Arabidopsis thaliana. Kinetic parameters of enzymatic steps in Scr cycling were identified by fitting model simulations to experimental data. A statistical analysis of the kinetic parameters and calculated flux rates allowed for estimation of the variability and supported the predictability of the model. A principal component analysis of the parameter results revealed the identifiability of the model parameters. We investigated the stability properties of Scr cycling and found that feedback inhibition of enzymes catalysing metabolite interconversions at different steps of the cycle have differential influence on stability. Applying this observation to futile cycling of Scr in leaf cells points to the enzyme hexokinase as an important regulator, while the step of Scr degradation by invertases appears subordinate.Plant carbohydrate metabolism comprises numerous metabolite interconversions, some of which form cycles of metabolite degradation and re-synthesis and are thus referred to as futile cycles. In this study, we present a systems biology approach to analyse any possible regulatory principle that operates such futile cycles based on experimental data for sucrose (Scr) cycling in photosynthetically active leaves of the model plant  Arabidopsis thaliana ::vInv), GVmax(t) values represent time-variant maximal velocities of enzyme reactions, Km are the Michaelis-Menten constants representing substrate affinity of the enzyme and Ki are the inhibitory constants. Changes in maximal velocities of enzyme reactions were described over a whole diurnal cycle by a cubic spline interpolation for Vmax(t). This course is defined by the sample tk = {3,7,11,15,19,23} h and values for Vmax(tk), which are subject to parameter identification. This description reflects changes of enzyme activity, mainly resulting from changes in enzyme concentration. Measurements of enzyme activities supported this assumption . In the case of allosteric inhibition by a product, as, for example, feedback inhibition of Hex on invertase enzymes, the corresponding element in θ assumes values within the range . Further details on θ for Michaelis-Menten kinetics can be found in . We analysed different modifications of the metabolic cycle by varying modes of activation and inhibition. Each particular metabolic cycle was simulated for 106 different sets of parameters, and resulting maximal real parts of the eigenvalues were plotted in histograms , positive real parts occurred, suggesting that the system may become unstable for certain parameter sets , e.g. by Hex or SP [First, we analysed the stability properties of a system without instances of activation and inhibition Figure , i.e. bys Figure . When adex or SP  were incv5 by SP (θ6 = -0.01) and strong inhibition of v4 by Hex (θ5 = -0.99), and (ii) strong feedback inhibition of v5 by SP (θ6 = -0.99) and weak inhibition of v4 by Hex (θ5 = -0.01). The histograms representing the corresponding results showed that stability of the system for all the samples was only achieved when v5 was assumed to be inhibited strongly by SP  weak feedback inhibition of P Figure . Applyinelopment . Our finθ6 = -0.0 and stroA. thaliana. Based on simulated fluxes in leaf cells, we could assign possible physiological functions of vacuolar invertase in carbohydrate metabolism. Here, we explicate this model in more detail and perform a statistical evaluation that proves reproducibility of the prediction of cellular metabolite concentrations and fluxes. The PCA revealed that the identifiability of the mutant parameters could be improved by more measurements. In addition, it was shown that this system's biology model exhibits the property of sloppiness [Recently, we presented a kinetic modelling approach to simulate and analyse diurnal dynamics of carbohydrate metabolism in oppiness , allowinThe authors declare that they have no competing interests.Frc: fructose; Glc: glucose; Hex: hexoses; ODE: ordinary differential equations; PCA: principal component analysis; Scr: sucrose; SKM: structural kinetic modelling; SP: sugar phosphates; St: starch.


TUNEL and Western blot assays showed that Ber not only decreased myocardial apoptosis, caspase-3 activation, AMPKα and p53 phosphorylation, but also increased Bcl-2 expression in myocardium of rats exposed to DOX for 84 h. These findings indicate that Ber attenuates DOX-induced cardiomyocyte apoptosis via protecting mitochondria, inhibiting an increase in the AMP/ATP ratio and AMPKα phosphorylation as well as elevating Bcl-2 expression, which offer a novel mechanism responsible for protection of Ber against DOX-induced cardiomyopathy.Cardiomyocyte apoptosis is an important event in doxorubicin (DOX)-induced cardiac injury. The aim of the present study was to investigate the protection of berberine (Ber) against DOX- triggered cardiomyocyte apoptosis in neonatal rat cardiomyocytes and rats. In neonatal rat cardiomyocytes, Ber attenuated DOX-induced cellular injury and apoptosis in a dose-dependent manner. However, Ber has no significant effect on viability of MCF-7 breast cancer cells treated with DOX. Ber reduced caspase-3 and caspase-9, but not caspase-8 activity in DOX-treated cardiomyocytes. Furthermore, Ber decreased adenosine monophosphate-activated protein kinase α (AMPKα) and p53 phosphorylation at 2 h, cytosolic cytochrome c and mitochondrial Bax levels and increased Bcl-2 level at 6 h in DOX-stimulated cardiomyocytes. Pretreatment with compound C, an AMPK inhibitor, also suppressed p53 phosphorylation and apoptosis in DOX-treated cardiomyocytes. DOX stimulation for 30 min led to a loss of mitochondrial membrane potential and a rise in the AMP/ATP ratio. Ber markedly reduced DOX-induced mitochondrial membrane potential loss and an increase in the AMP/ATP ratio at 1 h and 2 h post DOX exposure. In  Doxorubicin (DOX), as a broad-spectrum antitumor antibiotic, is frequently used in chemotherapy due to its excellent anticancer efficacy coptis chinensis species. It has long history of use for treating diarrhea in traditional Chinese medicine. A growing number of studies reveal that Ber has a wide variety of biological effects, including anti-tumor and cardiovascular-protective actions in vitro and in vivo, and further elucidated the underlying mechanism in the present study. The results showed for the first time that Ber inhibited DOX-induced cardiomyocyte apoptosis in neonatal rat cardiomyocytes. In addition, Ber also attenuated myocardial apoptosis and improved survival in rats challenged with DOX. The mechanisms responsible for this protection of Ber may be related to the protection of mitochondria, inhibition of AMPKα and p53 phosphorylation as well as increase in Bcl-2 expression.Berberine (Ber) is an alkaloid from the All experiments in the present study were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The animal experimental protocol was previously approved by the Animal Care and Use Committee at School of Medicine, Jinan University (Permit Number: AE2011042501). All surgery was performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.2 for 48 h. Cells were treated with Ber  at concentration of 0.06, 0.25, 1.0, 4.0 µM or vehicle for 20 min, and then exposed to 1 µM DOX  for indicated time. In separate experiments, cells were pre-incubated with Compound C , an inhibitor of AMPK phosphorylation, or 5-aminoimidazole-4-carboxyamide ribonucleoside , a AMPK activator, 90 min before addition of 1 µM DOX or/and 1.0 µM Ber. The same volumes of corresponding solvents were added to the controls. Additionally, In order to examine the effects of Ber and DOX at varying doses on AMPK phosphorylation in neonatal rat cardiomyocytes, cardiomyocytes were incubated with different concentrations of Ber or DOX for 2 h in independent experiments.The neonatal Sprague-Dawley rats (1–2 days old), obtained from the medical laboratory animal center of Guangdong province , were deeply anesthetized with pentobarbital sodium (100 mg/kg) and decapitated for cardiac tissue harvesting, left ventricular cardiomyocytes were then enzymentically dissociated, and cultured in DMEM, supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin, at 37°C in a humidified incubator with 5% CO4 cells/well, respectively. After treatment with DOX or/and Ber for 24 h, the MCF-7 cell viability was examined with the Cell Counting kit-8  as the manufacturer’s instructions. The absorbance (OD) values at 450 nm were examined, and MCF-7 cell growth inhibition rate (%) was calculated using the following equation: [(1–OD of drug-treated group/OD of control)×100%]. Cardiomyocyte cytotoxicity was assessed by measuring the release of lactate dehydrogenase (LDH) from the cells. After cardiomyocyte exposure completed, culture medium was collected and LDH activity in the supernatants was determined by biochemistry autoanalyser.To measure cytotoxicity, MCF-7 breast cancer cells and neonatal rat cardiomyocytes were plated at a density of 5×10R Vevo770™ High-Resolution In Vivo Imagine System  as described previously The male adult (8–10 weeks old) Sprague-Dawley rats were obtained from the medical laboratory animal center of Guangdong province  and allowed to acclimate to the new environment for 3 days prior to experiment in a standard experimental room  with free access to commercial standard chow and tap water. The rats were randomly assigned to the following groups: Control, DOX, DOX+Ber and Ber. These rats were intraperitoneally injected with normal saline (2 mL) or a single dose of DOX , followed immediately by intragastrical administration of 2 ml distill water or Ber at a dose of 30, 60 or 120 mg/kg, and then once a day for another three consecutive days. Then, the rats were returned to its original cage, afforded free access to food and water, and the survival was recorded every 12 h for 14 days. At the end of survival experiments, the rats were deeply anesthetized with pentobarbital sodium (100 mg/kg) and sacrificed. In separate experiments, rats were treated as described above, but a single dose of Ber (60 mg/kg) was used. Then, the cardiac function was assessed at 84 h after DOX administration with VisualSonicsRats were anesthetized with 1.5% isoflurane  and imaged in the supine position using a 17.5-MHz center frequency RMV 707 scanhead. Two-dimensional B-mode and M-mode imaging were acquired by a technician who was blinded to rat treatment, left ventricular (LV) end-diastolic and end-systolic diameters were measured on the M-mode parasternal short-axis tracing at papillary muscle level. Left ventricular end diastolic volume (LVEDV), stroke volume (SV) and LV ejection fraction (EF) were calculated with analysis software, and the data were averaged from at least three consecutive cardiac cycles.Cardiac tissues were fixed in 10% buffered formalin and embedded in paraffin. The paraffin sections were stained with hematoxylin and eosin for histological analysis under an optical microscopy.4 cells/well) for 2 days prior to the indicated treatments. At 24 h after DOX, Ber or/and vehicle treatment, the cytoplasmic mono- and oligonucleosomes (histone/DNA fragments) were extracted and assayed according to the manufacturer’s instructions. Absorbance was measured at 405 nm using a microplate reader. The specific enrichment of mono- and oligonucleosomes was expressed as enrichment factor and calculated using the following formula: enrichment factor = the ratio of absorbance of the sample to absorbance of negative control group.For apoptosis analysis, DNA fragmentation was determined by a Cell Death Detection ELISA kit . Neonatal rat cardiomyocytes were cultured , TUNEL-positive cardiomyocytes were counted in 5 randomly selected fields per section. The rate of apoptosis was expressed as the ratio of TUNEL-positive cardiomyocyte nuclei (green) to total number of cardiomyocyte nuclei (blue).The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay was also carried out using an In-Situ Cell Death Detection Kit . Neonatal rat cardiomyocytes were cultured on glass slides for 48 h. Cardiomyocytes were treated with compound C, DOX, Ber or/and vehicle for 24 h, then fixed in 95% ethanol at room temperature for 30 min, and permeabilized in 0.1% Triton-X 100. After blocking with normal goat serum for 30 min at room temperature, the cardiomyocytes were subjected to immunofluorescent staining with a mouse anti-cardiac troponin I (cTnI) antibody , and DAPI , followed by TUNEL reactions according to the manufacturer’s protocol. In To measure caspase-3, 8 and 9 enzymatic activities, neonatal rat cardiomyocytes were cultured for 48 h followed by treatment with DOX (1 µM ) for 12 h in the presence or absence of Ber (1 µM). APO LOGIX Carboxyfluoroscein Caspase Detection Kits  were used to detect active caspase-3, 8 and 9 according to the manufacturer’s instructions.g for 15 min. The supernatant lysates were diluted in 2× or 5×SDS sample buffer, boiled for 5 min. Unless otherwise mentioned, whole cell or tissue lysates