In addition, we discuss possible therapeutic small molecules that may improve pulmonary disease outcomes in the future.Pulmonary diseases such as asthma, chronic obstructive pulmonary disease (COPD) and acute respiratory disease syndrome (ARDS) are common throughout the world. Tobacco products can potentially lead to lung damage, which in turn contribute to worsening disease outcomes. The same can be said of E-cigarette (E-cig) use, which has recently gained attention due to its possible adverse side effects. Despite this information, little is known about the potential for causality between smoking and E-cig use, and lung damage and worsening disease outcomes. In this review, we focus on a potential inflammatory mechanism, that is, calcium  are considered major risk factors in the development of pulmonary disease as well as worsening disease prognosis. However, at present, relatively little is known about the mechanistic actions by which smoking and vaping may worsen the disease. One theory suggests that long-term vaping leads to Ca Respiratory diseases such as pneumonia, acute respiratory disease syndrome (ARDS)  and chro2+) signaling and, in particular, abnormal Ca2+ influx plays in cystic fibrosis i), leading to dysfunctional ion secretion. When agonists bind to ion channels, the channels open to allow the flow of cation ions  through the cell membrane, inducing a wide variety of biological responses. Given the large number of physiological processes governed by changes in [Ca2+]i, Ca2+ signaling clearly plays a pivotal role in pulmonary disease   . STIM pr as ORAI . STIM camembrane ,31. STIM regions . This de on SOCE ,34. In acurrents . The actchannels ,36.2+ channels (VGCC) are activated upon the depolarization of the membrane and mediate Ca2+ entry into the cell. This event generates electrical signals that mediate many cellular events, e.g., enzyme activation and gene expression   ,39. IP3Rctivated . Followictivated ,42. IP3  of time ,43. The lization . The hyd3Rs and are Ca2+ release channels located on the sarcoplasmic reticulum and endoplasmic reticula [2+ through plasma membrane Ca2+ channels activates the RyRs, resulting in rapid, massive releases of intracellular Ca2+ stores.Ryanodine receptors (RyRs) share 40% homology with IPreticula . In tiss2+-ATPase (SERCA) is responsible for pumping Ca2+ into the ER lumen. These receptors mediate the storage of Ca2+ in the ER. Voltage-dependent anion channels (VDAC) and the mitochondrial Ca2+ uniporter family mediate mitochondrial Ca2+ entry [2+ signaling.Sarcoendoplasmic reticulum Ca2+ entry . Figure 2+ entry  illustra2+ signaling is not only important in intracellular processes but also plays a large role in immune cell response and inflammation. Ca2+ plays a pivotal role in cells as a secondary messenger. Stimulation of Ca2+ is responsible for the activation and regulation of many immune cells. Macrophages are key immune cells that function through phagocytosis. Their phagocytic function is dependent on internal Ca2+ regulation [2+ are important for phagocytosis as well as phagosome maturation. In addition, Ca2+ binding to phagocytic receptors on macrophages triggers IP3 production. This release of Ca2+ from the ER triggers SOCE. The influx of extracellular Ca2+ is also required for macrophage polarization into the M1 state, which is pro-inflammatory and important for fighting pulmonary infections. Lowering the cytosolic Ca2+ concentration can modulate the switch of M1 macrophages to M2, which are termed anti-inflammatory.Cagulation . Increas2+ signaling is regulated mainly by SOCE channels. The influx of Ca2+ into the cytosol is responsible for neutrophil activation. In neutrophils, SOCE is initiated by ligand-receptor interactions, which trigger a complex cascade of molecules and results in ORAI1 stimulation. Stimulation of ORAI1 via STIM1 leads to Ca2+ influx, which increases the cytosolic Ca2+ concentration [In neutrophils, Cantration . This re2+ is present at low concentrations prior to activation [3) and diacylglycerol. IP3 binds to receptors, leading to the release of Ca2+ into the cytosol. This depletion of the internal Ca2+ store activates SOCE channels, which are lymphocytes’ main mode of Ca2+ influx. Ca2+ release-activated channels (CRAC) are the most common channel configuration for SOCE found in lymphocytes.In lymphocytes, Cativation . Cross-l2+ dysregulation is an attributable trait that was described for various diseases including pulmonary diseases such as asthma and ALI [2+ dysregulation in these diseases.Ca and ALI . HoweverCOPD is an inflammatory lung disease, a major global health threat and is characterized by obstructed airflow within the lungs . This ob2+ signaling dysfunction has gained attention as a potential cause for increased morbidity and disease mortality, with cytosolic Ca2+ concentrations increasing following smoking or the use of other tobacco products [2+ influx, as well as endoplasmic reticulum and mitochondrial stores, do not play a role in the Ca2+ release caused by tobacco use. The study suggests that the Ca2+ originated from lysosomes and was maintained in the cytosol during chronic tobacco use. Total particulate matter (TPM) from tobacco products induced an increase in Ca2+ unrelated to extracellular Ca2+ and did not require the activation of the IP3 and/or TRP channel pathways. Another study [2+ influx in bronchial epithelial cells was decreased in smokers’ lungs, which occurred simultaneously with a decrease in Ca2+ release from the ER because of the differential expression of ORAI3. This differential expression inhibited SOCE channels. As alluded to earlier, these data suggest that ORAI3 expression might play a large role in Ca2+ signaling impairment in smokers as well as in patients with COPD.Recently Caproducts . The stuer study  demonstr2+ and Annexin V, with Annexin V being a well-described marker for apoptosis [2+ dysregulation caused by e-liquids/vaping based upon observations of similar health impacts to those resulting from the use of tobacco products. With Ca2+ being integral for many physiological responses and processes, it stands to reason that its dysregulation would have negative effects. Rowell and colleagues [2+ homeostasis using human bronchial epithelial cells. Banana pudding flavored e-liquid caused ER Ca2+ release and activated SOCE. In particular, long exposures to the e-liquid depleted ER Ca2+ stores and inhibited store-operated Ca2+ entry, suggesting that e-liquids may alter Ca2+ homeostasis through short- and long-term mechanisms. Vaping was also shown to have severe effects on disease and disease severity. For example, our lab [2+ mobilization in inflammation and respiratory disease development in relation to vaping. Additional studies [Klebsiella pneumonia also indicate an increase in ALI, i.e., increased lung wet/dry ratio. These increases in wet/dry ratio were significant in co-exposure models. One theory surrounding vaping is that it may trigger M0 macrophage polarization to pro-inflammatory M1 macrophages [2+. These data indicate the potential side effects of vaping on health and disease outcomes and underlie the need for awareness of these effects as vaping continues to rise in popularity amongst minors and young adults.Electronic cigarette (E-cig) use or “vaping” has become more prevalent in countries worldwide as it was initially portrayed as a safer alternative to cigarette smoking. Despite this information, little is known if long-term vaping will have similar effects to cigarette smoking. Recent studies have indicated possible adverse side effects associated with vaping ,57. For poptosis . Other epoptosis . Cinnamopoptosis  utilizedlleagues  screened our lab  utilized studies  also indrophages . These M2+ signaling dysregulation is not only the mechanism of the chronic side effects due to smoking and vaping but also a driving factor in pulmonary disease. These data suggest that targets affected by inhibitors of certain Ca2+ channels may serve as novel therapeutic targets for mediating many pulmonary diseases. Ca2+ signaling can be inhibited by Ca2+ antagonists such as dihydropyridines, phenylalkylamines and benzothiazepines [2+ channel antagonists whose effects on Ca2+ signaling have been well-studied [2+ entry, non-store operated calcium signaling and IP3R, respectively. The antagonist 2-APB has been documented to inhibit IP3R-induced Ca2+ release [2+ inhibition in TRPC- and ORAI-mediated Ca2+ entry, respectively [2+ channel antagonists were also able to significantly decrease inflammatory cytokine production as well as decrease pulmonary inflammation in vape-treated mice receiving either 2-APB or Pyr10. This study suggests that the Ca2+ channel antagonists 2-APB, Pyr10 and Pyr6 have potential use as novel therapeutics for pulmonary disease. 2+ signaling components and their mechanism of action. Many other compounds exist but have not yet been tested in relation to pulmonary disease, thus offering a potential future avenue of research focused on investigating these Ca2+ signaling mediators.Recent research suggests Caazepines . Pyr6, P-studied ,66. Pyr6 release . Pyr6 anectively , the eff2+ dysregulation is involved in the manifestation of several pulmonary diseases [2+ signaling mechanisms and the role that Ca2+ signaling plays in disease pathology is important. However, relatively little is known about the detailed mechanistic roles of Ca2+ in disease. Currently, studies have investigated the potential negative effects of vaping in relation to pulmonary disease. Ca2+ signaling pathways have more recently garnered attention as a potential mediator of these negative effects. These studies have demonstrated that vaping results in Ca2+ dysregulation, leading to decreases in cell viability and increases in inflammation. However, mechanistic details are lacking and will require further investigation. Finally, while Pyr6, Pyr10 and 2-APB were demonstrated to ameliorate disease prognosis (as described above), the mechanism is not well-understood. These data underly a need for studies to better understand the mechanism of action of vaping upon the lungs and the resultant pathology, which will include deciphering the role that Ca2+ dysregulation plays within this disease paradigm.Cadiseases . Because2+ signaling dysregulation within the context of diseases that might develop due to either vaping and/or tobacco smoking. Understanding the inflammatory pathways involved via mechanistic dissection with the Ca2+ channel antagonists 2-APB, Pyr10 and Pry6 will bolster our appreciation of this field of study and potentially yield novel therapeutic targets for diseases related to Ca2+ dysregulation. Furthermore, other Ca2+ antagonists exist and must be investigated in relation to pulmonary disease. Another aspect of future studies will focus on investigating the role of M0 macrophage polarization to M1 macrophages as this process was recently theorized to play a role in vaping-dependent inflammatory responses.Future avenues of study will focus on further investigating the mechanistic role of Ca


We achieved encapsulation of niclosamide in PEG-microspheres and PEG-nanospheres (Niclo-spheres). When applied to asthmatic mice via intratracheal instillation, Niclo-spheres strongly attenuated overproduction of mucus, inhibited secretion of the major proinflammatory mediator CLCA1, and improved mucociliary clearance in tracheas ex vivo. These effects were comparable for niclosamide encapsulated in PEG-nanospheres and PEG-microspheres. Niclo-spheres inhibited the Ca2+ activated Cl− channel TMEM16A and attenuated mucus production in CFBE and Calu-3 human airway epithelial cells. Both inhibitory effects were explained by a pronounced inhibition of intracellular Ca2+ signals. The data indicate that poorly dissolvable compounds such as niclosamide can be encapsulated in PEG-microspheres/nanospheres and deposited locally on the airway epithelium as encapsulated drugs, which may be advantageous over systemic application.Repurposing of the anthelminthic drug niclosamide was proposed as an effective treatment for inflammatory airway diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Niclosamide may also be effective for the treatment of viral respiratory infections, such as SARS-CoV-2, respiratory syncytial virus, and influenza. While systemic application of niclosamide may lead to unwanted side effects, local administration via aerosol may circumvent these problems, particularly when the drug is encapsulated into small polyethylene glycol (PEG) hydrospheres. In the present study, we examined whether PEG-encapsulated niclosamide inhibits the production of mucus and affects the pro-inflammatory mediator CLCA1 in mouse airways in vivo, while effects on mucociliary clearance were assessed in excised mouse tracheas. The potential of encapsulated niclosamide to inhibit TMEM16A whole-cell Cl The values Rmax and Rmin (maximum and minimum ratios) and the constant f2/Sb2S (ratio between the fluorescence of free and Ca2+-bound Fura-2 at 380 nm) were determined using 2 µM ionomycin , 5 µM nigericin , 10 µM monensin , and 5 mM EGTA  to equilibrate intracellular and extracellular Ca2+ in intact Fura-2-loaded cells. The dissociation constant (Kd) for the Fura-2•Ca2+ complex was taken as 224 nM [Cells were seeded on coated glass coverslips and loaded with 2 µM Fura-2, AM Ester  and 0.02% Pluronic F-127  in standard bicarbonate-free Ringer’s solution for 1 h at room temperature. The measurement of intracellular Ca earlier . Cells ws 224 nM ). Contros 224 nM .All compounds used were of the highest available grade of purity and were bought from Sigma-Aldrich , unless indicated otherwise. Data are shown as individual traces/representative images and/or as summaries with mean values ± SEM, with the respective number of experiments given in each figure legend. The data have been tested for a normal distribution. As the effects have been examined in individual animals, which naturally vary with respect to their responses, outliers have not been removed. Tracings and current–voltage relationships are shown as original data and were not fitted. For the analysis of patch clamp data, PULSE software version 8.65  was used. For the quantitative analysis of images and mucociliary clearance, ImageJ software with the manual tracking plugin and Zen  were used.t-test or ANOVA were used as appropriate. A p-value of < 0.05 was accepted as a statistically significant difference.For statistical analysis, paired or unpaired Student’s


