Protein tyrosine phosphatase (PTP) 1B, a negative regulator of the insulin and leptin signaling pathways, is currently considered a promising target for the development of novel therapeutic approaches used to treat insulin-resistant type 2 diabetes mellitus (IR-T2DM). In this study, we examined the PTP1B inhibitory activity of 147 Japanese prescription Kampo formulations to evaluate their potential for clinical application in IR-T2DM treatment.We specifically defined the prescribed daily dose as 1 Unit (U), and 147 Japanese prescription Kampo formulations were screened for PTP1B inhibitory activity at a final concentration of 0.1 mU/mL. We investigated the dependence of the inhibitory activity on the concentration of the Kampo formulations that exhibited high PTP1B inhibitory activity. Their inhibition mode by kinetic analysis, inhibitory selectivities against four homologous PTPs  and cellular activity in the insulin-signaling pathway by increasing the insulin-stimulated Akt phosphorylation level in human hepatocellular liver carcinoma HepG2 cells, were also investigated. The statistical partial least squares regression method was used to identify the crude drugs with the greatest contribution to the PTP1B inhibitory activity of the Kampo formulations.Rhei Rhizoma provided the greatest contribution to the PTP1B inhibitory activity of these Kampo formulations.Daiokanzoto, Masiningan, Tokakujokito, Keimakakuhanto and Choijokito exhibited high PTP1B inhibitory activity, which was concentration-dependent. Daiokanzoto, Masiningan and Tokakujokito inhibited PTP1B by mixed inhibition modes and exhibited different inhibitory selectivities against four homologous PTPs. Masiningan also exhibited cellular activity. Statistical analyses indicated that the constituent crude drug High PTP1B inhibitory activity was predominantly associated with formulations that were classified as Jyokito in Kampo medicine and with a modern clinical indication of constipation. Currently, there is no clinical treatment for IR-T2DM that uses a mechanism of action based on PTP1B inhibition. Thus, we propose the Kampo formulations identified in this study as strong PTP1B inhibitors, which could be developed as clinical therapeutic agents to treat IR-T2DM. The incidence of type 2 diabetes mellitus (T2DM) is increasing worldwide . A majorin vivo to increase insulin and leptin activity and results in normalized blood glucose levels and reduced adiposity [PTP1B dephosphorylates the insulin receptor and leptin receptor linked enzyme Janus kinase 2. It is an intracellular phosphatase that is localized on the cytoplasmic surface of the endoplasmic reticulum and is ubiquitously expressed in insulin target tissues, such as liver, muscle and adipose tissues . PTP1B idiposity ,11. In adiposity . PTP1B iin vivo study [A total of 148 prescription Kampo formulations consisting of 185 crude drugs are currently covered by Japan’s national health insurance. There are 147 types of oral formulations among 148 prescription Kampo formulations. These Kampo formulations are in clinical use for a variety of diseases, which are primarily based on traditional clinical theories. However, due to the elucidation of the mechanisms of action of Kampo formulations in recent years, evidence-based clinical applications are starting to be developed, as exemplified by Daikenchuto . Kampo fvo study .In this study, we report the results of an examination of the PTP1B inhibitory activity of 147 Japanese prescription Kampo formulations to evaluate their potential for clinical application for the treatment of IR-T2DM.Rhei Rhizoma and Cannabisi Fructus were purchased from Tochimoto Tenkaido Co., Ltd. . PTP1B , T-cell protein tyrosine phosphatase (TCPTP)  and Vaccinia H1-related phosphatase (VHR)  were purchased from Enzo Life Sciences, Inc. , and ursolic acid (purity: 98.5%), sodium orthovanadate (purity: 90%), citrate buffer solution (pH 6.0), p-nitrophenyl phosphate (p-NPP), bovine serum albumin, Src homology domain 2-Containing Protein Tyrosine Phosphatase 1 (SHP-1) and Src homology domain 2-Containing Protein Tyrosine Phosphatase 2 (SHP-2) were obtained from Sigma-Aldrich Co., LLC. . Sodium chloride (NaCl), dithiothreitol (DTT), sodium hydroxide (NaOH), tris (hydroxymethyl) aminomethane, polyoxyethylene (23) lauryl ether and insulin  were purchased from Wako Pure Chemical Industries, Ltd. , and ethylenediaminetetraacetic acid (EDTA) was obtained from Dojindo Co., Ltd. . Human hepatocellular carcinoma cell lines were purchased from pG2 Japanese Cancer Research Resources Bank/Health Science Research Resources Bank Co., Ltd. , and fetal bovine serum was purchased from SAFC Biosciences, Inc. . Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Nissui Co., Ltd. . p-Akt1/2/3 (Ser 473) and Akt1/2/3 (H-136) antibodies were purchased from Santa Cruz biotechnology, Inc. . Goat anti-rabbit IgG-heavy and light chain antibody was purchased from Bethyl Laboratories, Inc. . Hybond-P PVDF membrane and ECL-plus were purchased from GE Healthcare Life-Science Co., Ltd. . PVDF Blocking Reagent, Can Get Signal Solution-I and Can Get Signal Solution-II were purchased from Toyobo Co., Ltd. . Anti-rabbit IgG-h + l goat antibody was purchased from Bethyl laboratories Inc. .This study examined 147 prescription Kampo formulations, which represent the entire panel of oral Kampo formulations covered by Japan’s national health insurance. These formulations were manufactured by Tsumura & Co. ; Kotaro Pharmaceutical Co., Ltd. ; Ohsugi Pharmaceutical Co., Ltd. ; Kracie Holdings, Ltd. ; Sanwa Shoyaku Co., Ltd. ; Taikoseido Pharmaceutical Co., Ltd. ; or Toyo-Kampo Pharmaceutical Co., Ltd. . Each Kampo formulation was dissolved at 1/1,000 of the daily dose (1 Unit), as indicated on the package insert, in 1 mL of purified water, and extracted by sonication at room temperature for 15 min. The mixture was then centrifuged at 12,000 rpm for 15 min, and the supernatant or supernatant diluent (in purified water) was used as the sample for the PTP1B inhibitory activity assay.Rhei Rhizoma or Cannabisi Fructus (60 g) was dissolved in distilled water  and decocted until the volume was reduced by half. The extract solutions were freeze-dried to obtain samples of the crude drug.The crude drug of p-NPP as the substrate. Next, 40-μL solution of PTP1B or VHR was dissolved in a buffer solution consisting of 0.06 M citric acid (pH 6.0), 0.1 M NaCl, 1 mM EDTA and 1 mM DTT. For TCPTP, SHP-1 and SHP-2, assay buffer (PH 7.0) was prepared using 25 mM Tris/HCl, 50 mM NaCl, 2 mM ethylenediaminetetraacetic acid, 5 mM dithiothreitol, 0.01% Brij35 and 1 mg/mL bovine serum albumin; 50 μL of 2 mM p-NPP for PTP1B, TCPTP, VHR and SHP-2; and 16 mM for SHP-1; and 10 μL of the sample or the sample diluent were added to a 96-well plate . After incubation at 37°C for 30 min, the reaction was terminated by adding 20 μL of 10 M NaOH. The reaction mixture was shaken on a microplate mixer for 30 s, and the amount of p-nitrophenol produced was determined by measuring the absorbance at 405 nm. The nonenzymatic hydrolysis of 2 mM p-NPP was corrected by measuring the increase in absorbance at 405 nm, which was obtained in the absence of PTP1B. In this study, ursolic acid, a known PTP1B inhibitor, was used as the positive control and exhibited an IC50 (50% inhibition concentration) of 4.3 μM, which was consistent with a previous report [The PTP1B inhibitory activity was measured using p-nitrophenol production measured as an increase in absorbance (ΔA) at 405 nm of the sample solution over the initial 30 min, using the following formula:The inhibition rate of PTP1B activity of each sample was determined on the basis of the 50 using linear regression analysis.An inhibition rate of 90% or higher was considered to indicate complete inhibition. The dependence of the inhibitory activity on concentration was investigated in Kampo samples that exhibited a complete inhibition over a final concentration range from 0.1 mUnit/mL to 0.001 mUnit/mL. In addition, the crude drugs were investigated in the concentration range from 0.5 μg/mL to 100 μg/mL. The inhibition rate at each concentration was then used to calculate the ICp-NPP . The initial reaction velocities were measured in the presence (the concentration from 2 to 7 μUnit/mL) and absence of the inhibitor [To elucidate the inhibition mode, the inhibition kinetics of high PTP1B inhibitory Kampo samples  were analyzed using the Lineweaver–Burk method with various substrate concentrations of nhibitor .2 at 37°C. The cells were incubated with the samples  from 0 to 50 mU/mL for three days. The number of living cells was determined using the Premix WST-1 Cell Proliferation Assay System , according to the manufacturer’s instructions.HepG2 cells was cultured in DMEM, supplemented with 10% heat-inactivated fetal bovine serum in a humidified atmosphere containing 5% CO2. After pretreatment with each compound at 37°C for 60 min, HepG2 cells  in a 48-well plate were stimulated with 50 nmol/L insulin for 5 min at 37°C. After washing with cold phosphate buffered saline, the cells were lysed using sonication. The lysate was centrifuged at 15,000 rpm for 15 min, and the supernatant was separated on 12% SDS-PAGE. After transfer to a Hybond-P PVDF membrane, the membrane was blocked with PVDF Blocking Reagent at 4°C overnight and was probed with 0.4 μg/mL anti-AKT1/2/3 rabbit polyclonal antibody or anti-phosphorylated AKT1/2/3 rabbit polyclonal antibody in Can Get Signal Solution-I for 1 h at room temperature, followed by further incubation with 0.025 μg/mL HRP-labeled anti-rabbit IgG-h + l goat antibody in Can Get Signal Solution-II for 1 h at room temperature. The membrane was then washed three times with phosphate buffered saline-T, incubated with ECL-plus for 5 min at room temperature and analyzed using a Typhoon 9410. Sodium orthovanadate was used as the positive control [HepG2 cells were cultured in DMEM, supplemented with 10% heat-inactivated fetal bovine serum, at 37°C in a humidified atmosphere containing 5% CO control .Xm) and the PTP1B inhibitory activity of the Kampo formulations as the response variable (Y), we generated a regression formula to compare the contributions from individual constituent crude drugs in this study, as follows:The partial least squares (PLS) regression method  was usedY is the response variable, and X is the regressor variable factor. The regression coefficient is a, and its components are expressed as aj (j = 1 2 3…m).where m is the number of factors, n is the number of samples, tk as the explanatory variable by the following formula:In the PLS regression model, regression was performed using the latent variable qk is the coefficient for the kth component in Y, pk is the kth weight vector in X, A is the number of latent variables for PLS, e is the difference of Y, and E is the difference of X. Here, T represents a transposed matrix. The number of latent variables for PLS, A, was determined using the cross-validation method. Cross-validation was performed as described below. The n − 1 set consisting of n data subtracted from 1 set was used to estimate the model parameters. The predictive value Ŷ for the response Y was obtained for the 1 set that was subtracted. The same procedure was performed for each of n sets, and the prediction error was computed using the index of the following formula:where PRESS) was minimized. In this study, statistical analysis was performed using EXCEL Multivariate Analysis Ver. 6.0 .The number of components was selected in such a manner that the predicted residual sum of squares  and 1/1,000 of this as 1 mU. Twenty-two of the 147 prescription Kampo formulations were demonstrated to completely inhibit the PTP1B activity (inhibition of >90%) at a final concentration of 0.1 mU/mL , in which water was used as a negative control. As shown in Figure 2) of the regression model was 0.947. The experimental value, predictive value and difference for each Kampo formulation are shown in Table Rhei Rhizoma exhibited the greatest contribution to PTP1B inhibitory activity among the 11 constituent crude drugs  and Daisaikoto  specifically exhibited higher inhibitory activity compared to Keishikashakuyakukanzoto  and Daisaikotokyodaio . In addition to our present study, other studies have reported that Rhei Rhizoma is a beneficial crude drug for the amelioration of metabolic diseases from various perspectives, including diabetic nephropathy, hypercholesterolemia and vascular disorders [Rhei Rhizoma may result in the discovery of new therapeutic agents for IR-T2DM. Such a finding could further expand the potential of IR-T2DM treatment. Furthermore, further understanding of how the Kampo drug functions may be clarified by investigating reformulations of the complex PTP1B-inhibiting crude drug compositions in Kampo formulations.Using the PLS method in multivariate analysis, ns Table , which wisorders ,24. IdenIn conclusion, we successfully identified Kampo formulations with high PTP1B inhibitory activity from 147 prescription Kampo formulations. Although PTP1B is an important target for the treatment of diabetes mellitus, Kampo formulations exert their therapeutic effects via multiple mechanisms of action. Nevertheless, our study demonstrated the potential of concurrent assessments of Kampo formulations for an action on a single target for the purpose of identifying a candidate Kampo drug effective for the treatment of target-related diseases based on relevant theories in Kampo medicine, current knowledge in modern medicine and relationships found among constituent crude drugs from various perspectives. This may represent an innovative step toward the redefinition of the proper use of Kampo and the accumulation of evidence. On the basis of the results of this study, we are currently investigating how these candidate Kampo formulations, which were identified in this study, affect patients with IR-T2DM.The authors declare that there is no competing interests.WL and KK conceived the study. WL, TO and KK designed this study. TO performed the experiments, and HK performed the cellular experiments. TO and WL performed data analyses and drafted the manuscript. All of the authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/14/64/prepub


