Objective. To investigate the vasorelaxation effect and related mechanisms of an estrone derivate EA204 on isolated arterial preparation from rabbit thoracic aorta. Methods. Aortic rings from rabbit thoracic aorta were prepared and held in small organ bath filled with Krebs solution; tension change was recorded by a multichannel physiological signal collection and handling system. Results. EA204 (10−5 to 10−3 M) induced a concentration-dependent relaxation of aortic rings with endothelium and without endothelium. In denuded arterial preparations, EA204 had a potent relaxing effect on isolated arterial preparations contracted with phenylephrine, norepinephrine, and high-K+ solution or BaCl2. Mechanism study indicates that EA204 relaxes aortic rings by inhibiting Ca2+ channels (both receptor-operating Ca2+ channels and the voltage-dependent Ca2+ channels were involved) to decrease extracellular Ca2+ influx and intracellular Ca2+ release. EA204 is different from verapamil, which is a noncompetitive inhibitor of Ca2+ channels. In addition, K+ channels opening may contribute to this vasorelaxation effect. Conclusion. EA204 had a potent endothelium-independent relaxing effect on isolated arterial preparation by inhibiting Ca2+ channels and opening K+ channels. The results suggest that EA204 is a potential compound for treatment of cardiovascular diseases in postmenopausal women.Estrogen and its derivatives exert vascular protective effects, but the underlying mechanisms remain to be studied fully. It is widely accepted that the low incidence of cardiovascular mortality and morbidity in women can be ascribed to differences in their hormonal status compared to men. Moreover, cessation of ovarian hormones after menopause is known to increase susceptibility to vascular dysfunction such as atherosclerosis  and possConsidering the serious side effects and untoward reactions of natural estrogen, it is reasonable and necessary to synthesize new estrogen derivatives for treatment. Vasorelaxation induced by estrogen and derivatives may be either endothelium-dependent or endothelium-independent , 6. EstrNew Zealand rabbits of both sexes (2.0~2.5 kg) were obtained from the Experimental Animal Center of Ningxia Medical University. All animal experiments in this study were approved by the Ethics Committee of Ningxia Medical University.2 (1.2), CaCl2 (2.5), NaHCO3 (15.5), KH2PO4 (1.2), and glucose (11.5). The pH of the solution was adjusted to 7.3–7.4 with 5% CO2: 95% O2. All solutions were stored at −4°C and fresh dilutions were made daily. These drugs were dissolved in distilled water.A modified Krebs solution with the following ionic composition (mM) was used : NaCl . KCl was from Shenyang Chemical Reagent Factory, China. CaCl2 and 5% CO2, one of which was fixed to the organ bath and the other to a force displacement transducer (Chengdu Instrument Plant). Isometric tension change was recorded by RM6240B Multichannel Physiological Signal Collection and Handling System (Chengdu Instrument Plant). Each preparation was stretched to an initial tension of 1 g [Rabbits were killed by overdose of sodium pentobarbital (25 mg/kg) injected via the ear vein. The thoracic aortas were dissected and isolated by removing the surrounding connective tissue. They were cut into rings (3 mm) and were mounted between two stainless-steel triangles in a small organ bath containing 10 mL Krebs solution (37°C), aerated with 95% OμM) and then acetylcholine (20 μM) was added into the organ bath to determine whether the aorta was intact or denuded. If the maximal relaxant effect was more than 80% of initial contraction, we considered functional endothelium to be present. Failure of arteries to relax to acetylcholine was considered to indicate denuded aorta [μM); (2) after the contraction had reached a stable plateau, the strips were washed by Krebs solution and 30 min later the strips were precontracted again; (3) when a stable contraction was obtained, the cumulative concentrations of EA204 (10−5 to 10−3 M) were added and relaxation produced by each concentration of EA204 was measured and expressed as a percentage of the maximum possible relaxation . In other three groups, the experiments were repeated with norepinephrine (3 μM), high-K+ (60 mM), and BaCl2 (2 mg/mL), respectively.The effects of EA204 on the rings with intact or denuded endothelium have been examined. In order to test the endothelium-independent vasorelaxation induced by EA-204, endothelium was mechanically removed by rubbing with a steel wire. Aortic rings were precontracted with phenylephrine  to obtain control concentration-response curves of Ca2+. Then, rings were washed by Krebs solution and equilibrated for 1 h. Following the incubation of EA204 (10−5 M) for 20 min, the concentration-response curves of Ca2+ were repeated. Verapamil (10−7 M) was used to compare with EA204 as a noncompetitive inhibitor of Ca2+ channels.To investigate how EA204 affects Ca+ channels are involved, the affection of glibenclamide on the vasorelaxation effect of EA204 was investigated. After 1 h equilibration, the isolated preparations were precontracted with BaCl2 (2 mg/mL) or KCl (60 mM). After the contraction had reached a stable plateau, EA204 was added in a cumulative fashion (10−5 to 10−3 M) to obtain control concentration-response curves of EA204. Then, the isolated preparations were washed by Krebs solution and equilibrated for 1 h. Following the incubation of glibenclamide (10−5 M) for 10 min, the concentration-response curves of EA204 were repeated [To determine whether Krepeated .n refers to the number of preparations from different animals used.The data are presented as the means ± SEM for the number of experiments indicated; −5 to 10−3 M) induced a concentration-dependent relaxation of rings with endothelium and without endothelium (as shown in −5 to 3 × 10−4 M) induced a weaker relaxation in denuded aorta, but the maximal responses were identical under high concentration (10−3 M) condition.The effects of EA204 on the rings with intact or denuded endothelium were examined. Results have shown that EA204  resulted in concentration-dependent contraction of the isolated aorta of rabbits. Both EA204 and verapamil could shift the concentration-response curves of CaCl2 to the right, and the rightward shifts were concentration-dependent. It suggested that EA204 and verapamil inhibited voltage-dependent Ca2+ channels and decreased Ca2+ influx. However, after incubation with verapamil the maximum responses of the cumulative concentration-response curves were significantly decreased, while EA204 did not affect the maximum responses (as shown in To investigate whether EA204 affects Ca+ channels are involved, we investigated the affection of glibenclamide on the vasorelaxation effect of EA204. Glibenclamide is a K+ channels antagonist which could selectively block ATP sensitive K+ channels to increase extracellular Ca2+ influx. It was found that the vasorelaxation effect of EA204 was significantly decreased after the incubation of glibenclamide (as shown in Figures + channels opening may contribute to the vasorelaxation effect of EA204.To determine whether Kβ-estradiol has been shown to augment endothelium-dependent vasodilation [The cardiovascular protective activity of estrogen is reported to be mediated by an effect on the vessel wall itself directly and an indirect effect on the lipoprotein metabolism. Recently, researchers have made numerous efforts to understand estrogen's cardiovascular actions, especially in its endothelial mechanisms , 14. Fordilation , promotedilation , 17, anddilation . However2+ channels: the voltage-dependent Ca2+ channels and receptor-operated Ca2+ channels. Phenylephrine and norepinephrine cause vasoconstriction by opening receptor-operated calcium channels [+ solution can excite voltage-dependent Ca2+ channels and increase extracellular Ca2+ influx; BaCl2 causes the depolarization of the cell membrane and intracellular Ca2+ release; moreover, BaCl2 can transit the cell membrane through the Ca2+ channels to bind with troponin directly. Researchers have demonstrated that the relaxant effect of 17β-estradiol on human saphenous vein was elicited by calcium-dependent pathways [2+ channels.In smooth muscle cells, there are two major kinds of Cachannels , 20. Higpathways . Consist2+ channels, a noncompetitive calcium antagonist verapamil was used to compare with EA204. CaCl2 was added in a cumulative fashion to obtain control concentration-response curves of Ca2+. As reported before, increasing concentration of CaCl2 resulted in concentration-dependent contraction of the isolated aorta [2 to the right, and the effects were dose dependent. The results suggested that EA204 and verapamil inhibited voltage-dependent Ca2+ channels and decreased Ca2+ influx. However, after incubation with verapamil, the maximum responses of the cumulative concentration-response curves to CaCl2 were significantly decreased, which were not affected after incubation with EA204. The result indicates that, different from verapamil, EA204 is not a noncompetitive calcium antagonist.In order to know more about the relationship between relaxation effect of EA204 and Caed aorta . Moreove+ channels antagonist, which can selectively block ATP sensitive K+ channels to increase extracellular Ca2+ influx [+ channels are usually involved in the relaxing effect; thus we investigated the effect of glibenclamide on EA204 induced vasorelaxation to discuss if K+ channels are responsible for this effect. We found that the vasorelaxation effect of EA204 was significantly decreased after the incubation of glibenclamide. The results indicate that K+ channels opening is involved in the mechanisms of vasorelaxation effect.Glibenclamide is a K+ influx . K+ chan2+ influx through voltage-dependent or receptor-operated Ca2+ channels, the inhibition of intracellular Ca2+ release, and the opening of K+ channels. These data suggest that EA204 is a potential compound to be used for the treatment of cardiovascular diseases in postmenopausal women.Taken together, the endothelium-independent vasorelaxant action and underlying mechanism of estrone derivate EA204 in rabbit aorta were elucidated for the first time in this study. The mechanism includes the inhibition of Ca


AML pathogenesis is attributed to several recurrent chromosomal abnormalities in nearly half of the AML cases while the rest of the AML cases show at least one or more molecular abnormalities. Activating mutations and/or high expression of receptor tyrosine kinase (RTK) family members  have been well documented in AML. Specific RTK inhibitors (TKI) have been developed for targeted therapy of AML and many are currently under study . Sunitin+ ALDH+ cells were stained using Aldefluor Kit (Stem Cell Technologies) and CD34-VioBlue antibodies (Miltenyi Biotech).Human primary AML patient bone marrow or peripheral blood mononuclear cells (AML-MNC) were isolated by density gradient centrifugation. Human normal hematopoietic stem/progenitors cells (HSPC) were isolated from umbilical cord blood using anti-CD34 antibodies on a magnetic separator (Miltenyi Biotech). AML-MNCs and HSPC were incubated in SFEM (Stem Cell Technologies) with varying concentrations of Sunitinib malate (Sigma) and DMSO as vehicle control for 24 hours. Clonogenic assays were performed by plating the drug treated cells in Methycellulose and the colonies were evaluated after 10-14 days. CD34+ALDH+cells and CD34- ALDH- cells were sorted and incubated with or without Sunitinib. AML colonies were detected only in CD34+ALDH+ population that showed total inhibition by 7 µM Sunitinib.With 7µM Sunitinib, there was a 75% reduction  of AML-CFC as compared to untreated, while 70µM Sunitinib treatment showed total eradication of AML-CFC. Treatment of normal HSPC with 7µM Sunitinib showed only 29% reduction  Figure  of normaSunitinib is effective in reduction of clonogenic AML cells. Sunitinib also inhibits normal HSPC derived CFCs albeit less effectively than AML-CFC. This study highlights the relevance of TKI inhibitors in targeting AML. Further studies are aimed at confirming the molecular mechanisms involved in the inhibition as well as taking this study further for targeting patient derived leukemic stem cells using immuno-deficient mouse models.Authors would like to thank KACST for funding this project (grant number 09BIO-693-03).


