CP-P protein inhibited the proliferation and induced apoptosis of breast cancer cells through the suppression of nuclear factor kappaB (NF-kB) activation. CP-P, when administered individually or in combination with cyclophosphamide  to rats with 7, 12-dimethyl benz(a)anthracene (DMBA)-induced breast cancer decreased tumor volume significantly without affecting the body weight. To elucidate the anticancer mechanism of CP-P, antioxidant activities such as superoxide dismutase (SOD), catalase (CAT), glutathione-s-transferase (GST) and non-enzymatic antioxidant - reduced glutathione (GSH), vitamin E and C generation in the breast were analyzed by various assays. SOD, CAT, GST, GSH, vitamin E and C levels were high in combination-treated groups (CP-P+CYC) versus the CYC alone-treated groups. Also, the combination was more effective in down-regulating the expression of NF-kB-regulated gene products (cyclin D1 and Bcl-2) in breast tumor tissues. Our findings indicate that CP-P possesses significant antitumor activity comparable to a commonly used anticancer drug, cyclophosphamide, and may form the basis of a novel therapy for breast cancer.Breast cancer is the most common cancer among women. To date, improvements in hormonal and cytotoxic therapies have not yet led to a sustained remission or cure. In the present study, we investigated the  Breast cancer (BC) is one of the leading causes of death in women worldwide Calotropis procera (L.) R.Br. belongs to the family Asclepidaceae and is well known for its various medicinal properties C. procera are effective in treating migraines C. procera are widely used in Asian and West African traditional therapy for dermatological and bronchial infections C. procera is commonly used in Saudi Arabian traditional medicine for treating various diseases including fever, joint pain, muscular spasm and constipation. Ethanol extract of the plant has been tested on laboratory animals for its antipyretic, analgesic, antibacterial, anti-inflammatory, purgative C. procera has been found to produce marked improvement in cases of diarrhoea and dysentery C. procera roots has been studied in albino rats to explore its antifertility and hormonal activities C. procera roots have also been analyzed upon the oestrous cycle in rats Several agents derived from natural sources exhibit anti-cancer properties without considerable adverse effects and play a vital role for developing new drugs C. procera latex have been well investigated C. procera reportedly exhibit potent anti-fungal C. procera root bark. Because Calotropis procera protein (CP-P) exhibits anti-inflammatory, anti-oxidant, and antipyretic effects, we postulated that CP-P may mediate its effects by modulating the NF-κB activation pathway. The latter has been closely linked to inflammation, tumorigenesis, proliferation, invasion, angiogenesis, and metastasis. The NF-κB pathway is activated in response to various inflammatory agents, carcinogens, tumor promoters, and growth factors. Hence, in the present study we aim to investigate whether the antiproliferative/pro-apoptotic in vivo effects of CP-P on 7, 12-dimethyl benz(a)anthracene (DMBA) induced breast cancer in rat models are mediated through the suppression of NF-κB signaling pathway. Our results indicate that CP-P inhibited the NF-κB activation pathway and the expression of various antiapoptotic and proliferative gene products, subsequently inducing apoptosis and suppression of tumor growth in a DMBA-induced breast cancer model.The milky latex from this plant has been used in traditional medicine to cure skin infections, poison, ulcer, enlargement of spleen, liver, abdominal glands, colic piles, worms and different inflammatory diseases C. procera extract was separated by gel-filtration chromatography on Superdex G-75 column, yielding five major peaks CP-1–CP-5  . The oppWe also investigated the apoptotic potential of CP-P apoptotic potential in breast cancer cells. Cell cycle analysis was used to analyze the distribution of MCF-7 cells in different phases of the cell cycle following exposure to CP-P alone, or in combination with CYC, after 24 h. We found that the apoptotic phase (Sub-G1 phase) was significantly increased in cells treated with CP-P+CYC after 24 h. In addition, CP-P+CYC induced apoptotic effects were statistically significant  after 24 h . HoweverBecause IκBα degradation is essential for the activation of NF-κB TNF-α induces the phosphorylation of p65, which is required for transcriptional activity TNF-α has been reported to activate TAK1 leading to the phosphorylation of both IKK-α and IKK-β We next investigated whether CP-P can modulate the expression of NF-κB -regulated gene products involved in the proliferation and survival of tumor cells. Pretreatment with CP-P inhibited TNF-α induced expression of cyclin D1 and Bcl-2 in MCF-7 cells as observed by western blot analysis . The supin vivo study showing a significant decrease in tumor volume (Each breast cancer control (BCC), rat received 1 mg of DMBA that induced 100% multiplicity of mammary carcinomas during a median latent period from 3 to 13 weeks . The antr volume , a signir volume . The tumr volume . There wHistopathological examination revealed a clear architecture of normal mammary glands , whereasAntioxidant enzyme levels were decreased in the BCC rats after 13 weeks (group II). The CP-P alone treated group (0.2 mg/kg) (III) showed significantly (P<0.01*) elevated levels of SOD following 20 days of oral treatment. The combination-treated group (IV) exerted tremendous changes in SOD levels. Within the CYC group (V), SOD led to pronounced level of increment as compared to the NC (group I).The GST levels were higher for the CP-P (group III) individually-treated groups than the CP-P+CYC treatment group (V) . The GSTNon-enzymatic antioxidants are also involved in the tumor reduction after the treatment with CP-P . GSH plaVitamin E is a naturally occurring compound known as tocopherols and tocotrienols. In this study, the vitamin E levels were reduced tremendously by DMBA in untreated BCC rats (group II). Group III treated with CP-P (0.2 mg/kg) showed significant elevated levels of vitamin E. More interestingly, the combination (0.1 mg/kg of CP-P+CYC each) treated rats (group V) greatly increased the vitamin E levels versus BCC control. Additionally, CYC (0.2 mg/kg) treated groups (IV) also exerted the higher level of vitamin E content. There was a gradual reduction of vitamin content due to the toxic side effects by CYC following 20 days treatment.in vitro cytotoxic activity of CP-P, with or without CYC, in breast cancer cell lines MCF-7 and MDA-MB-231. Second, we also found that CP-P induced significant apoptotic effects in MCF-7 cells, with or without CYC, which may account for its potential anti-cancer effects. Third, we also observed that CP-P suppressed NF-κB activation induced by TNF-α leading to the suppression of IκBα phosphorylation and degradation. We also observed that the suppression of TNF-α-induced IKK activation leads to the inhibition of IκBα phosphorylation and degradation in MCF-7 cells. CP-P also down-regulated the expression of various NF-κB-dependent gene products involved in cell proliferation (e.g. cyclin D1), and anti-apoptosis (e.g. Bcl-2). Several lines of evidence support the fact that NF-κB plays an essential role in the development and progression of breast cancer. First, studies have suggested the presence of constitutively active NF-κB at an early stage of neoplastic transformation of mammary cells In the present study, we investigated the effects of a natural protein product (CP-P) as a complementary therapy in DMBA-induced mammary tumorigenesis model. First, we found a significant Solanum pseudocapsicum (2.5 mg/kg doses) showed similar antitumor activity with increased survival time of tumor-bearing rats Additionally, we also observed the potential inhibitory effects of CP-P on the development of DMBA-induced breast cancer in rats. The 0.2 mg/kg dose of CP-P alone treated animals showed only 60.5% inhibition or reduction of tumor. A 0.2 mg/kg dose of CYC alone treated animals displayed at least 70% tumor reduction whereas the combination of CP-P (0.1 mg/kg)+CYC (0.1 mg/kg) treatment caused 74.5% reduction of tumor and effectively inhibits DMBA-induced tumors in rats. A single dose of DMBA induces mammary tumors in approximately 90 to 95% of female Sprague-Dawley (SD) rats Histopathological examination confirmed that the normal control rats displayed normal neoplasm, the CYC treated group showed low grade differentiation demonstrated by giant multinucleated cells, infiltration of tissues and necrotic effects due to CYC on rats as well as decreased cell density, infiltration, higher level of fibrosis, reduction of inflammation, faster neovascularization, non-invasiveness of tissue architecture observed in CP-P treated rats. However, the combination of CP-P+CYC treated group V showed glandular structures, an indication of functional differentiation due to maximal proliferative rates in the vasculature and epithelium as a result of antitumor effect. These data suggest that combination treatment prevents tumor progression significantly. As a result, the total number and volume of tumors per rat was decreased in the treated groups. In our previous study, pathological changes confirm the formation of tumor tubules and neovascularization after treatment Furthermore, the activities of enzymatic and non-enzymatic anti-oxidants were examined in the liver and mammary glands before and after 3 weeks treatment. CP-P treated groups  showed significantly (P<0.01*) elevated levels of SOD. The combination (CP-P+CYC)-treated group V exerted significant changes in the enzyme SOD levels than the CYC alone-treated rats. Glutathione S-transferases (GSTs) are a family of isoenzymes that play an important role in protecting cells from cytotoxins and carcinogens We also observed that in the BCC-group, the vitamin C content was drastically reduced after 90 days (13 weeks) due to carcinogen exposure. Whereas, in the CP-P administered group (III), a significant increase in vitamin C levels were noted, which may also account for its potential anti-cancer effects. Interestingly, it has been reported previously that the