However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays.Cow’s milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow’s milk allergic children, are being employed to assess An oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin  was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays.Mice were immunized with bovine β-lactoglobulin, and subsequently the variable domains of dissimilar anti-β-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains.After sensitization with this pool of anti-β-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa β-lactoglobulin, and 5–10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow’s milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-β-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on β-lactoglobulin, which were also recognized by serum IgEs from cow’s milk allergic children.Usage of our ‘unlimited’ source and well-defined pool of β-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow’s milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula. Food allergens, aeroallergens, medications and insect venoms are the most common allergens which are responsible for inducing Type I or immunoglobulin E (IgE)-mediated hypersensitivity reactions in vitro cellular degranulation assays is proposed as a strategy for the screening of new HA formulas aimed at preventing sensitization in atopic children and avoiding clinical symptoms in infants suffering from CMA in vitro cellular degranulation assays uses the rat basophilic leukaemia cell line RBL-2H3 stably transfected with the α-chain of the human FcεRI (RBL-huFcεRI) as target cells. This α-chain of human FcεRI associates with one β-chain and two γ-chains of rat FcεRI, and forms functional high affinity IgE receptor complexes on the surface of RBL-huFcεRI cells. The usefulness of RBL-huFcεRI cell degranulation after exposure to IgE containing serum from allergic patients, and subsequent cross-linking with either anti-human IgE antibodies in vitro model system for the detection of allergens and for the study of IgE-allergen interactions CMA is the most common allergy in early childhood, with an estimated prevalence of 3% in the pediatric population Whey proteins represent nearly 20% of the total CM proteins. The known major allergenic proteins of whey are β-lactoglobulin (BLG) and α-lactalbumin (ALA) In this study, the development of six BLG-specific chimeric human IgE monoclonal antibodies covering allergenic epitopes of BLG is described. The optimal concentration of our pool of BLG-specific chimeric IgE monoclonal antibodies for use in the RBL-huFcεRI degranulation assay was established and tested with whey hydrolysates with different degrees of hydrolysis to assess sensitivity and specificity.The Immunization protocol described below was approved  by the Animal Experiments Committee of the Faculty of Veterinary Medicine, Utrecht University, The Netherlands.The sera used for epitope mapping was from a cohort study that was performed in children successively referred with eosinophilic esophagitis to the pediatric GI department of Hospital Necker-Enfants malades, Paris, France. Participants were recruited from the ARSENE cohort, a collection of blood samples registered within the French Ministry of Health (number DC-2009-955) 5 cells/200 µl per well in aminopterin selection medium, i.e., opti-MEM I with GlutaMax/10% FCS that was supplemented with 50x Hybri-Max aminopterin (a de novo DNA synthesis inhibitor (Sigma)). From day 7 after fusion, aminopterin selection medium was replenished every 2–3 days, and at day 16, aminopterin selection medium was replaced by opti-MEM I with GlutaMax/10% FCS.BALB/c mice  were subcutaneously injected with bovine whey , each mouse received 200 µg bovine whey in 200 µl PBS mixed with 200 µl Complete Freund’s adjuvant (Sigma) at day 0. Antibody responses in mice were then boosted by subcutaneous injections of bovine whey, each mouse received 200 µg bovine whey in 200 µl PBS mixed with 200 µl Incomplete Freund’s adjuvant (Sigma) at day 21 and day 42. This was followed by intraperitoneal injections with bovine whey without adjuvant, each mouse received 200 µg bovine whey in 200 µL PBS at day 61 and day 62. At day 65, splenocytes from immunized mice were fused with SP2/0-Ag14 myeloma cells (DSMZ) using slightly modified hybridoma technology originally described by Köhler and Milstein 2SO4, optical densities were measured at a wavelength of 450 nm (reference wavelength of 655 nm) using a microplate reader (BioRad). Hybridomas positive in this whey ELISA were expanded and cryopreserved.From day 16 after fusion, supernatants from hybridomas were screened for anti-whey IgG antibody production using a standard ELISA with bovine whey (Lacprodan WPC80). Briefly, bovine whey was coated (100 ng whey/100 µl PBS/well) onto EIA plates (Corning) o/n at 4°C. After washing with PBS/0.05% Tween 20, plates were blocked with PBS/0.05% Tween 20/10% ELISA Blocking Reagent (Roche) for 1 hour at RT. Subsequently, plates were incubated with 100 µl serially-diluted hybridoma supernatant per well for 1 hour at RT. After washing in PBS/0.05% Tween 20, binding of IgG antibodies was determined with 1∶5,000 diluted HRP-conjugated goat anti-mouse IgG-specific antibodies (Jackson ImmunoResearch) for 1 hour at RT, followed by a ready-to-use solution of TMB substrate (Invitrogen) for colorimetric detection. After adding 1 M HFusions yielded 40 hybridomas with high IgG antibody titers against bovine whey. Next, supernatants of these anti-whey producing hybridomas were tested for their fine-specificity against bovine BLG and bovine ALA, the main constituents of bovine whey. Briefly, whey-purified BLG or ALA  was coated (10 and 50 ng/100 µl PBS/well) onto EIA plates (Corning) o/n at 4°C. After incubation, 10-fold diluted supernatants of the 40 anti-whey producing hybridomas were examined using the same whey ELISA method as described above. This screening resulted in 15 hybridomas that produced IgG antibodies specifically against bovine BLG, 7 hybridomas that produced IgG antibodies against bovine ALA, and 18 hybridomas against non-BLG/non-ALA proteins .N-hydroxysuccinimido-biotin (Pierce). Then, whey-purified bovine BLG was coated (200 ng/100 µl PBS/well) onto EIA plates (Corning) o/n at 4°C. After blocking with PBS/0.05% Tween 20/1% BSA fraction V (Roche), BLG was saturated with each individual unlabeled mouse anti-BLG IgG antibodies 7D5, 8C3, 8F7, or 13A5 (2.5 µg/100 µl PBS/well) for 1 hour at RT, followed by an incubation with suboptimal concentrations of each individual biotinylated mouse anti-BLG antibodies 7D5, 8C3, 8F7, or 13A5 for 1 hour RT. Binding of biotinylated antibodies was determined with 1∶5,000 diluted HRP-conjugated streptavidin (Jackson ImmunoResearch) for 1 hour at RT, followed by a ready-to-use solution of TMB substrate (Invitrogen) for colorimetric detection.Recognition of multiple dissimilar epitopes on BLG by BLG-specific IgE antibodies on sensitized FcεRI expressing RBL cells is a prerequisite for successful IgE/BLG cross-linking, and subsequent degranulation. In order to identify mouse anti-BLG IgG antibodies, which recognized similar or dissimilar parts on BLG, a competitive BLG ELISA test was performed. For this, a selection protein G-purified mouse anti-BLG IgG antibodies from hybridomas with highest antibody titers designated as 7D5, 8C3, 8F7, and 13A5, was labeled with 6 viable cells from each subcloned hybridoma were washed in PBS and frozen at −80°C. From these cell pellets, total RNA was isolated and purified using an RNeasy mini kit (Qiagen) according to manufacturer's instructions. Purified RNA (1–2 µg) was then used as templates for cDNA synthesis with a RevertAid H Minus First Strand cDNA synthesis kit (Fermentas). Next, variable light (VL) and variable heavy (VH) regions were amplified in PCR reactions comprising 25 ng cDNA, 10x PCR mix containing dNTPs (Life Technologies), 25 pmol 5′ and 3′ primers (Biolegio) described in After selection of the lead hybridomas that produced mouse (non)cross-blocking anti-BLG IgG antibodies , these hybridomas were subcloned (by limiting dilution), and designated as clone 5D6.1, 7D5.1, 8C3.3, 8F7.1, 11B6.2, and 13A5.2. Then, 5×102 under humidified conditions on an orbital shaker at 110 rpm. Supernatants were harvested for antibody purification when cellular viability dropped below 50%.Chimeric IgEκ or IgG1κ  anti-BLG antibodies were generated by combining expression plasmids containing cDNA encoding chimeric κ light chains with their corresponding plasmids encoding chimeric IgE heavy chains or chimeric IgG1 heavy chains. To this end, 300 ml CHO-S cells (Life Technologies) were transiently transfected using these plasmids (at a heavy-to-light chain plasmid ratio of 1∶1 (w/w); plasmids have similar sizes) with FreeStyle MAX reagent (Life Technologies). Cultures were incubated for several days at 36.5°C and 5% COSupernatants from transiently transfected CHO cells were collected, and then cleared by centrifugation at 3500 g for 10 minutes at RT. Chimeric anti-BLG antibodies were purified using either HiTrap KappaSelect (chimeric human IgE antibodies) or HiTrap protein A HP (chimeric human IgG1 antibodies) columns  on an ÄKTAprime plus liquid chromatography system . Briefly, culture supernatants were adjusted to pH 7.2 and loaded onto affinity columns. After washing with PBS pH 7.2, chimeric anti-BLG antibodies were eluted from affinity columns with 0.1 M glycine buffer pH 3.0, and instantly neutralized in 1 M Tris buffer pH 9.0. After dialysis against PBS pH 7.2, antibody concentrations were determined by measuring the absorbance at 280 nm. Extinction coefficients (1 mg/ml at 1 cm pathway) of 1.53 and 1.36 Binding of purified chimeric human IgE and IgG1 anti-BLG antibodies clones 5D6.1, 7D5.1, 8C3.3, 8F7.1, 11B6.2 or 13A5.2 was done by using the BLG ELISA as described above. For detection, however, 1∶1,000 diluted HRP-conjugated goat anti-human IgE-specific antibodies (Sigma) and 1∶5,000 diluted HRP-conjugated goat anti-human IgG-specific antibodies (Jackson ImmunoResearch) were applied.Bovine BLG (120 µg/480 µl non-reducing sample buffer) was loaded and electrophoresed in a one well pre-casted 4–12% BisTris gel/MOPS running buffer NuPage Novex system (Invitrogen). Then, bovine BLG was electro-blotted onto a polyvinylidene fluoride transfer membrane (Millipore). After blocking with PBS/0.05% Tween 