All cyt bc1 inhibitors act on either the ubiquinol oxidation (QP) or ubiquinone reduction (QN) site. The primary cause of resistance to bc1 inhibitors is target site mutations, creating a need for novel agents that act on alternative sites within the cyt bc1 to overcome resistance. Pyrimorph, a synthetic fungicide, inhibits the growth of a broad range of plant pathogenic fungi, though little is known concerning its mechanism of action. In this study, using isolated mitochondria from pathogenic fungus Phytophthora capsici, we show that pyrimorph blocks mitochondrial electron transport by affecting the function of cyt bc1. Indeed, pyrimorph inhibits the activities of both purified 11-subunit mitochondrial and 4-subunit bacterial bc1 with IC50 values of 85.0 μM and 69.2 μM, respectively, indicating that it targets the essential subunits of cyt bc1 complexes. Using an array of biochemical and spectral methods, we show that pyrimorph acts on an area near the QP site and falls into the category of a mixed-type, noncompetitive inhibitor with respect to the substrate ubiquinol. In silico molecular docking of pyrimorph to cyt b from mammalian and bacterial sources also suggests that pyrimorph binds in the vicinity of the quinol oxidation site.The respiratory chain cytochrome  It catalyzes the reaction of electron transfer (ET) from ubiquinol to cyt c and couples this reaction to proton translocation across the mitochondrial inner membrane, contributing to the cross-membrane proton motive force essential for cellular functions such as ATP synthesis bc1 in cellular energy metabolism makes it a prime target for numerous natural and synthetic antibiotics. More than 20 synthetic fungicides targeting cyt bc1 are in widespread use in agriculture with an annual sale exceeding $2.7 billion The cytochrome bc1 inhibitors target either the ubiquinol oxidation site (QP or Qo) or the ubiquinone reduction site (QN or Qi), which are defined by the Q-cycle mechanism of cyt bc1 function bc1 from various organisms, only three subunits are essential for ET-coupled proton translocation function: they are cyt b, cyt c1 and the iron-sulfur protein (ISP). The cyt b subunit contains two b-type hemes (bL and bH), the cyt c1 subunit has a c-type heme, and the ISP possesses a 2Fe-2S cluster. Both active sites are located within the cyt b subunit, as demonstrated by crystallographic studies of mitochondrial and bacterial bc1 complexes bc1 fungicides has been reported at an alarming rate, rendering many of these reagents ineffective. Most common mechanisms of resistance involve target site mutations and corresponding strategies to overcome drug resistance have been proposed P and QN sites of cyt bc1 is most attractive primarily because the new compounds presumably are able to circumvent existing fungal resistance.All cyt tert-butylphenyl)-acryloyl] morpholine, is a novel systemic antifungal agent that belongs to the family of carboxylic acid amide (CAA) fungicides Phytophthora infestans, Phytophthora capsici, and Rhizoctonia solani and is able to suppress zoosporangia germination of Pseudoperonospora cubensis with EC50 values in the range between 1.3 and 13.5 μM in vitro sensitivities of various asexual stages of Peronophythora litchii to pyrimorph were studied with four single-sporangium isolates, showing high sensitivity at the stage of mycelial growth with an EC50 of 0.3 μM Pyrimorph, (Z)-3- shows a trough centered around 565 nm (b hemes. This spectrum was compared to spectra with bound QP site inhibitor myxothiazol [(bc1+myx)-bc1] and QN site inhibitor antimycin A [(bc1+ant)-bc1], respectively  – (bc1+ant)] (bc1+pyr)-(bc1)] ]  looks al-(bc1)] . This exbc1 does affect subsequent binding of myxothiazol and vise versa, because the difference spectrum [(bc1+pyr+myx) – (bc1+myx)] (bc1+pyr)-(bc1)] (bc1+pyr+myx) – (bc1+pyr)] (bc1+myx)-(bc1)] ]  does not-(bc1)] , nor doe1+pyr)]  resemble-(bc1)] . These sbc1 function under pseudo first-order reaction conditions by measuring its activity with respect to changes in concentration of either substrates Q0C10BrH2 or cyt c in the presence of different amount of pyrimorph, allowing double reciprocal or Lineweaver-Burk plots to reveal the relationship between 1/V and 1/[S]. The measurements were done for both Btbc1 and Rsbc1, revealing nearly identical kinetic behavior  were subjected to extensive docking trials using Autodock Vina but no new locations showing improved affinity over the classic sites were identified. The QP site showed the highest binding affinity to pyrimorph. Unlike traditional inhibitors, pyrimorph does not enter the QP site, but rather blocks the entrance or portal to the quinol oxidation site  phenyl group enters the access portal, where it is stabilized by the aromatic side chain of F274 and partially by F128. The latter primarily interacts with the pyridyl moiety via aromatic-aromatic (Ar-Ar) interactions. The tert-butyl group that penetrates into the QP site is flanked by the residues Y273, Y131 and P270, establishing beneficial van-der-Waals contacts.Docking of pyrimorph to known inhibitor-binding sites in the cyt ion site . While ibc1 inhibitors has been extensively investigated, revealing a wide variety of underlying mechanisms including target site mutations bc1 inhibitors. Thus, extensive research has been focusing on how to overcome resistance caused by target site mutations. Finding inhibitors that target alternative sites seems to be an attractive strategy.Resistance to cyt P. capsici were selected in the presence of the inhibitor and the three most resistant strains share a common mutation (Q1077K) in the CesA3 gene, which is different from the one (G1104V) selected for mandipropamid resistance in vitro biochemical experiments that shows at the protein level the inhibition of CesA3 by pyrimorph.Pyrimorph is a fungicide containing a carboxylic acid amide (CAA) moiety and was shown to be cross-resistant with other CAA fungicides such as mandipropamid, dimethomorph and flumorph P. capsici mycelia  Direct comparison between results of two very different assays is not a fair comparison, because in isolated light mitochondrial fraction the estimation of cyt bc1 concentration is difficult in the presence of many different proteins. However, the inhibitory concentrations or IC values are directly related to the amount of enzyme in the assay solution. So the lower IC value could be due to a lower concentration of bc1 in the assay conditions. (2) Being a hydrophobic compound, pyrimorph may preferentially partition into the lipid bilayer of mitochondrial membranes, leading to a higher local concentration and in turn to the apparent 95% inhibition at 4 μM concentration . (4) The cyt bc1 of P. capcisi is more sensitive to pyrimorph than either bovine or bacterial bc1. However, we note that the purified bacterial complex is more sensitive to pyrimorph than bovine bc1 but only by a factor of 1.2. The difference might simply be a reflection of changes in the sequences and we do observe that bacterial bc1 exhibits slightly higher similarity to fungal than bovine mitochondrial cyt b.It did not escape our notice that cyt ntration , Fig. 1.Btbc1 and 4-subunit Rsbc1 demonstrates that the inhibitor acts on cyt b, cyt c1 or ISP subunits of the complex; it does not inhibit mitochondrial bc1 function through binding to the so-called supernumerary subunits  Single turn-over experiments show the reduction rate of cyt c1 and re-oxidation rate of cyt b in the presence of two different concentrations of pyrimorph -(bc1)] (bc1+myx)-(bc1)] (bc1+ant)-(bc1)]  Dif QP site , because-(bc1)]  resemble-(bc1)]  but not -(bc1)] .P pocket as the target site, it also suggests that pyrimorph has a non-overlapping binding site with the classic QP site inhibitor myxothiazol  on the side that binds to the QP entrance, modifications could significantly increase the affinity, making it a better competitive inhibitor. (2) Improvements in hydrophobicity or geometric factors on the side that stays outside the QP pocket, the inhibitor could enhance its non-competitive properties. Should it be possible to design a dual type inhibitor, fungal resistance may be stalled for an extended period. Suggestions for improvements might include modifications of the tert-butyl group to include polar groups  that are within the reach of E271 (both sidechain and backbone amide) as well as sterically demanding aromatic groups that may take advantage of the large, aromatic cavity of the QP site. On the side that stays outside of the QP pocket, variations of saturated and aromatic ring systems seem likely to improve binding properties, as it appears that the morpholino group and the chloro-pyridyl group can change places with minimal change in binding energy.Molecular modeling of other CAA-type fungicides such as dimethomorph indicate a very similar binding position and orientation to the Qhibitors . Since b


Allergy to plant-derived foods is variable in clinical presentation and in dangerousness due to properties of allergens involved. Clinical data permits to identify some markers of allergens involved and laboratory investigation completes this data.Caucasian 16-year-old boy was refered to Immunoalergology Unit for food allergy investigation. Since early childhood the patient avoids ingestion of peach although without any symptoms to mention. At the age of 7 he experienced labial and palpebral angioedema, oropharyngeal pruritis and dyspnoea immediately after ingestion of a non-peeled fresh apple and later with a cooked apple. At 8 years of age he experienced a similar reaction after ingestion of a melon. In the exposition to peanut powder he experiences nasal and oropharyngeal pruritis. The skin prick tests performed with extracts of common aeroallergens were negative. The skin prick tests were positive with extracts of peach, apple, melon, peanut, walnut, hazelnut and almond. Specific IgE was elevated for peach peel and pulp, apple and melon peel, walnut, raw and roasted peanut, hazelnut and for purified peach LTP. The molecular mass of the IgE binding bands was calculated by SDS PAGE immunoblotting with identification of 15-17 kDa bands in all the extracts assayed . Immunoblotting inhibition method was performed with apple peel extract in solid phase and purified LTP (Pru p 3) in inhibition phase with a total IgE binding inhibition. The proteomic study confirmed LTP nature of melon protein involved in allergic reaction. The patient had an indication of a strict avoidance diet  and use of IM epinephrine kit.We present a clinical case of severe allergy to botanically unrelated, plant-derived foods. LTP involvement was suggested by a clinical data and confirmed by EAST (elevated specific IgE to purified peach LTP), SDS Page immunoblotting inhibition and proteomic study. To our knowledge there are no previous descriptions of LTP proteins involved in allergy to melon.