were used for analysis. Mitochondrial proteins and cytosolic proteins were prepared using mitochondria isolation kit . Equal amounts of protein from each sample were resolved on SDS-polyacrylamide gel by electrophoresis, and transferred to immobilon polyvinylidene diflouride  or NC membrane, blocked with 5% BSA in TBST  at room temperature for 1 h. The membranes were incubated with primary anti-bodies over night at 4°C. Following incubation with HRP-conjugated secondary antibodies, then immunoblots were exposed on film with an enhanced chemiluminescence reagent . The bands were quantified by optical density ratio using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or voltage-dependent anion channel (VDAC) as a control. The primary antibodies included Bcl-2, Cytochrome c, Bax, cleaved caspase-3, VDAC, GAPDH, p53, phospho-p53(Ser-15), AMPKα, phospho-AMPKα (Thr172), acetyl-CoA carboxylase (ACC) and phospho-ACC, all of which were purchased from Cell Signaling Technology, Inc. .Neonatal rat cardiomyocyte or heart homogenates were harvested in RIPA buffer  containing 1 mM PMSF and incubated for 30 min on ice, then centrifuged at 4°C with 12000×Neonatal rat cardiomyocytes were treated with Ber at concentration of 1.0 µM or vehicle for 20 min, and then exposed to 1.0 µM DOX for 30 min, 1 h or 2 h. Mitochondrial membrane potential was visualized in cardiomyocytes stained with 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) by using a laser scanning confocal microscope. JC-1 is a dual-emission mitochondrial membrane potential sensing dye, which can be accumulated and aggregates in polarized  mitochondria, and aggregate JC-1 shifts its emission to red. The mitochondrial depolarization  prevent JC-1 entry into mitochondria, monomeric JC-1 remains in the cytosol and fluoresces green. Cardiomyocytes from different treatment groups were washed and incubated with JC-1 at 37°C for 15 min, then washed and mounted on the confocal microscopy for imaging. The aggregate JC-1 (red fluorescence) was detected at the emission wavelength of 590 nm, and the monomeric JC-1(green fluorescence) monitored at 529 nm. The ratio of aggregated and monomeric JC-1 was used to quantify changes in mitochondrial membrane potential, and decreased JC-1 ratio represented depolarization of the mitochondria indicating the decrease in mitochondrial membrane potential.g for 15 min at 4°C. Samples were neutralized with ice-cold 1 M K2HPO4 and analyzed by high performance liquid chromatography (HPLC). HPLC was performed on a Boston 250×4.6 mm C18 column. The flow rate was 1 ml/min at 25°C with a starting buffer of 50 mM potassium phosphate buffer (pH 6.5). The injection volume was 40 µL. Peaks were identified by comparison with retention times of standards and analysis of peak spectra from a recorded 3d-field with Chromeleon™ software. Adenosine triphosphate (ATP) and adenosine monophosphate (AMP) were determined by measuring peak area at 256 nm. The contents of AMP and ATP in samples were calculated according to standards of known concentration and expressed as nM/mg protein.Cardiomyocytes were extracted by adding 7% perchloric acid. The extracts were centrifuged at 12,000 P<0.05.Quantitative data are expressed as mean±standard error of the mean (SEM). Statistical differences among groups were evaluated with one-way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis. Mortality was analyzed using Kaplan-Meier survival analysis and compared by log-rank test. Differences were considered statistically significant at P<0.01). However, treatment with Ber at doses of 0.25, 1.0 and 4.0 µM attenuated DOX-induced increase in LDH activity . Ber alone has no obvious effect on LDH activity in the culture medium of cardiomyocytes .Apoptotic cells were also detected by TUNEL assay . As showIt has been reported that DOX-induced apoptosis of cardiomyocytes is a multifactorial process Previous studies have demonstrated that DOX induces AMPK and p53 activation leading to cardiomyocyte apoptosis P<0.01); Ber markedly suppressed DOX-stimulated ACC phosphorylation (P<0.05), but not AICAR-induced ACC phosphorylation; DOX did not affect AICAR-induced ACC phosphorylation. In addition, cardiomyocytes were exposed to 1.0 µM DOX for 12 h , or vehicle for 90 min, and then incubated with DOX (1.0 µM) or/and Ber (1.0 µM) for 2 h, the ACC phosphorylation was measured by Western blotting. As shown in for 12 h  or 24 h for 12 h , AMPKα pfor 12 h , but Berfor 12 h .As shown in P<0.01, n = 9–10). All rats survived at 72 h after DOX challenge in Ber (60 mg/kg)+DOX group.Echocardiographic examination demonstrated that DOX treatment significantly reduced LVEDV and SV at 84 h in rats. In contrast, LVEDV and SV were higher in Ber (60 mg/kg)+DOX group than those in DOX group . HoweverP<0.01) and Ber+DOX groups (P<0.05).Histological examination of cardiac sections stained with hematoxylin-eosin showed that DOX induced cardiomyocyte cytoplasmic vacuolization, an essential feature of DOX cardiotoxicity, which was attenuated by Ber treatment . As showP<0.05). Moreover, the level of cleaved caspase-3 protein in myocardium was higher at 84 h after DOX challenge in DOX group than that in control. This alteration of cleaved caspase-3 protein expression induced by DOX was reversed by Ber (60 mg/kg) treatment +DOX group compared with DOX group (To confirm our findings that inhibition of AMPKα and p53 phosphorylation contributes to the cardioprotection of Ber OX group . On the OX group .in vitro and in vivo.DOX-induced cardiomyopathy remains a lethal disease. Unfortunately, the clinically effective therapy and preventive treatment are yet to be discovered Numerous researches have demonstrated that Ber possesses antitumor activity and induces apoptosis in many cancer cell lines in a concentration-dependent manner It has been proved that Bax translocation from the cytosol to mitochondria results in cytochrome c release from the mitochondria, anti-apoptotic Bcl-2 can interfere with mitochondrial cytochrome c release and suppress apoptosis progression Numerous studies have demonstrated that DOX-induced cardiomyocyte apoptosis is associated with activation of p53 tumor suppressor protein More recently, it was reported that AMPK activation induced p53-dependent apoptosis However, some researchers reported that DOX inhibited AMPKα phosphorylation in cardiomyocytes More importantly, both DOX and AMPK activator, AICAR, significantly increased phosphorylation of AMPK downstream target ACC; Ber markedly suppressed DOX-stimulated ACC phosphorylation, but not AICAR-induced ACC phosphorylation; Additionally, DOX did not affect AICAR-induced ACC phosphorylation. Thus, DOX or Ber itself may not directly affect AMPK phosphorylation in the present study and inhibition of DOX-induced AMPK phosphorylation by Ber in cardiomyocytes at 2 h after DOX exposure may involve other initial events. It is well known that the increase in the ratio of intracellular AMP to ATP is a major regulator of AMPK activity in vitro observations, we also investigated cardiac AMPKα and p53 phosphorylation as well as Bcl-2 protein expression in DOX- challenged rats. The results showed that DOX- stimulated cardiac AMPKα and p53 phosphorylation were markedly attenuated by Ber. In addition, Ber enhanced myocardial Bcl-2 protein level in DOX-challenged rats. Therefore, the results from the in vivo experiments also indicate that Ber protects against DOX-induced cardiomyocyte apoptosis, at least in part, through inhibiting myocardial AMPKα and p53 phosphorylation and elevating Bcl-2 expression in the early stage of DOX treatment.Furthermore, we examined the effect of Ber on DOX-stimulated cardiomyocyte apoptosis in a rat model of acute DOX-induced cardiotoxicity. Our present results demonstrated that DOX administered at a single dose of 20 mg/kg increased mortality and serum LDH activity, induced myocardial injury and reduced LVEDV and SV at 84 h after DOX treatment. TUNEL and Western blotting analysis revealed that DOX significantly increased the number of TUNEL-positive cardiomyocytes and cleaved caspase-3 protein level in myocardium 84 h after DOX challenge, these effects of DOX were markedly reduced by Ber. These findings indicate that Ber improves survival and inhibits myocardial apoptosis and injury in an acute DOX-induced cardiotoxicity model, but the details of the mechanism by which DOX decreases LVEDV is unknown. However, DOX challenge did not reduce left ventricular EF 84 h after DOX challenge in the present study. It has demonstrated that elevated catecholamine can compensate for cytotoxic damage and preserve normal cardiac contraction in the early stages of DOX-induced cardiac dysfunction In summary, the present study provides evidence to show that DOX rapidly induces mitochondrial injury, increases the ratio of intracellular AMP to ATP, in turn activates AMPK phosphorylation and facilitates cardiomyocyte apoptosis. Ber suppresses DOX-induced cardiomyocyte apoptosis via protecting mitochondria, reducing the increased ratio of AMP to ATP and inhibiting AMPK phosphorylation as well as elevating Bcl-2 expression in the early stage of DOX treatment . Moreove


Salmonella replicate in membrane-bound compartments, which accumulate a dense meshwork of F-actin through the kinase activity of the Salmonella SPI-2 type III secretion effector SteC. We find that SteC promotes actin cytoskeleton reorganization by activating a signaling pathway involving the MAP kinases MEK and ERK, myosin light chain kinase (MLCK) and Myosin IIB. Specifically, SteC phosphorylates MEK directly on serine 200 (S200), a previously unstudied phosphorylation site. S200 phosphorylation is predicted to displace a negative regulatory helix causing autophosphorylation on the known MEK activatory residues, S218 and S222. In support of this, substitution of S200 with alanine prevented phosphorylation on S218 and S222, and phosphomimetic mutations of S200 stimulated phosphorylation of these residues. Both steC-null and kinase-deficient mutant strains displayed enhanced replication in infected cells, suggesting that SteC manipulates the actin cytoskeleton to restrain bacterial growth, thereby regulating virulence.After host cell entry,  Salmonella SteC reorganizes F-actin via a MEK/ERK/MLCK/Myosin IIB pathway ► SteC phosphorylates MEK1 S200, which is required for SteC-induced MEK activation ► MEK1 S200 phosphorylation induces autophosphorylation on residues S218/222 ► SteC also controls bacterial growth via its kinase function►  Salmonella enterica serovar Typhimurium (S. Typhimurium) causes gastrointestinal disease in humans and a typhoid-like systemic infection in certain mouse strains. Its virulence requires two type III secretion systems (T3SSs), encoded by the pathogenicity islands Salmonella Pathogenicity Island 1 (SPI-1) and SPI-2. T3SSs are multiprotein organelles assembled in the bacterial cell envelope with needle-like extensions   . The SCVle (SCV) . A few hle (SCV) . In the filament , and actfilament  networksfilament . Althougfilament , its tarfilament . Overexpfilament . This stfilament , which cfilament . This suSteC overexpression causes the formation of thick F-actin fibers connected to clusters of highly condensed F-actin, similar in appearance to those obtained when Myosin II is activated . Three iS. Typhimurium and examined for recruitment of the Myosin IIB heavy chain to SCVs. In cells infected for 8 hr with wild-type (WT) or complemented deletion mutant  strains, approximately 70% of microcolonies were associated with Myosin IIB (ssaV (SPI-2 T3SS defective) or steC mutant bacteria, Myosin IIB association with SCVs was much lower. The steC mutant translocated other effectors as efficiently as WT Salmonella (data not shown). This shows that SteC is responsible for the recruitment of Myosin IIB to bacterial microcolonies. PMLC colocalized with F-actin structures around WT S. Typhimurium vacuoles  bacteria. Therefore the Myosin inhibitors BDM or Blebbistatin were added during Latrunculin B washout. Exposure of cells to either drug decreased their ability to accumulate F-actin in the vicinity of SCVs , we pretreated infected cells with the actin-depolymerizing agent Latrunculin B, which completely prevents SCV-associated F-actin structures . After LssaV  or steC To study the requirement of Myosin II isoforms in the formation of SteC-dependent F-actin meshworks, we used mouse embryonic fibroblasts (MEFs) lacking Myosin IIB. These cells and their WT counterparts do not express Myosin IIC . ControlMLC phosphorylation and subsequent Myosin II activation is regulated by numerous kinases, the best described of which are MLCK and ROCK . We usedTo clarify the roles of MLCK and ROCK in SteC-dependent F-actin reorganization, we carried out RNAi-mediated knockdown of these proteins in Swiss 3T3 fibroblasts D. Single2+/calmodulin binding, and this activation was shown to be enhanced by ERK-mediated phosphorylation  or ERK (FR180204) inhibitors. Both inhibitors significantly reduced the percentage of microcolonies associated with F-actin    strain drylation B. Taken -32P] ATP and purified WT MEK1. Proteins were then separated by SDS-PAGE and analyzed by autoradiography. SteC underwent autophosphorylation as described previously .Phosphorylation of MEK1 by SteC was then analyzed by mass spectrometry with precursor ion scanning. Phosphorylation of either S218 or S222 was revealed by the presence of a weakly assigned phosphopeptide. However, a combination of precursor ion scanning and titanium dioxide enrichment clearly revealed phosphorylation of S200 D. This rStructural analysis of MEK revealed that amino acids 32–51 fold into an alpha helix (helix A) that is packed against the N-terminal lobe and the MEK1 kinase domain and maintains MEK1 in an inactive state . S200 isIn native MEK1, the T loop carrying the two activatory residues, S218 and S222, had a poorly defined conformation and appeared flexible C. When bHelix A displacement was previously shown to induce MEK1 activation, in part by inducing autophosphorylation on S218/222 . Since STo determine whether S200 is necessary for SteC-mediated MEK1 autophosphorylation, we used retroviral gene transfer of MEK1 KO MEFs to create cell lines expressing similar levels of WT MEK1 or a point mutant of MEK1 in which S200 was substituted with alanine (S200A). WT MEK1 and MEK1 S200A were immunoprecipitated and subjected to in vitro kinase assays in the presence of recombinant His-SteC. Whereas SteC caused phosphorylation of S218/222 of WT MEK, it was unable to induce efficient phosphorylation of S218/222 in MEK1S200A E  or glutamic acid (S200E). These constructs were expressed in serum-starved REF52 fibroblasts . Cells wsteC mutant strain in the mouse model of systemic infection (steC mutant bacteria and compared their growth after 4 days by competitive index (CI) analysis, which provides a sensitive measure of the relative degree of attenuation , which ADP ribosylates actin and inhibits the formation of SCV-associated actin structures  were added, or 1 mM ATP was used in cold assays. The mixture was incubated for 30 min at 30°C and subjected to SDS-PAGE, Coomassie blue staining, and autoradiography or WB with phospho-specific antibodies. Alternatively, MEK1 KO cells transduced with vectors encoding MEK1-Flag or MEK1-S200A-Flag were lysed in modified RIPA buffer . MEK1 variants were immunoprecipitated with agarose-conjugated mouse anti-Flag antibody . Kinase assays were carried out as described above, directly on the beads.SteC-6His and SteCK256H-6His were expressed and purified as described before . Protein3VO4 and 10 mM NaF. Samples were boiled for 5 min and separated by SDS-PAGE. Samples were then transferred onto PVDF Immobilon-P membranes (Millipore), and WB was carried out according to the manufacturer’s instructions. Animal procedures were conducted in accordance with UK Home Office Guidelines.Swiss 3T3 cells or MEFs were collected from a well in a 6-well plate 3 days after transfection with siRNAs. Infected Swiss 3T3 cells were collected 8 hr after invasion. Cells were lifted with trypsin and washed in PBS before lysis in 5× sample buffer  supplemented with 2 mM activated Na8 cfu of each strain in 0.2 ml. At least five mice were inoculated per strain mixture for each experiment. Mice were sacrificed 4 days after inoculation. The CI value is the mean of three independent experiments.Female BALB/c mice  of 18–22 g were inoculated by oral gavage with bacteria suspended in physiological saline solution. The bacterial inocula used were 1 × 10Please refer to the All results are reported as mean ± standard error of the mean (SEM). Statistical analyses were performed with Prism 5 software (GraphPad) with one-way ANOVA and Bonferroni post hoc analyses or two-tailed unpaired Student’s t test. Differences denoted as significant in the text fall below a p value of 0.05.


It has been noted that target sites located in the coding region or the 3′-untranslated region (3′-UTR) can be silenced to significantly different levels by the same siRNA, but little is known about at what specificity the silencing was achieved. In an exploration of positional effects on siRNA specificity by luciferase reporter system, we surprisingly discovered that siRNA had greatly elevated tolerance towards mismatches in target sites in the 3′-UTR of the mRNA compared with the same target sites cloned in the coding region. Assessment of changes in protein and mRNA levels suggested that the differential mismatch tolerance might have resulted from location-specific translational repression in the 3′-UTR. Ablation of argonaute proteins by AGO-specific siRNAs revealed that the AGO2 had major impact on siRNA silencing activity against sites in both coding region and 3′-UTR, while the silencing of nonnucleolytic AGO proteins  did not significantly affect silencing of sites in either region. This paper revealed the discovery that the specificity of an siRNA can be affected by the location of its target site. D. melanogaster or RDE-1 in C. elegans, and these two proteins are specialized for cleavage of perfectly matched targets. However, bulged miRNAs are sorted into AGO1 in files or ALG-1 in worms to function in miRNA pathway. Among human AGO proteins, only AGO2-containing ribonucleoproteins can guide the cleavage of complementary target mRNAs at the site opposite the 10–11th positions of the siRNA guide strand RNA interference (RNAi) is the process of post-transcriptional gene silencing mediated by double-stranded RNA RNAi has revolutionized many aspects of life science research as a powerful tool, but the specificity issue has been a matter of concern ever since the early days when siRNA was explored as a potential therapeutic reagent In our previous studies In this study, in order to compare the silencing specificity of siRNA on single-nucleotide mismatched target sites in the coding region versus 3′-UTR, we chose a highly potent siRNA (siR-04) and cloned the corresponding perfectly-matched target site and all possible 57 single-nucleotide mismatched target sites into the coding region or the 3′-UTR of firefly luciferase reporter gene in a eukaryotic expression plasmid . The absIn order to rule out the influence of siRNA size and sequence, we screened three additional siRNAs  under the same scrutiny. In total, 366 fusion constructs were prepared and tested. Although different siRNAs had the different extent of elevated mismatch tolerance at 3′-UTR sites compared to the coding region sites, the trend of compromised gene silencing at the 3′-UTR sites was consistent , approximating to the cleavage site (position 10 and 12) or 3′-end of the guide strand (position 17), and represented as different kinds of interaction between the siR-04 and the target site. Consistent with the finding for siR-04 towards the perfectly-matched sites, AGO2 contributed predominately to siR-04 activity in the two regions . The redThrough sufficient comparison of mismatch tolerance properties of siRNA on targeting sites in CDS versus 3′-UTR, it was found that the silencing activity of siRNA on mismatched sites was universally much higher in 3′-UTR than that in coding region. However, how about the silencing efficiency of siRNA towards perfectly-matched site in either region? Therefore, we compared the silencing efficacy of four siRNAs, siR-04, siR-26 and siR-40, as well as siR-206 on perfectly-matched sites located in both regions in the 100-fold dilution ranges of the siRNA. It showed that siR-04 had very tiny different activities on perfectly-matched site located in the coding region versus 3′-UTR, with only slightly better potency towards site in the 3′-UTR. However, siR-206 showed a reverse response on silencing its perfectly-matched target compared to siR-04. The silencing efficiency of siR-206 towards the coding region site was 1.5 times better than that towards the 3′-UTR site at the siRNA final concentration of 16.7 nM and 1.67 nM. siR-26 and siR-40 were more efficient when targeting 3′-UTR sites over two orders of magnitude of siRNA concentration . This diOur previous studies on siRNA specificity have established a practical guideline for allele-specific siRNA design. Recently, some SNPs located in the 3′-UTR of genes were found to affect mRNA stability or be associated with susceptibility to diseases By systematic comparison of mismatch tolerance properties of siRNA on targeting sites in the coding region versus 3′-UTR, it was demonstrated that siRNAs suffered universally from astonishing losses of specificity in terms of discrimination of single nucleotide mismatches. We went further to explore the mechanism governing the silencing efficacy on these sites. Our data demonstrated that for all mismatched sites, the silencing of AGO2 had the greatest impact on the silencing activity, which is consistent with previous reports DNA oligonucleotides were obtained from Invitrogen ; RNA oligonucleotides were from RiboBio  and Genepharma . The sequences of siRNAs and their matched targets, as well as human AGO1–4 specific siRNAs were included in Supplementary A previously reported siRNA validation vector siQuant BglII-ApaI cloning site was inserted downstream of the stop codon, and the original cloning site was destroyed. Furthermore, an irrelevant fragment of 1299 bp was amplified from the 3′-UTR of mouse Esrrb gene (NM_011934) and inserted upstream of the cloning site to space it from the stop codon. The same sequence was also inserted downstream of the cloning site to space it from poly(A) signal. Perfectly matched or single-nucleotide mutated target sequences were accordingly inserted into the cloning site, forming various fusion reporter genes.To assess the silencing activity in 3′-UTR, modifications were made in siQuant vector. For cloning target sequence in different positions of 3′-UTR, a 5 cells/well one day before transfection. Reporter plasmid (100 ng/well) carrying siRNA target was transfected into the cells at approximately 50% confluence using Lipofectamine™ 2000 , together with pRL-TK control vector (50 ng/well), with or without the siRNA (16.7 nM).Human embryonic kidney cells (HEK293A) were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 100 units/ml penicillin and 100 µg/ml streptomycin . The cells were seeded into 24-well plates at a density of ∼1×10firefly luciferase signal was normalized to the renilla luciferase signal for each individual well, and the silencing efficacy of each siRNA was calculated by comparison with a sample without siRNA treatment. All experiments were performed in triplicates and repeated at least twice.Twenty-four hours after transfection, the activities of both luciferases were determined by a fluorometer . The in vitro transcription system . Hybridization and stringent washing were performed according to DIG easy Hyb , and the signals were detected by Streptavidin-IRDye 800 CW  on an Odyssey® Infrared Imaging System . All experiments were performed at least twice.Twenty-four hours after transfection of reporter plasmid and siRNA, total RNA was harvested from HEK293A cells with TRIzol® Reagent  according to the manufacturer’s instructions. Total RNA (10–15 µg) was separated by electrophoresis on a 1% denaturing formaldehyde/MOPS agarose gel containing ethidium bromide then transferred onto a charged nylon filter . The intensities of 18S and 28S rRNA bands were checked under the ultraviolet light to verify that all samples were loaded equally and that no RNA degradation had occurred. The Biotin-16-UTP  labeled RNA probe  was generated by the Riboprobe® 5 cells/well or 1.2×105 cells/well, respectively. Cells in 6-well plates were cultured another 24 hours to detect protein levels of AGOs by western blot, while cells in 24-well plates were allowed to adhere to the plates for 6 hours then treated with reporter plasmid carrying siRNA target site, pRL-TK control vector, with or without siRNA (16.7 nM) to perform dual-luciferase assay on the following day.For the ablation of Argonaute proteins, AGO-specific siRNA (100 nM) was transfected into HEK293A cells, which were seeded into 6-well plates one day before, using Lipofectamine™ 2000 . Twenty-four hours after siAgo treatment, one portion of cells were lysed to detect mRNA levels of AGOs by RT-PCR, the others were split and seeded into 6-well plates or 24-well plates at a density of 5×10Twenty-four hours after transfection of 100 nM AGO-specific siRNAs, total RNA was harvested from HEK293A cells with TRIzol® Reagent (Invitrogen) and converted into cDNAs by ImProm-II™ Reverse Transcription System . Real time PCR was then performed using SYBR® Green I dye  to detect the mRNA expression levels of human AGOs and β-actin, an internal control. The results were normalized to that transfected by scrambled control siRNA. All experiments were performed in triplicates and repeated at least triples. Primer sets used in the detection assessed