20/1% BSA fraction V (Roche) for 1 hour at RT, this membrane was incubated with 1 µg/ml purified chimeric human IgE anti-BLG antibody clones 5D6.1, 7D5.1, 8C3.3, 8F7.1, 11B6.2 or 13A5.2 for 1 hour at RT. After washing in PBS/0.05% Tween 20, binding of these chimeric human IgE anti-BLG antibodies was determined with 1∶4,000 diluted HRP-conjugated goat anti-human IgE-specific antibodies (Sigma) for 1 hour at RT, followed by a ready-to-use solution of TMB substrate (Sigma) for colorimetric detection.6 cells/ml in ice-chilled PBS/0.1% BSA (Sigma)/0.01% NaN3 (Acros). Subsequently, RBL cells were incubated with 20 µg/ml purified chimeric human IgE anti-BLG antibody clones 5D6.1, 7D5.1, 8C3.3, 8F7.1, 11B6.2 or 13A5.2 for 30 minutes at 4°C. After washing in ice-chilled PBS/0.1% BSA/0.01% NaN3, binding of chimeric huIgE anti-BLG antibodies was detected using 1∶10 diluted FITC-labeled goat anti-human IgE-specific antibodies (KPL) for 30 minutes at 4°C. For human FcεRI expression levels, RBL cells were incubated with a 1∶5 diluted PE-labeled mouse anti-human FcεRI α chain-specific antibody (eBioscience) for 30 minutes at 4°C. RBL cells were fixed in PBS/0.1% BSA/0.01% NaN3/2% formaldehyde (Sigma) for 30 minutes at 4°C prior to flow cytometric measurements using FACSCalibur (BD Biosciences).RBL-huFcεRI cells (see below) were adjusted to 1×105/well) in 96-wells flat bottom culture plate were sensitized o/n with 5 µg/ml commercially available human purified IgE (Chemicon/Millipore) and stimulated with 10 µg/ml rabbit anti-human IgE antibodies (Dako) in Tyrode’s buffer at pH 7.4 with 0.1% HSA  for 1 hour. This release served as maximum release (100% degranulation). The sensitized cells with single BLG-specific chimeric IgE monoclonal antibodies or a pool of BLG-specific chimeric IgE monoclonal antibodies were stimulated with anti-human IgE (10 µg/ml), milk powder , whey , BLG  or whey hydrolysates  for 1 hour. Therapeutic anti-IgE humanized (huIgG1κ) antibody omalizumab , which is well-known for preventing the binding of huIgE on huFcεRI, was applied  during o/n sensitization of RBL-huFcεRI cells to determine the specificity of the pool of BLG-specific chimeric IgE monoclonal antibodies on degranulation using cross-linking with BLG (1 µg/ml). β-Hexosaminidase activity was determined by a fluorescence assay using 4-methylumbelliferyl-N-acetyl-α-D-glucosamine (Sigma-Aldrich) as a substrate The cell-line RBL-hεIa-2B12, which was transfected with the α-chain of human Fcε receptor type 1 (hu) FcεRI complex 2SO4  and absorbance was read at 450 nm. Values of 2x background OD were considered positive. Peptides recognized by 3 or more CMA patient’s sera were considered as relevant allergenic epitopes.Bovine BLG exists in a range of naturally occurring genetic variants that differ from each other by a few amino acids (AA) substitutions. Two predominant genetic variants of BLG protein are known, A and B t-test. P-values of less than 0.05 were considered statistically significant.Comparisons were made with (two-tailed and paired) the Student’s Generation of chimeric human/mouse IgE and IgG1 anti-BLG antibody 8F7.1: proof of principle) was able to prevent the binding of mouse anti-BLG antibodies 5D6 and 11B6 on BLG (data not shown).IgE molecules bind to high affinity FcεRIs present on mast cells and basophils, and, through the interaction with a multivalent allergen, trigger release of vasoactive mediators. This process is only feasible when allergen-specific IgE antibodies bind to dissimilar epitopes on a monomeric allergen. However, even when this is the case, allergen-specific IgE antibodies against dissimilar epitopes could prevent their mutual binding to an allergen by sterical hindrance. Therefore, we aimed to generate a panel of mouse anti-BLG antibodies, which did not recognize identical epitopes on BLG and/or did not show any cross-blocking or overlapping occurrence. To this end, we performed a competitive BLG ELISA test. As shown in Hybridoma 8F7 was used to provide proof of principle of our chimerization strategy. For this, hybridoma 8F7 cells were subcloned by limiting dilution, which resulted in several hybridoma clones with stable production levels of mouse anti-BLG IgG antibodies. From these stable hybridoma clones, clone 8F7.1 was selected for cloning and sequencing of the VH and VL regions. After chimerization, the binding capacity of purified chimeric human IgE and human IgG1 antibody clone 8F7.1 against BLG was examined. As shown in Hybridomas 5D6, 7D5, 8C3, 11B6 and 13A5 were also subcloned by limiting dilution. Then, VH and VL regions from stable clones 5D6.1, 7D5.1, 8C3.3, 11B6.2 and 13A5.2 were determined. After chimerization, purity of these chimeric human anti-BLG IgE antibodies was examined as was their binding to bovine BLG (through mouse variable regions of chimeric antibodies) and to human FcεRI α-chain expressing RBL cells (through human Fcε heavy chain tail of chimeric huIgE antibodies). All chimeric human IgE  and humaNext, the binding of our purified chimeric human anti-BLG IgE antibodies against BLG was investigated. As shown in Altogether, our generated panel of non- and cross-blocking (against domains I-III) chimeric human IgE anti-BLG antibodies seemed to be of (i) high purity, and showed (ii) a proper heavy and light chain assembly into intact human IgE antibodies, while (iii) retaining their binding potential to native bovine BLG and to membrane-bound human FcεRI α chains.To investigate whether the BLG-specific chimeric IgE monoclonal antibodies were biological active, RBL-huFcεRI cells were sensitized with a serially-diluted (0.065–40 µg/ml) individual BLG-specific chimeric human IgE monoclonal antibodies, and then cross-linked with anti-human IgE. All individual BLG-specific chimeric IgE monoclonal antibodies induced degranulation with an optimum at approximately 1.0 µg/ml (data not shown). To assess the optimal concentration of our pool of six BLG-specific chimeric human IgE monoclonal antibodies , RBL-huFcεRI cells were sensitized with serially-diluted pool of chimeric human IgE antibodies, i.e., 0.008–8 µg of each human IgE/ml for anti-human IgE, BLG and whey cross-linking, and 0.03–1 µg of each human IgE/ml for milk powder cross-linking. Cross-linking with anti-human IgE showed a maximal degranulation of 94% at a concentration of 0.25 µg of each chimeric human IgE/ml, whereas cross-linking with BLG showed a maximal degranulation of 84% at a concentration of 1.0 µg of each chimeric human IgE/ml . For furTo assess the sensitivity of the RBL-huFcεRI assay with the pool of six BLG-specific chimeric IgE monoclonal antibodies , RBL-huFcεRI cells were sensitized with BLG-specific chimeric IgE monoclonal antibodies (1.0 µg of each human IgE/ml) and cross-linked with whey hydrolysates with different degrees of hydrolysis as determined by gel permeation chromatography (data not shown). The degranulation of the BLG-specific chimeric IgE monoclonal antibody pool without cross-linking was comparable to the spontaneous degranulation (min) of the RBL-huFcεRI cells alone both 3%; . Cross-lDirect comparison of BLG-specific chimeric IgE monoclonal antibodies with a serum pool of CMA patients was done with HA<5 kDa as a cross-linker. As shown in In order to assess whether the BLG-specific chimeric IgE monoclonal antibodies recognized similar regions or epitopes on BLG as IgEs from CMA allergic patients did, individual BLG-specific chimeric IgE monoclonal antibodies and 10 individual CMA sera were tested for their binding on 25 synthetic peptides spanning the BLG of the B whey protein variant and 6 peptides of the A whey protein variant. The BLG-derived peptides that were recognized by chimeric human IgEs and CMA-derived IgEs are listed in ad infinitum availability, and hence, will improve the reproducibility of the degranulation assay.Accumulating concerns about food safety have increased the attention for legislation around health claims on food products. These health claims need to be substantiated with experimental data, and for HA formulas, the elimination of potential allergic epitopes needs to be confirmed. For safety assessment of CM hydrolysates, a robust and reproducible functional assay for determination of (non)allergenicity is essential. Currently, degranulation of human FcεRI-transfected RBL-2H3 cells after sensitization with IgE from pooled serum from CMA patients and exposure to hydrolysates is being used. However, serum samples from CMA patients may contain intervening or cytotoxic substances, which make these sera less suitable for usage in this degranulation assay. In addition, limited availability and inter-lot differences  of these serum pools are structural standardization issues. In the current study, we have tackled these serum-related problems by using recombinant DNA technology, and developed six BLG-specific chimeric human IgE monoclonal antibodies covering relevant allergenic epitopes of BLG, which resulted in enhanced sensitivity and specificity of the degranulation assay. Due to the recombinant nature of our BLG-specific chimeric human IgE antibodies, the sensitizing IgE pool is characterized by identicalness and in vitro RBL degranulation assay combined with our pool of chimeric IgEs is one of many tests, which is used for assessment of residual allergenicity of whey hydrolysates .The results of the current study unambiguously demonstrated that sensitization of FcεRI expressing RBL cells with our well-defined non-cross-blocking and cross-blocking chimeric human IgE anti-BLG antibodies - designated to recognize (arbitrary) domain I, II, and III on BLG - induced degranulation after cross-linking with BLG. Furthermore, we provided evidence that these chimeric human IgE anti-BLG antibodies bound to a number of regions on BLG alike to those that are recognized by serum IgEs from individual CMA patients , which iCurrent chimeric human IgE antibodies specifically recognize BLG, which is the major allergen (≈65%) present in bovine whey. ALA is another allergen (≈25%) in bovine whey. Antibodies against ALA were also raised during our immunization with whey. These mouse anti-ALA antibodies could be used to generate chimeric human IgE anti-ALA antibodies, and subsequently implemented in the RBL degranulation assay. CM protein content consists of ≈20% whey and ≈80% casein. We first focused on whey proteins to test whey-based hydrolysates, but currently we are also developing chimeric human IgE antibodies against casein  to investigate (non)allergenicity of casein-based hydrolysates in the RBL degranulation assay.Theoretically, cross-linking of FcεRI/IgE complexes by monomeric allergens on mast cells or basophils is achieved by at least two IgE antibodies whose binding sites on an allergen are not overlapping, although homodimeric (including BLG) or even oligomeric allergens have been described to enhance allergenicity As mentioned above, dimeric allergens are suggested to increase allergenicity in vitro model to assess the safety of and to improve hydrolysed formula for infants diagnosed with CMA.As shown in previous experiments in which whey protein degradation was followed during the hydrolysis process, a concurrent degradation of BLG and ALA was shown