intake of vitamin C significantly reduces the incidence and growth of mammary tumors, and therefore has strong potential as a useful therapeutic for inhibiting breast cancer development Additionally, we also observed that vitamin E levels were significantly reduced by DMBA in BCC rats. Interestingly, CP-P treated rats also had increased vitamin E levels, which may also account for the observed anti-proliferative effects of CP-P. CP-P also increased the hepatic UDP-glucuronosyltransferase (UDP-GT) and glutathione-s-transferase (GST) activities. There was a decrease in the mammary DNA-DMBA adducts under the same treatment regimen that led to suppression of DMBA-induced mammary carcinogenesis. In addition, the combination treatment induced an apoptotic effect evidenced by TUNEL staining and significantly decreased the expression of cyclin D1 and Bcl-2 in breast tumor tissues.Results from this study confirm that CP-P has potential anticancer activity which is mainly attributed to its effect on the proliferation, apoptosis and redox status in breast cancer cells. Anticancer action occurs through the inhibition of the NF-κB activation cascade. Further studies are warranted to elucidate the anticancer potential of CP-P in breast cancer and use for other inflammatory conditions.3), sodium tungstate, sodium nitrate, methanol  and other reagents were of analytical grade. The drug cyclophosphamide (CYC) was obtained from the Apollo pharmacy, Chennai-600034. Antibodies against p65, p50, IkappaBalpha (IκBα), cyclin D1, B-cell lymphoma 2 (Bcl-2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), IkappaB kinase alpha (IKKα), and poly (ADP-ribose) polymerase (PARP) were obtained from Santa Cruz Biotechnology . Phospho-specific anti-IκBα (Ser32), phospho-specific anti-p65 (Ser536), and phospho-specific IKKα/β (Ser-180/181) were purchased from Cell Signaling .Phosphotungstic acid, thiobarbituric acid, trichloroacetic acid, acetic acid, sodium salicylate, and ethylenediaminetetraacetic acid were purchased from Hi-media Chemicals (Pvt) Ltd, Mumbai, India. Reduced Glutathione and 5, 5′Dithiobis (2-nitrobenzoic acid (DTNB), carcinogen 7, 12-Dimethylbenz(a)anthracene (DMBA), and β-actin antibody were purchased from Sigma Chemicals Co . Ascorbate, iron (III) chloride (FeClC. procera (CP) was collected in Thiruthani, Thiruvalluvar district, Tamil Nadu, India and properly identified Root-bark of The root-bark extract was used for purifying antitumor protein. The dried material was powdered with an electric blender. Powdered material (50 g) was suspended in 250 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.4), and the suspension was centrifuged at 250 g at 4°C for 15 min. Aliquots (5 ml) of the brownish clear supernatant were applied on a Superdex G-75 column  previously equilibrated and eluted with the same buffer. Fractions (2 ml each) were collected at a flow rate of 12 ml/h. Absorbance of all fractions was monitored at 280 nm. Five fractions  were collected from the single pool of clear CP supernatant fractionated on the Superdex column, with aliquots taken for protein measurement. The CP-3 fraction with highest protein content and antitumor activity was further purified by reverse-phase high-performance liquid chromatography  into two peaks (CP-F1 and CP-F2). The CP-F1 fraction was further separated by a C8 column (Jupitor Phenomex) in 0.1% aqueous trifluoroacetic acid (TFA) , and eluted with a linear gradient of 80% acetonitrile (ACN) in 0.1% TFA. These fractions were monitored at 215 nm Analyses were performed primarily using a perspective biosystem matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/MS) Voyager-DE™ mass spectrometer operated in delayed extraction mode. The biologically active fraction was analyzed using a saturated solution of α-cyano-4-hydroxycinnamic acid  in acetone containing 1% TFA. The active fraction was selected in the mass range of 1–70 kDa. Spectra were calibrated using the sequazyme mass standards kit . The database search program for mass [MS-Fit], was used in National Center for Biotechnology Information (NCBI) and matrix science mascot was used in the matrix science-help-sequence database (MSDB). MALDI-TOF mass spectrometry was used for molecular mass determination.2 incubator at 37°C. Serum-free medium was used for the above experimental conditions. The cells were grown up to 80% confluence prior to an experiment.Human breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from the American Type Culture Collection . The cells were cultured under standard tissue culture conditions and tested negative for mycoplasma contamination. The cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium , supplemented with 10% w/v fetal bovine serum (FBS) (Gibco), 1 mM sodium pyruvate, penicillin (100 units/ml) and streptomycin (100 units/ml) (Gibco) in a 5% CO6/ml) were incubated in triplicate in a 96-well plate with or without different concentrations of CP-P or CYC alone, or in combination (800–12.5 µg/ml) in a final volume of 0.2 ml for indicated time intervals at 37°C. Thereafter, MTT (20 µl of 5 mg/ml in PBS) was added to each well. After a 