In LBD, α-synuclein oligomers exhibit a neuron-specific cytoplasmic distribution. Highly active neurons and neurons with a high Ca2+ burden are prone to damage in LBD. The neuronal vulnerability may be determined by transneuronal axonal transmission of the pathological processes; however, this hypothesis seems inconsistent with pathological findings that neurons anatomically connected to LBD-vulnerable neurons, such as neurons in the ventral tegmentum, are spared in LBD. This review focuses on and discusses the crucial roles played by α-synuclein oligomers and Ca2+ dyshomeostasis in early intraneural pathophysiology in LBD-vulnerable neurons. A challenging view is proposed on the synergy between retrograde transport of α-synuclein and vesicular Ca release, whereby neuronal vulnerability is propagated backward along repeatedly activated signaling pathway.α-Synuclein oligomers and Ca The pathological hallmarks of Lewy body disease (LBD), [also known as Parkinson’s disease (PD) and dementia with Lewy bodies] are Lewy bodies and Lewy neurites, which are composed of insoluble α-synuclein fibrils  receptors (IP3Rs) , which is mediated by direct binding with calcium binding protein 1 (CaBP1), a neuron-specific member of the calmodulin superfamily. This binding disrupts the normal Caα-Synuclein is highly expressed in the brain and is enriched at presynaptic terminals. It augments assembly of the SNARE machinery, and plays a role in neurotransmitter release and protection of nerve terminals against injury   during autonomous action potentials causes oxidative stress and cell damage , homer1a and amyloid-β through a patch pipette, how Ca2+ and K+ channels are involved in the regulation or pathophysiological change of neocortical pyramidal cell excitability and Ca2+ dynamics are demonstrated  channels under the application of blockers for the channels or receptors responsible for intraneural Ca2+ dynamics  from IP3Rs in neurons of the neocortex and amygdala, and contributes to the regulation of neuronal excitability and synaptic plasticity Yamamoto et al. 3 turnover is finely tuned by synaptic stimulation or neuromodulation and is necessary for spike-induced or IP3-induced Ca2+ release from IP3Rs in central neurons  blocker, U73122, failed to inhibit it  and decreased spike frequency  is the most promising candidate, because CaBP1 is (1) a Ca2+-binding protein expressed in the cytosol of rodent and human central neurons  and CaBP1 on αSNo-mediated change were compatible with this hypothesis. CaBP1 Ab occluded the effect of αSNo, which was blocked by nifedipine and heparin, indicating that αSNo-mediated capture of CaBP1 is necessary and sufficient for the aberrant CICR from IP3 and CaBP1 have opposing effects on the IP3R channel under high intracellular Ca2+ concentrations (Haynes et al. 3 obstructs the inter-subunit interaction of IP3R and encourages IP3R channel opening (Choe and Ehrlich 3R and prevents the inter-subunit interaction of IP3R when the somatic concentration of IP3 is low, thereby hindering IP3R channel opening in a Ca2+-dependent manner Choe and Ehrlich 3 and CaBP1 Ab occlude and heparin and CaBP1 inhibit the action of αSNo (Fig. 3R, but not the Ca2+ buffering effect of CaBP1, is essential for aberrant CICR from IP3R (Fig. IP3R is localized on ER membrane connected with mitochondria, known as the mitochondria-associated ER membrane, and increased Ca2+ transfer from the ER via IP3Rs to mitochondria inhibits autophagy (Szabadkai et al., 3R in the presence of intraneural α-synuclein oligomers may increase the risk of activity-dependent mitochondrial stress through the IP3R-mitochondrial connection, and may lead to neuronal fragility in oligomeric α-synuclein-bearing neurons. This notion is supported by previous studies indicating that α-synuclein can alter mitochondrial Ca2+ homeostasis by enhancing ER-mitochondria interactions (Cali et al. IP2+ homeostasis (Berridge 2+ signals can transmit through the cytosol by CICR from the ER, and induce regional or global communication within the cell, at a pace that is slower than action potential propagation in the plasma membrane. CICR can be mediated by IP3Rs or ryanodine receptors and can be augmented by elevated cytosolic Ca2+ (Berridge 3Rs propagates as a Ca2+ wave along the ER via IP3Rs and ryanodine receptors throughout the somatodendritic portion and the nucleus (Power and Sah 2+ wave propagating from dendrite to soma and nucleus, is initiated only when strong synaptic stimulation or synaptic stimulation concurrent with repetitive spikes occurs (Power and Sah 3R which is distributed in the soma and dendrites, thereby regulating neuronal excitability and synaptic plasticity (Power and Sah 3Rs in the presence of α-synuclein oligomers (Yamamoto et al. 2+ wave to be propagated along the soma-dendritic region (Yamamoto et al. BST1, encodes cyclic ADP-ribose hydrolase 2, which synthesizes cyclic ADP-ribose, a ryanodine receptor agonist (Satake et al. BST1 variants can disturb normal channel function of the ryanodine receptor, another Ca2+ release channel in the ER, and can enhance the aberrant CICR via IP3Rs to promote propagation of dysregulated Ca2+ waves (Yamamoto et al. 2+ wave may increase the risk of distinct activity-dependent Ca2+ dyshomeostasis and may lead to pathological intraneural spreading and neuronal fragility specific to repeatedly activated signaling path in oligomeric α-synuclein-bearing neurons, although this remains to be studied. Considering that oligomeric α-synuclein accumulates first at presynaptic terminals in LBD (Bridi et al. The ER is a continuous “neuron-within-neuron” network that extends to all parts of the neuron, including the spines, cell soma, and synaptic endings, and that supports regional and long-distance CaBerridge . Ca2+ siBerridge . The CIC2+ influx via CaV1.3 L-VDCC and inhibits degeneration of SNc neurons (Chan et al. 3Rs (Yamamoto et al. Immunohistochemical studies reveal that among central neurons, the expression level of CaBP1 is lowest in SNc neurons, which are the most fragile in PD (Laube et al.


Hepatitis E virus (HEV) is zoonotic and the leading cause of acute viral hepatitis worldwide. Rabbit HEV can infect humans and is prevalent globally. It is reported that laboratory rabbits are also naturally infected with HEV. Therefore, it is important to investigate in a large scale the prevalence of HEV in laboratory rabbits. Serum samples were collected from 649 laboratory rabbits of 13 different commercial vendors in Beijing, China, from 2017 to 2019, and anti-HEV and HEV antigen (Ag) were tested. Fecal samples were collected from 50 laboratory rabbits from one of the vendors for HEV RNA detection. Six laboratory rabbits with natural HEV infection were euthanized and their liver, kidney, bile and urine samples were collected for HEV RNA quantification. Liver tissues were subjected to histopathology analysis. The overall positive rates of anti-HEV antibodies and HEV-Ag are 2.6% (15/588) and 7.9% (51/649), respectively. HEV RNA was detected in 12.0% (6/50) of the rabbits. High viral load of HEV RNA was detected in liver and bile samples. Liver inflammation was observed. HEV is circulating in laboratory rabbit population in China. Strict screening is crucial to ensure experimental accuracy and prevent zoonotic transmission to research personnel. Hepatitis E virus (HEV) is a single-stranded, positive sense RNA virus and the major cause of acute viral hepatitis worldwide. It is estimated that 20 million infections occur annually, leading up to 3 million symptomatic cases and 70,000 deaths . There aSince the first discovery of HEV in pigs in 1997 , HEV hasIn 2009, researchers firstly reported in China that rabbit is a novel animal host of HEV harboring a novel HEV genotype, the rabbit HEV . SubsequA total number of 649 laboratory rabbits were detected during 2017–2019 in this study and their serum samples were collected . Thirty-6 copies/g to 2.45 × 107 copies/g. All samples were successfully sequenced. Phylogenetic analysis of partial ORF2 sequence showed that all sequences belong to HEV-3ra  and Real-Time quantitative PCR (RT-qPCR). Relatively high fecal HEV RNA load was detected ranging from 4.07 × 10 HEV-3ra . 9 copies/g. One kidney tissue and one urine sample from different individual rabbit was positive for HEV RNA, with viral load of 1.48 × 104 copies/g and 4.29 × 102 copies/mL, respectively.The six HEV RNA-positive rabbits were euthanized and tissues including liver and kidney, bile and urine samples were collected for HEV RNA quantification. High viral load of HEV RNA was detected in the liver tissues and bile samples . All livTo investigate the liver histopathology of the laboratory rabbits with HEV-3ra natural infection, liver tissues were selected from three rabbits  with the highest hepatic viral load for hematoxylin-eosin (HE) staining. Mild-to-moderate infiltration of lymphocytes was found in all liver tissues, indicating inflammation .In this study, we investigated the prevalence of HEV in laboratory rabbits in a large extent. The result demonstrated that the laboratory rabbits in almost all surveyed vendors  experienced a current or past HEV infection event. Six rabbits with current HEV infection were detected in one of the vendors. Sequences obtained from these rabbits all belong to HEV-3ra. The current study highlights the current situation of naturally occurring HEV infection in the laboratory rabbit population. In this study, we performed anti-HEV antibodies and HEV Ag (ORF2 proteins) tests by using two different ELISA kits. HEV Ag is a recently discovered novel indicator of HEV infection ,26 and iIn natural settings, HEV-3ra could infect various breeds of rabbits and the prevalence of anti-HEV and HEV RNA is high . Laborat6–107 copies/g) of naturally infected laboratory rabbits indicating active replication of HEV in these animals. The result was further confirmed by detecting high viral load of HEV RNA in these rabbits’ bile and liver tissues (approximately 109 copies/g) and inflammation in the liver. Phylogenetic analysis of the six obtained HEV sequences from one of the vendors demonstrated that they all belong to HEV-3ra and clustered closely with the HEV-3ra sequences isolated from rabbits in China. We did not collect serum samples from naturally infected rabbits as previous studies found that fecal virus shedding is longer and more stable in infected rabbits  and Peking University Health Science Center , China.Oryctolagus cuniculus) from 13 commercial vendors in China. Rabbits from each vendor were randomly selected and serum samples were collected from 649 laboratory rabbits during this period. Fifty fecal samples were collected for HEV RNA detection from one of the 13 vendors in 2020. The fecal samples were all diluted with phosphate buffered saline to prepare 10% suspensions and centrifuged at 5000× g for 30 min. The supernatants were harvested and immediately stored at −80 °C until use [From 2017 to 2019, we performed routine examination of 6-month-old laboratory white rabbits  kits . Signal-to-cutoff (S/CO) values for anti-HEV antibody were calculated and values >1 were considered positive. All experiments were carried out under the manufacturers’ instructions and detailed methods were described in a previous study .g for 15 min at 4 °C and subjected to RNA extraction [Approximately 100 mg of the fresh tissue collected was homogenized in 1 mL of TRIzol reagent  and clarified by centrifugation at 5000× traction . RNA wastraction ,38. HEV-9 to 101 copies) were constructed and used as the standard for quantification of viral genome copy number [Quantification of HEV RNA was carried out using a commercial One-Step RT-qPCR kit  with a previously established in-house method . To quany number . The finNucleotide sequences were assembled and analyzed using MEGA 6.0 . PhylogeThe rabbits’ liver tissues were fixed in 10% neutral buffered formalin and were subsequently embedded in paraffin. Specimens were cut into 4 μm sections. Slides were stained with HE and subjected to histopathological examination .We demonstrated that past or recent HEV infection existed in laboratory rabbits in Beijing, China, and the results strongly suggested that HEV is widespread in this special animal population. Researchers engage in, not only HEV study, but also other pathogens should be aware of the influence of pre-existing HEV infection on other pathogens infection. Further study should address this issue and the risk of HEV infection in research personnel should be investigated.


