We investigated the effects of ArgII activity on mitochondrial Ca2+ uptake through mitochondrial p32 protein (p32m) and on CaMKII-dependent vascular smooth muscle cell (VSMC) contraction. Native low-density lipoprotein stimulation induced an increase in [Ca2+]m as measured by CoCl2-quenched calcein-AM fluorescence, which was prevented by Arg inhibition in hAoSMCs and reduced in mAoSMCs from ArgII−/− mice. Conversely, [Ca2+]c analyzed with Fluo-4 AM was increased by Arg inhibition and ArgII gene knockout. The increased [Ca2+]c resulted in CaMKII and MLC 20 phosphorylation, which was associated with enhanced vasoconstriction activity to phenylephrine (PE) in the vascular tension assay. Cy5-tagged siRNA against mitochondrial p32 mRNA (sip32m) abolished mitochondrial Ca2+ uptake and induced activation of CaMKII. Spermine, a polyamine, induced mitochondrial Ca2+ uptake and dephosphorylation of CaMKII and was completely inhibited by sip32m incubation. In mAoSMCs from ApoE-null mice fed a high-cholesterol diet (ApoE−/− +HCD), Arg activity was increased, and spermine concentration was higher than that of wild-type mice. Furthermore, [Ca2+]m and p32m levels were elevated, and CaMKII phosphorylation was reduced in mAoSMCs from ApoE−/− +HCD. In vascular tension experiments, an attenuated response to vasoconstrictors in de-endothelialized aorta from ApoE−/− +HCD was recovered by incubation of sip32m. ArgII activity-dependent production of spermine augments Ca2+ transition from the cytosol to the mitochondria in a p32m-dependent manner and regulates CaMKII-dependent constriction in VSMCs.Although arginase II (ArgII) is abundant in mitochondria, Ca Researchers have illuminated how a protein, arginase II (ArgII), is involved in development of vascular diseases such as atherosclerosis, or narrowing of the arteries by plaque deposits. Blood vessel diameter is regulated by layers of muscle; the balance between constriction and relaxation is critical for blood flow and vascular health. Increased ArgII is known to be a factor in arterial disease; however, the details of regulation, and how they relate to plaque deposition, remain poorly understood. Sungwoo Ryoo at Kangwon National University, Chuncheon, South Korea and co-workers investigated how ArgII levels affect arterial constriction and relaxation in mice. Decreasing ArgII restored the muscle cells’ contraction response by preventing excessive calcium accumulation in the cellular powerhouse, mitochondria. These results may aid in developing treatments for one of the leading causes of death worldwide. Arginase I (ArgI) is found in the cytosol, and arginase II (ArgII) is targeted to the mitochondria. Although ArgI can stimulate proliferation through a polyamine-dependent mechanism in aortic smooth muscle cells6, the mechanism underlying the role of ArgII in mitochondrial function is yet unknown. We previously demonstrated that ArgII played an important role in the regulation of mitochondrial Ca2+ concentration in native low-density lipoprotein (nLDL)-treated hAoSMCs, although we did not define the target protein7. Together with the endoplasmic reticulum, the mitochondria play a critical role as a Ca2+-handling organelle in cells, and mitochondrial Ca2+ influx from the cytosol affects cellular physiology and pathophysiology through contributions to the spatial and temporal profiles of intracellular Ca2+ signaling.Arginase hydrolyzes L-arginine to urea and L-ornithine, a precursor for polyamines, including spermine, spermidine, and putrescine. Two isoforms of arginase are found in a variety of cells and may be expressed in species- and tissue-specific manners2+ binding sites and is a major Ca2+ sensor and regulatory protein. In vascular smooth muscle cells (VSMCs), increased cytosolic concentration of Ca2+ ([Ca2+]c) activates the multifunctional Ca2+/calmodulin-dependent kinase II (CaMKII)8. The activated CaMKII provokes vascular smooth muscle cell (VSMC) contraction through interactions with myosin light chain kinase (MLCK), leading to MLC phosphorylation and actin-myosin interaction10. Furthermore, increased levels of intracellular Ca2+ may function as a second messenger to activate other signaling molecules13.Calmodulin contains four EF-hand Ca14, as hyaluronan-binding protein 1 (HABP1), as receptor for globular head domains complement 1q (gC1qR), or as complement 1q binding protein (C1qbp)15. Moreover, p32 is a conserved eukaryotic protein that exists in multiple subcellular compartments18. However, recent studies have demonstrated that p32 is primarily targeted to the mitochondria because the 73 N-terminal amino acids contain a mitochondrial localization sequence19. Mitochondrial p32 protein has been studied previously in cancer development in the context of mitochondrion morphology20, the maintenance of oxidative phosphorylation21, and ARF-induced apoptosis22. However, the primary function of p32 in the mitochondrion is yet unknown.Initially, p32 was characterized as a pre-mRNA splicing factor SF-2 associated protein6, the protein regulated by arginase activity and its function in the mitochondrion are not yet known. To determine the physiological contribution of ArgII activity to vasoconstriction in VSMCs from humans and mice, we investigated how ArgII activity modulates [Ca2+]c and [Ca2+]m through p32. Furthermore, we tested the underlying mechanism of p32-dependent vasoconstriction in de-endothelialized vessels.Although arginase activity in VSMCs provokes cell proliferation in a polyamine-dependent mannerWe purchased 2(S)-amino-6-boronohexanoic acid (ABH) from Calbiochem . Antibodies against p-CaMKII (Cat. No. 12716), CaMKII (Cat. No. 3362), MLC20 (Cat. No. 3672), and p-MLC20 (Cat. No. 3674) were purchased from Cell Signaling Technology . Arginase II (sc-20151), eNOS (sc-645), HSP60 (sc-1052), and α-actin (sc-56459) antibodies were purchased from Santa Cruz Biotechnology . p32 antiserum was obtained from Abcam, Co. . siRNA against arginase II  and scramble RNA  were purchased from Santa Cruz Biotechnology. All reagents were purchased from Sigma  unless otherwise stated.−1) was prepared from the plasma of normocholesterolemic subjects by differential ultracentrifugation as previously described7. Isolated nLDL did not exhibit oxidative modification within 3 weeks compared with the oxidation of commercial nLDL and oxidized LDL from Intracel , as determined by thiobarbiturate-reactive substance assays using malondialdehyde as a standard.The nLDL  for 1 hr to remove the endothelial cells. The de-endothelialized aortic pieces were incubated with culture medium on gelatin (0.1%)-coated culture dishes for approximately 10 days. The cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 8 mM HEPES, and 2 mM glutamine at 37 °C in a humidified 5% CO2 incubator. Before all experiments, isolated mAoSMCs at passage numbers 1–3 were maintained for 24 h in starvation medium to maintain their contractile phenotype. mAoSMCs were identified by their ‘spindle-shaped’ pattern and further confirmed by double staining using platelet endothelial cell adhesion molecule-1, a specific marker for endothelial cells, and α-smooth muscle actin, a specific marker for SMCs. All cells were stained with anti-α-smooth muscle actin antibodies. Each experiment was performed in at least two different batches of VSMCs depending on the availability of the cells.Human aortic smooth muscle cells (hAoSMCs) from four different batches were purchased from Lonza, Co. , maintained in medium 231 supplemented with SMGS  according to the manufacturer’s protocol and treated with nLDL  after incubation in starvation medium  for 24 h. mAoSMCs of at least eight different batches were isolated from the thoracic and upper parts of the abdominal aorta of 10-week-old male mice (C57BL/6) as previously described−/− mice with a C57BL/6 background were gifted generously by Prof. Jaye Chin-Dusting to establish a colony. Mice were anaesthetized by 2.5% isoflurane in O2 and subjected to the following experiments. Aortic rings were studied from ten 10-week-old male C57BL/6 WT mice fed a normal diet (ND) and ten 10-week-old male ApoE−/− mice  fed a high-cholesterol diet  for 8 weeks24.The study was approved in accordance with the Guide for the Care and Use of Laboratory Animals . Studies were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Three pairs of ArgII4.Arginase activity was determined from urea concentration measurements as described previously2+ content ([Ca2+]m) was performed by an established loading procedure of the cells with calcein acetoxymethyl ester  and CoCl2 for mitochondrial localization of calcein fluorescence25. Briefly, cells were loaded with 500 nM calcein-AM at 37 °C for 30 min in starved media. Calcein-AM generates free fluorescent intracellular calcein upon cleavage of the ester bond. CoCl2 was then added to the medium to quench the cytosolic calcein fluorescence, while intramitochondrial calcein allowed visualization of the mitochondria as bright fluorescent bodies. Subsequently, the cells were washed free of calcein-AM and CoCl2 and incubated in Tyrode’s modified solution . For detection of calcein fluorescence, a 470 nm excitation and a 510 nm emission filter were used. We also tested [Ca2+]m using Rhod-2 AM . Briefly, cells were loaded with 2.5 μmol/L Rhod-2 AM at 37 °C for 1 h in starved media. Subsequently, the cells were washed free of Rhod-2 AM and incubated in Tyrode’s modified solution . For detection of Rhod-2 AM fluorescence, 552 nm excitation and 581 nm emission filters were used. MitoTracker green FM  was incubated to confirm the mitochondria at 100 nmol/L for 1 h and imaged at 490 nm excitation and 516 nm emission. [Ca2+]c was monitored using Fluo-4 AM  at 494 nm excitation, and emission at 506 nm was detected. Intensity values were normalized according to the initial fluorescence values after subtraction of background using the Metamorph program (Molecular Probe).Direct assessment of the mitochondrial Ca2+]m and [Ca2+]c were also determined using flow cytometry . The fluorescence intensity for each sample was determined using CellQuest software. The Ca2+ level was determined by comparing the fold changes in the fluorescence intensities of treated cells versus control cells.m, we isolated aortic smooth muscle cells (mAoSMCs) from WT and ArgII−/− mice . The [Ca2+]m level in mAoSMCs from ArgII−/− mice was significantly lower  and was not changed by nLDL stimulation . However, [Ca2+]c in mAoSMCs from WT mice was decreased by nLDL treatment , and [Ca2+]c in mAoSMCs from ArgII−/− mice was higher  and not affected by nLDL . The results from microscopic images were confirmed again with FACS analysis .We first wished to determine the effect of ArgII inhibition on mitochondrial Ca2+]c, we measured the activation of a Ca2+-dependent protein kinase, CaMKII, which was related to VSMC constriction. As shown in Fig. −/− . Elevated CaMKII phosphorylation by ArgII downregulation was associated with MLC20 phosphorylation. As shown in Fig. −/− and maintained at high levels even with nLDL stimulation . MLC20 phosphorylation in de-endothelialized vessels .To assess the hypothesis that nLDL treatment decreased c , while spermidine and putrescine had no effect. To confirm the association between spermine-dependent mitochondrial Ca2+ uptake and p32m, cells were preincubated with sip32m and scm siRNA and then stimulated with polyamine. Indeed, sip32m treatment prevented spermine-induced mitochondrial Ca2+ uptake . We next tested whether the incubation of spermine affects CaMKII phosphorylation. As shown in Fig. P < 0.01). The effect of spermine on CaMKII phosphorylation was prevented by sip32m incubation . Therefore, we suggest here that ArgII inhibition attenuates [Ca2+]m by decreasing the level of spermine inducing the p32-dependent transition of cytosolic Ca2+ into the mitochondria.To test whether ArgII activity impacts p32-dependent mitochondrial Caion Fig. , but scmion Fig. . BecauseP < 0.01; # vs. untreated, P < 0.01). To examine the movement of Cy5-conjugated siRNA into cells, the vessels were imaged with Cy5 fluorescence  and PE . scm siRNA incubation resulted in no difference compared to the untreated rings.We next tested whether sip32m could regulate VSMC constriction. First, sip32m incubation with de-endothelialized rings from WT mice significantly reduced the p32m level at 