in the previous report Fifty-six hours after transfection of 100 nM AGO-specific siRNAs, total proteins were extracted from HEK293A cells by cell lysis buffer . Isolated proteins (20–40 µg) were separated by electrophoresis on a 10% SDS polyacrylamide gel and transferred onto a nitrocellulose filter membrane . The membrane was blocked and incubated at 4°C overnight with an antibody reacting with human AGO1 , human AGO2 , or GAPDH . Then the membrane was washed and incubated at room temperature with secondary antibody conjugated with horseradish peroxidase . The signals were detected by ECL kit  on an imaging system , and the intensity of protein bands was quantified by ImageJ software . All experiments were performed at least twice.Figure S1The secondary structures of the transcripts bearing siRNA target sites in different locations. (A) siR-04 target site in CDS; (B) siR-04 target site in 3′-UTR; (C) siR-04 target site far from the stop codon within the 3′-UTR; (D) siR-04 target site far from the poly(A) signal within the 3′-UTR. The secondary structures of these mRNAs were predicted by software of RNAstructure 5.3 (TIF)Click here for additional data file.Figure S2Positional effects of elevated mismatch tolerance in 3′-UTR was a universal phenomenon. (A–H) The original silencing efficacies of siRNAs on single-nucleotide mismatched targets in CDS (left) or 3′-UTR (right). (A) siR-04 on CDS sites; (B) siR-04 on 3′-UTR sites; (C) siR-26 on CDS sites; (D) siR-26 on 3′-UTR sites; (E) siR-40 on CDS sites; (F) siR-40 on 3′-UTR sites; (G) siR-206 on CDS sites; (H) siR-206 on 3′-UTR sites. The figure was plotted against the position (numbered from the start of the siRNA antisense strand) and the identity of the mismatched nucleotides. The position and the wild-type target sequence were given under the x-axis. The y-axis represented the remained luciferase activity, a ratio of firefly luciferase signal/renilla luciferase signal. The lower of the bars indicated siRNA had stronger silencing activities. White bars indicate wild-type targets; colored bars indicate single-nucleotide mismatched targets. Error bars represented SD. Data were average values of assays in triplicates, and all experiments were repeated at least twice.(TIF)Click here for additional data file.Figure S3Comparison of gene silencing in mRNA and protein levels at position 12 (A) and 10 (B). The intensity of mRNA bands was quantified by ImageJ software, while silencing efficacy of siR-04 on protein level was reflected by reporter enzyme activity. The target site location and siRNA:mRNA match pattern were given under the x-axis. The y-axis represented the remaining reporter gene expression in protein and mRNA levels, respectively. Error bars represented SD. All experiments were performed at least twice.(TIF)Click here for additional data file.Figure S4AGO2 contributed predominately to silencing activity in both regions, while down-regulated AGO1/3/4 had little impact. (A) The original silencing efficacies of siR-04 on perfectly matched or single-nucleotide mismatched target in CDS versus 3′-UTR after AGOs ablation.  AGO3 and AGO4 do not contribute to the translational repression on single-nucleotide mismatched targets. Silencing of AGO3 and AGO4 expression was carried out by gene-specific siRNAs, and subsequently, influences of the gene silencing were evaluated on single-nucleotide mismatched targets at position 4 (B) and 10 (C). All data were normalized to mock. Error bars represented mean SD. Data were average values of assays in triplicates, and all experiments were repeated at least twice.(TIF)Click here for additional data file.Table S1BglII and ApaI (labeled in bold).Sequences of siRNAs, miRNAs and DNA oligos used in this study. The underlined sequences represented the interaction region between siRNA and its target site, and the two terminals of target oligos are cohesive ends of (DOC)Click here for additional data file.


The voltage-gated sodium channel β2 subunit (Navβ2) is a physiological substrate of BACE1 (β-site APP cleaving enzyme) and γ-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Navβ2 by BACE1 and γ-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navβ2.We found a major  and a minor (144145 L↓Q) BACE1 cleavage site in the extracellular domain of human Navβ2 using a cell-free BACE1 cleavage assay followed by mass spectrometry. Next, we introduced two different double mutations into the identified major BACE1 cleavage site in human Navβ2: 147LM/VI and 147LM/AA. Both mutations dramatically decreased the cleavage of human Navβ2 by endogenous BACE1 in cell-free BACE1 cleavage assays. Neither of the two mutations affected subcellular localization of Navβ2 as confirmed by confocal fluorescence microscopy and subcellular fractionation of cholesterol-rich domains. Finally, wildtype and mutated Navβ2 were expressed along BACE1 in B104 rat neuroblastoma cells. In spite of α-secretase still actively cleaving the mutant proteins, Navβ2 cleavage products decreased by ~50% in cells expressing Navβ2 (147LM/VI) and ~75% in cells expressing Navβ2 (147LM/AA) as compared to cells expressing wildtype Navβ2.in vitro and cell-based results clearly show that the 147-148 L↓M is the major BACE1 cleavage site in human Navβ2. These findings expand our understanding of the role of BACE1 in voltage-gated sodium channel metabolism.We identified a major (147-148 L↓M) and a minor (144-145 L↓Q) BACE1 cleavage site in human Navβ2. Our  Togethein vivo ,9-12. ClVoltage-gated sodium channels (Nav) are composed of central α subunits and one or two accessory β subunits . The porPreviously, we and another group found that ADAM10, BACE1, and γ-secretase cleave Navβ2 in neuronal cells and mouse brains ,12. In aNM_004588) containing a C-terminal V5-His tag and full-length human BACE1 (GenBank: AF190725) containing a C-terminal myc tag have been described previously [2 (147LM/VI): 5'-GGCAAGATCCATCTGCAGGTCGTCATTGAAGAGCCCCCTGAGCGG-3' 5'-CCGCTCAGGGGGCTCTTCAATGACGACCTGCAGATGGATCTTGCC-3'; Navβ2 (147LM/AA): 5'-GGCAAGATCCATCTGCAGGTCGCCGCGGAAGAGCCCCCTGAGCGG-3' and 5-'CCGCTCAGGGGGCTCTTCCGCGGCGACCTGCAGATGGATCTTGCC-3'. Effectene (Qiagen) was routinely used for transfecting cell lines. GL189  was used in 10 μM concentration.Expression constructs encoding full-length human Navβ2 (GenBank: eviously . Navβ2 (Navβ2 substrate peptide (β2-peptide) with N-terminal biotin was synthesized by CHI Scientific . A biotinylated tyrosine group was added to the N-terminus of the β2-peptide. Reaction mixtures containing 20 mg of β2-peptide, 0.1 M Na-Acetate (pH 4.0), and 2.5 mg human BACE1 (R&D systems), were prepared and incubated at 37°C for 16 h. Reactions were stopped by heating to 95°C with LDS-SDS-PAGE sample loading buffer (Invitrogen) for 5 min.Reaction samples were then resolved on 12% BisTris gels (Invitrogen), transferred to PDVF membrane for Western blot analysis or fixed directly for silver staining. Vector ABC kit (Vector Labs) was used to detect full-length and N-terminal fragment of β2-peptide in Western Blot while Silver SNAP II kit (Invitrogen) was used to detect total protein in the gel.in vitro cleavage assay were analyzed by MS using a QStarR Pulsar I (Applied Biosystems) equipped with a nanospray source (in collaboration with Proteomic core at Harvard Partners Center for Genetics and Genomics). Analyst software (Invitrogen) was used to determine the molecular weights of all cleavage products in the reaction mixture.Reaction samples from the β2-peptide g. 20-50 mg of protein were resolved on 12% BisTris gels (Invitrogen). The blots were visualized by enhanced chemiluminescence (ECL). Images were captured using BioMax film (Kodak) or VersaDoc imaging system (Biorad) and quantified with QuantityOne software (Biorad). The followings are antibodies used in this study: anti-V5 , anti-myc  anti-GAPDH , and antiflotillin-1 .Cell lysates were prepared by directly extracting cells in a buffer containing 10 mM Tris-HCl (pH 6.8), 1 mM EDTA, 150 mM NaCl, 0.25% Nonidet P-40, 1% Triton X-100, and a protease inhibitor cocktail (Roche) followed by a centrifugation at 16,000 in vitro generation of Navβ2-CTFβ were performed as described earlier [Membrane preparation and  earlier . In brieCells were grown to 80 - 90% confluency in three 150-mm dishes, washed twice in phosphate buffered saline and scraped into 1.2 ml extraction buffer containing 0.5% Lubrol WX , protease inhibitor cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were then homogenized by five passages through a 25-gauge needle. Cell lysates were adjusted to 45% final concentration of sucrose  and loaded to the bottom of a 12-ml SW41 ultracentrifuge tube. A discontinuous sucrose gradient was established by sequentially layering 35% sucrose (4 ml) and 5% sucrose (4 ml) on top of the sample. Tubes were subjected to ultracentrifugation at 39,000 rpm for 18 h in Beckman SW41 rotor at 4°C. Twelve 1 ml fractions were collected from the top of the gradient and equal volume of each fraction was analyzed by Western blotting. V5 antibody was used to detect Navβ2 and flotillin-1 antibody was used as a lipid raft marker.Cells were grown on coverslips to 20 - 40% confluency and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were then rinsed three times with PBS and blocked for 1 h in PBS containing 1% BSA (Sigma-Aldrich) 0.1% Triton X-100 (FisherBiotech), 0.1% Gelatin (Sigma-Aldrich) and 0.05% Tween 20 . Cells were incubated with anti V5 antibody for 1 h, washed three times with PBS and incubated for 30 min with rabbit anti-mouse Alexa Fluor 488 antibody (1:200: Serotec) at room temperature. After washing three times with PBS containing 0.1% Triton X-100 and 0.05% Tween-20 for 3 min the coverslips were mounted onto glass slides using Prolong Gold antifade reagent with DAPI (Invitrogen) and analyzed with a Olympus florescence microscope equipped with a confocal disk scanning unit.To identify BACE1 cleavage sites in human Navβ2, we synthesized a substrate peptide (β2-peptide) corresponding to a 32 amino acid sequence at the juxtamembrane region of human Navβ2 Figure . A biotiTo specifically measure BACE1-mediated cleavage of Navβ2, we used a cell-free BACE1-cleavage assay . We intrTo exclude the possibility that the mutations introduced into Navβ2 caused its retention in early compartments, we studied the subcellular localization of the mutated versus wildtype Navβ2. We established enriched cultures of CHO cells transfected with wildtype and mutated Navβ2 and investigated localization of Navβ2 by florescence confocal microscopy. We could not detect any significant difference in the subcellular localization of mutated Navβ2 as compared to wildtype Navβ2 Figure . These dNext, we tested whether the distribution of Navβ2 into cholesterol-rich domains (lipid rafts) is altered by the 147LM/AA mutation. As previously reported, Navβ2 was detected in lipid raft enriched fractions, confirmed by flotillin-1 staining , we tested whether 147LM/VI and 147LM/AA mutations would also reduce Navβ2 cleavage in cells. Wildtype and mutated Navβ2 were transfected into B104 rat neuroblastoma cells stably overexpressing BACE1. α-secretase-mediated processing of Navβ2 would not be affected by mutations in the BACE1 cleavage site. Therefore, we did not expect complete inhibition of Navβ2 processing in cells expressing the mutated form of the protein. Indeed, we found that Navβ2-CTF production was decreased by ~50% in cells expressing Navβ2 (147LM/VI) and reduced by ~75% in cells expressing Navβ2 (147LM/AA) as compared to wildtype Navβ2 expressing cells Figure  and 4B. et al. previously reported that 144-145 L↓Q is the major BACE1 cleavage site of mouse Navβ2 [Using a similar synthetic peptide substrate and MALDI-MS, Wong se Navβ2 . It is ise Navβ2 . Furtherin vitro cleavage assay using synthetic substrate peptides [in vitro studies [A recent study identified 68 BACE1 substrates, underscoring the role of BACE1 in various cellular processes [peptides . The maj studies . These sIn our previous studies, we have found that elevated BACE1-mediated cleavage of human Navβ2 increased mRNA and protein levels of Nav1.1 α subunit by increasing the release of the β2 intracellular domain (Navβ2-ICD) . TherefoBACE1 levels and activities are significantly elevated in AD brains, possibly contributing to the disease progression -28. BACEin vitro assay and a cell based model. Our data clearly demonstrate that the BACE1 cleavage site (147-148 L↓M) is mainly responsible for BACE1 cleavage of human Navβ2.We identified a major (147-148 L↓M) and a minor (144-145 L↓Q) BASE1 cleavage site in human Navβ2 by using a synthetic β2-peptide and MS. We also found that mutations of the major BACE1 cleavage site (147LM/VI and 147 LM/AA) dramatically decreased BACE1-mediated cleavage of human Navβ2 in an APP: amyloid precursor protein; Aβ: amyloid β peptide; Nav: voltage-gated sodium channel; MS: mass spectrometry; Navβ2: voltage-gated sodium channel β2 subunit; BACE1: β-site APP cleaving enzyme; β2-peptide: Navβ2 substrate peptide; CHO: Chinese hamster ovary; CTF: C-terminal fragment; FL: full-length; ICD: intracellular domainThe authors declare that they have no competing interests.in vitro cleavage assay, immunofluorescence imaging and Western blotting. DYK did the in vitro cleavage assay and MS. RB carried out the lipid raft fractionation and helped to draft the manuscript. DMK conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.MTG and DYK participated in the design of the study and drafted the manuscript. MTG carried out the


