulates repair.Calcium  cells juxtaposed to the damaged cell. Interestingly, organoids exhibited a similar pattern of gene expression of these calcium‐related transcripts to uninjured, intact and ulcerated tissues  pathway based upon expression of SOCE‐associated genes .To identify potential Ca) Figure ; Table 12.22+ sensor mice and monitored repair by live imaging  in the presence of verapamil   compared to the control repair rate   Figure . Further) Figure ,d. These2.32+ as an essential source to aid in Ca2+ mobilization during gastric repair, an inhibitor of store operated Ca2+ entry  was added to the media 1 hr prior to PD. The presence of YM58483 dampened the Ca2+ mobilization following PD  compared to the control FRET/CFP peak at 1.37 ± 0.02   to 0.33 ± 0.06 min−1 (n = 4)  Figure . Additio) Figure ,d. These2.42+ release in mediating wound repair. We inhibited PLC by adding U73122 (10 μM) into the media and monitored the response following injury. Following PD, U73122 dampened the Ca2+ response during repair  compared to the control of 1.32 ± 0.03   compared to control   Figure . Further) Figure ,d. These) Figure , identif2+ from the endoplasmic reticulum (ER) via the downstream receptor inositol triphosphate receptor (IP3R)  to test the role of IP3R during restitution. Following PD, 2‐APB dampened the Ca2+ mobilization  compared to the control   compared to the control   Figure . Further) Figure ,d. These32+ dynamics has been well studied in other systems, much remains unclear within the context of gastric epithelial cells, especially during repair. The general role of Ca2+ as an effector of gastric wound repair has been established for 30 years  and PLC, IP3 (intracellular) pathways to promoting repair. We have previously shown in vivo the importance of localized changes in both extracellular and intracellular Ca2+ during gastric epithelial repair . In vivo models are also limited in the ability to apply drugs while avoiding more systemic effects. To circumvent these issues, we used the gastric organoid model. Similar to previous in vivo work, this study found that inhibition of voltage‐gated channels, PLC and IP3R, caused significantly decreased Ca2+ mobilization in addition to delayed restitution. Our functional findings are parallel to what has been witnessed in vivo, indicating the reliability of gastric organoids to mimic native tissue and confirming the role of both voltage‐gated channels and the PLC pathway during Ca2+ driven repair. The concordance of highly expressed calcium‐signaling genes further support these conclusions. This study extends our prior report that restitution in gastric organoids is dependent upon intracellular Ca2+ mobilization, acting downstream of several important factors triggering proper repair  and diacylglycerol (DAG), metabolites that can stimulate Ca2+ release from intracellular stores and activate protein kinase C (PKC). PKC is also a conventional target of Ca2+‐dependent regulation. Both PLC and PKC have been shown in primary, immortalized, and cancer‐derived cell lines to stimulate epithelial cell migration  is a known initiator of Ca2+ stores from the endoplasmic reticulum (ER) involves PLC. Either in response to activation via a G‐protein coupled receptor or tyrosine kinase receptor, PLC hydrolyzes the IP3 precursor, phosphatidylinositol 4,5‐bisphosphate (PIP2) to produce diacylglycerol (DAG) and IP3 , are the principal intracellular Ca2+ channels responsible for the release of Ca2+ from the ER membrane stores , and YC‐Nano), we have observed that all cells respond to damage in a similar manner , or overall organoid structure. Growth rates and histology were not considered due to the relatively short‐term exposure to the drugs. Since calcium is an important physiological response for homeostasis, we anticipate that long‐term exposure to these pharmacologic agents would affect organoid growth, size, and histology. In the future, studies of long‐term inhibitor exposure may be beneficial as patients are often treated with drugs for extended periods.2+ dynamics by demonstrating the importance of two classes of plasma membrane channels and the role of intracellular ER release in the observed intracellular Ca2+ mobilization  expressing the Yellow Cameleon‐Nano15 (YC Nano) Ca4.22+ and Mg2+ in 8‐well or 2‐well Lab‐Tek chamber with cover glass (Thermo Fisher Scientific) to grow gastric organoids. Gastric organoids were cultured in a 5% CO2 incubator at 37°C for 3–4 days prior to experiments.Gastric organoids were generated from mouse gastric corpus as previously described  on an inverted confocal microscope (Zeiss LSM 510 NLO) and imaged with a C‐Achroplan NIR 40x objective lens. To assess repair of damage area, images of YFP  in the gastric organoid were collected simultaneously with transmitted light and a confocal reflectance image (reflecting 730 nm light to show cell/tissue structure). For assessing intracellular Ca2+ changes in YC Nanogastric organoids, images of YFP‐FRET  and CFP  were collected simultaneously with a transmitted light image. Wavelength selections for Ca2+ imaging were guided by previous work with YC sensors  of a single cell was repetitively scanned at high Ti‐Sa laser power (730 or 840 nm: 630 mW average) for 500 iterations (requiring ≈3 s).Experiments were performed in organoid culture medium under 5% CO2O, or dPBS added to the medium; vehicle was dependent on the solvent the inhibitors were dissolved in. During experiments with inhibitors, all observed gastric organoids maintained their shape, size and overall integrity, and no increase in shedding (apoptotic) cells was observed compared to untreated gastric organoids. Concentrations were determined based upon in vitro studies and tested to assess effectiveness and potential toxicity.Experiments examined gastric organoids embedded in Matrigel, located approximately 100–300 μm from the cover glass. Inhibitors were preincubated at least 1 hr prior to experimentation to assure equilibration in Matrigel and inhibitors were kept in the medium during experiments. Inhibitory reagents included: Verapamil , YM58483 , U73122 , and 2‐APB . Final DMSO concentration in experiments was <0.1%. Solvent control groups contained 0.1% DMSO added to medium. Vehicle control groups contained either 0.1% DMSO, ddHDamage–repair cycle was measured independently once per gastric organoid, and outcomes from at least three different gastric organoids , were compiled for each experimental protocol.4.42) was quantified from the time course of images as described . Changes in intracellular Ca2+ were measured as FRET/CFP ratio using YC Nano gastric organoids. Background images were subtracted from FRET‐YFP and CFP images, the resultant images were divided on a pixel‐by‐pixel basis to calculate the FRET/CFP ratio image. All time course ratio images were then normalized to the averaged predamage baseline images. Regions of interest were determined by transmitted light and 514 nm excited YFP images to define cellular structures for whole cell and lateral region measurements.Damaged area (units of µm4.5http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE73336)  from 'n' organoid experiments. Statistical significance was determined using unpaired Student's T‐test or one‐way ANOVA with Dunnett's multiple comparison post hoc test. A The authors declare no competing or financial interests.K.A.E and M.H.M contributed to conceptualization: K.A.E also contributed to Methodology, Formal analysis, and Investigation and Y.O., R.A.K. contributed to Resources; Writing – the original draft: K.A.E. and M.H.M; Supervision: M.H.M.; Project administration: K.A.E. and M.H.M; and Funding acquisition: K.A.E., M.H.M.


Myocardial infarction (MI) is one of the leading causes of death worldwide. However, there is no effective treatment for MI. In this study, trimetazidine (TMZ) and Danhong injection (DHI), representing western medicine and traditional Chinese medicine for MI, were used as tools to identify vital processes in alleviating MI injury. Administration of DHI and TMZ obviously decreased myocardial infarct size, improved ultrasonic heart function, and reduced creatine kinase (CK), lactate dehydrogenase (LDH), and glutamic oxaloacetic transaminase (AST) levels after MI. RNA-seq results indicated calcium ion handling and negative regulation of apoptotic process were vital processes and DHI and TMZ obviously reduced the expression of CaMK II and inhibited cleaved caspase-3 and Bax. Furthermore, DHI and TMZ increased p-S16-PLB, p-S16T17-PLB, CACNA1C, p-RyR2, and p-PKA expression but did not affect SERCA2a expression. In addition to the enhancement of cardiac myocyte shortening amplitude, maximum shortening velocity, and calcium transients, DHI and TMZ increased sarcoplasmic reticulum calcium content and enhanced SERCA2a calcium uptake capability by upregulating the phosphorylation of PLB but did not affect calcium exclusion by NCX. In conclusion, DHI and TMZ protect against MI through inhibiting apoptosis by downregulating CaMKII pathway and enhancing cardiac myocyte contractile functions possibly through the PKA signaling pathway. Myocardial infarction (MI), caused by a sudden decrease in or interruption of coronary blood supply and oxygen, is a leading cause of death worldwide, resulting in acute and persistent ischemia in the myocardium, followed by myocardial apoptosis and necrosis . A largeSalvia miltiorrhiza and Carthamus tinctorius, is widely used for cardiovascular diseases and ischemic encephalopathy, including myocardial ischemia  and housed in a room with proper temperature and humidity under a 12/12-hour light-dark cycle. All of the experiments were conducted according to the guidelines of the Committee on Animal Care and Use of Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. The rats were given free access to water and food and allowed to acclimatize for 7 days before any experiments.Salvia miltiorrhiza and Carthamus tinctorius and was provided by Shandong Danhong Pharmaceutical Co., Ltd. .After anesthesia with pentobarbital sodium , myocardial ischemia was induced in animals by tightening a slipknot around the left anterior descending coronary artery with a 6-0 silk suture. The same procedures were performed on sham group rats, but the silk suture was not tied after passing beneath the left anterior descending coronary artery. The rats were randomly divided into six groups: (i) sham; (ii) myocardial infarction ; (iii) TMZ + MI ; (iv) D0.42 + MI ; (v) D1.05 + MI ; and (vi) D2.1 + MI . The dose of TMZ was determined according to previous research , 16. TheRat hearts were harvested and sliced into five sections of 1 mm thickness across the left ventricular long axis. To identify the infarct area, the heart sections were incubated with triphenyltetrazolium chloride  for 15 min at room temperature. Infarction rates were measured using Image-J software. The infarction rate is shown as a percentage of the infarct area over the total area. After 24 hours of infarction induction, rats were anesthetized with isoflurane, and echocardiographic measurements were performed. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured and calculated using Vevo 770  and related software through detection along the short axis of the left ventricle at the papillary muscles.Rat blood samples were collected after 24 hours of ischemia. The kits for creatine kinase , lactate dehydrogenase , and aspartate transaminase  were purchased, and signals were detected using a microplate reader  using the detection kit purchased from Nanjing Jiancheng (China).v/v) polyformaldehyde and incubated with Triton X-100 (0.5% v/v) for 30 min before serum blocking for 60 min. Then, antibodies against Bax (ab182733) and Bcl-2 (ab196495) were added for 24 hours at 4°C before incubation with a secondary antibody (1: 200) in the dark at 37°C for 60 min. After washing with PBS, 4, 6-diamidino-2-phenylindole  was used to stain the nuclei, and photos were taken under a confocal microscope . One-Step TUNEL Apoptosis Assay Kit  was employed to assess myocardial apoptosis through specific detection of DNA breaks in apoptotic cells according to the instructions included in the kit.For immunofluorescence staining, samples were fixed with 4% (2) normal Tyrode's (NT) solution ). Then, the perfusate was switched to Ca2+-free Tyrode's solution for 5 min, followed by perfusion for approximately 25 min with the same solution containing 30 μm CaCl2 and 0.6 g/ml type II collagenase . Next, ventricular tissue at the edge of the infarct area was removed (tissue from the same location was collected for the sham group) and minced in KB solution ) to obtain single myocytes. The cells were filtered through a nylon mesh and resuspended in centrifuge tubes for centrifugation (400 rpm for 30 s), and the supernatant was then discarded. The myocytes were resuspended, and extracellular Ca2+ was slowly added back up to 1.2 mm. Calcium-tolerant and rod-shaped cells showing clear cross striations were used for Ca2+ transient and sarcomere shortening measurements, respectively.SD rats were grouped and pretreated as described above, and LV myocytes were enzymatically isolated as previously described . In brieμm Fura-2 AM in the dark for 30 min at room temperature, and fluorescence and sarcomere length measurements were recorded with Fluorescence Measurement and Cell Dimensioning Systems . After loading, cells were washed and resuspended twice in NT solution and then placed in a cell chamber, stimulated with a 4 ms duration electrical stimulation at 1 Hz, and superfused at room temperature. Myocytes were exposed to 340 or 380 nm excitation wavelength. The emitted fluorescent signal was detected at 510 nm. The IonWizard software (IonOptix) was used to synchronously record fluorescence and sarcomere length in myocytes. At the end of the recording, the background fluorescence for individual myocytes was detected by moving the view to a nearby blank area. IonWizard was used to correct the Fura-2 AM ratio by subtracting the background and analyzing the calcium transient and sarcomere shortening.Isolated cardiac myocytes were loaded with 2 2+ release in response to the rapid application of caffeine. The process to assess sarcoplasmic reticulum calcium content has been described by Bassani et al. i) promotes calcium binding to the protein troponin C to drive myofilament sliding, and then myocytes are shortened. Immediately, [Ca2+]i decrease to allow Ca2+ to dissociate from troponin and the myocytes to relax. Intracellular Ca2+ ions are mainly removed by four calcium transporters: SR Ca2+-ATPase, sarcolemmal Na+/Ca2+ exchanger, sarcolemmal Ca2+-ATPase, or mitochondrial Ca2+ uniporter. In normal rat ventricular myocytes, the SR Ca2+-ATPase promotes the uptake of [Ca2+]i into the SR and contributes to 92% of calcium removal from the intracellular space. However, the Na+/Ca2+ exchanger removes [Ca2+]i out of myocytes and only contributes to 7% while others contribute less than 1% to the decrease in [Ca2+]i i . Therefo2+ transport and heart contraction [2+ overload and further lead to myocardial cell death, ventricular arrhythmia, and even heart failure [2+ homeostasis, CaMK II inhibition is used to prevent myocardial injury, such as in myocardial ischemia-reperfusion, myocardial infarction, and Ca2+-related heart injury [CaMK II is a central regulator of intracellular Catraction . Recent  failure , 25. The failure , 27. Cont injury . In thist injury . In thisAccording to the CaMK II paradox, myocyte contractility should be immediately reduced by the CaMK II pathway after MI. However, we found that cardiac myocyte contractility increased after DHI treatment compared with that with MI alone; thus, we hypothesized that DHI can upregulate other pathways to enhance cell shortening. One of all the major symptoms of myocardial infarction, reduced heart contractility, is accompanied by decreased SERCA2a in SR. Recent studies have indicated that enhanced SERCA2a/PLB activity could rescue myocardial contractility and improve heart function. Calcium transient data showed that calcium removal capabilities were elevated in the DHI- and TMZ-treated groups. Because calcium removal occurs mainly by SERCA2a and NCX , caffein2+ homeostasis could be improved by TMZ, consistent with previous data [2+ homeostasis, and the effect of DHI was comparable to the effect of TMZ. Our results demonstrated that both TMZ and DHI inhibited cell apoptosis by downregulating the CaMKII pathway, while they enhanced cardiac myocyte contractile ability and maintained calcium homeostasis possibly through the PKA signaling pathway. In this study, only a little difference in modulating calcium was observed during MI between treatment of DHI and TMZ. According to On the one hand, DHI and TMZ reduced apoptosis; on the other hand, they increased cell shrinkage, and these two aspects of regulation maintained the normal function of the heart after myocardial infarction. Heart contractility and Caous data . ImportaTaken together, the results in this study demonstrated that DHI and TMZ protects against MI by inhibiting cell apoptosis by inhibiting the CaMKII pathway and enhancing cardiac myocyte contractile function after myocardial infarction, possibly through the PKA signaling pathway, to enhance calcium handling capability.


The transformation of acute promyelocytic leukemia (APL) to acute mononuclear leukemia during treatment is a rare clinical phenomenon, and no CCAAT/enhancer-binding protein alpha (CEBPA) double mutations have been reported.A 42-year-old male was hospitalized for ecchymosis of the left lower limb for more than 1 month, gingival bleeding, and fatigue for 10 days, with aggravation of symptoms for 2 days.A diagnosis of APL was based on bone marrow (BM) morphology, immunophenotyping, fusion gene analysis, and fluorescence in situ hybridization. At a 1-year follow-up of maintenance treatment, he developed thrombocytopenia and was diagnosed with acute myeloid leukemia (AML) with a CEBPA double mutation by BM morphology, immunotyping, chromosomal analysis, polymerase chain reaction, and next generation sequencing.Complete remission of APL was achieved after all-trans retinoic acid and arsenic trioxide double induction therapy, followed by 2 cycles of mitoxantrone and cytarabine, and 1 cycle of idarubicin and cytarabine. Thereafter, sequential maintenance therapy of arsenic trioxide + all-trans retinoic acid + methotrexate was started. In the fourth cycle of maintenance therapy, APL was transformed into AML with a CEBPA double mutation. After 1 cycle of idarubicin and cytarabine, the patient achieved complete remission and received 3 cycles of idarubicin and cytarabine and three cycles of high-dose cytarabine as consolidation therapy.At present, the patient is in continuous remission with minimal residual disease negative for both of APL and AML.AML with a CEBPA double mutation after APL treatment is very rare, thus the prognosis of this event will require further observation. Hence, the clinical diagnosis and treatment of APL remain challenging. Here, we report a case of acute myelomonocytic leukemia with a double mutation to the CCAAT/enhancer-binding protein alpha (CEBPA) gene during maintenance therapy for APL and include a review of the current literature.Acute promyelocytic leukemia (APL) is a common subtype of acute myeloid leukemia (AML) and was formerly one of the most fatal forms of AML. The introduction of all-trans retinoic acid (ATRA) in combination with arsenic trioxide (ATO) and other chemotherapies has resulted in complete remission rates of >90% and long-term remission rates of >80%.2The patient was a 42-year-old male with no previous exposure to industrial toxins or radioactive substances. In November 2018, he was hospitalized for ecchymosis of the left lower limb for more than 1 month, gingival bleeding, and fatigue for 10 days, with aggravation for 2 days. On physical examination, the patient appeared anemic with scattered spots of bleeding of the mucosa and the skin over the whole body, no abnormality in heart rhythm or lung auscultation, a soft abdomen, no palpation of the liver or spleen under the ribs, and no edema of either lower limb. The findings of initial blood chemistry analysis were as follows: white blood cell count, 8.1 × 109/L; hemoglobin concentration, 69 g/L; platelet count, 16 × 109/L; prothrombin time, 14.6 s, activated partial thromboplastin time, 21.4 s; thrombin time, 21.5 s; K+, 3.71 mmol/L; Na+, 141 mmol/L; Ca2+, 1.89 mmol/L; γ-glutamine acylase, 144 U/L; total protein, 57.2 g/L; and albumin, 33.2 g/L. Bone marrow (BM) analysis revealed abnormal promyelocytes (89.2%) of different sizes with quasi-round shapes and tumor-like processes on the edge of the cytoplasm, medium amounts of cytoplasm, blue staining, purplish red granules in the cytoplasm, round nuclei, visible depressions, loose and fine chromatin, and visible nucleoli. In addition, erythroid differentiation was inhibited, megakaryocytes were absent, and platelets were rare ,4 leading to changes in DNA structure and secondary changes, most of which are treatment-related myelodysplastic syndrome or AML;,6the use of cytotoxic drugs, especially topoisomerase inhibitors, alkalinizing agents, and anthracyclines,(ii)APL accompanied by other clones in the early stage of the disease, but masked by the dominant clones, as the application of ATRA, ATO, and chemotherapy eliminate abnormal promyelocytes, so that other clones have a better chance of survival; and(iii),8 and more frequent lineage switching with specific genetic subtypes of leukemia, such as mixed lineage leukemia rearrangement, which may have greater inherent plasticity. In the present case, the onset of secondary myeloid leukemia was earlier, as the switch to AML occurred only 17 months after complete remission of APL, which is a much shorter period than the median time of 44 months reported in the literature. A retrospective study conducted by Wang et al reported treatment-related myeloid tumors in 67 cases that most commonly occurred at about 3 years after complete remission of APL. In view of the short period of remission during treatment in this case, it was difficult to differentiate treatment-related secondary myeloid tumor formation from APL recurrence with a series of transitions. Moreover, a retrospective analysis of 651 AML patients conducted by Wang et al found fewer favorable genetic molecular events in elderly patients with AML and secondary AML patients, such as a CEBPA double mutation, runx1-runxt1t1, etc. A double mutation to the CEBPA gene is detected in leukemia cells during AML transformation, and some cases before transformation to APL were accompanied with a double mutation to the CEBPA gene. However, our patient was negative for the CEBPA double mutation at the first onset, which does not support the possibility of evolution from APL to AML. The CEBPA double mutation is often associated with a good prognosis of AML. Patients with a CEBPA double mutation are classified as low risk. However, in this patient, AML was secondary to APL. As reported in the literature, the prognosis of APL secondary to treated-acute myeloid leukemia is poor and survival is relatively short. Although the patient achieved complete remission after 1 course of chemotherapy, his long-term survival remains to be further investigated, as it is unclear whether the choice of follow-up consolidation therapy should be medium dose cytarabine-based chemotherapy or allogeneic hematopoietic stem cell transplantation.the inherent lineage plasticity of primitive leukemia cells determines the potential for reprogramming under lineage-specific pressureThe formation of a secondary myeloid tumor after APL treatment is a rare clinical phenomenon, which usually occurs at about 3 years after disease remission, although early development of a secondary myeloid tumor is not impossible. Also, it remains uncertain whether the prognosis of secondary AML with a CEBPA double mutation after APL is equivalent to that of newly diagnosed AML with a CEBPA double mutation, thus additional clinical cases are needed. The mechanism underlying secondary AML after APL therapy is unknown, but it is believed that with the development of genetic cytology and the accumulation of clinical cases, more accurate mechanisms will be identified and individualized prevention and treatment regimens will be clinically available.Conceptualization: Guifang Ouyang.Data curation: Ye Sun, Chong Wang, Yongcheng Sun, Jiaping Wang, Lixia Sheng.Formal analysis: Chunmeng Rong, An Wu.Funding acquisition: Lixia Sheng.Methodology: Lixia Sheng.Writing – original draft: Ye Sun.Writing – review & editing: Lixia Sheng.