100 nM, but scm siRNA had no effect on the protein level  fed a high-cholesterol diet (HCD). Arg activity was increased in de-endothelialized aortas of ApoE−/− mice fed an HCD . In addition, the level of L-arginine was decreased , while the concentrations of polyamine, spermine , spermidine , and putrescine  were increased  and [Ca2+]c was reduced  in isolated mAoSMCs from ApoE−/− fed an HCD, which was consistent with the augmented p32m expression  and attenuated CaMKII activation  in de-endothelialized aortas of ApoE−/− mice fed an HCD. The increased p32m expression in ApoE−/− fed an HCD might be dependent on the diet type because mice fed an ND did not show changes in p32m expression . Consistent with these data, the responses to NE were similar to PE responses . All groups showed no response to an endothelium-dependent vasorelaxant, acetylcholine . Our study showed that nLDL stimulation exerted an increase in [Ca2+]m that was prevented by Arg inhibition  is activated by elevated [Ca2+]c and autophosphorylated to participate in the cell cycle and growth regulation in VSMCs. In addition, CaMKII has been implicated as a regulator of VSMC contraction. Consistent with these observations, ArgII−/− and p32m knockdown demonstrated augmented activation of CaMKII and enhanced vasoconstrictive responses to agonists in de-endothelialized vessels.In VSMCs, intracellular Ca2+]c was maintained at higher levels by downregulating Arg activity than that of untreated cells . Therefore, higher [Ca2+]c by transient inhibition of Arg activity (Fig. 2+]m in mAoSMCs from ArgII−/− mice was maintained at lower levels than that of WT cells and not increased under nLDL-stimulated conditions. As shown in Supplementary Fig. −/− mice. Therefore, the nLDL-dependent Ca2+ transition through p32m may be attenuated and then could induce a reciprocal increase in [Ca2+]c.In this current study, two major points should be explained. The first is why m levels were increased in VSMCs from ApoE−/− mice. Finally, downregulation of p32m using the siRNA technique augmented CaMKII phosphorylation and agonist-induced vasoconstriction activity.In conclusion, we demonstrated that ArgII activity, coupled with spermine production, contributes to mitochondrial dysfunction through increased p32m-dependent mitochondrial CaSupplemental figures


Using in vitro experiments and ex vivo cultures of human PCa slices, we demonstrated that LA and EPA exert anticancer effects, by modulating Ca2+ entry, which was involved in Zeb1 regulation and cancer cellular migration. This functional approach using human prostate tumors highlights the clinical relevance of our observations, and may allow us to consider the possibility of targeting cancer spread by altering the lipid microenvironment.The composition of periprostatic adipose tissue (PPAT) has been shown to play a role in prostate cancer (PCa) progression. We recently reported an inverse association between PCa aggressiveness and elevated PPAT linoleic acid (LA) and eicosapentaenoic acid (EPA) content. In the present study, we identified a new signaling pathway with a positive feedback loop between the epithelial-to-mesenchymal transition (EMT) transcription factor Zeb1 and the Ca Prostate cancer (PCa), which is the most commonly diagnosed non cutaneous cancer in men, is a highly heterogeneous disease with variable clinical outcomes . The proWe recently demonstrated an association between aggressive PCa and the FA PPAT composition. A specific FA profile was associated with PCa aggressiveness, characterized by low levels of both the essential n-6 polyunsaturated FA (PUFA) linoleic acid (LA) and the n-3 PUFA eicosapentaenoic acid (EPA). Moreover, in vitro migration potential of PCa cell lines supplemented with FA extracts obtained from PPAT was inversely correlated with adipose tissue LA content . Taken tCancer progression is dependent on the epithelial-to-mesenchymal transition (EMT) process, which allows the de-differentiation of epithelial cells to a mesenchymal-like phenotype and increases cancer cell invasion and migration. Zeb1 (zinc finger enhancer binding protein) is a key EMT driver that can be induced not only by microenvironmental factors, including TGFβ and hypoxia, but also by other EMT transcription factors such as Snail . Zeb1 ha2+) signaling plays a major role in several events driving cancer progression mainly by regulating cytosolic Ca2+ concentrations involved in cell migration [2+ entry has been associated with EMT in breast cancer cells [2+ channels have been shown to induce the expression of EMT markers [2+ channels has already been studied in different processes of tumor development and progression, including proliferation, differentiation, apoptosis and migration [2+ entry from extracellular medium is mainly supported by a capacitive Ca2+ entry mechanism, also known as store-operated Ca2+ entry (SOCE), which is mediated by store-operated Ca2+ channels [2+ activated K+ channel regulating Ca2+ entry and cancer cell migration [2+ channels, thereby leading to increased cytosolic Ca2+ concentrations in cancer cells and promoting metastases development [Calcium  promoter–luciferase reporter constructs in PC3-transfected with the pCIneo-Zeb1 plasmid, which allows constitutive transcription of Zeb1. As predicted, overexpression of Zeb1 increased KCNN3 promoter activity (by 3-fold)  D. These  3-fold) E. 2+ concentration [2+ channel inhibitors GSK7975A and Synta66 . Ca2+ channel inhibitors had no effect on Zeb1 basal expression  induced a robust and rapid increase of intracellular Ca2+ concentrations, which was followed by a plateau phase (curve control). As already observed in our previous study [2+ entry was increased in tumor compared to nontumor prostate slices. Interestingly, PCa slices supplemented with LA and EPA exhibited a reduced responsiveness to extracellular Ca2+ variations to a level close to that observed in nontumor tissues. Accordingly, the increase of intracellular Ca2+ concentration was of a reduced amplitude in LA- and EPA-treated slices  compared to vehicle alone-treated slices. In PA-treated slices, no significant change in Ca2+ entry was observed   . Figure 2+ concentrations mediated by major EMT inducers such as hypoxia, EGF [2+ concentration.Expression levels of the key EMT transcription factor Zeb1 is increased with the stages of PCa progression, and is at its highest level in metastases. This factor has a major impact on patients’ survival . We showxia, EGF , TGFβ [1xia, EGF ,20,21, oxia, EGF . In the 2+ channel expression as a consequence of EMT induction in cancer cells [KCNN3 (SK3 encoding gene) could be a target gene for Zeb1. Experiments on promoter activity demonstrated a transcriptional control of the SK3 channel gene by Zeb1. This finding is novel, as, up until now, little was known about the transcriptional regulation of the SK3 channel gene. Previous studies had shown a regulation by members of the Sp transcription factor family and estrogens [2+ entry that promotes PCa cell migration.Several studies have reported a modulation of Caer cells . We repostrogens . Taken tTGFβ can act as both activator and repressor of prostate cancer progression. TGFβ exerts a tumor suppressor role at the early stages. However, during tumor progression, TGFβ is involved in migration and invasion processes via the activation of EMT, which is involved in tumor progression and metastases ,25. In t2+ entry involved in Zeb1 regulation. This finding is crucial as SK3 is a target gene of Zeb1. Indeed, we reported in a previous study a pivotal role for the SK3 channel in human cancer cell migration and bone metastases [2+ channels complexes, and this disruption may lead to decreased SK3-dependent Ca2+ entry and cell migration.The present in vitro and ex vivo results suggest that LA and EPA exert their “anticancer” effects by reducing Catastases . The mecNo clinical trial has reported the use of FA as adjuvant treatment in PCa. We have previously showed that, by analyzing FA profile of periprostatic adipose tissue (PPAT) obtained from PCa patients, the least aggressive tumors were those with the highest levels of LA and EPA . FA conc2+ [2+ concentrations between tumor and adjacent nontumor prostate slices and reported increased intracellular Ca2+ concentrations in malignant compared to benign tissues (about 20% increase). We also found greater intracellular Ca2+ concentrations in tissues sample obtained from aggressive PCa compared to indolent tumors. This finding suggested that Ca2+ entry may contribute to cancer progression [2+ during tumorigenesis and cancer progression [2+ entry not only in vitro, but also ex vivo, in human PCa slices. As these FA may reduce PCa aggressiveness through “normalization” of Ca2+ entry in cancer cells, they could therefore be used for therapeutic applications.As animal models do not perfectly reproduce the clinical situations of PCa, we developed ex vivo tissue cultures of human PCa slices, which preserve both tumor microenvironment and cancer cell heterogeneity. We reported that this model reproduces the characteristics of human PCa, including its sensitivity to androgen deprivation and response to hypoxia and extracellular Ca2+ . In our gression . Importagression . In the 2. Slices were incubated in a humidified incubator at 37 °C with 5% CO2, and treated or not with fatty acids, LA, EPA, and PA (60 µM) for 48 h. Slices were used for either ex vivo culture and immunohistochemistry (3 patients) or for ex vivo culture and intracellular Ca2+ measurements (11 patients).Prostatic tissue samples were obtained from 14 patients undergoing radical prostatectomy for PCa. The characteristics of patients and tissues are summarized in Tissue slices or cells were fixed in 10% formalin, embedded in paraffin, and cut in serial 3 µm sections. One section was stained with hematoxylin-eosin-saffron (HES), and the other sections were deparaffinized, rehydrated, and heated in citrate buffer pH 6 for antigenic retrieval. After blocking for endogenous peroxidase with 3% hydrogen peroxide, Zeb1 primary antibody  was incubated 1 h. Immunohistochemistry was then performed using the streptavidin–biotin–peroxydase method with diaminobenzidine (DAB) as the chromogen . Slides were finally counterstained with haematoxylin. Positive staining was expressed as a percentage of total cancer cells.2 2, KCl 4, D-glucose 11.1, HEPES 10, and CaCl2 2, adjusted to pH 7.4 with NaOH. The dye was excited at 545 ± 20 nm with LED illumination (CoolLED pE-300 white) and fluorescence was collected at 605 ± 40 nm with a sCMOS Zyla 4.2 PLUS camera and a Macro Zoom System Microscope MVX10. Image acquisition rate was 0.1 Hz, and exposure time was 600 ms. For measurement, human prostate samples were incubated in 2 mM CaCl2 PSS. After a stabilizing time (~100 s), CaCl2 was added to the bath to reach a final concentration of 5 mM Ca2+. Analyses were performed with ImageJ Software. The Ca2+ fluorescence signal was normalized by the initial fluorescence signal obtained in the 2 mM (CaCl) to correct differences of dye loading across the preparation, and then the variations of the Ca2+ signal was determined by calculating the difference between the basal, at 2 mM CaCl2, and the maximal Ca2+ fluorescence value after 5 mM CaCl2 application.Intracellular calcium variations were assessed with rhod-2 calcium-sensitive dye. Briefly, human prostate slices were incubated for 30 min with 5 µM of Rhod-2 AM (at room temperature). Then, slices were washed for 20 min with a physiological saline solution (PSS) before experiments. The PSS had the following composition (in mM); NaCl 140, MgCl2. All experiments were performed with mycoplasma-free cells. DU145 and PC3 PCa cell lines have been chosen for their high migration capacity.Human PCa DU145 (HTB-81) and PC3 (CRL-1435) cell lines was obtained from American Type Culture Collection  and was received on 2016/2018. This cell line has been tested and authenticated by DNA fingerprinting by the ATCC. After reception, cells were amplified in order to make a large reserve of cryopreserved cells. Every 3 months, a new cryopreserved bulb was thawed and used for this study. DU145 and PC3 cell lines were grown in RPMI medium , supplemented with 5% FBS  and 1% (v/v) penicillin–streptomycin in a humidified incubator at 37 °C with 5% COLA (L1876), EPA (17266), palmitic acid (PA) (P5177), and TGFβ1 (H8541) were from Sigma-Aldrich. GSK7975A (GLXC03243) and Synta66 (GLXC03244) were from GLXX Lab IMC. 1-Ohexadecyl-2-O-methyl-sn-glycero-3-lactose (Ohmline) was synthetized as previously described .2+ concentrations were studied using the ratiometric fluorescent dye Fura-2-AM (Molecular Probes) . For SOCE measurement, free-Ca2+ PSS (composition in 2+ depletion (~500 s), PSS with 2 mM CaCl2 (2Ca), was added. Fluorescence emission was measured at 510 nm with an excitation light at 340 and 380 nm. Analyses were performed using the SoftMax Pro Software. Results were expressed by the value at the peak 2Ca normalized by the value of the area of the Ca2+ Tg responses.Cytosolic CaKCNN3 promoter sequence has been delineated following publication of Sun et al. that described limits of functional promoter (2540 bp) [® Reporter Assay System (Promega).2540 bp) . Transfe2 and O2. Analyses were performed using the NIS Element AR software. Results were analyzed by measuring the area of the injured area to 0 h and 48 h after injury. Values were plotted as the percentage of wound closure.Transwell assays were performed in cell culture inserts with 8µm pore size . Migrated cells to the bottom side were fixed, stained, and automatically counted. Wound healing was performed with a sterile 2-mm-wide tip on confluent cells. Phase-contrast images were performed on a Nikon microscope coupled to a Nikon camera (DS Qi2). The system included a cage incubator  that controlled temperature and levels of COCells seeded at a density of 250,000 cells/well were incubated with a mix of siRNA and PepMute  in medium for 6h. SiRNA used in this study have been validated in previous reports and are available in Total RNAs from cultured cells were extracted using the Nucleopsin RNA kit . RNA was reverse-transcribed with an RT kit . mRNA levels were quantified using LightCycler 480 . For each reaction, SYBR Green mix  was mixed with specific primers 0.5 µM,  and cDNACells were lysed in RIPA buffer (50 mM Tris (pH 7.4), 1% NP-40, 150 mM NaCl, 1mM EDTA, 1 mM EGTA, 0.1% SDS, 0.5% sodium deoxycholate, and 10% glycerol) with phosphatase inhibitors  and protease inhibitors . Protein concentration was determined by BCA protein assay kit . Proteins were separated by denaturating SDS-PAGE and transferred to a polyvinylidene difluoride membrane . Primary antibodies were incubated overnight. The antibodies used were rabbit anti-Zeb1 , mouse anti-Ecadherin , and goat anti-β-adaptin . Antibody binding was revealed with horseradish peroxidase conjugated anti-rabbit, anti-mouse, and anti-goat antibodies (Santa Cruz). The bands were detected with the Amersham ECL SelectTM Western Blotting Detection Reagent kit , visualized with a CDD camera (DNR Bio-imaging Systems MF-ChemiBis 3.2.) and quantified using Multi Gauge software.Analyses were made using the Mann–Whitney test and Kruskal–Wallis one-way ANOVA, followed by Dunn’s test and the nonparametric Wilcoxon (as indicated in figure legends).2+ entry, and this pathway is associated with increased disease aggressiveness. Several steps of this process are strongly inhibited by LA and EPA supplementation. This observation underlines the therapeutic potential of these PUFAs as adjuvant for cancer treatments. The present study identified in PCa a signaling pathway leading to Zeb1 and SK3 induction through increased Ca


Additionally, a recent proteomics study revealed that RGS14 interacts with two key Ca2+-activated proteins in CA2 neurons: calcium/calmodulin and CaMKII. Here, we investigated whether RGS14 regulates Ca2+ signaling in its host CA2 neurons. We found that the nascent LTP of CA2 synapses caused by genetic knockout (KO) of RGS14 in mice requires Ca2+-dependent postsynaptic signaling through NMDA receptors, CaMK, and PKA, revealing similar mechanisms to those in CA1. We report that RGS14 negatively regulates the long-term structural plasticity of dendritic spines of CA2 neurons. We further show that wild-type (WT) CA2 neurons display significantly attenuated spine Ca2+ transients during structural plasticity induction compared with the Ca2+ transients from CA2 spines of RGS14 KO mice and CA1 controls. Finally, we demonstrate that acute overexpression of RGS14 is sufficient to block spine plasticity, and elevating extracellular Ca2+ levels restores plasticity to RGS14-expressing neurons. Together, these results demonstrate for the first time that RGS14 regulates plasticity in hippocampal area CA2 by restricting Ca2+ elevations in CA2 spines and downstream signaling pathways.Pyramidal neurons in hippocampal area CA2 are distinct from neighboring CA1 in that they resist synaptic long-term potentiation (LTP) at CA3 Schaffer collateral synapses. Regulator of G protein signaling 14 (RGS14) is a complex scaffolding protein enriched in CA2 dendritic spines that naturally blocks CA2 synaptic plasticity and hippocampus-dependent learning, but the cellular mechanisms by which RGS14 gates LTP are largely unexplored. A previous study has attributed the lack of plasticity to higher rates of calcium (Ca These results define a previously unrecognized role for RGS14 as a natural inhibitor of postsynaptic Ca2+ signaling in hippocampal area CA2.Recent studies of hippocampal area CA2 have provided strong evidence in support of a clear role for this apparently plasticity-resistant subregion of the hippocampus in social, spatial, and temporal aspects of memory. RGS14 is a critical factor that inhibits synaptic plasticity in CA2, but the molecular mechanisms by which RGS14 limits LTP remained unknown. Here we provide new evidence that RGS14 restricts spine CaPyramidal neurons in hippocampal area CA2 differ markedly from neighboring subfields in that synaptic long-term potentiation (LTP) is not readily induced . A numbeRGS14 is a complex scaffolding protein with an unconventional protein architecture that allows it to integrate G protein and ERK/MAPK signaling . RGS14 i2+ transients due to higher Ca2+ extrusion rates and buffering compared to CA1 and CA3 pyramidal neurons , which then potently activates many plasticity-inducing pathways, including CaMK, Ras/ERK, and PKA  were crossed with RGS14 WT/KO mice to label CA2 dendrites for field recordings.Animals in all experiments were house under a 12-h:12-h light/dark cycle with access to food and water escribed . Both mam choline chloride, 2.5 mm KCl, 26 mm NaHCO3, 3.3 mm MgCl2, 1.2 mm NaH2PO4, 10 mm glucose, and 0.5 mm CaCl2, pH 7.4, equilibrated with 95% O2/5% CO2. Coronal slices (400 µm) were prepared on a vibratome (Leica VT1200), and slices were maintained in a submerged holding chamber at 32°C for 1 h and then at room temperature in oxygenated artificial CSF (ACSF).Adult RGS14 WT or KO;Amigo2-EGFP+ mice [postnatal day 20 (P20)–P50] were sedated by isoflurane inhalation and perfused intracardially with a chilled choline chloride solution. Brains were removed and placed in the same choline chloride solution composed of 124 mm CaCl2, 2 mm MgCl2, and 100 µm picrotoxin. One or two glass recording electrodes (resistance ∼4 MΩ) containing the same ACSF solution was placed in the stratum radiatum of CA2 or CA1, respectively (∼100–200 µm away from the soma) while stimulating Schaffer collateral fibers with square current pulses (0.1-ms duration) using a concentric bipolar stimulation electrode (FHC) every 30 s. For clarity of display, every other data point was removed from averaged time-course field recording experiments; all data points were included in the statistical analyses. Area CA2 was detected by Amigo2-EGFP fluorescence. The initial slope of the excitatory postsynaptic potential (EPSP) was monitored with custom software. The stimulation strength was set to ∼50% saturation. LTP was assessed by applying three sets of high-frequency stimuli  with 20-s intervals. All data were analyzed with an in-house program written in Matlab (MathWorks). Data are presented as mean ± SEM. For all LTP experiments, predetermined statistical comparisons were made between the normalized average field EPSP (fEPSP) slope 40–60 min following LTP induction to assess long-lasting changes in synaptic efficacy. Statistical comparisons were performed using two-way ANOVA, and Sidak’s multiple comparisons test was used to compare the same CA region between RGS14 WT and KO animals. Differences between datasets were judged to be significant at p ≤ 0.05. Statistical analyses were performed in GraphPad Prism 7.Experiments were performed at room temperature (∼21°C), and submerged slices were perfused with oxygenated ACSF containing 2 mm, Sigma), KN-62 , or PKI (14–22) amide myristoylated , to inhibit NMDA receptors, CaMK, or PKA, respectively. For KN-62 experiments, RGS14 KO control slices were perfused with ACSF containing 0.01% DMSO as a vehicle control. Electrophysiological recordings and LTP induction protocol were performed as described above. Data are presented as mean ± SEM. Statistical comparisons were performed using Student’s t test to compare each inhibitor with respective KO control, and differences between datasets were judged to be significant at p ≤ 0.05. Statistical analyses were performed in GraphPad Prism 7.To assess whether LTP could be inhibited pharmacologically, slices from RGS14 KO/Amigo2-EGFP+ animals were perfused with either ACSF for controls or ACSF supplemented with either APV  diluted in 0.1% PBS-X (0.1% Triton X-100 in PBS) at room temperature, and incubated in primary antibodies diluted in the same buffer overnight. A chicken polyclonal anti-GFP antibody (Abcam) was used at a 1:2000 dilution to enhance Amigo2-EGFP fluorescence with either a rabbit polyclonal anti-PCP4 antibody (Santa Cruz) or a rabbit polyclonal anti-Wfs1 antibody (ProteinTech). Sections were thoroughly washed in 0.1% PBS-X and incubated in secondary antibodies  diluted at 1:500 for 2 h at room temperature. Finally, sections were washed in 0.1% PBS-X and mounted under ProLong Gold Antifade fluorescence media with DAPI (Invitrogen). Sections were then imaged on a Zeiss 710 meta confocal microscope using a 40× oil-immersion lens.2 and 5% CO2. The culture medium was exchanged with fresh medium every 3 d. After 7–10 d in culture, neurons were sparsely transfected with ballistic gene transfer  was uncaged with a train of 4–8-ms laser pulses  near a spine of interest. Experiments were performed at room temperature in ACSF solution containing (in mm): 127 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 4 CaCl2, 25 glucose, 0.001 tetrodotoxin (Tocris), and 4 MNI-caged l-glutamate, bubbled with 95% O2 and 5% CO2. We examined secondary/tertiary branches of apical dendrites of CA1 and CA2 pyramidal neurons (located in stratum radiatum) in organotypic cultured hippocampus slices at 10–22 d in vitro.Glutamate uncaging and imaging of live neurons were performed under a custom-built two-photon microscope with two Ti:Sapphire lasers . In brief, the lasers were tuned at the wavelengths of 920 and 720 nm for imaging and uncaging, respectively. The intensity of each laser was independently controlled with electro-optical modulators (Conoptics). The fluorescence was collected with an objective , divided with a dichroic mirror (565dcxr), and detected with photoelectron multiplier tubes (PMTs) placed after wavelength filters . MNI-caged 2+ imaging, we performed whole-cell patch recordings of CA2/CA1 pyramidal neurons in cultured hippocampus slices, with the patch pipette containing the Ca2+ indicator Fluo-4FF  and Alexa Fluor 594 (500 µm) diluted in potassium gluconate internal solution . Both dyes were excited simultaneously by a Ti:Sapphire laser  at 920 nm. Fluo-4FF (green) and Alexa Fluor 594 (red) fluorescence were used to quantify the change in [Ca2+] (Δ[Ca2+]) using the following formula:G/R)sat is the ratio of green and red fluorescence at a saturating Ca2+ concentration measured in the patch pipette (after each day of imaging), and the dissociation constant (DK) for Fluo-4FF is 10.4 µm (2+] (µm) for all 30 glutamate uncaging pulses from baseline ± SEM. To analyze the Ca2+ decay kinetics from CA2 spines, the maximum and minimum values from the uncaging-triggered averages were normalized, and decay time constants were extrapolated by curve fitting (nonlinear regression) in GraphPad Prism 7.For Ca 10.4 µm . Images Z stack of five slices with 1-µm separation, averaging five