It takes a special sort of case to spur attorneys into a debate over the drooling habits of toddlers. Yet that’s where lawyers from the Natural Resources Defense Council (NRDC), the U.S. Environmental Protection Agency (EPA), and Swiss chemicals company HeiQ found themselves in January 2013 as they debated in a federal appeals court the extent to which 1-year-olds and 3-year-olds chew, salivate, and swallow.At issue in the NRDC’s suit against the EPA, which is still awaiting ruling, was whether the agency was right in granting a conditional registration in December 2011 to a nanosilver-based antimicrobial fabric treatment manufactured by HeiQ.NRDC lawyer Catherine Rahm, however, begged to differ with the agency’s methods. In the January hearing, she argued that the agency record shows infants are more likely than any other subset of children to chew on fabrics that could contain the pesticide, and that if the agency were to recalculate its risk assessment based on the body weight of a 1-year-old, nanosilver concentrations in HeiQ’s product could result in potentially harmful exposures.It’s an obscure but critical distinction as far as risk assessment goes. And given the implications for HeiQ and other companies looking to follow in its footsteps, the case has landed at the center of a prolonged conflict over the regulation of nanosilver and the growing deployment of this antimicrobial ingredient in a variety of commercial and consumer products.Yet regardless of which side prevails in the case, the truth about nanosilver is not black and white. Even the loudest voices joining the NRDC’s call for strict regulation of nanosilver concede that context is key.“I’m not somebody who is for or against nano,” says Ian Illuminato, who directs Friends of the Earth’s U.S. nanotechnology campaign, which has produced a series of reports on the potential dangers of nanosilver and other nanomaterials. “I’m just saying we need to do this in a mature and adult way. That’s not to say this isn’t a helpful technology and it doesn’t have a place in our future, but in the way it’s evolving right now, it’s very risky.”Greg Lowry, a researcher at Carnegie Mellon University who studies the fate of nanoparticles in the environment, agrees that the use of nanosilver is a matter of balancing risk and reward. “There’s a tremendous potential benefit to the use in some of these products,” he says. “What one has to do is to ask yourself whether the benefits outweigh the risk.”Nanosilver itself is nothing new. It has been used for different reasons in consumer and commercial products over the past century, although “nano” terminology does not always appear in the patent or scientific literature.Silver Nanotechnologies and the Environment, a report published by the Pew Project on Emerging Nanotechnologies.Recent advances in the ability to synthesize nanosilver particles have led to a surge of even more innovations.As a result, nanosilver has appeared in an increasingly wide range of products on U.S. shelves, among them electric shavers, athletic clothing, bed and bath linens, cosmetics, baby bottles, stuffed animals, keyboards, paints, and food containers. It’s also used in hospital equipment including catheters, stents, bandages, and wound dressings, as well as on surfaces including wheelchair seats and door handles. In Southeast Asia, nanosilver is used even more commonly and often openly; it has been sprayed in Hong Kong subwaysDespite its widespread use, nanosilver remains a fairly poorly understood material to both regulators and scientists. Consensus remains elusive on subjects as essential as how it behaves in the human body and the environment, and the extent to which its use may contribute to bacterial resistance.Watchdog groups have seized on this murkiness in their continued call for tighter regulation of nanosilver. The NRDC, for example, points out that conditional registration of pesticides such as HeiQ’s product is based on the condition that complete toxicity data will be provided in a timely manner—meaning the product is allowed on the market while the company conducts the required studies.Recent evidence suggests that nanosilver’s form may not, in and of itself, be a factor in its germ-killing ability.Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) within minutes, says ABL Medical managing director Keith Moeller. SilvrSTAT is also approved by the Food and Drug Administration (FDA) for use in elder-care centers to help treat diabetic and septic ulcers, surgical wounds, and grafted sites, Moeller says, and by the EPA for use as a disinfectant for hard nonporous surfaces.But with antibiotic-resistant infections a growing concern around the world, there’s a powerful incentive to develop new antimicrobial tools, and nanosilver offers great promise in this arena. A new nanosilver-based gel known as SilvrSTAT™, for example—introduced in fall 2012 as a wound-care antimicrobial by Utah-based ABL Medical—was proven in laboratory tests to kill strains of methicillin-resistant “We’ve never actually found a pathogen we couldn’t kill,” Moeller says. “That’s why silver is being used all over. It’s an incredibly safe, very broad-spectrum antimicrobial agent. It’s naturally occurring, and it’s just really effective.”Nanosilver also plays a preventive role in special socks, shoes, and bandages developed for diabetics to prevent foot, ankle, and leg ulcers from progressing; in socks for military troops working in less-than-sanitary conditions to prevent trench foot, athlete’s foot, and other fungal infections; and in common athletic clothing to slow the spread of MSRA and other bacterial infections associated with close-contact sports.Rosalind Volpe, executive director of the Silver Nanotechnology Working Group, says nanosilver also deserves a place in common household goods. A paper by the working group explains, “Nanosilver antimicrobial treatments can bring a number of functionalities to consumer articles, including longer shelf life  giving more safety, less waste and ultimately lower prices for consumers; plastics that are protected against the degrading action of bacteria ; and textiles that are protected against colonization of bacteria that can lead to odors , ultimately giving greater comfort and prolonged use. Additional benefits such as reduced washing frequency at lower temperatures can give significant water and energy savings.”But the expansion of nanosilver into new applications may also contribute to unique risks. Looming large among them is the possibility that widespread use of nanosilver will contribute to silver resistance in bacteria, as has happened with other common antibiotics including penicillin,Nanosilver impregnated into consumer products and coatings will slowly, through laundering, be abraded from its substrate material at varying volumes and over varying periods of time, depending on concentrations in the material and the strength with which it is bonded, says Bernd Nowack of the Swiss Federal Laboratories for Materials Science and Technology. In consumer textiles, he notes, the industry standard is for the antimicrobial effect to persist over at least 50 washes, although some research suggests nanosilver can leach from certain products within the first few washes.Critics have questioned whether it’s wise to dispatch such a powerful weapon against bacteria in everyday contexts where bacteria pose a relatively minor concern. “It’s one thing if we’re using a little bit of nanosilver in the shoes of diabetics,” says Jaydee Hanson, policy director for the nonprofit International Center for Technology Assessment. “It’s another thing if you’re putting it in all underwear, all socks, every bed, every bed sheet. It’s a huge, exponential increase in the amount of nanosilver we’re putting into the environment.”Gregory Crocetti, a Melbourne-based microbiologist who has worked on Friends of the Earth Australia’s nanosilver campaign, takes a stricter position. “Nanosilver should remain in a hospital setting only,” he says. “Those clinical uses will be diminished by completely hysterical and frivolous uses in homes. ... Nanosilver has a high likelihood of promoting not just silver resistance but also antibiotic resistance because of the process of co-selection.” Co-selection occurs when bacteria challenged with one antimicrobial find a resistance gene to it by swapping DNA with bacteria that are resistant to a different antimicrobial.in vitro studies rather than clinical or environmental settings.,,,Salmonella typhimurium had been isolated from a Massachusetts burn ward;Up to this point there has been limited documentation of silver resistance. These few cases largely have been isolated in Some researchers and industry groups argue that silver and microbes have coexisted for billions of years and that resistance would have become evident by now if it were a viable threat. But Crocetti points out that silver was not historically used in the context of the widespread antibiotic resistance seen today.sil operon—the major set of silver resistance genes—is regularly found alongside the cassettes of antibiotic resistance genes on many clinically important plasmids. This means that silver resistance has been regularly co-selected by, and can itself co-select for, other antibiotic resistance genes.”“It is widely thought that different types of antimicrobial resistance genes have not been assembled by bacteria into such large collections on mobile genetic elements, particularly plasmids, until the last few decades,” Crocetti says. “It is important to note here that the Other research has shown that exposure to silver nanoparticles may, in some cases, improve bacterial survival rates. The authors of a widely cited study out of Rice University warned that low doses of silver may promote survival in what the authors called “an apparent hormetic effect.”,,,in vitro.,,Studies have also investigated nanosilver’s toxicity to aquatic organismsRecent findings have suggested that some nanosilver particles are rapidly converted to more stable silver sulfides in certain oxygen-free environments where sulfates are present, such as wastewater plants. This significantly reduces the particles’ ability to release silver ions and kill bacteriaAccording to Luoma, environmental surveillance is a “critical requirement for a future risk management strategy.” Given the lack of methodologies that currently exist for routine monitoring of nanomaterials, he suggests that monitoring of silver itself in water, sediment, or biomonitors “could be a viable interim approach until methods specific to the nanomaterial are developed.”Underlying this discussion so far is the assumption that what we’re doing actually matters—that the amount of silver, including nanosilver, that’s released into the environment and our bodies from consumer and hospital products is significant in relationship to background levels in the environment.The Silver Nanotechnology Working Group isn’t so sure that’s the case. Nanosilver production has been estimated at 2.8–20 tons per year in the United StatesThus, Volpe says, new applications of nanosilver are not expected to have a significant impact on the level of silver demand over the next 10–15 years, even though many applications may achieve significant commercial success. The NRDC, in contrast, argues that “if nanosilver proves to be much more toxic than conventional silver, the smaller quantities released will not necessarily cause less harm.”Silver Nanotechnologies and the Environment that silver is rare in the Earth’s crust and background concentrations extremely low. “Thus,” he wrote, “the addition of only a small mass of silver to a water body from human activities will result in proportionally large deviations from the natural conditions.”Luoma noted in Actual production data for nanosilver are not publicly available. Lowry believes the only way to get an accurate handle on how much nanosilver is entering the environment now and years down the line is to develop a comprehensive inventory of synthesis and production data. “With that kind of process, we can figure out what the loading on our waterways would be,” he says. “But without an inventory, it’s really hard to understand what our concentrations are. ... If you don’t know what the loading will be, you don’t know what the environmental concentrations will be.”