Xenopus homologue of Math3/NeuroM, that functions downstream of Ngn2 in the neurogenic cascade.Basic Helix Loop Helix (bHLH) proneural transcription factors are master regulators of neurogenesis that act at multiple stages in this process. We have previously demonstrated that multi-site phosphorylation of two members of the proneural protein family, Ngn2 and Ascl1, limits their ability to drive neuronal differentiation when cyclin-dependent kinase levels are high, as would be found in rapidly cycling cells. Here we investigate potential phospho-regulation of proneural protein NeuroD4 , the Xenopus embryo system, we show that NeuroD4 is expressed and phosphorylated during primary neurogenesis, and this phosphorylation limits its ability to drive neuronal differentiation. Phosphorylation of up to six serine/threonine-proline sites contributes additively to regulation of NeuroD4 proneural activity without altering neuronal subtype specification, and number rather than location of available phospho-sites is the key for limiting NeuroD4 activity. Mechanistically, a phospho-mutant NeuroD4 displays increased protein stability and enhanced chromatin binding relative to wild-type NeuroD4, resulting in transcriptional up-regulation of a range of target genes that further promote neuronal differentiation.Using the developing Multi-site phosphorylation on serine/threonine-proline pairs is a widely conserved mechanism of limiting proneural protein activity, where it is the number of phosphorylated sites, rather than their location that determines protein activity. Hence, multi-site phosphorylation is very well suited to allow co-ordination of proneural protein activity with the cellular proline-directed kinase environment.The online version of this article (doi:10.1186/s13064-015-0044-8) contains supplementary material, which is available to authorized users. Drosophila [Xenopus [Basic helix-loop-helix (bHLH) transcription factors are master regulators during development, acting in all three embryonic germ layers and establishing a complex network of interactions that direct cell fate towards specified lineages –4. Withiosophila  and thro[Xenopus , chick [[Xenopus , rodent [Xenopus . A dynam[Xenopus . Moreove[Xenopus –13.Xenopus embryos and mammalian cell models [Proneural protein activity may lie at the heart of mechanisms that allow cell cycle exit to be co-ordinated with differentiation. For instance, we have previously demonstrated that both Ngn2 and Ascl1 undergo multi-site phosphorylation on serine-proline sites, and this directly reduces their ability to drive neuronal differentiation under conditions of high cyclin dependent kinase (cdk) activity –16. Accol models , 16. ThiXenopus protein also known as Xath3, and homologue of mammalian Math3/NeuroM. Focussing on Xenopus primary neurogenesis, we show that NeuroD4 is expressed and phosphorylated at neural plate stages in the developing embryo, and this limits its ability to drive primary neurogenesis. Phosphorylation of up to six sites additively contributes to regulation of NeuroD4 proneural activity, without altering neuronal subtype specification. Mechanistically, we demonstrate that phospho-mutant NeuroD4 has both enhanced protein stability relative to wild-type NeuroD4 in vivo, and also increased chromatin binding at neural plate stages. Our findings indicate that cell-cycle mediated multi-site phosphorylation is a widely conserved mechanism of regulating proneural protein activity.Here, we investigate phospho-regulation of the proneural protein NeuroD4 , the XenNeuroD4 is grouped in the Atonal family, related to the Drosophila proneural gene atonal [Xenopus (genbank accession number NP 001081213.1) NeuroD4 homologues show not only the strong sequence conservation in the bHLH domain, but also sequence conservation in potential proline-directed kinase sites  NeuroD4 . We set Xenopus, NeuroD4 expression is first detected from stage 12 in the presumptive neural plate [Xenopus embryos. Extracts were prepared from stage 13 embryos injected with an HA-tagged wild type (WT) NeuroD4 mRNA, and western blot analysis shows the presence of a broad band of NeuroD4 protein  8.5 and then later in the developing brain and retina . Similaral plate , becominal plate . NeuroD4al plate . Firstlyein Fig. . ProteinXenopus embryos, and neural-β-tubulin expression was assayed at stage 18 , creating 6T/S-A NeuroD4 Fig. . In orde 18 Fig. -d. ConsiPreventing multi-site phosphorylation of Ngn2 and Ascl1 enhances the transcriptional activation of a number of downstream targets. However, not all targets show a similar level of up-regulation by the phospho-mutant proteins compared to their wild-type counterparts , 16. ForWT NeuroD4 over-expression results in a four-fold increase in both xMyt1 and xNeuroD1 expression, but does not significantly up-regulate p27Xic1 or xDelta at this level of overexpressed mRNA. In contrast, 6T/S-A NeuroD4 up-regulates all four transcripts relative to both uninjected controls and WT NeuroD4 Fig. . Of noteWe have previously demonstrated that the cdk inhibitor (cdki) p27Xic1 is required for primary neurogenesis, playing a dual role in slowing the cell cycle and independently promoting differentiation by stabilisation of the Ngn2 protein . HoweverXenopus, and in contrast to phospho-mutant Ngn2 [In summary, a phospho-mutant form of NeuroD4 displays substantially enhanced ability to drive neuronal differentiation in ant Ngn2 , shows aIn some bHLH proteins where phosphorylation of SP and/or TP sites has been shown to regulate protein activity, individual phosphorylation sites have particularly important regulatory significance, for example S200 in MyoD  or S147 Mutation of individual SP sites generates mutants with activity similar to WT NeuroD4, while mutation of individual TP sites results in a small increase in activity Fig. -d. HowevXenopus embryos as described above. All three paired mutants produce a similar six to seven fold increase in neural-β-tubulin expression that is significantly higher than that induced by WT NeuroD4, but still substantially lower than that induced by the full phospho-mutant 6T/S-A NeuroD4 , which can be distinguished by expression of xHox11L2 [The bilateral stripes of xHox11L2 , xVsx1 [xHox11L2 , and xHbxHox11L2 . All thrxHox11L2 , so thisxHox11L2 , 40 and xHox11L2 . This maXenopus embryos, and mutation of phosphorylation sites additively enhances the ability of NeuroD4 to drive neuronal differentiation. We then investigated the mechanisms by which 6T/S-A NeuroD4 may exert its increased neurogenic activity. We show above  acts downstream of Ngn2 in primary neurogenesis  and its Xenopus Ascl1 has no TP sites, so NeuroD4 is the first example of TPs playing a role in proline-directed multi-site phospho-regulation of proneural protein activity.Previous work investigating multi-site phospho-regulation of Ascl1 and Ngn2 indicates that the number of sites available for phosphorylation is a more important determinant of proneural protein activity than the exact location of those sites , 16. We SP and TP sites in NeuroD4 are clustered into three pairs of sites, two pairs of which are found within stretches of highly conserved residues. To see if SP/TP sites in these pairs are redundant to each other, we tested mutants where both members of each pair were mutated simultaneously. While NeuroD4 with the mutated pairs of sites show more activity than the wild-type protein or single SP/TP site mutants, each of the paired mutants results in similar enhancement of activity (approximately 1.5 to 2-fold compared to WT NeuroD4) and none show activity approaching the full phospho-mutant. Thus, mutation of single or pairs of SP/TP sites does not result in a NeuroD4 protein with the high level of activity of the full phospho-mutant. These findings support the hypothesis that it is the number of phospho-sites available and not phosphorylation at a particular location that controls proneural protein activity. To test this hypothesis further, we generated a phospho-mutant series where SP/TP sites were cumulatively mutated from the N-terminus towards the C-terminus of the protein. This cumulative phospho-mutant series can also be used to test the alternative of a threshold versus a rheostat model of phospho-regulation. Under a threshold model, mutation of a specific number of sites above a threshold would be enough to give full activation of NeuroD4. Such threshold regulation has been described for NFAT transcription factors . A rheosHow does phosphorylation control NeuroD4 activity? Direct downstream targets of NeuroD4 have not been systematically identified. However, xNeuroD1 and xMyT1 are clearly up-regulated by both Ngn2 and NeuroD4 in a neurogenic cascade , 23, 27 In contrast to Ngn2 targets that drive differentiation such as xNeuroD1 and xMyT1, we have seen that phospho-status of Ngn2 has very little effect on xDelta expression, which promotes cell non-autonomous progenitor maintenance via Notch signalling . We haveXenopus, p27Xic1 [Cdks can phosphorylate proneural proteins on SP/TP sites , 16. Our p27Xic1  is not tin vitro [in vivo compared to the wild-type protein. This reinforces our findings in vitro and in tissue culture cells showing greater association of phospho-mutant proneural proteins with E box-containing DNA [We have previously shown than un(der)phosphorylated Ngn2 shows enhanced DNA binding compared to its phosphorylated counterpart . Westernin vitro , but 9S-in vitro . Our datin vitro . As wellning DNA .Together with our previous work on Ngn2 and Ascl1 phospho-regulation, the results presented above