frames/slice. Using multiposition imaging of spines with high-throughput automation, dendritic spines at four positions on separate secondary/tertiary dendrites were imaged simultaneously, employing algorithms for autofocusing and drift correction to maintain position and optimal focus during long imaging experiments as recently described , and thoroughly washed in PBS. Slices were blocked for at least 1 h at room temperature in a blocking solution containing 10% NGS (Vector Labs) diluted in 0.1% PBS-X before incubation in primary antibodies diluted in the same blocking solution for 42 h at room temperature. A rabbit polyclonal anti-PCP4 antibody (Santa Cruz) was used at a 1:500 dilution to delineate area CA2 in all slice culture experiments. For sLTP experiments with mEGFP-expressing neurons, a chicken polyclonal anti-GFP antibody (Abcam) was used at a dilution of 1:1000 to visualize imaged neurons. For Ca2+ imaging experiments, Alexa Fluor 594 fluorescence was used to identify imaged neurons. Sections were thoroughly washed in 0.1% PBS-X and incubated in secondary antibodies  diluted at 1:500 for 2 h at room temperature. After rinsing samples thoroughly in 0.1% PBS-X, slices were optically cleared by incubating in a 60% 2,2′-thiodiethanol solution  for 30 min at room temperature . Images were only adjusted for brightness/contrast and cropped for presentation.post hoc Sidak’s multiple comparison test between genotypes . For pharmacological experiments, LTP in the presence of each inhibitor was compared with paired KO CA2 controls using two-tailed, unpaired t tests. For RGS14 overexpression experiments, CA2 neurons transfected with RGS14-GFP were compared to mEGFP-transfected CA2 neurons by unpaired t test. For RGS14 overexpression experiments in CA1 neurons, all groups were compared by one-way ANOVA followed by Fisher’s least significant difference (LSD) test. Complete results of the statistical analyses for each experiment are reported in the Results, and sample sizes are noted in the figure legends. In figures * denotes p < 0.05, ** denotes p ≤ 0.01, *** denotes p < 0.001, **** denotes p < 0.0001.All statistical analyses were performed in GraphPad Prism 7. Statistical analyses of CA2/CA1 neurons from RGS14 WT/KO mice were made using two-way ANOVA (non-repeated measures) with RGS14 has been previously shown to inhibit LTP induction at Schaffer collateral synapses onto CA2 pyramidal neurons by performing whole-cell recordings in brain slices prepared from young mice while stimulating in stratum radiatum . HoweverF = 6.64, p = 0.0127; results for CA region were F = 2.478, p = 0.1212; and results for interaction were F = 3.992, p = 0.0507. Sidak’s post hoc WT CA2–KO CA2 p = 0.0036, WT CA1–KO CA1 p = 0.9028). Baseline synaptic responses were not altered, as input-output curves of fEPSP slope and amplitude were not different in CA2 or CA1 between adult RGS14 WT and KO mice (data not shown).We next performed field potential recordings in acute brain slices prepared from adult RGS14 WT and KO (both Amigo2-EGFP+) mice and replicated previous findings that high-frequency stimulation (3 × 100 Hz) of stratum radiatum induces robust LTP in CA2 neurons of RGS14 KO mice, which is absent in WT mice, and similar to CA1 controls ; Fig. 1C2+ handling mechanisms in spines  mice in the presence of pharmacological inhibitors of Ca2+-dependent signaling pathways  as well as inhibitors of CaMK  or PKA  = 2.868, p = 0.0075; for KN-62 t(37)=2.59, p = 0.0136; for PKI t(37) = 2.502, p = 0.0169). These findings indicate that the nascent LTP in CA2 neurons of RGS14 KO mice requires NMDARs and subsequent CaMK and PKA activity, which can be activated—directly or indirectly in the case of PKA—by Ca2+  = 1.676, p = 0.1998; results for CA region were F = 4.624, p = 0.0350; and results for interaction were F = 3.174, p = 0.0792. Sidak’s post hoc WT CA2–KO CA2 p = 0.0749, WT CA1–KO CA1 p = 0.9236). CA2 spines from slices prepared from RGS14 WT mice exhibited significantly less enlargement compared to RGS14 KO CA2 spines during the later sustained phase of sLTP  = 2.172, p = 0.1451; results for CA region were F = 0.1954, p = 0.6598; and results for interaction were F = 4.198, p = 0.0443. Sidak’s post hoc WT CA2–KO CA2 p = 0.0360, WT CA1–KO CA1 p = 0.8953.). These results indicate that RGS14 naturally restricts the structural plasticity of dendritic spines in CA2 pyramidal cells, similar to the case with functional plasticity.Dendritic spines of RGS14 WT CA2 neurons exhibited significantly smaller volume change following repetitive glutamate uncaging to induce sLTP compared to RGS14 KO CA2 neurons or CA1 controls B–D. Comp2+ is critical for the induction of synaptic plasticity  across all 30 glutamate uncaging pulses  = 6.698, p = 0.0115; results for CA region were F = 2.367, p = 0.1279; and results for interaction were F = 6.675, p = 0.0116. Sidak’s post hoc WT CA2–KO CA2 p < 0.0001, WT CA1–KO CA1 p > 0.9999). Nonlinear regression analysis of normalized uncaging-triggered average Ca2+ responses reveals similar decay kinetics in CA2 spines of RGS14 WT and KO mice . These results demonstrate that spines of CA2 neurons from WT mice display reduced spine Ca2+ transients, which correlates with the attenuated sustained spine structural plasticity; genetic deletion of RGS14 unmasks synaptic plasticity and restores Ca2+ responses to similar levels as those observed in CA1. Together, these data suggest that RGS14 may have a previously unrecognized role in regulating spine Ca2+ to constrain CA2 synaptic plasticity.Uncaging-evoked peak Cat test, **, p = 0.01). RGS14-GFP overexpression similarly blocked the transient and sustained phases of spine plasticity in CA1 pyramidal neurons . Given the low probability of transfecting CA2 pyramidal neurons using ballistic gene transfer, we attempted to reverse the plasticity blockade in RGS14-expressing CA1 pyramidal neurons by increasing the extracellular Ca2+ concentration from [4 mm] to [8 mm] as a similar manipulation increased spine Ca2+ and restored LTP to WT CA2 neurons . These results demonstrate that RGS14 is able to block long-term structural plasticity in hippocampal neurons, and increasing extracellular [Ca2+] restores sustained phase sLTP to RGS14-expressing CA1 neurons. Together, these data suggest that limited Ca2+ signaling is central to RGS14’s ability to gate the structural plasticity of dendritic spines as boosting Ca2+ can unmask plasticity.To more directly test if there is a causal relationship between RGS14 and the diminished capacity for spine plasticity, we acutely overexpressed GFP-tagged RGS14 (RGS14-GFP) or GFP (control) in organotypic slice cultures prepared from RGS14 KO mice and induced spine plasticity by glutamate uncaging. RGS14-GFP overexpression significantly reduces spine volume enlargement in the sustained phase of sLTP in CA2 pyramidal neurons as expected ( neurons . We find2+ signaling in hippocampal CA2 neurons. Specifically, we found that the synaptic potentiation present in CA2 neurons lacking RGS14 requires NMDAR activation along with downstream CaMK and PKA activity, revealing a striking similarity to Ca2+-driven mechanisms underlying LTP in CA1 (2+ transients, and plastic spines from CA2 neurons of RGS14 KO mice display larger spine Ca2+ transients that are comparable to CA1 controls. Lastly, we found that acute re-expression of RGS14 in brain slices from RGS14 KO mice blocks sustained phase plasticity in CA2 and CA1 pyramidal neurons, and raising the extracellular Ca2+ concentration can reverse the blockade of long-lasting sLTP induced by RGS14. Taken together, these results show that RGS14 is a critical regulator of CA2 plasticity and Ca2+ signaling that seemingly differentiates CA2 pyramidal neurons from the neighboring CA1 subfield, best known for its robust plasticity.In this study, we have identified a previously unknown role for RGS14 in the regulation of postsynaptic CaP in CA1 . We furtP in CA1 , to our P in CA1 . We also+/CaM upstream of CaMKs (Here, we used a pharmacological approach to reveal the nascent plasticity of CA2 synapses in brain slices of adult RGS14 KO mice requires intact NMDAR, CaMK, and PKA activity , and we of CaMKs , Gαi/o sof CaMKs , and H Rof CaMKs . Thus, Rof CaMKs  and PKA of CaMKs . Future 2+ levels in WT CA2 neurons to levels comparable to those found in CA1 spines, by brief application of high extracellular [Ca2+] or blocking Ca2+ extrusion through plasma membrane pumps, permits LTP in CA2 pyramidal neurons (2+ transients we observed in WT CA2 neurons is consistent with previous findings that action potential-evoked spine Ca2+ transients in CA2 are smaller than in CA3 and CA1, owing to higher rates of Ca2+ extrusion and buffering (2+ elevation. We found highly similar decay kinetics of spine Ca2+ responses between WT and KO CA2 neurons, which could be attributed to the Ca2+ handling that has been previously observed in CA2 spines (2+ extrusion rate or buffering alone, but it may regulate both processes in CA2 spines to reduce peak Ca2+ without affecting decay time. One possible mechanism by which RGS14 could regulate spine Ca2+ levels is through direct interactions with Ca2+/CaM (2+ dynamics in CA2 and CA1 pyramidal neurons (2+ handling properties of CA2 pyramidal neurons, in addition to RGS14, is PCP4/Pep-19, a CA2-enriched protein that modulates calmodulin, which could underlie the robust spine Ca2+ extrusion (2+ levels in CA2 spines was unexpected and indicates a shared mechanism of robust Ca2+ regulation to inhibit synaptic plasticity in hippocampal CA2.Similar to the nascent plasticity of CA2 neurons in RGS14 KO mice shown here and before , boostin neurons . These ruffering . These t2 spines . From thCa2+/CaM , which h neurons . Anotherxtrusion . Our fin2+ through NMDA receptor influx, although it has been shown that CA2 NMDAR currents from whole-cell recordings did not differ between RGS14 WT and KO mice (2+ influx in dendritic spines can be modulated by cAMP-PKA signaling (2+ levels in hippocampal CA2 neurons.RGS14 may additionally limit peak Ca KO mice . Howeverignaling , which m2+/CaM inhibitors block both the transient and sustained phases of spine plasticity (2+ signals—either by regulating the Ca2+ levels in spines (2+/CaM or CaMKII (2+ during sLTP induction is sufficient to restore sustained, but not transient, phase plasticity to RGS14-expressing CA1 neurons (2+/CaM, as transient phase plasticity was not restored. Consistently, the LTP of CA2 synapses in brain slices of RGS14 KO mice requires intact CaMKII signaling (Previous studies in hippocampal CA1 neurons have shown that Caasticity , while iasticity . Therefon spines  or throur CaMKII —to achie neurons B could pignaling . Future 2+ as well as NMDAR, CaMKII, and ERK activity (2+ (RGS14 also likely modulates forms of plasticity in area CA2 in addition to tetanus-induced LTP. RGS14 is well placed to regulate the CA2 synaptic potentiation mediated by oxytocin/vasopressin, which requires postsynaptic Caactivity , similaractivity . RGS14 mvity (2+ . Lastly,vity (2+ ; acute dWhy would multiple repressive mechanisms exist to block synaptic plasticity in stratum radiatum of area CA2? A study of the expression and localization of RGS14 across development in the postnatal mouse brain showed that RGS14 levels gradually increase with age until reaching the highest, stable levels in adulthood . Given t2+ levels revealing a mechanism upstream of Ras-ERK whereby RGS14 controls synaptic plasticity in CA2. These results provide new insight into the cellular regulation of plasticity in area CA2 and the central role that RGS14 plays in this process.Although much remains to be explored in the mechanistic underpinnings of restricted plasticity in hippocampal CA2, here we define an unexpected role for RGS14 as a novel regulator of spine Ca