MIR233 is genetically or epigenetically silenced in a subset of acute myeloid leukemia (AML). MIR223 is normally expressed throughout myeloid differentiation and highly expressed in hematopoietic stem cells (HSCs). However, the contribution of MIR223 loss to leukemic transformation and HSC function is largely unknown. Herein, we characterize HSC function and myeloid differentiation in Mir223 deficient mice. We show that Mir223 loss results in a modest expansion of myeloid progenitors, but is not sufficient to induce a myeloproliferative disorder. Loss of Mir223 had no discernible effect on HSC quiescence, long-term repopulating activity, or self-renewal capacity. These results suggest that MIR223 loss is likely not an initiating event in AML but may cooperate with other AML associated oncogenes to induce leukemogenesis. MIR223 gene in a male patient with therapy-related AML . Al. Al- c-kobserved . Moreovels Figs. , suggestrol HSCs . Likewisobserved . Finallyent HSCs . Thus, MJAK2 or FLT3 ,16. The pathways leading to myeloid expansion associated with activating mutations of tyrosine kinases genes and miR-223 loss are likely to be distinct. However, it also is possible that the severity of myeloid expansion determines whether HSCs are affected. In support of the latter possibility, mice carrying a D835Y FLT3 mutation have a less aggressive myeloproliferative phenotype compared with FLT3-ITD, and HSC loss is only seen in the FLT3-ITD mice [Mir223 is not sufficient to induce a myeloproliferative disorder in mice, suggesting that loss of this gene is likely not an initiating event in AML. MiR-223 silencing is often seen in association with specific mutations, such as AML-ETO. Whether miR-223 loss cooperates with AML-ETO or other oncogenes to induce AML will require further study.Collectively, these data show that miR-223 loss results in a modest expansion of myeloid progenitors without significant effects on HSCs. This contrasts with the HSC exhaustion that is observed in mice with a myeloproliferative disorder caused by expression of activating mutations of ITD mice . Our dat


The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca2+, or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca2+ but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca2+ than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca2+. Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca2+ entry in sperm through the Ca2+/CaM/CaMKKs/CaMKI pathway. The Ca2+/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca2+ entry in the cells.Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca Biological sperm functions such as motility and ability to undergo acrosome reaction (AR) are central to male fertility. These functions are highly dependent on energetic metabolism which is itself largely controlled by 5’-AMP activated protein kinase (AMPK) signaling. The activity of this kinase is regulated by calcium through signaling pathways –2 that aThe PKA, PKB and PKC signaling pathways have previously been shown to be involved in chicken motility regulation –8 and PK2+/calmodulin-dependent protein kinase kinases (CaMKKs) were initially identified as novel members of the protein serine/threonine kinases CaMK family, with 2 forms, CaMKKα and CaMKKβ , both expressed in the nervous system, in endothelial cells of many areas of the brain, in hematopoietic cells, and at lower levels in testis, spleen, lung, liver and skeletal muscle -methyl-2-aminoethanesulfonic acid) (318 mOsm and pH 7.4) containing 5mM Ca2+ for 5 min. The samples were then centrifuged at 400g for 5 min and the pellets resuspended in 100μl NaCl-TES. FITC-PNA was then added (1mg/ml) and the sperm were incubated for 10 min in the dark at 4°C, then washed in 440μl of NaCl-TES and centrifuged at 400g for 5 min. The pellets were resuspended in 200μl NaCl-TES for analysis. The sperm having completed their acrosome reaction were identified and counted under fluorescence microscopy . A minimum of 100 sperm were counted for each sample (two slides/sample = 1 replicate) and a total of six replicates/treatment examined. Acrosome reaction was characterized by the green fluorescence of the acrosomal region [The completion of the acrosome reaction was detected by FITC-conjugated peanut agglutinin (FITC-PNA) binding . Sperm . Cell lysates were centrifuged at 12000g for 30 min at 4°C and the protein concentration in each supernatant was determined by a colorimetric assay . The proteins were then separated by 10% SDS-PAGE (SDS Polyacrylamide Gel Electrophoresis) and transferred onto nitrocellulose membrane . Afterwards, the membranes were incubated in anti-CaMKKα (50kDa), anti-CaMKKβ (65kDa) or anti-AMPKα (62kDa), anti-phospho-Thr172-AMPKα (62kDa) or anti-CaMKI (41kDa), anti-phospho-Thr177-CaMKI (41kDa) or anti-CaMKIV (60kDa), anti-phospho-Thr196-CaMKIV (60kDa) diluted in 5% BSA in TBS-Tween 0.1%  as primary antibodies overnight at 4°C. Finally, the membranes were further incubated for one hour in anti-rabbit IgG (H+L) (DyLight 680 Conjugate) . The band intensity was analyzed using Odyssey Software, version 1.2 . AMPKα, CaMKI, or CaMKIV were used as loading controls. The secondary antibody alone control was used to check for non-specific labeling by the secondary antibody.6/ml) were fixed in paraformaldehyde (4%) for 4 min, spread onto a poly-L-lysine slide and then air-dried at room temperature. Afterwards, sperm were washed in PBS (3 x 3 min) and then permeabilized for 10 min with 0.35% Triton X-100 (Sigma-Aldrich) in PBS. Non-specific binding was blocked with PBS supplemented with 10% goat serum (Sigma–Aldrich) for 30 min at room temperature. Samples were then incubated overnight at 4°C with anti-CaMKKα, anti-CaMKKβ, anti-CaMKI or anti-CaMKIV, diluted 1:100 in PBS-1% goat serum, rinsed (3 x 3 min) with PBS and then incubated with biotinylated goat anti-rabbit IgG (H+L)  (1:200 in PBS-1% goat serum) for 1 hour at 4°C. After 3 rinses with PBS, sperm cells were incubated with Cy2 conjugated-Streptavidin (Southern biotech) (1:200 in PBS) for 45 min at room temperature in the dark, rinsed (3 x 3 min) with PBS and incubated with 4’,6’-diamidino-2-phenylindole (DAPI) (Sigma–Aldrich) (0.05μg/ml) for 10 min. The presence of CaMKKα, CaMKKβ, CaMKI and CaMKIV in sperm was examined by fluorescence microscopy . Negative controls were performed by omitting primary antibodies.CaMKKα, CaMKKβ, CaMKI and CaMKIV were localized in chicken sperm by immunocytochemistry. Sperm (500 x 10The data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using the GraphPad InStat program . The significance of the difference between treatments was calculated by Student's t-test or analysis of variance (ANOVA). The level of significance was at P < 0.05.The presence of CaMKKs (α and β) was assessed by western-blotting and indirect immunofluorescence using specific primary antibodies against CaMKKα and CaMKKβ respectively. Proteins of the predicted size of CaMKKα (∼50kDa)  and CaMKA band of approximately 41kDa was detected using the anti-CaMKI antibody, showing the presence of CaMKI in chicken sperm . CaMKI wA band of approximately 60kDa was detected using the anti-CaMKIV antibody, showing the presence of CaMKIV in chicken sperm . CaMKIV Collectively, the results in Figs 2+ . The maximum percentage of motile sperm was observed with 5mM Ca2+  at 5 min, the percentage of motile sperm then decreased  and lasted for over 60 min .To further characterize Ca2+-dependent AMPK phosphorylation, we studied CaMKI and CaMKIV phosphorylation at Thr177 and Thr196 respectively in the presence or absence of 5mM Ca2+ and/or of the CaMKK inhibitor 10μM STO-609. Results reported in 2+, showing that CaMKKs inhibition decreases CaMKI phosphorylation. After 15 min, STO-609 inhibited CaMKI phosphorylation in the presence as well as in the absence of extracellular Ca2+, suggesting a secondary involvement of Ca2+ intracellular store in CaMKK catalysis of CaMKI phosphorylation. However, STO-609 did not significantly affect the phosphorylation of CaMKIV . Over the whole 1-50μM range, STO-609 decreased the percentage of sperm motility and AR in a dose-dependent manner, both in the presence and absence of Ca2+. The initial motility and AR levels were even higher in the presence of Ca2+  in both the presence or absence of Ca2+ .2+, 10μM STO-609 significantly decreased motile, rapid and progressive cells after only 5 min of incubation compared to the control, while the other parameters, VAP, VSL, VCL, LIN and STR, were significantly decreased compared to the control after 60 min of incubation. There was about 28% reduction of the percentage of motile sperm in the medium without 5mM Ca2+ at 5 min and 62% at 60 min, and 16% reduction of the percentage of motile sperm in the medium with 5mM Ca2+ at 5 min and 38% at 60 min. Moreover, the motility parameters were even higher in the presence than in the absence of Ca2+.In the presence or absence of Ca2+, the respective decreases after 5 min and 60 min were ~15% and ~60% .The addition of 10μM STO-609 significantly decreased the sperm acrosome reaction capacity in a time-dependent manner, and acrosome reaction was dramatically reduced after 60 min of incubation in the presence or absence of calcium in the medium . In the and ~60% . Thus th2+ was present or not.After 5 min of incubation in 10μM STO-609, the percentage of sperm viability assessed with SYBR14/PI was not affected. However, after 60 min of incubation in 10μM STO-609 significantly decreased sperm viability by 9% in both the absence and presence of calcium in the medium compared to their respective controls. After 60 min of incubation, the rate of viable cells decreased by 20% for the controls and by 26% with 10μM STO-609 in both media compared to the measurements taken after 5 min . The 10μCalcium plays fundamental and diversified roles in cell metabolism. The sperm are highly motile cells with a silent genome –4 but th2+ in the sperm medium increased by 29% AMPK phosphorylation and 22% CaMKI phosphorylation and sperm functions as expected in case of activation of the Ca2+/CaMKKs/CaMKI pathway while the inhibition of this pathway through STO-609 returned to the basal levels observed in absence of Ca2+. AMPK is a central actor in energy homeostasis regulation having a key role in activation of sperm function [2+/CaM complexes and are thus members of the CaM kinase family [For the first time in germ cells, we localized CaMKKα and CaMKKβ and showed that CaMKKβ and phospho-AMPK  were loce family . CaMKKs e family –2. The le family –2. On th2+ and/or of the selective CaMKK inhibitor STO-609 provided further evidence that Ca2+/ CaMKK signaling is effective in the stimulation of AMPK and CaMKI phosphorylations. Both CaMKI and AMPK phosphorylations were stimulated in the presence of extracellular Ca2+ concentrations corresponding to normal physiological levels in chicken oviducts [2+ was close to that which had been previously observed with boar sperm [2+, the phospho-Thr172-AMPK was not affected by STO-609 even at the highest concentration of 50μM, showing the dependence of CaMKKs towards extracellular Ca2+ for AMPK phosphorylation. STO-609 is a selective inhibitor of both CaMKKα and CaMKK β [in vitro [2+-induced AMPK activation in chicken sperm is mediated through CaMKKs.Experiments with the presence or absence of extracellular Caoviducts . The AMPar sperm . In the in vitro . Such ac2+ [in vitro in less than 5 min [CaMKK-dependent AMPK activation was found to be very rapid in chicken sperm, in less than 5 min of incubation at 35°C. This is consistent with our previous results showing fast AMPK activation by AICAR in the absence of extracellular Ca2+ . These a2+ . Unlike 2+ –52. Bird2+ . They do2+ . The sigan 5 min . We thus2+ stimulation, and that this effect was inhibited when the cells were pre-treated with STO-609. This strongly suggests that CaMKKs catalyzed CaMKI phosphorylation. Interestingly, the STO 609-dependent decrease of CaMKI phosphorylation was observed even in the absence of extracellular calcium, albeit more slowly (15 min instead of 5 min). This suggests that CaMKI can be activated in chicken sperm in response to the release of internal stores of cellular