MRD1 and MRP1 in the target cells. These findings suggested a novel role of EVs in mediating the acquired chemo-resistance in AML patients by inducing the expression of the drug efflux pumps; however, further investigations will be required to elucidate other underlying mechanisms of resistance that are mediated by EVs.In spite of successful initial remission, chemo-resistance and relapse are still concerning points in acute myeloid leukemia (AML) treatment strategies. Multidrug resistance (MDR) appears to be the major contributor of chemo-resistance, arising in some sub-clones of cancers and could be developed in others. The aim of this study was to investigate the role of extracellular vesicles (EVs) derived from AML patients on the transmission of chemo-resistance phenotype. Ultracentrifugation was employed to isolate EVs from healthy controls, new cases, and relapsed AML patients. The EVs size, morphology, and immunophenotype were determined by dynamic light scattering, TEM, and flow cytometry respectively. Bradford assay was performed to measure the protein content of EVs. MTT assay and flow cytometry analysis were also used to determine the viability index, induction of apoptosis, and ROS generation in U937 cells. The expression level of two efflux pumps was assessed using qRT-PCR analysis. Findings of TEM, DLS, and flow cytometry confirmed that EVs had a desirable shape, size, and surface markers. EVs derived from both new cases and relapsed AML patients significantly reduced idarubicin-induced apoptosis in the U937 cells. The analysis of drug efflux pumps gens revealed that EVs over-express  Acute myeloid leukemia (AML) is the commonest acute leukemia in adults. Despite basic advances in the understanding of AML pathogenesis, the patient’s clinical situation is still dismal and their therapeutic outcome has not kept pace in parallel with molecular findings , 2. AlthEVs are bi-layer lipid membrane particles released by both normal and neoplastic cells that could deliver their cargos to recipient cells by a variety of mechanisms, including ligand/receptor signaling, phagocytosis, and/or endocytosis . The amoMRP1 and MDR1 were evaluated. Among a variety of drug resistance molecules, the multi-drug resistance gene (MDR1), also known as P-glycoprotein 1 (Pgp), and multidrug resistance protein 1 (MRP-1) are contributed to the treatment failure of anthracyclines in AML patients , 17. In Study populationEVs were obtained from peripheral blood of 11 patients with AML, including 7 new cases before treatment and 4 relapsed AML patients who were referred to Taleghani hospital . Sequentially, 10 apparently healthy subjects were included in this study as a control group. Informed consent was obtained from all individuals and was included in the study. All patients were diagnosed with a history of non-M3-AML. Samples were collected in heparin tubes. The detailed demographic and biological characteristics of patients were presented in Isolation of Extracellular vesicles×g. The collected supernatants were then centrifuged at 12,000 ×g for 20 min. The ultimate supernatants were transferred into 5 mL ultracentrifuge tubes (Beckman model. L5-50-USA) and were ultra-centrifuged at 110,000 ×g for 90 min . The supernatants were then discarded, the pellet containing EVs were dissolved in 500 µL PBS and transferred to the -80 °C for further analyses. The volume of the plasma samples was variable. At first, the plasma samples were diluted with a similar volume of Phosphate Buffered Saline (PBS) and were centrifuged for 15 min at 3000 EVs surface CD markers detectionEV-enriched and suspended pellets were analyzed by a attune NxT cytometer (Thermo Fisher Scientific-USA). The system was calibrated using standard microbeads with diameters of 0.3–0.1–3 μm (BD-USA) to define the precise size of particles, which shall be gated as EVs in flow cytometry . EVs werTEM and DLSFifty microliter EVs from AML patient and healthy subjects were put on a copper grid coated with 0.125% Formvar in chloroform. The grids were stained with 1% v/v uranyl acetate in ddH2O and the samples were examined immediately. A JEOL 1011 transmission electron microscope was used for imaging . The size distribution of EVs was determined using DLS by a Malvern Nano ZS instrument (UK). Briefly, the volume of each sample (patients and cell line) was increased up to 1 mL by PBS, loaded into a quartz cuvette, and measured at 25 °C. Finally, the results were analyzed using the Zetasizer software (version 7.11). Bradford assayThe determination was carried out according to the manufacturer’s instructions  and spectrophotometric quantification of the protein content was measured at 595 nm. Protein levels were first measured separately for all the samples, then the samples from each group  were pooled separately and used for subsequent tests.DrugIdarubicin hydrochloride  is an anthracycline anti-leukemic drug, which is commonly used in combination with cytosine arabinoside as a first-line treatment of AML. To obtain a 1 mg/mL stock solution, this drug was dissolved in phosphate-buffered saline (PBS) and the stock was kept at −20 °C. Actinomycin D, a chemotherapy drug with the ability to inhibit transcription, was provided from Minatajhiz Aria Company . This drug was dissolved in Dimethyl sulfoxide (DMSO) to obtain a 0.5 mg/mL stock solution and stored at −20 °C.Cell cultureThe human AML cell line U937 was obtained from the Pasteur Institute  and cultured in suspension in RPMI medium supplemented with 2 mM L-glutamine, 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin in a humidified 5% CO2 at 37 ºC. For Idarubicin (IDA) treatment, a relevant amount of stock solution (0.5 mM in RPMI) of IDA was added to a culture medium to attain the concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 μM. All drug treatments were carried out in three independent experiments and all assays were performed in triplicate.MTT assayU937 cells (5 × 103) were seeded into a 96-well plate in the presence of IDA. After 36 h, the MTT solution (5 mg/mL in PBS) was added to each well and incubated at 37 °C for the next 3 h. The percentage of the metabolic activity of the cells was evaluated by dividing the optical densitometry (OD) of a resulting formazan measured by an enzyme-linked immunosorbent assay (ELISA) reader in the drug-treated groups by the OD of the control group. Evaluation of Apoptosis by Annexin V/Propidium Iodide (PI) AssayTo explore the effect of IDA, IDA-plus-healthy-EVs, IDA-plus-nAML-EV, and IDA-plus-rAML-EV on the induction of apoptotic cell death, the cells were subjected to Annexin-V/PI staining analysis. Drugs-treated cells were collected after 36 h of treatment and suspended in 100 μL of the incubation buffer. Then, 2 μL annexin-V-Flous was added to each sample and incubated for 20 min in the dark. The intensity of fluorescence was measured by flow cytometry. ROS assayTo determine the effect of IDA on the production of intracellular reactive oxygen species (ROS) in U937 cells, we used a fluorogenic dye DCFH-DA, for measuring hydroxyl, peroxyl, and other ROS activity within the cell. After incubation with IDA, the cells were incubated with DCFH-DA at 37 °C for 30 min. Finally, fluorescence intensities of the samples were detected by a fluorescence spectrophotometer  with excitation at 485 nm and emission at 530 nm.RNA extraction, cDNA synthesis, and quantitative real-time PCRst strand cDNA Synthesis Kit  and the synthesized cDNA were kept at -20 °C until use. Next, to examine the effect of IDA-plus healthy-EVs, IDA-plus-nAML-EV, and IDA-plus-rAML-EV on the expression of MRD1 and MRP1 in U937 cells, control group, and patients samples, thawed cDNAs were subjected to quantitative real-time PCR (qRT-PCR). The fold change values were calculated based on 2−ΔΔCt relative expression formula. Selected primers are shown in Table 2Total RNA from U937 cells, AML patients, and AML derived EVs were extracted by RiboEx (GeneAll®- South Korea), and RiboEx LS TM (GeneAll®) respectively, according to the Protocol. After confirming the quantity of each extracted RNA by Nanodrop instrument, the reverse transcription reaction was performed using the PrimeScript™ 1Statistical analysis t-test and one-way variance analysis. A probability level of P < .05 was considered statistically significant.The results obtain from three independent tests and presented as mean ± SD. Statistical analyses were analyzed using GraphPad Software  and the significance of differences between experimental variables was determined by the use of a two-tailed student’s Evaluating the characteristics of isolated EVs by flow-cytometry, DLS, and electron microscopy imagingFlowcytometryEVs isolated from the plasma of AML patients expressed CD34, HLA DR, and CD33, as well as CD9 and CD63. The presence of these markers confirmed that they were originated from myeloblasts. Although the expressional pattern of new cases and relapsed-relevant CD markers, CD9 and CD63 on EVs were similar, the expression of other markers on EVs was proportional to the percentage of blasts. While CD9, CD63, and CD33 were positive in the control group, the expression of CD34 and HLA DR were negative (data were not shown) .TEM and DLS analyze of EVsTo investigate whether the structure of isolated EVs was completely conserved during the isolation process, a TEM technique was performed. As presented in The comparison between EVs protein levels in healthy individuals, new cases, and relapsed patientsP < 0.05). Ten apparently healthy subjects as controls were included in this study and plasma EVs were isolated and mean protein content was 17.5 ± 3 µg, which was significantly lower than both groups (at diagnosis and relapsed patients),   . There wAML-EVS protected U937 cells from the cytotoxic effects of the chemotherapeutic agent50 values of 0.2 μM  and to correspond EVs, the expression of these genes was evaluated by qRT-PCR. Results demonstrated that EVs expressed MDR-1 and MRP1 in parallel with corresponding cells. Moreover, the higher levels of MDR-1 and MRP1 mRNA expression were detected in relapsed AML as compared to new cases AML  (To verify the presence of MDR-1 and ctively) . EVs-treated cells increased the expression of drug export pumps in U937 cellsMRD1, MRP1, in leukemic cells upon exposure to the single concentration of healthy-EVs, new case AML-Evs, and relapsed AML-EVs (30 µg/mL). Our results showed that the treatment of the cells with new case AML-EVs and relapsed AML-EVs increased the mRNA expression level of MRD1 and MRP1 as compared to the cells, which were treated with healthy EVs . MRP1 expression was increased by 1.9- and 2.8-fold changes in 30 µg new case AML-EVs and relapsed AML-EVs compared to control, respectively . To identify what processes, at the molecular level, explain the reduction of IDA- induced apoptosis in U937 cells, we evaluate the expression of two important drug efflux pumps, lthy EVs . As showThe effects of EV on gene expression were due to de novo cell regulation rather than RNA transfer MRD1 and MRP1 were assessed. The resulting data showed that the expression of these genes was comparable prior and post EVs treatment, indicating that the elevation of these genes in the U937 cells untreated with actinomycin D was independent of direct RNA transfer by EVs  prior to EV incubation. After 36 h of EV integration, expression of r by EVs . AML-derived EVs