Phosphoinositide 3-kinase (PI3K) inhibitor wortmannin suppressed pAKT, pERK, pmTOR, and their downstream targets, indicating PI3K to be a common upstream mediator. In addition, α-TEA induced increased levels of pIRS-1 (Ser-307), a phosphorylation site correlated with insulin receptor substrate-1 (IRS-1) inactivation, and decreased levels of total IRS-1. Small interfering RNA (siRNA) knockdown of JNK blocked the impact of α-TEA on pIRS-1 and total IRS-1 and impeded its ability to downregulate the phosphorylated status of AKT, ERK, and mTOR. Combinations of α-TEA+MEK or mTOR inhibitor acted cooperatively to induce apoptosis and reduce basal levels of pERK and pmTOR. Importantly, inhibition of MEK and mTOR resulted in increased levels of pAKT and IRS-1, and α-TEA blocked them.α-TEA and α-TEA+MEK or mTOR inhibitor-induced apoptosis in human MCF-7 and HCC-1954 breast cancer cells.Downregulation of IRS-1/PI3K pathways via JNK are critical for  The PI3K, AKT, ERK, and mTOR prosurvival mediators are important therapeutic targets, as they are constitutively activated in many cancers and contribute to cancer progression by promoting cellular proliferation and inhibiting cell death signalling pathways . PhosphoFor example, the mTOR substrate p70S6K can ultimately diminish prosurvival signalling via PI3K/AKT by catalysing an inhibitory phosphorylation site on insulin receptor substrate-1, an upstream mediator of PI3K (α-Tocopherol ether-linked acetic acid (α-TEA) is a promising agent for cancer prevention/therapy based on its antitumour actions reported in several in vitro and in vivo studies on a variety of cancers, including human oestrogen-responsive and nonresponsive breast cancers . Lessons learned from this study are that α-TEA in combination with ERK or mTOR inhibitors is an effective anticancer strategy and lays the groundwork for a mechanism-based combinational therapeutic strategy for employing ERK and mTOR inhibitors more effectively in the clinic.α-Tocopherol ether-linked acetic acid (FW=488.8) was prepared in-house as described previously (α-Tocopherol ether-linked acetic acid (40 mM) dissolved in ethanol served as stock solution. Equivalent final concentrations of ethanol (0.05–0.1%) for concentration of α-TEA used served as vehicle controls (VEH).The oestrogen receptor-negative human breast cancer cells, HCC-1954, were obtained from the American Type Culture Collection . The oestrogen-responsive human breast cancer cells, MCF-7, were originally provided by Dr Suzanne Fuqua . The MCF-7 cells were cultured as previously described . Antibodies to the following proteins were used: poly (ADP-ribose) polymerase (PARP), phospho-JNK (pJNK thr-183/tyr-185), phospho-ERK (pERK thr-202/tyr-204), total ERK and phospho-caspase-9 (pcaspase-9 ser-196) , Phospho-AKT (pAKT ser-473), pAKT (ser-308), total AKT, phospho-GSK (pGSK α/β ser-21/9), total GSK α/β, phospho-Bad (pBad ser-136), phospho-Bad (pBad ser-112), total Bad, total casapse-9, phospho-p90RSK (pp90RSK ser-380), total p90RSK, phospho-mTOR (pmTOR ser-2448), total mTOR, phospho-p70S6K (pp70S6K thr-389), total p70S6K, phospho-4E-BP1 (p4E-BP1 thr-37/46), total 4E-BP1, phospho-IRS (pIRS ser-307), total IRS, and glyceraldehyde-3-phosphate dehydrogenase .Whole-cell protein lysates and western blot analyses were conducted as described previously . Protein2 cell culture dishes at a density of 2 × 106 cells per dish using Lipofectamine-2000  and siRNA duplex, resulting in a final siRNA concentration of 30 nM following the company's instructions. At 1 day after transfection, the cells were re-cultured in 100 mm2 dishes at 2 × 106 cells per dish and incubated for 1 day followed by treatments.A scrambled RNA duplex that does not target any known genes was used as a nonspecific negative control for RNAi (referred to as control siRNA) . Transfection of siRNAs to AKT1, AKT2, JNK1/2, or control (Ambion) was performed in 100 mmApoptosis was quantified by Annexin V-FITC/propidium iodide (PI) assay following the manufacturer's instructions. The Annexin V-FITC/PI assay measures the amount of phosphatidylserine on the outer surface of the plasma membrane  and the amount of PI, a dye that does not cross the plasma membrane of viable cells but readily enters dead cells or cells in the late stages of apoptosis and binds DNA. Fluorescence was measured using fluorescence activated cell sorter (FACS) analyses with a FACSCalibur flow cytometer, and data were analysed using CellQuest software . Cells displaying phosphatidylserine on their surface  were considered to be apoptotic.t-test for comparison between two treatments to determine statistical differences. Differences were considered statistically significant at P<0.05.Apoptosis data were analysed using a one-way analysis of variance (ANOVA) followed by Tukey's test for comparison of more than two treatments or a two-tailed Student's α-TEA at different doses  and MCF-7 cells (40 μM) with α-TEA for 9, 15, and 24 h: (1) reduces levels of pAKT (both Ser-473 and Ser-308) as well as its downstream substrates pGSK (α/β) (Ser-21/9), pBad (Ser-136), and pcaspase-9 (Ser-196) and increases cleaved caspase 9 in a time-dependent manner  and phosphorylated downstream substrates GSK (pGSK), Bad (pBad), and caspase 9 (pcaspase-9). Treatment of HCC-1954  dowt manner , and (3)t manner . These dμM wortmannin (PI3KI) for 4 and 8 h. Wortmannin reduced the phosphorylated forms of: (1) AKT and its substrates pBad (Ser-136) and pcaspase-9 (Ser-196)  , (2) ERKSer-196) , and pmTSer-196) , indicatα-TEA suppresses pAKT, pERK, and pmTOR in unison, the effect of α-TEA on phospho-IRS-1 (pIRS-1 Ser-307) was examined. Ser-307 is a phosphorylation site regulated by JNK for inactivation of IRS , and reduced levels of total IRS-1 in both cell lines (α-TEA to increase pIRS-1 (Ser-307) and decrease levels of total IRS-1 protein, and suppressed the ability of α-TEA to reduce pAKT (Ser-473), pERK1/2, and pmTOR (Ser-2448). These data suggest that downregulation of IRS-1 is involved in the ability of α-TEA to suppress AKT, ERK, and mTOR signalling and demonstrated that JNK is involved in this event via phosphorylation of IRS-1 at Ser-307, leading to its degradation.To identify a common upstream factor that might account for how ll lines . Knockdoll lines  or siRNAll lines  blocked α-TEA-induced apoptosis, HCC-1954 and MCF-7 cells were cultured with 10 and 20 μMα-TEA, respectively, plus 1 μM PI3K inhibitor (PI3KI) wortmannin, 10 μM MEK inhibitor (MEKI) U01260, and 50 nM mTOR inhibitor (mTORI) rapamycin for 18 h. Single treatments with α-TEA or inhibitors induced low levels of apoptosis, whereas combinations of α-TEA plus each of the three inhibitors individually significantly enhanced induction of apoptosis in both cell types in comparison with single treatments (α-TEA and PI3K inhibitors reduced levels of pAKT (Ser-473), pERK1/2, and pmTOR (Ser-2448), and the combination of α-TEA+PI3K inhibitor acted cooperatively to enhance PARP cleavage and to reduce levels of pAKT (Ser-473), pERK1/2, and pmTOR (Ser-2448) when compared with individual treatments or control . Phosphorylation of Bad at Ser-112 and Ser-136 disrupts its association with Bcl-2 or Bcl-xl, promoting cell survival  and its downstream mediators p70S6K and 4E-BP1. In addition, our data show that α-TEA not only enhances rapamycin suppression of mTOR and induction of apoptosis, but also suppresses rapamycin-mediated feedback activation of AKT, providing a rationale for developing a combination regimen of mTOR+α-TEA for breast cancer treatment.Mammalian target of rapamycin is a downstream mediator of PI3K/AKT signalling, regulating proliferation, survival, mobility, and angiogenesis via targeting p70S6 kinase (p70S6K) and 4E-BP1 in breast cancers that exhibit constitutively activated PI3K/AKT signalling . Accumulα-TEA functions as an IRS-1 suppressor in human breast cancer cells via JNK-dependent phosphorylation of IRS-1 at ser-307. Thus, α-TEA-mediated phosphorylation of IRS-1 at ser-307 is correlated with downregulation of total protein levels of IRS-1, and both α-TEA-mediated phosphorylation of IRS-1 and downregulation of total protein level of IRS-1 are JNK dependent, suggesting that α-TEA downregulation of total protein level of IRS-1 may be subsequent to phosphorylation of IRS-1 at ser-307, as phosphorylation of IRS-1 at ser-307 has been reported to decrease IRS-1 stability  stress in α-TEA-induced apoptosis (c-Jun N-terminal kinase, a proapoptotic mediator, has been reported to be activated by α-TEA to target PI3K-mediated prosurvival factors via JNK-mediated inhibition of IRS-1 not only has a role in enhancing α-TEA-induced apoptosis by inhibition of AKT, ERK, mTOR, and their downstream mediators, but also suggests a novel means for preventing procancer impact of AKT activation induced by ERK and mTOR inhibitors.In summary, the ability of