Ca2+. The sensitivity of CaMKKβ to STO-609 being 5-fold higher than that of the CaMKKα [2+ internal stores, whereas the faster CaMKI phosphorylation in the presence of extracellular Ca2+ is mediated through CaMKKβ activation. However, we did not observe any change of CaMKIV phosphorylation while using STO-609. This suggests that the localization of CaMKIV in the sperm flagellum is consistent with a putative role in chicken sperm motility regulation, but not through the mediation of CaMKKs.Furthermore, we show that CaMKI phosphorylation was increased upon extracellular Cae CaMKKα , it migh2+, in parallel with a decrease in AMPK and CaMKI phosphorylations. This suggests that CaMKKs affect chicken sperm motility and acrosome reaction through AMPK and CaMKI activation. STO-609 also quickly (after 5 min) decreased chicken sperm functions in the absence of extracellular Ca2+. However, in the absence of extracellular Ca2+, the decrease of CaMKI phosphorylation was only observed after 15 min of incubation, indicating the involvement of intracellular Ca2+ in CaMKI phosphorylation through CaMKKs, but the decrease was slower than in presence of extracellular Ca2+. It can thus be hypothesized that the direct Ca2+/CaMKKs/AMPK and indirect Ca2+/CaM/CaMKKs/CaMKI/AMPK signaling pathways are rapidly involved together in chicken sperm functions in the presence of extracellular Ca2+. In its absence, the Ca2+/CaM/CaMKKs/CaMKI signaling pathway could be involved in chicken sperm motility, and AR in another way independent of extracellular Ca2+ entry after 15 minutes of incubation. Alternatively, the levels obtained without extracellular Ca2+ could also take other routes that do not pass through Ca2+/CaMK/CaMKKs/CaMKI signaling pathway since the decrease of sperm functions observed from 5 to 15 min of incubation in the presence of STO-609 reached a basal level lower in the absence of Ca2+ than in Ca2+ presence. Both CaMKI and AMPK can be phosphorylated by CaMKKs, suggesting direct and/or indirect AMPK activation through its Thr172 phosphorylation. However, the possibility that the Ca2+/CaM complex directly affects sperm functions cannot be ruled out since the addition of W-7 or trifluoperazine (inhibitors of CaM) inhibited the motility of fowl spermatozoa [2+ dependent sperm functions in other species [2+ is also known as a regulator of the predominant flagellar adenylate cyclase variant, soluble adenylyl cyclase (sAC) [We also demonstrate here that appropriate doses (10μM) of the CaMKK inhibitor, STO-609, decreased chicken sperm motility and acrosome reaction without affecting sperm viability. STO-609 rapidly decreased sperm AR as well as several sperm motility parameters  after 5 min of incubation in the presence of extracellular Carmatozoa . Moreovermatozoa  and is i species –57, but se (sAC) –60, but se (sAC) .2+ levels from extracellular Ca2+ entry in the sperm activates the Ca2+/CaMKKs/AMPK signaling pathway with further stimulating effects on sperm motility and AR. In the absence of extracellular calcium, the Ca2+/CaMKK signaling pathway is less solicited to phosphorylate AMPK and other ways of action are opened. In all cases, the signaling pathways are quicker with extracellular calcium AMPK pathway (5 min) than with stimulation by intracellular calcium CaMKI pathway (15 min). Taken together, our data bring new information that highlight the complex relationships between calcium signaling pathways and energetic metabolism needed to ensure germ cell functions.All our results suggest the action of new actors in the regulation of sperm functions that are activated in the presence of extracellular calcium and are summarized in 2+/CaM/CaMKKs/CaMKI, involved in birds’ sperm functions which can be activated with extracellular calcium. These results improve our knowledge of signal transduction in highly reactive cells with silent genomes.This study provides the first evidence of the localization and action of CaMKK α and β and CaMKI on extracellular calcium dependent AMPK regulation in sperm cells. We have also shown a new signaling pathway, Ca


There is an undeniable mystique about the position, and many academicians may find themselves wondering: “Maybe I could do this? Maybe I should do this? If it were me, I could do this so much better….”In some respects, the Chair is like any other or many other jobs. But the reality is that a medical school Department Chair is a unique position, with distinctive relationships and responsibilities. The scope of the position can be breathtaking, ranging from oversight of medical student training to maintenance of the highest quality patient care. Despite the importance of this position, there has been little attention in the literature to the realities of chairmanships and virtually nothing in terms of practical advice.For eight years I was a medical school Department Chair. When I look back I can only wonder how anybody manages to do the job. My formal preparation was basically nonexistent. There was, of course, on the job training, but there should be a better way.One way to approach a difficult situation requiring a new set of behaviors is a 12-step program. This approach has been widely utilized for alcoholism and drug addiction, but has also been incorporated in programs involving work behavior for psychiatrists and nurse practitioners.I arrived my first day as Chair, at Grand Rounds, shortly after the beginning of the conference. My in-house rival for the job promptly stood up and left the room. It was the beginning of my real exposure to a Department in which (among other things) clinical programs were rudimentary or nonexistent, and the residency program was on probation and seemed ready to be disbanded. In fact, several years earlier there were serious discussions to merge the Department into another (and far better) Department at a nearby medical school. By the end of my Chairmanship, about thirty faculty members had been recruited, half a dozen clinical programs were established, the residency program was off probation and on solid footing, and the Department had achieved national recognition including entering the top ten in NIH research funding.The 12 steps:1.The Chair position is truly middle management. You are the interface between faculty, residents, and staff (on the one hand) and the Dean’s Office (on the other). This is a middle management administrative position. That’s all it is. And, despite what you may have imagined, there is no throne or crown that comes with the position. You may wish to periodically remind yourself: middle management, administrative position. As mantras go, that one is not too bad for a Chair.2.This seems obvious, but will often be quickly forgotten. Chairs are chosen because they have been successful. They have run successful clinical programs, successful laboratories, successful program projects, successful residency programs. Success, success, success…. So, a Chairmanship is going to be just one more success story, right? Well, maybe…but it can also be an illustration of the Peter Principle, which, as you may recall is as follows: Everyone rises to their level of incompetence.The best way to avoid being a walking, talking illustration of the Peter Principle is to approach the position with a degree of humility. And humility may not be easy, given the past successes of the new Chair. But being Chair will be quite different from anything tackled before.The most important thing about the learning curve is to realize…there is a learning curve! The way you handle an issue when you start out as Chair will be different than how you would deal with it after being Chair for a number of years. On the job growth can occur, if you genuinely realize that you have a lot to learn.3.Different institutions will have different attitudes and expectations for the Chair. That is pretty obvious. What is less obvious is that different classes of institutions will view Chairs differently. Specifically, the differences between a public and private institution have not been that well appreciated, at least as they pertain to the Chair.Chair authority at private institutions tends to be far greater than at public institutions. Accountability to faculty will be substantially more at public institutions, where a Chair can easily feel squeezed between the Dean’s expectations and the demands of the faculty. Private institution Chairs can be more dictatorial, I mean authoritarian, and get away with it. Formal Chair reviews are common in public institutions, but may be absent at private institutions.The private/public distinction for Chair authority is particularly important at the beginning of the Chairmanship. Chairs are often recruited to and enter an institution as Department Chair. The transition from being a faculty member at a private institution to being Chair at a public institution will create (at the very least) some significant culture shock.4.More likely than not, your predecessor Chair will remain in the Department as a faculty member. And not just as any faculty member, but as the person who has been through it and seen just about all of it before. Some of the faculty will still see your predecessor as the true leader of the Department, title or no title.Your predecessor will view you with some serious ambivalence. Your successes will not generate wholehearted admiration, and your failures may not create much sympathy. Your predecessor will be intently interested in what is happening in the Department, but is likely to be at least as interested in their own Chair legacy. Unfortunately, the reputation of your predecessor may be enhanced by your difficulties, and your successes may even be viewed as diminishing your predecessor’s legacy.Given these murky attitudes, what is the best approach? That’s easy. Treat your predecessor with due respect. Set up meetings, lunches or breakfasts, to catch up. Let your predecessor mentor you. Nothing is more flattering to your predecessor than the belief that you want their mentoring. The good news is: You will really benefit from what your predecessor has to say. And the act of mentoring will engage your predecessor and give a stake in your success. And you will have an ally, which is always a good thing.5.You have a good idea of what you want to accomplish as Chair. You have seen the workings of a Department for quite a while now, and you know what needs to be done. So, you set about accomplishing this, achieve it, and voila, you are a successful Chair! Right? Well, not necessarily.As Chair, you will be reviewed. It may be a formal review, it may be an informal review. But your performance will be reviewed. And the criteria of this Chair review may have absolutely nothing to do with what you thought was important.So, one of the first things to do is to find out exactly how the review process will unfold. What are the institution’s criteria for a successful Chair? The review criteria may well be available on paper, for your own perusal. Or it may require a detailed discussion with the Dean.But be very clear on this: You will need to come to grips with your institution’s review criteria, even if they are vastly different from what you think they should be. What you want to avoid is a situation where by your own criteria you are doing a great job, but by the institution’s criteria and expectations, you are not so hot.The tough part may come when you realize that what you think is important for the Chair, what you want to accomplish as Chair, may simply not be what the institution had in mind. You will then be left with adjusting your expectations to the realities of your institution. Either that, or you will be left with a very unhappy Chair experience.6.You have role models for the Chair, of course. There are Chairs out there who you admire, respect, and want to imitate. But are they the ones who are really your role models?It is entirely possible, even likely, that you don’t know who your real role models are. Here’s a clue: think about your own Department Chair. Whether you like it or not, whether you are aware of it or not, you are likely to be re-enacting your former Chair(s), at least some of the time.I once joked (sort of) that when I was faced