Pasteurella multocida (P. multocida) type B infection in rabbits. Fifty New Zealand rabbits, 5 weeks old, were divided equally into five groups. Except for group 1, all groups were intranasally infected with P. multocida type B (2 × 105 colony forming units/ml/rabbit). Then, group 3 rabbits were orally treated with allicin (50 mg/kg BW) for 5 days, group 4 rabbits received a single oral dose of norfloxacin 30% (100 mg/kg BW), while group 5 rabbits were treated with a combination of norfloxacin and allicin. Hematological, serum biochemical, inflammatory cytokine, immunological, and histopathological analyses were performed. Results revealed that rabbits, infected with P. multocida type B, exhibited macrocytic hypochromic anemia and leukocytosis with a significant elevation in the phagocytic percentage and index. Moreover, significant reductions in serum total protein, albumin, globulin, and immunoglobulin (IgG and IgM) levels were observed in infected rabbits. Infected rabbits showed significant increases in serum inflammatory cytokine (TNF-α and IL-6), alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, and serum bilirubin  levels. Further, P. multocida infection induced oxidative stress as demonstrated by the significant reduction in serum levels of reduced glutathione and superoxide dismutase enzyme and marked elevation in serum malondialdehyde. Treatment with allicin, norfloxacin, or their combination significantly ameliorated the alterations in all studied parameters. In conclusion, allicin could ameliorate the inflammation and oxidative stress, induced by P. multocida type B infection in rabbits.The present study investigated the efficacy of allicin as an antibacterial, anti-inflammatory, antioxidant, and immunostimulant agent in reducing the severity of  Pasteurella multocida (P. multocida) is a microbe, which occurs naturally in the respiratory tract of some animal species; however, it can be a virulent pathogen that infects other animal hosts [P. multocida may be asymptomatic [al hosts . Pasteural hosts ; howeverptomatic .Norfloxacin is a second-generation fluoroquinolone with a broad spectrum of activity, potent bactericidal action, and high tissue penetration , 5. DespAllium sativum) is considered the oldest medicinal herb and had been used for the treatment of several diseases. It is effective against several gram-positive, gram-negative, and acid-fast bacteria [Garlic  effects of allicin alone or combined with norfloxacin in the treatment of Male New Zealand white rabbits  were obtained from the Faculty of Veterinary Medicine, Zagazig University. Rabbits were housed in a pathogen-free facility, maintained at 24 ± 2°C with a 50–60% relative humidity and a 12 h light: dark cycle. Rabbits had ad libitum access to tap water and basal ration. All rabbits were acclimatized for one week before beginning the experiment. Rabbit handling and treatment procedures were performed according to the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH) and approved by a research ethics committee at the Faculty of Veterinary Medicine, Zagazig University. All efforts were exerted to reduce animal suffering.Pasteurella multocida type B was obtained from the National Research Centre, Dokki  and used for experimental infection with a final concentration of 2 × 105 colony-forming units (CFU) prior to inoculation.Norfloxacin was purchased from ATCO Pharma . Allicin was obtained from Anhui Ruisen Biological Technology Co., China. All used kits were marketed by Spinreact, Spain.5 CFU/ml/rabbit of P. multocida type B at day 1 of the experiment [After acclimatization, fifty rabbits (6 weeks of age) were randomly allocated into five equal groups (10 rabbits for each group). Group 1 rabbits were given intranasal (IN) phosphate-buffered saline (1 ml) and kept as a negative control group. Rabbits in other groups were IN infected with 2 × 10periment . Five daperiment , group 4periment , while gBlood samples were collected at the end of the experiment (24 hours after last dose) from the ear vein: 2 ml of blood in a heparinized test tube for evaluation of phagocytic percent and index, 0.5 ml in a test tube with the anticoagulant (EDTA) for hematological studies, and 5 ml in a glass tube without anticoagulant for serum separation to assess biochemical parameters. The lung and liver tissues were dissected out after cervical dislocation, washed with physiological saline, and fixed with 10% neutral buffered formalin for histopathological examination.Candida albicans  was prepared and used for evaluation of the phagocytic activity by the method of Wilkinson [C. albicans (phagocytic%) that attached to or were ingested by 100 phagocytes in each individual preparation was determined by light microscopy. Moreover, the phagocytic index was calculated by determining the average number of attached and engulfed C. albicans multiplied by the phagocytic percent [The heparinized blood samples of rabbits from different groups were used for leukocyte separation. ilkinson . The num percent .Blood samples, collected in tubes containing 10% EDTA solution, were used for determination of red blood cells (RBCs), hemoglobin (Hb), hematocrit/packed cell volume (HCT/PCV%), white blood cells (WBC: leukogram), and differential leukocyte counts (DLC) according to Coles . The meaSerum samples were analyzed for determination of alanine aminotransferase (ALT) by the method of Reitman and Frankel , alkalinα) and interleukin-6 (IL-6) were measured using standard kits obtained from Genorise  and CUSABIO (CSB-E06903Rb), respectively.The serum concentrations of immunoglobulin G (IgG) and immunoglobulin M (IgM) were determined using the commercial IgG and IgM ELISA kits, purchased from Bethyl Laboratories, USA , respectively. The serum concentrations of tumor necrosis factor-alpha (TNF-Serum samples were used to assay the reduced glutathione (GSH) level according to Jollow et al. , superoxSpecimens from the lungs and liver of different groups were collected and fixed in 10% neutral buffered formalin, transferred in ethanol (70%), then cleared in xylene and embedded in paraffin. Five-micron-thick sections of paraffin were prepared then stained with hematoxylin and eosin dyes  and examp value < 0.05 was considered statistically significant.All data were expressed as the mean ± standard error of mean (SEM) and were statistically analyzed by the SPSS/PC software (2001) using one-way analysis of variance (ANOVA), followed by post hoc Tukey's test. A On the third day of the experiment, gp.2 rabbits (infected but untreated) started to show the acute form of the disease  with a mortality rate of 60%. However, rabbits treated with allicin (gp.3) and norfloxacin (gp.4) showed less severe clinical signs than did gp.2 with mortality rates of 40 and 30%, respectively. Rabbits treated with both allicin and norfloxacin (gp.5) were nearly healthy with much less severe clinical signs and a mortality rate of 10% till the time of scarification at the end of the experiment.p < 0.05) in RBC count, Hb concentration, PCV%, and MCHC values, while MCV values exhibited a significant increase (p < 0.05) in rabbits infected with P. multocida (gp.2), compared to controls (gp.1). However, rabbits of gps.3, 4, and 5 showed non-significant changes in the RBCs count, Hb concentration, PCV%, MCV, and MCHC% except Hb concentration and MCHC% showed significant increases in gp.5 compared to gp.2  with a significant decrease (p < 0.05) in the lymphocytic count and non- significant changes in eosinophil count in P. multocida-infected rabbits (gp.2), compared to controls (gp.1). The treated gps.  showed a non significant decrease (p < 0.05) in the previous parameters except the neutrophil count, which showed a significant decrease (p < 0.05) while total leukocyte count that showed a significant decrease (p < 0.05) in gp.5 compared to gp.2, while gp.4 showed a significant increases (p < 0.05) in the total leukocytic, neutrophil and eosinophil count with a non-significant changes in lymphocyte and monocyte counts compared to gp.2. The phagocytic activities (phagocytic% and phagocytic index) were significantly increased (p < 0.05) in P. multocida-infected rabbits (gp.2) compared to controls. The treated rabbits  exhibited significant increases in both parameters (p < 0.05), compared to the infected nontreated group; however, none of these treatments could restore the normal range concentrations as in the control group (gp.1)  .p < 0.05) in gp.2, while gps.3, 4, and 5 demonstrated significant increases (p < 0.05), compared to infected rabbits. The combination treatment restored the serum total protein concentration to the normal control level (as gp.1 rabbits).α) showed significant increases (p < 0.05) in gp.2 and significant decreases (p < 0.05) in the treated groups (4 and 5), except in the group receiving allicin (gp.3), which failed to improve serum TNF-α concentration, compared to infected nontreated rabbits .Serum inflammatory cytokines (IL-6 and TNF- rabbits . The comp < 0.05) in the P. multocida-infected group compared to controls and significantly decreased (p < 0.05) in treated groups  compared to gp.2. The total, direct, and indirect bilirubin concentrations showed significant increases (p < 0.05) in infected rabbits; however, it exhibited significant reductions (p < 0.05) in the treated groups , compared to infected nontreated rabbits  with a significant increase (p < 0.05) in MDA serum concentration, compared to controls, while the treated rabbits  exhibited significant increases (p < 0.05) in GSH and SOD concentrations with a significant decrease (p < 0.05) in MDA concentration, compared to infected nontreated rabbits  showed mild edema between alveoli with scanty inflammatory cells and no necrosis or tissue destruction , probably due to enhanced erythropoiesis as a response of the bone marrow to the increased blood loss in trachea-pulmonary hemorrhage, caused by septicemia [P. multocida-infected rabbits. Rabbits treated with a combination of allicin and norfloxacin showed an improvement in the picture of anemia, especially Hb level, which reflects reduction of the bacterial toxic effect on the bone marrow with decreasing or stopping the hemorrhage. The leukogram investigation revealed leukocytosis with heterophilia, and monocytosis in the P. multocida-infected group, which can be attributed to the body inflammatory response [Rabbits, infected with pticemia . Our respticemia  who reporesponse ; leukocyP. multocida are usually associated with leukocytosis as a physiological response from the body to minimize the spread of infection [α [Infections with nfection –43. Lympnfection . In contnfection . Neutropction [α –48.P. multocida-infected rabbits. In agreement with El-Deeb and Elmoslemany [α and IL-6) were markedly elevated to enhance leukocyte migration into the infection site [The cellular immune response in the body increases physiologically in case of infection to destroy infective agents and minimize the spread of infection . This stoslemany , proinflion site –48. It hion site , 51.+ T cells [α and IL-6 [Allicin treatment significantly increased the phagocytic activity in infected rabbits, probably due to the ability of allicin to modulate the peripheral leukocytes' immune functions  through  T cells . Furtherand IL-6 –58. The P. multocida-infected rabbits, which may be due to protein loss during hemorrhage. Anorexia and fever that result from the infection lead to increased protein catabolism and reduced protein synthesis by degenerated hepatocytes [P. multocida infection [Hypoproteinemia and hypoglobulinemia were observed in atocytes . Allicinnfection , hemorrhP. multocida bacteria and limit the systemic infection [Allicin-treated groups had an elevation in globulin, IgG, and IgM serum levels, reflecting the increased production of Igs from lymphoid organs to opsonize the nfection . It has nfection . SimilarPasteurella-infected rabbits, possibly related to injury and degeneration of hepatocytes [Regarding enzyme activities and liver functions, ALT, ALP, LDH, and bilirubin showed significant elevations in atocytes –63. Howeatocytes .P. multocida, showed marked reductions in serum GSH and SOD with elevation of MDA levels, reflecting the increased lipid peroxidation and production of reactive oxygen species by bacterial infection [Rabbits, infected with nfection . Our resnfection , 65, 66.nfection –69. TreaP. multocida can be ameliorated by allicin administration, probably through its antioxidant, anti-inflammatory, and immunostimulant effects. This protective effect could reduce the use of antibiotic in pets and livestock, reducing human exposure to antibiotic residues and bacterial resistance to antibiotics. Further, norfloxacin can be used for the effective treatment of pasteurellosis in rabbits. The combination of allicin and norfloxacin was more powerful in improving the P. multocida-induced alterations than each treatment alone.The results of this study show that the clinically observed damage in rabbits infected with