strengthen and widens our model whereby un(der)phosphorylation of multiple proneural transcription factors enhances promoter dwell-time on their downstream target promoters. Enhanced dwell-time disproportionately promotes activation of targets that require extensive chromatin modification for transcription to occur, particularly genes associated with differentiation such as xNeuroD1 and xMyT1; proneural proteins can associate with chromatin modification enzymes such as histone acetyltransferases and swi/snf-family nucleosome remodellers, and underphosphorylated proneural proteins will recruit them more effectively to target promoters , 49. ConXenopus embryos. Homologues of NeuroD4 in other species show strong sequence conservation of potential proline-directed kinase sites (SP/TP). We have used the Xenopus embryo model to demonstrate that NeuroD4 proneural activity is regulated by phosphorylation on up to six SP/TP sites. A multiply phospho-mutant version of NeuroD4 is able to drive significantly enhanced neurogenesis compared to the wild-type protein, although the subtype of neurons generated is not altered. Extensive mutational analysis demonstrates that it is the number rather than location of available phospho-sites that is the critical factor for regulation of NeuroD4 activity. NeuroD4 dephosphorylation promotes neuronal differentiation through a combination of increased protein stability, increased chromatin binding, and consequent up-regulation of a range of target genes that also promote differentiation. Taken together with previous findings about regulation of the proneural proteins Ngn2 and Ascl1, we propose that a conserved rheostat-like mechanism of phospho-regulation enables proneural protein activity to control the balance between progenitor maintenance and differentiation in response to the cellular kinase environment.Here we show that proneural bHLH protein NeuroD4 is expressed and phosphorylated during primary neurogenesis in Xenopus laevis were housed, bred and treated according to the guidelines approved by the UK Home Office under the Animal (Scientific Procedures) Act 1986. All animal work has been carried out under UK Home Office Licence and has passed an Institutional ethical review committee assessment, undertaken by the Animal Welfare and Ethical Review Committee (AWERC) at the University of Cambridge.X. laevis wild-type (WT) NeuroD4 in pCS2+ (Genbank accession number NM 001087744) was a kind gift from Professor Shin-Ichi Ohnuna . For western blot detection, HA-tagged WT NeuroD4 in pCS2+ was generated by PCR. Primers 5′-GATCGAATCCACCATGTCAGAGATGGTCAATGTG-3′ and 5′- GATCCTCGAGTTAAGCGTAATCTGGAACATCGTATGGGTATTCAGTGAATATAGCATGGTGTG-3′ were used to amplify the coding region of NeuroD4, adding a single HA tag at the 3′ end before the stop codon, and introducing EcoR1 and Xho1 restriction enzyme sites to the extreme 5′ and 3′ ends respectively. This PCR product was digested with EcoR1 and Xho1 and ligated into a blank pCS2+ vector. All phospho-mutant constructs, including the HA-tagged 6T/S-A NeuroD4, were subsequently generated from either the untagged WT NeuroD4 or HA-tagged WT NeuroD4 constructs by site directed mutagenesis. Single or multiple site mutation were performed using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies) or the QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies) respectively, each according to manufacturer’s instructions and using the PCR primers as shown in Table X. laevis eggs were obtained by standard hormone methods of induction, and subsequently fertilised in vitro. pCS2+ plasmids containing the various NeuroD4 constructs were linearised with Not1 enzyme and capped mRNA was transcribed in vitro using the SP6 mMessage mMachine® kit (Ambion). Unless otherwise stated, two-cell-stage embryos were unilaterally injected into the animal pole with 100 pg NeuroD4 mRNA in a total volume of 10 nl, with co-injection of 500 pg GFP and β-gal mRNA to enable lineage tracing. Embryos were subsequently cultured at 16 °C in Ficoll solution  and staged according to the methods of Niewkoop and Faber [4, pH 7.5) for 90 min, washed twice in phosphate-buffered saline (PBS), followed by PBS supplemented with 2 mM MgCl2, and embryos were stained with 1 mg/ml X-gal  in X-gal mixer (5.35 mM K3Fe(CN)6, 5.35 mM K4Fe(CN)6, 1.2 mM MgCl2, 0.1 % sodium deoxycholate, 0.2 % NP-40 in PBS). Embryos were again washed twice in PBS and dehydrated in methanol prior to storage at −20 °C.nd Faber . At stagX. laevis neural-β-tubulin [X. laevis p27Xic1 [X. laevis xMyt1 [X. laevis xNeuroD1 ; linearised with BamH1 and transcribed from T7) and X. laevis xHox11L2 [2O2, 5 % formamide, 0.1 % SSC), prior to PBS washes and storage in MEMFA at room temperature. Embryos were scored for neurogenesis as described in Additional file Dig-oxigenin-labelled anti-sense probes were synthesised from the following plasmids: -tubulin ; X. laev p27Xic1 ; X. laevxHox11L2 . Whole mxHox11L2 , with soEmbryos for qRT-PCR analysis were co-injected with 500 pg mRNA encoding GFP, and GFP expression was subsequently used to confirm successful injection. Samples of four embryos were snap-frozen at stage 18 and whole embryo RNA was extracted using the Qiagen RNeasy® Mini Kit. Template cDNAs were synthesised using the QuantiTect® Reverse Transcription Kit (Qiagen), and qRT-PCR was performed using the Quantifast® SYBR Green PCR Kit (Qiagen) in a LightCycler® 480 (Roche). Thermal cycling conditions: 95 °C for 5 min, then 45 cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C for 20 s. Primer sequences were as shown in Table For detection of NeuroD4 phosphorylation, embryos were injected bilaterally at the two-cell stage with 300 pg WT NeuroD4-HA mRNA. For comparison of WT and 6T/S-A NeuroD4 protein stability, embryos were injected at the one cell stage with 200 pg of the respective HA-tagged mRNA. Samples of five embryos were snap-frozen on dry ice at stage 13 and subsequently lysed in 50ul of pre-chilled IP-buffer ). Lysed embryos were centrifuged at 4 °C for 5 min at 16 000 xg and the supernatant re-spun and final supernatant used as the protein extract. Protein was quantified in the extract using a BCA assay (Thermo Scientific) according to manufacturer’s instructions, and 30ug was subsequently used for Western blot analysis in a total volume of 10 ul. For detection of NeuroD4 phospho-status, samples were incubated at 30 °C for 1 h in the presence or absence of Lambda protein phosphatase (NEB). 3.5 ul of SDS-loading buffer containing reducing agent was then added to each sample and incubated at 70 °C for 10 min prior to brief 15 s centrifugation. Samples were loaded on to a pre-cast BioRad Criterion™ TGX™ 18 % gel using the BioRad precision plus protein dual colour standard as a ladder. The gel was run for 2 h at 200 mV in running buffer . Transfer was conducted at 100 mV for 45 min at 4 °C, using pre-chilled transfer buffer  and PVDF membrane activated in methanol. Blocking was performed for over 1 h in 4 % milk in TBS with 0.1 % tween and the membrane was cut at just below the 50KDa level to enable separate antibody incubation to detect either the NeuroD4 protein or Tubulin loading control. Primary antibodies were used at 1:2000 dilution in 2 % milk in TBS-T and applied for at least 1 h at room temperature or 4 °C overnight: rat HRP-conjugated anti-HA (clone 3 F10) antibody (Roche) and mouse anti-α-tubulin antibody (Sigma). Membranes were washed at least 5 times in TBS-T and anti-tubulin antibody was detected using an HRP-conjugated anti-mouse antibody at 1:10000 dilution in 2 % milk in TBS-T for 1 h at room temperature. Membranes were washed again at least 5 times prior to Amersham ECL detection reagent reaction  according to manufacturer’s instructions. Experiments were conducted in independent duplicate and representative images are shown. For protein quantification in Fig. Embryos were injected at the one cell stage with 200 pg HA-tagged mRNA as before. At stage 14, embryos were transfered to glass vials and rinsed 3 times with distilled water. Embryos were then incubated at room temperature for 30 min in 1 % formaldehyde in 0.1 % MBS solution, prior to quenching in 0.25x MBS containing 125 mM glycine for 30 min. Embryos were then washed twice for 30 min total in 0.25x MBS, and 30 embryos per condition were snap-frozen on dry ice. Nuclei were extracted using a modified protocol for chromatin isolation. Embryos were homogenised in 50ul of Buffer E1 ) and spun for 2 min at 3500 rpm at 4 °C. The supernatent was collected and retained as the cytoplasmic fraction, and 10 ul of this was subsequently used for Western Blot. The lipid residue was removed and the remaining pellet was resuspended in 1 ml of Buffer E1, followed by a repeat centrifugation step. The pellet was again resuspended in 1 ml of Buffer E1, and samples were rested on ice for 10 min prior to centrifugation. The resulting pellet was then resuspended in 50ul Buffer E2 ), and the process was repeated with 2 cycles of centrifugation and resuspension, followed by 10 min rest on ice before final centrifugation. The resulting pellet was resuspended in 250ul of Buffer E3 ), and this represents the chromatin fraction. 20ul of the chromatin fraction was then DNAse treated (Qiagen) at room temperature for 15 min and Western Blot analysis was conducted as described above using 10ul samples. A 4-20 % gel was used and antibodies were as above, with the additional use of Anti-Histone H3 antibody (Abcam) and anti-rabbit secondary antibody (Sigma). ImageJ software was again used to quantify protein bands and the density of WT or 6T/S-A NeuroD4 protein was calculated relative to tubulin protein or histone H3 protein for cytoplasmic and chromatin fractions respectively.Elongation Factor 1α (EF1α), and for analysis, mRNA levels in the injected embryos were calculated relative to stage-matched uninjected control embryos. Mean values and the standard error of the mean (s.e.m.) were calculated from at least three independent experiments (n = 3). Statistical significance was determined using a paired two-tailed Student’s t-test with (p < 0.05) = *; (p < 0.025) = **; (p < 0.0125) = ***. For in situ hybridisation data, embryos were scored for the extent and pattern of marker gene expression on the injected side of the embryo relative to the uninjected side and uninjected control embryos. Additional file p values are illustrated as described above.For qRT-PCR data, mRNA expression was normalised to expression of the housekeeping gene