with a difficult problem, I would ask myself “what would (my former Chair) do in this situation?” And then I thought it through, carried it out, and when I did it…it was a disaster!Of course it didn’t work, because of the vastly different personalities and circumstances. But the point here is this: You may or may not realize it, you may not like it, but you are going to be viewing events through the prism of your experience and relationship with your former Chair. At least, you will at the onset of your Chairmanship. So the quicker you realize this, the better off you will be.7.Your attention will no doubt focus on your faculty, your residents, and medical students. That’s fine. But you will probably overlook the most easily overlooked members of your Department: your staff.Staff can make a Department or Chair look good, or look terrible. As Chair, you will be spending more time with staff than you ever have previously. And one of your biggest roles is to be supportive of your staff. That may not sound too exciting, and yet it can be the most satisfying part of your job as Chair. That’s right, better than Department rankings and recognition and all that other stuff. You will have a degree of emotional intimacy (and I mean that in a good way) with your staff unlike any other relations in the Department. So make the most of it, and you will be amazed how much you and the Department will benefit.8.You are used to having casual conversation with colleagues in your Department, of course. You engage in casual conversations and make casual comments. But now you are Chair, and you will find that some casual comments aren’t so casual anymore. Your words are taken far more seriously. Your words are repeated, sometimes mistakenly, and sometime even distorted.Academic physicians tend to have pretty fragile egos. Motivation for many physicians to enter academia is at least partially related to a desire for recognition, maybe to assuage that fragile ego. This is part of what has been termed a narcissistic personality, which is an interesting topic in itself for academicians. But the larger and more immediate issue for the Chair is this: It doesn’t take much to damage that fragile ego of your Department faculty member. And a thoughtless comment coming from the Chair is doubly damaging (and infuriating).Narcissistic injury generates narcissistic rage. In other words, it is easy for a Chair to make an enemy. And that enemy will return the favor of narcissistic injury, this time directed back at the Chair. And so an academic feud is born, a feud arising out of who knows what. And resolving that feud will take motivation and understanding that is beyond the skill set of most academicians.9.One of your first (and often overlooked) tasks as Chair is to establish a certain climate. Actually, you will be establishing a climate, whether you intend to or not. Ideally, that climate will encompass collegiality, respect, and integrity. Easier said than done….The best way to establish the right climate is by meetings. And this is what being Chair is all about: meetings, meetings, and more meetings. Not emails…meetings!I know, I know…meetings are boring! Of course they are. And here’s the secret: They are supposed to be boring!That’s right, the meetings are supposed to be boring, or at least most of the meetings are supposed to be boring. The fact that meetings are boring  is actually a good sign. It means that the important issues are being addressed, and that there is little excitement left in issues.On the other hand, if the meetings are exciting, well, you are in trouble! You need to have enough meetings to digest the tough issues to a sufficient extent that any significant excitement is largely drained out of them.So, have your meetings, and expect them to be boring. And to get there, you can never have too many meetings.10.Probably the most exciting thing about being Chair is recruiting. This is the real building part of the job. New people, new energy, new ideas…and maybe new problems.What are you looking for in that recruit? Raw academic talent? An academic pedigree that will look good (on paper at least) for the Department? Energy and ambition?Probably the best way to think about it is to view this as if you are bringing someone into your family. An academic department can create extraordinary closeness, connections, emotions.Your emotional reaction to the recruit will tell the story. Do you like this person or not? Can you trust them? Would you want to be in the Department with them if you weren’t Chair?Far too many talented recruits turn out to be world-class headaches. In administrative jargon, they are “high maintenance” faculty. They don’t need a Chair, they need treatment. When recruiting, trust your gut.11.One of my predecessor Chairs is fond of zen-like aphorisms, and one of his favorites is that the Chair “is not a life sentence”. You may have started as Chair with a predetermined limited time frame for the position. But the perks of the position, the sense of entitlement, the stipend, and the  deference can prove to be addictive. What seemed like a few years becomes, well, a life sentence.But the reality is that only you know if you are enjoying the experience. Excitement may be replaced by dread and boredom. Your mind wanders incessantly during the meetings. You have mastered the job, but wonder if Chair mastery really means all that much. Your relations with the Dean have changed, and maybe there is a new Dean in place. The rather benign father figure at the onset has become an increasingly difficult taskmaster. Your meetings with the Dean have an edge, and are sometimes adversarial.There are, of course, a wide variety of variations on this theme. But regardless of the circumstances, it is essential to remember that you are working at the pleasure of the Dean. And if the Dean really is no longer pleased, it’s over.12.I became convinced some time ago that the person who becomes Chair will experience a rapid decline in intelligence, at least a 10% drop of IQ (I know I certainly did). Why do I say such a thing? That’s easy: just look at all the dumb things Chairs do!The good news is, this intellectual decline is reversible…so, enjoy your rebound!--,--The 12-step program described herein provides a unique and practical approach to issues a Chair must face. While casual if not irreverent at times, it has the advantage of accessibility and easy engagement. The question remains, however, how this approach may compare to current and traditional views of the Chair’s position. The need for substantial input to new Chairs is clearly recognized.The author declares that he has no conflict of interest.


Only upon wounding do HF cells migrate from the follicle to contribute to the neo‐epidermis. However, the identity of the first‐responding cells, and in particular whether this process involves a direct contribution of K15+ve bulge cells to the early stage of epidermal wound repair remains unclear. Here we demonstrate that epidermal injury in murine skin does not induce bulge activation during early epidermal wound repair. Specifically, bulge cells of uninjured HFs neither proliferate nor appear to migrate out of the bulge niche upon epidermal wounding. In support of these observations, Diphtheria toxin‐mediated partial ablation of K15+ve bulge cells fails to delay wound healing. Our data suggest that bulge cells only respond to epidermal wounding during later stages of repair. We discuss that this response may have evolved as a protective safeguarding mechanism against bulge stem cell exhaust and tumorigenesis. Stem Cells2016;34:1377–1385The cutaneous healing response has evolved to occur rapidly, in order to minimize infection and to re‐establish epithelial homeostasis. Rapid healing is achieved through complex coordination of multiple cell types, which importantly includes specific cell populations within the hair follicle (HF). Under physiological conditions, the epithelial compartments of HF and interfollicular epidermis remain discrete, with K15 In response to skin injury, peri‐wound hair follicles become activated to make a significant contribution to the healing process. Until recently, the bulge region, which houses the best‐characterized population of stem cells within the hair follicle, has been considered the major contributor to wound repair. Our research contradicts this widely accepted paradigm and reveals bulge inactivity during the first stages of wound healing. This may represent an evolutionarily programmed mechanism to protect against injury‐induced cancer formation.+ve bulge is considered the primary HF SC niche, housing “the most quiescent and long‐lived” population of cells +ve junctional zone The hair follicle (HF) is a dynamically remodeled miniorgan comprising multiple adult stem cell (SC) populations that underlie its tremendous growth and regeneration potential +ve cells persist for up to a year in the healed IFE, whereas Ito et al. +ve lineage within 20 days postwounding. Studies have also demonstrated that HF contribution may be important for healing rate immediately after wounding. Specifically, Langton et al. Under specific conditions of perturbed epidermal homeostasis, HF‐derived keratinocytes actively contribute to the IFE. For example, following injury HF‐derived keratinocytes proliferate and migrate into the neo‐epidermis (IFE) +ve cells in HF regeneration and healing However, the temporal kinetics of the HF‐derived cell contribution to epidermal healing remains unclear. It is particularly important to understand the identity and location of the cell populations that are first activated and responsible for the early contribution of HFs to wound repair. Conversely, whether specific HF progenitor cell niches might be dispensable for epidermal repair remains unknown. The latter issue is particularly important in light of recent work demonstrating (a) plasticity both within and between HF SC niches +ve bulge cells in the early stages of epidermal repair. Here we present the first detailed analysis of HF keratinocyte proliferation over an extensive early wound healing time course. While many studies have analyzed the involvement of HFs in wound re‐epithelialization during the later stages (7 days onwards) to our knowledge until now, the kinetics of the early HF response to wounding has not been studied. We show that while upper HF ORS keratinocytes rapidly proliferate following epidermal injury, with differential induction kinetics between anagen and telogen HFs, K15+ve bulge cells are not activated to proliferate by epidermal injury and appear to be largely dispensable for timely wound re‐epithelialization. We suggest that these unexpected findings reveal an evolutionarily conserved mechanism to protect against tumorigenesis.In this study we clarify the role of K15All animal procedures were approved by the University of Manchester ethical review panel and performed under UK home office licence (number 40/3020 and 70/8136). Mice were housed in groups prior to experimentation with access to food and water ad libitum.PR transgenic strain The previously described K15CreMale C57BL/6 mice at either 32 days old (anagen) or 49 days old (telogen) were anaesthetized with isofluorane by inhalation. A single 6 mm full thickness excision was made on the left hand side of the dorsal flank, with the right hand side left unwounded as an internal contralateral control . The control was used to identify any systemic wound induced changes to hair cycle (none were observed). Buprenorphine was administered for analgesia, and wounds were left to heal by secondary intention, with mice being housed individually postoperatively.PR;DTA mice and sex‐matched littermate controls were anaesthetized, and 2 × 6 mm full thickness excisions were made on the dorsal flank. The biopsy tissue obtained at the time of wounding was collected and fixed for histology, to confirm efficacy of bulge ablation treatment at the time of injury . The two wounds per animal were assessed as independent replicates K15cre2 at 2, 4, 6, 12, 18, 24, or 72 hours postwounding. All mice received 40 mg/kg of 5‐bromo‐2‐deoxyuridine (BrdU) via i.p. injection 2 hours prior to