Drosophila melanogaster Held Out Wings (HOW) is a conserved RNA–binding protein (RBP) belonging to the STAR family, whose closest mammalian ortholog Quaking (QKI) has been implicated in embryonic development and nervous system myelination. The HOW RBP modulates a variety of developmental processes by controlling mRNA levels and the splicing profile of multiple key regulatory genes; however, mechanisms regulating its activity in tissues have yet to be elucidated. Here, we link receptor tyrosine kinase (RTK) signaling to the regulation of QKI subfamily of STAR proteins, by showing that HOW undergoes phosphorylation by MAPK/ERK. Importantly, we show that this modification facilitates HOW dimerization and potentiates its ability to bind RNA and regulate its levels. Employing an antibody that specifically recognizes phosphorylated HOW, we show that HOW is phosphorylated in embryonic muscles and heart cardioblasts in vivo, thus documenting for the first time Serine/Threonine (Ser/Thr) phosphorylation of a STAR protein in the context of an intact organism. We also identify the sallimus/D-titin (sls) gene as a novel muscle target of HOW–mediated negative regulation and further show that this regulation is phosphorylation-dependent, underscoring the physiological relevance of this modification. Importantly, we demonstrate that HOW Thr phosphorylation is reduced following muscle-specific knock down of Drosophila MAPK rolled and that, correspondingly, Sls is elevated in these muscles, similarly to the HOW RNAi effect. Taken together, our results provide a coherent mechanism of differential HOW activation; MAPK/ERK-dependent phosphorylation of HOW promotes the formation of HOW dimers and thus enhances its activity in controlling mRNA levels of key muscle-specific genes. Hence, our findings bridge between MAPK/ERK signaling and RNA regulation in developing muscles. Drosophila RNA–binding protein Held Out wings (HOW). Specifically, we show that HOW undergoes phosphorylation by MAPK/ERK, which increases its ability to form dimers and enhances its RNA–binding capacity. We further demonstrate that HOW is phosphorylated in embryonic and larval muscles by MAPK in vivo and that this event is important for its ability to regulate the levels of a giant sarcomeric gene homologous to vertebrate titin, thus contributing to the maintenance of muscle sarcomeric architecture. Importantly, HOW is a close homolog of mammalian Quaking, an essential protein in embryonic development and nervous system myelination, a reduction of which is correlated with Schizophrenia. Thus, our results raise the possibility that MAPK/ERK phosphorylation similarly regulates RNA profiles in other tissues controlled by proteins of the conserved Quaking family.Somatic muscles are huge cells that feature highly organized sarcomeric architecture, whose formation and maintenance are not fully understood. Multiple signals play a role in these cells, including the highly conserved MAPK/ERK pathway, which often serves as a cue for cellular proliferation or differentiation. In this study, we reveal a role for MAPK/ERK signaling in the regulation of multiple muscle genes at the level of mRNA, through the control of the activity of the  Drosophila melanogaster Held Out Wings (HOW) is an RBP that belongs to a family of evolutionarily conserved “Signal Transduction and Activation of RNA” (STAR) proteins C. elegans, the STAR protein GLD-1 promotes germ cell differentiation Regulation of gene expression at the level of RNA is often mediated through the activities of RNA-binding proteins (RBPs), which control different aspects of RNA metabolism of target genes Drosophila protein orthologous to mammalian QKI, is highly expressed in muscles, tendons stripe A, a transcription factor essential for tendon cell maturation nrxIV, thereby controlling glial cell maturation cdc25/string, a cell cycle promoting phosphatase, is essential to inhibit cell division in invaginating mesodermal cells HOW, the Structurally, HOW contains a single maxi-KH RNA binding motif that is flanked by two additional conserved domains, QUA1 and QUA2 Consistent with their expression in a wide range of tissues during development, the activity of STAR proteins is highly regulated at distinct post-translational levels, including phosphorylation by various kinases  (TP) at residues 59 and 64 . These c species .85RKQLAA, 121KKEPLTL, and 245KKRQLMELAI, as well as a single DEF domain motif, 151FNF. All four motifs are fully conserved throughout Drosophila species, and partially conserved in other STAR proteins , rendering it non-phosphorylatable.To start investigating phosphorylation of HOW by MAPK/ERK, we generated a HOW construct in which the putative phosphoacceptor sites T59 and T64 were mutated to Alanine WT and HOW(L)TTAA were transcribed and translated in vitro in the presence of S35-Methionine, incubated with recombinant activated ERK2, and run on an SDS gel. Incubation of in vitro translated Yan, an established MAPK substrate protein WT but not HOW(L)TTAA also displayed a MAPK-dependent mobility shift , indicating that MAPK phosphorylates HOW and that the phosphorylation event occurs on the predicted residues WT and HOW(L)TTAA were expressed in these cells, precipitated with an anti-HOW antibody, and subjected to Western blot analysis using an anti-phospho-Thr-Pro (anti-pTP) antibody TTAA form did not. This indicated that in unstimulated cells HOW is phosphorylated at least on one TP site.We next tested whether HOW is phosphorylated on its MAPK/ERK consensus sites using antibody . HOW(L)WWT possesses all of the sites predicted to be phosphorylated by, and to bind MAPK, it did not react with the anti-pTP antibody when tested in a similar manner , which differs from HOW(L) only at the C terminus r manner . This diWe further confirmed the identity of the kinase phosphorylating HOW in S2R+ cells by employing the specific MAPKK/MEK inhibitor, U0126 In a complementary approach, we stimulated MAPK/ERK signaling using Phorbol 12-Myristate 13-Acetate (TPA/PMA). This compound is an activator of protein kinase C Drosophila S2R+ cells.Taken together, these experiments demonstrate that HOW is phosphorylated by MAPK/ERK in in vitro translated HOW by activated ERK  protein using an anti-GFP antibody, and tested, using anti-HOW antibody, the co-precipitation of HOW(L)-HA (WT-HA readily co-immunoprecipitated with GFPWT-HOW (TTAA co-immunoprecipitated less efficiently (lane 2). Quantification of the experiments, while normalizing the co-precipitated HOW-HA levels to its levels in the crude extract as well as to the immunoprecipitated HOW-GFP, showed that co-precipitation of the phospho-mutant HOW was reduced by about 40% compared to that of HOWWT (TTDD). However, this mutation did not appear to mimic phosphorylated HOW, in this as well as in other assays (data not shown). As a control for the dimerization assay, we mutated HOW on a Glutamic acid residue, changing it to Glycine  and similarly tagged it with HA or GFP. This residue was previously shown to be essential for QKI EG-HA was reduced to about 10% of wild-type levels -HA . We diffFPWT-HOW , lane1, FPWT-HOW , lane 4.of HOWWT . In addie levels , confirmEGTTAA is significantly reduced.Collectively, the above experiments strongly suggest that MAPK-dependent phosphorylation occurs only on HOW dimers, although we cannot exclude a possible change of conformation in the monomeric structure of HOWEG, the mutant form that does not dimerize, exhibited an extremely low RNA binding activity, supporting a role for the dimerization of HOW in RNA binding  was affinity purified from S2R+ cells that were either grown in normal medium TTAA relative to HOW(L)WT WT . Thus, Hin vivo, we generated a polyclonal antibody designed to specifically recognize HOW only when it is phosphorylated on the more conserved Thr residue, T64 (we refer to the antibody as anti-pHOW (pT64) (see WThow(l), TTAAhow(l), and by treating a sample of the HOW(L)WT expressing cells with the MAPKK/MEK inhibitor U0126. We performed immunoprecipitation (IP) with an anti-HOW antibody and used anti-pHOW (pT64) antibodies in Western blot analysis (WT (lane 2) but not with HOW(L)TTAA (lane 4) nor with non-transfected cells (lane 1). In the sample treated with U0126 (lane 3), the reactivity of the antibody was significantly reduced, suggesting the antibody indeed detects HOW phosphorylation, particularly on T64.To follow the pattern of HOW phosphorylation T64) see ). We firanalysis . The antDrosophila embryos, employing how mutant (stru−/−how) embryos, in which zygotic HOW is not produced, as a control for antibody specificity. The embryos were also co-stained with a general anti-HOW antibody. Specific anti-pHOW staining was detected in the nuclei of somatic muscles (struhow mutants (We used the anti-pHOW (pT64) antibody to identify tissues in which HOW is phosphorylated . To this mutants . ImportaWT or HOW(L)TTAA under UAS-GAL4 binding sequences. The expression of HOW(L)WT or HOW(L)TTAA was driven in embryonic tendon cells by the tendon-specific driver, sr-GAL4, and the embryos were stained for pHOW  antibody did not label tendon cells, which exhibited a strong anti-HOW staining  is not efor pHOW  Whereas antibody  arrows, staining  arrows. WT or HOW(L)TTAA in muscles using the mef2-GAL4 driver. The HOW variants were subsequently immunoprecipitated using anti-HA conjugated beads and reacted with anti-pHOW (pT64) antibodies on Western blots (WT but not HOW(L)TTAA (lane 3), was detectable by the anti-pHOW antibody. In addition, an anti-HOW antibody identified an additional upper band that might represent phosphorylated HOW (lane 2). To conclude, these findings strongly indicate that HOW(L) undergoes phosphorylation on T64 in embryonic somatic and cardiac muscles and that phosphorylated HOW in muscles is localized specifically to the nucleus.Based on these results, we conclude that HOW(L) is phosphorylated on T64 in the nuclei of somatic muscles and heart cardioblasts. To further verify that HOW undergoes Thr phosphorylation in embryonic somatic muscles, we also expressed HA–tagged HOW(L)rn blots , lane 2.how was knocked down , the sarcomeric organization is aberrant. In these larvae, the Z discs appear discontinuous and spotty, a phenotype similar to that caused by the knock down of several genes encoding sarcomeric proteins rd instar larvae, in which we reduced HOW levels using RNAi mediated knock-down in muscles. To enhance the effect of how down-regulation, the larvae used were also heterozygous for struhow.To further characterize the physiological significance of MAPK/ERK-dependent phosphorylation of HOW, we focused on somatic muscles, a tissue in which a high degree of HOW phosphorylation was detected. Recently, HOW was shown to be essential for muscle sarcomerization WT) versus non-phosphorylated HOW (HOWTTAA) in this developmental process.Under these conditions, we observed an increase in the 500 kDa isoform of Sls, a giant protein that serves as a scaffold in the sarcomere, linking the Z-discs to the thick filaments WT or HOWTTAA in muscles (using the mef2-GAL4 driver) and followed the expression of Sls and MSP-300 by fluorescent labeling WT resulted in reduced levels of Sls , or together with HOWWT or HOWTTAA (WT overexpression. A much milder effect was observed following HOW(L)TTAA overexpression .sls RNA levels, and if so, whether phosphorylation is important for this type of regulation. To this end, we purified total RNA from the body walls of single 3rd instar larvae and performed real-time PCR to quantify the levels of the sls and how transcripts (rp49 RNA served as control) using the SYBR green method. We compared larvae from a wild-type background (w−), to larvae heterozygous for struhow which expressed how RNAi in muscles, and to larvae over-expressing either HOW(L)WT or HOW(L)TTAA in muscles. In larvae expressing lower levels of how (sls mRNA was upregulated (WT was more