2 h incubation at 37°C, 0.1 ml lysis buffer  was added; incubation was continued overnight at 37°C and optical density (OD) at 490 nm was measured by a BenchMark Plus plate reader . The percent of cytotoxicity was calculated by comparing with the untreated control.The antiproliferative effect of CP-P against MCF-7 and MDA-MB-231 cells was determined by the 3--2, 5-diphenyltetrazolium bromide  dye uptake method as described previously The lactate dehydrogenase (LDH) release into media was an indicator of cell death detected according to the manufacturer's instructions . Briefly, after incubation, 200 µl of cell medium was transferred to a sterile 96-well plate with 50 µl of LDH reagent. After the reaction, the brownish red color development was measured at 440 nm by ELISA reader . Results are expressed as the percentage of LDH release versus control cells 5 cells/ml) were seeded onto sterile cover glass. Cells were initially fixed in 2.5% glutaraldehyde followed by post-fixation with 1% osmium tetroxide. Samples were then dehydrated with an ascending series of ethyl alcohol baths (50–100%) before subsequent embedding in araldite resin. Semi- and ultra-thin sections were cut using a glass knife. The ultrathin sections were doubly stained with uranyl acetate and lead citrate before viewing under an Olympus EM2085 microscope .TEM of CP-P alone, or in a combination with CYC, treated MCF-7 cancer cells were performed as previously described 50 dose, 24 h) MCF-7 cells were spun out of culture medium, washed with cold PBS, and fixed in 70% ethanol. The cells were then washed again, treated with DNase-free RNase A (10 µg/ml) at 37°C for 30 min, and stained with PI (200 µl from 50 µg/ml) for 15 min at 37°C. Cell cycle phase distribution of nuclear DNA was measured in a FACS caliber single laser flow cytometer  using a 488 nm argon laser light source and 623 nm band pass filter using Cell Quest software (n = 4).The phases of cell cycle distribution following CP-P alone, or in combination with CYC, treatment of MCF-7 cells were analyzed after propidium iodide (PI) staining by flow cytometry To determine protein expression levels, we prepared extracts and fractionated them by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously in situ with the use of exogenous terminal deoxynucleotidyl transfer (TdT). This is commonly defined as the TUNEL assay 6 cells/well) were grown in a 96-well plate and exposed to CP-P or CYC alone or in combination, for 24 h. Cells without treatment served as controls. Cells were fixed with 4% paraformaldehyde for 10 min. After fixation at room temperature, cells were washed twice with PBS for 5 min each and then incubated with 3% hydrogen peroxide (H2O2) to block endogenous peroxidase activity. After 10 min, the cells were incubated in the labeling reaction mixture containing TdT at 37°C for 1 h. The reaction was terminated by adding stop buffer and cells were washed 3 times with PBS for 2 min each. Cell nuclei were counterstained with Hoechst 33342  and mounted upon a cover slip with Dako fluorescent mounting medium. Cells were observed under fluorescence microscope .Activation of endonucleases that cleave chromosomal deoxyribonucleic acid (DNA) is a hallmark of apoptosis. DNA fragmentation revealed by a multitude of DNA strand breaks, therefore, is considered the gold standard for identifying apoptotic cells. Several methodologies are based on fluorochrome-labeling of 3-OH termini of DNA strand breaks Six week old female Sprague Dawely rats (250–300 gm body weight) were obtained from the National Institute of Nutrition,  and divided into five groups (n = 5 per group), rats subsequently maintained in a controlled condition including 12 h light and 12 h dark cycles. Standard diet and tap water was ad libitum. The experiment was conducted according to the guidelines of the Institutional Animal Ethical Committee for Experimentations on Animals (IAECEA), and approved by the University of Madras, India. Group I served as a normal control animals (NC), group II - breast cancer bearing-animals treated with saline served as the breast cancer control (BCC), group III - breast cancer-bearing animals treated with CP-P alone , group IV - breast cancer-bearing rats treated with CYC alone 0.2 mg/kg, i.p) and group V - breast cancer-bearing animals treated with CP-P +CYC . Induction of breast cancer: 1 mg of DMBA was dissolved in 0.5 ml of sunflower oil and 0.5 ml saline injected by subcutaneous injection beneath the mammary gland on either side (group II–V). The solid tumor developed after 90 days. After 13 weeks, the breast tumor-bearing rats were treated for 20 days Macroscopic mammary tumors, fixed in 10% buffered formalin, were embedded in paraffin using a conventional automated system. The blocks were cut to obtain 5 µm thick sections and stained with hemotoxylin-eosin (H & E) for histological examination The overnight-fasted animals were sacrificed by cervical decapitation; breast tissues were excised and quickly removed. The tissue (50 mg) was washed thoroughly with chilled PBS, cut into small pieces with a sterile heavy-duty blade, and finally