cull to assess proliferation within a defined period , with the exception of the continual proliferation study which received BrdU at the time of wounding and subsequently every 12 hours until cull at 72 hours (or every 12 hours from 108 hours until cull at 7 days) . All wounds were bisected along the caudal to rostral plane to present HFs in a longitudinal orientation . Bisected wounds were fixed and processed as previously described PR DTA studies, controls tissue was taken from the biopsy (time zero), plus also treated and untreated regions at the time of cull.Mice were culled by rising concentration of COhttp://www.vectorlabs.com). Antigen retrieval was via the citrate (pH 6) method with primary antibody incubation performed at 4°C for 16 hours . Sections were counterstained with Gills haematoxylin (Vector). TUNEL assay was performed using the in situ cell death detection kit  as per manufacturer's instructions, antigen retrieval with Proteinase K (Sigma) at 20 μg/ml for 20 minutes at 37°C. Keratin 15 CD34 and α6 integrin were quantified as number of outer bulge cells present in control and K15crePR;DTA mice.Approximately 5 μm tissue sections were used for immunohistochemistry. Protocols for BrdU, Keratin 6, and Cd34 have been previously described Wound edge tissue sections were analyzed for BrdU incorporation within the basal layer of the IFE (excluding neo‐epidermis) and cells within the ORS of HFs. HFs that had been partially amputated during wounding were excluded from analysis. In order to directly compare the same cellular material from anagen VI HFs we only counted ORS within the upper HF . Supporting Information Figure S1E indicates zones of analysis, with BrdU counts presented as a % of total measureable basal cells in that region. Quantification of BrdU positive cells per HF region is presented as a raw number . Sections were washed with Phosphate buffered saline (PBS) supplemented with 0.1% Tween‐20, then incubated in secondary antibody . Sections were washed, counterstained with DAPI, and mounted.Dual immunofluorescence of BrdU and Cd34 was conducted on paraffin‐embedded tissue, with antigen retrieval via the citrate method. Sections were blocked with rabbit serum before incubating for 16 hours with primary antibody at 4°C , during the first 24 hours postinjury. Our methodology, using a single 6 mm circular excision, permitted the first detailed proliferation analysis of longitudinally sectioned wound edge HFs, with corresponding unwounded contra‐lateral flank skin . These analyses revealed that injury induces upper ORS keratinocyte proliferation in peri‐wound HFs from 6 hours postwounding in both anagen and telogen HFs  and a clear increase in TUNEL+ve cells in the bulge , cell death was preferentially induced in the Cd34 SC compartment, while the non‐SC K6 compartment remained relatively intact To functionally probe the importance of the HF bulge for early epidermal repair, we next established an inducible K15‐CreSCs Fig. A. As explge Fig. B–2F. Whi+ve HF bulge cells  prior to wounding , when combined with current literature our data strongly suggest that bulge SCs only respond to epidermal wounding during later stages of healing. Indeed, the seminal work of Ito et al. +ve lineage cell contribution to murine neo‐epidermis at 5–8 days postwounding, has been followed by a plethora of studies revealing the influence of HFs on wound repair (Table Collectively, our data suggest that wounding does not induce substantial bulge activation in unwounded peri‐wound HFs during the first 3 days of healing. Specifically, bulge cells of uninjured HFs do not proliferate or migrate from the bulge SC niche and partial ablation of K15ir Table  12, 13.+ve bulge SC population prior to injury‐induced migration. However, we suggest that these findings could be the result of analysing directly injured HFs, where bulge proliferation/migration may be specifically induced to regenerate the HF itself We recognize that our observations may be seen to conflict with earlier reports by Tumbar et al. and Mardareyev et al. +ve labeled cells from the bulge SC niche 2 days after wounding. In fact, Page et al. report that the initial response to injury is by Lrig1 positive cells in the junctional zone, where we also observe high levels of cell proliferation (Fig. +ve or K19+ve bulge subsets contributed to IFE re‐epithelialization In support of our data, Page et al. Several papers have reported bulge cell proliferation during later healing +ve bulge cells The maintenance of epithelial SC quiescence shortly after wounding may also serve as a safeguarding mechanism against tumorigenesis +ve cells are particularly sensitive to changes in Lef1 Indeed, a number of studies have now linked bulge SCs to carcinogenesis namely to the development of basal carcinoma We conclude that HFs respond rapidly and substantially to skin injury. However, during the earliest phase of wound healing, K15+ve bulge cells are excluded.C.L.G.: concept and design, collection and assembly of data, data analysis and interpretation, manuscript revisions, manuscript writing. D.M.A.: concept and design, collection and assembly of data, data analysis and interpretation, manuscript editing. D.J.H.: provision of study material, concept and design. R.P.: concept and design, manuscript editing. M.J.H.: finance, concept and design, manuscript writing, final editing and approval of manuscript.The authors indicate no potential conflicts of interest.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


Our concept for a multiscale mathematical model of Ca2+ -induced Ca2+ release (CICR) and whole cardiomyocyte electrophysiology incorporates stochastic simulation of individual LC- and RyR-channels, spatially detailed concentration dynamics in dyadic clefts, rabbit membrane potential dynamics, and a system of partial differential equations for myoplasmic and lumenal free Ca2+ and Ca2+-binding molecules in the bulk of the cell. We developed a novel computational approach to resolve the concentration gradients from dyadic space to cell level by using a quasistatic approximation within the dyad and finite element methods for integrating the partial differential equations. We show whole cell Ca2+-concentration profiles using three previously published RyR-channel Markov schemes.Mathematical modeling of excitation-contraction coupling (ECC) in ventricular cardiac myocytes is a multiscale problem, and it is therefore difficult to develop spatially detailed simulation tools. ECC involves gradients on the length scale of 100 nm in dyadic spaces and concentration profiles along the 100 μm of the whole cell, as well as the sub-millisecond time scale of local concentration changes and the change of lumenal Ca The timing of contraction is controlled through electrical excitation via a process known as excitation-contraction-coupling (ECC). ECC is mediated through Ca2+, and is facilitated through an amplification process known as Ca2+-induced Ca2+ release (CICR). In ventricular myocytes, CICR is controlled locally by the colocalization of L-type Ca2+-channels (LCCs) in the T-tubule membrane on the one side of a dyadic cleft [aka Ca2+ release unit (CRU)] and ryanodine receptor channels (RyRs) in the junctional sarcoplasmic reticulum (jSR) membrane on the other side. Depolarization of the plasma membrane leads to the activation of LCCs, which causes Ca2+ entry from the extracellular space into the dyadic space. The influx of Ca2+ activates RyRs, which release Ca2+ from the sarcoplasmic reticulum (SR)  that through a mechanism of local control of CICR, a graded LCC current produces a graded RyR Ca2+] changes in the dyad to tens of seconds for SR dynamics) are involved. To account for that multi-scale character, recently several models with spatially distributed Ca2+ release sites have been developed  and time scales  to simulate a computationally efficient mathematical model of CRUs with spatially resolved  is visualized in Figure We simulated 16 z-discs, each with 320 CRUs, and the Ca2+ transients from the initial concentration profile in the dyadic space, via sparks to the whole cell transient. The cytoplasmic [Ca2+] is rather heterogeneous, due to randomness of release events as well as variations in CRU size. The concentration of 0.6 μM is reached in almost the whole volume, but 2.4 μM only in the proximity of the the z-discs. The currents reproduce basic features of the Mahajan-model . Gain (the ratio of Jrel to JCa) was ~4 during the first 50 ms of the AP and ~3 for the remainder of the AP . With the  detailed molecular stochastic modeling of the CRU at a continuous spatial scale; (2) a whole-cell ODE electrophysiology model, which describes the potassium channels, the voltage gated sodium channels, the sodium-potassium pump, and integrates all membrane fluxes to derive the total membrane current and voltage; (3) a PDE FEM calcium diffusion model representing myoplasmic and nSR CaTaking gradients in the dyadic cleft into account in a simulation with thousands of CRUs would be impossible without the use of the stationary Green function. An estimate of the advantage of the quasi-static approximation with respect to computational speed can be obtained from estimating the number of operations per cleft. Our approach requires to solve a linear system of equations whenever channels open or close or the boundary condition or the jSR concentration has changed significantly. That means and spatial resolution of intra-CRU [Ca2+] with realistic steep [Ca2+] gradients. Other approaches include the multiscale model of Restrepo and Karma  nd Karma  which sind Karma . This wand Karma  and was nd Karma  incorpornd Karma , based ond Karma , developl et al.  investigl et al.  model hal et al.  modeled o et al.  which in2+]  can result in an early local maximum of the [Ca2+]i transient  in the first phase of an action potential, followed by forward mode (negative current) during the V-plateau and decline. However, in our approach the INaCa current turns negative much later during the plateau than in the ODE model. The Na+/Ca2+-exchanger depends on the [Ca2+] in sub-membrane space in the ODE model and on the cytoplasmic concentration c in our simulations, including of course the regions with high c-values at and close to CRUs. The main reason for the differences is that the sub-membrane [Ca2+] in the ODE model exhibits a sharp rise and decline while the average myoplasmic concentration shows slower dynamics. We have experimented with localizing the Na+/Ca2+-exchanger molecule density in proximity to the CRUs to reflect the observed higher abundance of Na+/Ca2+-exchanger near the CRU  to run it. We expect some improvement of simulation efficiency by more specifically tailored numerical methods, however the requirement for HPC will remain. The use of the Green function inside the dyadic cleft requires linearity of the reaction diffusion equations there. That excludes non-linear buffering terms and allows for linear buffering only. Some of the limitations of our model arise from its early state of development and will be removed with inclusion of more detail like e.g., anisotropic diffusion, mitochondria, more detailed buffering, and non-junctional RyR. We used modular programming as far as possible to ease the exploration of a variety of ion channel models and other species specific membrane potential dynamics.NC implemented the PDE model and coupled this with the CRU model and cell electrophysiology ODE model. JV co-implemented the cell electrophysiology ODE and PDE model. WN and SG implemented the RyR Markov-models. WN implemented the CRU model and co-implemented the PDE model. NC, WN, JV, SG carried out simulations, processed data from simulations and produced results figures. SG, NC, WN, JV, MF drafted the manuscript. MF conceived the CRU model, the PDE model and the coupling of the model scales. All authors approved and assisted in redrafting the final manuscript.This study has been supported by DFG grant FA350/9-1, DFG GRK 1772, BMBF grant eMed:SMART and a collaborative grant from the German Centre for Cardiovascular Research to MF and S. Luther, MPI DS Göttingen.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.