Mentors reported statistically significantly improved mentoring competency after workshop participation. Junior faculty most frequently identified mentors (61%) and collaborators (23%) as resources for goal attainment. Top barriers included insufficient time and time-management issues (57%), funding limitations (18%), work–life balance issues (18%), including inadequate time for self-care and career development activities. Our MA can serve as a model and roadmap for providing resources to faculty across traditional tracks within medical schools.Formal mentoring programs are increasingly recognized as critical for faculty career development. We describe a mentoring academy (MA) developed for faculty across tracks  within a “school of health” encompassing schools of medicine and nursing. The program is anchored dually in a clinical and translational science center and a school of health. The structure includes the involvement of departmental and center mentoring directors to achieve widespread uptake and oversight. A fundamental resource provided by the MA includes providing workshops to enhance mentoring skills. Initiatives for junior faculty emphasize establishing and maintaining strong mentoring relationships and implementing individual development plans (IDPs) for career planning. We present self-report data on competency improvement from mentor workshops and data on resources and barriers identified by junior faculty ( Mentoring, formal and deliberate guidance in career development planning, is recognized as critical for junior faculty to achieve success in academic health centers ,2–4. IndWe developed and launched a mentoring program, the mentoring academy (MA), to recognize and advance excellence in mentoring, as well as develop mentoring skills informed by best practices. Thus, from its inception, the MA acknowledged and addressed the need to reorient the culture of the institution to support, reward, and enhance quality mentoring in the human health sciences across all faculty tracks. Leadership and senior faculty envisioned that by building cadres of skilled mentors, it would facilitate the development of the next generation of independent, highly successful academic faculty and advance the overall mission of our School of Medicine (SOM). As the School of Nursing at (SON) was developing during this time period, the SON increasingly participated in the MA, thus broadening the scope even more. This article describes how we developed our mentoring program to serve a wide variety of faculty members across different academic tracks. We also present data on two of its primary resources that were initiated to change the culture, workshops on mentoring (Initiative I), and information derived from the primary career planning tool, the individual development plan (IDP), a process that was expected for junior faculty in the health system (Initiative II).The MA originated, simultaneously, in two different “homes” on our health system campus see . One homA critical component of the MA includes departmental and center resources via departmental mentoring directors (DMDs) and center mentoring directors (CMDs). These directors provide an administrative and executive link to ensure that a functioning mentoring team is in place for each junior faculty member. The DMD and CMD roles include: (1) assisting new faculty in identifying potential mentors and guiding them to resources; (2) facilitating and tracking IDP completion; (3) implementing departmental mentoring activities; (4) facilitate faculty engagement in MA activities and resources. The CMDs partner with DMDs for those mentees having a significant center association. DMDs/CMDs are provided modest financial incentives for their roles. Compensation is divided between support from the Dean’s office and the home department or center.There are two levels of membership in the MA, General Member and Master Mentor levels. To qualify as a general member, the faculty member must apply to the MA for membership, have achieved associate (advanced) or full professor rank, participated in a minimum of two workshops on mentoring, and have experience in mentoring others. Additionally, the faculty member’s department chair must send a recommendation affirming the applicant’s skills and experience as a mentor and confirm that there are no reservations about the faculty member’s skills or history as a mentor. In order to build an “Academy” of mentors, the Master Mentor track was also created. We considered the Master’s level status as critical to signify a change in the institution’s culture by raising the recognition and reward for mentoring, with those achieving unique dedication and skill in mentoring invited into the Academy at this elevated level. The initial group of Master Mentors was involved in designing the structure, guidelines, and selection of resources. Prospective Master Mentor members are invited to apply for the Master’s level status and are expected to have achieved an advanced academic rank, present evidence of a sustained track record of excellence in mentoring, have externally recognized mentoring expertise  or be a director of a funded training program, participated in all five MA workshops on mentoring, obtain recommendation from their department chair, and have approval by the other currently active Master Mentors. The Master Mentors must also commit to providing service to the MA after they have been awarded the Master Mentor status.Mentees are expected to take an active role in the mentoring process . We instThe opportunity to share best practices in mentoring with peers in the context of mentoring workshops was the earliest resource made available to our mentors. We employ a flexible approach to reach the large group of interested faculty mentors and mentees working in a busy academic–clinical organization by offering workshops during the work week at a variety of times  and the weekend. The curriculum consists of a core module of five workshops, based on the curriculum developed by Pfund et al. ,10, modiWe also developed a workshop for mentees on establishing successful mentorship . WorkshoMA events have been attended by more than 500 SOH faculty, staff, and students. A total of 331 faculty have attended at least one of the 88 1-h MA workshops since 2011, representing approximately 46% of mid- and senior faculty. The majority of MA workshop participants (58%) completed online entry surveys prior to attending their first workshop that asked them to rate their current level of mentoring skills in several competency areas, based on surveys used in previous studies on mentoring ,10. Chant-tests to evaluate perceived improvement in mentoring skills before and after participating in the workshops for prerating and postrating (n = 347 participants). Participants consistently rated their mentoring skills higher after each workshop despite the fact that they rated their preworkshop mentoring skills relatively high . A composite score based on averaging presurvey  and postsurvey  ratings across the five workshop modules was also statistically significant . A majority of participants (92%) (328 of 355 responses) in the core workshops said they would recommend the MA workshops to their colleagues.To facilitate an inductive content analysis, the authors created a database containing all postworkshop comments which were reviewed by each member of the group. The group then convened to develop a set of themes pertaining to workshop content, format, and overall satisfaction. Several themes emerged including that the participants found both the content and format of the MA workshops to be valuable and that they served to reinforce best practices for mentoring. Another common theme was the value of the workshops in delivering information on topics not covered elsewhere in the health system, such as how to mentor for diversity and inclusion .In this replication of implementing the curriculum from the Pfund et al. , we foun. The use of IDPs as a formal tool to communicate career objectives between mentors and mentees is also increasing in academic settings [Goal setting is recognized as a critical step in achieving one’s objectives in the work environment  and incrsettings , particusettings  policy rsettings  includinsettings . Researcsettings . Howeversettings .An initiative driven by the MA included the expectation for junior faculty  to annually complete their IDP with their mentors. Modest stipends are given to DMDs, when their department achieves a minimum of 60% of the faculty submitting their IDPs. DMDs with high rates of IDP submissions for their department  have received recognition at an annual dean’s award ceremony.During this phase of the MA, templates for developing IDPs were made available to mentees, mentors, and DMDs. It was expected that the templates would be adapted to best meet the requirement of structured career planning between a mentor and mentee. The resulting IDPs typically included a section on goal setting, time distribution between roles , and fields for describing resources needed and challenges experienced. To better understand how IDPs were being used at our institution and to formulate some ways the MA could address common challenges, we examined the content of IDPs that were completed by health faculty from 25 departments and one research center during a 4-year period (2014–2018).To facilitate an inductive content analysis, individually identifying information was removed by one author (REG) from paper copies of 222 IDPs completed by assistant professors (mentees) between 2014 and 2018. The number of IDPs represents approximately 65% of the 340 individuals with junior faculty (assistant or early associate rank) appointments during the time period. Ten IDPs were initially coded for mentions of supports and resources received. After review by the authorship group, all the IDP mentions were then entered into an Excel database. This served as the data source of the frequency of supports, resources, and barriers experienced and to summarize them into a set of salient themes. The primary fields in the final analytic database included the mentee’s self-reported: (a) career track and academic rank; (b) percent effort distribution ; (c) constraints and resources to achieve their current activities; (d) barriers and/or resources to achieve new goals. We report here on the barriers and resources. The authors read through the full set of de-identified data and arrived at a set of summarized themes through group discussion. It should be noted that the analysis of IDPs was not intended to create a data set for research, but to serve as a potentially rich source of information to inform the development of resources to meet the needs of our junior faculty.Junior faculty, regardless of employment track, most commonly noted their mentors and departmental collaborators as their primary resources for career development. Examples included help with time management and prioritizing workload to enable them to focus on their career and goals. Exposure to outside institutions and interdisciplinary teams was also frequently mentioned as a resource. Collaborators were noted for assisting in finding out about new funding opportunities to support research or career goal efforts. Finally, access to other clinicians, networking, and access to listservs emerged as themes related to career support See .The majority of IDPs (74%) included reported challenges such as lack of time or difficulty with optimal time management. Specifically, junior faculty mentioned lacking protected time for research or other tasks and lacking the experience to select activities or tasks that would clearly benefit their career development and advance their goals. Achieving an optimal or healthy work–life balance was also frequently mentioned. Junior faculty noted in their IDPs that they had difficulties in achieving the self-care necessary to prevent burnout and having sufficient time and/or energy to dedicate to their family, especially if they had young children. Equally frequent were challenges related to limits in funding and lack of financial support, including insufficient funding to participate in career development activities