homogenized on ice. The homogenate was centrifuged at 10,000 rpm for 15 min and supernatant diluted with PBS to a final concentration of protein (30 µg/ml). The biochemical anlysis of enzymatic antioxidant such as superoxide dismutase, catalase, glutathione-s-transferase, and non-enzymatic antioxidants such as reduced glutathione, vitamin C and E were estimated in the treated and untreated breast tissues of control rats.Superoxide activity was assessed by the nitroblue tetrazolium (NBT) reduction method Glutathione-S-transferase was assayed by the method described by Habig et al, 2O2) 0.4 ml was added to the supernatant. The reaction was stopped at 15, 30, 45 and 60 seconds by adding 0.2 ml of dichromate acetic acid. Tubes were boiled for 10 min, cooled and read at 620 nm. The standards for H2O2, ranging from 20–100 µm, were processed as above. Catalase activity was expressed as µmoles of H2O2 decomposed/min/mg of tissue protein.The catalase activity was determined spectrophotometrically according to the method described by Johansson and Borg, 2O2 was made up to 10 ml with double distilled water). Phosphate buffered solution (0.3 mM) and 50 mg of tissue homogenate or lysate were mixed and incubated at 37°C for 10 min. The reaction was arrested by adding 0.5 ml of trichloroacetic acid (TCA) (10%) and tubes were centrifuged 0.5 ml of supernatant. 0.3 ml of phosphate solution and 1.0 ml of DTNP-2,2′-dithiobis-(5-nitropyridine) (6 mM), 40 mg of DTNB dissolved in 100 ml of 1% trisodium citrate was added and colour development was read at 420 nm using a spectrophotometer. Glutathione activity was expressed as µg glutathione utilized/min/mg protein.Glutathione peroxide was assayed by the method described by Forstrom et al, Ascorbic acid level was estimated by the method described by Omaye et al, Vitamin E level was determined by the method described by Gupta et al, t-test. Multiple comparisons were done using ANOVA. The differences were statistically significant at *P<0.01; **P<0.05 levels.Statistical comparisons between control and treatment mean values of two parameters were analyzed using the Student's File S1Detailed Methodology.(DOC)Click here for additional data file.Table S1Detailed methodology for the supplementary materials of the amino acid sequence of the apolipoprotein A-I to which CP-P has been matched. A peptide summary report and clearer picture of MS/MS by mascot search results.(DOC)Click here for additional data file.Table S2Showed the highest score of apolipoprotein A-I matched with the CP-P protein.(DOC)Click here for additional data file.


Phytophthora infestans, remains a threat to potato production worldwide because newly evolved pathogen strains have consistently overcome major resistance genes. The potato RB gene, derived from the wild species Solanum bulbocastanum, confers resistance to most P. infestans strains through recognition of members of the pathogen effector family IPI-O. While the majority of IPI-O proteins are recognized by RB to elicit resistance , some family members are able to elude detection (e.g. IPI-O4). In addition, IPI-O4 blocks recognition of IPI-O1, leading to inactivation of RB-mediated programmed cell death. Here, we report results that elucidate molecular mechanisms governing resistance elicitation or suppression of RB by IPI-O. Our data indicate self-association of the RB coiled coil (CC) domain as well as a physical interaction between this domain and the effectors IPI-O4 and IPI-O1. We identified four amino acids within IPI-O that are critical for interaction with the RB CC domain and one of these amino acids, at position 129, determines hypersensitive response (HR) elicitation in planta. IPI-O1 mutant L129P fails to induce HR in presence of RB while IPI-O4 P129L gains the ability to induce an HR. Like IPI-O4, IPI-O1 L129P is also able to suppress the HR mediated by RB, indicating a critical step in the evolution of this gene family. Our results point to a model in which IPI-O effectors can affect RB function through interaction with the RB CC domain.Despite intensive breeding efforts, potato late blight, caused by the oomycete pathogen  Phytophthora infestans, is able to rapidly evolve to overcome resistance genes. The pathogen accomplishes this by secreting an arsenal of proteins, termed effectors, that function to modify host cells. Although hundreds of candidate effectors have been identified in P. infestans, their roles in pathogenicity or virulence remains basically unknown. Our results showed that one of these effectors functions to turn off resistance mediated by the potato gene RB. This effector accomplishes this by directly interacting with RB, which likely modifies its ability to turn on host resistance. Further molecular analysis identified two amino acids within the effector that determine interaction, which can assist in developing appropriate disease control strategies.The potato late blight pathogen,  Plant resistance to microbial pathogens is a complex process and includes a variety of constitutive and inducible defense mechanisms The majority of plant R proteins have nucleotide binding (NB) and leucine-rich