The enzyme Rho kinase (ROK) inhibits MLCP, favouring vasoconstriction. We tested the hypothesis that ROK-dependent MLCP inhibition was attenuated in late sepsis and associated with reduced contractile responses to certain vasopressor agents.Sepsis is characterised by diminished vasopressor responsiveness. Vasoconstriction depends upon a balance: Ca+ assessed voltage-gated Ca2+ channels ), (ii) thromboxane A2 receptor activation assessed the activation state of the LTCC and ROK/MLCP axis, (iii) direct PKC activation (phorbol-dibutyrate (PDBu), 5 μM) assessed the PKC/CPI-17 axis independent of Ca2+ entry and (iv) α1-adrenoceptor stimulation with phenylephrine (10−8 to 10−4 M) and noradrenaline (10−8 to 10−4 M) assessed the sum of these pathways plus the role of the sarcoplasmic reticulum (SR). ROK-dependent MLCP activity was indexed by Western blot analysis of P[Thr855]MYPT. Parametric and non-parametric data were analysed using unpaired Student's t-tests and Mann-Whitney tests, respectively.This is a prospective, controlled animal study. Sixteen-week-old C57/BL6 mice received laparotomy or laparotomy with caecal ligation and puncture (CLP). Antibiotics, fluids and analgesia were provided before sacrifice on day 5. Vasoconstriction of the femoral arteries to a range of stimuli was assessed using myography: (i) depolarisation with 87 mM Kn = 6 to 7) and stimulated (n = 8 to 12) vessels from mice that had undergone CLP (p < 0.05). Vessels from CLP mice demonstrated reduced vasoconstriction to K+, thromboxane A2 receptor activation and PKC activation . α1-adrenergic responses were unchanged (n = 7 to 12).ROK-dependent inhibition of MLCP activity was attenuated in both unstimulated (+ depolarisation, thromboxane A2 receptor activation and PKC activation were diminished in vitro whilst α1-adrenergic responses remained intact. Inhibiting MLCP may present a novel therapeutic target to manage sepsis-induced vascular dysfunction.In a murine model of sepsis, ROK-dependent inhibition of MLCP activity in vessels from septic mice was reduced. Responses to K Septic shock arises when infection causes direct tissue injury and a maladaptive host response culminating in refractory vasodilation and loss of vasopressor sensitivity. Clinically, this manifests as septic shock and multi-organ failure, which even in modern intensive care units heralds a 90-day mortality rate of over 30% ,2.2+) binding to calmodulin. Cystosolic Ca2+ is derived from either the sarcoplasmic reticulum (SR) or the extracellular space through, for example, voltage-gated L-type Cav1.2 Ca2+ channels (LTCC). Vasoconstrictors variably modulate the cytosolic [Ca2+] and the degree of inhibition of MLCP to regulate vascular tone. MLCP can be inhibited either by (i) RhoA activation of Rho kinase (ROK), which inhibits MLCP by phosphorylating Thr855 of the MYPT regulatory subunit of MLCP [2+] - ‘Ca2+ sensitisation’. On the other hand, increasing MLCP activity by reducing Thr855 phosphorylation of MYPT or reduced Thr38 CPI-17 phosphorylation shifts the [Ca2+]:force relationship to the right - effectively reducing Ca2+ sensitivity. Individual vasopressors vasoconstrict through different signalling pathways; for instance, thromboxane is bimodal, activating LTCC and RhoA/ROK : force relationship [2+]  , suggest+ , suggehip [2+] ,14. Mechrestored ,16, as ilephrine . Howeverood flow , has not pathway ,19. Dimiood flow ,20.2+ desensitisation and that this activity would be associated with a reduction in in vitro vascular responses to vasoconstrictors such as thromboxane A2, phorbol ester and high K+ which are uni- or bimodal, engaging one or two signalling pathways. We hypothesised that responses to α1-adrenergic agonists which invoke multimodal mechanisms of Ca2+ entry (LTCC and SR Ca2+) and Ca2+ sensitisation (PKC/CPI-17 and Rho/ROK) would be relatively preserved in vitro.To address this gap in knowledge, we used a murine caecal ligation and puncture (CLP) model of sepsis that included an intensive care treatment regimen that simulated the clinical experience of a septic patient ,22. CLP ad libitum. Changes in clinical status were determined by cumulative clinical record scores as outlined previously MYPT, proteins were extracted from each 2-mm arterial segment using 100 μl of sample buffer containing 50 mM Tris-HCl, pH 6.8, 1% (v/v) Tween-20) containing either a mouse-derived affinity-purified anti-MYPT antibody  made in-house or a commercially available  rabbit-derived anti-P[Thr855]MYPT antibody  for 60 min. Nitrocellulose membranes were washed three times in TBS-T and incubated with a 1:10,000 dilution (in TBS-T) of biotin-conjugated secondary antibodies : anti-mouse IgG for MYPT and anti-rabbit IgG for P[Thr855]MYPT , for 60 min before another three washes with TBS-T. The nitrocellulose was then incubated for 60 min in TBS-T with a 1:10,000 dilution of streptavidin conjugated to the 800-nm DyLight fluorochome (Pierce Thermo Scientific); fluorescence was detected and quantified using the LI-COR Odyssey system. Western blot signals of the Thr855 phosphorylation state of MYPT of untreated vs. U46619 (0.1 μM)- or PDBu (5 μM)-treated rat caudal artery as previously published MYPT (less MLCP inhibition) in response to TxA2 receptor stimulation than sham-treated mice .Thromboxane activates the RhoA/ROK pathway leading to inhibitory phosphorylation of Thr855 MYPT, the regulatory subunit of MLCP, favouring contraction by Catisation . To esta2 elicits VSM contraction both through Rho kinase-mediated MLCP inhibition and by increasing the open probability of LTCC. If RhoA/ROK activity was disrupted during sepsis, decreasing [Thr855]MYPT phosphorylation, it would be expected that activation of TxA2 receptors would fail to fully restore MLCP inhibition. The consequence would be reduced Ca2+ sensitivity and a diminished contractile response to the TxA2 mimetic U46619. The contraction of isolated femoral artery segments in response to U46619 (0.1 μM) was attenuated in CLP compared to sham mice , consistent with the hypothesis that reduced MLCP inhibition contributes to septic shock.TxAn = 8; p < 0.05). As expected, treatment of artery segments from control mice with PDBu did not directly alter the Thr855 phosphorylation of MYPT (data not shown) cyt and a force:intracellular Ca2+ relationship shifted to the right, consistent with Ca2+ desensitisation cyt through SR Ca2+ release, a mechanism not shared by TxA2 receptor, PDBu activation or high-K+-mediated contraction   . In addierfusion .The role of RhoA/ROK-mediated inhibition of MLCP extends beyond vascular smooth muscle: thrombin, a key mediator of the cross-talk between coagulation and inflammation in sepsis has, been shown to inactivate MLCP and therefore contraction in a RhoA-dependent manner in human endothelial cells . Given tWe studied an animal model of sepsis at a single time point, and translating our results to patients mandates caution. To reflect human sepsis as closely as possible, we used a CLP model, which has advantages over bacterial inoculum and lipopolysaccharide models , incorpo2 and membrane depolarisation. Approaches aimed at directly inhibiting MLCP might prove to be an effective therapeutic strategy to counter the pathological vasodilatation of septic shock.Mechanisms of pathological vasodilation and vasopressor resistance in septic shock are diverse. Our observations in a murine caecal ligation and puncture model are consistent with the hypothesis that during sepsis disinhibition of myosin phosphatase promotes insensitivity to certain physiologically important vasoconstrictor mechanisms, such as thromboxane A


In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs) is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE) in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same linear consensus motif, and provide evidence suggesting IRS-1 S265 as a novel Akt site. KSR-LIVE is an open-access algorithm that allows users to dissect phosphorylation signaling within a specific biological context, with the potential to be included in the standard analysis workflow for studying temporal high-throughput signal transduction data. Cells use intricate signaling networks to monitor and respond to environmental cues and to appropriately regulate specialized biological functions such as differentiation, metabolism and proliferation. A significant portion of signal transduction is mediated via the posttranslational modification (PTM) of proteins. One of the most prevalent and acute PTMs is phosphorylation, particularly on Ser/Thr residues. Phosphorylation is mediated by protein kinases, each of which targets a specific subset of protein substrates. The specificity of these interactions is governed by a range of factors such as the structure of the kinase catalytic site, subcellular localization and the formation of regulatory scaffolds and adaptor proteins . This spThe study of cell signaling networks has been revolutionized by high throughput proteomics methods and analytical workflows, enabling collection, analysis and quantification of protein phosphorylation on a global scale  . CurrentA key development in identifying the relationships between kinases and their substrates was the recognition that short stretches of amino acid sequence (consensus sequence motifs) could be used to predict kinase-substrates . This hahttps://cran.r-project.org/package=ksrlive).Here, we developed an approach to enable automatic identification of biologically relevant KSRs–substrates that are phosphorylated by a kinase within the biological context of the experiment–using Large scale In Vivo temporal Experiments (KSR-LIVE). KSR-LIVE sources previously reported experimentally-derived KSR data from a comprehensive knowledgebase, and uses a clustering procedure on temporal data to filter for biologically relevant KSRs. KSR-LIVE can be easily integrated into standard bioinformatics workflows, and is available as an R package on CRAN  profile for the kinase. For some kinases multiple exclusive clusters could be identified and in those cases all exclusive clusters were included in the analysis. The substrates that form the CTA are listed in This two-step clustering procedure identifies the activation pattern for each kinase in an unbiased, automated fashion.As an example, we sourced substrates from our KSR