RT-PCR, immunohistochemistry and electrophysiology suggested that Kv7.5 was the predominant Kv7 subunit in both rat and human arterial SMCs; CACNA1C  and CACNA1 G  were the most abundant voltage-gated Ca2+ channel gene transcripts in both types of VSMCs. This study establishes Kv7 channels as key regulators of Ca2+ signalling in VSMCs with Kv7.5 playing a dominant role.Voltage-gated Kv7 (or KCNQ) channels control activity of excitable cells, including vascular smooth muscle cells (VSMCs), by setting their resting membrane potential and controlling other excitability parameters. Excitation-contraction coupling in muscle cells is mediated by Ca Thus, depolarisation of VSMCs membrane potential (Em) results in the opening of voltage-gated Ca2+ channels (VGCCs), predominantly L-type i) via the influx of Ca2+ from L-type Ca2+ channels [Em in A7r5 rat aortic smooth muscle cells [2+]i in VSMCs still remain to be clarified i in VSMCs could identify novel approaches for the treatment of cardiovascular disease, including pulmonary hypertension. In the present study, we examined the influence of Kv7 channel activity on [Ca2+]i in rat and primary human arterial SMCs.The clinical use of AVP has increased significantly in recent years , but its22.12+ regulation i and Em by microfluorimetry. For fluorescence measurements, confluent cell monolayers grown on glass coverslips were used 2–5 days after plating.Rat A7r5 VSMCs derived from rat thoracic aorta were obtained from the European Collection of Cell Cultures. The A7r5 cell line retains many characteristics of VSMCs, which provides a convenient model system to study mechanisms of Cagulation . Human Vgulation ,31. Vess2.22). Once the cell monolayer was confluent, the coverslips were transferred to a 35 mm petri dish and washed with the Ca2+ containing buffer twice. The Ca2+ containing buffer was composed of (in mM) 135 NaCl, 5 KCl, 1.2 MgSO4, 2.5 CaCl2, 5 HEPES, and 10 glucose (pH7.4 with NaOH). The osmolality was adjusted to 300 mOsm with sucrose. The cells were loaded with 4 μM Fura 2-AM  dissolved in Ca2+ containing buffer without pluronic acid for 40 min in the dark at 37 °C. Loading solution was pipetted out and the cells were washed with Ca2+ containing buffer. Coverslips were then incubated in Ca2+ containing buffer to de-esterify for 10 min. For simultaneous recordings of Ca2+ transients and action potentials, rat A7r5 cells were also loaded with the voltage-sensitive dye FluoVolt  in Ca2+ containing buffer supplemented with the dye (1:1000), PowerLoad Concentrate (1:100) and Background Suppressor (1:10) in the dark at 37 °C. Before recording, each coverslip was broken into fragments with a diamond pencil, and fragments were then transferred to a perfusion chamber. Cells were constantly perfused with standard bath solution at 2−3 ml/min using gravity perfusion system via lengths of Tygon tubing . The composition of standard bath solution was (in mM): 135 NaCl, 5 KCl, 1.2 MgSO4, 2.5 CaCl2, 5 HEPES, and 10 glucose (pH7.4 with NaOH); the osmolality adjusted to 300 mOsm with sucrose. The 50 mM K+ bath solution was made with isotonic Na+ substitution; all drugs were applied in standard bath solution via the perfusion system. Intracellular Ca2+ levels were measured ratiometrically using a Cairn Research ME-SE Photometry system . Changes in [Ca2+]i were evaluated as the ratio of intensities of fluorescence emitted at 510 nm following alternating excitation at 340 and 380 nm  using a monochromator. For measurement of Em, cells were sequentially excited at 340, 380 and 488 nm (Fura 2-AM and FluoVolt loaded samples). Acquisition Engine 1.6.1 software was used to record and analyse the traces. Magnitude of changes in [Ca2+]i was calculated either as peak F340/F380 ratio or as the area under the curve . Change of Em was presented as ΔF/F0 in cells.Cells were plated on circular glass coverslips  in 24-well plates and kept in a humidified incubator . A cDNA template was generated using the iScript cDNA synthesis instructions  with 1 μg RNA. End-point PCR and RT-PCR were performed using TaqMan gene expression assays . The probes used in this study are listed in the Supplemental Table I. All samples consisted of 1 μl of cDNA and 19 μl of RT-PCR reaction mix (as per manufacturer’s instructions). Quantitative analysis of mRNA expression was determined by using a CFX Connect System . The reaction profile was 2 min at 50 °C, 10 min at 95 °C, 15 s at 95 °C for 50–60 cycles, then 1 min at 60 °C. PCR amplification products were separated by the 2.0 % agarose gel electrophoresis with gels containing SYBR safe DNA stain . Quantitative analysis of mRNA expression was carried out using a CFX Connect System , and relative gene expression calculated using the 2−ΔΔCt method with hypoxanthine phosphoribosyltransferase 1 (Hprt1) as the housekeeper gene. Statistical analysis was performed on the ΔCt values i followed by a slow decline, presumably due to VGCCs inactivation and extrusion of Ca2+ from cytosol by Ca2+ ATPases and/or Na+/Ca2+ exchanger. A limited number of rapid oscillations was often observed during the transient. Smooth muscle cells mainly express L- and T-type VGCCs, while other isoforms are not significantly expressed [2+ channel blocker [2+ channels [+-induced [Ca2+]i transients by 95.9 ± 2.3 % (n = 5) and by 85.0 ± 13.3 % (n = 5), respectively  and T-type  VGCC subunits. We found that Cacna1c (Cav1.2) L-type and Cacna1g (Cav3.1) T-type Ca2+ channel genes were the predominant subtypes in A7r5 cells  induced a sustained [Ca2+]i elevation and evoked Ca2+ oscillations persisting for the duration of XE991 application; both effects recovered upon XE991 washout  was applied first to induce oscillations, then nifedipine (2 μM) was applied in the continued presence of XE991. Nifedipine virtually abolished the oscillations and reverted [Ca2+]i, elevation induced by XE991 . We then performed a triple-wavelength  simultaneous recordings of [Ca2+]i and Em changes in response to XE991. 2+ trace (black) and FluoVolt Em trace (red); XE991 induced slow Em depolarisation followed by a burst of sharp spikes which were temporally well-correlated with [Ca2+]i (upper panel). Peak cross correlation between the FluoVolt Em and the ratiometric Ca2+ signal was 0.6 ± 0.18, occurring at a time lag of 3.0 ± 0.5 s (n = 5) (lower panel). Both, [Ca2+]i oscillations and Em spikes were abolished by retigabine.We next asked what is the nature of i elevation followed by a period of repetitive Ca2+ oscillations lasting throughout the AVP application; washout of the AVP was followed by a gradual return to the baseline ; peak cross correlation between the FluoVolt Em and the ratiometric Ca2+ signals was 0.84 ± 0.01, occurring at a time lag of 2.5 ± 1.4 s (n = 4) (lower panel). These [Ca2+]i oscillations and Em spikes were abolished by retigabine. Collectively, these data indicate that Kv7/KCNQ channels are present and functional in A7r5 cells and that treatment with a physiological concentration of AVP (100 pM) leads to depolarisation and Ca2+ influx through both L- and T-type Ca2+ channels; these effects were abolished by retigabine and were qualitatively very similar to these produced by the XE991.To investigate if the control over the currents . AVP is  pathway . Exposurbaseline A. Retigabaseline B,F. To tal level C,G. In tt manner D,H. We ae to AVP E. Simila3.42+]i and Em, suggesting a common mechanism, a Kv7 channel inhibition, the action of these two agents on the Kv7 channels is different. While XE991 is a direct ion channel blocker, the action of AVP on the Kv7 channels depends on the PLC-mediated signalling cascade, which inhibits Kv7s by a combination of PIP2 hydrolysis, PKC activation and ER Ca2+ release [2+]i response to AVP (100 pM) in A7r5 cells (p = 0.051) in peak Ca2+ levels between the control (untreated) and edelfosine-treated groups  significantly attenuated the amplitude of the sustained [Ca2+]i elevation induced by AVP (100 pM); it did not completely abolished oscillations but reduced their amplitude. RyRs inhibitor, tetracaine, effectively inhibited the amplitude and frequency of Ca2+ oscillation caused by AVP, and when applied together, 2-APB and tetracaine significantly attenuated [Ca2+]i response to AVP (2+ oscillations evoked by Kv7 channel inhibitor. A7r5 cells were pre-treated (10 min) with edelfosine and the effect of XE991 (10 μM) was explored with Ca2+ imaging. Edelfosine (10 μM) did not inhibit Ca2+ oscillations: neither the amplitude of sustained [Ca2+]i elevation nor spike frequency were different from control  receptors i, however, there were no oscillations  L-type and CACNA1 G (Cav3.1) T-type Ca2+ channel genes were the predominant subtypes in IMA SMCs  L-type Ca2+ channels and low voltage-activated (LVA) T-type Ca2+ channels [2+]i through the L-type Ca2+ channels i were completely blocked by the Kv7 opener, retigabine, indicating a strong control over the excitability and Ca2+ signalling in VSMCs exerted by the Kv7 channels. The inhibitory effect of retigabine was even stronger than that of the L-type VGCC blocker nifedipine, which is widely prescribed to treat cardiovascular disease i that are very similar to these of XE991. Indeed, the main mechanism of action of such hormones to stimulate rhythmic constrictions of arteries has been recognized as depolarisation of the Em to open VGCCs i oscillations. The effect of AVP took longer to develop but resulted in a similar spike frequency to that of XE991  i elevation and mean spike frequency induced by AVP. Moreover, inhibition of IP3Rs and RyRs with 2-APB and tetracaine, respectively, also attenuated the AVP-induced Ca2+ oscillations  and late (sustained) phases of constriction via IPectively . Higher r5 cells ,74. PLC-led GPCR . Within epletion ,19, IP3-modulin)  and actirolysis)  are the rolysis) . Our finllations , suggest neurons ,76. As p2+ signalling in primary human VSMCs as well. AVP has marked vasoconstrictor effect and gained attention as a possible tool against septic shock. However, it has been reported to induce graft vessel spasm during open-heart surgery [2+]i which may be a characteristic feature in human IMA SMCs. Importantly, nifedipine, NNC 55-0396 and retigabine all significantly supressed the AVP-induced [Ca2+]i transients, with retigabine being the most efficacious. Notably, there were no obvious oscillations in in human IMA SMCs in response to AVP or XE991. The reason for this difference between A7r5 and human primary SMCs cells remains to be elucidated, but it could be due to the more pronounced depolarisation and Ca2+ overload in the latter cell type. Despite this difference, these results indicated that Kv7 channels may have important clinical implications in cardiovascular disease and retigabine or its successors (e.g. drugs tailored to selectively activate Kv7.5) could have a potential in containment of the AVP-related perioperative vasospasm.Finally, we present evidence that Kv7 channel activity exerts a strong control over the intracellular Ca surgery . Here weYuan-Ming Tsai: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Visualization, Writing - original draft. Frederick Jones: Methodology, Software, Validation, Formal analysis. Pierce Mullen: Software, Formal analysis. Karen E. Porter: Conceptualization, Resources, Writing - review & editing. Derek Steele: Methodology, Writing - review & editing, Supervision. Chris Peers: Conceptualization, Methodology, Resources, Supervision. Nikita Gamper: Conceptualization, Methodology, Supervision, Resources, Funding acquisition, Writing - review & editing, Supervision.The authors have no conflicts of interest to report pertaining to this study.