decreased ROS level in U937 cells To assess the effect of AML-derived EVs on the production of ROS in U937 cells, the cells were treated with IDA, either alone or combined with healthy-EVs, new case AML-EVs, and relapsed AML-EVs. Then, the production of ROS was evaluated using flow cytometry analysis. As shown in One of the main obstacles in the way to successful treatment of leukemia is drug-resistance, which is mediated through several mechanisms. Dysregulation in the expression level of both oncogenes and tumor suppressors, alteration in apoptotic pathways, as well as disruption in DNA repair responses are the most important mechanisms that are participated in decreasing the sensitivity of cancer cells to the conventional treatment strategies . To date. Previously reported that the amount of EVs was elevated in various hematologic malignancies  cells could modify the gene expression even after exposure to actinomycin D, which abolished the gene expression system in CLL cells (We also examined whether the overexpression of drug efflux pumps in U937 cells was mediated by a direct transfer of LL cells . On the LL cells  and 42. Under normal conditions, ROS production and ROS elimination are balanced; however, it has also been reported that the production of ROS could be an adaptive response to age, sex, tumor growth or stress conditions during the cell´s lifespan . EVs andMRD1 and MRP1 gene expression, but not a direct transfer of these genes. We also found that ROS reduction may be another mechanism, which could result in the generation of resistant cells upon exposure to relapsed AML-derived EVs. A better understanding of other plausible mechanisms involved in arising of resistant phenotype may lead to the development of more effective treatment for AML patients in the future. Arising resistant diseases is of an utmost important issue in medicine, particularly in the path of cancer treatment. Our data revealed that EVs could confer IDA resistance in a sensitive AML-derived cell line. With more depth analyses, we found that resistant phenotype was mediated, at least in part, through induction of


Treatment with RvD2 significantly blocked the histamine-induced [Ca2+]i increase as well as secretion. RvD2 and RvD1 counter-regulate different histamine receptor subtypes. RvD2 inhibited the increase in [Ca2+]i induced by the activation of H1, H3, or H4 receptors, whereas RvD1 inhibited H1 and H3 receptors. RvD2 and RvD1 also activate distinct receptor-specific protein kinases to counter-regulate the histamine receptors, probably by phosphorylation. Thus, our data suggest that the counter-regulation of H receptor subtypes by RvD2 and RvD1 to inhibit mucin secretion are separately regulated.Resolvin (Rv) D2 and RvD1 are biosynthesized from docosahexaenoic acid (DHA) and promote resolution of inflammation in multiple organs and tissues, including the conjunctiva. Histamine is a mediator produced by mast cells in the conjunctiva during the allergic response. We determined the interaction of RvD2 with histamine and its receptor subtypes in cultured conjunctival goblet cells and compared them with RvD1 by measuring intracellular [Ca Allergic conjunctivitis is a predisposition to hypersensitivity reactions upon exposure to specific environmental antigens and is characterized by ocular symptoms, such as itching, blurry vision, redness, and watery eyes, which are frequently combined with nasal and skin symptoms ,2. This 2+]i ([Ca2+]i) via phospholipase C (PLC) i, cultured CGCs derived from both rats and humans were incubated with RvD2 (10−10 M–10−8 M) for 30 min before histamine 10−5 M was added . Treatment with RvD2 (10−10 M) did not alter the histamine-stimulated increase in [Ca2+]i, while treatment with RvD2 (10−9 and 10−8 M) significantly blocked the histamine-stimulated increase in [Ca2+]i to 147.5 ± 96.8 nM (p = 0.02) and 157.8 ± 43.4 nM (p = 0.02), respectively. Similar results were obtained in cells derived from humans as in those derived from rats. Histamine significantly increased peak [Ca2+]i by 330.3 ± 47.4 nM above basal (p < 0.001) . To determine the action of RvD2 on the histamine-stimulated increase in [Caas added a. In CGC [Ca2+]i b. The i increases in both rat- and human-derived CGCs.Our published data showed that RvD1 counter-regulates histamine response in human-derived CGCs . We treat 10−5 M c,d. TreaScansite4, where protein kinase A (PKA) phosphorylation sites were predicted on all four histamine receptor subtypes. To determine if RvD2 uses PKA to counter-regulate the histamine-stimulated increase in [Ca2+]i, PKA inhibitor H89 (10−5 M) was given 15 min prior to RvD2 treatment . Treatment with RvD2 at 10−8 M significantly decreased the histamine-stimulated increase in [Ca2+]i to 127.8 ± 25.3 nM (p = 0.02). Treatment with H89 10−5 M alone did not alter the histamine-stimulated response. The counter-regulation by RvD2 to the histamine-stimulated response was successfully reversed by treatment with H89 (10−5 M) to 262.5 ± 46.2 nM. This finding indicates that RvD2 uses PKA to counter-regulate the histamine receptor-stimulated increase in [Ca2+]i. The phosphorylation sites for individual protein kinases on histamine receptor subtypes were predicted using reatment a,b. Hist−8 M for 30 min before agonists specific to the histamine receptors H1-H4 were added individually  (−8 M significantly decreased the H1-stimulated increase in [Ca2+]i to 133.6 ± 22.6 nM (p = 0.02). Amthamine dihydrobromide (H2 agonist) at 10−5 M increased peak [Ca2+]i by 345.7 ± 49.7 nM above basal (p < 0.001). Treatment with RvD2 10−8 M did not alter the H2-stimulated increase in [Ca2+]i (p = 0.3). (R)-(-)-α-methylhistamine (H3 agonist) at 10−5 M increased peak [Ca2+]i by 416.5 ± 50.1 nM above basal . 4-methylhistamine dihydrochloride (H4 agonist) at 10−5 significantly increased peak [Ca2+]i by 421.6 ± 73.6 nM above basal (p < 0.001) (−8 M significantly blocked the H4-stimulated increase in [Ca2+]i to 156.1 ± 26.7 nM (p = 0.003). As a positive control, histamine-stimulated increase in [Ca2+]i was significantly downregulated by RvD2 treatment (p < 0.001)  a,b. Trea [Ca2+]i a,b (p = < 0.001) c,d. Trea< 0.001) a,d.−6 M significantly increased peak [Ca2+]i by 198.7 ± 23.5 nM above basal (p < 0.001), which is consistent with our previous findings [−8 M significantly decreased the H1-stimulated increase in [Ca2+]i to 123.2 ± 10.4 nM (p= 0.01). H2 agonist at 10−5 M increased peak [Ca2+]i by 259.7 ± 28.4 nM above basal (p < 0.001). Treatment with RvD1 at 10−8 M did not alter the H2-stimulated increase in [Ca2+]i (p = 0.07). The H3 agonist at 10−5 M increased peak [Ca2+]i by 282.9 ± 53.1 nM above basal (p < 0.001). Treatment with RvD1 at 10−8 M significantly blocked the H3-stimulated increase in [Ca2+]i to 141.4 ± 14.7 nM (p = 0.03). The H4 agonist at 10−5 M increased peak [Ca2+]i by 212.8 ± 33.6 nM above basal, while RvD1 at 10−8 M did not significantly alter the H4-stimulated increase in [Ca2+]i (p = 0.1).A similar experiment was conducted using RvD1 to compare its counter-regulatory function on histamine receptor subtypes with that of RvD2 e,g. The findings . TreatmeIn summary, RvD2 and RvD1 each counter-regulates the action of histamine via different histamine receptor subtypes with RvD2 acting on H1, 3, and 4 and RvD1 acting on H1 and 3. Neither resolvin counter-regulates the H2 receptor subtype.4 (LXA4) activate β-ARK1 to counter-regulate the action of the H1 receptor subtype agonist, histamine dimaleate, on CGCs to increase [Ca2+]i [−6 M) was given 15 min before H receptor subtype agonist or RvD2 (10−8 M) administration. RvD2 stimulation was followed 30 min later by H receptor subtype agonist. The H1 agonist at 10−6 M increased [Ca2+]i by 320.9 ± 47.4 nM above basal (p < 0.001) (2+]i increase to 121.1 ± 11.3 nM (p = 0.01). β-ARK1I treatment did not alter stimulation by H1 agonist (a control for a direct effect on the H1 receptor subtype), but reversed the inhibitory action of RvD2 on H1 agonist. These results indicate that RvD2 activates β-ARK1 to downregulate the H1 receptor subtype. β-ARK, later named G-protein-coupled receptor kinase, was initially described as phosphorylating β-adrenergic receptors and promoting the interaction of the receptor with β-arrestin to counter-regulate the activation of G-proteins and associated signaling pathways . We publ [Ca2+]i ,26. To d< 0.001) a,b. RvD22+]i induced by the H3 agonist at 10−6 M (p = 0.01) (p = 0.01).When CGCs were stimulated by the H3 agonist, RvD2 successfully downregulated the increase in i (p = 0.3) A result similar to that of the H1 agonist was obtained with the H4 agonist at 10 [Ca2+]i e,f. β-ARThese results indicate that RvD2 activates β-ARK1 to downregulate the H1 and H4, but not the H3, receptor subtypes.−6 M increased [Ca2+]i in cultured rat CGCs by 255.0 ± 36.4 nM (p < 0.001) (−8 M for 30 min significantly attenutated the increase in [Ca2+]i induced by the H3 agonist to 98.2 ± 10.7 nM (p = 0.003). β-ARKI treatment alone did not alter the H3-triggered [Ca2+]i increase. β-ARKI 15 min treatment successfully reversed the downregulation by RvD1 on H3 to 262.8 ± 62.5 nM (p = 0.9) (Our published data showed that RvD1 uses β-ARK1 to counter-regulate activity of H1 receptor subtype . We dete< 0.001) a,b. Pre-p = 0.9) a,b. ThesScansite4 predicted PKA and PKC phosphorylation sites on histamine receptors, the PKA inhibitor H89 at 10−5 M or the PKC inhibitor Ro-317549 at 10−7 M were used in rat CGCs, 15 min prior to histamine receptor subtype agonists or RvD2 (10−8 M) treatment that was then followed 30 min later by stimulation by individual histamine receptor subtype agonists. Activation of the H1 receptor by its agonist at 10−6 M increased [Ca2+]i by 435.4 ± 33.2 nM above basal (p < 0.001) (2+]i to 119.1 ± 15.2 nM (p < 0.001). Neither H89 nor Ro-317549 treatment altered stimulation by the H1 agonist. Preincubation with H89 successfully reversed the inhibition of H1 response by RvD2. In contrast, the inhibition caused by RvD2 was not reversed by Ro-317549 (As < 0.001) a,b. Treao-317549 a,b.