knowledgebase for the kinase Akt, an intensely-studied kinase that is activated in response to insulin ,25. FromWe next expanded our analysis to explore substrates for other kinases in the Insulin Dataset. To be included in the KSR-LIVE analysis, kinases had to have more than 2 exclusive substrates; 23 kinases fulfilled these criteria, and using KSR-LIVE, we extracted CTAs for 9 of these 23 kinases , all of which are implicated in insulin signaling ,26–28. NAmong the kinases whose CTA profiles could be determined, their CTAs could be divided into three distinct groups based on their temporal responses . Akt andWe compared the identified biologically relevant substrates to a standard reference. We chose substrates for Akt and mTOR  that hadFurther, we investigated the sensitivity of KSR-LIVE to the number of time points sampled in the phosphoproteomics data by removing time points one at a time, and comparing the identified CTA substrates to the manually curated substrates . The KSRKinases are often regulated by phosphorylation so kinase phosphorylation is often used as a proxy for activity. Thus, we compared the kinase CTAs to the phosphorylation profiles of the kinases themselves . We expeIn contrast, it is known that Eef2k and Gsk3b are deactivated upon phosphorylation at S78/366 and S9, respectively –32. WithIn conclusion, we observed instances where the temporal relationship of phosphorylation of the kinase does not align precisely with the phosphorylation of its substrates, suggesting that kinase CTA profiles are a more suitable marker of kinase activity than phosphorylation of kinases themselves.We next applied KSR-LIVE to another recently published temporal phosphoproteomics dataset–a time-course of NGF stimulation in a human neuroblastoma cell line (SH-SY5Y) , hereaftKSR-LIVE was able to extract a CTA for Mapk1 (Erk2) as well as mTOR, and both were active after 10 min and remained active throughout the experiment . ActivatIn addition to revealing the dynamics of kinase activation, CTAs can also be used to train kinase substrate prediction algorithms, for example the ensemble algorithm described previously ,35. To eWe performed leave one out cross validation and achieved a specificity of 0.94+-0.06 for Akt predictions. A candidate substrate of interest was Irs1 S265, which contains an RxRxxS/T motif, and was reported to be an Rps6kb1 substrate , but basWe conclude that using high resolution temporal phosphoproteomics data, KSR-LIVE can dissect phosphorylation signaling within specific biological contexts. KSR-LIVE provides key information on dynamic kinase activation and downstream signaling, and enables the prediction of substrates and biological functions for kinases of interest.Applying KSR-LIVE to the Insulin Dataset we were able to identify CTAs for 9 kinases. Six of the kinases are activated/inhibited by phosphorylation which follows their CTA with the exception of Mapk1/3. Although the known activating sites on Mapk1/3 are phosphorylated its substrates follow with a time delay of 1 min. This may reflect additional steps in the regulation of this kinase–for instance, phosphorylation of Mapk occurs in the cytoplasm, triggering its nuclear import to target nuclear substrates .Interestingly, for the kinases that are inhibited by phosphorylation, the inverse relationship between kinase and substrate phosphorylation is not maintained for the whole time-course. For Eef2k, S78 phosphorylation decreases with the CTA. Indeed, pS78 coincides with reduced phosphorylation at S392/S396, which is found within the linker site and required for kinases to access S78 . HoweverWe calculated the accuracy of KSR-LIVE by comparing the substrates in the CTA of Akt and mTOR to manually curated gold standard substrates and found that it can achieve an accuracy of over 70% for both kinases offering a significant improvement over the accuracy of the KSR knowledgebase alone. Interestingly, the best accuracy was achieved when the peak of activation was the second time point (30 s for Akt and 5 min for mTOR). Thus, it is crucial to measure phosphorylation at the peak of activation. Studies where the time-course begins after this point will therefore likely miss early activation events, decreasing the accuracy in predicting substrates of early-activated kinases. To achieve highest accuracy it is crucial to study time points that center on the peak of activation of the kinases of interest.In addition to mapping kinase activity, the extracted kinase CTAs greatly facilitate the prediction of new substrates by providing an automatically generated training set which can be used by learning algorithms, eliminating the resource-intensive and curator-biased process of manually generating a positive training set. Where expert knowledge about substrates is available, manually curated substrates can be added to the automatically generated list, adding more power to the KSR prediction.The primary advantage of KSR-LIVE over KSR prediction methods using consensus motifs is the consideration of biological context, utilizing biologically relevant KSRs in the training set. This is achieved using time-series data, on the premise that kinase activity can be inferred from the phosphorylation of its substrates. The substrates are not phosphorylated with identical time profiles, due to numerous factors such as copy number, localization and additional regulatory mechanisms, but they may be clustered together to distinguish one kinase CTA from another Figs  and 2. FIn this context, network inference would be a powerful tool to uncover network topology. However, while the datasets used here possessed many time points and proteome coverage to enable clustering, we could not utilize Bayesian inference because the number of data points was insufficient (data not shown). While other studies have demonstrated this approach is able to elucidate signaling topology, only a small number of phosphorylation sites were considered in their networks –substantGeneralizing substrate profiles using clustering enables simple and efficient measurement of kinase CTAs Figs  and 3. Ohttps://cran.r-project.org/package=ksrlive).The KSR knowledgebase covers a significant portion of the kinome. As it expands, KSR-LIVE will not only be able to assess the activity of additional kinases, but shed more insight into their regulation. For instance, we identified a single cluster for each kinase in the Insulin Dataset, but knowledge of additional KSRs may reveal several substrate clusters for each kinase. Overall, KSR-LIVE has the potential to be included in the standard analysis workflow to study temporal high-throughput signal transduction data. This will further improve our understanding of complex diseases caused by dysregulated signaling, including cancer and type 2 diabetes. KSR-LIVE is publicly available as an R package (The information from four databases were combined into one integrated ‘KSR knowledgebase’. The databases used were: PhosphoSitePlus (retrieved 06/2014), PhosphoELM (release 9.0), PhosphoPOINT (04/2014) and Human Protein Reference Database (release 9). Data from human and mouse was used, and the mouse proteins were mapped to human proteins using the Inparanoid ortholog database (version 8.0). The integrated KSR knowledgebase consisted of 11,666 interactions between 396 kinases and 8,035 phosphosites on 2,431 proteins. Due to redundant UniProt IDs referring to the same protein, Blast (version 2.2.30) was used to map between the integrated database and phosphoproteomics data. The sequences of the proteins were downloaded from UniProt 10/2014.Phosphosites with no missing values in two or more replicates were used for clustering. The clustering consisted of two steps, first with only exclusive substrates and in a second step using all available kinase substrates. Both taken together resulted in the final kinase activity marker substrates. {Formatting Citation}et al.). In the Humphrey et al. data only substrates with multiple replicates were analyzed to assure reliability of the time profiles. The resulting tight clusters formed the core substrates for a kinase. In the second step, clustering was performed using all available substrates for a kinase. All tight clusters containing the original exclusive substrate clusters, were subsequently used to calculate the mean and standard deviation for the characteristic temporal activation profile for the kinase.In the first step, substrates exclusive to a kinase were extracted from the integrated KSR knowledgebase and clustered using tight clustering . The use2. Differentiation was induced at 100% confluence by addition of 250 nM dexamethasone, 350 nM insulin, 0.5 mM 3-isobutyl-1-methylxanthine and 400 nM biotin for 72 h. Cells were then incubated in media containing 350 nM insulin for a further 72 h, and refreshed with naïve media every 2 d after. Adipocytes were used between days 10–12 after initiation of differentiation.3T3-L1 fibroblasts were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) Foetal Bovine Serum (Life Technologies) and GlutaMAX (Life Technologies) at 37° C with 10% CO3T3-L1 adipocytes in12-well plates were washed twice with PBS and incubated with serum-free DMEM containing GlutaMax and 0.2% (w/v) BSA. After 1.5 h, cells were treated for 30 min with 10 μM MK2206 , 10 μM GDC-0068 , 100 nM rapamycin  or solvent control. Cells were then stimulated where indicated with 100 nM insulin (Sigma-Aldrich) for 20 min before being washed thrice with cold phosphate-buffered saline (PBS) on ice and lysed with 1% (w/v) sodium dodecylsulfate (SDS) in PBS. Lysates were sonicated and subjected to SDS-PAGE and Western blotting as described previously . AntibodS1 FigA) Overlap of Akt (left) and mTOR (right) training sets. B) Scatter plot of prediction scores using the KSR-LIVE training set (y-axis) and the manually curated training set (x-axis). KSR-LIVE training set is shown in blue, the manually curated training set in green and sites that are contained in both are shown in red. Dashed lines represent the top 50 prediction score threshold.(TIF)Click here for additional data file.S1 TableThis table lists all substrates that make up the characteristic temporal response of a kinase.(XLSX)Click here for additional data file.S2 TableTable of all sites and the kinase prediction score for Akt and Rps6kb1 as well as the sites used as training sets.(XLSX)Click here for additional data file.


MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X), a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel finding in HEK293T cells and then use Drosophila to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. In vitro studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80) and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in Drosophila in vivo, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2.Methyl-CpG binding protein 2 (MeCP2) is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire  MECP2 Duplication Syndrome, respectively, which are characterized by severe cognitive, language, and motor impairments. While mutations causing Rett Syndrome and related disorders have been identified across the entire length of the MECP2 locus . Bo. BoMECP2ding MN5 .Drosophila by expressing the truncated human MECP2 allele p.Arg294* (R294X), which accounts for about 5% of typical Rett patients [R294X, a phenomenon not observed with expression of full-length MECP2 (MECP2FL) or MBD mutated MECP2. While we have previously found that expression of MECP2FL reduces MN5 dendritic length and branching, somata are not affected and have normal membrane currents [MECP2FL expressing flies   . To dete 0.0001) .Drosophila caspases will cleave the human PARP protein tethered to a membrane bound Venus tag, and caspase activity can be visualized using an antibody specific to human cleaved PARP (cPARP). Following expression of R294X we detected caspase activity throughout residual degenerating arborizations of MN5 even after the somata had already disappeared at early adult stages  but no difference compared to R294X alone .Between P10 and eclosion, cell death patterning and timing is variable between flies, but no intact cell bodies were found beyond one day post-eclosion in over 30 flies analyzed. We further determined that the effects of R294X exert a dominant negative effect over full-length, intact MeCP2, as concomitant expression of 4X alone . AnalysiMECP2 has been shown to enhance apoptosis in various cell lines and in primary neuron cultures [MECP2 expression, we transfected HEK293T cells with GFP-tagged R294X, MeCP2FL, or control vector. Consistent with previously published data [MECP2FL increased the number of apoptotic cells as compared to GFP-transfected controls  is missing in the R294X allele, this mutation also disrupts the TRD by deleting the last 16 amino acids. Additionally, a proline-rich WW domain binding region (WWBR) has been identified spanning much of the CTD, from AA 325–486 [Val312*/V312X (with intact TRD but truncated CTD), p.Lys431*/K431X , and p.Val312*/V481X (truncation eliminating the last five amino acids of the WWBR). New flies, including also new MECP2FL and R294X transgenes, were made using PhiC31 mediated site specific integration into the Attp2 landing site on chromosome III [Upon establishing validity of the  325–486 . While t 325–486 . In addi 325–486 , 58, cor 325–486 . Both ph 325–486 , which a 325–486 . To testsome III  to elimiR294X, V312X and K431X  as UAS-transgene. Expression of MECP2FLS80E increased cPARP reactivity, indicative of higher caspase activity, in adult MN5s at one day post-eclosion compared to normal MECP2FL . Thus, while MeCP2 S80 phosphorylation likely contributes to apoptosis, or at least caspase activation, C-terminal truncations cause apoptosis independent of S80 phosphorylation status , and more often both, MN5 somata at 1–2 days post eclosion (67% of preparations). However, even in flies with both intact MN5s cPARP immunoreactivity revealed high caspase activity throughout the somata and neuronal arborizations in either one or both MN5s at this time point  also increases caspase activity in Drosophila motoneurons. However, we further find that C-terminal truncations cause apoptosis independently of S80 phosphorylation status. This suggests that the effects of S80 phosphorylation may be upstream to the consequences of C-terminal truncation. This is consistent with a recent report on the role of the CTD in microRNA processing [In support of the latter, previous th MeCP2 . This isocessing . This stocessing .MECP2 overexpression in primary neuron culture [Drosophila slp1) to MeCP2, which in turn acts to suppress MeCP2 neurotoxicity. However, further experiments are needed to test whether internal MeCP2 N-C-terminal interactions promote cell survival.Our data are consistent with a model in which activity induced de-phosphorylation at S80 facilitates MeCP2 N-C terminal interactions, which in turn mediate a “healthy” conformation of MeCP2 promoting cell survival. Upon S80 phosphorylation, which is promoted in the absence of activity , N-C int culture . Thus, iR294X at, near, or beyond the end of the TRD, are seen with high frequency in Rett patients [R294X in vitro Click here for additional data file.S2 FigDrosophila MN5s  expressing MECP2FL or C-terminal truncated alleles. Transgenic flies were generated using phiC31 site-specific integration into the attp2 landing site to control for possible positional effects on transgene expression. MeCP2 expression was confirmed by immunohistochemistry with an antibody towards the N-terminus of human MeCP2. Nuclear location and localization of MeCP2 was confirmed by comparison with numerous images with nuclear labeling collected previously, including those previously published [MECP2FL or the K431X truncation. No significant differences were detected (Student’s t-test). N = 4/group, each N consists of ten pooled fly heads. Scale bar depicts 10 μm. Error bars show mean +/- SEM.a. Representative images of ublished . b. Repr(TIF)Click here for additional data file.S3 FigFOXG1 (human and mouse) with Drosophila ortholog slp1 shows high conservation in this sequence. The MeCP2 binding region (234–254 of mouse Foxg1) as determined by (2) is outlined in black.Alignment of the DNA binding domain of (TIF)Click here for additional data file.


This paper presents the development of the capacitive sensor to measure the growth of ice on a fuel pipe surface in real time. The ice sensor consists of pairs of electrodes to detect the change in capacitance and a thermocouple temperature sensor to examine the ice formation situation. In addition, an environmental chamber was specially designed to control the humidity and temperature to simulate the ice formation conditions. From the humidity, a water film is formed on the ice sensor, which results in an increase in capacitance. Ice nucleation occurs, followed by the rapid formation of frost ice that decreases the capacitance suddenly. The capacitance is saturated. The developed ice sensor explains the ice growth providing information about the icing temperature in real time. The monitoring of ice growth is an important issue in aviation and transportation systems because ice can cause severe accidents. For instance an emergency landing of a British Airways Boeing 777 occurred in Gatwick airport on 17 January 2008. It was revealed that thick layers of ice originated from dissolved water in the fuel had built up within the fuel pipes as the aircraft passed over Siberia. As the aircraft approached the airport, this ice was released as a so-called “snow ball” which blocked the fuel flow at the fuel-cooled oil-cooler leading to a sudden loss of power. The problem occurred in the engines when the aircraft was close to the airport and it was unable to reach the runway. It crash-landed just short of the runway—thankfully without any casualties [In a sub-zero temperature environment, the presence or formation of ice must be detected in real time. This will allow the pilot or driver to take appropriate action. However, there are no available ice detectors for directly detecting the thickness of ice within fuel pipes rather than just the presence of ice. This paper presents a cost-effective and small size capacitive ice sensor. It is also challenging to measure ice growth in real time. A specially designed environmental test system to simulate the sub-zero ice conditions is also presented.d, the distance between two confronting electrodes and N, the number of electrode pairs [−12 F/m, and the relative static permittivity for water is 80 and for the kerosene jet fuel it is 2.0 at 273 K and 4.2 for ice at 263 K in an input frequency of 105 Hz [The capacitive ice sensor uses interdigitated comb electrodes, which have been widely used for electrostatic actuators , accelerde pairs . The dieAs the ice grows, jet fuel in the medium surrounding the confronting electrodes is replaced with ice. In Equation (1), the relative static permittivity of the jet fuel is changed to that of the ice. As a result, the ice sensor detects in real time the change in capacitance, which is proportional to the increase in ice thickness.The ice sensor consists of paired electrodes on the front surface to measure the ice growth by using the capacitance change and a K-type thermocouple temperature sensor on the back surface to measure the temperature at which the ice forms. To examine the effect of the substrate on the ice growth, Pyrex 7740 glass with a low thermal conductivity of 1.05 W/mK and a silicon wafer with a high thermal conductivity of 149 W/mK were compared.Three masks were designed for fabrication. Firstly, the interdigitated comb-type electrodes for the capacitive sensor were made directly on the front surface by a standard image reversal process as shown in Secondly, to fabricate the K-type thermocouple temperature sensor, Alumel was patterned, followed by patterning of Chromel . Both maA feasibility test of the fabricated ice sensor was performed in water. The ice sensor was placed in a reservoir with a scale. As deionized water was poured into the reservoir and the level of water raised linearly from 0 to 6 mm with an increment of 1 mm, the capacitance change was measured for an input frequency of 100 kHz and a voltage of 1000 mV at an ambient temperature of 22 °C.Also, the fabricated K-type thermocouple temperature sensor was calibrated by comparing with a reference K-type thermocouple temperature sensor. In a thermal cycler, the environmental temperature was set from −40 °C to 50 °C, analogous to the temperature that an airplane might experience. As shown in In the real engineering situation, the water is dissolved in the jet fuel and the ice nucleates on the inner surface of the fuel pipe. However, to examine the ice growth in the fuel pipe, the test environment requires an expensive and complicated setup for the accurate control of the amount of water in the fuel and temperature. Thus, the measurement was performed in air for the experimental evaluation.To nucleate the ice, humidity was used as a water source. Firstly, a commercial environmental chamber was tested to control the temperature and humidity. However, as soon as the ice started to form, the humidity changed in the chamber. Thus, for the feasibility test of the ice growth measurement in real time we specially designed an environmental test system, which can control temperature and humidity separately and simultaneously. In the experiment, the