Acute myeloid leukemia (AML) is a high remission, high relapse fatal blood cancer. Although mTORC1 is a master regulator of cell proliferation and survival, its inhibitors have not performed well as AML treatments. To uncover the dynamics of mTORC1 activity in vivo, fluorescent probes are developed to track single cell proliferation, apoptosis and mTORC1 activity of AML cells in the bone marrow of live animals and to quantify these activities in the context of microanatomical localization and intra-tumoral heterogeneity. When chemotherapy drugs commonly used clinically are given to mice with AML, apoptosis is rapid, diffuse and not preferentially restricted to anatomic sites. Dynamic measurement of mTORC1 activity indicated a decline in mTORC1 activity with AML progression. However, at the time of maximal chemotherapy response, mTORC1 signaling is high and positively correlated with a leukemia stemness transcriptional profile. Cell barcoding reveals the induction of mTORC1 activity rather than selection of mTORC1 high cells and timed inhibition of mTORC1 improved the killing of AML cells. These data define the real-time dynamics of AML and the mTORC1 pathway in association with AML growth, response to and relapse after chemotherapy. They provide guidance for timed intervention with pathway-specific inhibitors. The role of mTORC1 in AML has not yet been proven due to the mixed results of its inhibitors in clinical trials. Here the authors show the real-time dynamics of the mTORC1 pathway in association with AML growth and response to chemotherapy with fluorescent markers, providing guidance for timed intervention with pathway-specific inhibitors. Although conventional chemotherapies can induce remission, the majority of patients experience relapse of the disease. Therefore, identifying targetable vulnerabilities of AML following standard chemotherapy is warranted.Acute myeloid leukemia (AML) is a highly lethal hematological malignancy. Approximately 20,000 people are newly diagnosed per year in the U.S. and more than half of them ultimately die of the disease4. It is a master regulator of cell growth and is widely reported to be a critical pathway in cancer cells including AML3. mTORC1 inhibition has been reported to abrogate the progression of malignancies including AML in vitro and in vivo8. In addition, recurrent mutations, including FLT3-ITD and other tyrosine kinases, can result in activation of mTORC1 signaling, making it an attractive target for AML treatment rather than targeting each specific mutation10. Therefore, mTORC1 inhibition has been considered for potential treatment strategies for AML12, but clinical use of mTORC1 inhibitors has shown limited efficacy12.mTORC1 signaling regulates the proliferation and survival of cells in response to environmental changes in nutrients, growth factors, and oxygen16, it is likely that mTORC1 activity changes dynamically depending on cell anatomical location and, perhaps, the dramatic environmental shifts accompanying chemotherapy.Since mTORC1 activity depends on growth signals and nutrient availability in the microenvironmentIn this study, we seek to monitor mTORC1 activity over time in live animals, reasoning that mTORC1 activity may be very different depending on the in vivo context of cells. Combining intravital imaging and a dynamic probe of mTORC1 activity during growth, treatment and relapse of an AML model in mice, we define distinct temporal features of mTORC1 activity that argue for time-specific targeting of it.2. Once mTORC1 is activated, PDCD4 is rapidly phosphorylated by S6 kinase (S6K), ubiquitinated and degraded by the proteasome17.  is a ubiquitously expressed nuclear localization signal (NLS)-containing protein and a downstream target of mTORC117. Fig. . Therefo17, with or without a nuclear localization signal (NLS). These were evaluated in the mouse fibroblast cell line NIH 3T3 in which PDCD4 is rapidly degraded after induction of mTORC1 signaling17. We assessed the different fusion constructs after serum refeeding to starved NIH 3T3 for induction of mTORC1 activity. Among these transfectants, NLS with the N-terminal 80 amino acids of PDCD4 (NLS+1–80) appeared to be the most sensitive probe for mTORC1–activity ity Fig. . TherefoThe inverse relationship of mVenus signal intensity to mTORC1 activity was demonstrated by flow cytometry and western blot analysis of phosphorylated downstream targets of mTORC1 over time following serum refeeding of the transduced cells Fig. . The mVeRaptor. We transduced mCherry-TOSI or control mCherry into CreERT-Raptorfl/fl AML cells and induced Raptor knock-out by adding hydroxytamoxifen (HTM). This resulted in an increase of mCherry-TOSI without affecting the WT control cells . This probe allowed us to analyze mTORC1 activity in GFP-expressing cells and measured its response to knocking out 18, transplantation of the cell lines FM4, FM4-mVenus or FM4-VT into sublethally irradiated mice induced AML which was lethal in all recipients within 2 months after transplantation.To evaluate mTORC1 activity in a mouse model of AML, we used mVenus-TOSI in the context of cells bearing the potent leukemogenic fusion MLL-AF9. We retrovirally transduced mVenus-TOSI into a mouse AML cell line (FM4) that expresses the retrovirally transduced MLL-AF9 oncogene and iRFP. From these, several single cell derived clones were generated to assure uniformity of mVenus signal intensity. mVenus-TOSI transduced clones with the brightest mVenus signal intensity were used for further experiments (FM4-mVenus). For in vivo imaging, we also retrovirally transduced TdTomato fluorescent protein, which produces a much brighter signal than iRFP, into FM4-mVenus cells and made single cell clones expressing both fluorophores . As expected from prior reports−/TdTomato+) and mTORC1 low  populations could be visualized. As shown in Fig. +/TdTomato+) similar to the changes observed using flow cytometry. Therefore, the probe is useful for assessing mTORC1 activity in isolated cells or in vivo. Cells with high mTORC1 activity showed higher colony-forming capacity compared to cells with low mTORC1 activity 28 depending on the expression of highly expressed genes (mouse mTORC1 high gene signature (mTOR-GS)) in mTORC1 high compared to mTORC1 low mouse AML cells. The human AML samples were stratified into two distinct clusters by the differential expression of the mouse mTORC1 high gene signature  in mTORC1 high AML patients (2800 genes) (human mTOR-GS) with those in mouse mTOR-GS (158 genes) and found that 44 genes overlapped . Pathway analysis on these 44 overlapped genes in MSigDB revealed Myc and mTORC1 related signatures were enriched 29 and found there was intratumor heterogeneity in mTORC1 signaling in some AML cases  in vitro. Ara-C selectively killed the mTORC1 high cells in vitro . Cells were sorted based on their apparent mTORC1 activity and clonal diversity was measured by DNA sequencing. We reasoned that selection would be accompanied by a more restricted pool of barcodes after chemotherapy33. Selection by chemotherapy should cause limited clonal abundance in the mTORC1 high population compared to the mTORC1 low population. In contrast, induction would result in relatively constant representation of clones before and after chemotherapy since in that case, barcodes could exist in and transition between each state, eventually resulting in a stationary barcode distribution.There are two possible mechanisms for the shift of gene expression to a high mTORC1 activity signature after chemotherapy; one is the selection of mTORC1 high cells and the second is the induction of mTORC1 activity following chemotherapy. We assessed these two possible scenarios by introducing genetic barcodes using the ClonTracer libraryWe found that a significant proportion of unique barcodes are represented in both the mTORC1 high and low cell populations   and tested it in our AML treatment model. This probe is a modified version of an established FRET probe with a caspase-3 sensitive DEDV sequence, SCAT3. When excited near the peak of ECFP excitation , the SCAT3 probe emits mVenus or YFP (FRET) signal when it is intact, but emits ECFP signal (no FRET) when its DEDV sequence is cleaved37. However, these mTORC1 inhibitors were mainly used as monotherapy for established AML, settings our data would suggest are likely to be mTORC1 low and therefore likely unaffected. We hypothesized that timed mTORC1 inhibition might change its anticancer efficacy. We tested this hypothesis using inducible deletion of Raptor, a critical component of mTORC138. A primary MLL-AF9 AML cell line derived from Raptorfl/fl mice was established and retrovirally transduced with CreERT2. The Cre recombinase was induced in animals