The complete remission rate (including CRp and CRi) in FLT3-ITD-positive patients was 54% (50/92) and the corresponding partial remission rate (PR) was 17% (16/92)2 Thus, although the FLT3-ITD mutation status correlates with response, the error rate in stratification of patients into responders and non-responders is high, as still 29% of the FLT3-ITD-positive patients failed to respond. Exclusion of FLT3-ITD-negative patients from AC220 treatment also seems critical, as the total response rate (CR+PR) in FLT3-ITD-negative patients is substantially lower . As AC220 is a tyrosine kinase inhibitor, we hypothesized that investigating phosphorylation-based signaling on a system-wide scale in AML cells allows for identification of markers enabling more accurate prediction of therapy response as compared to commonly used genetic markers. Hence, we applied quantitative mass spectrometry to decipher a multivariate phosphorylation site marker, which we refer to as phospho-signature, in patient-derived AML blasts that might be useful as predictive biomarkers for AC220 treatment.Treatment with inhibitors of the receptor tyrosine kinase FLT3 are currently studied as promising therapies in acute myeloid leukemia (AML). However, only a subset of patients benefit from these treatments and the presence of activating mutations within FLT3 can predict response to a certain extent only. AC220 (quizartinib) is an example of a potent FLT3 inhibitorWe first collected bone marrow aspirates of 21 patients enrolled in the phase II clinical trial of AC220 monotherapy in AML  with FLT3-ITD before treatment . We proc3 and further bioinformatics tools as outlined below. In total, 13 236 phospho-sites were identified in the training group. Of these, 7831 were confidently assigned to specific serine, threonine or tyrosine residues (class I sites).To monitor quantitatively the phospho-proteomes of the patient-derived AML blasts, we used super-SILAC in combination with quantitative mass spectrometry see . Data an4 revealed three significantly different sites at a false-discovery rate of 10% (see Supplementary Table 2). The first regulated site (S160) is located on the endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1). The protein carrying the second phosphorylation site (S630) was B-cell lymphoma/leukemia 11A (BCL11A), which functions as a myeloid and B-cell proto-oncogene and may play a role in leukemogenesis and hematopoiesis.5 Furthermore, the expression of BCL11A is associated with a poor outcome of AML patients.6 The third phosphorylation site (S333) is located on Ran-binding protein 3 (RANBP3). RANBP3 mediates nuclear export of Smad2/3 and thereby inhibits TGF-β signaling.7 Furthermore, the Ras/ERK/RSK and the PI3K/AKT signaling pathways regulate the activity of RANBP3.8 Both the pathways are activated in FLT3-ITD-positive cells.9 To our knowledge, no function has been described for these phospho-sites in AML so far. Interestingly, other phosphorylation events that are downstream of FLT3-ITD, such as phosphorylation of Y694 in STAT5A, were not differentially regulated between the responder and the non-responder group , which harbored the fifth site (S458) from the phospho-signature, form the nuclear lamina and has an important role in cell cycle-dependent regulation of nuclear structure and gene transcription.12 All five sites were identified and localized with high confidence  were correctly classified , correspWe finally applied the identified phospho-signature to test its predictive power on nine additional validation samples . These sr=0.92, P=0.03). The correlation for EEPD1 is smaller and not significantly different from 0  due to one outlier sample .Differences in phosphorylation of a specific site may be caused by either a difference in phosphorylation site stoichiometry, a difference in expression of the corresponding protein, or by a combination of both. In order to distinguish between these three possibilities, we analyzed the proteome of six validation samples . For twor sample . Furtherr sample  and agai13 could be applied instead of global analysis strategies. Such targeted methods can reproducibly detect and quantify given peptides from relatively low sample amounts and can be routinely applied to large number of samples. We also showed that at least one of the phosphorylation markers, LMN1 (S458), strongly correlates with the expression of the corresponding protein. This creates the further option to measure LMN1 protein expression rather than performing targeted phosphorylation site analysis.These results show the utility of a global and unbiased analysis to enable the identification of non-obvious but highly predictive markers that have no known association with the drug's main target. For clinical application of the biomarker signature, it would be sufficient to detect and quantify five phosphorylation sites. Notably, economic targeted detection methods, such as immunological methods or the mass spectrometry-based multiple reaction monitoringIn summary, phosphoproteomic analyses of primary AML bone marrow by high-resolution quantitative mass spectrometry is feasible and offers the opportunity to discover posttranslational modifications as pre-therapeutic response parameters. A signature consisting of five phosphorylation sites predicted the response to treatment of AML patients with AC220.


The model predictions were confirmed through further experiments in human hepatocellular carcinoma (HepG2) cells and the results were used to update the model and our current understanding of the signaling induced by palmitate.Palmitic acid, the most common saturated free fatty acid, has been implicated in ER (endoplasmic reticulum) stress-mediated apoptosis. This lipoapotosis is dependent, in part, on the upregulation of the activating transcription factor-4 (ATF4). To better understand the mechanisms by which palmitate upregulates the expression level of ATF4, we integrated literature information on palmitate-induced ER stress signaling into a discrete dynamic model. The model provides an in silico simulation and experimental results are: 1) palmitate induces different signaling pathways (PKR (double-stranded RNA-activated protein kinase), PERK (PKR-like ER kinase), PKA (cyclic AMP (cAMP)-dependent protein kinase A) in a time dependent-manner, 2) both ATF4 and CREB1 (cAMP-responsive element-binding protein 1) interact with the Atf4 promoter to contribute to a prolonged accumulation of ATF4, and 3) CREB1 is involved in ER-stress induced apoptosis upon palmitate treatment, by regulating ATF4 expression and possibly Ca2+ dependent-CaM  signaling pathway.The three key things from the in silico model helped to delineate the essential signaling pathways in palmitate-mediated apoptosis.The  Elevated serum free fatty acids (FFA) and hepatic lipid accumulation in non-adipose tissues can lead to cellular dysfunction or cell death, due in part to the diversion of unoxidized FFAs to nonoxidative pathways, resulting in lipoapoptosis . Excess The ER is one of the largest organelles in the cells, and perturbing ER homeostasis or inducing ER stress has profound effects on cell survival. Cellular perturbations, such as alterations in calcium storage in the ER lumen or an imbalance in the luminal-oxidizing environment will cause ER stress. This stress is sensed by the cells through three ER transmembrane proteins, inositol requiring enzyme (IRE) 1α, PKR-like ER kinase (PERK), and activating transcription factor (ATF) 6α . They acOf the ER transmembrane proteins, PERK is involved in regulating both translation and transcription to return the folding demand and capacity to homeostasis. Like PKR , one of the eukaryotic translation inhibition factor 2α (eIF2α) kinases, PERK (eIF2ak3) also phosphorylates eIF2α at Ser 51, and phosphorylated eIF2α impedes global translation initiation to decrease the protein load in the ER . ATF4 biSaturated long chain-FFAs, especially palmitic acid, have been implicated in ER stress-induced apoptosis in liver cells ,13. SincTo evaluate the dynamics of the conventional ER signaling pathway, we measured the protein expression levels of PERK, eIF2α, and ATF4 at 3, 6, and 24 hrs upon palmitate treatment. As shown in Figure In addition, our group recently found that cAMP promoted apoptosis in palmitate-treated HepG2 cells, suggesting that cAMP-CREB1 signaling may be involved in palmitate-mediated ER stress . Furtherin silico to provide insight into the regulatory mechanisms involved.To resolve these discrepancies from the conventional pathway and incorporate our findings with the current knowledge, we collected and integrated information from the literature on palmitate-induced ER stress signaling, with an emphasis on how ATF4 could be activated by palmitate. We constructed a network of palmitate-induced signaling processes by collating the literature information Figure . We formin silico results suggest an inconsistency with the current knowledge of the palmitate-induced signaling processes mediated by eIF2α and ATF4.Our simulation results suggest plausible dynamic profiles of the network upon palmitate-stimulation Figures A, 4B, 4CSince PKR, another eIF2α kinase, has been shown to be involved in ATF4-dependent ER stress in human embryonic kidney cells , we quesin silico simulation and the experimental observation lies in the prolonged activation of ATF4. The experimental measurements  which contains one CRE site  and performed an EMSA assay. As shown in Figure Atf4 promoter. Thus, the ChiP-chip data and our EMSA data confirmed our potential models in Figure Since ChIP-chip data for pCREB1 are available, we determined whether the CREB1 protein interacts with the database . A cut-odatabase  for detaCGTCA; -14 to -160probe -17 to -147 2+ activation regulated by palmitate are essential for CREB1 phosphorylation. The in silico knock-out results  plays an important role in the activation of the CREB1 pathway [To identify the essential signaling pathways of ATF4 and CREB1 activated by palmitate, we simulated a model that incorporated our experimental findings and then evaluated the dynamics of eIF2α phosphorylation, ATF4 expression, and CREB1 phosphorylation, by systematically deleting specific pathways. We addressed whether PKA, p38, Ca pathway ,27, and in-silico simulations  and the positive-feedback stabilizes the ATF4 expression. Upon palmitate treatment, PKA or Ca2+-dependent CaM signaling pathways could enhance the interaction of CREB1 alone or the ATF4/CREB1 heterodimer with the Atf4 promoter to prolong ATF4 accumulation. Although these remain open questions, our findings suggested that both ATF4 and CREB1 binding on the Atf4 promoter plays an important role in prolonging ATF4 accumulation upon palmitate exposure.ATF4 is known to form homo and heterodimers for DNA binding . One stupromoter . Along wpromoter , one groignaling . Consistin silico model provides a framework to integrate regulatory information into a complex network and test the network for consistency by comparing the dynamic simulations with experimental measurements. Our simulations show inconsistencies between current knowledge of the network and our observation of palmitate-induced ER stress in the liver cells, which led us to explore the temporal response of the different pathways  to palmitate stimulation. This led to the identification of an essential