−6 M increased [Ca2+]i by 255.5 ± 38.9 nM above basal (p < 0.001) (−8 M) significantly blocked the H3-stimulated increase in [Ca2+]i to 111.6 ± 19.4 nM (p = 0.01). Neither H89 nor Ro-317549 treatment altered stimulation by the H3 agonist. Treatment with H89 before RvD2 and then H3 agonist addition reversed the inhibition of H3 response by RvD2. In contrast, the inhibition of H3 caused by RvD2 was not reversed by Ro-317549 (Results similar to H1 response were obtained with the H3 agonist. The H3 agonist at 10< 0.001) c,d. Prioo-317549 c,d. −5 M increased [Ca2+]i by 300.0 ± 66.9 nM above basal (p < 0.001) (−8 M) significantly downregulated the H4-stimulated increase in [Ca2+]i to 90.4 ± 22.5 nM (p = 0.01). Neither H89 nor Ro-317549 treatment altered stimulation by the H4 agonist. H89 15 min pretreatment did not reverse the inhibition of H4 response by RvD2. In contrast, the inhibition caused by RvD2 was reversed by Ro-317549 (H89 and Ro-317549 were next studied on the H4 response. The H4 agonist at 10< 0.001) e,f Prioro-317549 e,f. 2+]i induced by H1 and H3 receptor activation. In contrast, RvD2 activated PKC, but not PKA, to counter-regulate the H4 response. These results indicate that RvD2 activated PKA, but not PKC, to counter-regulate the increase in [Ca−6 M increased [Ca2+]i by 268.2 ± 47.8 nM above the basal (p < 0.001) (−8 M) significantly blocked the H1 agonist-stimulated increase in [Ca2+]i to 114.9 ± 10.9 nM (p = 0.01). Neither H89 nor Ro-317549 treatment altered stimulation by the H1 agonist. Treatment with H89 successfully reversed the inhibition of H1 response by RvD1  a–c. Prio by RvD1 a,c. The  by RvD1 b,c. 2+]i by 229.7 ± 32.3 nM above basal (p < 0.001) (−8 M significantly blocked the H3-stimulated increase in [Ca2+]i to 98.9 ± 8.8 nM (p = 0.002). Neither the H89 nor Ro-317549 treatment altered stimulation by the H3 agonist. Addition of H89 or Ro-317549, respectively, reversed the inhibition of H3 response by RvD1 to 205.8 ± 55.7 and 176.1 ± 20.4 nM, respectively,  d–f. Prioctively, d–f. Thes2+]i and mucin secretion in CGCs by acting via different histamine receptor subtypes. The action of histamine via H1 and H3 receptors is inhibited by both RvDs, while the H4 receptor is only attenuated by RvD2. Neither RvD2 nor RvD1 blocks the action of the H2 receptor. Each receptor was modulated by phosphorylation by different kinases, while β-ARK1 was generally relatively used by both RvDs to counter-regulate histamine receptors , or timing . These distinct activities could explain why RvD1 and RvD2 and other SPMs used alone stimulate secretion, but when used before the addition of a pro-inflammatory mediator inhibit its action and block secretion. These dissimilar effects of RvD1 and RvD2 could also explain how SPMs can function in maintaining the mucous layer in health by stimulating secretion and returning the overproduction of mucins to homeostasis in inflammatory diseases by inhibiting secretion.In the present study, we observed that RvD2 and RvD1 use different protein kinases to modulate the same H receptor subtype . Among tecretion c,d. We pRvD2 is biosynthesized from one of the ω3-fatty acids docosahexaenoic acid (DHA) that is converted to 17S-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-DHA (17S-HpDHA) by 15-lipoxygenase type I (15-LOX). 17S-HpDHA next transforms to 7(8) epoxide-containing intermediate by 5-lipoxygenase (LOX) this then converted into both RvD1 or RvD2 . Both Rv2+]i increase by interacting with inositol trisphosphate (IP3) receptor on the ER leading to mucin secretion i and mucin secretion; RvD1 also activates PLA2 and PLD, that result in the activation of extracellular regulated kinase (ERK)1/2 i increase by RvD2 in both human and rat CGCs. There is an 84.89% similarity in the RvD2 receptor GPR18 sequence between humans and rats, according to the Ensembl database [2+]i, increase -5-isoquinolinesulfonamide dihydrochloride (H89) was purchased from MilliporeSigma . Ro 317549 (3-(1-(3-Aminopropyl)-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione acetate monohydrate) was purchased from Tocris Bioscience . β-ARK1 inhibitor (Methyl 5-[(E)-2-(5-nitrofuran-2-yl)ethenyl]furan-2-carboxylate) was purchased from Santa Cruz Biotechnology . 2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 10 mM HEPES, and 5.5 mM glucose (pH 7.45)] for [Ca2+]i measurements. Agonists and inhibitors were diluted in RPMI 1640 medium for secretion experiments and Krebs-Ringer bicarbonate buffer with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid i. Inhibitors were added for 15 min and resolvins 30 min before stimulation with agonists. Data are shown as the actual [Ca2+]i with time or as the change in peak [Ca2+]i, calculated by subtracting the average of the basal value from the peak [Ca2+]i.Goblet cells were passaged and seeded onto glass-bottom dishes. Cells were incubated at 37 °C with KRB-HEPES containing 0.5% bovine serum albumin, 0.5 μM Fura-2/AM, 8 μM pluronic acid F127, and 250 μM sulfinpyrazone for 1 h. Before use, cells were washed in KRB-HEPES with sulfinpyrazone. A ratio imaging system  with excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm was used to measure [Cat-test was used for comparison of two groups and one-way ANOVA with Dunnett’s multiple comparisons was used to perform statistical analysis in multiple groups. p < 0.05 was set as statistically significant.The data are presented as average ± SEM. Student’s


Ca2+ (1.00 Å) as a new B-site doping ion can successfully boost CsPbI3 NC performance with both improved phase stability and optoelectronic properties. With a Ca2+/Pb2+ ratio of 0.40%, both phase and photoluminescence (PL) stability could be greatly enhanced. Facilitated by increased tolerance factor, the cubic phase of its solid film could be maintained after 58 days in ambient condition or 4 h accelerated aging process at 120°C. The PL stability of its solution could be preserved to 83% after 147 days in ambient condition. Even using UV light to accelerate aging, the T50 of PL could boost 1.8-folds as compared to CsPbI3 NCs. Because Ca2+ doping can dramatically decrease defect densities of films and reduce hole injection barriers, the red light-emitting diodes (LEDs) exhibited about triple enhancement for maximum the external quantum efficiency (EQE) up to 7.8% and 2.2 times enhancement for half-lifetime of LED up to 85 min. We believe it is promising to further explore high-quality CsPbI3 NC LEDs via a Ca2+-doping strategy. It should be noted that the high-performance optoelectronic properties of CsPbX3 NCs are dominated by their crystal phase, especially for CsPbI3 [α) CsPbI3  exhibits good optoelectronic performance. However, the tolerance factor (τ) of α-CsPbI3 is too small to stabilize its cubic phase at room temperature. The metastable state of α-CsPbI3 can be easily transformed into the orthorhombic (δ) phase (Eg = 2.82 eV) with poor optoelectronic performance [α-CsPbI3 is the foundation for achieving high-performance CsPbI3 NC LEDs [All-inorganic perovskite  function for perovskite (ABX3), doping ions with suitable size can realize precise tuning of τ to stabilize cubic phase [α-CsPbI3. It should be noted that common methods to synthesize CsPbI3 NCs need high temperature (>170°C) [α-CsPbI3. Due to the ionic nature of CsPbI3, X-site ions are at the corner of the PbI64− octahedron, which can easily migrate. With Br− doping as an example, though CsPbBrxI3-x NCs exhibit a cubic phase, the ion migration of Br− and I− easily leads to phase separation and degradation of optoelectronic performance [64− octahedron, which can hardly migrate. Therefore, B-site doping is the efficient way to stabilize α-CsPbI3 without any side effects, such as thermal decomposition, or phase separation [α-CsPbI3 (Pb2+ radius 1.19 Å). For example, Mn2+ (0.67 Å) doping by direct synthesis or posttreatment methods can improve the stability of α-CsPbI3 NCs from a few days to nearly a month under ambient conditions [3 NCs but also boost their optoelectronic performance. Several B-site doping ions have been studied to promote the development of CsPbI3 NC LEDs, such as Mn2+ [2+ [2+ [3+ [2+ [2+ [2+ [2+, Cu2+, and Sr2+, can reduce charge injection barriers and enhance carrier transport properties. To date, the state of the art for CsPbI3 NC LEDs is facilitated by alkaline earth metal ion doping such as Sr2+ doping [2+ (1.18 Å) is almost the same as Pb2+ (1.19 Å). In other words, both stability and optoelectronic performance have more room to improve by doping other small size alkaline earth metal ions, such as Ca2+ (1.00 Å).According to the Goldschmidt tolerance factor  . As a re(>170°C) . Currentformance , whereasparation –30. On tnditions , 32. In  as Mn2+ , Zn2+ [3Mn2+ [2+ , 35, Zr2 [2+ [2+ , Y3+ [35 [3+ [2+ , Ni2+ [3 [2+ [2+ , and Sr2 [2+ [2+ –41. It s+ doping –41, whic+ doping . It shou2+ as a new B-site doping ion to boost CsPbI3 NC performance with both improved phase stability and optoelectronic properties. Doping Ca2+ to partly replace Pb2+ must increase τ. Therefore, Ca2+ doping can dramatically improve the phase stability of CsPbI3 NCs, which suppresses the decreasing PLQY resulting from phase transition [2+/Pb2+ ratios . For the case of 0.40% Ca2+/Pb2+ ratios, Ca2+-doped CsPbI3 NCs showed enhanced phase stability to long-term storage and heat. Its solid film could exhibit the cubic phase after 58-day storage in ambient condition or 4 h accelerated aging process at 120°C. Additionally, its PL intensity in solution decayed less than 17% after 147-day storage in ambient condition. Even using UV light to accelerate aging, the half-lifetime of PL could boost 1.8-folds as compared to that of CsPbI3 NCs. Furthermore, the Ca2+-doped CsPbI3 NCs were employed as the emission layer to fabricate LED. Benefitting from the Ca2+ doping, the defect densities were decreased to 21%, its valence band maximum went up to -5.55 eV to reduce hole injection barriers, and its lower Fermi level enhanced hole transport efficiency. As a result, Ca2+-doped CsPbI3 NC-based LED exhibited about triple enhancement for maximum EQE up to 7.8%, and 2.2 times enhancement for half-lifetime of LED up to 85 min. We believe it is promising to further explore high-quality CsPbI3 NC LED via Ca2+-doping strategy.Herein, we explored Caansition , 40, 42.2+-doped CsPbI3 NCs were synthesized by using different feed ratios of Ca(Ac)2/PbI2  via hot injection method . As shown in Table 2+/Pb2+ ratios for Ca2+-doped CsPbI3 NCs were determined by ICP-MS. According to ICP-MS results, the real Ca2+/Pb2+ ratios were 0%, 0.35%, 0.40%, and 1.20% for the condition of 0%, 15%, 25%, and 35% feed ratios of Ca(Ac)2/PbI2. Such results implied that Ca2+ was successfully doped into CsPbI3 NCs. The Pb2+ is located in the center of the octahedron (PbI64-). The exchange and migration of the B-site ion is more difficult as compared to that of A-site and X-site ions. Therefore, B-site ions do not easily incorporate into the CsPbI3 lattice, and the actual doping ratio of the B-site ion is much lower than the feed ratio [2+ (1.00 Å) is smaller than Pb2+ (1.19 Å), and Ca2+ doping must induce the lattice contraction, which can be confirmed by XRD. 2+-doped CsPbI3 NC films with different real Ca2+/Pb2+ ratios. The patterns for all films match well with α-CsPbI3 (PDF#97-018-1288), and the diffraction peaks at 14.09° and 28.58° correspond to the (100) and (200) planes, respectively [γ-CsPbI3 [γ-CsPbI3 from 25-30° cannot be observed. It is possible that a slight lattice distortion occurred due to the influence of water and oxygen during the test [2+/Pb2+ ratio, no additional diffraction peaks were observed, which indicated that doping Ca2+ did not significantly change the CsPbI3 crystal phase. Their fine XRD patterns are shown in α-CsPbI3 (200) plane , which verifies the lattice contraction resulted from the partial replacement of Pb2+ (1.19 Å). An enhanced peak at 20° is attributed to the (110) crystal plane for CsPbI3. Based on the HRTEM images, the cubic NCs become truncated cubic NCs  plane of α-CsPbI3 [2+ is successfully doped into CsPbI3 NCs.Caed ratio , 40, 43.ectively . It shouγ-CsPbI3 , 45, whithe test . With ins Figure . Therefom Figure , which i Figures  reveal tα-CsPbI3 . This is2+-doped CsPbI3 NCs in a high-angle annular dark field scanning transmission electron microscopy image (HAADF-STEM)  . ElementDF-STEM)  can be oDF-STEM) , which s2+-doped CsPbI3 NCs are shown in 2+-doped CsPbI3 NCs are compared in 2+ orbitals to influence the electronic structure of NCs [2+ doping, their PLQYs can be improved. With increasing Ca/Pb ratios from 0% to 0.40%, their PLQYs increased from 89% (±1.5%) to 93% (±1.3%), whereas further increasing the Ca/Pb ratio to 1.20% led PLQY lower to 91% (±1.4%). This is due to that the smaller NCs (Ca/Pb = 1.20%) with larger surface-to-volume ratio must have more surface defects. In addition, Ca2+ cannot be as emission centers. Therefore, excess Ca2+ doping may decrease PLQY. 2+-doped CsPbI3 NCs. All of the decay curves can be fitted by a double-exponential (Table 2+-doped CsPbI3 NCs are 71.76 ns (0%), 77.47 ns (0.35%), 119.47 ns (0.40%), and 92.62 ns (1.20%). Such results demonstrate that the improved τ facilitated by Ca2+ doping can reduce the lattice distortion and phase transition to maintain PLQYs at a relatively high level.The optical properties of Cae of NCs , 50, 51 al Table . The aveτ for Ca2+ doping in CsPbI3 can much improve their stability. We measured the stability of their solutions and solid films. Firstly, all samples were stored in the conditions of 20 ± 5°C and 40−50% humidity. 