with established leukemia by tamoxifen injection at different time points in relation to chemotherapy  demonstrated marked increased killing of AML cells compared with later Cre induction 39.While mTORC1 inhibitors were found to repress AML cell proliferation in vitro40. Interestingly, our analysis showed that AML cases with mTORC1 high activity correlated with a poor prognosis. Chemotherapy in vitro selectively killed mTORC1 high cells without activating mTORC1 signaling likely due to the persistence of the cell cycle dependent killing agents in the culture continuing to eliminate mTORC1 high cells. On the other hand, chemotherapy treatment in vivo is followed by clearance of AML and by the secondary increased availability of nutrients and/or oxygen as the leukemic burden decreases42. Our barcoding experiments indicate that this is due to induction of mTORC1 activity post chemotherapy rather than selection of mTORC1 high cells. In aggregate, these data point to the value of dynamic measurement of pathways in vivo to understand changing activation and therefore, vulnerabilities to inhibitors.Using this probe, we found mTORC1-high and low populations in an AML model with functional and gene expression correlates of those populations. In addition, in vivo imaging and flow cytometric analysis revealed that there was intra-tumoral heterogeneity of mTORC1 activity. Imaging data also implied that mTORC1 activity might depend on the location in the bone marrow. We further found that mTORC1 activity was negatively correlated with AML burden, perhaps due to the limited availability of key nutrients. Similarly, there is heterogeneity in mTORC1 activity among human AML cases as shown from analysis of microarray data and from early phase clinical trialsRaptor, an essential component of mTORC1, we could observe improved killing of AML cells when Raptor was deleted with chemotherapy. While a very different set of circumstances apply, it is interesting to note that a recent clinical trial used a Rapalog, Everolimus, in combination with chemotherapy in childhood acute lymphoid leukemia and observed a favorable outcome43, though there were failures of mTORC1 inhibitors used as monotherapy. Similarly, a single-arm study showed that the combination of the mTORC1 inhibitor, Sirolimus with chemotherapy in adult high-risk AML patients could be beneficial for AML with higher mTORC1 activity. Sirolimus administration was started 3 days earlier than chemotherapy, a time we found to be suboptimal40. The combination of mTORC1 inhibition at specific times following specific types of chemotherapy may be worth testing in AML.The shift in mTORC1 activity after chemotherapy suggests that mTORC1 inhibition has the greatest potential for AML cell killing at particular time points. By applying timed genetic deletion of Further analysis is needed to reveal the contributions of the microenvironment to mTORC1 activity during AML progression/regression and appropriate timing and inhibitor combinations of mTORC1 inhibition for AML treatment. Nonetheless, we propose mVenus-TOSI as a useful biosensor for dynamic imaging of mTORC1 activity that reveals mTORC1 signaling to be dynamic in vivo depending on the state of disease or treatment. The dynamic monitoring of molecular functions in AML and perhaps other cancers may enable more effective combinations of agents in a timed manner to improve disease outcomes.−22 using primers with 5′-EcoRI and 3′-XhoI site by PCR and subcloned into pMXs-IP with PDCD4 fragments. MSCV-IRES-KO and MSCV-IRES-KO-hRPTOR plasmids were kind gifts from Dr. Takayuki Hoshii6. The 5′half of SCAT3.2 probe was generated by amplifying Amcyan using primers with 5′-BamHI and 3′-EcoRI sites from pMXs-Amcyan-hGEMININ22 and pCDNA3-SCAT334. The 3′ half of SCAT3.2 were generated by cleaved from pCDNA3-SCAT3 with EcoRI and NotI. Both 5′ and 3′ half of SCAT3.2 were subcloned into pMX-IP.Full length or partial fragments , PDCD4 (1–100), PDCD4 ) of mouse PDCD4 were amplified from mouse spleen-derived cDNA using primers with 5′-XhoI and 3′-Not I site by PCR. These fragments were subcloned into a pMXs-internal ribosome entry site (IRES)-Puro vector (pMXs-IP). The fragments for mVenus or mVenus with SV40NLS (mVenusNLS) were amplified from pMXs-IP-mVenus-p27K18. These cell lines were transplanted into mice and AML cells were harvested after the mice developed AML. AML cells with mVenus-TOSI (FM4-mVenus or FM4-mVenus-TdT) were generated by retrovirally transducing with pMXs-IP-mVenus-TOSI and pLenti-iRFP with or without pMXs-IRES-TdTomato and were selected with drug and FACS sorting followed by picking single cell derived clones. Similarly, FM4-SCAT3.2 and FM4-mVenus-p27K− were developed. Other mouse pre-leukemic cell lines were generated similarly from mouse GMP (CL3) or hematopoietic stem cells (KLS-1). For generation of the Raptor KO mouse model, AML cells (Raptorfl/fl AML) were similarly generated by transducing MLL-AF9 to GMP cells derived from Raptorfl/fl mice and transplanted into recipient mice. Then CreERT2 was retrovirally introduced into Raptorfl/fl AML cells with MSCV-IRES-CreERT2 (CreERT2 Raptorl/flf AML). A mouse AML cell line with MLL-Septin6 (HF6) were a kind gift from Dr. Toshio Kitamura44.Mouse granulocyte macrophage progenitor (GMP)-derived pre-leukemic cells were generated with retroviral transduction of the MLL-AF9 oncogene as described previouslyAnti-mouse S6K, S6, 4EBP, PDCD4, beta-Actin, phospho-S6K, phosphor-S6, phosphor-4EBP, and phospho-4EBP-Alexa647 antibodies were purchased from Cell Signaling Technologies . Anti-mouse phoshoS6-PE, anti-mouse CD45.1 Pacific Blue, and anti-mouse Ki67 Brilliant Violet 605 antibodies were purchased from Biolegend . Anti-GFP antibody purchased from Abcam . Cytarabine arabinoside (Ara-C) and doxorubicin were purchased form Sigma-Aldrich . Rapamycin, Rad001, Torin2, AZD0855, and MLN(0128) were purchased from Selleckchem .NIH 3T3 cells were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin (DMEM/10%FBS). Mouse AML cell lines were cultured in RPMI1640 supplemented with 10% FBS and 3 ng/ml IL-3, 10 ng/ml SCF and penicillin/streptomycin (the AML culture medium). For proliferation assays, relative cell numbers before and after culture were analyzed with CellTiter-Glo . For colony formation assays, 1000 AML cells were suspended in 1 ml MethoCultTM GF M3434 , plated in 3.5 cm dishes and counted after 7 days.18. For rapamycin treatment, the transplanted mice were treated with intraperitoneal injection (i.p.) of 4 mg/kg rapamycin or vehicle as described6. For chemotherapy treatment, the transplanted mice were treated with i.p. of cytosine arabinoside 100 mg/kg for 5 days and doxorubicin 3 mg/kg for 3 days concurrently as described18 and the treatment was started 21 days after bone marrow transplantation. I.p. injection of 10 mg/kg tamoxifen or vehicle for 4 days was started at day −3, 0, or 5 of chemotherapy in order to delete Raptor. For flow cytometry analysis, AML cells were harvested from bone marrow by bone marrow aspiration or sacrificing animals. The animal study protocol was approved by Massachusetts General Hospital Institutional Animal Care and Use Committee (IACUC).Mouse AML was developed by transplanting mouse AML cell line into sublethally irradiated (4.5 Gy) mice as described− or negative Ki67 staining as reported22. For apoptosis cell detection, FRET and Amcyan signal from FM4-SCAT3.2 were detected similarly as described previously34.Flow Cytometry analyses were conducted with BD FACS Aria II, BD LSRII, and BD FACS caliber ) following manufactures protocols. For mTOR signal detection, mVenus with/without iRFP signal intensity were measured by flow cytometer. mTORC1 low (mVenus high) cells was defined by the cells with mVenus intensity higher than lower 10th percentile of mVenus intensity of the reference cells (FM4-venus cells which was independently treated with 100 nM rapamycin for 24 h). For cell cycle analysis, G0 cell were detected by intensity of mVenus signal from FM4-mVenus-p27K45. At least 2 mice were imaged for each condition and representative data shown. Each dot represents an individual cell. For analysis of the distance from bone, mTORC1 activity low (mVenus high = yellow) cells were determined by the ratio between signal intensity of mVenus/TdTomato (channel 2/channel 1) >=0.4 and mTORC1 high (mVenus low = red) were defined by the ratio <0.4. The distance between the bone surface and the center of the individual cell was determined based on the z-stack images as described46. Briefly, 3D models of these cells and the bone (second harmonic generation) were developed through z-stack reconstruction. In Supplementary Fig. Intravital imaging of mouse calvarial bone marrow was performed with a custom made 2-photon or confocal microscope, or Olympus 2-photon microscope system ) as describedhttp://rsbweb.nih.gov/ij/) and further analysis was done with R (https://www.r-project.org) or Matlab .For survival and apoptosis imaging, FRET (mVenus) signal (channel 2) and Amcyan signal (channel 1) were collected and the ratio between mVenus/Amcyan was calculated in order to normalize FRET signal. The images were analyzed using Olympus FV10-SW software and/or Image J software  for gene set enrichment analysis (GSEA) as described49. Gene Signatures used here were collected from MSigDB (http://software.broadinstitute.org/gsea/msigdb/index.jsp) or publications25 and gene-set overlap analysis was conducted with the Investigate Gene Sets fuction of MSigDB.Fifty thousand mVenus-high and mVenus-low AML cells with mVenus-TOSI (FM4-Venus) were sorted from bone marrow cells of each of 3–4 mice with AML treated with vehicle or chemotherapy regimen at day 7 (3 days after the end of chemotherapy). RNAs were extracted from samples with a DNA/RNA micro kit ), used for making a sequencing library with Illumina Truseq kit, and subjected to sequencing with Next-seq (core at Boston University). RNAseq data was analyzed with STARGSE12417)28 were used for clustering and survival analysis with R. The p-values for the difference between mTORC1 high and low group at different time points were also automatically calculated with logrank test. Differentially expressed