and conferences. Other challenges cited were inadequate space, laboratory equipment, and experience/skill set  See .Our experience suggests that a comprehensive mentoring program can be implemented to address the breadth of academic tracks present in traditional academic health centers. We found that many, but not all, of the concerns among mentors and mentees are common regardless of track. In order to resonate with faculty from the different tracks, we refined our curriculum and tools to include case studies reflecting potential challenges and characters across clinical, research, and teaching settings. We recommend that workshop content be updated on a regular basis to resonate with the changing demands of health system faculty. Findings from our analysis on our mentoring workshops suggest that while mentors’ self-ratings are fairly high on their preworkshop skills, intense, small group workshops can lead to perceived, significant improvement after workshop participation.The findings from the IDP analysis reinforce the value that junior faculty place on robust mentoring and the need for a mentoring program to develop and strengthen mentoring skills. Furthermore, our junior faculty frequently noted the contribution of their peers as a mentoring resource. Thus, both junior faculty and their mentors should actively consider peer mentors as part of a mentoring team. There are many successful peer mentoring models ,26, inclThe IDP analysis also suggests that mentors should be prepared to discuss and suggest resources and tips for helping junior faculty manage and prioritize their time. Work–life balance and self-care issues were another prevalent theme in the IDPs, not surprisingly, as these issues are already fairly well recognized as concerns in academic medicine –30. MentUltimately, many of the challenges raised by junior faculty may need to be addressed on a systems level by the institution. For example, leadership may not only ensure that the relevant workshops on time management are offered, but also provide release time for faculty members from their clinic or teaching responsibilities in order to attend the workshops. The need for on-site child care was one of the most common requests from our junior faculty, but can only be accomplished if leadership considers it a priority. Human resource administrators may also be able to assist with suggesting childcare resources that are shared during the faculty onboarding process and they could share those resources with mentors who are interested in mentoring faculty around work–life integration challenges. One example of low-cost, grass-roots resource is our recently initiated Facebook group for sharing information and reviews of childcare options, recreational activities for families, and baby-sitting co-ops.The strength of this analysis is that it is one of the first to systematically review reports of resources and barriers in the context of goal attainment in junior faculty in an academic health setting. We recognize that our experience is representative of only one institution; however, our program is likely generalizable to other institutions with similar leadership support at comparably sized academic health institutions. We acknowledge the limitations in drawing inferences about how changes in perceived skills as rated by participants in the workshops might reflect changes in actual behavior; however, Pfund et al. ,10 did dOur MA can serve as a model for other academic health institutions. Such programs require strong, visible support from leadership to modify and sustain a mentoring culture  as well


Their coordinated action maintains a low Ca2+ baseline, allows a fast increase in free Ca2+ concentration upon a stimulus, and terminates this Ca2+ elevation by an exponential decrease – hence forming a Ca2+ signal. In this respect, the Ca2+ signaling machinery is conserved in different fungi. However, does the similarity of the genetic inventory that shapes the Ca2+ peak imply that if “you’ve seen one, you’ve seen them all” in terms of physiological relevance? Individual studies have focused mostly on a single species, and mechanisms elucidated in few model organisms are usually extrapolated to other species. This mini-review focuses on the physiological relevance of the machinery that maintains Ca2+ homeostasis for growth, virulence, and stress responses. It reveals common and divergent functions of homologous proteins in different fungal species. In conclusion, for the physiological role of these Ca2+ transport proteins, “seen one,” in many cases, does not mean: “seen them all.”The key players of calcium (Ca Ca2+ signals are usually characterized by a sharp rise in cytosolic free Ca2+ ([Ca2+]cyt) followed by an exponential decrease   . Ca2+/H+2+ signals are decoded and modulated by Ca2+-sensitive proteins, such as calmodulin (CaM) and calcineurin (CN). CaM binds to CaM-dependent proteins and modulates their activity by Ca2+-induced conformational changes. The protein phosphatase CN is activated by Ca2+ itself and by CaM. CN activates the transcription factor Crz1 by dephosphorylation, thus triggering its translocation into the nucleus  is formed by Cch1, Mid1, and Ecm7. Cch1 is a homolog of the α subunit of mammalian L-type voltage-gated Ca2+ channels  is still unclear. Fig1, a plasma membrane protein, has been proposed to be either the LACS Ca2+ channel itself or an important regulator of it. It is needed for Ca2+ influx and normal mating in all fungi analyzed so far. In S. cerevisiae and Candida albicans, Fig1 was shown to be important for Ca2+ influx during mating and cell fusion  is a Carevisiae . It is arevisiae  and ampl by CICR . In thess lactis , C. albialbicans , and theminearum  resembleosphates . The phye stress ; no othee stress . In conte stress . In C. aalbicans  and Aspeumigatus , TRPY1 ihomologs . In thisirulence . In summ2+/H+ antiporter in the vacuolar membrane of S. cerevisiae. It can also sequester Mn2+. Therefore, Vcx1 allows cells to grow at very high extracellular concentrations of these ions  maintains the basal [Ca2+]cyt prior to a signal . A deletion of PmcC is lethal. Deletion of PmcA results in impairment of spore germination, growth at high [Ca2+] in rich  media, and virulence . Both locate to the vacuolar membrane network (VMN) and subapically also to the plasma membrane, as their mammalian homolog. Nca2 is needed to supply the VMN and export Ca2+ out of the cell. The protein is beneficial for growth in minimal media with low [Ca2+] and essential when [Ca2+]ext is high. Female spores of nca2Δ strains are infertile, and both genders produce less spores. Nca3 seems to be dispensable for growth, sporulation, and stress tolerance . Therefore, phylogenetic similarity does not necessarily correlate with biological function.Albeit examined in only few species yet, the function of Spf1 is, as known so far, very similar in different fungi as well as compared to the Pmr1/Eca1 family. This is contrasted by the clearly more diverse functions of Pmc1 in different species  must exist in the fungal plasma membrane. The LACS component Fig1 may be one interesting candidate in this respect.A wealth of data on transport proteins that maintain Cain fungi , althougin fungi  and expein fungi  indicate2+ signals  appears to be quite conserved during fungal evolution, whereas in others , there appear to be striking differences between species. Therefore, the “seen one, seen them all” principle should be applied very cautiously, in particular in translational studies aiming to develop antifungal drugs. An ensuing question remains to be answered however: What are the causes for this evolutionary divergence? Therefore, future work needs move from the description of phenotypes to the deciphering of mechanisms in phylogenetically diverse fungal species.Regarding the known players, the physiological role of some of the proteins involved in shaping CaML and EP conceived, drafted, and finalized the manuscript, approved the final version of the article, and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Downregulation of suppressor of cytokine signalling-1 (SOCS1) is one of the vital reasons for JAK1-STAT3 pathway activation in acute myeloid leukaemia (AML). CUE domain-containing 2 (CUEDC2) was a novel interacting partner of SOCS1 and a positive correlation between the expression of CUEDC2 and SOCS1 was confirmed in primary AML cells and AML cell lines without SOCS1 promoter methylation. We aimed to explore roles of CUEDC2 in regulating ubiquitin-mediated degradation of SOCS1 in the leukaemogenesis of AML.According to in vitro experiments, CUEDC2 overexpression increased the level of SOCS1 protein, suppressed JAK1-STAT3 pathway activation. The suppression of this pathway inhibited AML cells’ proliferation by causing G1 arrest and enhanced AML cells’ sensitivity to cytarabine and idarubicin. Similarity, downregulation of CUEDC2 produced opposite results. Knockout or low expression of CUEDC2 in mouse or AML patients displayed lower overall survival and event-free survival rates, compared with these mouse and AML patients had high-CUEDC2 expression. Mechanistic studies revealed that CUEDC2 overexpression attenuated SOCS1 ubiquitination, facilitated its stabilisation by enhancing SOCS1, Elongin C and Cullin-2 (CUL2) interactions, thus inhibited JAK1-STAT3 pathway and leukaemogenesis of AML. Therefore, our novel findings indicated that CUEDC2 interacted with SOCS1 to suppress SOCS1’s ubiquitin-mediated degradation, JAK1-STAT3 pathway activation and leukaemogenesis of AML. The inhibition of JAK1-STAT3 pathway represents a promising therapeutic strategy for AML patients. Many JAK1-STAT3 pathway inhibitors have been developed based on its known activation mechanism. However, the efficacy was not confirmed in recent clinical trials4. Thus, other mechanisms underlying JAK1-STAT3 signalling hyperactivation in AML need to investigate.Despite of the improved outcomes of acute myeloid leukaemia (AML) in recent years, most patients will suffer relapse receiving chemotherapy alone. Deep explore of the molecular mechanism of AML is very important for translational research to improve the survival of patients. The hyperactivation of JAK1-STAT3 pathway plays vital roles in leukaemogenesis and relapse of AML5. More importantly, downregulation of SOCS1 is a key reason for JAK1-STAT3 pathway activation and leukaemogenesis of AML7. SOCS1 negatively regulates JAK1-STAT3 pathway through three mechanisms. First, SOCS1 binds to the activation loop of JAK1 via its SH2 domain and inhibits JAK1’s kinase activity8. Second, SOCS1 regulates the activity of this pathway by SOCS box-mediated proteasomal degradation of JAKs9. Third, SOCS1 binds to the phospho-tyrosine residues on the receptors and physically blocks STATs from binding to their receptors10. Hypermethylation of SOCS1 promoter and elevated ubiquitin-mediated degradation were main mechanisms of SOCS1 downregulation in AML12. The mechanism of SOCS1 promoter hypermethylation has been intensively studied and almost completely clarified. Although the Eongin BC complex, which interacts with the SOCS box, has been shown to increase the SOCS1 content by inhibiting its degradation13, the mechanism how SOCS1 degradation is regulated in AML remains unclear. Thus, studies aiming to elucidate which gene or protein might be involved in regulating SOCS1’s ubiquitin-mediated degradation and its degradation regulating mechanism in AML are of great importance.The suppressors of cytokine signalling (SOCS) proteins are important for regulating of JAK-STAT pathway14, inflammation, tumour development15, and chromosomal instability16. Identified as ubiquitin-binding motifs, CUE domains interact with both mono and polyubiquitin and play dual roles in recognising mono and polyubiquitin as well as in facilitating intramolecular monoubiquitination17. CUEDC2 might be a novel regulator of SOCS1’s ubiquitin-mediated degradation and an inhibitor of the JAK1-STAT3 pathway. However, whether CUEDC2 was involved in regulating SOCS1’s ubiquitin-mediated degradation and the leukaemogenesis of AML remains unclear.The CUE domain-containing protein 2 (CUEDC2), a novel interacting partner and a potential regulator of the ubiquitin-mediated degradation of SOCS1, is a promising target of treatment. CUEDC2 plays key roles in protein