humidity was set to a targeted humidity as a water source to form ice on the sensor and then liquid nitrogen was flown to the cold plate to simulate an ice formation temperature of below 0 degrees. As the ice formed, the capacitance and the temperature of the ice formation were measured simultaneously.To control the temperature of the cold plate from ambient to −40 °C for the ice growth, liquid nitrogen  with a boiling temperature of −195.79 °C at atmospheric pressure was used. The LN2 was filled in the reservoir with a volume of 1000 mL. The reservoir was connected with a sudden expansion-sudden contraction tube and the flow rate could be controlled by regulating the valve. The center of the extended part in the tube was machined to insert the ice sensor. The valve opening allowed maintaining the temperature at −45 °C for 30 min when the reservoir was fully charged. The humidity must be constant while the ice grows. It is very difficult to use ambient humidity as an ice nucleation source, since it always changes. In order to obtain a constant relative humidity, a humidifier and a fan were used to introduce microdroplets into the test section. Here, the humidity was controlled by regulating the amount of microdroplets for the constant fan speed. Finally, real time measurement of the ice growth was performed. The capacitive ice sensor was packaged with external electrodes, as shown in f is the input frequency and cX is the capacitance reactance.The capacitance was measured for an AC input frequency using an LCR-meter. In the capacitance measurement, the relative permittivity depends on the temperature and the input frequency :(2)C=12In order to eliminate their effect on the capacitance measurement, various input frequencies were tested. To prevent ice formation on the capacitive ice sensor at subzero temperatures, it was wrapped with a plastic bag and charged with nitrogen gas to remove humidity. As reported in previous work ,10, at tFinally, the ice growth was measured using a capacitive ice sensor on the glass substrate. After the ice sensor was installed in the cold plate, the LN2 valve was opened. When the temperature approached −45 °C, the humidifier and fan were turned on. The capacitance and temperature were measured from the sensor simultaneously over time.As shown in The first increase in the capacitance could be explained by the formation of a water film on the ice sensor. The water droplets generated by the humidifier attached on the sensor surface and accumulated to form a liquid film by hydrophilicity. The air on the ice sensor was replaced by the liquid, resulting in an increase in the capacitance because the relative static permittivity of water is 80 times higher than that of air. More water droplets attached and the water thickness grew with time. As a result, the capacitance rose.The sudden decrease in the capacitance could be explained by the formation of ice. The nucleation of ice started to occur and the frost ice grew quickly. Thus, the capacitance in the graph dropped sharply. Then, there was a general descent over time and temperature. The fluctuating decrease reflected the combination of the formation of frost and the attachment of water droplets.The measured capacitance was saturated under the condition of a continuous supply of water droplets. It is interesting to examine the variation of saturation temperature with respect to environmental humidity. Different relative humidities of 30%, 35%, 44%, 52%, and 60% were tested, as shown in The second increase in the capacitance in the graph could again be explained by water film formation. The frost ice was melted by an air blower. The melted water formed a film on the ice sensor and, as a result, the capacitance increased.The fabricated capacitive ice sensor measured the ice growth in real time. The measured capacitance provided meaningful information to indicate the existence of water in jet fuel and to alarm about possible ice growth. However, it could not simulate the ice growth from the fuel pipe surface because the frost ice did not grow only from the surface of the cold plate but also from the sensor surface. To improve this, the formation of the water film on the sensor surface must be prevented by using a hydrophobic treatment.A measurement of the growth of ice thickness in real time was successfully performed using a capacitive ice sensor. Especially, the measured capacitance could explain the situation of ice formation. Contrary to expectations, as the water film on the sensor surface froze, the capacitance decreased and became saturated at different levels depending on the concentration of the water supply.To simulate ice formation in jet fuel, not only the test environment but also the ice sensor should be improved to allow the ice growth only from the surface of the jet fuel pipe. One possible suggestion is to treat the surface of the ice sensor with a hydrophobic coating to prevent the attachment of water.


Objectives: Herbal medicine is known as a valid alternative treatment. Salvia Reuterana, which has been used in the Iranian traditional medicine, is mostly distributed in the central highlands of Iran. Salvia Reuterana is a medicinal herb with various therapeutic usages. The aim of the present review is to take account of pharmacological properties of Salvia Reuterana. Materials and Methods: The present review summarizes the literature with respect to various pharmacological properties of Salvia Reuterana. Results:Salvia Reuterana possesses neurological, antimicrobial, antioxidant, chemotherapeutic, and antidiabetic properties. Conclusions: Salvia Reuterana can be used as an alternative for treatment of several disorders. In the recent years we have witnessed rapidly increasing advances in the field of herbal medicine. World health organization (WHO) has reported that more than 80% of the world's population takes advantages of traditional medicine as their primary therapeutic needs on the grounds of their natural origin and low side effects. In the long history of the use of herbal medicines, human interactions with the environment have been thought to be a key element and it appears that systematic screening of medicinal herbs may discover novel effective compounds  includes 58 species, of which 17 are known to be endemic. The name Salvia also refers to the Latin word Salvare, which means healer. Species of Salvia have traditionally been utilized in folk medicine to preclude stomach ailments and the common cold  have been used as hypoglycemic, tonic, antimicrobial, anti-inflammatory, and anti-anxiety herbal drugs, respectively -beta-ocimene (32.3%), alpha-gurjunene (14.1%), germacrened-D (11.2%), and hexyl acetate (7.6%) were the major constituents  was much lower than those exerted by diazepam, elsewhere . Main drawbacks of anxiety drug therapy include co-morbid psychiatric disorders and increase in the medication dose, giving rise to deleterious side effects. Recently, there has been an increasing interest in assessing plants employed for treatment of sleep disorders and related diseases in traditional and alternative systems of medicine . Broadly speaking, it is difficult to ignore plant extracts and essential oils as potentially useful sources of antimicrobial compounds. The crude herbal extracts of aromatic plants have traditionally been used for food preservation and also as antimicrobials , Raji (Burkitt's lymphoma), Fen (bladder carcinoma), K562 (myelogenous leukemia), and Jurkat (T cell leukemia), which were obtained from Iranian cell bank, was investigated by Amirghofran et al.    and antioxidant defense mechanisms, and gives rise to potential tissue dysfunction and damage. ROS are produced in the early and advanced stages of glycation. The glycation phenomenon is attributed to the nonenzymatic and non-oxidative covalent linkage of glucose molecule to protein. It was demonstrated that non-enzymatic protein glycation initially occurs by the creation of a Schiff base between amino groups of the protein chain and the aldehyde (or keto) groups of the sugars. Afterwards, the Schiff base enters a number of reactions to produce fluorescent brown pigments that is called advanced glycation products  up to 53.3% with a concentration of 50 µg/ml as the same concentration of aminoguanidine (AG) that leaded to 60.4% inhibition, using different in vitro glycation models . It was shown that an increase in graded concentrations of the S. Reuterana extract diminished α-amylase activity from 1.19±0.45 to 25.01±1.68%. Nevertheless, the inhibitory activity of S. Reuterana on α-amylase was weak and failed to reach the 50% inhibition level of enzyme activity .α-Amylase refers to one of the enzymes contributing to catalyzing the breakdown of starch to maltose and subsequently to glucose, the only sugar able to be consumed by the body. The inhibition of such enzymes diminishes blood glucose level. Herbs and their constitutions have recently attracted much attention because they can abate diabetes, and a host of researchers has worked on hypoglycemic agents from medicinal plants. The evidence from the published literature suggests that polyphenols and flavonoids are among natural active antidiabetic compounds. It has been demonstrated that such compounds possess various biological properties, involving carbohydrate hydrolyzing enzyme inhibition and antioxidant activity. Nickavar et al. studied α-Amylase inhibitory activities of ethanol extracts derived from six Iranian Effect of S. Reuterana aerial parts on serum parameters in normal and streptozotocin-induced diabetic ratsSalvia Reuterana upon serum parameters , and alanine aminotransferase (ALT)) in normal and diabetic rats . Pharmacological studies have thus far confirmed its antibacterial, antifungal, carminative, antianxiety, and antioxidant properties. Neurological, antimicrobial, chemotherapeutic, antioxidant, α-amylase inhibitory, and antidiabetic properties of S. Reuterana extract have been demonstrated by recent studies (ref). The anxiolytic mechanism affects GABA receptors, while the mechanism of inhibition of acetylcholinesterase contributes to its sedative effects (ref). Methanolic extract of S. Reuterana also causes a significant decline in protein carbonyl formation and increase in protein thiol levels and protected structural changes of BSA  from glycation processes and this may be involved in such antioxidative activity (ref). Little, however, is known about the active component and mechanism of the antimicrobial, chemotherapic, α-amylase inhibitory, and serum parameters’ lowering properties of S. Reuterana. Thus, further research regarding S. Reuterana would be of great value in discovering such unknowns.Medicinal plants are utilized by approximately 80% of the population of the world (ref). In spite of successes of synthetic drugs, they have a number of adverse effects. Natural remedies are becoming popular alternatives owing to fewer side effects (ref). Salvia is one of the largest genuses of the Lamiaceae including roughly 900 species throughout the world.