Myocardial ischemia-reperfusion injury (MIRI) results in the aggravation of myocardial injury caused by rapid recanalization of the ischemic myocardium. In the past few years, there is a growing interest in investigating the complex pathophysiological mechanism of MIRI for the identification of effective targets and drugs to alleviate MIRI. Currently, pyroptosis, a type of inflammatory programmed death, has received greater attention. It is involved in the MIRI development in combination with other mechanisms of MIRI, such as oxidative stress, calcium overload, necroptosis, and apoptosis, thereby forming an intertwined association between different pathways that affect MIRI by regulating common pathway molecules. This review describes the pyroptosis mechanism in MIRI and its relationship with other mechanisms, and also highlights non-coding RNAs and non-cardiomyocytes as regulators of cardiomyocyte pyroptosis by mediating associated pathways or proteins to participate in the initiation and development of MIRI. The research progress on novel small molecule drugs, clinical drugs, traditional Chinese medicine, etc. for regulating pyroptosis can play a crucial role in effective MIRI alleviation. When compared to research on other mature mechanisms, the research studies on pyroptosis in MIRI are inadequate. Although many related protective drugs have been identified, these drugs generally lack clinical applications. It is necessary to further explore and verify these drugs to expand their applications in clinical setting. Early inhibition of MIRI by targeted regulation of pyroptosis is a key concern that needs to be addressed in future studies. Acute myocardial infarction (AMI) is a severe and common medical emergency, resulting in irreversible damage to the heart . AMI leaPyroptosis, a form of lytic programmed cell death induced by inflammasomes, has gradually attracted more attention in recent years. Previous studies have shown that it was involved in the pathogenesis of many cardiovascular diseases and also played a vital regulatory role in the occurrence and development of MIRI. Therefore, elucidating the pyroptosis mechanism could help in MIRI alleviation by targeting this pathway ,10.Since its discovery, pytoptosis has been thoroughly studied for a long time. Zychlinsky et al. discovered a Shigella flexneri-mediated cytotoxic killing mechanism in 1992 after human colon mucus was invaded by the Gram-negative bacterial pathogen. At that point in time, this was regarded as an invasive strain-induced programmed cell death and was characterized by chromatin condensation, DNA fragmentation, and a caspase-dependent apoptosis pathway .In 2001, “pyroptosis” was defined as caspase-1-dependent pro-inflammatory programmed cell death . The 201Pyroptosis is the mechanism by which the human immune system responds to pathogens as well as endogenous damage. Nod-like receptor protein 3 (NLRP3) senses cardiomyocyte injury and recruits an apoptosis-associated speck-like protein containing a carboxy-terminal CARD (ASC) and procaspase-1 to form inflammasomes, which initiate the pyroptosis pathway. This, in turn, activates gasdermin D (GSDMD), which triggers cell death, and is also the classical pathway of pyroptosis .+ efflux, and lysosome destruction, while the changes in both calcium concentration and reactive oxygen species (ROS) production can lead to the activation of the NLRP3 to form NLRP3 inflammasomes. Moreover, upon activation, the NLRP3 protein recruits ASC and cysteine-recruiting proteins to complete the inflammasome assembly, and subsequently activates caspase-1 [The NLRP3 inflammasome is an important component of innate immune response-dependent pattern-recognition receptors and is a key protein that triggers pyroptosis in MIRI. Toll-like protein-sensing receptors derived from danger-associated molecular patterns (DAMPs) activate the nuclear factor kappa B (NF-κB) pathway, Kaspase-1 ,16,17. Oaspase-1 . The N taspase-1 ,20,21. ODuring the process of myocardial ischemia and reperfusion, a large amount of ROS is produced, especially in the initial stage of reperfusion. The excessive ROS induces cell damage by disrupting the cellular signaling pathway, activating the inflammatory factors, and inducing lipid peroxidation and even cell death . The masDuring MIRI, dysfunctional calcium homeostasis and disordered calcium distribution are observed in the cells, resulting in an abnormal increase in intracellular calcium concentration, also known as calcium overload. Calcium overload can disturb of the oxidative phosphorylation cycle in mitochondria, thereby leading to a decrease in mitochondrial membrane potential, a decrease in tissue ATP content, and metabolic disturbance in cardiomyocytes.Firstly, calcium overload is shown to be closely related to pyroptosis in other non-cardiomyocytes. It was observed that the accumulation of inflammatory metabolites in chondrocytes of joint tissues in rats led to a decrease in the PH of the joint tissues and activation of acid-sensing ion channel 1a, which promoted the influx of calcium ions into cells, thereby leading to increased intracellular calcium ion concentrations. This, in turn, stimulated NLRP3 expression, polymerization, and inflammasome assembly. These changes further lead to the activation of caspase-1 by cleavage of pro-IL-1β and pro-IL-18 into their biologically active forms, followed by the initiation of pyroptosis. Therefore, it can be concluded that the increased intracellular calcium ion concentration promoted cell pyroptosis by upregulating the expression of the inflammasome . In addiSignificant progress has been gradually made to understand the protective effect of calcium overload-mediated pyroptosis in cardiomyocytes.Mo et al. developed an adult rat cardiomyocyte hypoxia/reperfusion (H/R) model and observed that calcium overload could result in H/R-induced cardiomyocyte pyroptosis by regulating the NLRP3/caspase-1 pathway. A study observed that the intracellular ion channel inositol 1,4,5-triphosphate receptor IP3R1 activated calcium overload by increasing the intracellular calcium ion concentration and also aggravated pyroptosis caused by MIRI . AconitiParticularly, calcium overload in MIRI acts as an excitatory factor of oxidative stress, which further stimulates the formation of inflammasomes. Moreover, it has been proven that oxidative stress and pyroptosis promote each other during MIRI. Thus, it is evident that there is a strong relationship between them during the progression of MIRI.So far, the relationship between pyroptosis, apoptosis, and necroptosis in the MIRI model has not yet been fully explored. However, current studies have indicated that the cell death-regulated pathways can cross-regulate each other , and casCaspase-8 is a protease that regulates both cell death and survival. The extrinsic pathway of apoptosis is triggered by the transmembrane death receptors belonging to the tumor necrosis factor (TNF) family. Caspase-8 is involved in executing this extrinsic pathway . MoreoveRecently, the role of caspase-8 in pyroptosis was demonstrated in embryonic intestinal tissue. Briefly, the deficiency of caspase-8 induced ASC formation, thereby upregulating GSDMD, caspase-3, and caspase-1-dependent cleavage of caspase-7. In other words, higher levels of caspase-8 could suppress pyroptosis-related proteins by inhibiting ASC formation . CaspaseThe above findings demonstrated that apoptosis, necroptosis, and pyroptosis are overlapping pathways and exhibit overlapping effects in coordinating programmed cell death. That indicates that, if one pathway is activated or deactivated, other pathways would also be affected due to a chain reaction . TherefoThere are few studies on the relationship between pyroptosis and other mechanisms of MIRI, such as autophagy, endoplasmic reticulum stress, and ferroptosis. Thus, those studies are not considered here.Pyroptosis has been shown to act on non-cardiomyocytes, thereby leading to MIRI. Pyroptosis aggravates MIRI by acting on non-myocardial cells, including fibroblasts, vascular endothelial cells, and macrophages, to activate the inflammatory pathways.Fibroblasts are the central cells producing extracellular matrix and account for 60% to 70% of the total cells in the normal myocardial tissue. They are also the major constituent of non-cardiomyocytes in the heart. A study by Kawaguchi demonstrated that the activation of inflammasomes by cardiac fibroblasts is critical for MIRI. Importantly, in vitro experiments revealed that H/R stimulated inflammasome activation in cardiac fibroblasts and not in cardiomyocytes. This activation was mediated by the production of ROS and potassium efflux, thereby inducing the secretion of IL-1β, which ultimately led to cardiac fibroblast pyroptosis. Cardiac fibroblasts are more capable of detecting injury signals than cardiomyocytes, and thus enhance the inflammatory response to MIRI . In addiThe leukocyte aggregation and activation mediated by microvascular injury play an important role in MIRI. The expressions of various adhesion molecules of neutrophils and vascular endothelial cells are enhanced, and this leads to high adhesion and aggregation of these cells. Neutrophils can chemotactically migrate through the vascular wall, thereby aggravating the infiltration of inflammatory cells. The free radicals and lysosomal enzymes cause damage to the endothelial, eventually forming microthrombi and tissue edema, and even leading to a no-reflow phenomenon ,50. The MIRI leads to a release of large amounts of cardiac extracellular vesicles (EVs), which, in turn, promote local aseptic inflammation and can be shunted to distant organs. EVs can deliver miR-155-5p to M1 macrophages, promote M1-like macrophage polarization, increase the expressions of pro-inflammatory cytokines, and induce a pro-inflammatory phenotype by activation of the JAK2/STAT1 pathway .Several studies have demonstrated that exosomes may activate the cardioprotective signaling pathway . For exaIn recent years, epigenetics has gradually become a research hotspot, and several studies have revealed that it plays an important role in MIRI regulation and pathogenesis. Moreover, a large of studies have indicated that ncRNAs are involved in MIRI regulation. They can be used as potential biomarkers and therapeutic targets for MIRI. Moreover, they also have broad prospects in disease diagnosis and clinical treatment . NcRNAs The miRNAs are non-coding single-stranded RNA molecules with a length of approximately 22 nucleotides and are involved in the regulation of post-transcriptional gene expression. The miRNAs regulate the expression of different genes by inhibiting the translation of messenger RNA (mRNA), thereby promoting mRNA degradation . The expThe lncRNAs are non-coding RNAs having a length of more than 200 nucleotides. The abnormal expression of lncRNAs in the cardiovascular system is associated with abnormal cardiac development and