We investigated the effects of ascending aortic banding (AoB) on Ca2+-handling in rat isolated atrial myocytes in comparison to age-matched sham-operated animals (Sham). Myocytes were either labelled for ryanodine receptor (RyR) or loaded with fluo-3-AM and imaged by confocal microscopy. AoB myocytes were hypertrophied in comparison to Sham controls (P<0.0001). RyR labeling was localized to the z-lines and to the cell edge. There were no differences between AoB and Sham in the intensity or pattern of RyR-staining. In both AoB and Sham, electrical stimulation evoked robust SR Ca2+-release at the cell edge whereas Ca2+ transients at the cell center were much smaller. Western blotting showed a decreased L-type Ca channel expression but no significant changes in RyR or RyR phosphorylation or in expression of Na+/Ca2+ exchanger, SR Ca2+ ATPase or phospholamban. Mathematical modeling indicated that [Ca2+]i transients at the cell center were accounted for by simple centripetal diffusion of Ca2+ released at the cell edge. In contrast, caffeine (10 mM) induced Ca2+ release was uniform across the cell. The caffeine-induced transient was smaller in AoB than in Sham, suggesting a reduced SR Ca2+-load in hypertrophied cells. There were no significant differences between AoB and Sham cells in the rate of Ca2+ extrusion during recovery of electrically-stimulated or caffeine-induced transients. The incidence and frequency of spontaneous Ca2+-transients following rapid-pacing (4 Hz) was greater in AoB than in Sham myocytes. In conclusion, elevated afterload causes cellular hypertrophy and remodeling of atrial SR Ca2+-release.Atrial remodeling due to elevated arterial pressure predisposes the heart to atrial fibrillation (AF). Although abnormal sarcoplasmic reticulum (SR) function has been associated with AF, there is little information on the effects of elevated afterload on atrial Ca This value for Kd was chosen to allow direct comparison of the data with a previous study of Ca2+ transport in rat atrial cells[2+ extrusion from the cytosol during the recovery of twitch and caffeine-induced transients were quantified by fitting the decaying phase of the transient with the equation:t represents time, k the rate constant, A a constant representing the notional peak of the transient at t = 0 and c the resting level.Atrial myocytes were re-suspended in a Tyrode’s solution containing 0.75 mM Ca = 100 nM, where Fial cells. Ca2+ ex2+ that would result from diffusive processes in cells a computer model was used to solve the general reaction-diffusion equation:DX is the diffusion coefficient for diffusible species X, JX the reactive flux for all buffers B and JS the source (and sinks) of Ca2+. Consideration of the elliptical cross-section of a cylindrical cell . An ana in cells. Calcium2+ transport across the cell to see whether Ca2+ signals in the center of the cell could be explained by purely passive diffusion or whether they required regenerative Ca2+ release.By solving the model reaction-diffusion equations and fitting results to experimental data, the requirement to deconvolve the fluorescent signals before trying to solve the inverse problem posed by the system of equations was overcome . It shoun/N, where n = number of cells and N = the number of hearts. P<0.05 was considered statistically significant.Data are expressed as mean ± standard error of the mean (SEM). Data sets were subject to D’Agostino and Pearson omnibus normality test. Statistical analyses applied are indicated in the figure legends. Sample sizes are reported as Staining of the sarcolemma of isolated left atrial (LA) myocytes from both Sham and AoB rats with the lipophilic dye, di-8-ANEPPS, was located exclusively to the cell edge, consistent with a sparsity of t-tubules in these cells. From co2+ transport was investigated by Western blotting of LA tissue homogenates from Sham and AoB rats  or in the regularity of the staining associated with the striations  as quantified by, respectively, the position and height of the peak from the Fourier transform. Western blot analysis confirmed no difference in RyR protein expression between Sham and AoB atria (Ryanodine receptor (RyR) labeling showed a clear striated pattern , as prevoB atria . The banoB atria , consist2+-transients that were initiated at the periphery of the cells . In contrast to the cell edge, transients at the center of the cells had a much smaller amplitude  and were often barely detectable (e.g. n = 10), and AoB, 50.4±21.4 ms, . Thus, the cells generally showed a ‘U’-shaped profile of Ca2+ changes across the width of the cell[2+ transients at the cell edge or at the cell center, nor was there any significant difference in the delay of the peak at the center of the cell relative to the cell edge.Electrical stimulation of fluo-3-loaded cells elicited Cahe cells . Ca2+-trble e.g. . The del the cell. However the cell. In summ2+ transient at the cell center clearly suggests that in the majority of cells there was little, if any, regenerative propagation of Ca2+ release to the center. On the other hand, the short delay between the transient at the edge and center of the cell might be consistent with the propagation of a Ca2+ wave involving calcium-induced calcium release[2+ transient  increas2+ )  further decreased the rate of decline of Ca2+ ) . In the ate Ca2+ . In comp of Ca2+  by a facansients  or in thents , the Ca2+ that is taken up must return to the cell surface by diffusion within the SR lumen (for subsequent release and/or extrusion), a process reminiscent of the SR vectorial transport and buffer barrier described for smooth muscle cells[2+ release events in Sham cells (The modeling shows that without SR Cas slower , showingcle cells. This wiam cells  if the d2+ regulation in a purely afterload-induced atrial hypertrophy. Spontaneous Ca2+ release has been reported to be increased in AF in association with changes in RyR function, although SR Ca2+ content was reported to be unchanged and Na+/Ca2+ exchange function increased[1c subunit has also been reported in human AF, associated with a decreased L-type Ca2+ current density [2+ release has been shown to lead to atrial arrhythmogenesis in a canine model of heart failure [2+ release in heart failure has been associated with increased SR Ca2+ load despite reduced LTCC expression and Ca2+ current[2+ release was not increased in atrial myocytes despite an increased SR Ca2+ load [2+ transient was not different between AoB and Sham myocytes, despite the reduced SR Ca2+ load and Ca2+ trigger via LTCC. That atrial effective refractory period has been reported to be unaffected in AoB hearts indicates that atrial action potential duration was unchanged[2+ was reduced in AoB cells, as has been recently reported in an ovine model of heart failure[2+ load, a reduction in cytosolic buffering would have resulted in an increase in the amplitude of the caffeine-induced transient. Thus, the reduced amplitude of the caffeine-induced Ca2+ transient in AoB compared with Sham cells in the present study provides robust evidence of a reduction in atrial SR Ca2+ load through elevated afterload-induced remodeling.The present study is the first explicitly to examine Caincreased–11, 43.  density , 15, 44. failure . The inc+ current, 45, 46.a2+ load . While, a2+ load . In the 2+ transients, demonstrating that the activity of the major Ca2+ extrusion systems, the SR Ca2+ ATPase and the Na+/Ca2+ exchanger, were not affected by the afterload-induced atrial hypertrophy. Moreover, the values of ktwitch and kCaff were similar to those reported by Trafford and colleagues in normal atrial myocytes[The absence of differences in the present study between Sham and AoB in SERCA and NCX expression was reflected in the lack of difference in the rate constants of recovery of twitch and caffeine-induced Ca myocytes.2+ in regulating spontaneous release [2+ release in AoB compared with Sham atrial cells in the present study occurred despite reduced SR Ca2+ load. Presumably, the increased incidence of spontaneous release events in AoB cells reflects the increased volume of the cell: As there was no change in RyR distribution in the hypertrophied cells, the number of RyRs and potential release sites must have been increased across the cell (2+ transients, was increased ~6.4-fold in AoB compared to Sham cells, a figure that corresponds well with the ~5.3-fold increased cell volume of AoB relative to Sham cells.Given the key role played by SR luminal Ca release , it is nthe cell , with th2+ transport and RyR2 distribution were unaffected but there was an increased susceptibility to spontaneous Ca2+ release, presumably reflecting maladaptive changes in cell geometry. We suggest that the increased susceptibility to spontaneous Ca2+ release reported here may underly the increased incidence of atrial tachyarrhythmias in conditions of elevated afterload.In summary, we have shown in an aortic banded model in rats that elevated afterload causes atrial cellular hypertrophy in which cellular Ca