ubiquitin-mediated degradationIn this study, we found that CUEDC2 overexpression attenuated SOCS1 ubiquitination, facilitated its stabilisation by enhancing SOCS1, Elongin C and cullin-2 (CUL2) interactions, thus inhibited JAK1-STAT3 pathway and leukaemogenesis of AML. Therefore, our novel findings indicated that CUEDC2 interacted with SOCS1 to suppress SOCS1’s ubiquitin-mediated degradation, JAK1-STAT3 pathway activation and leukaemogenesis of AML.The expression and methylation of SOCS1’s promoter in primary AML cells and AML cell lines were detected to analyse mechanisms underlying its downregulation. In approximately 48.4% of primary AML cells and 50% of AML cell lines, the mRNA level of SOCS1 was lower Fig.  and its 18, this study also suggested that downregulation of SOCS1 in AML cells was caused by SOCS1 promoter methylation and ubiquitin-mediated degradation.In order to determine whether SOCS1 expression in these AML cells without SOCS1 promoter methylation was regulated by ubiquitin-mediated degradation, the same AML cells and samples used in SOCS1 protein levels detected Fig.  were tren = 56) displayed the most obvious reduction in SOCS1 levels among all detected primary AML cell subtypes, followed by t AML (n = 63), t(11q23)/MLL AML (n = 34), t AML (n = 77) and complex AML (n = 45) (data not shown). Similarly, correlations between SOCS1 expression and its promoter methylation with primary AML cells subtypes were also analysed; leukaemia with normal karyotype displayed the highest level of SOCS1 promoter methylation  among all detected primary AML cells subtypes, followed by t AML , complex AML , t(11q23)/MLL AML , t AML  and inv(16)/t AML  (data not shown). Thus, our result indicated that the SOCS1 expression and its promoter methylation had a closely relationship to AML subtypes and lineages of primary AML cells.After exploration downregulation mechanisms of SOCS1 in AML, correlation between SOCS1 expression and its promoter methylation to AML subtypes and lineages of primary AML cells were analysed. The SOCS1 expression reduction was most pronounced in U937 and THP-1 monocytic cell lines among all analysed AML cell lines, followed by K562 and HL-60 APL cell line, the human erythroleukemia cell (HEL) and KG-1 erythroleukaemia cell lines, and ML-1 and ML-2 myelomonocytic cell lines  was used to investigate specific proteins that might be involved in regulating the ubiquitin-mediated degradation of SOCS1. Using a shotgun analysis, 289 proteins were identified, and only 87 proteins that might actually interact with SOCS1 are shown Table , which wIn order to determine interaction domains between SOCS1 and CUEDC2, GST-pulldown experiments were done. A series of GST-SOCS1 and GST-CUEDC2 deletion mutations were constructed Fig. . As indiSince CUEDC2 was involved in protein ubiquitin-mediated degradation and interacted with SOCS1, the correlation between SOCS1 and CUEDC2 in AML cells without SOCS1 promoter methylation was analysed. Pearson’s correlation coefficients were calculated to determine the correlation between the decreased levels of CUEDC2 protein  and the decreased levels of SOCS1 protein in primary AML cells and AML cell lines without SOCS1 promoter methylation. Our result indicated that the coefficient of correlation between decreased protein levels of CUEDC2 and decreased protein levels of SOCS1 in primary AML cells without SOCS1 promoter methylation was >0.9 Table . CombineA luciferase reporter assay was performed to elucidate whether CUEDC2 affected the JAK1-STAT3 pathway. HEL and KG-1 cell lines were cotransfected with the pACT-Luc luciferase reporter plasmid, HA-STAT3 and Flag-CUEDC2 or the CUEDC2 siRNA, respectively. Since CUEDC2 regulated SOCS1 expression at posttranscriptional level, AML cell lines without SOCS1 promoter methylation were used in subsequent studies. Compared to bone marrow cells from healthy donors, four AML cell lines, KG-1, HEL, ML-1 and ML-2, presented lower protein levels and had no SOCS1 promoter methylation. More importantly, HEL cell line had the lowest CUEDC2 protein level; whereas KG-1 cells had the highest CUEDC2 protein level among these four cell lines. Therefore, HEL and KG-1 cells were used to overexpression and knockdown of CUEDC2 expression, respectively. The expression of CUEDC2 increased 5–6-fold in CUEDC2 overexpressed cells, while the expression of CUEDC2 in CUEDC2 knockdown cells only had about 10% of those in WT KG-1 cells Fig. .Fig. 3CU19.Luciferase assays suggested that CUEDC2 overexpression inhibited IL-6-induced STAT3 activation Fig. . In contHEL and KG-1 cells in which CUEDC2 stably overexpressed or knockdown were obtained by lentivirus-mediated transfection. Western blots revealed an approximately fivefold increase in CUEDC2 expressed HEL cells Fig. , but CUE20.No significant differences in proportions of late apoptotic and total apoptotic cells were observed between CUEDC2 overexpressed HEL cells and control cells. Whereas knockdown the expression of CUEDC2 in KG-1 cells decreased the proportion of early apoptotic cells compared with that in control cells Fig. . This reEffects of CUEDC2 on the cell cycle transitions of AML cells were determined. In the presence of overexpressed CUEDC2, a greater percentage of HEL cells were in G0/G1 phase, suggesting that overexpression of CUEDC2 caused G1 phase arrest Fig. . In contEffects of knockdown or overexpression of CUEDC2 on the sensitivity of AML cells to chemotherapeutic drugs was determined. When CUEDC2 was knockdown in KG-1 cells, its resistance to cytarabine and idarubicin was significantly increased Fig. . At the MLL-AF9 bone marrow transplant experiments were performed to determine whether CUEDC2 was involved in MLL-AF9-induced AML in vivo. A detailed description of protocols was summarised in Fig. floxed) or mice transplanted with TAM-treated Cre+ cells (without CUEDC2floxed) had similar latencies to AML development  displayed a significantly shorter latency  counts than that of WT mice  and Flag-CUEDC2 (Flag vector) in HEK293T cells, and a greater amount of SOCS1 was pulled down in the presence of overexpressed CUEDC2 and CUL2  1640 medium, and transferred as an overlayer to centrifuge tubes containing 10 ml of Ficoll-Hypaque . After centrifugation at room temperature for 30 min, the interface layer, containing predominantly leukaemic blasts, was extracted with a sterile Pasteur pipette, suspended in RPMI medium, and washed three times. These mononuclear cells isolated from patients with AML were used to detect SOCS1 and CUEDC2 expression. Luciferase activities were determined using a Dual-Luciferase Reporter Assay System (Promega). The MSCV-MLL-AF9 plasmid was a kind gift from Dr. Tao Cheng . The CUEDC2floxed, CreER+ mice were generated by Guangzhou Cyagen Biosciences Inc.The AML cell lines HEL, U937, THP-1, KG-1, K562, HL-60, ML-1 and ML-2 used in the present study are preserved in our laboratory. For primary AML cells, leukaemic blasts were obtained from the peripheral blood of AML patients after obtaining informed consent. These studies have been sanctioned by the Investigational Review Board of Xuzhou Medical University, and all patients provided informed consent. Mononuclear cells were isolated as previously described16.Total RNA was isolated from AML cell lines and mononuclear cells isolated from patients with AML using Trizol reagent (Invitrogen). Reverse transcription was performed with the Transcriptor First Strand cDNA Synthesis Kit (Roche). Primers and TaqMan probes for SOCS1 were purchased from Life Technologies . Levels of specific mRNAs in AML cells were detected. The methylation of the SOCS1 promoter in AML cell lines and primary AML cells was detected using a methylation-specific polymerase chain reaction, as previously describedLevels of the SOCS1 and CUEDC2 proteins in the AML cell lines were detected by western blotting. However, levels of the SOCS1 and CUEDC2 proteins in the mononuclear cells isolated from patients with AML were detected using both the protein expression antibody array protein chip from Shanghai H. Wayen Biotechnology Inc., and western blotting. Levels of the SOCS1 and CUEDC2 protein in the mononuclear cells isolated from the healthy donors were set to 1.0. The decrease in levels of CUEDC2 and SOCS1 proteins in AML cells was used to determine the correlation between the two by calculating Pearson’s correlation coefficient.™ RNAi Designer from Life Technologies. Sequences of the CUEDC2 shRNA, scrambled RNA and pWPXLd-CUEDC2 are listed in Table Lentiviral pLV-shRNA and pWPXLd plasmids were used to knockdown and overexpress CUEDC2 in AML cells. Two CUEDC2-targeting sequences were designed by BLOCK-iTAML cell lines were transfected with the CUEDC2 shRNA, pWPXLd-CUEDC2 or control lentiviruses for 72 h. Next, cells were continuously cultured in the medium containing 1.2 μg/mL puromycin for approximately 15 days. Finally, AML cells transfected with CUEDC2, shRNA or control lentiviruses were sorted by flow cytometry.Total proteins were extracted using Mammalian Protein Extraction Reagent (Thermo) supplemented with a complete protease inhibitor cocktail (Roche). Immunoprecipitations were performed by incubating whole cell extracts with the indicated antibody for 6 h at 4 °C with rocking after a preincubation with Protein A/G-Sepharose (Abcam). Immunoprecipitates were washed and resolved by SDS-PAGE. For MS, after Coomassie Brilliant blue staining (Sigma-Aldrich), total protein bands in the lane were excised and analysed by ion-trap MS at Shanghai Genechem Co., Ltd. Antibodies against Flag (No. 8146), Myc (No. 2272), anti-HA (No. 3724), SOCS1 (No. 3950), STAT3 (No. 9139), pSTAT3 (No. 9131), JAK1 (No. 3332), pJAK1 (No. 3331) and Ub (No. 3936) were purchased from Cell Signalling Technology.3 cells per well, and viability was detected every 24 h for 5 days. Approximately, 2.0 × 104 cells were treated with various concentrations of cytarabine and idarubicin for 48 h, and 10 μL of CCK-8 were then added to each well to determine the cells’ sensitivity to chemotherapeutic drugs. Following 4 h incubation, the absorbance was measured at 450 nm.Cell proliferation was assessed with the Cell Counting Kit-8 (CCK-8) assay. AML cells were seeded into 96-well plates at a density of 3 × 106 cells and cultured in 2 mL of serum-free IMDM medium for 24 h to synchronise the cell cycle for cell cycle analysis. The phases of the cell cycle were determined by staining cells with 7-AAD (Sigma), according to the manufacturer’s protocols. AML cell apoptosis was detected using an Annexin-V-APC and 7-AAD kit, according to the manufacturer’s instructions. The analysis was performed on a FACScan cytometer (BD) using CellQuest software.Cells were plated in 6-well plates at a density of 2 × 10Escherichia coli BL21 strain and purified according to manufacturer’s instructions . The Myc-SOCS1 protein, which was obtained from the whole cell lysates of the indicated cells transfected with the Myc-SOCS1 plasmid, was incubated with GST and GST-CUEDC2 or its truncated fusion protein bound to GST beads in 1 mL of binding buffer containing protease inhibitor cocktail at 4 °C for 6 h. GST beads were then washed three times, suspended in 20 μL of 1× SDS-PAGE loading buffer and detected by immunoblotting.GST and GST fusion proteins were expressed in the floxed/floxed CreER− and CUEDC2floxed/floxed CreER+ mice were administered 150 mg/kg 5-fluorouracil . Recovered mononuclear bone marrow cells (MNBCs) were transduced with recombinant MLL-AF9-expressing retroviruses and transplanted into lethally irradiated F1 C57BL6, Ly-45.2 mice. Recipient mice developed leukaemia within 40–60 days, with a median survival time of 51 days. The survival rates of the different groups were determined.Both CUEDC2P values for all Kaplan–Meier survival curve analyses. The correlations between the levels of the CUEDC2 and SOCS1 proteins in AML cells were analysed by calculating Pearson’s correlation coefficients. Comparisons of mean values between the control and treated groups were analysed using Student’s t test. P  < 0.05 was considered significant.Statistical analyses were performed using the SPSS version 16.0. Data are presented as the means ± SEM of three independent experiments. The log-rank test was used to determine Figure S1Table S1Table S2Table S3Summary and Figure legend for supplemnbtary date