cardiovascular diseases ,64. The The circRNAs are endogenous biomolecules forming covalently closed loops with tissue- and cell-specific expression patterns in eukaryotes, and their biogenesis is regulated by the specific cis-acting elements and trans-acting factors. Several circRNAs exert important biological effects by acting as miRNAs or protein inhibitors , thereby regulating protein functions or gene expression . Recent Therefore, several nc RNAs regulate pyroptosis in MIRI. . In addiMCC950 is a small molecule inhibitor that selectively blocks NLRP3 inflammasome activation . MoreoveINF4E is also a potent NLRP3 inflammasome inhibitor and can inhibit the activities of caspase-1 and NLRP3-ATPase. A study by Mastrocola et al. demonstrated that the time-dependent attenuation of MIRI by INF4E via activation of reperfusion injury rescue kinase (RISK) and mitochondrial pathways . FurtherGenerally, VX-756 is metabolized to the active molecule VRT-043198, a highly selective prodrug caspase 1 inhibitor and a potent caspase-1 selective inhibitor.During reperfusion, the co-administration of VX-765 with ticagrelor or cangrelor (P2Y12 receptor antagonists) led to a reduction in the myocardial area and an improvement in ventricular function in an ischemia-reperfusion mouse model. VRT-043198, an active derivative of VX-765, improved myocardial function at the onset of reperfusion, implying that caspase-1 tends to damage the heart only upon reperfusion. Furthermore, the studies observed that VX-765 reduced circulating IL-1β, prevented the loss of cardiac glycolytic enzymes, preserved mitochondrial complex I activity, and suppressed the release of lactate dehydrogenase release ,81. CarmColchicine leads to a reduction in the activity, adhesion, and chemotaxis of neutrophils by interfering with the lysosomal degranulation, and it also inhibits the granulocyte migration to the inflammatory area, thereby exerting an anti-inflammatory effect. In the past few years, colchicine has been demonstrated to exhibit anti-inflammatory effects and has been proved to be effective in a wide range of cardiovascular diseases . SeveralIn this study, the focus is mainly on the anti-inflammatory and anti-pyroptotic effects of colchicine in the MIRI model. Colchicine was found to be associated with increased levels of systemic interleukin-10 (IL-10) and decreased levels of cardiac transforming growth factor-β . The preMetformin is the first-line drug for the treatment of type 2 diabetes, and several studies have demonstrated that it plays a crucial role in ischemia-reperfusion injury in different organs such as the heart, gut, brain, kidney, bladder, testis, ovary, and liver ,95,96,97Dexmedetomidine is a high selective α2-adrenoceptor agonist that is commonly used for perioperative sedation. Dexmedetomidine can reduce MIRI and exerts a protective effect on ischemia-reperfusion injury by improving myocardium function, which is not regulated by the central nervous system ,105. A sTrimetazidine is an inhibitor of free fatty acid oxidation playing a crucial role in myocardial and muscle glucose metabolism and has been demonstrated to provide cardioprotection without any associated side effect in patients with angina, diabetes, and undergoing revascularization surgery ,107. TriNatural substances primarily include traditional Chinese medicinal compounds, crude extracts of traditional Chinese medicines, and active monomer components. Generally, they have multiple components, multiple channels, and multi-target effects, and show fewer side effects on patients. Numerous studies have shown that natural substances offer unique advantages and disadvantages for ischemic cardiomyopathy and have complex pathological mechanisms and therapeutic potentials in cardiovascular diseases.Emodin exhibits excellent anti-inflammatory effects, which can protect the myocardium from MIRI. It was observed that emodin inhibited the GSDMD-mediated TLR4/MyD88/NF-κB/NLRP3 inflammasome pathway for MIRI alleviation in cardiomyocytes . MoreoveCinnamyl ethyl acetate extract has been shown to protect the heart of rats from MIRI by inhibiting NLRP3 inflammasome activation and pyroptosis . The resMyriocin is a specific inhibitor of serine palmitoyl transferase (SPT) and of ceramide de novo synthesis. During MIRI, SPT promotes the synthesis of ceramide, an inflammatory mediator, and makes it accumulate in myocardial cells to aggravate the damage. Myriocin can reduce inflammatory reaction and oxidative stress by downregulating ceramide and reducing the production of ROS ,112. MyrSevoflurane is one of the most commonly used inhalation anesthetics, which helps in rapid recovery and leads to a few adverse effects on circulatory disorders and respiratory depression in patients. Several studies have demonstrated that sevoflurane has a protective effect on MIRI by suppressing the inflammatory response. A review by Wu et al. indicated that the inhibition of cardiomyocyte inflammation and pyroptosis by regulation of the P2X7-NLRP3 signaling pathway is a potential mechanism of sevoflurane against MIRI . BrieflyHydrogen is an extremely flammable, colorless, transparent, odorless, and tasteless gas that is insoluble in water. Previous studies have indicated that hydrogen exerts antioxidant, anti-apoptotic, and anti-inflammatory effects in different types of diseases ,118,119.The protective effect of small molecular substances on MIRI is governed by their chemical properties. Generally, these substances have dose-dependent or time-dependent characteristics and show only a few adverse effects on humans or rats. NLRP3 inhibitors may also be indirectly involved in MIRI regulation because of their action on other pathways. This can provide new insights into the treatment and prevention of ischemic cardiovascular diseases. The molecular inhibitors involved in other signaling pathways may also contribute to the inhibition of inflammatory responses and cell pyroptosis, thus further advocating the need to evaluate the efficacy and mechanism of action of acute myocardial infarction . TherefoMyocardial ischemia-reperfusion is a complex pathophysiological process involving multiple molecular signaling pathways. A complete understanding of MIRI development and progression can help to identify targets for the treatment of patients with associated cardiovascular diseases and improve the clinical prognosis of patients with acute myocardial infarction and coronary heart disease after PCI. Several mechanisms of MIRI are closely linked and dependent on each other. Some of the mechanisms, such as calcium overload, energy metabolism disorders, oxidative stress, autophagy, etc., have been largely studied and are demonstrated to exhibit a regulatory role in MIRI development. The role of pyroptosis in MIRI is still under investigation, and the associated signaling pathways need to be elucidated further. Recently, a majority of studies on MIRI alleviation have focused on improving pyroptosis by inhibiting NLRP3 inflammasome, and NLRP3 is proven to be a potentially effective key molecule in MIRI treatment. A cascade of inflammatory responses in MIRI is triggered by the release of several inflammatory factors, leading to myocardial infarction and myocardial fibrosis. However, the inflammatory response is indispensable to promoting repair sites of damage. A proper balance of inflammatory and anti-inflammatory responses can help in the timely removal of necrotic tissue cells from the human body. The studies on the regulatory role of pyroptosis in MIRI alleviation revealed that the degree of pyroptosis can be controlled for the regulation of the inflammatory responses in tissues and repairing the damaged myocardium to obtain an optimal degree for MIRI alleviation. It is noteworthy that cardiac fibroblasts act as sentinel cells, and NLRP3 inflammasome is upregulated in cardiac fibroblasts to mediate MIRI, whereas cardiomyocytes are mere “victims” of pyroptosis. However, further experimental studies are required to support and verify this conclusion.Although several inhibitors of pyroptosis have been used in MIRI animal models and have achieved good efficacy in reducing myocardial infarction size and improving cardiac functions, the present studies are still limited to animal experimental models, and the human clinical trials to understand the effect of these inhibitors remain insufficient. At present, all of these drugs are not approved for use in MIRI patients. Therefore, it is expected that more clinical trials to verify the results can be helpful to promote the research on potential drugs. This study also elucidated the effects of different drug categories, including small molecule drugs, clinical drugs, proprietary Chinese medicines, and even gases, on MIRI and cell pyroptosis. They act in different overlapping signaling pathways and ultimately affect cells. Therefore, the combined effect of two different drugs on MIRI treatment needs to be further studied to promote their applications in clinical trials. Perhaps, with the advancements in the pharmacodynamics, pharmacokinetics, toxicology, and corresponding mechanisms of drugs, studies can be conducted to understand the applications of binding the useful and effective monomer components of different drugs, which may provide a breakthrough innovation in MIRI treatment. Pyroptosis is involved in the occurrence and development of several cardiovascular and inflammatory-related diseases. Further research on the pyroptosis signaling pathway of diabetics with MIRI can help us clearly understand the specific mechanism of pyroptosis in such disease and provide a new direction for MIRI prevention and treatment.Myocardial ischemia-reperfusion injury is a deleterious process involving multiple signaling pathways, and affecting multiple genes, multiple molecules, multiple cells, and multiple tissues. The underlying mechanisms are driven by biological signal transduction pathways and epigenetics. Pyroptosis interacts with other models of cell death in MIRI, and out of these, the potential therapeutic effects of inhibiting the major targets of NLRP, caspase, and GSDMD have gradually been elucidated. However, at present, no available drug can inhibit the expression of proteins associated with pyroptosis for the clinical treatment of myocardial ischemia-reperfusion syndrome. Furthermore, sevoflurane and hydrogen have great potential applications in MIRI treatment. Targeting pyroptosis helps to strictly control the degree of inflammatory response; however, a few issues still need to be resolved to realize such applications. Due to the complex and diverse characteristics of MIRI pathogenesis, single-target therapy for reperfusion injury may be less effective. In such cases, multi-target combination therapy may be an effective approach for the treatment of reperfusion injury. Therefore, it is necessary and urgent to discover promising therapeutic targets for maximizing the benefits of revascularization.