genes were analyzed with genefilter package (https://bioconductor.org/packages/release/bioc/html/genefilter.html). The single cell RNA-seq data for human AML patients (GSE116256)29 were used for clustering and sub-population analysis for mTORC1 related pathway analysis with MSigDB. Single cell RNA-seq data analysis was performed with R and Seurat Package 51.The microarray data for human AML patients ) with MOI < 10%. They were incubated for 3 days and sorted with FACS AriaII (BD). One million sorted cells were obtained. The sorted cells were expanded up to 1 billion by culturing for 10 days. One million cells were transplanted into each of the four groups of the four mice. Four hundred thousand mVenus-high or low AML cells were sorted from the mouse bone marrow by sacrificing the mice immediately before (day 0) or at either of the 3 different time after chemotherapy . Genomic DNA was extracted with the DNA/RNA mini kit (Qiagen) and sequencing libraries were constructed according to the protocol from Addgene (https://www.addgene.org/pooled-library/clontracer/). The resulting sequencing libraries were pooled and subjected with Illumina High-seq to DFCI sequencing core. The data was analyzed with the Python program provided by Addgene for alignment  and further analysis was conducted by R.Two million FM4-Venus cells were subjected to lentiviral infection of the ClonTracer library plasmidt test or Analysis of Variance (ANOVA) and paired t test for independent variables. ANOVA was used for initial analysis for Figs. t test. Survival was analyzed with log rank test.Statistical significance was calculated using the Student, Welch Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Reporting Summary


The increasing of intracellular calcium concentration is a fundamental process for mediating osteoclastogenesis, which is involved in osteoclastic bone resorption. Cytosolic calcium binds to calmodulin and subsequently activates calcineurin, leading to NFATc1 activation, a master transcription factor required for osteoclast differentiation. Targeting the various activation processes in osteoclastogenesis provides various therapeutic strategies for bone loss. Diverse compounds that modulate calcium signaling have been applied to regulate osteoclast differentiation and, subsequently, attenuate bone loss. Thus, in this review, we summarized the modulation of the NFATc1 pathway through various compounds that regulate calcium signaling and the calcium influx machinery. Furthermore, we addressed the involvement of transient receptor potential channels in osteoclastogenesis. Bone remodeling is balanced by the coordinated activities of osteoclastic resorption and osteoblastic formation . Imbalan2+ signaling in osteoclasts is important for multiple cellular functions, including proliferation, differentiation, gene transcription and bone resorption i) through inducing its release from the endoplasmic reticulum (ER). Ca2+ influx through store-operated Ca2+ entry (SOCE) and transient receptor potential (TRP) channels causes RANKL-induced [Ca2+]i oscillations during osteoclastogenesis [2+ balance in osteoclasts [2+ release and reuptake into the ER stores is also necessary for [Ca2+]i oscillations. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) transports Ca2+ from the cytosol into the ER, and SERCA activity is also essential for [Ca2+]i oscillations. Disruption of SERCA2 expression impairs RANKL-induced [Ca2+]i oscillations [2+]i oscillations i is a fundamental process mediating osteoclastogenesis , vanilloid (TRPVs), melastatin (TRPMs), mucolipin (TRPMLs), polycystins (TRPPs), and ankyrin (TRPA) i oscillations activate NFATc1 and promote osteoclast differentiation i oscillations at the early differentiation stage of osteoclasts, whereas TRPV4 is highly associated with the Ca2+ response at the late stage of differentiation under FSS simulation i i . It is p [Ca2+]i . TRPV5 k [Ca2+]i . In cont [Ca2+]i . Althougeoclasts ,62. The eoclasts . Song etby NF-κB . Thus, Tntiation .2+ [2+-permeable channel in lysosomes and plays vital roles in lysosomal trafficking and functions [2+]i oscillations, which reduces osteoclastogenesis and bone remodeling.The TRPML family has three members: TRPML1, TRPML2, and TRPML3. Among these, TRPML1 is a non-selective cation channel that permeates Ca2+ . TRPML1 unctions . Erkhembunctions  showed t2+ signaling. Thus, in this section, we mentioned by listing diverse compounds depending on their mode of action. 2+ signaling in osteoclastogenesis. Osteoclasts are responsible for bone resorption and are therefore considered targets of anti-osteoporosis therapies. Novel treatment strategies aimed at preventing excessive bone resorption have been studied . The stu2+]i oscillations, and subsequently, inhibits calcineurin-NFATc1 signaling [KMUP-1 (7-[2-[4-(2-chlorophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine), a chemical synthetic xanthine-based derivative, effectively suppresses RANKL-induced osteoclast differentiation in vitro, and also attenuated ovariectomized (OVX)-induced osteoclast differentiation and prevented bone resorption in vivo . Especiaignaling .2+-calcineurin-NFATc1 signaling pathway in vitro and in vivo [2+]i oscillations, without decreasing PLCγ phosphorylation. In addition, it was proposed that zinc inhibits calcineurin in the early stage of osteoclast differentiation and [Ca2+]i oscillations in the middle or late stage of osteoclast differentiation i oscillations without decreasing PLCγ phosphorylation [Praeruptorin A is isolated from the dried root of rylation .2+]i oscillations, which leads to the suppression of the activation of NFATc1 in cultured primary bone marrow-derived macrophages (BMMs) i oscillations in BMMs. Furthermore, lumichrome suppresses NFATc1, NF-κB, and MAPK signaling activation and decreases bone loss in OVX-mice by inhibiting osteoclastogenesis.Lumichrome is a natural metabolite of riboflavin, a member of the B family of vitamins, and has been shown to have a beneficial effect on bone formation ,100. ChuAsiaticoside, a natural compound, is extracted from Centella asiatica and is a member of the triterpenoid family [2+]i oscillations and NFATc1 expression in BMMs. Therefore, Asiaticoside suppresses the differentiation and function of the osteoclast via inhibiting the NF-κB and NFATc1 pathways [d family . It signpathways .Vigna angularis (azuki bean). Kim et al. [2+]i oscillations, which leads to NFATc1 activation. In vitro, OAA inhibits RANKL-induced osteoclast differentiation through PLCγ2-Ca2+-NFATc1 signaling. OAA administration also suppresses lipopolysaccharide (LPS)-induced bone loss in vivo.Oleanolic acid acetate (OAA) is a compound isolated from m et al.  have repHarpagophytum procumbens (devil’s claw), inhibits [Ca2+]i oscillations via inactivation of several kinases such as Bruton’s tyrosine kinase (Btk), spleen tyrosine kinase (Syk), and PLCγ2, which leads to the suppression of RANKL-induced osteoclast differentiation i oscillations by inhibiting phosphorylation of PLCγ2 and blocks the downstream activation of Ca2+/calmodulin-dependent protein kinase (CaMK) type IV and cAMP-responsive element-binding protein (CREB) in BMMs. Moreover, physalin D protects RANKL-induced bone loss in vivo i, through the involvement of Ca2+ influx via voltage-gated Ca2+ channels (VGCCs), resulting in the abrogation of RANKL-induced [Ca2+]i oscillations and the inhibition of NFATc1 expression in BMMs. However, GH-induced intracellular [Ca2+]i elevation was independent of Ca2+ release from intracellular Ca2+ stores in BMMs. Taken together, these findings suggest that GH abrogates RANKL-induced [Ca2+]i oscillations, inhibits NFATc1 expression, and reduces osteoclast differentiation by inactivating VGCCs.g et al.  have shoPortulaca oleracea (PO), also known as verdolaga, red root, or pursley, has been widely used as traditional medicine. PO ethanol extract (POEE) has dual and contrary effects on RANKL-induced osteoclast differentiation. The POEE inhibits RANKL-induced [Ca2+]i oscillations and NFATc1 activation, while it enhances RANKL-induced osteoclast differentiation by reducing RANKL-mediated cytotoxicity. Erkhembaatar et al. i oscillations and the activation of downstream targets of Ca2+-CaMKK /Pyk2 (Proline-rich tyrosine kinase 2) signaling during osteoclast differentiation, resulting in the inhibition of NFATc1 and c-FOS in BMMs. In addition, an in vivo study showed that XAT treatment prevents bone loss and increases new bone formation in OVX-mice.Xanthotoxin (XAT) is isolated from the seeds of a plant of the carrot family mi majus . XAT hasmi majus ,117. Intskeleton . Dou et skeleton  showed tSinomenium acutum. SIN has been used for the treatment of rheumatoid arthritis (RA) in China i oscillations in RAW264.7 cells and BMMs. CSA also protects the bone loss of OVX- induced C57BL/6 mice [Cajaninstilbene acid (CSA) is a bioactive compound derived from pigeon pea leaves . It suppL/6 mice .2+]I, mitochondrial biogenesis, mitochondrial membrane potential, and glyoxalase I, resulting in the inhibition of RANKL-induced osteoclast differentiation and bone resorbing activity in RAW264.7 cells [Methylglyoxal is derived from organic compounds and is a precursor of advanced glycation end products. Its formation involves several metabolic pathways . The for.7 cells .2+]i by blocking Ca2+ channels except two pore segment channel 2 (TPC2) and inositol 1,4,5-triphosphate receptor type 1 (IP3R1). TPC2 is a Ca2+-permeable channel expressed in lysosomes, and IP3R1 is a Ca2+ channel that mediates Ca2+ release from the ER, following IP3 stimulation. APO inhibits osteoclast differentiation by decreasing [Ca2+]i.The catechol apocynin (APO) is used as a NADPH oxidase (NOX) inhibitor . Soares Sanguis draxonis, which is a Chinese traditional herb also known as Dragon’s Blood [2+]i oscillations, ROS production, and NFATc1 translocation into the nucleus in BMMs. Therefore, LrB can inhibit osteoclast differentiation and function by suppressing [Ca2+]i oscillations, ROS, and NFATc1 activities. LrB also exerts a protective effect on OVX-induced osteoporosis in a mouse model [Loureirin B (LrB) is an active component isolated from ’s Blood . Yuhao e’s Blood  investigse model .2+-binding protein that regulates intracellular Ca2+ homeostasis by modulating cytoplasmic and ER Ca2+ levels [2+]i oscillations, but not ionomycin-induced Ca2+ influx in BMMs. Recombinant CRT also blocks expression of NFATc1 and c-Fos, but not CREB and NF-κB in RAW264.7 cells.Calreticulin (CRT) is a Ca+ levels ,130,131.+ levels  found th2+]i oscillations, ROS production, and NFATc1 activities in BMMs. Furthermore, 6-shogaol attenuates osteoclastogenesis and alveolar bone resorption in a ligature-induced periodontitis model in vivo i oscillations in BMMs [Amyloid beta peptide (Aβ) is the principal component of the accumulations of β-amyloid found in the brains of Alzheimer’s patients . Various in BMMs .2+ signaling in osteoclastogenesis have highlighted its role in bone biology. Considering the involvement of Ca2+ signaling in bone biology, the relatively few studies available to date suggest the importance of TRP channels for modulating osteoclastogenesis and bone loss. Therefore, most of the therapeutic potentials remain open. We estimate that pharmacological targeting of this membrane channels may result in the development of therapeutics that facilitate or inhibit Ca2+ influx.The crucial studies on Ca