repeat (LRR) motifs, and can be divided into two sub-classes based on a variable N-terminal domain, which typically contains coiled-coil (CC) motifs or includes homology to the Toll-interleukin-1 receptor (TIR) The mechanisms of pathogen effector perception by R proteins include both direct and indirect protein interactions. The R proteins Pi-ta Phytophothora infestans (Mont.) de Bary. The late blight R gene, RB , from the wild potato species S. bulbocastanum, encodes a CC-NB-LRR protein and confers broad-spectrum, partial resistance to most strains of the pathogen due to the almost ubiquitous presence of the corresponding effector IPI-O Phytophthora effectors with a highly conserved N-terminal RXLR motif and a C-terminal W motif P. infestans isolates, are recognized by RB, while class III variants (e.g. IPI-O4) are not P. infestans strains lacking a class I IPI-O are virulent on plants carrying RBP. infestans strains with class III variants are more aggressive on plants with RBRB, but is also capable of inhibiting the HR elicited IPI-O1 Potato and tomato late blight is caused by the oomycete pathogen RB-mediated HR. Our findings suggest a model in which IPI-O4 is able to affect RB function through interaction with the CC domain, possibly disrupting interactions that would otherwise lead to R protein activation.In the present study, we investigated intra- and intermolecular interactions of the RB protein using specific R protein domains and IPI-O variants. We identified amino acids that play a key role not only in the interaction between the RB CC domain and IPI-O4, but also in elicitation or suppression of an To investigate physical interactions between RB domains, pair-wise combinations of protein domains were assayed using a directed yeast two-hybrid interaction assay. Our results showed that none of the separate or fused domains of RB interacted with dissimilar domains . Self-asNicotiana benthamiana leaves. Expression of versions of the RB CC domain, IPI-O1, and IPI-O4 fused with green fluorescent protein (GFP) showed proteins of the expected sizes in leaves after detection with GFP antibodies, although some degradation products were observed  gained the ability to elicit the HR in  with RB . In contRB-transgenic N. benthamiana leaves with IPI-O1 and IPI-O1 L129P to test whether this mutant is able to suppress RB-mediated HR elicitation  suppresses resistance mediated by the tomato I-2 and I-3 genes, but tomato plants with either I or I-1 are able to recognize Avr1 to elicit resistance P. syringae expressing AvrPtoB, a ubiquitin ligase N. benthamiana expressing RBin planta might result in a direct interaction. The ability of the IPI-O1 L129P mutant to suppress IPI-O1 elicitation of the HR in the presence of RB demonstrates a critical step in the evolution of this effector family. This finding suggests that a simple mutation at this location in IPI-O1 could alter pathogen virulence when RB is present. Our previous analysis of IPI-O variants from P. infestans isolates identified two alleles outside of the IPI-O4 class that contain a proline at amino acid 129 RB gene indicating its ability to suppress resistance responses Effectors from different types of pathogens have been found to suppress host basal defense or hypersensitive cell death induced by elicitors or effectors even in the presence of cognate R proteins N. benthamiana has allowed us to rapidly identify specific amino acids within a pathogen effector that condition host resistance. Four amino acids in IPI-O play an important role in RB CC/IPI-O4 interaction and one of them also determines IPI-O1 recognition by RB. All four amino acids are located C-terminal to the RXLR motif, which is consistent with the fact that the RXLR motif is responsible for translocation The utilization of transient effector expression in in planta if the effector molecule itself is recognized by RB and triggers the HR. The important role of CC domain interaction is further demonstrated by the fact that IPI-O4 mutants that are compromised in RB CC domain interactions are also compromised in suppressing HR induced by IPI-O1.The two single amino acid mutants of IPI-O4, P129L and G135S, lost the ability to interact with the RB CC domain. However, the double mutant IPI-O4 P129L/G135S elicited the HR and regained the ability to interact with the CC domain, demonstrating a key link between IPI-O/CC interaction and resistance elicitation. These results also suggest that HR elicitation by IPI-O4 P129L/G135S is epistatic to interaction with the CC domain. In other words, interaction with the CC domain is not sufficient to inhibit the HR in planta, but not in yeast. The IPI-O1 fusion protein is stable in yeast, suggesting that perhaps an additional accessory protein, found only in planta, is necessary for stability of the interaction with the RB CC domain. Our simplified model does not account for this. Combined with the fact that GFP:IPI-O1 fusion protein was not as abundant as GFP:IPI-O4 in N. benthamiana leaves, we hypothesize that IPI-O1 protein accumulation is affected by factors in the plant cell that interfere with its ability