BackgroundIn this study, we aimed to provide a descriptive overview of the utilization of hematopoietic stem cell transplantation (HSCT) for the treatment of acute myeloid leukemia (AML), determine the rates of HSCT use stratified by patients’ demographic characteristics, and measure the hospitalization outcomes.MethodologyWe conducted a cross-sectional study using the Nationwide Inpatient Sample (NIS) obtained from hospitals in the United States. Our sample included 21,385 adult patients (aged ≥18 years) with a primary discharge diagnosis of AML. The sample was further grouped by inpatients who were managed with HSCT and chemotherapy as the primary procedure. We compared the demographic characteristics and hospital outcomes in AML inpatients across treatment cohorts by performing descriptive statistics and Pearson’s chi-square test. Next, we measured the differences in continuous variables (length of stay and cost) using the analysis of variance (ANOVA). All analyses were conducted using SPSS version 26 .ResultsThe hospital-based utilization rate of HSCT was 0.4% in AML inpatients. The utilization rate of HSCT was higher in females (0.5%), African Americans (0.6%), those with median household incomes above the 50th percentile (0.5%), and those covered by private insurance (0.8%). A significantly higher proportion of AML inpatients with HSCT had depression . AML inpatients receiving HSCT had significantly longer hospitalization stays and higher treatment costs than those receiving chemotherapy. The all-cause inpatient mortality was 11.6% in AML inpatients. Statistically, there were no significant differences by treatment.ConclusionsHSCT appears to be underutilized for the treatment of AML. This treatment had a higher utilization rate in females and those from high-income families and was covered by private insurance. The utilization of chemotherapy and HSCT did not significantly differ in the presence of comorbidities, except for depression and hypertension having a higher utilization of HSCT. Acute myeloid leukemia (AML) is a cancer of the blood stem cells in the bone marrow, most prominently affecting myeloid cells . Among aThe standard therapy for AML involves the induction of chemotherapy with a combination of cytarabine and anthracycline . The patThe burden of AML is not only seen in clinical settings but also financially. Across all treatment episodes, the average cost has been estimated to be $439,104 for relapsed and refractory, followed by HSCT at $329,621, high-intensity induction chemotherapy at $198,657, high-intensity consolidation chemotherapy at $73,428, and low-intensity chemotherapy at $53,082. Inpatient hospitalization is the largest contributor to cost, accounting for roughly 70% of the cost across all treatment groups [In this study, we provide a descriptive overview of the utilization of HSCT for the treatment of AML and determine the rates of HSCT use stratified by patients’ demographic characteristics using a Nationwide Inpatient Sample (NIS). Our second goal is to measure the hospitalization outcomes, including length of stay (LOS), cost, the severity of illness, and inpatient mortality in inpatients managed with HSCT.Study sampleWe conducted a cross-sectional study using the NIS. The NIS is hospital-based administrative data obtained from non-federal hospitals across 48 states and the District of Columbia in the United States. As the NIS is de-identified data, we were not required to take institutional review board approval for our study .We included 21,385 adult patients (aged ≥18 years) with a primary discharge diagnosis of AML. The sample was further grouped by inpatients who were managed with HSCT and chemotherapy as the primary procedure.VariablesDemographic variables including age, sex, race, primary payer, and median household income were collected. The comorbidities noted in the patient records included alcohol abuse, arthropathies, metastatic cancer, depression, diabetes with complications, hypertension, chronic pulmonary disease, obesity, and hypothyroidism. Hospital outcomes drawn from the NIS included the LOS and cost during hospitalization for the treatment of AML, the severity of illness which was measured using the all-patient refined drugs (APR-DRGs), and in-hospital mortality  .Statistical analysisWe compared the distributions of demographic characteristics and hospital outcomes in AML inpatients across treatment cohorts by performing descriptive statistics and Pearson’s chi-square test. Next, we measured the differences in continuous variables, i.e., age, LOS, and cost using the analysis of variance (ANOVA). All analyses were conducted using SPSS version 26.0 . Statistical significance was set at a two-sided p-value <0.05.The hospital-based utilization rate of HSCT was 0.4%  in AML inpatients. The inpatients in the HSCT cohort were younger compared to chemotherapy and total AML inpatients (mean age: 51.7 vs. 54.6 vs. 62.7 years). Females accounted for the majority of HSCT recipients (55.6%), and the utilization rate of HSCT was also higher in females (0.5%) compared to males (0.3%) when males formed 55.8% of total AML inpatients. Although Whites (72.2%) accounted for the majority of HSCT recipients, the rate of utilization was the highest in African Americans (0.6%), with Whites having a utilization rate of 0.4%.The inpatients were closely distributed by median household income but the HSCT utilization was higher in those with a median household income above the 50th percentile (64.7%) for a utilization rate of 0.5%. Private insurance was the dominant primary payer to cover the HSCT inpatients (66.7%) with a utilization rate of 0.8%, which was much higher than that seen in total AML inpatients who were majorly covered by Medicare (48.7%).A significantly higher proportion of AML inpatients with HSCT had depression  and hypertension , whereas there existed a statistically non-significant difference across other comorbidities by treatment, as shown in Table AML patients who received HSCT had significantly longer LOS and higher treatment costs compared to those who received chemotherapy and the total inpatients. When 73.2% of total AML inpatients had major severity of illness, about four-fifths were HSCT recipients, with a utilization rate of 0.5%. The all-cause inpatient mortality was 11.6% in AML inpatients, and, statistically, there existed no significant difference by treatment, as shown in Table The patients who achieved complete remission on treatment had much better survival rates than those who did not . Along sAccording to an evidence-based review from the American Society of Transplantation and Cellular Therapy, the cytogenic risk is the most important parameter when considering suitable candidates for allogeneic HSCT. Moreover, allogeneic HSCT in patients with complete remission is best suitable for adverse or intermediate-risk cytogenetics but not for favorable-risk cytogenetics. The results from their meta-analysis stated that allogeneic HSCT recipients showed significantly better relapse-free survival than those whose cytogenetics were adverse risk (hazard ratio (HR) = 0.69; 95% confidence interval (CI) = 0.57-0.84) or intermediate risk  rather than favorable risk  . While cExisting literature on AML does not speak much about the healthcare and financial burden. Our cross-sectional study identified that the average LOS for patients with AML was significantly higher among patients receiving HSCT. Along similar lines, a study by Pandya et al. stated that the duration of each episode of care was the longest in patients with refractory or relapsed AML 14.77 months), followed by patients with HSCT (6.35 months). Another important finding from our study was that most people who utilized HSCT were covered by private insurance, and medicare covered only 0.2% of the patients. This finding is in line with the findings of the study by Pandya et al., which stated that the total mean episode costs were the highest in refractory or relapsed episodes  [7 months,AML costs are significantly driven by hospitalization and medical costs, the cost of stem cell transplants, and the cost of medications. With AML treatment moving to the outpatient setting, hospitalization and medical costs are likely to decline. As more patients become eligible for improved transplant procedures, the cost of stem cell transplantation may increase, although the overall cost may decrease if new procedures lead to reduced hospital stay in patients . OverallThere was a statistically significant difference in the percentage of AML patients with depression undergoing HSCT compared with those not receiving HSCT (22.2% in comparison to 11.4%) in total oncological conditions that often coexist with depression. Patients receiving allogeneic HSCT are at risk of poor treatment survival outcomes in addition to depression, which affects the quality of life. Consequently, most stem cell transplantation centers require pre-transplant psychiatric evaluations for patients. Depression has been identified as a major risk factor for post-transplant mortality by numerous prognostic scoring systems. A highly useful prognostic tool in allogeneic HSCT is the hematopoietic cell transplantation-specific comorbidity index . Due to Our study should be viewed with some limitations in mind. To conduct our study, we used a data sample from the NIS, which does not contain individual patient-level information. It is important to note that the participants in our study were selected from an inpatient sample, so their comorbidities may differ from those in the general population. We could not include certain comorbidities, including arrhythmia, coronary artery disease, stroke, inflammatory bowel disease, and liver disease, which may impact the utilization of HSCT. There is also the possibility that the results of our study might not represent people from other races or ethnicities because our study population largely consists of Whites. Lastly, due to the nature of the NIS data, we did not have further information if LOS could impact the utilization of chemotherapy versus HSCT and post-HSCT complications, including GVHD. One of the strengths of the study is that the NIS has the capacity to build a population-based inpatient representation of associations between diseases and comorbidities. The chances of recall bias are minimal given that the NIS has primary and secondary diagnostic codes and other clinical information obtained at the time of hospitalization. Another strength of this study is its large sample size of 21,385 inpatients and data reliability as the information was coded independently of the individual practitioner, minimizing reporting bias. Moreover, the large sample size increased the power to detect differences.HSCT seems to be underutilized for the treatment of AML. This treatment was used in comparatively younger inpatients with a higher utilization rate in females and those from high-income families and covered by private insurance. The utilization of chemotherapy and HSCT did not significantly differ by the presence of comorbidities except for depression and hypertension as there was a higher utilization rate of HSCT in at-risk inpatients. AML patients who received HSCT had significantly longer hospitalization stays and higher treatment costs compared to those who received chemotherapy. The all-cause inpatient mortality was 11.6% in AML, but the utilization of chemotherapy versus HSCT did not have a statistically significant difference in mortality rates. Future studies may examine the potential factors, including GVHD and post-HSCT complications, that may affect the hospitalization stay. Moreover, it is important to understand and compare the long-term survival rate in inpatients undergoing HSCT versus chemotherapy.