Enterococcus faecium EF2019 (CCM7420) and its enterocin (Ent7420) in broiler rabbits. The antibacterial and anticoccidial effect of additives was observed, with good colonization ability of the CCM7420 strain. Both additives showed a tendency to modulate the serum biochemistry parameters and to improve the immunity, jejunal morphology, weight gains, feed conversion ratio and meat quality .Weaning is the most important and critical period in rabbits breeding; the cecal digestion is very complex and only small dietary and/or environmental changes can disturb the stable microbial population/fermentation and gut health, leading to digestive dysbiosis and increased morbidity, often with fatal outcome and big economic losses. Control of the microbiota, prevention of digestive disturbances and improving gut health and immunity can be achieved through the natural substances application in rabbit nutrition. While probiotics are frequently used in rabbit farms, the in vivo administration of bacteriocins  in these animals is often limited and has become an area of research activity. Moreover, the most of probiotic strains used in rabbits are non-autochthonous (have a different origin than the rabbits ecosystem). Therefore, our study focused on improving rabbits’ health using the autochthonous strain Enterococcus faecium CCM7420 with probiotic properties and its bacteriocin Ent7420. The experiments included varying duration of application (14 and 21 days), form of application (fresh culture and lyophilized form), combination with herbal extract and application of the partially purified enterocin—Ent7420, produced by this strain. Results from these studies showed that E. faecium CCM7420 strain was able to colonize the gastrointestinal tract (caecum) of rabbits , to change the composition of intestinal microbiota , to modulate the immunity (significant increase of phagocytic activity), morphometry  ratio), physiological  and parasitological (Eimeria sp. oocysts) parameters and to improve weight gains , feed conversion ratio and meat quality .The present review evaluates and compares the effects achieved after application of rabbit-derived bacteriocin-producing strain  Coccidia and bacteria—Clostridia sp. and enteropathogenic Escherichia coli) and gastrointestinal infections . These To overcome this period, to reduce economic losses and to improve and stabilize the health status and gastrointestinal tract development, antibiotic growth promoters (AGPs) have been widely used for years. Although, these synthetic drugs showed good effects on production indicators, on the other hand, there was a risk of increasing resistance to antibiotics and transferring of antibiotic resistance genes from animal to human, which also threatened the human health and quality of meat and food . For thiThe last two decades have seen a substantial increase in the use natural supplements and/or additives in animal nutrition, in which their antimicrobial activity has been highlighted many times. The EU in its Regulation EC 1831/2003 defined the terms “feed additives” as “substances, microorganisms or preparations, other than feed material and premixtures, which are intentionally added to feed or water” and the “antimicrobials” as “substances produced either synthetically or naturally, used to kill or inhibit the growth of microorganisms, including bacteria, viruses or fungi, or of parasites, in particular protozoa” . AccordiThe use of several natural feed additives has already been reviewed in rabbit breeding ,13,14,15Enterococcus faecium CCM7420  form rehydrated in water  as well as mixed in feed and pelleted (15 g/100 kg feed), application of its partially purified bacteriocin (PPB)—enterocin (Ent) EF2019 (applied into water) and fresh culture in combination with natural substance (Eleutherococcus senticosus).The aim of this review was to summarize all achieved properties and physiological effects of the bacteriocin-producing strain with probiotic properties abeling, ) isolateEnterococcus faecium EF2019 (CCM7420) is a bacteriocin-producing strain [gelE gene) or hemolytic activities with low ability to form biofilm (0.092). The strain was deponed into Czech Collection of Microorganisms in Brno, Czech Republic to have number CCM7420. This strain showed the broadest inhibitory activity from all tested rabbits enterococcal strains against the indicators E. avium EA5, Listeria innocua LMG13568 and L. monocytogenes CCM4699 and against other tested enterococci and staphylococci tested such as clostridia, pseudomonads, enterobacteria and coliform bacteria [L. innocua LMG13568 (after 4 h) inhibited its growth already at 1 h after enterocin addition with a difference of 1.5 log cycles (5 h of cultivation). This effect was prolonged up to 24 h. The Ent7420 was tested against more than 300 strains of enterococci, staphylococci, clostridia, pseudomonads, enterobacteria and coliforms [g strain , which wg strain ,48 and pg strain . Other ubacteria . The molp < 0.001 compared to control data; or freeze dried-lyophilized—numerical increase) and its application time (2 or 3 weeks). The feed conversion ratio (FCR) was influenced only through the Ent7420 administration and the combinative application of the CCM7420 strain with Eleutherococcus senticosus extract. The effect of probiotics, including registered probiotic preparations and new beneficial microorganisms on the growth performance of rabbits have been already described/reviewed; they usually confirmed the increased body weight [Saccharomyces cerevisiae, S. boulardii, Bacillus licheniformis, B. cereus, B. cereus var. toyoi, Pediococcus acidilactici, Lactococcus lactis, Lactobacillus acidophilus, L. plantarum, L. helveticus, L. delbrueckii and L. sporogenes [Enterococcus faecium as a component of the probiotic bacterial mix (Lactina—E. faecium NBIMCC 8270 [E. faecium CCM7420 [E. faecium DSM 10663/NCIMB 10415 [E. faecium AL41 (CCM8558) and CCM4231 strains application in rabbits. Improved body weight in rabbits was also noted after probiotics administration by Bovera et al. [The results from all these experiments indicated that the CCM7420 strain could improve the average daily weight gain . In allMB 10415 ). SimilaMB 10415  and SzabMB 10415  also repa et al. , Bhatt ea et al. . On the a et al. ,52,53,54E. faecium CCM7420 was associated with increased enterococci (p < 0.01) and lactic acid bacteria (LAB) counts during the treatment period by a 1.3 log cycle on average  in sufficient counts. The microbiological examinations in fecal samples confirmed the presence of E. faecium CCM7420 strain. This strain was able to colonize the digestive tract of rabbits, reaching counts in the range 2.8–6.7 log cycle during the 2 or 3 weeks treatment. These numbers are comparable with the level of autochthonous probiotic strains of several Enterococcus spp. after their application in rabbits [E. faecium AL41 (CCM8558) strain in rabbits [p < 0.01; [S. aureus counts were also reduced by 0.5–1.2 log; p < 0.001 [p < 0.001 [p < 0.05 [S. aureus and clostridia were presented in probiotic-treated rabbits [S. aureus was noted [In all experiments, administration of  average , while t rabbits ,29,56. Olication . Outgoinlication ,59,60 anlication , enteroc rabbits . Obvious rabbits . Importa < 0.01; ); the S. < 0.001 ,26. The p < 0.05 ,26). Sim rabbits ,23,29. Ias noted . Our resas noted , who tesas noted ).Pseudomonas-like sp., Clostridium-like sp. and S. aureus—during the additives supplementation confirm the antibacterial effect of CCM7420 strain. The inhibitory effect of the bacteriocin-producing and probiotic enterococci and their enterocins on rabbits’ intestinal microbiota was already reported in our previous studies [E. coli and C. perfringens in rabbits treated by probiotic. The dominancy in antimicrobial activity of CCM7420 strain combined with E. senticosus can be confirmed by the fact that the E. senticosus extract possesses slight or no antimicrobial activity [Changes in bacterial composition—decrease of fecal coliforms,  studies ,28,29,52 studies  also desactivity . We suppEimeria species, especially in young animals and the highest incidence of oocysts was usually found around the weaning period [Eimeria sp. oocysts. The different treatment period had no impact on oocysts reduction. When the fresh culture of the CCM7420 strain at concentration ×109 CFU/mL/g was applied only for 2 weeks to compare its effect with the commonly used probiotic strain Lactobacillus rhamnosus GG, the decrease in oocysts counts was observed after probiotic application  compared to control data (1.5 × 104 OPG), but also compared to the initial counts in experimental group (7.5 × 102 OPG). This reduction effect was maintained until the end of the experiment, also after cessation of the CCM7420 strain. Moreover, at the end of the experiment (at day 42), the difference one order of magnitude in Eimeria sp. oocysts was found comparing the control (1.5 × 104 OPG) and the experimental