feedback structure in the downstream ATF4/CREB1 regulation that differs from the conventional understanding of the mechanism. The study demonstrates an iterative learning process in which we begin by integrating the current knowledge to build a model to generate hypothesis, which is tested with experiments to obtain novel information. The new information is then incorporated into the network model to correct and update our understanding of the regulation. The updated model can be used to guide new experiments, thereby forming an iterative process that can systematically be applied to study biological processes. The discrete dynamic modeling approach developed in [In this study, our loped in  for signAtf4 promoter to contribute to a prolonged ATF4 accumulation and their feedbacks appear necessary for ER-stress induced apoptosis. Third, palmitate responds later to PKA activation and possibly along with the Ca2+ dependent-CaM signaling pathway to increase CREB1 phosphorylation. Thus this systems biology approach helped provide insight into the molecular mechanisms by which palmitate induces ER stress in liver cells.Integrating experiments and computational simulations helped to identify several notable findings, summarized in Figure We applied the approach of discrete dynamic modeling proposed in our previous study . The modTwo novel properties are introduced to the original modeling approach in  to extenSi = f(Si-1), in which the current system state Si depends on the system state of the last step Si-1, our new approach does not apply this Markov assumption, and instead Si = f, which could present a history-dependent dynamics. In practice, we implement the idea by simply introducing multiple intermediate  components in between the regulator and its targets to delay the signal transduction.1) Delayed processes. To deal with certain regulatory interactions that are expected to respond much slower than other signaling processes, such as transcription, translation, and the slower response of PERK upon palmitate treatment, we introduce a delay on these interactions such that the current state of the targeted gene depends on not the last state but an earlier previous state of its activator/inhibitor. Therefore, different from the original state transition: 2) Encode posterior information into the model. Novel information, such as time-separation of the interactions, or the measurement of the activity of particular component in the network, could be incorporated to refine the model by adding constraints to the simulation. The constraints could be a delay of a certain interaction, or impose a constant value of activity on certain components (e.g. PP1) during the simulation.The discrete dynamic model is based on the simple logic (activation/inhibition) in a regulatory network, which corresponds to our current  understanding in this biological system. We measure the average gene expression level in an “in silico” cell population that has varying initial states and order of reactions, to enhance the robustness of the simulation results. The Additional file 2. Sodium palmitate was purchased from Sigma. In all experiments, palmitate (0.7 mM) was complexed to 2% (w/v) fatty acid free BSA  dissolved in regular medium. For palmitate treatment, HepG2 cells were seeded in the 6-well plate and cultured until 90% confluence.HepG2/C3A human hepatocellular carcinoma cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% of penicillin–streptomycin  in a humidified incubator at 37°C and 5% CO′-GAGAGAAUAUACUACAAUUTT-3′ and 5′-AAUUGUAGUAUAUUCUCUTT-3′), human PKR (5′-GGUGAAGGUAGAUCAAAGATT-3′ and 5′-UCUUUGAUCUACCUUCACCTT-3′), human PERK (5′-GUGACGAAAUGGAACAAGATT-3′ and 5′-UCUUGUUCCAUUUCGUCACTA-3′), human CREB1 (5′-CCGUAACUCUAGUACAGCUTT-3′ and 5′-AGCUGUACUAGAGUUACGGTG-3′) were purchased from Ambion. SiRNAs targeting human ATF4 (siGENOME SMARTpool #M-005125-005) was purchased from Dharmacon. The scrambled non-targeting siRNA (Ambion), used as a negative control, or the siRNA targeting proteins was diluted in Opti-MEM (Invitrogen) and then mixed with the transfection reagent, Lipofectamine RNAiMAX (Invitrogen). The mixture of siRNA and Lipofectamine RNAiMAX in Opti-MEM was then added into six well plates and incubated at room temperature for 20 min. 6 × 105 HepG2 cells were suspended in antibiotic-free medium, seeded into 6-well plates, and cultured for 24 hr. After transfection, the cells were incubated in regular medium or palmitate-containing medium and then collected for further analysis.SiRNAs targeting human PACT (5′-TGGACTTCGAGCAAGAGATG-3′ and 5′-AGGAAGGAAGGCTGGAAGAG-3′) and human actin  were synthesized by Eurofins MWG Operon. Amplifications of the cDNA templates were detected by SYBR Green Supermix (Bio-Rad) using RT-PCR Detection System (Bio-Rad) and the cycle threshold values were determined by the MyIQ software (Bio-Rad). Each sample was performed in triplicate and normalized to the actin expression levels.Total RNA was extracted from cells with the RNeasy mini kit (Qiagen). One microgram of total mRNA was reverse-transcribed using an iScript cDNA synthesis kit (Bio-RAD). The first-strand cDNA was used as a template. The primers used for quantitative RT-PCR analyses of human ATF4 (5The HepG2 cells were washed twice with cold PBS and treated with CelLyticM cell lysis buffer (Sigma-Aldrich) or RIPA buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (Sigma-Aldrich) for 10 min on ice. The cell lysate was clarified by centrifugation at 13000 rpm for 10 min, and the supernatant was collected. Total protein levels were quantified by Bradford assay (Bio-Rad). Thirty micrograms of total protein was loaded onto 9% SDS-PAGE gel, transferred to nitrocellulose membranes, and probed with antibodies for target proteins: PERK , p-PERK (BioLegend), eIF2a , p-eIF2a  ATF4 (Santa Cruz Biotechnology), CREB (Santa Cruz Biotechnology), p-CREB , PKR , and p-PKR . The image was analyzed using the Molecular Imager ChemiDoc XRS System from Bio-Rad.2, 10 mM KCl, 1 mM DTT, 50 mM NaF, 1 mM orthovanadate, 1 complete protease inhibitor mini tablet/10 mL), and left to swell on ice for 15 min. Cells were then lysed by forcing them through a 25-gauge hypodermic needle 6-8 times. Lysate was centrifuged at 4000 g for 5 min to pellet the crude nuclear fraction. The nuclear pellet was then resuspended in Buffer (20 mM HEPES (pH 8.0), 420 mM NaCl, 1.5 mM MgCl2, 25% glycerol, 0.2 mM EDTA, 1 mM DTT, 50 mM NaF, 1 mM orthovanadate, 1 complete protease inhibitor mini tablet /10 mL) and incubated on ice for 30 min with gentle agitation. The nuclear extract was spun down at 16000 g for 15 min to pellet the nuclei. The supernatant was saved and used for EMSA.Nuclear extracts were prepared as described in the literature . 0.7 mM ′-end DNA Labeling Kit from Thermo Scientific. EMSA method was modified from the previous study [2, 1 mM EDTA, 0.1% NP-40, 50 mM NaF, 1 mM Orthovanadate) with 2 μg of ATF4 antibody for 1 hr on ice. The biotinylated DNA probe was added to each mixture (with the DNA probe only as a negative control and the DNA probe with nuclear extract as positive control) and incubated for 30 min at room temperature. The reactions were loaded on a 4-20% TGX gel and separated by gel electrophoresis. The gel was then transferred on to a nylon membrane and UV crosslinked with a hand held crosslinker for 10 min at 254 nm. The probe was then visualized using the Chemiluminescencent Nucleic Acid Detection Module from Thermo Scientific.A synthetic oligo representing −175 to −147 (ACTCCTTTTCTCGTCACAGCTACGCCCT) of the ATF4 promoter was used for EMSA. The probe was biotinylated with Biotin 3us study . 5 μg ofATF4: Activating transcription factor-4; Bcl-2: B cell lymphoma 2; CaM: Calmodulin; CARE: CCAAT/enhancer binding protein (C/EBP)/ATF response element; CHOP: C/EBP-homologous protein; CREB1: cAMP-responsive element-binding protein 1; EMSA: Electrophoretic mobility shift assay; eIF2α: Eukaryotic translation inhibition factor 2α; ER: Endoplasmic reticulum; FFA: Free fatty acid; IRE1: Inositol requiring enzyme 1; NASH: Non-alcoholic steatohepatitis; PACT: PKR activating protein; PERK: PKR-like ER kinase; PKA: cAMP-dependent protein kinase A; PKR: Double-stranded RNA-activated protein kinase; PUMA: p53-upregulated modulator of apoptosis; UPR: Unfolded protein response.The authors declare that they have no competing interests.HC and LZ conducted all experiments except for EMSA. MW performed the computational simulations. RT carried out the EMSA experiments. AN participated in the analysis of ChIP-chip data. HC and MW drafted the manuscript. CC guided and directed the overall design and coordination of the study and edited the manuscript. All authors read and approved the final manuscript.Contains experimental and computational simulation results: Figure S1. The protein expression levels of PKAc  upon palmitate treatment, Figure S2. The protein expression levels of PP1 upon palmitate treatment, Figure S3. In silico knock-out of signaling pathways for CREB1 activation, Figure S4. Simulation of the discrete dynamic model with different amount of independent sampling size. (DOCX 763 kb)Click here for file