It is now well established that BK elicits Ca2+ signals in the pancreatic stellate cells, but not in the quantitatively dominant acinar cells.5 In this context, it is interesting that Kusiak et al.6 have now shown that the TRPA1 channel is expressed in human pancreatic stellate cells and mediates Ca2+ signals of pathophysiological importance.It has been shown that TRPA1 can be activated by a number of noxious chemical agents as well as by the inflammatory peptide bradykinin (BK).7 In spite of this designation, intracellular Ca2+ signals are elicited by concentrations of BK that are only very slightly above the normal resting plasma level.5 It is well established that BK plasma levels are elevated in acute pancreatitis and that at such concentrations, BK elicits vigorous Ca2+ signal generation in the stellate cells.5 It is also known that in pancreatitis, the stellate cells can become transformed into an activated state and that in chronic pancreatitis, these activated stellate cells become a large component of the exocrine pancreas, contributing to the production of an inflammatory microenvironment that is potentially cancer promoting.7The stellate cells form a relatively small component of the normal exocrine pancreas, where they exist in a so-called quiescent state.6 now present data that provide a plausible explanation for the well-established fact that whereas alcohol-related acute pancreatitis often develops into chronic pancreatitis, this is not the case for bile-related acute pancreatitis.8 Bile acids are capable of inducing acute pancreatitis elicit large and sustained Ca2+ signals, not only in the acinar cells, but also in the normal quiescent stellate cells. These Ca2+ signals cause both acinar and stellate cell necrosis.5 When most of the stellate cells are dead, chronic pancreatitis cannot develop because only these cells are capable of producing the fibrotic microenvironment. With regard to alcohol-related pancreatitis, it turns out that in vivo, but not in isolated cells, alcohol and fatty acids promote the transition of quiescent to activated stellate cells, although the mechanism responsible for this has not yet been clarified.6 Alcohol and fatty acids, generating fatty acid ethyl esters inside the cells, elicit large Ca2+ signals in both acinar and quiescent stellate cells,6 but in the activated stellate cells, the Ca2+ signals elicited by this combination are very much smaller than in the quiescent cells, preventing necrosis.6 Why do the activated stellate cells generate much smaller, and therefore less toxic, Ca2+ signals in response to alcohol and fatty acids than the quiescent ones? Kusiak et al. show that the principal cause of the diminished Ca2+ signals is downregulation of the TRPA1 channel. This reduces Ca2+ influx and thereby protects against the development of toxic Ca2+ signals that overload the mitochondria with Ca2+ and reduce ATP generation. This protection makes the activated stellate cells markedly resistant to cell death. In alcohol-related acute pancreatitis, the stellate cells therefore largely survive and in their activated state they can produce the fibrotic microenvironment that is potentially cancer promoting (Kusiak et al.romoting .2+ signal generation evoked by alcohol and fatty acids in the quiescent stellate cells. It has previously been shown that BK-elicited opening of TRPA1 channels depends on phospholipase C activation.3 Kusiak et al. now show that Ca2+ signal generation in the stellate cells depends on primary release of Ca2+ from the endoplasmic reticulum, almost certainly mediated via IP3 receptors, which are expressed in the stellate cells.6 However, the precise mechanism by which alcohol and fatty acids cause opening of TRPA1 channels is unknown. The toxic Ca2+ signals elicited by alcohol and fatty acids in the pancreatic acinar cells have also been shown to depend on primary Ca2+ release from the endoplasmic reticulum, mostly through IP3 receptors but, in order to develop a sustained elevation of the cytosolic Ca2+ concentration, opening of Ca2+ release activated Ca2+ channels of the Orai1 type is required.5 The toxic effects of alcohol and fatty acids on the acinar cells can be prevented by pharmacologically blocking the Orai1 channels.5 The BK-elicited Ca2+ signals in stellate cells have also been shown to depend on Orai1 channel activity.5 It is therefore somewhat confusing that the Ca2+ signals generated by alcohol and fatty acids in the stellate cells would seem to depend on TRPA1 channels rather than Orai1 channels,6 since in both cases the primary event is release of Ca2+ from the endoplasmic reticulum.6 In this context, it is a difficulty that in the new study of Kusiak et al., all Ca2+ signaling experiments were carried out on cultured human stellate cells, whereas previous work5 was done on stellate cells in their normal environment in the exocrine mouse pancreatic tissue. Cultured cells may have different properties from normal cells in their normal environment, but species differences can also not be excluded. Specifically with regard to TRPA1 channels, it is interesting that caffeine has been shown to activate mouse TRPA1 channels, whereas it suppresses human TRPA1 channels.9The new results also indicate that TRPA1 plays an important role in the Ca2+ signals generated in the acinar cells by the combination of alcohol and fatty acids or by bile acids. These signals cause acinar necrosis and release of proteases, including trypsin and kallikrein, which in turn, via various intermediary steps, generate Ca2+ signals in stellate cells and macrophages thereby amplifying the inflammatory processes.5 The new results presented by Kusiak et al., showing that alcohol and fatty acids can also directly elicit Ca2+ signals in the stellate cells,6 raise the intriguing possibility that acute pancreatitis may be initiated by simultaneously occurring Ca2+ signaling events in both acinar and stellate cells. It may be important to note that the SARS-CoV-2 spike protein has very recently been shown to elicit Ca2+ signals primarily in the stellate cells. This leads secondarily to Ca2+ signal generation in adjacent macrophages, most likely due to cytokine secretion from the stellate cells.10 Mounting evidence therefore now indicates that the stellate cells play a more direct role in the initiation of acute pancreatitis than originally thought.Although it is now accepted that both stellate cells and macrophages play a crucial role in the development of acute pancreatitis, the actual initiation of the disease process has generally been attributed to the toxic Ca6 Clearly extensions of the work of Kusiak et al. to pathophysiological situations in vivo will be important in order to determine the potential general importance of this new concept.Although activation of TRPA1 is generally accepted to be proinflammatory,


The present study was conducted to investigate the potential effects of dietary supplemented propolis in two growing rabbit breeds on growth performance, immune response, blood parameters, carcass characteristics, and cecal microflora composition.A total of 90 growing rabbits aged 6 weeks from two breeds  were randomly allocated to 3 dietary propolis experimental treatments. The experimental treatments consisted of a 2×3 factorial arrangement with two rabbit breeds and three levels of dietary propolis supplementation . Each sub-treatment has 15 rabbits. The experimental period lasted six weeks.E. coli, and Salmonella spp. was significantly lower  in supplemented rabbit groups compared to the control group according to the microbiological analysis of cecal digesta. Based on breed effect, the results indicated that Jabali rabbits  performed better than V-line rabbits (foreign) in the majority of the studied traits.There were no significant differences in growth performance and carcass characteristics due to propolis administration. Propolis supplementation at a high level significantly increased  cellular-mediated immunity compared with the unsupplemented group. Furthermore, the rabbits receiving propolis exhibited a significant increase  in IgM immunoglobulins compared to the control. The current study provides further evidence that the dietary inclusion of propolis can significantly reduce pathogenic bacterial colonization in growing rabbits. The total count of microflora, Dietary propolis is promising for further investigation into improving intestinal health and enhancing immunity in growing rabbits. Recently, utilizing natural materials in livestock nutrition instead of antibiotics is alternatively being developed. Due to adverse effects of antibiotics on both animal and human health, their use in animal feeding as a growth promoter has been entirely banned by the European Union since 2006. There are many natural feed additives used in animal and poultry feeding, e.g. probiotics, prebiotics, organic acids, phytogenic compounds, and zeolites –4. PropoUtilizing supplemented propolis in livestock production via feed or drinking water to improve productive performance and health status has been investigated ,7,15,16.This experiment was carried out at the experimental rabbit farm at the College of Agriculture and Veterinary Medicine, Qassim University, Saudi Arabia in early spring season of 2019. The experiment started at 6 weeks of age and lasted 6 weeks. During the experimental period, minimum and maximum ambient temperatures were 13°C±0.6°C and 29°C± 0.7°C, respectively (mean±standard error). All experimental procedures, animal care, and handling were performed according to the animal care instructions of scientific research deanship and approved by the committee of health research ethics and animal care, Qassim University, Saudi Arabia.ad libitum to the animals.A total of 90 healthy growing rabbits representing the Saudi local breed  and an imported Spanish rabbit line  were used in the current experiment. Forty-five rabbits aged 6 weeks of each breed were randomly distributed into three dietary propolis treatments . Rabbits were housed individually in wire mesh cages (50 cm×40 cm×40 cm) equipped with feeding hoppers and drinking nipples. All the rabbits were kept under similar housing and management conditions. Propolis was administrated in feed in the concentration of 0, 250 mg/kg (low level), 500 mg/kg (high level). A commercial basal diet for growing rabbits containing 18.5% crude protein, and 9.4 MJ metabolizable energy/kg was used. Feed and water were available All rabbits were weighed at the beginning and end of the experiment. Body weight gain, feed intake, and feed conversion ratio (FCR) were determined for the overall experimental period (42 days). Data analysis was performed based on the individual records.in vivo response induced by injecting a mitogen was evaluated by injecting Phytohemagglutinin (PHA-P) into the left ear. At 12 weeks of age, 60 rabbits were randomly assigned  for cell-mediated immune response. Each animal was injected intradermally with 100 μg PHA-P  in 0.1 mL of sterile saline. Upon injection, the site of the needle was marked with permanent black ink to facilitate further measuring. The resultant swelling response in the ear was measured with a constant tension dial micrometer  before injection and at 24, 48, and 72 h after PHA-P injection. Ear swelling was expressed as the difference between the thickness of the ear before and after injection.The Haemagglutination assay was performed for assessing humoral immune response. Sheep red blood cells (SRBC), as foreign antigens were adopted to determine the total immunoglobulin in the blood. At the end of the eleventh week, the 10 rabbits per sub-group were assigned to challenge SRBC. Each rabbit was injected with 1 mL/kg body weight of 10% SRBC solution. One week later, blood sample was taken for determining antibodies formed against SRBC in the serum using 96-well plates. The antibody levels against SRBC were measured by a hemagglutination test using a 2% SRBC suspension. The serum was heat-inactivated at 56°C for 30 min and analyzed for total, mercaptoethanol (ME)- sensitive (presumably immunoglobulin M [IgM]) and ME-resistant (IgG) anti-SRBC antibodies, as previously mentioned in Fathi et al . The antwww.bio-diagnostic.com, as described by Fathi et al [2O2). The determination was performed by the reaction of antioxidants in the blood sample with a defined amount of exogenously provided H2O2. The antioxidants in the sample eliminate a certain amount of the provided H2O2. The residual of H2O2 is quantified colorimetrically by an enzymatic reaction which involves the conversion of 3,5,dichloro-2-hydroxy benzensulphonate to a colored product. Lipid peroxidation was determined by the quantification of MDA levels. The level of MDA was determined from the MDA equivalence standard. Samples and standards were first reacted with thiobarbituric acid in acidic medium at high temperature (95°C) for 30 min to form a reactive pink product. Optical density was measured using an ultraviolet spectrophotometer at 532 nm against blanks prepared by using distilled water. Glutathione peroxidase was determined in erythrocytes. The red blood cells were collected from blood samples and washed with saline solution three times. Cold deionized water (4°C) was added to lyse cells. The resulting clarified supernatant was used in GSH-Px assay. Enzyme activity (reduction of organic peroxide) was spectrophotometrically monitored by decreasing absorbance at 340 nm.Approximately 5 mL of blood was obtained from each slaughtered rabbit into a heparinized tube. The blood samples were centrifuged  and the resulting plasma was collected and stored