to suppress RB oligomerization and elicit resistance.Collectively, our data, combined with data from other CC-NB-LRR proteins, is consistent with the model shown in P. infestans delivery of IPI-O effectors into the plant cell could be responsible for the pathogen's ability to elude recognition and cause disease. If this is true, S. bulbocastanum RB is one of the latest in a line of R proteins that are able to recognize the presence of IPI-O1 while escaping suppression by this effector. Our ability to understand the molecular interactions that condition resistance and suppression will hopefully allow us to design or identify RB alleles that can continue to effectively control disease epidemics. A RB homolog that recognizes IPI-O4 has been found in the wild species of potato S. stoloniferumS. pinnatisectum showed no interaction with either IPI-O1 or IPI-O4. We are currently pursuing research focused on answering whether an engineered RB containing the CC domain from S. pinnatisectum may also escape suppression by IPI-O4. Despite the fact that P. infestans isolates that overcome RB resistance have been found RB remains valuable for potato breeding due to the almost ubiquitous existence of IPI-O1 in strains from major potato growing regions. However, due to the adaptive nature P. infestans and the continual proliferation of strains of P. infestans, we expect that the benefits of using the RB gene for late blight resistance will be ephemeral. Significant efforts must continue to be made to identify and integrate novel resistance. Pyramiding of RB, RB orthologs, and modified RB recognizing different IPI-O variants is one strategy that could be used to expand the durability of this gene.Our data demonstrates a co-evolutionary arms race between IPI-O and RB. The fact that several late blight susceptible potato species contain RB-like proteins with CC domains that interact with both IPI-O1 and IPI-O4, suggests that BamHI and SalI digested pSos or EcoRI and SalI digested pMyr, respectively.The CytoTrap system (Agilent) was used to detect protein-protein interactions. IPI-O1, IPI-O4, full length RB [amino acids (aa) 1–970], the CC domain of RB (aa 1–165), the NB domain of RB (aa 166–521), the CC-NB domain of RB (aa 1–521) and the LRR domain of RB (aa 522–970) were amplified with primers listed in Table S1 using Platinum PCR SuperMix High Fidelity (Invitrogen). IPI-O1 (PexRD6-1) and IPI-O4 (PexRD6-3) targets were those described in Vleeshouwers et al. 600 = 0.6 at 24°C in 10 ml SD/glucose (-L) media. Yeast cells with no pSos vector were grown in YPAD broth. Protein purification was performed according to the CytoTrap system instruction manual. Protein pellets were resuspended in 300 µl of SU buffer. 20 µl of each protein sample was heated at 65°C for 3 minutes, separated by 10% SDS-PAGE and transferred to Hybond-P . Immunoblotting was performed using a mouse monoclonal anti-Sos primary antibody  and an anti-mouse secondary antibody produced in goat . Antibody detection was carried out using an ECL Western blotting detection system  according to the manufacturer's instructions.Cdc25 yeast strains containing RB domains or IPI-O variants in pSos were grown to ODRB homologous CC fragments using primers RBFORBAMHI and RBCCREVSALI (Table S1). PCR conditions and cloning into pSos vectors was carried out as described above.Seeds from 10 wild species of potato, shown in P. infestans effectors IPI-O1 and IPI-O4 were generated by circular PCR using IPI-O1 and IpiO4 in pMyr as templates. A pair of oligonucleotide primers containing the desired nucleotide substitution (Table S1), each complementary to the opposite strands of the same target sequence, were extended during temperature cycling using PfuTurbo DNA polymerase (Agilent). After the temperature cycling, the PCR products were treated with DpnI for 2 hr at 37°C to digest the methylated parental DNA template and then transformed into E. coli. The resultant mutated plasmid constructs were verified by sequencing.Mutants of BamHI and SacI restriction enzyme sites. Primer sequences are listed in Table S1. PCR products were ligated into BamHI and SacI digested binary vector pBI121. All constructs were introduced into A. tumefaciens strain GV3101 by electroporation.The wild type and mutant IPI-O1 and IPI-O4 in pMyr were used as templates for PCR amplification to incorporate RB-transgenic N. benthamiana plants Wild type and BamHI and XhoI restriction enzyme sites. Primer sequences are listed in Table S1. PCR products were ligated into BamHI and XhoI digested pENTR1a (Invitrogen). Gateway LR reactions (Invitrogen) were performed to introduce wild type IPI-O1 and mutants into the binary vector pEG201 N. benthamiana leaves were harvested 21 hours after agroinfiltration. To prepare total protein, plant material was ground in liquid nitrogen and thawed in extraction buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, and a protease inhibitor cocktail according to the manufacturer's instructions (Sigma). Samples were incubated on ice for 1 hour. The suspension was centrifuged at 4000 g at 4°C 6 times, each time for 10 min. In the protein