PP2Cδ was inactive and the other retinal phosphatases also had much less or no effect. Similar results were observed for centrins 2 and 4. Centrin3 was not a substrate for CK2. The results suggest PP2C α and β to play a significant role in regulating the phosphorylation status of centrins in vivo.In the present study, we identified protein phosphatases dephosphorylating centrins previously phosphorylated by protein kinase CK2. The following phosphatases known to be present in the retina were tested: PP1, PP2A, PP2B, PP2C, PP5, and alkaline phosphatase. PP2C  In the highly specialized vertebrate photoreceptor cells, centrins are components of the ciliary apparatus localized in the connecting cilium and their basal bodies –3. In fuThe present study was designed to identify protein phosphatases that serve as counterparts for the CK2-mediated light-dependent phosphorylation of centrins in mammalian photoreceptor cells.μg) or GST-BAD (0.6 μg) were incubated in 30 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 5 mM β-glycerophosphate, 0.2 μg CK2, 0.06% 2-mercaptoethanol, 1 mM EGTA, and 100 μM ATP including 1 μCi [γ − 32P]ATP in a volume of 10 μL for 15 minutes at 37°C. Then unincorporated ATP was removed by centri-SEP spin colums.GST-centrins .Phosphorylated proteins were incubated with 0.16 μM of the CK2-inhibitor TBB . itor TBB .138 of centrin1 is conserved in centrin2 (Thr137) and centrin4 (Thr134) whereas centrin3 (Ser135) carries a serine residue instead . Therefore, we conclude that if there is crosstalk and hierarchy among the two phosphorylation sites identified in centrin proteins, PP2C α and β are playing a most decisive role. Overall, dephosphorylation of P-centrins by PP2C α and β should increase the affinity of centrins to Gtβγ and finally reduce transport of the G-protein transducin through the connecting cilium.Many proteins are phosphorylated at several distinct sites. Knowledge on the reversible phosphorylation of centrins currently comprises PKA at Ser167 –17, PKC 167 , Cdc2 [1167 , and CK2167 . This re


Arctium lappa L. (Compositae) strongly increased AMPK phosphorylation and subsequently up-regulated its downstream pathway in both H9C2 and C2C12 cells. It was discovered that arctigenin phosphorylated AMPK via calmodulin-dependent protein kinase kinase (CaMKK) and serine/threonine kinase 11(LKB1)-dependent pathways. Mice treadmill based in vivo assay further indicated that administration of arctigenin improved efficiently mice endurance as reflected by the increased fatigue time and distance, and potently enhanced mitochondrial biogenesis and fatty acid oxidation (FAO) related genes expression in muscle tissues. Our results thus suggested that arctigenin might be used as a potential lead compound for the discovery of the agents with mimic exercise training effects to treat metabolic diseases.Physical inactivity is considered as one of the potential risk factors for the development of type 2 diabetes and other metabolic diseases, while endurance exercise training could enhance fat oxidation that is associated with insulin sensitivity improvement in obesity. AMP-activated protein kinase (AMPK) as an energy sensor plays pivotal roles in the regulation of energy homeostasis, and its activation could improve glucose uptake, promote mitochondrial biogenesis and increase glycolysis. Recent research has even suggested that AMPK activation contributed to endurance enhancement without exercise. Here we report that the natural product arctigenin from the traditional herb  Physical inactivity is considered as one of the risk factors for the development of type 2 diabetes and other metabolic diseases. It is known that endurance exercise training could lead to fiber type transformation, mitochondrial biogenesis, angiogenesis and other adaptive changes in skeletal muscle AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase with three subunits  Recently, it was reported that AMPK activation could improve mice endurance in the absence of exercise training Arctium lappa L. (Compositae) with anti-cancer and anti-inflammatory effects in vitro and in vivoArctigenin ATG,  is a phein vitro and in vivo, subsequently leading to the mice treadmill endurance enhancement. Cell based assay revealed that arctigenin increased AMPK phosphorylation targeting the CaMKK and LKB1-dependent pathways. Our results thus demonstrated that arctigenin might be used as a lead compound for the discovery of the agents with mimic exercise training effects to treat metabolic diseases.In the present study, we discovered that arctigenin could increase AMPK phosphorylation and up-regulate its downstream-pathway related genes mRNA levels to promote mitochondrial biogenesis and fatty acid synthesis and oxidation As reported, AMPK activation switched on ATP-producing processes to enhance endurance PGC-1α was known to act as the master regulator in mitochondrial biogenesis and skeletal muscle adaptation PGC-1α as a key regulator in energy metabolism could respond to varied physical stimuli, such as muscle contraction, cold stress and overfeeding These results thereby indicated that arctigenin could induce PGC-1α transcription in skeletal muscle and cardiac muscle cell lines via up-regulating AMPK phosphorylation.Since arctigenin has been determined to induce PGC-1α transcription via up-regulating AMPK phosphorylation, this result thereby implied that arctigenin might play a potential role in promoting mitochondrial biogenesis and function. To further evaluate this hypothesis, the mRNA levels of the related genes were detected in both H9C2 and C2C12 cell lines with results listed in To clarify whether arctigenin regulated mitochondrial biogenesis and FAO related genes mRNA levels through its effect on AMPK phosphorylation, we examined the influence of arctigenin on these genes together with AMPK inhibitor (compound C) incubation in C2C12 and H9C2 cells. As indicated in Therefore, all our results suggested that arctigenin could activate PGC-1α transcription and increase mitochondrial biogenesis and fatty acid oxidation genes expression in both skeletal muscle and cardiac muscle cells.As reported, AMPK catalytic subunit over-expression evidently promoted basal and ligand-dependent transcription of PPARδ Therefore, all above-mentioned results suggested that arctigenin regulating the relative gene expression is related to the enhancement of AMPK phosphorylation, while might not to the promotion of PPARδ transcriptional activity.in vitro to investigate whether arctigenin could influence the conformation of AMPK thus inducing AMPK activity. As indicated in Considering that arctigenin could activate AMPK phosphorylation, we subsequently investigated the potential regulation of this natural product against the relevant AMPK-involved pathways. As determined, directly activating AMPK in an allosteric manner and/or indirectly promoting Thr172 phosphorylation of AMPK both contributed to the AMPK activation. Bear that in mind, we examined the recombinant AMPK enzyme activity with or without arctigenin incubation As reported, AMPK has an obligate requirement for phosphorylation by an upstream kinase on Thr-172 in the α-subunit catalytic domain Taken together, our results thereby suggested that arctigenin stimulated AMPK phosphorylation via CaMKK and LKB1-dependent pathways.AMPK was reported as an “exercise mimetic”, whose pharmacological activator-AICAR was ever tested to provide several benefits of exercise in sedentary mice ip administration. Therefore, these results demonstrated that arctigenin elevated sedentary mice endurance efficiently while no significant toxicity was observed.During the treatment, mice showed similar basal behavior, food consumption and body weight . In addiAs reported, endurance exercise, AMPK mutation with persistent activation and PGC-1α over-expression in transgenic mice driven by muscle creatine kinase (MCK) promoter could induce myofiber type switch To further investigate the potential regulative mechanism of arctigenin regarding its improvement of mice treadmill endurance, we addressed the relevant tissue-based assays. Compared with vehicle group, arctigenin (8 mg/kg) administration enhanced AMPK phosphorylation in gastrocnemius , quadricAs fatty acid synthesis and storage related genes were up-regulated, we thereby hypothesized that arctigenin treated mice might contain more fatty acid in skeletal muscle for instant oxidation to supply energy source during exercise. Bearing that in mind, we thus detected fatty acid levels in skeletal muscles (gastrocnemius and quadriceps) and found that arctigenin treatment could enhance fatty acid storage in gastrocnemius evidently while in quadriceps without significance .in vitro and in vivo results thereby suggested that arctigenin could enhance AMPK phosphorylation, mitochondrial biogenesis and FAO pathway related genes expression, finally leading to the enhancement of exercise-free endurance.Therefore, all those Over the past decades, aerobic endurance exercise has been potently highlighted concerning its significance in the clinical amelioration of many disease symptoms, such as glucose metabolism in type 2 diabetes Actual exercises could result in activation of kinases/phoshatases signaling pathway, nuclear translocation of transcription/translation factors, up-regulation of NUGEMPs and mtDNA-encoded proteins, and augmentation of muscle aerobic capacity in vitro  drug discovery.In summary, we demonstrated that arctigenin could efficiently increase rodent sedentary treadmill endurance via enhancing AMPK phosphorylation. This natural product induced the accommodation of mitochondrial biogenesis and FAO pathway to promote mitochondrial oxidative capacity without actual physical activities as summarized in All animal experiments were carried out in accordance with the Regulations of Experiments Animal Administration issued by the State Committee of Science and Technology of the People's Republic of China. Permit numbers: SCXK (HU) 2008-0017; SYXK (HU) 2008-0049. This study was approved by Science and Technology Commission of Shanghai Municipality.Restriction enzymes were purchased from New England Biolabs. Cell culture plastic ware was purchased from Corning Inc. DMEM, fetal bovine and horse serums were purchased from Invitrogen. Compound C and STO609 were obtained from Sigma. Calcium Phosphate Cell Transfection Kit was obtained from Beyotime. RNAiso, RT reagent Kit and SYRB Premix Ex Taq were purchased from TaKaRa. Dual Luciferase Assay System was obtained from Promega. Anti-cytochrome c, anti-phospho-AMPK (Thr172), anti-AMPKα1/α2, and anti-LKB1 antibodies were purchased from Cell Signaling Technology. Anti-CaMKK antibody was purchased from Senta Cruz Biotechnology. HEK293T, H9C2 and C2C12 cells were obtained from ATCC.As a typical cardiac muscle cell line, H9C2 was derived from embryonic BD1X rat heart tissue and not differentiated in our study. C2C12 was a subclone of the mouse myoblast cell line and differentiated in DMEM with 2% horse serum, forming contractile myotubes and expressing