at −20°C for further analyses. The concentrations of total protein, albumin, cholesterol, and triglycerides were determined in the plasma using commercial kits . The difference between total protein and albumin concentrations was used to calculate globin concentration. Then, the albumin to globulin ratio was calculated. Total antioxidant capacity (TAC), the activity of malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) activity were determined using commercial kits purchased from Biodiagnostic for diagnostic and research reagents, Dokki, Giza. Egypt, hi et al . Total aAt the end of the experimental period, 10 rabbits from each sub-group  were assigned for carcass evaluation. The rabbits were fasted for 12 h with free access to clean drinking water, and then sacrificed in the morning and weighed. After bleeding, they were dissected according to Blasco et al  and FathE. coli and Salmonella spp. were analysed in the cecal digesta according to the procedures described by McDonald et al [E. coli. Each dilution was cultured on selective media for each bacterial strain to be counted or detected. Nutrient agar was used for the total aerobic bacteria count, and MacConkey agar was used for the E. coli count. The culture plates for total aerobic bacteria and E. coli were incubated at 37°C in an aerobic environment. The colony-forming units (cfu) in log10 per gram for total aerobic bacteria and E. coli within digesta were counted based on the colony morphology and characteristics. For the detection of Salmonella spp., a sample (25 g) of cecal contents was pre-enriched in 225 mL of peptone water and incubated at 37°C for 16 to 24 h. For selective enrichment, 1 mL of peptone broth was transferred to 9 mL tetrathionate broth and incubated at 42°C for 24 h. From each selective enrichment broth, a 5-mm loop was streaked on selective plates of bismuth sulfite agar and incubated at 37°C for 24 h. Salmonella spp. was expressed as a percentage of detection.Cecal contents of the same slaughtered rabbits were collected. The contents were carefully hand-stripped into sterile containers. Total aerobic bacteria, ld et al  and Hornld et al . A samplTwo-way analysis of variance was performed using JMP software version 13.0  with breijk = the observation taken on the kth individual; μ = overall mean; Pi = the fixed effect of the jth propolis supplementation level; Bj = the fixed effect of the ith breed; (PB)ij = interaction between breed and propolis supplementation level; eijk = random error assumed to be independent normally distributed with mean = 0 and variance = σ2.where: YAll results were presented as mean, and the variability in the data was expressed as pooled standard error of the mean. The significance of difference among the groups was assessed using Tukey’s test. Statistical significance was considered when p<0.05. Polynomial contrasts and linearity were examined using regression procedures to describe the shape of the response to increasing concentrations of propolis supplementation and to determine the model of best fit, either linear or quadratic. The responses in optimal parameters to the propolis supplementation level can be modeled using the following quadratic equation:Where: Y = optimal response; a = intercept; b = coefficients of the quadratic equation; X = propolis level, and e = error.As shown in The results of the blood parameters and antioxidant status are summarized in As shown in E. coli of the caecum. Moreover, the results showed that feeding rabbits with diets supplemented with increasing levels of propolis linearly and quadratically (p<0.01) reduced the Salmonella spp. population in caecum content. Regarding the breed factor, there were no significant differences in caecum morphology (weight and length) due to rabbit breed. However, a significant decrease (p<0.01) in the relative weight of the caecum was found in the Jabali breed. Interestingly, the Jabali rabbits recorded significantly lower (p<0.01) bacterial count parameters compared with the V-line rabbits. Furthermore, the interaction between propolis level and breed type was highly significant (p<0.05 or p<0.01) for all bacterial count measurements.The results of morphological description and microbial counts of caecum as affected by the addition of different levels of propolis in the diets of rabbit breeds are presented in Propolis supplementation is one of the most important growth promoters used in farm animals and immuno-enhancer for human health. Based on the statistical analysis, there were no significant differences in either growth performance or feed efficiency due to propolis supplementation. Although the body weight gain was not statistically different among treatment groups, we found a numerically increase (4% and 8%) in groups receiving 250 mg/kg and 500 mg/kg propolis, respectively compared to those in the control group. This result is in harmony with the findings of Piza et al , who fouPasteurella multocida vaccine, enhanced specific and nonspecific immune response and reduced mortality rate in rabbits [The results of cell mediated immune response revealed that propolis administration at a high level significantly increased the response to PHA-P injection in a linear manner compared to the other groups. The response of rabbits receiving a low level was intermediate. Several studies have shown that propolis supplementation activates the immune system in different animal species ,29,30. A rabbits . In broi rabbits ,38. In a rabbits . In lymp rabbits . They fo rabbits . The pos rabbits  reportedThe results of blood biochemical analysis showed that propolis administration did not induce significant alterations in parameters. However, a numerical increase in globulin level (14% and 18%) was recorded in rabbits fed a diet supplemented with low or high levels of propolis, respectively compared to the control group. A significantly increase in blood globulin was found in rabbits fed a diet supplemented with 300 mg propolis as compared to the unsupplemented group . A similThe dressing percentage and carcass parts, as an important economic issue for the rabbit meat industry, showed no difference due to propolis supplementation compared to the control group. Studies involving the use of propolis as a feed additive have shown consistent results in terms of carcass quality. However, the effects of dietary propolis supplementation on carcass yield of fattening rabbit were insignificant ,15,24. AE. coli, and Salmonella spp. was clearly observed. A significant reduction was recorded in all bacterial population parameters, and that effect was linearly dose-dependent. Moreover, Salmonella spp. does not exist in rabbits fed a diet containing a high level of propolis. Propolis has been reported to have antibacterial activity against a wide range of pathogens. An improvement in health status was noticed in rabbits suffering from diarrhea symptoms after supplying the drinking water with propolis for 10 days [Clostridium spp, Staphylococcus aureus and bacteriosides spp. [Similar to the effect of dietary propolis on caecum morphology in our study, Coloni et al  showed t 10 days . All pro 10 days . Many st 10 days –47. In a 10 days . The pre 10 days –51. The  10 days ,53. Prop 10 days ,54. Besi 10 days . Caffeic 10 days . Phenolides spp. . RegardiEscherichia coli and Salmonella spp. in cecum of treated rabbits. The improvement in cecal microbiota was pronounced in Jabali rabbits compared V-line counterparts. Propolis supplementation can greatly improve the immune function of growing rabbits via the beneficial modulation of cecal microflora. Meanwhile, the growth performance and carcass quality remained unaffected by the dietary inclusion of propolis. Although, propolis is suggested as an effective alternative feed additive in rabbit feeding to enhance immunity and health status, further research is recommended to examine high inclusion rates of propolis in rabbit diets for improving growth performance as well.The results revealed that the addition of propolis to rabbits’ diets has a beneficial effect in reducing the colonization of


Ca2+/CaM is predicted to bind to a domain in the C-terminal portion of the intracellular loop (CL2) in the vast majority of Cx isoforms and for a number of Cx-s this prediction has proved correct. In this study, we investigate and characterise both Ca2+/CaM and apo-CaM binding to selected representatives of each of the α, β and γ connexin family to develop a better mechanistic understanding of CaM effects on gap junction function. The affinity and kinetics Ca2+/CaM and apo-CaM interactions of CL2 peptides of β-Cx32, γ-Cx35, α-Cx43, α-Cx45 and α-Cx57 were investigated. All five Cx CL2 peptides were found to have high affinity for Ca2+/CaM with dissociation constants (Kd(+Ca)) from 20 to 150 nM. The limiting rate of binding and the rates of dissociation covered a broad range. In addition, we obtained evidence for high affinity Ca2+-independent interaction of all five peptides with CaM, consistent with CaM remaining anchored to gap junctions in resting cells. However, for the α-Cx45 and α-Cx57 CL2 peptides, Ca2+-dependent association at resting [Ca2+] of 50–100 nM is indicated in these complexes as one of the CaM Ca2+ binding sites displays high affinity with Kd of 70 and 30 nM for Ca2+, respectively. Furthermore, complex conformational changes were observed in peptide-apo-CaM complexes with the structure of CaM compacted or stretched by the peptide in a concentration dependent manner suggesting that the CL2 domain may undergo helix-to-coil transition and/or forms bundles, which may be relevant in the hexameric gap junction. We demonstrate inhibition of gap junction permeability by Ca2+/CaM in a dose dependent manner, further cementing Ca2+/CaM as a regulator of gap junction function. The motion of a stretched CaM–CL2 complex compacting upon Ca2+ binding may bring about the Ca2+/CaM block of the gap junction pore by a push and pull action on the CL2 C-terminal hydrophobic residues of transmembrane domain 3 (TM3) in and out of the membrane.Ca Gap junction permeability is a critical factor for the precision of intercellular communications, such as the wave-like flux of second messengers and synchronicity of cell response in cardiac muscle. Voltage and pH are well-characterised regulators of gap junction permeability. In addition, gap junction function is modulated by interaction with a broad range of proteins  value of ~550 nM at half-maximal fluorescence change (defined as Kd for Ca2+)   A, while μM range . Mixture binding B–F. The lues (n) . TA-CaM 2+ dependency curve to Kd-s of 311 nM, 392 nM and 340 nM, respectively, indicating Ca2+-dependent binding in which Ca2+ binding to CaM strengthens peptide binding and vice versa. The high affinity of peptide binding—but an only mildly increased affinity for Ca2+ of the C-lobe (2+ binding is significantly increased in affinity by peptide binding. The lack of a change in TA-CaM fluorescence at resting [Ca2+] of ~50 nM taken together with evidence of binding in the absence of Ca2+ (see above) indicates Ca2+-independent apo-CaM binding to Cx32 and Cx35 and Cx43 CL2 in resting cells , CaM anchoring to the Cx45 CL2 in a gap junction would be based on CaM with a very tightly bond Ca2+ rather than apo-CaM. The higher Kd2 of 597 nM indicates N-lobe binding upon Ca2+ stimulation  and 18-β-glycyrrhetinic acid , to confirm that changes in gap-junctional coupling could be detected and quantified. 