RAS mutations are frequently found among acute myeloid leukemia patients (AML), generating a constitutively active signaling protein changing cellular proliferation, differentiation and apoptosis. We have previously shown that treatment of AML patients with high-dose cytarabine is preferentially beneficial for those harboring oncogenic RAS. On the basis of a murine AML cell culture model, we ascribed this effect to a RAS-driven, p53-dependent induction of differentiation. Hence, in this study we sought to confirm the correlation between RAS status and differentiation of primary blasts obtained from AML patients. The gene expression signature of AML blasts with oncogenic NRAS indeed corresponded to a more mature profile compared to blasts with wildtype RAS, as demonstrated by gene set enrichment analysis (GSEA) and real-time PCR analysis of myeloid ecotropic viral integration site 1 homolog (MEIS1) in a unique cohort of AML patients. In addition, in vitro cell culture experiments with established cell lines and a second set of primary AML cells showed that oncogenic NRAS mutations predisposed cells to cytarabine (AraC) driven differentiation. Taken together, our findings show that AML with inv(16) and NRAS mutation have a differentiation gene signature, supporting the notion that NRAS mutation may predispose leukemic cells to AraC induced differentiation. We therefore suggest that promotion of differentiation pathways by specific genetic alterations could explain the superior treatment outcome after therapy in some AML patient subgroups. Whether a differentiation gene expression status may generally predict for a superior treatment outcome in AML needs to be addressed in future studies. RAS genes [NRAS is most frequently affected [RAS genes are not mutated [12% to 19% of acute myeloid leukemia patients carry a gain-of-function mutation within the AS genes ,4, whileaffected ,6. In ad mutated ,8,9,10.  mutated ,14,15.RAS mutations benefitted most from post-induction dose-escalation of cytarabine [Common AML treatment regimens include the nucleoside analog cytarabine . AraC incorporation into DNA during S phase gives rise to a replication blocking lesion. As a consequence, the DNA damage response, that also involves p53, is activated ,17. In Atarabine . To deepRAS mutations has not been described in AML. This may be due to the genetic heterogeneity of this disease as several genetic aberrations have been described in AML [CBFB-MYH11 (cohort 1), which is frequently associated with oncogenic RAS mutations [In order to ask if these findings  were alsd in AML  which trutations ,19.NRAS is associated with a more differentiated gene expression signature in human inv(16) AML. Secondly, we analyzed whether AraC and oncogenic NRAS cooperate in promoting differentiation. To this end, we used myeloid cell lines as well as a second cohort of 22 primary AML cases (cohort 2). AML cells with oncogenic NRAS mutations showed more pronounced differentiation after AraC treatment ex vivo.We here show that indeed oncogenic CBFB-MYH11. The CBFB-MYH11 fusion gene/transcript was detected by classical cytogenetic analysis or PCR [NRAS12/13, 61 or KRAS mutation Two different AML patient cohorts were used in this study . Sampless or PCR . The stus or PCR . Writtens or PCR . MononucSamples from Marburg (cohort 2): Mononuclear cells were isolated from BM or peripheral blood (PB) of 22 untreated AML patients  by densiSamples from Dresden (cohort 1): high-performance liquid chromatography based method or peptide nucleic acid-mediated PCR clamping as described previously ,22,23. SNRAS12/13, 61 or KRAS12/13 mutation (cohort 1). MIAME compliant microarray data are available in the ArrayExpress database . GSEA was conducted only with mtNRAS  and U937  were seeded at a density of 5x104/ml. After incubation with 100 nM or 350 nM AraC (controls with corresponding amount of DMSO) for 48h, cytospin slides (2x104 cells/spot) were prepared and stained according to the May-Grünwald-Giemsa method.The AML cell lines HL-60  were treated with AraC and analyzed for differentiation by flow cytometry in the same manner and used as positive control , CD11c–PE , CD15-FITC , CD34-PE ; HLA-DR-FITC , CD14-PE  or CD117-APC . DNA was counterstained with 4',6-Diamidinophenyl-indole (DAPI) before analysis on an LSRII cytometer (BD Biosciences). Results were analyzed independently in a blinded manner with FLOWJO 9.6.4 or FACS-DIVA 6.0 software. Myeloblast/immature leukemias  were considered as „differentiating (positive)“, if either CD15 or CD11c increased, or HLA-DR or CD34 decreased. Monocytic leukemias  were considered positive for differentiation when CD15 declined, CD14, HLA-DR or CD11c increased, because immature monocytes often express CD15 highly and CD14 weekly. HLA-DR is strongly expressed on immature blasts and on mature monocytes. CD15 is weekly expressed on immature monocytes but strongly expressed in mature granulocytes [http://www.quantitativeskills.com/sisa/statistics/fisher.htm.Mann Whitney test and student’s t-test were calculated by Prism software , Fisher’s exact test via CBFB-MYH11 fusion gene or expressed a CBFB-MYH11 fusion transcript. 12 of 31 AML patients additionally carried oncogenic NRAS (12/13 or 61) mutations.We were interested whether our previous observation  that oncNRAS, NRAS regulated genes in mtNRAS samples, which corroborates the validity of our data. A gene set representing differentiation and maturation of hematopoietic cells was found to be enriched in mtNRAS samples . 12 patients harbored oncogenic NRAS, 19 patients carried wtRAS genes. Indeed, MEIS1 expression was significantly higher (1.9-fold) in blasts with wtRAS than in mtNRAS blasts (Homeobox (HOX) genes, among which is  markers . This prS blasts .RAS and mtNRAS cells to AraC treatment. RAS) and U937 (wtRAS). While brightening and granulation of the cytoplasm indicate differentiation of HL-60 cells after AraC treatment, this effect could not be observed in U937 cells. The morphological changes of HL-60 cells were accompanied by increased expression (32.6-fold) of the differentiation marker CD14  and lymphocytes (LC). The analyzed wtRAS blasts (upper 2 panels) were obtained from a treatment-naïve, 77-year-old Caucasian man suffering from AML type FAB M2  mtNRAS samples displayed an AraC-triggered change of at least one differentiation-indicating marker, whereas only 2 of 12 (17%) wtRAS samples did  . In ordeene sets  Tables, ntiation ,13.NRAS cells. Interestingly, internal tandem duplications of the FLT3 gene, also known to activate the MAPK signaling pathway, did not cause the same effect on differentiation. We conclude that AML cells with oncogenic NRAS are more prone to AraC-driven differentiation as compared to AML cells with wildtype NRAS, and that the NRAS mutation is the pivotal lesion for this effect.If oncogenic RAS is associated with differentiation, we next sought to study whether this effect could be enhanced by additional replication stress, for instance caused by AraC. Our data show that AraC induces myeloid differentiation in terms of morphology and protein surface expression preferentially in mtTherefore, depending on the cellular context where it is expressed, oncogenic RAS may trigger differentiation that is even more enhanced when cells are exposed to the replication blocking agent AraC. We therefore suggest that induction of myeloid differentiation may be a rational target of chemotherapy that has previously not been appreciated in AML other than acute promyelocytic leukemia (APL).S1 Materials and Methods(PDF)Click here for additional data file.S1 TableRAS or mtRAS and samples were used for cDNA array; GSEA and MEIS1 analysis using quantitative real time PCR.Patients carried inv(16) and wt(PDF)Click here for additional data file.S2 Tablein vitro cell culture and analysis of differentiation by flow cytometry.Samples were obtained before treatment of the patient and used for (PDF)Click here for additional data file.S3 TableHL-60 positive control, exemplifying live gating and differentiation response. An HL-60 sample was carried along with each experiment with patients’ samples. The occurrence of the HL-60 response in terms of cell death and differentiation induction by AraC confirmed successful performance of the experiment.(PDF)Click here for additional data file.S4 TableFor differentiation positive samples, the differentiation indicating marker is depicted. For differentiation negative samples (i.e. no shift with any marker observable), one of the analyzed markers is shown representatively for all markers.(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.


Non-calcified echogenic renal cortical nodules are commonly detected with abdominal ultrasound (US). The majority of these nodules represent benign angiomyolipomas (AMLs), which are present in 0.3–2.1 % of the population at autopsy . Renal cGiven that small RCCs are commonly echogenic and may mimic renal AMLs at US and that sonographic differentiating features are insensitive, an imaging quandary occurs. Confirmatory imaging with computed tomography (CT) is generally accepted for larger lesions to detect the presence of gross fat and confirm the presumed diagnosis of AML Fig. . For smaThe diagnosis of AML with CT is predicated on the ability to demonstrate gross fat within the nodule, which is essentially pathognomonic of AML; with only rare case reports of gross fat within both papillary and clear cell RCC . A repreThe MRI diagnosis of AML also relies on the ability to detect gross or macroscopic fat within the nodule. Gross fat can be detected by comparison of T1 signal intensity with and without the application of a variety of fat suppression techniques, with the suppression of signal intensity within the nodule diagnostic of gross fat . Using tThe use of in-phase (IP) and opposed-phase (OP) chemical shift imaging (CSI) can also be used to diagnose AML . CSI expImportant considerations for diagnosis of AML with CSI include: nodule size, base voxel resolution, and the relative amount of fat and water in each imaging voxel. If the nodule is extremely small (<5 mm) and/or the base voxel resolution (voxel size) is large, india ink artefact may completely obscure the centre of the nodule such that only a spot of signal loss  is appreciated on the OP image Fig. . It is eWith CSI, the degree of signal loss on OP imaging is dependent solely on the relative amounts of fat and water within each imaging voxel. One can therefore expect a greater loss of signal  at the interface of large Fig.  or even In conclusion, the incidental finding of an echogenic renal cortical nodule is a commonly encountered clinical scenario with an imaging differential diagnosis of renal AML or small RCC. Differentiating sonographic features are specific but lack sensitivity. Management strategies vary and are controversial. Based on the best available evidence, CT has been recommended for further characterisation of all echogenic renal cortical nodules regardless of size. While this is generally widely accepted for larger nodules, whether CT is required for the characterisation of all lesions (including those <1 cm) is uncertain due to a lack of available evidence. In the opinion of these authors, if further imaging is requested or performed for small (<1 cm) lesions, chemical shift MRI and not CT is the preferred imaging test.