3 NCs decreased to 36% of the initial one, whereas PL stabilities of Ca2+-doped CsPbI3 NCs exhibited much improvement. After 147-day storage, an obvious color bleaching was observed for the CsPbI3 NC solution, while the red color of Ca2+-doped CsPbI3 NC solutions can be maintained (2+-doped CsPbI3 NC solutions can be preserved to 80% (0.35%), 83% (0.40%), and 76% (1.20%) of the initial intensities after 147-day storage , and their PL intensities decayed with UV irradiation time increasing. We periodically measured their PL spectra, which were used to calculate their half-lifetimes (T50) under UV light , 74 min (0.35%), 92 min (0.40%), and 85 min (1.20%), separately. About 1.8 times enhancement of UV stability was observed for the 0.40% Ca/Pb ratio. Further increasing UV irradiation time to 100 min, it was hard to observe the red color for CsPbI3 NCs, while Ca2+-doped CsPbI3 NCs could still emit red PL  and heating condition (120°C). As shown in α-CsPbI3 (cubic phase) film gradually converted to δ-CsPbI3 (orthorhombic phase) film after 28-day storage time, and its color changed from red to yellow  could be maintained to the cubic phase after 58-day storage, which exhibited good PL performance /TPBi (40 nm)/LiF (1 nm)/Al (100 nm) shown in Figure 2+-doped CsPbI3 NCs (Ca/Pb = 0.40%) exhibited obviously enhanced current density, which implied the decreasing of defect density as well as the improvement for carrier transport efficiency.To identify the positive effect of Ca3 NCs or Ca2+-doped CsPbI3 NCs/MoO3/Al  is calculated according to the following equation:ε0 and ε are the vacuum dielectric constant and the relative dielectric constant, respectively; VTFL is the trap-filled limit voltage; L is the thickness of the NC film; and e is the elementary electronic charge. By assuming that ε = 6.3 [3 and Ca2+-doped CsPbI3 NC films were 0.71 × 1017 and 0.15 × 1017 cm−3, respectively. This means that the defect density could be decreased to 21.1% facilitated by 0.40% Ca2+ doping. Such results match with those results of PLQY and TRPL. In addition, the hole migration rates were estimated by fitting the space-charge-limited-current region (SCLC) with Child's law:ε0 is the vacuum permittivity; εr is the average relative dielectric constant of CsPbI3 (εr ≈ 6.32); L is the thickness of the perovskite film; and J, μ, and V are the measured current density, carrier migration rates, and applied voltage, respectively [3 and Ca2+-doped CsPbI3 NC films were 2.8 × 10−8 and 6.0 × 10−8 cm2V−1 s−1, respectively. With the Ca2+ doping, the hole mobility increased, which enhanced hole transport efficiency.Firstly, hole-only devices with a structure of ITO/PEDOT:PSS/CsPbIl Figure  were use ε = 6.3 , the defectively . The holg) for Ca2+-doped CsPbI3 NC film , its CBM, VBM, and Fermi level shifted to -3.73 eV, -5.55 eV, and -4.05 eV, respectively. The higher VBM reduces the energy barrier between PEDOT:PSS and the emission layer to boost hole transport efficiency. Additionally, the lower Fermi level reveals the transformation of CsPbI3 NCs from n-type to a more nearly ambipolar nature facilitated by Ca2+ doping, which also enhances hole transport efficiency [Then, the improvement for carrier transport efficiency can be confirmed by ultraviolet photoelectron spectra (UPS). Combining with their optical bandgaps , which was doubled as compared to the nondoping one. In addition, the maximum and average EQEs for Ca2+-doped CsPbI3 NC (0.40%) LEDs were 7.8% and 6.3%, both of which enhanced to about 3 times as compared to the nondoping one, respectively  LEDs could significantly increase to 85 min. A 2.2-fold improvement for the half-lifetime of LED confirmed that Ca2+ doping is a powerful strategy to promote both stability of CsPbI3 NCs and their LEDs. In a word, the boosting of both the efficiency and the stability of LEDs is mainly attributed to a decrease in defect density, improvement on hole injection efficiency, and reduction in phase transition.Benefitting from the enhanced current density, the Cage range . In the ectively . Therefong Table . Their eng Table . A brighng Table , and itsng Table . Further3 NCs based on Ca2+ (1.00 Å) doping. With a Ca2+/Pb2+ ratio of 0.40%, the phase stability could be greatly enhanced. Ca2+-doped CsPbI3 NC solid films could maintain the cubic phase after 58-day storage in ambient condition or 4 h accelerated aging process at 120°C. Moreover, the PL stability could be also improved. The PL intensity of Ca2+-doped CsPbI3 NC solutions could be preserved to 83% after 147-day storage in ambient condition. Even using UV light to accelerate aging, the T50 of PL could be boosted 1.8-folds as compared to that of CsPbI3 NCs. Red LED based on Ca2+-doped CsPbI3 NCs exhibited about triple enhancement for maximum EQE up to 7.8% and 2.2 times enhancement for half-lifetime of LED up to 85 min. These were mainly attributed to the decreased defect densities of films and reduced hole injection barrier facilitated by Ca2+ doping. Ca2+ as a new B-site doping ion can efficiently boost both stability and performance for CsPbI3 NC LEDs, which has the potential to promote the development of CsPbI3 NC LEDs.In summary, we explored stable and high-performance CsPbI2CO3, 99.99%), calcium acetate (Ca(Ac)2, 95%), and lead iodide  were purchased from Aladdin. Poly:poly(4-styrenesulphonate)  was purchased from Heraeus Materials Technology Co. Ltd. 2,2′,2″--tris(1-phenyl-1-H-benzimidazole) (TPBi) was purchased from Nichem Fine Technology Co. Ltd. All the chemicals were directly used without further purification.Oleic acid , 1-octadecene , methyl acetate , hydriodic acid  oleylamine , cesium carbonate , Ca(Ac)2 , and 10 mL ODE were mixed into a 100 mL three-neck flask. The mixture was degassed and dried in vacuum for 1 h at 120°C. Then, 1.0 mL OLA, OA, and preheated OLA-HI were injected into the reaction flask, separately. The mixed solution became clear and was kept under vacuum for 30 min at 120°C. Finally, the temperature was increased to 260°C, and 1.0 mL Cs-OA solution was injected immediately. After 1 min, the reaction flask was placed into an ice-water bath to stop the reaction.In a typical synthesis, PbIThe as-prepared NC solution was mixed with the same volume of MeOAc and was centrifuged at 15000 rpm for 5 min at 19°C to remove the supernatant. The precipitate was redispersed into toluene. Then, the NC toluene solution was mixed with the same volume of MeOAc and was centrifuged at 15000 rpm for 5 min at 19°C. After removing the supernatant, the precipitate was dispersed into toluene. Finally, the redispersed NC solution was centrifuged at 15000 rpm for 5 min at 19°C to discard the precipitate, and the supernatant was preserved for characterization and device fabrication.−4 Pa). The active area of the devices was 10 mm2 as defined by the overlapping area of the ITO and Al electrodes.Indium tin oxide- (ITO-) coated glass substrates were cleaned by ultrasonic cleaning for 15 min in acetone, ethanol, and deionized water, separately, and dried with nitrogen flow. The clean ITO glasses were treated by UV-ozone for 15 min. Then, PEDOT:PSS solutions were spin-coated onto the ITO substrates at 3000 rpm for 30 s and annealed at 120°C for 15 min in air. NCs were spin-coated onto the PEDOT:PSS layer at 1500 rpm for 30 s. This process was repeated five times. Finally, 40 nm of the TPBi layer, 1 nm of the LiF layer, and 100 nm of Al electrode were deposited in sequence by a thermal evaporation system under a high vacuum (∼2 × 10α radiation (λ = 0.154 nm). Photoluminescence fluorescence quantum yield (PLQY), which is defined as the ratio of emitted photons to absorbed ones, was determined by a FLS920 fluorescence spectrometer equipped with an integrating sphere. The morphology and size of NCs were confirmed by transmission electron microscope (TEM) , high-resolution TEM (HRTEM) , and energy-dispersive X-ray spectroscopy (EDS). The electroluminescent (EL) spectra and luminance- (L-) current density- (J-) voltage (V) characteristics were collected by using a Keithley 2400 source and PR-655 spectra scan spectrophotometer (Photo Research). The characterization for LED devices was measured at room temperature in air.The ultraviolet-visible (UV-Vis) absorption spectra of NC solutions were measured by a PerkinElmer Lambda 35S instrument in transmission mode. PL spectra were collected by a RF6000 spectrofluorometer with an excitation wavelength of 500 nm. The PL lifetimes of NCs were measured by a FLS920 fluorescence spectrometer with a pulse laser at 375 nm. The chemical compositions were measured by a PerkinElmer NexION 2000 inductively coupled plasma mass spectrometry (ICP-MS). X-ray diffraction (XRD) data were collected by a Bruker D8 Advance X-ray powder diffractometer with Cu K


The apoptosis of lens epithelial cells plays a key role in the development of cataract. We investigated a potential role for transient receptor potential vanilloid 2 (TRPV2) in the development of diabetic cataracts. Immunohistochemical and Western blotting analyses showed that TRPV2 expression levels were significantly increased in the lens epithelial cells of patients with diabetic cataracts as compared with senile cataract, as well as in both a human lens epithelial cell line (HLEpiC) and primary rat lens epithelial cells (RLEpiCs) cultured under high-glucose conditions. The [Ca2+]i increase evoked by a TRPV2 channel agonist was significantly enhanced in both HLEpiCs and RLEpiCs cultured in high-glucose media. This enhancement was blocked by the TRPV2 nonspecific inhibitor ruthenium red and by TRPV2-specific small interfering (si)RNA transfection. Culturing HLEpiCs or RLEpiCs for seven days in high glucose significantly increased apoptosis, which was inhibited by TRPV2-specific siRNA transfection. In addition, ROS inhibitor significantly suppressed the ROS-induced increase of TRPV2-mediated Ca2+ signal and apoptosis under high-glucose conditions. These findings suggest a mechanism underlying high-glucose–induced apoptosis of lens epithelial cells, and offer a potential target for developing new therapeutic options for diabetes-related cataracts.Cataracts are a serious complication of diabetes. In long-term hyperglycemia, intracellular Ca To further assess the role of TRPV2 in this enhanced Ca2+ influx, we used specific siRNA to inhibit TRPV2 expression. Transfection of TRPV2 siRNA significantly inhibited TRPV2 expression and 2-APB–induced Ca2+ influx under both NG and HG conditions, but the inhibition was stronger in the HG medium. In addition, the HG culture significantly enhanced ROS production, and the ROS inhibitor significantly