2+-dependent closure of Cx43 GJs in cells exposed to the Ca2+ ionophore ionomycin [2+]i that declined to resting levels in the absence of external Ca2+ (n = 11 cells) or to a lower but maintained plateau elevation in the presence of external Ca2+ (n = 9 cells)  A. Pre-trormation B,C. The 2+-dependent GJ uncoupling observed in HUVEC following ionomycin treatment is mediated by CaM, we incubated cells with the membrane permeant CaM inhibitory peptide mTrp (7–17.6 µM) or an inactive control peptide at the same concentrations . In control, peptide treated cells ionomycin pre-treatment significantly reduced LY spread following scratch wounding (p < 0.0001). However, treatment with mTrp reduced the inhibition of LY spread compared to the control peptide in a dose-dependent fashion .To determine whether the Catrations . PeptideKd in low 10 s of nM) Ca2+/CaM binding to the Cx CL2 peptides of Cx32, Cx35, Cx43, Cx45 and Cx57. In structural terms, our data show partial collapse of Ca2+/CaM around the peptides. As illustrated in 2+/CaM binds to target peptide domains by embracing them with the two Ca2+ binding lobes, the connecting flexible loop can adjust to different distances depending on where the residues of strong hydrophobic interaction are in binding motif sequence. Our data show complete collapse of Ca2+/CaM when binding to the smooth muscle (sm) MLCK derived Trp and mTrp peptide, in agreement with NMR and crystal structures  levels ~50 nM, we conclude that no such exposure occurs which, taken together with evidence from the kinetic studies of apo-CaM binding, is consistent with Ca2+-independent CaM binding in resting cells, likely by the C-lobe. This scenario applies to Cx32, Cx35 and Cx43.TA-CaM fluorescence increases in response to Ca2+ site, with apparent Kd-s of 75 and 32 nM, respectively  that is present in almost all connexins  in HUVEC that expresses a number of connexin isoforms, some of which only have CL2 as calmodulin binding domain. Furthermore, we demonstrated the concentration and time dependence of inhibition, thereby establishing an assay that can be used in the future—for example, for comparing the ability of endogenous calmodulin variants to block gap junction coupling. Previous studies have shown that CaM causes a Caonomycin ,15. Hereu et al.  that Ca22+-dependent and -independent binding between Cx CL2 peptides and CaM appear to be of high affinity, albeit via very different molecular interactions. We propose a model in which the C-terminal, highly hydrophobic section of the CL2 domain of the intracellular loop remains buried in the membrane in resting cells, with the CaM C-terminal domain anchored to the N terminal part of the CL2 domain. Ca2+ binding to CaM allows the N-lobe to capture the C-terminal peptide binding site by pulling a stretch of TM3 residues out of the membrane and thereby allowing blocking of the gap junction pore. Ca2+ dissociation releases the TM3 hydrophobic stretch of CL2 which slides back into the membrane, resulting in the gap junction pore opening.In conclusion, both CaPeptides were obtained from Genosphore Biotechnologies, Boulogne-Billancourt, France. They had >95% purity and their sizes were analysed by mass spectrometry. The amino acid sequences of the peptides are: Cx32 CL2 peptide: KRHKVHISGTLWWTYVISVV; Cx32 CT peptide: EINKLLSEQDGSLKDILRRS ; Cx35 CLε0(280nm) = 12,660 M−1cm−1; for Cx35, Cx43, Cx45, and Cx57 CL2 peptides, ε0(278nm) = 1280 M−1cm−1 and for mTrp peptide, ε0(280nm) = 5700 M−1cm−1. Myr-Ctrl peptide and Cx32 CT peptide concentrations were determined by weight.Peptide concentrations were determined spectroscopically using extinction coefficients calculated by Protein calculator v3.4: for Cx32 CL2 peptide, E. coli, purified and double-labelled DA-CaM  and Bmax represents the maximal signal (max fraction bound = 1), using GraphPad Prism 9.The titrations were performed using a Flurolog-3 fluorimeter . The sample was excited at 365 nm and the fluorescence emission was recorded at 415 nm. TA-CaM (10 nM) in 50 mM HEPES-K2+/DA-CaM and DA-CaM interactions with Cx CL2 peptides was measured using a TgK Scientific KinetAsyst™ double-mixing stopped-flow apparatus  in single mixing mode. The fluorescence was excited at 340 nm and the emission was detected by a photomultiplier tube with a long pass filter (>400 nm). For Ca2+-dependent association, 500 nM DA-CaM and different concentrations of a Cx CL2 or mTrp peptide were rapidly mixed in assay buffer and 1 mM CaCl2. For Ca2+-dependent dissociation, pre-mixed 500 nM DA-CaM with Cx CL2 peptide at saturating concentration  or 0.5 µM mTrp peptide in assay buffer and 1 mM CaCl2 was rapidly mixed with 20 µM CaM. For Ca2+-independent association, 500 nM DA-CaM in assay buffer containing 5 mM EGTA at 20 °C was rapidly mixed with different concentrations of a Cx CL2 or mTrp peptide. For Ca2+-independent association in the presence of trifluoroethanol (TFE), 500 nM DA-CaM in assay buffer containing 5 mM EGTA and 15% TFE was rapidly mixed with 5 µM or 17.5 µM Cx43 and 5 µM or 30 µM Cx57. For Ca2+-independent dissociation, 500 nM DA-CaM with Cx CL2 peptide  or 10 µM mTrp peptide in assay buffer containing 5 mM EGTA was rapidly mixed with 300 µM CaM. All concentrations given are those in the mixing chamber. Data shown are averages of 3 to 5 repeats. All experiments were carried out at 20 °C. Data were analysed using Kinetic Studio software . The kinetics of CaKd, the [Ca2+] concentration at which half-maximum fluorescence intensity is achieved) of TA-CaM in the presence of the different Cx CL2 peptides was determined by following the change in fluorescence emission . The mixture of 10 nM TA-CaM and 600 nM Cx32, 450 nM Cx35, 250 nM Cx43, 1.1 µM Cx45 or 900 nM Cx57 CL2 and 30 nM mTrp peptide in assay buffer and 5 mM EGTA were titrated with 325 mM Ca2+ in assay buffer using a syringe pump (10 µL/min) while constantly stirring. The fluorescence was corrected for dilution and [Ca2+] concentration was calculated using Maxchelator software. The data were fitted to the Hill equation with specific binding (y = Bmax∗xn/(Kdn + xn)) using GraphPad Prism 9.The apparent dissociation constant (w/v) for 30 min at 37 °C. Cells were cultured for 4 days in a 5% CO2 incubator at 37 °C until fully confluent as assessed by visual examination by light microscopy.Cryopreserved pooled primary HUVEC were purchased from Lonza . HUVEC were grown in a human growth medium (HGM) which comprised media M199 supplemented with 20% FCS. The antibiotic gentamicin (50 µg/mL) along with ECGS  and heparin (10 U/mL) were added to the media. HUVEC were grown on 24 well trays or 9 mm glass coverslips that had been pre-treated with porcine skin gelatine (1% 4Cl and incubated at room temperature (RT) for 15 min. The NH4Cl solution was then removed and replaced with PBS, supplemented with the detergent TX-100 (30 µL/10 mL) for 5 min at RT. The coverslips were then washed into a phosphate gelatine and saponin solution (PGAS) that contained (w/v): 0.2% gelatin, 0.02% saponin and 0.02% NaN3 in PBS and stored at 4 °C until processed for immunocytochemistry. For immunolabelling, a 40 μL droplet of PGAS solution containing single or multiple primary antibodies at the dilutions described above was placed on a sheet of parafilm within a humidified chamber and a 9 mm coverslip of fixed and permeabilised HUVEC placed cell side down on the droplet for 60 min at RT. The coverslips were then transferred to a well of a 24-well plate and washed 3 times with PGAS before being removed and placed cell-side down on a 40 µL droplet of PGAS containing species appropriate secondary antibodies coupled to FITC, RedX or Cy5 . Coverslips were then incubated for 60 min at RT in the dark before being washed 3 times in PGAS, followed by 3 washes in PBS and then mounted cell-side down in a 5 µL droplet of Mowiol-488 on a glass microscope slide. Mounted coverslips were kept in the dark at RT for 24 h before viewing on a confocal microscope. For analysis of Cx43, VE-cadherin localisation and expression in HUVEC, we used a pre-validated mouse anti-Cx43 monoclonal antibody directed to a C-terminal epitope  at a 1:25 dilution and a rabbit polyclonal anti-VE-cadherin antibody  at 1:400 dilution. HUVEC grown on 9 mm porcine gelatine coated coverslips were first washed carefully with phosphate buffered saline (PBS) and then placed into a solution of 3% paraformaldehyde (PFA) in PBS for 15 min. The coverslips were then washed into PBS supplemented with 50 mM NHFixed and immune-labelled HUVEC were imaged by confocal microscopy ) using a 60× 1.4 NA oil immersion objective. Light for FITC, RedX and Cy5 excitation were 488 nm (50 mW Kr/Ar laser), 542 nm (green laser diode) and 637 nm (5 mW red laser diode), respectively. Sequential image capture was used for multi-labelled samples and images were acquired using the same laser settings at each wavelength using a Kalman filter (4 frames). Images (1024 × 1024 pixels) were acquired at zooms 1.4 or 2.6. Data was recorded in BioRad pic format and converted to tif format in ImageJ. Figures were made in Photoshop CC (Adobe).2, 2 mM MgCl2, 10 mM glucose, 10 mM HEPES-NaOH pH 7.4) supplemented with 2.5 µM Fura-2AM  diluted from a stock solution of 5 mM Fura-2AM in DMSO with 2.0% Pluoronic F-127. Cells were incubated at room temperature in the dark for 20 min before being transferred into fresh Hanks solution and further maintained in the dark at RT until use. Cells were transferred to the stage of an Olympus IX71 inverted fluorescence microscope and maintained at 32–34 °C. The imaging system comprised an excitation monochromator , U-Plan-Apo ×100 1.35 NA objective and an Ixon EMCCD camera (Andor). Fura-2 was illuminated sequentially with 355 ± 5 nm and 380 ± 5 nm light and wavelength switching synchronised with image capture using WinFluor software . The EMCCD camera was operated in frame transfer mode at full gain and cooled to −70 °C. Full frame (512 by 512 pixels) images were acquired at 30 frames/s. Ionomycin was applied by droplet application.HUVEC were grown on 24 mm square uncoated glass coverslips in growth medium. For Fura-2 loading, the cells were transferred into Hanks solution  using an EVOS M5000 microscope  equipped with a 10 × 0.3 NA air objective and EVOS LED light cubes for bright field imaging, FITC, RedX and DAPI. Images were acquired sequentially and exported to ImageJ for processing. 2+ dependency of gap junction coupling, HUVEC were incubated in either +Ca+Mg-PBS (+Ca) or -Ca+Mg +0.2 mM EGTA (-Ca) containing PBS and stimulated with vehicle (0.001% DMSO), or 2 μM ionomycin for 8 min. Cells were then transferred to dye transfer buffer made using +Ca or −Ca PBS, supplemented with vehicle or 2 μM ionomycin, and 3 parallel cuts were made in cell monolayers in each well. Cells were incubated for a further 8 min prior to fixation. The cell permeant CaM inhibitory peptide, mTrp, and the control peptide mCtrl are described in [Phorbol 12-myristate 13-acetate (PMA) or 18-β-glycyrrhetinic acid (18BGRR) was made up in dimethysulphoxide (DMSO) to a 10 mM or 50 mM stock, respectively, and diluted into HGM to a final concentration of 100 nM or 100 µM, respectively. Cells were incubated in PMA or 18BGRR acid containing solution for 60 min at 37 °C. The PMA or 18BGRR solution was then carefully removed and the cells were washed once in +Ca+Mg-PBS. The PBS was then replaced with the dye transfer buffer (see above) and immediately 3 parallel cuts were made in the cell monolayer in the central part of each well using a micro scalpel knife with a flat edge stainless steel blade. For each experiment drug treatments were carried out in triplicate wells each with 3 cuts per well. Cells were then processed as described above. For analysis of the Caribed in . A stockData analysis was carried out in the open-source-based software program Fiji ImageJ. A custom-made macro  published in the t-tests, as appropriate in GraphPad Prism.Data are presented as mean ± s.e.m., unless otherwise stated. Statistical analysis of the effect of gap junction blockers and calmodulin inhibitory peptides on dye transfer, cell viability or cell count was carried out using ANOVA or