efficient in the down-regulation of sls RNA relative to HOW(L)TTAA  phosphorylation, we hypothesized that HOW(L) activity will be lowered in these larvae, resulting in elevated levels of Sls. Indeed, using a Western blot analysis  close to the polyadenylation site, possibly recruiting an E3 ubiquitin ligase complex to this site. Ubiquitination of the polyadenylation complex would subsequently lead to shortening of the poly-A tail EG mutant). This is also in line with our findings that HOW binding to RNA is significantly reduced when it is hypophosphorylated, and that phosphorylated HOW has a higher tendency to form dimers. We conclude that dimeric HOW binds RNA with higher affinity than monomeric HOW. This conclusion is in line with the observations that GLD-1 lacking its QUA1 domain has a lower affinity to RNA We show that a HOW mutant unable to form dimers exhibits a greatly diminished RNA binding capacity , it likely represents a true direct RNA target of HOW.A requirement for HOW in developing muscles had been demonstrated previously sls mRNA to promote fast exchange of newly formed Sls protein at the Z-disc. Indeed, in muscles where MAPK signaling was downregulated using rolled RNAi, a significant elevation in Sls levels is clearly evident  were created by site directed mutagenesis (Stratagene), following the manufacturer's protocols. pUAST-HOW(L) constructs tagged with HA or fused with GFP were generated in the pTWH and pTGW Drosophila Gateway vectors, respectively  using the Gateway cloning system (Invitrogen).Mutant HOW(L) constructs , mef2-GAL4, UAS-CD8 GFP , stripe-GAL4 , struhowhow RNAi line (ds-HOW) rolled RNAi . WT-3HAUAS-how(l) and TTAA-3HA were injected to flies by Genetic Services, Sudbury, MA, USA.Fly stocks used in this study include wPrimary antibodies used in this study include mouse anti-phospho-Thr-Pro (p-Thr-Pro-101) , rabbit and rat anti-HOW how DNA. Phorbol 12-Myristate 13-Acetate (TPA/PMA) (Sigma) treatment  was performed on starved cells, 20 h after transfection, for 15 min.S2R+ cells were grown and transfected essentially as previously described Cells were scraped off the flasks and collected in PBS, washed twice and lysed in 1% NP40 lysis buffer , 0.5% Phosphatase Inhibitor Cocktail 1, 20 mM β -glycerol phosphate).Embryos and larvae were collected and crushed in RIPA buffer .For IP, equal amounts of protein lysate were incubated with protein A/G beads  coupled with either rabbit anti-HOW polyclonal antibody or mouse anti-GFP monoclonal antibody, or agarose HA conjugated beads  for 1–2 h at 4°C. Beads were washed three times with the NP40 lysis buffer, and boiled in protein sample buffer to elute the proteins.in vitro phosphorylation assay was performed as described MAPK/ERK Fixation and staining of embryos were done following standard procedures. Larval Flat Preparations were performed essentially as described 6 cells per well), fixed with 3% PFA for 5 min, permeabilized using 3% PFA+0.1% TritonX-100, stained with primary antibody (1∶200) for 30 min, and secondary antibody (1∶400) for 30 min.S2R+ cells were seeded on Ibidi u-Slide 8 well  according to the manufacturer's instructions. Equal amounts were used as a template for cDNA preparation using the Verso cDNA kit (Thermo Scientific). Real time PCR was carried out using Fast SYBR Green Mastermix (Applied Biosystems) in a StepOne plus machine (Applied Biosystems). The following primers were used:sls (TGCCCATGCCGAAGACA) and (TGTCTTGTTTGCTGTTACGTTTACAG),rp49 (GACCATCCGCCCAGCATAC) and (CCATTTGTGCGACAGCTTAGC),how (AACTTTGTCGGTCGCATTTT) and (CGTCCTCCTTCTTCTTGTCG).In vitro RNA–protein binding assay was performed essentially as described in vitro translated, but were purified from S2R+ cells using immunoprecipitation with HA conjugated beads, followed by elution with an HA peptide .Phosphorylation sites were predicted by the GPS2.1 program


The seasonal prediction of the coastal oceanic warm event off West Australia, recently named the Ningaloo Niño, is explored by use of a state-of-the-art ocean-atmosphere coupled general circulation model. The Ningaloo Niño/Niña, which generally matures in austral summer, is found to be predictable two seasons ahead. In particular, the unprecedented extreme warm event in February 2011 was successfully predicted 9 months in advance. The successful prediction of the Ningaloo Niño is mainly due to the high prediction skill of La Niña in the Pacific. However, the model deficiency to underestimate its early evolution and peak amplitude needs to be improved. Since the Ningaloo Niño/Niña has potential impacts on regional societies and industries through extreme events, the present success of its prediction may encourage development of its early warning system. In austral summer of 2010/11, an unprecedented oceanic warm event was observed off the west coast of Australia. Sea surface temperature (SST) anomaly averaged in February-March 2011 reached about 3°C off the west coast of Australia, which is above four times of the standard deviation of its interannual variation in recent 30 years2467Study on the mechanism of the Ningaloo Niño has just started. Feng et al.Together with efforts to understand the mechanism of the Ningaloo Niño/Niña, exploring its prediction is important. Hendon and Wang11We begin by exploring historical time series of the Ningaloo Niño Index (NNI), which is defined as SST anomalies off the west coast of Australia  on a basis of the first empirical orthogonal function (EOF) mode in the monthly SST anomaly off Western Australia , which explains about 50% of the total variance. At a first glance, the SINTEX-F1 CGCM seems to be skillful in predicting most of significant events at least one season ahead, though the onset stage and the amplitude of the events are not well predicted . Figure Here we focus on the extreme warm event in early 2011 see . The SINTo examine details of the evolution, horizontal maps of both predicted and observed SST and surface wind anomalies are shown in the Clarke-Meyers effect14−1 in February 2011  up to 5-month lead when initialized on the first day of each month form May to November. In particular, the Ningaloo Niño is predicted very well when the model is initialized in austral winter-spring. Also, we have focused on the prediction of the unprecedented extreme warm event of the 2011 Ningaloo Niño. The SINTEX-F1 prediction initialized on June 1st, 2010 successfully predicted this extreme warm event in February 2011, i.e. 9 months in advance. The model reasonably predicted the rapid development of the La Niña condition, the easterly wind anomalies over the equatorial western Pacific in December 2010. It also predicated successfully warm SST anomalies off the west coast of Australia with northerly wind anomalies, the intensification of the Leeuwin Current, and the coastal downwelling in February 2011.The Ningaloo Niño has serious impacts on marine ecosystems, but also it may influence on Australian summer precipitation through induced anomalous atmospheric circulations. As the 2011 Ningaloo Nino matured in February 2011, most part of Australia experienced wetter-than-normal condition, which was also reasonably predicted by the model initialized on June 1st, 2010. Further studies are necessary for understating the Ningaloo Niño's influences on Australian summer precipitation in more detail, because Australian climate is also influenced by the Australian Monsoon, El Niño/Southern Oscillation (ENSO), and Indian Ocean Dipole16171819202123The potential source of the predictability of the 2011 Ningaloo Nino lies in the fact that the SINTEX-F1 reasonably predicts the quick transition from El Niño to La Niña in the tropical Pacific in late 2010 . Althoug2627the Clarke-Meyers effect. The model is skillful in predicting sea level anomalies off the west coast of Australia at 4–9 months lead due to the high prediction skill of ENSO. This is consistent with our results. Also, the coarse-resolution may fail to capture the local ocean-atmosphere feedback. Therefore, it is important to improve climate prediction models to capture such regional variations and thus to make seasonal climate information more beneficial to the regional societies. The current initialization method adopting a simple coupled SST-nudging initialization scheme may have limited the predictability, too. We expect that adoption of a suitable three-dimensional ocean data assimilation method will improve the preconditioning phase and the seasonal predictability of the Indian Ocean variations. We are developing the new high-resolution version of SINTEX-F2 GCM with new initialization scheme to challenge the improvement of the prediction skill for regional climate modes as typified by the Ningaloo Niño.Although the SINTEX-F1 prediction of the 2011 Ningaloo Niño 2 seasons ahead is in good qualitative agreement with the observation, the predicted amplitude of the Ningaloo Niño is only 50% of the observed. The coarse horizontal resolution of the ocean component of the SINTEX-F1 may be not enough to simulate the Leeuwin Current and coastal downwelling off the west coast of Australia qualitatively. To minimize such a model bias, Hendon and Wanghttp://www.jamstec.go.jp/frcgc/research/d1/iod/index.html. The SINTEX-F1 prediction system has demonstrated high performance in predicting ENSO and Indian Ocean Dipole2627282930The JAMSTEC seasonal prediction system was built on the basis of the Scale Interaction Experiment-Frontier (SINTEX-F1) fully coupled global ocean–atmosphere GCM11To evaluate the SINTEX-F1 prediction results, we use the NOAA OISSTv2T.D., S.B. and T.Y. contributed to designing the research, interpreting results, and writing the paper. T.D. performed the seasonal prediction system on a basis of the ocean-atmosphere coupled model and analyzed the observation data and model prediction outputs.Supplementary Figures


An important question pertaining to glycoproteins is whether the covalently-bound carbohydrate moieties contribute to its immunogenic activity. Ovomucoid (OM) is, for these kinds of studies, a model compound in view of its relatively high carbohydrate content but the role of the carbohydrate moieties on its immunogenic activity still remains controversial. The aim of this study was to establish the effect of glycosylation on the IgE-binding properties and biological activity of OM.Carbohydrate moieties of OM were removed by enzymatic deglycosylation with PNGase F and the degree of deglycosylation of the protein was evaluated by SDS-PAGE with PAS staining and RP-HPLC. Changes in secondary and tertiary structure of OM due to deglycosylation were evaluated by circular dichroism. Immunoreactivity of native and deglycosylated OM was evaluated by inhibition ELISA and western blot using the sera from ten egg-allergic patients with IgE levels from 37 to more than 100 kU/L. Using PBMCs from egg-allergic patients, the cellular proliferative potency and the capacity to stimulate the secretion of interleukins of native and deglycosylated OM were assessed. Basophil activation by OM, with and without carbohydrates, was assessed using human basophils passively sensitized with serum from children with egg allergy.PNGase F effectively released carbohydrates attached to OM after five days at 37° without apparent structural changes. Immunoassays showed that, in eight out of ten sera of egg-allergic patients, the IgE binding to deglycosylated OM was less effective than to the native protein. The results of immunoblotting showed that six out of ten sera, previously incubated with the deglycosylated protein, kept their IgE binding capacity to the glycosylated protein, supporting the idea that carbohydrates may play a role in antigen recognition. In spite of these results, no influence of glycosylation on T cell activation and proliferation was found.The carbohydrate chains attached to OM play a role in the reactivity against IgE, but their clinical relevance might be low.None declared.