suppressed ROS-induced increase of TRPV2-mediated Ca2+ signal and apoptosis under high-glucose conditions. These findings suggest that an HG environment stimulates ROS production, which enhances TRPV2 expression. High expression of TRPV2 in lens epithelial cells, and the resulting enhanced Ca2+ influx, leads to intracellular Ca2+ homeostasis and signal transduction abnormalities and Ca2+ overload. Eventually, the apoptosis of lens epithelial cells is increased, and may be related to the development of diabetes-related cataracts.In the present study, we first found elevated expression of the TRPV2 channel protein, which is primarily permeable to Ca2+ concentration in the diabetic lens is increased, and that Ca2+ antagonists exhibit anticataract properties i in lens epithelial cells through the SOCE pathway in a high-glucose environment [2+ influx was suppressed by a SOCE inhibitor, we still recorded an increased TRPV2-mediated Ca2+ influx in the HG environment. Thus, besides Orai3, TRPV2 may also involve in HG-induced Ca2+ overload in lens epithelial cells.Cataracts are the leading cause of blindness in the world, and people with diabetes are at higher risk of both cataract occurrence and more rapid progression . Previouoperties ,12,22,23Previous studies have shown that TRPV2 has important roles in the proliferation, invasion, and apoptosis of tumor cells ,25,26,272+ overload, and following cell apoptosis in lens epithelial cells. TRPV2 may be a potential target in high-glucose environment-induced lens epithelial cell apoptosis and diabetic cataracts. However, in the present study, we also found that under NG-cultured conditions, ROS and TRPV2 were both not significantly suppressed by antioxidant, but 2-APB-induced Ca2+ increase was dramatically reduced. The phenomenon may be caused by some TRPV2 activation component suppression in Tempol treatment, but further study is needed in the future.Increased blood glucose in diabetes induces oxidative stress in cells, and ROS has been reported to increase the expression of TRPV2, which mediates cell death in human hepatoma cells ,34. Our 2+ overload in an HG environment, suggesting that TRPV2 may be one of key ion channels mediating Ca2+ influx in lens epithelial cells, and may provide a potential therapeutic target for diabetic or HG-induced cataracts.In conclusion, our study showed that TRPV2 expression was significantly increased in the epithelial lens tissue of patients with diabetic cataracts, and that enhanced ROS induced lens epithelial cell apoptosis by upregulating TRPV2 expression and TRPV2-mediated Ca


Contrast media in CT is widely used in dogs and cats to provide superior tissue delineation and increase the diagnostic capabilities. These contrast‐enhanced imaging techniques are gaining popularity in rabbits; published studies reporting the safety of doing so are lacking.This retrospective observational study aimed to determine the incidence of adverse events following the intravenous administration of iodinated non‐ionic contrast medium in 350 rabbits. The medical records of this subset of rabbits admitted between January 2009 and November 2018, that underwent CT examination and received intravenous contrast media, were evaluated.From the 350 rabbits, 342 rabbits were still alive 7 days after the scan. A total of eight rabbits died within 7 days of the scan, seven of which within the first 24 h. All deaths were presumed to be sequelae to the diseases they were being investigated for and not thought to be related to the administration of intravenous contrast medium.The results indicated that the use of intravenous non‐iodinated contrast medium was well tolerated and safe in rabbits undergoing conscious CT examination. Oryctolagus cuniculus) as companion animals has increased in recent years,The popularity of domestic rabbits  School of Veterinary studies, for pet rabbits that underwent a CT study for diagnostic purposes between January 2009 and November 2018. This study was approved by the Ethical Committee at the University of Edinburgh (Veterinary Ethics and Welfare Committee reference 70.19).For rabbits meeting inclusion criteria, the following medical record data were recorded: age at time of imaging, breed, sex, weight at time of imaging, date of imaging, contrast medium administration and if deceased within 7 days after the CT scan, cause of death and results of a postmortem examination if available.Images were acquired with the use of two different CT scanners: a four‐row Multi‐detector row computed tomography (MDCT) unit  from January 2009 to October 2016 and a 64‐row MDCT unit  from October 2016 to November 2018. The cross‐sectional images were acquired on conscious rabbits, restrained within a device, the VetMouseTrap. Scan settings included a pitch of 1.5, tube potential of 120 kVp, reference tube current of 160 mA, slice thickness of 1.5 mm, matrix 512 × 512, and reconstruction with low and high‐frequency algorithms. A bolus of 740 mg iodine/kg of non‐ionic iodinated contrast medium  was injected through an angiocatheter placed within the marginal auricular vein. The rabbits examined from 2009 to 2015, had the contrast agent administered manually, followed by 1.5 ml of saline solution flush and post‐contrast images were acquired within 1 min after the contrast administration. From October 2016 onwards, the contrast was administered via a power injector pump  with a flow velocity of 0.8 ml/s and post‐contrast images were acquired within 18 s of contrast administration.N = 26; 2 years—N = 41; 3 years—N = 55; 4 years—N = 44; 5 years—N = 47; 6 years—N = 45; 7 years—N = 44; 8 years—N = 29; 9 years—N = 13; 10 years—N = 6) with a mean age of 4.8 years. There was no significant sex bias with 180 males (147 neutered and 33 entire) and 170 females (139 neutered and 31 entire).In total, 350 rabbits were included in the study and received intravenous contrast medium. All of the rabbits were scanned consciously in a purpose‐built restraint device. A variety of breeds were represented: dwarf lop, mini lop, lionhead, Dutch, French lop and mini rex. The age range of the rabbits varied between 1 and 10 years (1 year—N = 350), with no registered immediate adverse reactions or sudden death. From the 350 rabbits, 40 (≅11%) were euthanised within 7 days of the CT scan due to poor prognosis based on clinical deterioration and/or imaging findings which included neoplasia, severe dental disease, complex femoral fractures and severe spinal disease.The contrast medium administration was well tolerated in all the individuals  died within 7 days of the intravenous administration of the contrast medium. Of these eight rabbits, no deaths were believed to be linked directly to the administration of the contrast medium. Rabbits A–G died within 24 h of contrast administration, while Rabbit H died 6 days later. Rabbit A, diagnosed with a liver lobe torsion, died in recovery from emergency surgery for a haemo‐abdomen and liver lobe resection. Haemodynamic instability was believed to be a main factor. Rabbit B presented with non‐specific signs of gastrointestinal stasis and was referred for advanced imaging. Postmortem examination led to a diagnosis of rabbit haemorrhagic disease virus strain 2 (RHDV‐2). Rabbit C underwent CT evaluation for investigation of a suspected retrobulbar abscess. Death resulted after iatrogenic administration of penicillin by an intravenous route. Rabbits D and E both died following complications during a general anaesthetic, performed after the CT studies. Rabbit D underwent a CT scan to investigate the presence of gross metastatic disease, pre‐neutering, following palpation of a uterine mass. There were no gross signs of metastases and the ovariohysterectomy went ahead the following day, however the animal deteriorated when recovering from the general anaesthesia. Rabbit E underwent successful resuscitation following cardiac arrest during a general anaesthetic for a dental procedure. After recovery, the rabbit underwent a conscious CT evaluation and was diagnosed with lung pathology and serosanguinous pleural effusion, which was drained under ultrasound guidance. Respiratory signs continued to worsen within 24 h and the animal collapsed for a second time, without successful resuscitation. Rabbits F and G both presented with neurological signs and CT was performed to evaluate the brain, middle and internal ears. With no structural abnormalities noted, a presumptive diagnosis of N = 76) and the rabbits scanned between 2016 and 2018 (N = 274), received the contrast via power injector pump. Two of the 76 rabbits (≅2.6%) and five of the 274 rabbits (≅2%) died acutely, within 24 h after the CT examination. One of the 76 rabbits (≅1.3%) died 6 days after CT examination, due to colonic obstruction, confirmed on postmortem examination.The rabbits examined between 2009 and 2015, received the intravenous contrast media administered manually .With the development of non‐ionic low‐osmolar iodinated contrast media, the incidence of adverse reactions in human patients has greatly diminished and about 70% of the reported adverse reactions are dermatological and self‐limiting, with manifestations of pruritus and urticaria.Chronic kidney disease is the most well‐known risk factor for the use of iodinated ionic contrast agents in humans, yet other recognised conditions can increase the risk of adverse reactions, including diabetes, cardiac disease (particularly coronary artery disease) and dehydration.In humans, asthma, allergic conditions and atopic skin disease appear to correlate with an increased risk of contrast medium‐related reactions.Repeated use of contrast media has been documented in human literature as a possible predisposing factor to hypersensitivity reactions; this is a risk that may be higher when ionic contrast agents are used (17%–35%) in comparison to the risk of using non‐ionic agents (5%).Limitations of the present study include its retrospective nature and the lack of data such as pre‐ and post‐contrast administration serum biochemical analysis, haematology, heart rate and blood pressure values. In one study,The concentration of the contrast agent used was the same for all the rabbits. However, there was a bimodal administration of the contrast, performed both manually and via a pressure injector pump, which also poses as a limitation for the present study. The rate of the bolus injection should be standardised to allow the appropriate enhancement pattern of the various organs and potential lesions. It has been suggested that higher speed injections are linked to higher rates of contrast reactions in humans.The results of our study suggest that the intravenous administration of iodinated non‐ionic contrast media is well tolerated and safe in rabbits undergoing conscious CT examination. The incidence of adverse events and mortality within 7 days after the administration of the contrast agent in rabbits was low. The risk of developing adverse events following the administration of non‐ionic contrast media may be higher in rabbits with cardiovascular and renal diseases; further prospective studies are needed to evaluate the degree of correlation.The authors declare they have no conflicts of interest.The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. The research was approved by the Ethical Committee at the University of Edinburgh (Veterinary Ethics and Welfare Committee reference 70.19).