characteristic muscle proteins. Differentiated myotubes were used in our experiments.2. To induce myoblast fusion and myotubes differentiation, C2C12 myoblasts were switched to differentiation medium when 100% confluent in 6-well plate. Differentiation medium was exchanged every 2 days for 6 days before experimental manipulation.H9C2 and C2C12 cell lines were maintained in DMEM supplemented with 10% fetal bovine serum and the cells were grown at 37°C in an environment of 5% CO3VO4, 1% Triton X-100 and a protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined using a BCA protein assay kit . Equal amounts of lysates or supernatants of the whole cell extracts were fractionated by SDS-PAGE and transferred to Hybond-c nitrocellulose membrane (Amersham Bioscience). The membranes were blocked for one hour at room temperature and then incubated overnight at 4°C in TBST buffer (5% milk) containing related antibody. The membranes were then incubated for an hour at room temperature in TBST buffer (5% milk) containing anti-rabbit IgG or anti-mouse IgG . Blots were visualized by incubation with SuperSignal West Dura chemiluminescence kit (Pierce Biotechnology) and exposing to light-sensitive film.Tissues were lysed with lysis buffer containing 25 mmol/L Tris-HCl (PH 7.5), 150 mmol/L NaCl, 1 mmol/L NaThe bands were quantified as “intensity×area” using Image-Pro Plis software (MediaCybernetics) and statistically analyzed. SE was calculated from three repeats of the experiments or five individuals of arctigenin treated and vehicle groups.Total RNA was extracted from administrated cells or mice tissues using RNAiso (TaKaRa) reagent in accordance with the Kit instruction. cDNA was synthesized by RT reagent Kit (TaKaRa) and real-time PCR was performed using SYRB Premix Ex Taq (TaKaRa) on DNA Engene Opticon TM2 system . The primers were listed as follows:PGC-1α: sense 5′- gcccggtacagtgagtgttc-3′, anti 5′- ctgggccgtttagtcttcct-3′.ERRα: sense 5′- ctcagctctctacccaaacgc-3′, anti 5′- ccgcttggtgatctcacactc-3′.Cytochrome c: sense 5′- cagcttccattgcggacac-3′, anti 5′- ggcactcacggcagaatgaa-3′.PDK4: sense 5′- agggaggtcgagctgttctc-3′, anti 5′- ggagtgttcactaagcggtca-3′.FAS: sense 5′- ggaggtggtgatagccggtat-3′, anti 5′- tgggtaatccatagagcccag-3′.mCPT1b: sense 5′- tgggactggtcgattgcatc-3′, anti 5′- tcagggtttgtcggaagagaga-3′.SCD1: sense 5′- ttcttgcgatacactctggtgc-3′, anti 5′- cgggattgaatgttcttgtcgt-3′.GAPDH: sense 5′- acagcaacagggtggtggac-3′, anti 5′- tttgagggtgcagcgaactt-3.MHCI: sense 5′- ccttggcaccaatgtcccggctc-3′, anti 5′- gaagcgcaatgcagatgcggtg-3.MHCIIa: sense 5′- atgagctccgacgccgag-3′, anti 5′- tctgttagcatgaactggtaggcg-3.MHCIIx: sense 5′- aaggagcaggacaccagcgccca-3′, anti 5′- atctctttggtcactttcctgct-3.MHCIIb: sense 5′- gtgatttctcctgtcacctctc-3′, anti 5′- ggaggaccgcaagaacgtgctga-3.For evaluation of the effects of arctigenin on PGC-1α promoter, HEK293T cells (24-well plate) were transfected with pGL3-PGC-1α-luc together with PRL-SV40 and refreshed with normal medium 5 hours later. After transfection, HEK293T cells were incubated with indicated concentration of arctigenin for 24 h. Luciferase activity was measured using Dual Luciferase Assay kit.The recombinant AMPK activity was assayed using the modified conventional approach in non-radioactive way , casein kinase 1, Protein kinase A (PKA), etc.) In the assay, the substrate SAMS was used according to the literature methods C57BL/6J male mice at 6 weeks of age were purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences, and acclimated to SPF microisollators for 2 days before any experimental intervention. Mice were accommodated under standard conditions  in plastic cages and provided with water and food ad libitum.All 30 mice were adapted to treadmill running for 10 min at 10 m/min at week -1 (5 days/week) avoiding unexpected accidents and the first fatigue test was conducted at week 0. For fatigue test, mice ran at 10 m/min for 5 min and 15 m/min for 10 min. After the initial warm-up period, exercise intensity was increased by 5 m/min every 30 min from 20 m/min until mice could not be prompted to continue running by moderate electric stimulation (less than 0.1 milliampere) and stayed at electrode for at least 10 sec. After the first fatigue test, 20 mice with moderate endurance capacity were selected from total 30 mice and divided into arctigenin administration and vehicle treatment groups (n = 10/group). Before final fatigue assay, sedentary mice were treated with arctigenin  or vehicle (sterilized 0.9% Sodium Chloride containing 5% Tween-80) daily via intraperitoneal injection for 6 weeks. Mice endurance capacity was estimated by treadmill  running time and distance until fatigue Animals were euthanized 72 h after the last bout of exercise. Gastrocnemius, quadriceps and heart muscles were thus isolated, frozen, and stored at −80°C until further analysis.All data were reported as mean ± standard deviation of the mean (SD). Data were analyzed in either one-way ANOVA with an appropriate post hoc test for comparison of multiple groups or unpaired student's t test for comparison of two groups as described in figure legends (Graphpad Prism software).Figure S1Arctigenin (ATG) enhanced AMPK phosphorylation in HEK293T cells. HEK293T cells were incubated with indicated concentrations of arctigenin (0-40 µM) for 30 min, phospho- and total AMPK were then detected by western blotting. The results shown are representative of three independent experiments. The bands were quantified using Image-Pro Plus software. Values are means ± SE. **, p<0.05; ***, p<0.005; one-way ANOVA.(TIF)Click here for additional data file.Figure S2Arctigenin (ATG) activated PGC-1α transcription via up-regulating AMPK phosphorylation.A. When the confluence reached 30∼40% (24-well plate), HEK293T cells were transiently transfected with pGL3-PGC-1α promoter-Luc and SV40. 5 hours later, cells were refreshed with medium supplemented with arctigenin  or DMSO and incubated for 24 hours before Luciferase assays as described in “B. After transfection, HEK293T cells were administrated with or without 20 µM compound C for 1 hour before and during the incubation with actigenin (40 µM) for 24 hours before Luciferase assays as described in “(TIF)Click here for additional data file.Figure S3Effects of arctigenin (ATG) on ERRα, cytochrome c, PDK4, SCD1, FAS and mCPT1b were subjective to AMPK phosphorylation in H9C2. H9C2 cells were treated with or without 20 µM compound C for 1 hour before and during the incubation with actigenin (20 µM) for 24 hours. After harvested, mRNA levels of ERRα (A), cytochrome c (B), SCD1 (C), PDK4 (D), FAS (E) and mCPT1b (F) were analyzed. The results shown are representative of three independent experiments. Values are means ± SD. *, p<0.05. #, p<0.05: for compound C and arctigenin co-incubation group versus arctigenin treated group; student's t test.(TIF)Click here for additional data file.Figure S4Effects of arctigenin (ATG) on ERRα, cytochrome c, PDK4, SCD1, FAS and mCPT1b were subjective to AMPK phosphorylation in C2C12. Differentiated C2C12 cells were administrated with or without 20 µM compound C for 1 hour before and during the incubation with actigenin (20 µM) for 24 hours. After harvested, mRNA levels of ERRα (A), cytochrome c (B), SCD1 (C), PDK4 (D), FAS (E) and mCPT1b (F) were analyzed. The results shown are representative of three independent experiments. Values are means ± SD. *, p<0.05; **, p<0.01; ***, p<0.005. #, p<0.05; ##, p<0.01; ###, p<0.005: for compound C and arctigenin co-incubation group versus arctigenin treated group; student's t test.(TIF)Click here for additional data file.Figure S5Arctigenin (ATG) failed to regulate the co-activator recruitment and transcriptional activity of PPARδ.A. HEK293T cells were transfected with UAS-TK-Luc, pCMX-Gal4DBD-PPARδ-LBD and pRL-SV40 followed by treatment of DMSO, GW501516 (PPARδ agonist), and varied concentrations of arctigenin for 24 hours. B. HEK293T cells were transfected with pAdTrack-PPARδ, pcDNA3.1-RXRα, pSV-PPRE-Luc and pRL-SV40 and then incubated with DMSO, GW501516 (PPARδ agonist), and varied concentrations of arctigenin for 24 hours. C. HEK293T cells were transfected with pSV-PPRE-Luc and pRL-SV40, and incubated with DMSO, GW501516 (PPARd agonist), and varied concentrations of arctigenin for 24 hours. Relative luciferase activities were measured as described in (TIF)Click here for additional data file.Figure S6Effects of arctigenin (ATG) on diet, weight, inflammation and liver toxicity of mice.A. Daily food intake of each group was analyzed (n = 10/group). B. Weight change in each group was measured (n = 10/group). C. D. Serum from mice in each group was collected and levels of TNFα (C) and IL-6 (D) were analyzed (n = 10/group). E. F. Activities of ALT (E) and AST (F) were measured (n = 10/group). Values are means ± SE. *, p<0.5; **, p<0.01; student's t test.(TIF)Click here for additional data file.Figure S7Arctigenin (ATG) failed to induce skeletal muscle fiber-type change. Metachromatical staining of frozen cross-sections from gastrocnemius and quadriceps in vehicle and arctigenin treated groups. The results shown are representative of three independent experiments. Dark-brown stained type I fibers were indicated by arrows.(TIF)Click here for additional data file.Figure S8Arctigenin (ATG) enhanced fatty acid storage in gastrocnemius. Free fatty acid in gastrocnemius (A) or quadriceps (B) of each group was analyzed (n = 7/group). Values are means ± SD. *, p<0.5; student's t test.(TIF)Click here for additional data file.Figure S9Arctigenin (ATG) failed to impact the phosphorylation of AMPK on Ser485/491 sites. HEK293T, H9C2 and differentiated C2C12 cells were incubated with indicated concentrations of arctigenin (0–40 µM) for 30 min, AMPK (Thr172), AMPK (Ser485/491) and total AMPK were then detected by western blotting. The results shown are representative of three independent experiments.(TIF)Click here for additional data file.Figure S10A scheme demonstrating recombinant AMPK activity assay approach.(TIF)Click here for additional data file.Text S1Supporting documents.(DOC)Click here for additional data file.