This is particularly remarkable since Ca2+ acts as a central modulator in various signaling pathways, including, but not limited to, cell differentiation and survival. Despite this, the mechanisms underlying this modulation have yet to be unraveled. Here, we assessed the effect of EMFs on intracellular [Ca2+], by exposing HEK 293 cells to both radio‐frequency electromagnetic fields (RF‐EMFs) and static magnetic fields (SMFs). We detected a constant and significant increase in [Ca2+] subsequent to exposure to both types of fields. Strikingly, the increase was nulled by administration of 10 μM Thapsigargin, a blocker of sarco/endoplasmic reticulum Ca2+‐ATPases (SERCAs), indicating the involvement of the endoplasmic reticulum (ER) in EMF‐related modulation of Ca2+ homeostasis.Biological effects of electromagnetic fields (EMFs) have previously been identified for cellular proliferation and changes in expression and conduction of diverse types of ion channels. The major effect elicited by EMFs seems to be directed toward Ca Each well was checked for full confluence before the experiments.The human embryonic kidney cell line HEK 293  was cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) , supplemented with 10% heat inactivated fetal bovine serum , 1% Penicillin/Streptomycin  and 1% L‐Glutamine  stock solutions. On the day prior to each experiment cells (confluence 60–70%) were divided and seeded in 96‐well plates  on a volume of 100 µl/well. For some experiments, cells were directly seeded in Standard Extracellular Physiological Saline . Cells were seeded in two 96‐wells microplates  on the day prior to the experiment to obtain full confluence on experiment day. On experiment day, culture media was removed, and each plate washed with Standard Extracellular Physiological Saline  to remove any trace of dead cells or culture media. Both plates were then incubated for 1 h  in Standard Extracellular Physiological Saline containing 1 μM Fluo‐4AM , 50 μl/well. After incubation time, plates were washed again and resuspended in 50 μl of Standard Extracellular Physiological Saline. A first measurement of intracellular [Ca2+] was acquired using a Clariostar Plate reader , pre‐adjusted for gain and focus level, and used as baseline. Plates then underwent exposure protocols (details in the below section). Fluorescence intensity reflecting intracellular [Ca2+] was measured at T1 (15 min) and T2 (30 min) . To evaluate minimum responses, 10 µM of EGTA  was also added to dye solution in some experiments. To interfere with intracellular and extracellular Ca2+ dynamics various compounds were also used and added to the dye incubation solution ; to block SERCAs on the ER, 10 μM Thapsigargin ; to block RyRs, 10 μM Dantrolene . Data were recorded as individual well scans.To assess the origin of the detected increase, some experiments were performed in Ca2.32+] evaluated every 15 min. For maximum radiation, the EMF source was placed directly on the top of plate's lid  was used as a source of radiation Figure . The mobd Figure . The Sped Figure  powered 2.4® software version 6 for Windows (p < 0.05). Error bars represent standard error of the mean (SEM). T‐tests were used to determine whether a difference is seen between the means of two unrelated groups of data. The “n” value reported refer to microplate wells. For each condition 3 independent experiments were carried out.Statistical analysis was performed using Graph Pad PrismAll experimental procedures were approved by and adhered to the guidelines of ethical committee of University of Surrey, Guildford, UK.33.1To compare the impact of the electric and the magnetic component of the exposure, the effect of different frequency fields was assessed. Specifically, the exposures consisted of oscillating radiofrequency fields (RF‐EMFs) and static magnetic field (SMFs), constant in time and direction, and thus, with a 0 Hz frequency. For the first we choose to use a mobile phone, closely resembling the exposure deriving from smartphones and wireless devices usage. For the second, a static magnetic field generator consisting of two Helmholtz coils connected to a DC power supply. As we were interested in the rapid effects of EMFs, we focused our investigations on early timepoints (15 and 30 min).2+], was observed as result of both types of exposures. In the RF‐EMF exposed cohort, this was significantly different at T1 (15 min) and T2 (30 min)  when compared to sham. Additionally, in this group when compared between T1 and T2, the increase in intracellular Ca2+ was significantly increased  in T2 when compared to T1 suggesting a time dependent effect on intracellular increases albeit acute.A rise in dye fluorescence, reflecting an increase in intracellular , we decided to focus our investigation on this type of fields. In order to characterize the origin of the detected increase, the RF‐EMF experiments were repeated in Ca2+‐free solutions, aiming to evaluate the involvement of intracellular Ca2+ stores. We observed no significant difference between Ca2+‐free and standard physiological solution at both T1 and T2. This suggested an intracellular origin for the RF‐EMF‐elicited increase in Ca2+.As, contrary to SMFs, acute exposure to RF‐EMFs is not commonly reported to increase cytosolic  , confirming the presence of nifedipine‐sensitive channels on the membrane of HEK 293 cells , suggesting the involvement of these channels in the modulation of the increase triggered by EMFs exposure . Hence, we assessed the impact of Dantrolene (10 μM), a commonly used antagonist of RyRs. Dantrolene treated group displayed no difference when compared to the control, both in the sham . These transporters are responsible for the replenishment of ER stores by consequence of the release of Ca2+ in the cytoplasm. To inhibit the activity of SERCAs, cells were treated with 10 μM Thapsigargin, a commonly used SERCA blocker. An initial intracellular Ca2+ increase was detected , in line with the blockage of ER replenishment as with the activation of non‐selective Ca2+‐permeable cation channels triggered by depletion of intracellular stores. Strikingly, in this group the increase at both T1 and T2 exposure was minimal, and no significant difference was observed between the two timepoints. Moreover, the relative increase was significantly different  to that of control and dantrolene‐treated group, both at T1 and T2   that, along with mitochondria, is primarily involved in the control of cytoplasmic [Ca2+ in the external solution, the intracellular Ca2+ was mostly abolished, suggesting a direct involvement of the ER release in the detected Ca2+ raise. Moreover, no difference was observed between sham and exposed group, indicating a direct involvement of the ER internal stores in the EMFs modulation. These results are in line with both the involvement of ER in the intracellular Ca2+ increase detected and with the involvement of a membrane independent mechanism, modulating EMF effects, as already observed as consequence of exposure to extremely low‐frequency fields in entorhinal cortex neurons . Interestingly, altered oxidative stress has been linked to both ER abnormal function and different fields exposure  exposure to different frequencies and intensities of EMFs, but different, and sometimes opposite, effects are reported for chronic exposure. This controversy could be maybe resolved by considering the time‐dependent cellular adaptation to perturbances of Ca2+ homeostasis, that could involve both fast and slow regulations, as different (positive and negative) feedback loops.It is important to stress the early timepoints on which we focused this study. The rapid effects observed are in line with a broad literature reporting basal Ca2+ internal stores in EMF‐elicited alteration of Ca2+ homeostasis is still missing, as diverse mechanisms involving different cellular compartments could have a different impact depending on the tissue. Nonetheless, this research contributes to the understanding of the cellular effect elicited by EMF on internal Ca2+ dynamics of HEK 293 cells and sheds a light on the key involvement of the ER in this modulation.A consensus on the specific contribution of CaThe authors declare no competing interests.F.B., K.J., R.L., J.M., and S.R.P.S. designed the research. F.B., K.J., and R.L. analyzed the data. F.B. performed experiments. F.B., K.J., and R.L. wrote the paper. All authors approved the final manuscript.


Adaptor molecules play a crucial role in signal transduction in immune cells. Several adaptor molecules, such as the linker for the activation of T cells (LAT) and SH2 domain-containing leukocyte protein of 76 kDa (SLP-76), are essential for T cell development and activation following T cell receptor (TCR) aggregation, suggesting that adaptor molecules are good therapeutic targets for T cell-mediated immune disorders, such as autoimmune diseases and allergies. Signal-transducing adaptor protein (STAP)-2 is a member of the STAP family of adaptor proteins. STAP-2 functions as a scaffold for various intracellular proteins, including BRK, signal transducer, and activator of transcription (STAT)3, STAT5, and myeloid differentiation primary response protein (MyD88). In T cells, STAP-2 is involved in stromal cell-derived factor (SDF)-1α-induced migration, integrin-dependent cell adhesion, and Fas-mediated apoptosis. We previously reported the critical function of STAP-2 in TCR-mediated T cell activation and T cell-mediated autoimmune diseases. Here, we review how STAP-2 affects the pathogenesis of T cell-mediated inflammation and immune diseases in order to develop novel STAP-2-targeting therapeutic strategies. T cells are essential players in acquired immune responses. Antigen-presenting cells, such as dendritic cells and macrophages, phagocytose pathogens and digest them into peptides, which are then presented to the major histocompatibility complex (MHC) for recognition by T cells. The T cell receptor (TCR) aggregates, and the TCR signal cascade is activated. T cell activation after TCR aggregation is initiated by the lymphocyte-specific protein tyrosine kinase (LCK)-mediated phosphorylation of the TCR-associated CD3 complex and ζ chain. Zeta-chain-associated protein kinase 70 (ZAP-70) is recruited to phosphorylated CD3 molecules, resulting in the phosphorylation of the linker for the activation of T cells (LAT) ,2,3,4,5.T cells express several adaptor proteins, such as LAT, SLP-76, VAV, and Grb2. Following ZAP-70 phosphorylation by LCK, activated ZAP-70 phosphorylates the LAT through the LCK-mediated bridging of ZAP-70 to the LAT . ZAP-70 The signal-transducing adaptor protein (STAP) family of proteins consists of two members: STAP-1 and STAP-2 . Both STHuman STAP-2 was originally identified as BKS, i.e., a substrate of BRK tyrosine kinase . We idenIn addition, STAP-2 has four putative tyrosine residues that are phosphorylated by several kinases . The tyrT cells are essential for acquired immune responses to eliminate pathogens such as viruses and cancers. We previously reported that STAP-2 is crucial for certain T cell activation events. STAP-2 deficiency results in the reduction of SDF-1α-induced T cell migration, whereas Jurkat cells overexpressing STAP-2 show increased cell migration toward SDF-1α . MechaniIntegrin signaling is required for many cellular processes, such as cell adhesion, cell migration, proliferation, differentiation, and metastasis . One of STAP-2 is also involved in T cell survival , which iSTAP-2, in addition to its contributions to non-TCR-mediated T cell function, also enables TCR-mediated T cell activation . STAP-2 The Toll-like receptor (TLR) family is an important pattern recognition receptor for the elimination of pathogens, such as bacteria and viruses . In certPropionibacterium acnes (P. acnes)-induced granuloma formation is an infection model in which mice show splenomegaly and granuloma formation in the liver in response to heat-killed P. acnes in a Th17 response-dependent manner [P. acnes administration than WT mice. The granuloma area was significantly reduced in STAP-2 KO mice than in WT mice after the P. acnes injection. In contrast, lymphocyte-specific STAP-2 transgenic (Tg) mice showed severe splenomegaly and granuloma formation after the P. acnes treatment compared with WT mice [t manner ,44. STAP WT mice . TherefoIn autoimmune diseases, the immune system attacks the body’s own organs and destroys healthy tissues. The dysregulation of T cells appears to be a cause of certain autoimmune diseases, such as multiple sclerosis. Using experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, the Th17 response was proposed to be necessary for the pathogenesis of EAE . The Th1Understanding the mechanisms underlying leukemia and solid tumors is necessary for the development of new therapeutic strategies for patients. One of the recent innovative therapies is the use of immune checkpoint inhibitors, such as ipilimumab (anti-CTLA-4 mAb) and nivolumab (anti-PD-1 mAb). Although these new therapeutic medicines constitute a significant breakthrough, the response rate of patients with solid tumors is approximately 30%, indicating that other targets need to be identified to cure patients with malignancies. In this regard, STAP-2 may be a good target for treating patients with certain types of leukemia and solid tumors.BCR–ABL is a chimeric protein derived from the Philadelphia chromosome. More than 90% of patients with CML have Philadelphia chromosome abnormalities and BCR–ABL expression, which causes the onset of CML. STAP-2 binds to BCR–ABL through its SH2-like domain . STAP-2-STAP-2 also participates as a scaffold protein in the EGF receptor (EGFR) signaling cascade . STAP-2 + T cells [+ T cell generation, resulting in the promotion of tumor antigen-reactive memory CD8+ T cell development, although the detailed mechanism remains to be elucidated. Thus, STAP-2 is likely to be a suitable target for the development of novel therapeutic drugs for patients with certain types of cancer, specifically leukemia.STAP-2 is also involved in the growth of breast cancer cells through its interaction with Brk and STAT3 . During  T cells . STAP-2 As described here, STAP-2 interacts with a variety of key molecules involved in cell signaling and transcription. The manipulation of STAP-2 expression was shown to contribute to inflammatory and immune diseases and some malignancies critically. Hence, we hope that a novel STAP-2-targeting drug is developed in the future.