With improvements in acute myeloid leukemia (AML) diagnosis and treatment, more patients are surviving for longer periods. A French population of 9453 AML patients aged ≥15 years diagnosed from 1995 to 2015 was studied to quantify the proportion cured (P), time to cure (TTC) and median survival of patients who are not cured (MedS). Net survival (NS) was estimated using a flexible model adjusted for age and sex in sixteen AML subtypes. When cure assumption was acceptable, the flexible cure model was used to estimate P, TTC and MedS for the uncured patients. The 5-year NS varied from 68% to 9% in men and from 77% to 11% in women in acute promyelocytic leukemia (AML-APL) and in therapy-related AML (t-AML), respectively. Major age-differenced survival was observed for patients with a diagnosis of AML with recurrent cytogenetic abnormalities. A poorer survival in younger patients was found in t-AML and AML with minimal differentiation. An atypical survival profile was found for acute myelomonocytic leukemia and AML without maturation in both sexes and for AML not otherwise specified (only for men) according to age, with a better prognosis for middle-aged compared to younger patients. Sex disparity regarding survival was observed in younger patients with t-AML diagnosed at 25 years of age (+28% at 5 years in men compared to women) and in AML with minimal differentiation (+23% at 5 years in women compared to men). All AML subtypes included an age group for which the assumption of cure was acceptable, although P varied from 90% in younger women with AML-APL to 3% in older men with acute monoblastic and monocytic leukemia. Increased P was associated with shorter TTC. A sizeable proportion of AML patients do not achieve cure, and MedS for these did not exceed 23 months. We identify AML subsets where cure assumption is negative, thus pointing to priority areas for future research efforts. Acute myeloid leukemia (AML) is an aggressive cancer currently classified into 23 morphology codes in the latest WHO classification of tumors of hematopoietic and lymphoid tissues . The worSince the year 2000, AML has been categorized into subtypes that take into account differences in clinical presentation, disease biology, prognosis and, for some, treatment guidelines ,6. AlthoThe concept of statistical cure assumes that a part of the patient population will not experience mortality due to the diagnosed cancer. In other words, the mortality observed in this group of patients becomes equal to that observed in the general population. Simultaneous analysis of the proportion of “cured” patients, the time to cure and the median survival of uncured AML patients provides a more detailed assessment of the progress in diagnostic and/or therapeutic management in a given population . In thisThis retrospective population-based study included all AML cases diagnosed between 1 January 1995 and 31 December 2015 in patients aged ≥15 years and recorded in the database of the French Network of cancer registries (FRANCIM). The diagnosis of AML was defined according to the third edition of the International Classification of Diseases for Oncology (ICD-O-3) . Cases dTo ensure sufficient statistical power, only AML subtypes and sex combinations for which the number of cases at diagnosis was higher than 500 or for which the number of deaths within 10 years after the diagnosis was higher than 130 were considered for survival analyses using a strategy to check for complex effects related to age. In situations where the number of deaths within 10 years after the diagnosis was between 70 and 130, an adapted strategy was made to simplify the model by adapting the number of parameters according to the number of deaths see . AML subNet survival at time t since diagnosis (NS(t)) was estimated using the flexible parametric model (without cure assumption) of the cumulative excess mortality rate proposed by Nelson et al. . The comFor each AML subtype and sex, the assumption of statistical cure was accepted when the NS curve reached a plateau and the EMR approached zero within 10 years (less than 0.05). When the assumption of statistical cure was acceptable, the cumulative EMR was modeled using the flexible parametric cure model proposed by Andersson et al. . The modThirdly, we estimated the median survival (MedS) of “uncured” patients, corresponding to the time since diagnosis at which the net survival (NS) of uncured patients reached 50%.NS estimated from both models described above (without and with cure assumption) was compared to NS obtained using the non-parametric estimator proposed by Pohar Perme . EMR estTM, version 15 .All statistical analyses were performed using STATAThe study cohort comprised 9453 patients diagnosed with AML classified into the following subtypes: AML-NOS (2201 patients), AML with maturation , AML-RCA (981 patients), AML-MRC (969 patients), acute myelomonocytic leukemia , AML without maturation , acute monoblastic and monocytic leukemia , t-AML (613 patients), APL (575 patients), AML-RCA (406 patients) and 961 cases for seven other AML subtypes overall . A slighFor the whole AML study population, except for APL and AML-RCA, the EMR was very high just after the diagnosis, which implied that NS dropped quickly during the first year and stabilized thereafter . The 10-Modeling the influence of age  showed aIn older patients (75 years old), diagnoses of AML-M4 and AML-M6 in men and diagnoses of AML-MRC, AML-M0, AML-M6 and AML-NOS in women presented the worst prognosis . In the Associations between age and excess mortality differed according to AML subtype. Three situations were observed in men: (1) a non-significant non-linear effect was observed in AML-MRC, t-AML, AML-M6 and AML-M0 (NS decreased linearly with increasing age at diagnosis); (2) a significant non-linear effect in APL, AML-RCA, AML-M2, AML-M5 and AML-NOS was observed, as NS was relatively similar from 15 to 45 years of age and decreased thereafter; (3) a significant non-linear effect was observed in AML-M4 and AML-M1, as NS was the highest for middle-aged compared to both younger and older patients .Note that in women, the effect of age was similar to that observed in men, except in AML-NOS .p = 40%; 95% CI 25–52 in men and p = 55%; 95% CI 41–68 in women). P decreased linearly with age in AML-MRC, AML-M6, AML-M0 and AML-NOS (for women), while age had a non-linear effect on P in other AML subtypes. In men and women with AML-M1 and in men with AML-M4, P was higher in patients aged 50 years at diagnosis  than it was in younger or older patients  to 31% , respectively, in men, and from 90%  to 10% , respectively, in women . In oldepatients . Except The TTC was always less than 10 years regardless of AML subtype, sex and age. For patients aged 25 years, TTC varied from 2 years  for APL to 8 years  for AML-M2 in men and from 1 year  for APL to 7 years  for t-AML in women . For patAccording to our assumption of cure testing, a large proportion of patients with a diagnosis of AML will not be “cured”. The MedS for “uncured” young patients (25 years) varied in men from less than 1 month for APL to 23 months  for AML-M2, and, in women, from 6 months  for t-AML to 21 months  for AML-M2. No major change in MedS was observed in the elderly (75 years old); MedS was less than 5 months regardless of AML subtype and sex .According to sex, the largest difference in the MedS for “uncured” patients was observed in the youngest patients with t-AML, varying from 17 months in men to 6 months in women .In this study, by using flexible modeling methods, we estimated the long-term net survival and proportion of cured patients according to AML subtype diagnosed during the 1995–2015 period in a French population-based cohort. AML survival and cure indicators varied according to AML subtype, age at diagnosis and sex.Our results highlight various phenomena in AML. First, we confirmed that long-term survival is different according to AML subtype, with APL presenting the best prognosis. These results highlight progress in APL management with better knowledge of cytogenetic abnormalities and disease biology and the use of tailored treatments particularly ATRA/ATR as standard of care from the early 2000s ,19.The worst situation was observed in t-AML and AML-MRC. AML-NOS, probably because of its less-defined molecular profile, also presented poor prognosis. Our results for AML-NOS and AML-RCA are comparable to those observed in the Surveillance Epidemiology and End Results (SEER) database in the USA .Taken together, t-AML, AML-MRC and AML-NOS represent 40% of AML cases, and thus, new clinical strategies need to be developed. Several clinical trials using new combination of chemotherapy report encouraging results in these entities ,22.A second point raised by our study is the comparatively poor survival of older patients. Even in AML-RCA, where specific treatment strategies are available, the 10-year NS did not exceed 11% in older patients (75 years). The presence of multiple comorbidities in the oldest patients could make access to more aggressive treatment difficult . NeverthMore surprisingly, an atypical survival trend by age was found for AML-M4 and AML-M1 in both sexes and for AML-NOS (only for men), in which age had a non-linear effect on excess mortality, with a better prognosis for middle-aged than for both younger and older patients. We found also poor survival in younger patients with t-AML and AML-M0. Progress has been made in the outcome of childhood and young adult AML. While the AML outcome of infants (under the age of one year) was worse than that of older children, there have been improvements in recent years. Notably, the use of allogeneic hematopoietic stem cell transplantation in first complete remission has contributed to improved outcome of these particular patients compared to the oldest childhood patients . HoweverOur analysis highlights sex differences in the prognosis of younger AML patients. This appears to be in keeping with results from Hossain et al., who have already found a significant sex effect on the survival of pediatric and young adult AML patients in the US population, with a substantially increased risk of death in younger male patients diagnosed with APL and AML-inv (16) (20–24 years age) .The most surprising results in our study were observed in younger patients with t-AML, where better prognosis was seen in men compared to women  vs. 12% , respectively) and MedS of “uncured” patients from 17 months  in men to 6 months  in women, with P being similar in both sexes. A UK population-based study of patients diagnosed with AML in the period 2004–2013 showed that men with t-AML show better survival compared to women  in men and 1.2  in women) . In a USThe major finding of our study was the quantification of the number of AML patients that can be considered as cured. The marked decrease in the excess mortality rate observed 2 years after diagnosis, leading to relatively stable survival thereafter for the 10-year follow up period, allowed to accept the cure assumption in each AML subtype for a part of patients with different age groups. This could be demonstrated by using a flexible parametric tool that allows to keep the quantitative form of covariates while considering their complex effects. In the cure framework, several models were developed, but the flexible cure model proposed by Andersson et al. appears to be the most appropriate tool, especially in older cohorts . CurrentIn several AML subtypes, age groups concerned by cure are not the same according to sex. In AML-M0, one in two of the younger female patients are considered as cured (P = 44% in those aged 25), whereas the assumption of cure was not accepted in younger men. In patients aged 25 years with t-AML, P ranging from 10% to 31% in women and men, respectively. Even if few patients are diagnosed at an early age, few hypotheses could explain these sex differences.Even if concerning a minority of AML patients, the finding that a proportion is considered as “cured” during their follow-up is of great importance. This is particularly true in the youngest patients, where it could help them to recover a normal life, at least regarding social status and access insurance and finance.We observed an inverse relationship between the TTC and the proportion of “cured” patients: the highest P was found in patients with a shorter TTC. This important finding is likely to be related to changes in clinical practice relating to studies that report the importance of a rapid decrease of leukemic blasts in blood and bone marrow, as measured by flow cytometry, to reach complete remission, sustained control or clearance of minimal residual disease and consequentlong term event-free survival ,33. The Complete remission was obtained in 60% of AML cases, and of these, 59% relapsed with a median duration of first complete remission of 7.2 months. The median all-cause survival from relapse was 6 months . The allStudies conducted in different European countries have shown disparities in AML cure and survival of uncured patients. In Sweden, for example, an increase in the proportion of “cured” patients and a similar age disparity for cure as in our study were found, but the MedS in “uncured” patients was higher for younger patients than that in France . By contp < 0.001) [In order to indirectly appreciate the impact of changing clinical practices on survival, we conducted a specific survival analysis by period of diagnosis, splitting the database into two periods of diagnosis: 1995–2005 and 2006–2015. This analysis strategy did not show significant differences in 5-year AML NS between the two study periods by either AML subtype or sex for all ages. A previous study based on population-based cancer registries in Switzerland between 2001 and 2013 found modestly improved survival over the period of diagnosis for all ages, with significantly improved outcomes only being seen in patients aged 65–74 years  . These r< 0.001) . This go< 0.001) .Our work allowed to identify AML subtypes where cure assumption is negative and/or settings where no progress has been achieved in AML cure or survival for uncured patients, thus pointing to priority areas for future research efforts. Mutational profiling has been integrated into AML classification  and in c