Activation of ErbB2/4 receptor tyrosine kinases in cardiomyocytes by neuregulin treatment is associated with improvement in cardiac function, supporting its use in human patients with heart failure despite the lack of a specific mechanism. Neuregulin infusion in rodents increases cardiac myosin light chain kinase (cMLCK) expression and cardiac myosin regulatory light chain (RLC) phosphorylation which may improve actin-myosin interactions for contraction. We generated a cMLCK knockout mouse to test the hypothesis that cMLCK is necessary for neuregulin-induced improvement in cardiac function by increasing RLC phosphorylation.The cMLCK knockout mice have attenuated RLC phosphorylation and decreased cardiac performance measured as fractional shortening. Neuregulin infusion for seven days in wildtype mice increased cardiac cMLCK protein expression and RLC phosphorylation while increasing Akt phosphorylation and decreasing phospholamban phosphorylation. There was no change in fractional shortening. In contrast, neuregulin infusion in cMLCK knockout animals increased cardiac performance in the absence of cMLCK without increasing RLC phosphorylation. In addition, CaMKII signaling appeared to be enhanced in neuregulin-treated knockout mice.Thus, Neuregulin may improve cardiac performance in the failing heart without increasing cMLCK and RLC phosphorylation by activating other signaling pathways. Neuregulin is a growth factor which signals to the ErbB family of receptor tyrosine kinases 2+ sensitivity of myofilament contraction and maximal force development Cardiac myosin exists as a hexamer, each comprised of two heavy chain subunits, each with two types of light chain subunits, an essential light chain and regulatory light chain (RLC). RLC is phosphorylated in cardiac muscle by its dedicated kinase cMLCK at a specific serine in the N-terminus RLC is phosphorylated in normal beating hearts. The extent of RLC phosphorylation is about 0.4 mol phosphate per mol RLC in different animal species, and this basal phosphorylation is sufficient to enhance contractile force in permeable fibers 2+/calmodulin to homologous calmodulin binding sequences to displace an autoinhibitory segment from the catalytic cleft 2+/calmodulin-dependency of cMLCK activity is controversial even though this kinase has a calmodulin binding sequence and autoinhibitory segment similar to the skeletal and smooth muscle MLCKs Although the physiological importance of RLC phosphorylation is demonstrated with genetically modified mice, determinants for cMLCK activity have yet to be identified. There is agreement that the kinase has a low specific activity neo cassette, floxed gene, and Cre recombinase transgene were removed. This knockout mouse line has no cMLCK, and shows attenuated RLC phosphorylation and diminished cardiac function in young mice, similar to previously described results for the hypomorph in vivo, and have investigated potential alternative mechanisms.Ablation of cMLCK in a hypomorphic mouse model previously described by our laboratory attenuated RLC phosphorylation All procedures were performed in accordance with the Institutional Animal Care and Use Guidelines. The protocol was approved by the University of Texas Southwestern Medical Center Institutional Animal Care and Use Committee (APN#0166-06-11-1). Animals were housed under specific pathogen free conditions in our AAALAC certified rodent facilities; surgeries were performed under isoflurane anesthesia and mice were monitored during recovery.Mylk3+/−, but not the CAG-Cre gene, were selected for subsequent breeding. Littermate wildtype mice were used as controls. For all studies, 12–15 week old male mice were used.A conventional knockout of cMLCK was generated from the floxed mice previously described Mylk3+/+.Total RNA was purified from isolated heart ventricles with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used to synthesize cDNA from 0.5 microgram of RNA. Quantitative PCR was performed using the following TaqMan® probes purchased from Applied Biosystems: ANP, Mm01255748_g1; BNP, Mm01255770_g1; Col1a2, Mm00483888_m1; Myh6, Mm00440359_m1; Myh7, Mm00600555_m1; and 18S, 4333760F. Analyses were performed by the comparative CT method. Initial data were normalized to 18S; relative values were obtained by normalizing to the median for Echocardiograms were performed on conscious, gently restrained mice using either a Sonos 5500 system with a 15-MHz linear probe or Vevo 2100 system with a MS400C scanhead. Left ventricular internal diameter at end-diastole (LVIDd) and end-systole (LVISd) were measured from M-mode recordings. Percent fractional shortening was calculated as ((LVIDd − LVISd)/LVIDd)x100. Measurements of interventricular septum thickness, left ventricular internal diameter, and left ventricular posterior wall thickness were made from two-dimensional parasternal short axis views in diastole Mice were treated with 0.2% BSA/PBS or neuregulin  5ug/kg/h in 0.2% BSA/PBS for 7 days through surgically implanted Alzet® mini-osmotic pump  Data are expressed as mean ± S.E. Statistical evaluation was carried out in GraphPad Prism using an unpaired Student's t-test for two comparisons or paired t-test for comparison of same sample before and after treatment. Analysis of variance and Newman-Keuls post-test were used for multiple comparisons. Significance was accepted at a value of p<0.05.Frozen ventricles were ground in liquid nitrogen and an aliquot thawed in 10% trichloroacetic acid containing 10 mM dithiothreitol. Precipitated protein was washed free of acid with three 5-minute washes in ethyl ether and resuspended by vigorous agitation in urea sample buffer  using an orbital shaker (IKA Vibrax VXR) set at 1400 rpm for 30 minutes at room temperature. Complete denaturation and solubilization was achieved by addition of urea crystals and prolonged agitation. Protein samples were subjected to centrifugation at 10,000 x g for 2 minutes and protein concentrations in supernatant fractions measured by Bradford assay. Proteins (2 – 40 µg) were subjected to SDS-PAGE after boiling in Laemmli buffer, transferred to PVDF  or nitrocellulose , and blotted by standard procedures. The amount of protein loaded was optimized empirically for each antibody to ensure density measurements were proportional to the amount of protein.Measurements of RLC phosphorylation in heart homogenates were performed by urea/glycerol-PAGE and immunoblotting, as previously described 2+ transport ATPase, SERCA2a (MA3-919); total protein kinase D, PKD (2052P); and pPKD_Ser916 (2051P) were purchased from Pierce. Antibody to phosphorylated cardiac myosin binding protein C, pMybp-C_Ser282 (ALX 215-057), was purchased from Enzo Life. Antibody to total CaMKIIδ was a generous gift from Donald Bers .Antibodies specific to cardiac RLC and cMLCK were reported previously A mouse line of cMLCK knockout animals was generated by breeding cMLCK floxed mice In order to ascertain whether cMLCK is necessary for neuregulin-associated increase in RLC phosphorylation and thereby improved cardiac function, we infused neuregulin into cMLCK knockout mice for seven days Comparison of the interventricular-septal and posterior wall thickness at diastole before and after treatment shows significant increases in thickness associated with neuregulin treatment for hearts from wildtype or cMLCK knockout mice . While aThere are several reports of protein kinases being upregulated with neuregulin treatment To test possible associations of neuregulin with regulation of sarcomeric proteins that increase myocardial contractility, sarcomeric protein phosphorylation was measured. Total cMLCK and RLC phosphorylation both increased in wildtype neuregulin-treated hearts, but not in the cMLCK knockout group . The con2+ handling by the sarcoplasmic reticulum via the Ca2+-ATPase pump (SERCA2a) and its regulator, phospholamban (PLB). Measurement of phosphorylated PLB showed a decrease in both the cyclic-AMP and CaMKII-regulated sites, Ser16 and Thr17, in neuregulin-treated hearts from wildtype mice (C (cytoplasmic CaMKIIδ), the kinase which phosphorylates that residue. Total CaMKIIδ , CaMKIIδC and phosphorylated CaMKII were all increased only in hearts from cMLCK knockout mice after treatment with neuregulin  showed its ratio over total is significantly increased only in the cMLCK knockout, neuregulin-treated group. Overexpression studies of CaMKIIδ isoforms in the heart associates increased CaMKIIδC levels with poor cardiac function Comparison of the PKA-dependent and CaMKII-dependent phosphorylation sites in PLB showed that in the cMLCK knockout hearts, only the CaMKII-dependent site was modestly decreased in neuregulin-treated when compared with vehicle-treated hearts. However, the amount of phosphorylated PLB Thr17 in the neuregulin-treated cMLCK knockout hearts was significantly greater than in the neuregulin-treated wildtype hearts. This increase is consistent with CaMKIIδ measurements, where only in the cMLCK knockout, neuregulin-treated group, total and pCaMKII levels were increased. Although the antibody to phosphorylated CaMKII is not specific to CaMKIIδ, parallel signal increases in the neuregulin-treated group supports its activation. Measurement of the cytoplasmic isoform of CaMKIIδ (CaMKIIδThere are numerous differences between rodents and human hearts which must be considered when mouse models are used to study human cardiac diseases. The studies reported here used an engineered heart failure model caused by the ablation of cMLCK and thereby phosphorylated RLC, not yet described in the human population. This model was used to address the specific question of whether neuregulin-induced improvement of cardiac performance was dependent on cMLCK. In summary, heart failure may be improved by neuregulin treatment without cMLCK and increased RLC phosphorylation. The neuregulin-ErbB signaling pathways that improve cardiac function independent of RLC phosphorylation need to be identified.