Amyotrophic lateral sclerosis (ALS) is a fatal neurologic disease characterized by progressive motor neuron degeneration. Clinical disease management is hindered by both a lengthy diagnostic process and the absence of effective treatments. Reliable panels of diagnostic, surrogate, and prognostic biomarkers are needed to accelerate disease diagnosis and expedite drug development. The cysteine protease inhibitor cystatin C has recently gained interest as a candidate diagnostic biomarker for ALS, but further studies are required to fully characterize its biomarker utility. We used quantitative enzyme-linked immunosorbent assay (ELISA) to assess initial and longitudinal cerebrospinal fluid (CSF) and plasma cystatin C levels in 104 ALS patients and controls. Cystatin C levels in ALS patients were significantly elevated in plasma and reduced in CSF compared to healthy controls, but did not differ significantly from neurologic disease controls. In addition, the direction of longitudinal change in CSF cystatin C levels correlated to the rate of ALS disease progression, and initial CSF cystatin C levels were predictive of patient survival, suggesting that cystatin C may function as a surrogate marker of disease progression and survival. These data verify prior results for reduced cystatin C levels in the CSF of ALS patients, identify increased cystatin C levels in the plasma of ALS patients, and reveal correlations between CSF cystatin C levels to both ALS disease progression and patient survival. Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease that affects approximately 1.5 to 2.5 per 100,000 individuals of all races and ethnicities throughout the world Biomarkers also hold promise to monitor disease progression and to stratify patient populations for use in clinical trials. One reason new drug therapies have not been successfully translated from ALS model systems to humans is ALS disease heterogeneity One protein that has shown potential for ALS diagnostic utility is cystatin C, a widely expressed cysteine protease inhibitor that is approximately five times more abundant in CSF than in plasma Two prior surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) studies reported significant decreases in CSF cystatin C levels in ALS patients relative to healthy controls The objective of this study was to use quantitative ELISA to further evaluate the utility of cystatin C as a biomarker for ALS using a large subject population. Our subject group size was based on power analysis of previously published mass spectrometry reports on cystatin C in ALS We collected longitudinal CSF and plasma samples from 104 ALS and control subjects  and evalIn order to assess the diagnostic utility of cystatin C, we first compared the mean first-draw cystatin C levels among ALS patients, neurologic disease controls, and healthy controls. A generalized linear model was used to estimate the mean total and percent cystatin C for each diagnostic category, with both gender and age included as co-factors in the model. This statistical design controls for between-group differences in each co-factor when generating estimated means. Therefore, the differences in age and gender among our diagnostic groups should not have affected our results, even in the case that cystatin C varies with these factors. We found that the estimated means for both measures of cystatin C were lower in ALS patients than in disease controls and healthy controls, similar to prior studies . Howeverp-values were reduced for the ALS vs. mimic disease control subgroup comparison . However, the p-values when compared to the analysis including all ALS patients. However, the pair-wise comparisons between these ALS subgroups and mimic disease controls still fell short of statistical significance.We next compared two ALS subgroups to the disease mimic group. Limb-onset ALS (ALS-L) and ALS patients greater than one year from symptom onset both exhibited reduced levels of cystatin C in the CSF when compared to disease mimics , with imFinally, we calculated the diagnostic sensitivity and specificity for several cutoff concentration values of CSF cystatin C. Total cystatin C concentration measurements were found to have better diagnostic parameters than percent cystatin C values. A cutoff value of 2.20 µg/ml identified a small subset of ALS patients (sensitivity: 23%) with relatively high specificity . A cutoff value of 2.70 µg/ml identified a modest subset of ALS patients (sensitivity: 32%) while maintaining high specificity versus controls . A less conservative cutoff value of 3.50 µg/ml identified a majority of ALS patients (sensitivity: 52%), but demonstrated lower specificity .As noted above, cystatin C was previously reported to be significantly reduced in the CSF of ALS patients using mass spectrometry-based proteomics, but the between-group differences based on our ELISA data were less robust. To explore the relationship between CSF cystatin C levels measured by these two techniques, we compared our ELISA results with SELDI-TOF-MS data for the same CSF samples. We found significant, positive correlations between the 13.3 kDa SELDI-TOF-MS mass peak intensity for cystatin C and both total cystatin C and percent cystatin C protein levels as measured by ELISA  or percent cystatin C levels  in CSF and plasma samples drawn from individual patients on the same day . The absWe next examined whether cystatin C levels change over time in ALS patients, and if these changes are associated with clinical disease progression. We compiled the first CSF draws for each of the ALS patients in our study and carried out linear regressions comparing both total and percent cystatin C with the time from symptom onset. Similar to a prior study Next, we collected longitudinal CSF samples from ALS patients and assessed the effect of time on cystatin C levels using a statistical model for repeated measures. This experimental design controls for individual differences in baseline cystatin C levels, but not for individual differences in rate of disease progression. We collected at least three longitudinal CSF draws from 15 ALS patients over a 1–2 year time period for each patient. When all 15 values were averaged for each time point, we did not observe a significant change in cystatin C levels over time , but both measures of CSF cystatin C levels showed a direct correlation, with the results for total cystatin C almost reaching statistical significance . These findings suggest that cystatin C levels in CSF but not plasma may be useful as prognostic indicators of patient survival time.Finally, we assessed the relationship between first-draw cystatin C levels (in CSF and plasma) and patient survival time. Neither measure of plasma cystatin C showed a correlation with subsequent survival time  and a larger high cystatin C group (n = 21). This analysis revealed significantly longer patient survival in the high cystatin C group than in the low cystatin C group . Next, bThe present study represents a comprehensive evaluation of cystatin C as a candidate biomarker in ALS, and is unique in its assessment of two distinct biofluids (CSF and blood plasma), two different measurements of the protein of interest , and longitudinally collected CSF samples. Prior studies using SELDI-TOF-MS found significantly lower cystatin C abundance in the CSF of ALS patients relative to healthy controls To be clinically useful as a diagnostic biomarker, cystatin C must also be able to differentiate between ALS patients and individuals with neurologic diseases that closely resemble ALS, or ALS “mimic diseases.” A recent study reported a significant reduction in CSF cystatin C levels in ALS patients relative to polyneuropathy patients We also assessed the diagnostic utility of plasma cystatin C levels. Plasma cystatin C has been extensively characterized as a peripheral biomarker for kidney function and as a prognostic indicator of the risk of morbidity and mortality relating to cardiovascular disease A recent study examined a single CSF draw per ALS patient, taken at varying times from symptom onset, to indirectly infer the average longitudinal change in cystatin C concentration in the group as a whole, and they reported that cystatin C levels do not change over time We also examined longitudinal CSF data from multiple patients to more accurately assess the changes in cystatin C over time. We found that longitudinal cystatin C concentrations were relatively constant in ALS patients as a combined group. In contrast, the subgroup of patients with slow or absent clinical disease progression exhibited longitudinal increases in cystatin C concentration, and the subgroup with more typical, continuous clinical deterioration exhibited longitudinal decreases in total cystatin C. Interestingly, slow progressors often exhibited lower initial levels of CSF cystatin C than fast progressors . SimilarWe also conducted an analysis to determine the relationship between longitudinal changes in CSF cystatin C levels and time-matched changes in three functional clinical measures of disease progression . However, no significant correlations were found (data not shown). This indicates that cystatin C levels may change independently of the clinical parameters used for monitoring disease progression. However, this finding does not eliminate the possibility that changes in CSF cystatin C levels correlate with more subtle biochemical changes associated with disease progression, as these may not be accurately reflected by overt functional measures of clinical disease status We recently demonstrated a correlation between CSF cystatin C levels and patient survival by SELDI-TOF-MS in vitro, in order to clarify its potential mechanistic role in ALS pathogenesis.The results of this comprehensive biomarker assessment also have implications for the potential mechanistic involvement of cystatin C in the pathogenesis of ALS. The function of cystatin C within the CNS has not been extensively studied, but it appears to have both neurotoxic and neuroprotective properties In summary, we have completed a comprehensive evaluation of cystatin C as a candidate ALS biomarker, including assessments of two complementary measures of cystatin C in two distinct biofluids as well as examinations of both longitudinal CSF samples and patient survival data. Our findings indicate that cystatin C levels, as determined by ELISA, are increased in the plasma and decreased in the CSF of ALS patients relative to healthy controls. CSF cystatin C measurements may possess a more limited diagnostic capacity for ALS than previously proposed, but may still have the potential to improve the diagnostic parameters of a biomarker panel. Additionally, longitudinal changes in CSF cystatin C levels may be useful as a biomarker of fast versus slow rates of disease progression. Our data also demonstrate that CSF cystatin C concentration has prognostic utility in estimating patient survival time. Further validation studies are necessary to confirm these findings and ultimately determine if cystatin C measurements can be used to enhance clinical disease management and clinical trial design. Finally, the association of high or increasing cystatin C levels with slower disease progression and increased survival time suggests a potential neuroprotective role for this protein in the pathobiology of ALS.This study was approved by the institutional review board (IRB) at the University of Pittsburgh, and written informed consent was obtained from all participating subjects. ALS subjects were diagnosed by experienced neurologists specialized in motor neuron disease, using revised El Escorial criteria The disease control group included six patients with multiple sclerosis, one with bilateral facial palsies, one with neurosarcoidosis, one with viral encephalitis, one with CNS lymphoma, one with brain metastases, one with pseudotumor cerebri, one with a seizure disorder, one with complicated migraine, one with paresthesis and possible myelopathy, one with a probable conversion disorder, and nine with neurologic diseases that can clinically resemble ALS at presentation. This ALS-mimic disease subgroup included two patients with primary lateral sclerosis (PLS), two with chronic inflammatory demyelinating polyneuropathy (CIDP), two with progressive muscular atrophy, one with spinocerebellar ataxia, one with small fiber neuropathy, and one with idiopathic sensorimotor polyneuropathy. CSF samples were obtained by lumbar puncture, immediately centrifuged at 450 g for five minutes at 4°C to remove cells and debris, aliquoted, and then frozen at −80°C. Intravenous blood samples were collected in EDTA containing tubes, inverted to mix, and centrifuged at 1,733 g for 10 min at 4°C. The plasma was decanted, aliquoted, and frozen at −80°C. CSF and plasma were aliquoted into small volumes for single use in experiments in order to eliminate any freeze/thaw effects. Samples of either biofluid were thawed on ice immediately prior to use.CSF and plasma samples from individual patients were assigned to random 96-well plate positions, and evaluated in duplicate wells for each ELISA. All samples were independently assayed at least twice. For all experiments, we used a human cystatin C sandwich ELISA kit , according to the manufacturer's instructions. Briefly, diluted CSF and plasma samples  were applied to antibody pre-coated ELISA plates for 30 min with gentle agitation. The wells were washed thoroughly and then the secondary antibody conjugate solution was applied for 30 min with gentle agitation. After a second wash, the 3,3′,5,5′ Tetramethylbenzidine substrate solution  was applied for 10 min, color development was stopped with an acidic stop solution, and the optical density was measured at 450 nm using a plate reader. Total protein concentrations for each sample were calculated using a BCA protein assay (Pierce), according to the manufacturer's instructions.For each ELISA plate, a standard curve was generated by plotting the logarithm of the cystatin C concentration against the logit log of the adjusted optical density . This procedure produced a linear standard curve, which was then used to calculate sample cystatin C concentration from sample optical density. The data points were averaged to determine the absolute cystatin C concentration, or “total cystatin C,” for each sample. Sample cystatin C concentrations were normalized to the sample total protein concentration to determine the percent of total biofluid protein accounted for by cystatin C, or “percent cystatin C.”For the analyses of diagnostic utility, we included only the initial sample collected from each patient, representing the time point closest to symptom onset. Differences between group and subgroup means were identified using the SPSS generalized linear model, with diagnosis and sex as factors in the model and age at draw as a covariate. This model was subsequently used to calculate and compare the estimated marginal group means, in order to determine which pairwise differences among the levels of each factor were responsible for the significant main effects.The relationship between first-draw cystatin C levels and the length of time from symptom onset was assessed by linear regression using GraphPad Prism 5.0 software . For this analysis, the “time from symptom onset” data were transformed by the natural logarithm (Ln) in order to achieve normality as required by the selected statistical test.The effect of time on longitudinal cystatin C levels in ALS patients with multiple biofluid draws was assessed with SPSS software, using the general linear model for repeated measures. The model was applied for all patients combined and for patient subgroups sorted by progression speed. Fast progressors were defined as patients exhibiting above average rates of ALSFRS decline . The Spearman's rank correlation coefficient (r) was calculated and the permutation test was applied to determine if r was significantly different from zero.The relationship between first-draw cystatin C levels and post-draw survival time (for deceased patients only) was assessed by Spearman correlation analysis (GraphPad Prism 5.0). The prognostic utility of CSF cystatin C was further explored by generating Kaplan-Meier survival curves for patients falling above or below several cut-off values of cystatin C. SPSS software was used to calculate the p-values for differential survival time by three different methods: the Log Rank (Mantel-Cox) test, the Breslow  test, and the Tarone-Ware test. For all statistical analyses in this study, the significance level was set at p<0.05.Figure S1Correlation of ELISA-based cystatin C levels and SELDI-TOF-MS 13.3 kDa mass peak intensity levels by Spearman correlation analysis. Both total (A) and percent (B) cystatin C ELISA measurements correlated to the 13.3 kDa cystatin C mass peak.(TIF)Click here for additional data file.Figure S2Correlation analysis for cystatin C levels in CSF and plasma. There was no correlation between total cystatin C concentrations (A)  or percent cystatin C levels (B)  between CSF and plasma.(TIF)Click here for additional data file.