To trigger exocytosis, Ca2+ ions enter the cytosol from intracellular Ca2+ stores or from the extracellular space. The molecular events of late stages of exocytosis, and their dependence on [Ca2+]c, were extensively described in isolated single cells from various endocrine glands. Notably, less work has been done on endocrine cells in situ to address the heterogeneity of [Ca2+]c events contributing to a collective functional response of a gland. For this, β cell collectives in a pancreatic islet are particularly well suited as they are the smallest, experimentally manageable functional unit, where [Ca2+]c dynamics can be simultaneously assessed on both cellular and collective level. Here, we measured [Ca2+]c transients across all relevant timescales, from a subsecond to a minute time range, using high-resolution imaging with a low-affinity Ca2+ sensor. We quantified the recordings with a novel computational framework for automatic image segmentation and [Ca2+]c event identification. Our results demonstrate that under physiological conditions the duration of [Ca2+]c events is variable, and segregated into three reproducible modes, subsecond, second, and tens of seconds time range, and are a result of a progressive temporal summation of the shortest events. Using pharmacological tools we show that activation of intracellular Ca2+ receptors is both sufficient and necessary for glucose-dependent [Ca2+]c oscillations in β cell collectives, and that a subset of [Ca2+]c events could be triggered even in the absence of Ca2+ influx across the plasma membrane. In aggregate, our experimental and analytical platform was able to readily address the involvement of intracellular Ca2+ receptors in shaping the heterogeneity of [Ca2+]c responses in collectives of endocrine cells in situ.The release of peptide hormones is predominantly regulated by a transient increase in cytosolic CaNEW & NOTEWORTHY Physiological glucose or ryanodine stimulation of β cell collectives generates a large number of [Ca2+]c events, which can be rapidly assessed with our newly developed automatic image segmentation and [Ca2+]c event identification pipeline. The event durations segregate into three reproducible modes produced by a progressive temporal summation. Using pharmacological tools, we show that activation of ryanodine intracellular Ca2+ receptors is both sufficient and necessary for glucose-dependent [Ca2+]c oscillations in β cell collectives. Resulting elevation in [Ca2+]c ultimately produces a complex spatio-temporal pattern of [Ca2+]c events, membrane potential changes, and exocytosis of insulin -trisphosphate (IP3) receptors (IP3R) and the ryanodine receptor (RYR), contribute to Ca2+-dependent insulin release in β cells (2+ channels (VACCs). RYRs are primarily located on endoplasmic reticulum (ER) membranes and can be activated by Ca2+ ions, ATP, and modulated by PKA  phenomena in a broader sense could occur as cluster firing of fields of RYRs (or IP3Rs)  . And foull types . Furtherll types . Moreoveolerance –21.2+ release to [Ca2+]c changes requires a much higher temporally resolved imaging. Fortunately, the advancement in the synthesis of novel fluorescent Ca2+-sensing markers  and the administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection (No: U34401-12/2015/3) approved the experimental protocol.2 and cervical dislocation, we accessed the abdominal cavity via laparotomy and distally clamped the common bile duct at the major duodenal papilla. Proximally, we injected the low-melting-point 1.9% agarose (Lonza) dissolved in extracellular solution  at 40°C into the common bile duct. Immediately after injection, we cooled the agarose-infused pancreas with ice-cold ECS and extracted it. We prepared tissue slices with a thickness of 140 µm with a vibratome  and collected them in HEPES-buffered ECS at RT . For staining, we incubated the slices for 50 min at RT in the dye-loading solution , 0.03% Pluronic F-127 (wt/vol), and 0.12% dimethylsulfoxide (vol/vol) dissolved in HEPES-buffered ECS. The Ca2+ fluorescent dyes from a Calbryte series are highly fluorescent and photostable, allowing long-term recording even in deeper layers of the tissue slice. The linear part of the Ca2+-binding curve for Calbryte 520 (KD of 1.2 µM) captures the [Ca2+]c changes in β cells better than the high-affinity sensors we routinely used in our imaging experiments before. All chemicals were obtained from Sigma-Aldrich  unless otherwise specified.C57BL/6J mice, 8–20 wk of age, and of either sex (Jackson Laboratories), were kept on a 12:12-h light:dark schedule in individually ventilated cages (Allentown LLC) and used to prepare pancreatic tissue slices, as described previously . In brie2 to allow for a precise quantification of [Ca2+]c oscillations. Calbryte 520 was excited by a 488 nm argon laser (Leica SP5 and Nikon A1R) or 490 nm line of a white laser (Leica SP8). The emitted fluorescence was detected by HyD hybrid detector in the range of 500–700 nm using a photon counting mode (Leica) or GaAsP PMT detectors (Nikon).We transferred individual pancreatic slices to a perifusion system containing 6 mM glucose in HEPES-buffered ECS at 34°C. We show representative traces for each experimental condition. Each of these conditions has been tested on at least three separate experimental days. Slices were exposed to a series of square-pulse-like stimulations characterized by exposure to 8 mM glucose for 25 min, followed by a washout with substimulatory 6 mM glucose concentration until all the activity switched off. Imaging was performed on standard confocal microscopes equipped with resonant scanners (Leica Microsystems TCS SP5 and SP8 or Nikon A1R) both upright and inverted with their respective sets of ×20 high numerical aperture (NA) objective lenses. Acquisition frequency was set to at least 20 Hz at 256 × 256 pixels, with pixels size close to 1 µmT × X × Y) numpy array  rely on loading the recording, or its part, into memory, for viewing, analysis, and processing. It also requires laborious and long human engagement. We have developed a set of interdependent tools to automatize as much as possible the analysis pipeline  are then distributed according to the Poisson distribution, with standard deviation (σ1) being equal to the square root of the mean µ1, z-score  x in the units of standard deviation from the expected mean.Trace processing was conducted as follows. In an ideal detector, recording a time trace of a single pixel in the absence of any signal would consist of independent values of the number of photons detected during the dwell time. The values  filtering, implemented in scipy.signal module , with means µa and µb, would have a standard deviation as expected z-score for a ROI trace, we always considered the sum, rather than the average trace of the underlying pixels. When we visualized traces, we show them averaged, only to keep the scales comparable. The same reasoning holds for rebinning a single trace, where the resulting trace, rebinned by a factor of n, had a standard deviation All aforementioned refers also to a sum of pixel traces, but, crucially, not to their mean. A sum of two pixels Experiments discussed in this manuscript were recorded on standard Leica SP5 or SP8, as well as NIKON A1R confocal microscopes. In cases where we used a Hybrid detector in the photon counting mode (Leica), we saw no significant departure from our assumption of Poisson distribution. Even with nonunit gain, the linear dependence between variance and mean remains, though the slope was different from 1 . Other types of detectors introduce additional sources of noise other than Poisson , but mostly they were still dominated by Poisson in our experience, at least as long as the values between neighboring pixels and frames were independent.z-score, and identify regions where z > 4 as candidate events.Traces contain features spanning orders of magnitude in time: from tens of milliseconds, to tens of minutes. We aimed to investigate how these events at different timescales and a connection between them and the islets’ environment interact. For this, we devised a two-step algorithm to identify intrinsic timescale of events and to minimize false positives . In the first step, we performed a sequential filtering of the traces at timescales starting from 0.5 s, and increasing by a factor of t0), its maximal height, and the width at the half of the height , which is our measurement of its duration. For simplicity, we defined end time as tend = t0 + δt, although events arguably last much longer after the intensity drops at half of the peak.Events were characterized by the start time . For shorter events, we required only that an event is not unique to a single timescale.t/2), which we ascribed to artifacts from zero-padding for the filtering. We termed this procedure event distilling   B and D. ntration B–D. The ntration B.2+ events was to determine to what extent these multiple time scales of [Ca2+]c events depended on the activity of L-type VACCs. Previous experiments performed on β cell-selective Cav1.2 Ca2+ channel null mice showed that, in the absence of the Ca2+ channel, [Ca2+]c events were only moderately affected during the first minutes of the glucose stimulation, with a characteristic change in bursting pattern observed during acute stimulation  during the first section and 3.4 s  in the second section of the double protocol, where we co-applied isradipine. However, in the continuation of the plateau phase, the pattern of [Ca2+]c events was characterized by shorter and smaller events with the median value of halfwidth duration of 2.4 s  [2+]c events, the events disappeared completely.Our next step toward elucidating the molecular mechanisms of the aforementioned cytosolic Camulation . In the 2+]c events, with progressively shorter and smaller events, before events subsided completely . A complete inhibition of the glucose-dependent [Ca2+]c events was obtained when slices were exposed to 100 µM diazoxide before the onset of glucose dependence . At this concentration, diazoxide clamps the membrane potential close to the resting membrane potential, which is around the diffusion equilibrium potential for K+  (Blocking L-type VACCs with isradipine during the plateau phase produced a similar change in the phenotype of c events resulted in a series of smaller, but higher frequency events  in comparison to 184 20 s  during the control section. Our results suggest that in the presence of sufficient extracellular Ca2+ concentration, the CICR-like [Ca2+]c oscillations are likely to occur in glucose-stimulated β cells in situ and that these events decompose at subphysiologically low extracellular [Ca2+]. The spectrum of recorded patterns of β cell activities confirms previously described role of CICR through intracellular Ca2+ channels in glucose-dependent activity of these cells  as reported earlier  to supraphysiological levels (15–25 mM). This is consistent with previous observations suggesting that closure of KATP channels is not the sole mechanism to depolarize β cells  supports ER Ca2+ release through IP3  and inducing insulin release via the production of IP3 and Ca2+ release from the intracellular stores (3 receptors due to pre-emptying of intracellular Ca2+ stores in too low (0–3 mM) extracellular glucose.Glucose plays an essential role in the filling, and therefore also emptying, intracellular Ca+ stores . Forced llations , 49. Slosigargin , 62 or wsigargin –65. Simiignaling . Increasough IP3  and RYR r stores , 67–69. 2+ release had strong early support from 45Ca2+ flux studies   . Both ERmellitus .+ channels can protect β cells from cytokine-induced death, and reduces diabetes incidence in NOD mice . M.S.R., C.E.-M., and J.D.J. received financial support from National Institutes of Health (NIH) R01DK127236. M.S.R., A.S., and D.K. further received financial support from the Slovenian Research Agency . J.D.J. received Grants from CIHR and Diabetes Canada.No conflicts of interest, financial or otherwise, are declared by the authors. S.S. and M.S.R. conceived and designed research; S.P., J.P., V.P., L.K.B., N.S., M.S.K., and J.D. performed experiments; S.P., S.S., J.P., N.S., D.K., and M.S.R. analyzed data; S.P., S.S., J.P., L.K.B., D.K., A.S., C.E.-M., J.D.J., and M.S.R. interpreted results of experiments; S.P., S.S., J.P., and M.S.R., prepared figures; S.P., S.S., and M.S.R. drafted manuscript; S.P., S.S., J.P., V.P., L.K.B., N.S., M.S.K., J.D., D.K., A.S., C.E.-M., J.D.J., and M.S.R edited and revised manuscript; S.P., S.S., J.P., V.P., L.K.B., N.S., M.S.K., J.D., D.K., A.S., C.E.-M., J.D.J., and M.S.R. approved final version of manuscript.