groups  at the end of probiotic application (day 21). This finding could be explained by the irregular excretion of oocysts. In the group with Ent7420 addition, a decreasing tendency of oocysts occurrence (not significant) was observed up to the end of enterocin substance application. This could lead to consideration that longer application of CCM7420 strain did not influence more the Eimeria sp. oocysts counts in rabbits.Coccidiosis is one of the most frequent and prevalent parasitic diseases in rabbit farms. The most markedly and typical symptoms are weight loss, diarrhea (from mild intermittent to severe) with the presence of blood and/or mucus in feces, which leads, through dehydration, to the mortality of animals. The high morbidity and mortality rates among all ages, especially in the young rabbits may be responsible for important economic losses . The oocg period . The EU g period . Today, 103 OPG; ). On the103 OPG; ) in bothEimeria sp. are still limited and focused on mainly the avian coccidiosis [Eimeria sp. oocysts was also documented by Strompfová et al. [p < 0.05) were found in this experiment. The in vivo administration of bacteriocin-producing and probiotic strains decreased Eimeria sp. oocysts in rabbits. Outgoing from these results, we supposed that anticoccidial effect could be done due to the lactic acid production or by the effect of bacteriocins produced by the mentioned strains. Moreover, the potential protective effect of the CCM7420 strain against zoonotic Trichinella spiralis infection was also investigated in the framework of a new therapeutic strategy aimed at using probiotics to control parasitic zoonoses [T. spiralis infection and female fecundity ex vivo and in vitro (about 60%) throughout the CCM7420 administration.While probiotics are widely used in animals because they improve the growth performance, productivity, health status and stimulate the immunity, studies concerning the protective effect against cidiosis ,66. In rcidiosis ,67,68,69cidiosis ,53,55,70á et al.  and no dzoonoses , when thThe measurement of biochemical parameters is data mostly used for diagnostic investigations and presents a useful way for controlling the health of animals. However, in some cases, several “components” of the host biochemistry are less specific because of the reparation/compensation ability of healthy tissues/organs and/or metabolic processes.E. faecium CCM7420 application, increased (even though not significantly) concentration of the total protein (TP) was noted in most experiments, and remained stable or higher also three weeks after the strain ceasing (cessation). The highest increase in TP was measured through the fresh CCM7420 culture administration in rabbits . It could be that the glucose concentration was reduced by increased H+ concentration due to higher organic acid values in the cecum content, which inhibited gluconeogenesis [+ (lactate accumulation) in the organism first stimulates physicochemical mineral dissolution by increasing the osteoclast and osteoblast activity (bone resorption) and mostly the Ca2+ and Mg2+ reabsorption in renal tubules for pH neutralization; it is usually confirmed also with higher serum calcium levels. This hypothesis was confirmed also by us, when the slight increase of calcium content was noted during three weeks CCM7420 fresh culture and its Ent7420 application. The rabbits’ blood calcium levels fluctuate widely, dependent upon the level of calcium in their diet and the intestinal absorption as well [E. senticosus [The tested serum parameters were in the range of normal values defined for these parameters in previous studies with rabbits ,74,75, a rabbits  [24,25,2ntration ; similarntration  and Kalmntration  who obseod serum ,29,38. Bogenesis . The inc as well ; this is as well  and Szab as well  describe as well  presente as well ,34,51; snticosus ,55 in rap < 0.05; [Exogenous factors such as manipulation, nutritional, weather and temperature changes  often induce physiological oxidative stress, which is avoided by the host’s defense system. The host’s reaction to stress can be marked mostly by the glutathione-peroxidase (GPx) enzyme activity in blood. In addition, there were no significant differences in GPx activity in blood among experimental groups whereas they were differently affected by CCM7420 . Applica < 0.05; ). Compar < 0.05; ,36. Outg < 0.05; ,29,38.2, CH4 and H2; [E. faecium CCM7420 to rabbits led to an increase of fecal levels of acetic acid (p < 0.001) compared to control animals [E. faecium CCM4231; [L. plantarum spray application [In rabbits, approximately 40% of digested organic matter of the feed is digested in the caeco-colic segment; so the caecum and the proximal colon are the primary fermenters . The dig and H2; ). The st and H2; ,13,14. T and H2; ,30,38,78 animals , while oCCM4231;  or bacteCCM4231; ,38) in rlication . ConcludE. faecium CCM7420 alone and in combination with Eleutherococcus senticosus demonstrated significant increases in total phagocytic activity (PA) of leukocytes and PA of neutrophils at the end of the treatment period (21 days) and also after three weeks of the ost-treatment period  and AL41  also showed stimulation of non-specific immune reaction in rabbits; the significant increase of PA (p < 0.001) was noted in both experiments during the treatment and increased several weeks after the strain’s cessation. Fathi et al. [p < 0.05) compared also to the control data [Knowledge of the immune response and homeostasis in farm animals represents important information to protect animals from especially bacteria-derived diseases, to improve their health and productivity. The overall organization of the rabbit’s digestive immune—lymphoid system is mostly similar to other species, but at the same time it is very special. There are two additional structures identified only in this species, the sacculus rotundus and the vermiform appendix (a place of lymphoid cells differentiation and maturation); they generally act synergistically. The gut microbiota contribute to intestinal homeostasis via inducing the intestinal immune cells and also influence the systemic host immunity. The probiotic consumption shows a beneficial effect in several ways, including intestinal microbiota balance and ability to modulate host innate and specific immune response. Their effect on non-specific immunity was reported as enhanced phagocytosis of pathogenic bacteria and modified cytokine production , while t 0.0001; ). Anothei et al.  also repi et al.  noted norol data . The samt period  days androl data ) as wellrol data . We expeE. faecium CCM7420 strain administration [It is well known, that probiotic supplementation improves not only the growth rate and enhances the efficiency of feed conversion but also may positively influence the health status via enhancing gut health in rabbits . Gut heastration . These rstration ,85). Thip = 0.0011) in treated groups with freeze-dried CCM7420 strain , in contrary to other findings with reduced iron content during microbial fermented feed utilization with Lactobacillus plantarum and Pediococcus acidilactici [p = 0.0004) and calcium (p < 0.0001) were noted. Similarly to these results, the copper concentrations in rabbit meat also reduced during the enterocin M addition to rabbits, however, not significant but only numerically changes were recorded. Similarly to us, lower concentrations of copper, zinc and manganese was found by Shah et al. [Rabbit meat is greatly valued for its high nutritional and dietary quality, especially its low amounts of cholesterol, fat and sodium and high content of polyunsaturated fatty acids (PUFA), potassium, phosphorus and magnesium ,86; for ilactici  and afteilactici . This acilactici  and coulilactici  and lantilactici . The enlh et al.  after prh et al. ,93. Regah et al. . Despiteh et al. . InferriE. faecium CCM7420 in different application forms  was lyophilized (freeze-dried) from rehydrated water  as well as mixed in feed and pelleted (15 g/100 kg feed), either alone and in combination with Eleutherococcus senticosus and its enterocin Ent7420  were tested in rabbits. During these experiments, the following effects of the strain and its enterocin were observed: improved average daily weight gain and feed conversion ratio, good colonization ability of the tested strain with maximum counts in the first 2–3 weeks of application, increased lactic acid bacteria and reduced coagulase-positive staphylococci including S. aureus, coliforms and clostridia population as well as the Eimeria sp. oocysts counts in the rabbit’s gut. Improved biochemical blood parameters  have been noted during the CCM7420 strain application; however, the glutathione-peroxidase level was not disturbed and oxidative stress was not evoked through the additives application. Another interesting finding was the significant stimulation of blood phagocytic activity and also the improved morphometry parameters (enlargement of the absorption surface in jejunum and higher villi height: crypt depth (VH:CD) ratio). The physicochemical properties of rabbit meat were not negatively affected by the CCM7420 strain, while the meat iron content significantly increased during its application, which improved the rabbit meat quality. It could be also emphasized that knowing the probiotic properties and the ability of Enterococcus faecium CCM7420 to produce enterocin Ent7420 with an antimicrobial effect is of great interest mainly in the case of several disease/pathologies, such as epizootic rabbit enteropathy, which are difficult to prevent and combat because their etiology is not known and there is no vaccine. This strain is the main component of the Prorabbit probiotic preparation, which is often used in Slovak rabbit farms . Moreover, to the best of our current knowledge, our team is the first that deals with experiments regarding the morphological changes in the jejunum of rabbits during enterocins administration; the first reports regarding the effect of beneficial/probiotic strains and bacteriocins on the mineral and amino acid concentrations as well as the effect of bacteriocins on fatty acid content of rabbit meat were also published by our team.The strain