We found that the formation of TEP from precursor materials studied was essentially determined by the Ca2+ concentration at a fixed sodium concentration, while Na+ at high Na+/Ca2+ ratio out-competed Ca2+ for the binding sites on the precursor molecules, leading to a significantly decreased trend of TEP formation. Our results indicate that a more abundant TEP could be expected in freshwater than in seawater, and we also discuss the engineering implications of the findings.Transparent exopolymer particles (TEP) are planktonic, organic microgels which play significant roles in cycling of carbon and trace elements, aggregation of particles, feeding and accommodating microbes as well as development of biofilms. However, few studies are available on the mechanism of TEP formation in various water environments. Here we investigate the formation of TEP with alginate blocks as precursors at various Na TEP have been described as a class of transparent particulate acidic polysaccharides that can be visualized via staining with alcian blue, typically with a size larger than 0.4 μm135679103412In general, TEP may be derived from organic substances released by organisms in aquatic environments. Two pathways have been proposed to illustrate the formation of TEP from dissolved organic matter (DOM) in aquatic environments, i.e. abiotic and biotic5141516514185115+/Ca2+ ratios. Therefore, this study aimed to offer new insights into the TEP formation from different precursor materials at various concentrations of calcium and sodium ions. For this purpose, previously studied alginate blocks, namely 1,4-linked β-d-mannuronic acid (MM) and α-l-guluronic acid (GG) and heteropolymeric block (MG), were chosen as model precursors in this study221It has been observed that the formation of TEP can be promoted by divalent cations  through bridging with neighboring TEP molecular chains5et al.21MG-, MM- and GG-blocks were fractionated from sodium alginate  according to the method by Leal 2  and NaCl  were used to adjust Ca2+ and Na+ concentrations in the precursor solutions. All precursor solutions were freshly prepared right before experiments. Although pH was not specifically adjusted, it remained approximately at 6.7 ± 0.3 in all the precursor solutions.Precursor solutions were prepared by dissolving MG-, MM- and GG-blocks respectively into ultrapure Milli-Q water (resistivity: 18.2 MΩ.cm at 25 °C) with continuous stirring for 2 hours to make up a final concentration of 50 mg/L. CaClet al.34In this study, TEP was determined as described in 2SO4 solution with regular shaking over a period of 2 hours. Alcian blue dissolved in sulfuric acid exhibited a light green color, and the absorbance of the sulfuric acid solution with eluted alcian blue was measured using a UV–Vis spectrophotometer  at 787 nm wavelength in a 1 cm cuvette. Absorbance corrections due to stain adsorption onto filter and interference associated with solution salinity were also performed according to the procedures proposed by Villacorte et al.4eq.L−1. Total organic carbon (TOC) measurements were employed to determine the amount of xanthan gum retained by the polycarbonate filters when performed the calibrationThe prepared precursor solutions were first filtered through a series of polycarbonate filters  of pore size 0.05, 0.1, 0.2 and 0.4 μm at a constant vacuum of 0.2 bars, followed by filtration of 1 mL of Milli-Q water for washing out the remaining salinity2To visualize the TEP derived from the precursors , a bright-field microscope  was employed. The fresh sample solutions were prepared prior to observation as described above. In order to visualize TEP with the bright-field microscope, precursor solutions were stained by freshly pre-filtered (0.05 μm polycarbonate filter) alcian blue solution as presented above. Stained samples were then observed under the microscope. For each sample, about 20 images were randomly taken.+/Ca2+ ratios were also observed by a field emission scanning electron microscopy (FESEM) . Although TEP were freeze dried prior to microscopic observation, this microscopic technique could still provide direct visualizations of evidence of TEP micro-structures. 10–50 mL of sample solutions prepared as described above were filtered through 0.1 μm polycarbonate filters  at a constant pressure of 0.2 bars and was then rinsed by 1 mL of Milli-Q water. Filters with retained alginate blocks were completely freeze-dried completely in a freeze dryer  for further examination. All samples were observed at least three times and 8–10 images were randomly recorded each time.The micro-structures of TEP derived from MG-, MM- and GG-blocks at different Na+/Ca2+ ratios. For force measurements on TEP, TEP films were prepared by filtering 1 mg Xeq TEP onto 0.1-μm polycarbonate filters at a constant pressure of 0.2 bars. Subsequently, the filter with retained TEP was carefully glued onto a holder using double-sided sticky tape with TEP film upwards. All TEP samples were prepared right before the force measurements. To prevent TEP film dehydration, the TEP samples were analyzed in a liquid cell filled completely with the test solution of interest. The chemistry composition of the test solution was the same as that used in the corresponding TEP samples, while a clean 0.1-μm polycarbonate filter was used as a reference surface. The clean filter was first soaked in Milli-Q water for 24 hours and 50 mL Milli-Q water was then filtered through it just before the measurement. Force measurements on the clean filters were conducted with all test solutions.A XE-100 AFM  was employed to determine the stickiness and micromechanical characteristics of TEP developed from GG-blocks as precursor at different Na2 at a respective forward and backward speed of 0.3 μm/s. A minimum of four different places on the TEP film was measured and 16 force measurements were conducted at each location. XEI 1.8.0.Build32 software was used to flatten image as well as to calculate the maximum retract force (adhesion) and total adhesion energy.The force measurements were conducted at room temperature of 22 ± 2 °C. Surface architecture was first imaged using non-contact mode to minimize the contact of the tip with the TEP film and subsequently a suitable position on the surface was located for force measurements. For the force measurements, the scan rate was set at 1 Hz with image resolution at 512 data number per trace. The force measurements were approached for an area of 5 × 5 μm2+ concentration of 1 mM and Na+ concentration of 10 mM. For TEP with different sizes ranging from 0.05 μm to 0.4 μm and above, the highest concentration was observed in GG-blocks solution, while the lowest concentration was achieved in MG-blocks solution. For example, the TEP concentrations developed in the solution of GG-blocks are 10-, 75-, 97- and 138-times higher than those in MG-blocks solution in respective TEP size range of 0.05–0.1 μm, 0.1–0.2 μm, 0.2–0.4 μm and >0.4 μm. The total TEP concentrations (TEP retained by 0.05 μm polycarbonate filters) on average in MG-blocks solution was about 1.29 mg Xeq/L, while 9.59 mg Xeq/L and 73.25 mg Xeq/L found in MM-and GG-blocks solutions respectively. At given Na+ and Ca2+ concentrations, such tendencies of TEP formation from MG-, MM- and GG-blocks is probably due to the differences in the the stiffness of these precursor materials, which indeed have shown to be in the order MG<MM<GG252+, known as “egg-box” structure202+ concentrations with a fixed Na+ concentration of 10 mM. According to Ca2+ concentration, the phases pertaining to TEP development can clearly be derived from 2+ concentration (0 to 0.015 mM), TEP formation was not sensitive to Ca2+ concentration; (ii) at medium Ca2+ concentration of 0.15 to 0.5 mM, the formation of TEP from GG blocks appeared to be proportionally related to Ca2+ concentration, and as the result, a sharp increase in TEP concentration was observed in this phase; (iii) at high Ca2+ concentration of 0.5 to 2.0 mM, further increase of the Ca2+ concentration had insignificant effect on TEP formation from GG-blocks at a given concentration of GG-blocks. These results indeed provide direct evidence showing the dependence of TEP formation on calcium ion for a given precursor material, i.e. Ca2+ should play a critical role in TEP formation.+ concentration at a fixed Ca2+ concentration of 1 mM leads to a remarkable reduction in TEP formation from all the three precursor materials, i.e. MM-, MG-, and GG-blocks. Such a trend is completely different from that observed at various Ca2+ concentrations with a fixed Na+ concentration . In contrast to Ca2+  are negatively charged, thus an electrical competition between positively charged Na+ and Ca2+ exists for the binding sites. Increased Na+ concentration can out-compete Ca2+, thus prevents the Ca2+ bridging and decreases the TEP formation. As shown above, in the cases where MG- and MM-blocks served as precursors, the bindings between calcium ions and these precursors are weaker than that between GG-blocks and calcium ions. Therefore, the bridging effect by calcium ion in MG-and MM-blocks is weakened or even disappeared at elevated Na+ concentration, and this in turn explains significantly reduced TEP formation as shown in + on TEP formation from GG-blocks is less significant  often contains less Na+ concentrations with a fixed Ca2+ concentration of 1 mM were directly visualized by means of a light-field microscope. As can be seen in + concentration of 10 mM, the largest TEP in size and the highest concentration  Ca2+ was essential for the formation of TEP from precursor materials studied; (iii) Na+ itself had no contribution to the development of TEP, while high-concentration Na+ tended to out-compete Ca2+ for the binding sites on the precursor molecules, leading to a significantly decreased trend of TEP formation at high Na+/Ca2+ ratios and (iv) the AFM force measurements provided useful information on viscoelasticity of TEP formed at different Na+/Ca2+ ratios, revealing that the TEP formed at high Na+/Ca2+ ratio was softer and sticker than that formed at low Na+/Ca2+ ratio. The present study offers experimental evidence showing that a more abundant TEP can be expected in freshwater than in seawater. This in turn implies that TEP-associated biofilm development and membrane fouling would be more significant in membrane filtration of freshwater than seawater.TEP formation was determined under various solution chemistries with alginate blocks as model precursors. The salient points derived from the present study are (i) TEP formation was dependent on physico-chemical properties of precursor materials at a given NaHow to cite this article: Meng, S. and Liu, Y. New insights into transparent exopolymer particles (TEP) formation from precursor materials at various Na+/Ca2+ ratios. Sci. Rep.6, 19747; doi: 10.1038/srep19747 (2016).


Computational and mathematical modeling played a crucial role in the understanding of spatio-temporal Ca2+ dynamics that drives these processes, and showed that Ca2+ concentration around a single Ca2+ channel reaches a quasi-stationary distribution (known as the Ca2+ "nanodomain") within tens of microseconds after the opening of the channel, and collapses as rapidly after the closing of the channel. Such localization of Ca2+ in time and space is achieved by its rapid diffusion as well as its binding to its multiple interaction partners collectively called Ca2+ buffers and Ca2+ sensors. One of the successes of mathematical modeling was the development of several analytic approximations describing the equilibrium concentration of Ca2+ as a function of distance from the open Ca2+ channel, such as the Rapid Buffering Approximation (RBA), the Linear Approximation (LA) and the Excess Buffering Approximation (EBA) [2+ buffers, in particular their mobility and Ca2+ binding rates, and the strength of the Ca2+ current. Here we present a novel approximation method which does not rely on a specific range of the relevant Ca2+ and buffer parameters, and is based on matching the low-distance and large-distance asymptotic behavior of the concentration function. Even at low orders, the resulting approximation is as accurate as the second-order RBA and EBA approximations [2+ nanodomain properties on the relevant buffering parameters, and second, it constitutes an efficient numerical approximation tool in the modeling of the Ca2+ signals underlying presynaptic and postsynaptic phenomena.Localized calcium (Caon (EBA) -4. Each