blot assay, 100 mg of ground plant material was thawed in 100 µl of extraction buffer, and 25 µl of the supernatant was resolved on a 12% SDS-PAGE gel and transferred to Hybond-P  according to the manufacturers recommendations. Protein was detected using an anti-HA-peroxidase antibody (Roche) with an ECL Western blotting detection system  according the manufacturer's instructions. In the co-immnunoprecipitation assay, 1 gram of ground plant material was thawed in 1 ml of extraction buffer. Seven µl of anti-GFP antibody (Clontech) was added to each protein sample, followed by incubation for 1 hour with end-to-end shaking at 4°C. 50 µl of pre-equlibrated protein A agarose (Roche) was then added to each sample and incubated for 4 hours with end-to-end shaking at 4°C. The beads were spun at 4000 g at 4°C and washed with 1 ml of wash buffer (50 mM Tris (pH 7.5), 150 mM NaCl) 6 times (15 min each time) with end-to-end shaking at 4°C. After washing, the beads were eluted with 45 µl of 2× SDS loading buffer and heated at 95°C for 7 min. The eluted protein was separated on a 10% SDS-PAGE gel and transferred to PVDF membrane and detected with anti-Myc-HRP antibodies (Sigma) using an ECL Western blotting detection system .The wild type and mutant IPI-O1, IPI-O4 and the RB CC domain in pMyr were used as templates for PCR amplification to incorporate Figure S1Yeast two-hybrid screening of RB domain interactions. Each panel shows three independent transformants of identical genotypes. Positive and negative controls were provided by the CytoTrap system manufacturer. RB = full-length RB; CC = RB coiled-coil domain; NB = RB nucleotide binding domain; LRR = RB leucine-rich repeat domain. All spots contained similar quantities of yeast at the time of plating. Pictures were taken after 8 days of growth on selective media.(TIF)Click here for additional data file.Figure S2Protein blotting showing stability of IPI-O and CC SOS-domain fusions in yeast. A–D) Total yeast proteins were separated on separate acrylamide gels, blotted, and probed with SOS-specific antibody. Ponceau S stained PVDF membranes are shown below the results of protein blotting and antibody detection.(TIF)Click here for additional data file.Figure S3Protein blotting of the input protein for co-immunoprecipitation. Total protein from leaf sections agroinfiltrated with the indicated constructs was extracted and separated on an acrylamide gel. Protein was blotted and detected using a GFP-specific antibody (A) or a Myc-tag specific antibody (B). Ponceau S stained PVDF membranes are shown to demonstrate equal loading.(TIF)Click here for additional data file.Figure S4Amino acid sequence identity chart showing the pairwise percent identity between RB CC domains amplified from wild potato species. Sequence names contain the three-letter abbreviation for each species. Numbers after the species name represents the PCR clone number.(TIF)Click here for additional data file.Figure S5Sequence alignment of RB CC domain deduced amino acid sequences. Letters in black boxes are identical to the consensus sequence (shown at top of each row). Sequence names contain the three-letter abbreviation for each species. Numbers after the species name represents the PCR clone number.(TIF)Click here for additional data file.Figure S6RB-transgenic N. benthamiana leaves. A protein blot was performed using total protein extracts following agroinfiltration with constructs expressing indicated HA-IPI-O1 mutants. The 18-kDa protein band represents the expected size of recombinant IPI-O1 mutants. Lane1: wild type IPI-O1; Lane2: non-infiltrated N. benthamiana; Lane3: IPI-O1 L129P; Lane4: IPI-O1 Y82K/L129P; Lane5: IPI-O1 V86G/L129P; Lane6: IPI-O1 L129P/S135G.IPI-O1 mutants containing L129P are stable in (TIF)Click here for additional data file.Figure S7A. tumefaciens strains expressing IPI-O mutants or the indicated controls were infiltrated into leaves of RB transgenic N. benthamiana plants. Leaves were photographed at 6 days after infiltration.IPI-O1 L129P inhibits the HR induced by IPI-O1. (TIF)Click here for additional data file.Figure S8A. tumefaciens strains expressing IPI-O mutants or the indicated controls were infiltrated into leaves of RB transgenic N. benthamiana plants. Leaves were photographed at 6 days after infiltration.IPI-O4 inhibits the HR induced by the IPI-O4 P129L/G135S double mutant. (TIF)Click here for additional data file.Figure S9A. tumefaciens strains expressing IPI-O mutants or the indicated controls were infiltrated into leaves of RB transgenic N. benthamiana plants. Leaves were photographed at 6 days after infiltration. Note that the inhibitory effect of these mutants is not as strong as that of IPI-O1 L129P since some cell death was still observed in the area coinfiltrated with IPI-O1 and IPI-O4 K82Y (A), G86V (B), or G135S (C).IPI-O4 K82Y, G86V, and G135S inhibit the HR induced by IPI-O1. (TIF)Click here for additional data file.


Peucedanum praeruptorum Dunn and has several biological activities, but its anti-osteoporotic activity has not been studied yet.A decrease of bone mass is a major risk factor for fracture. Several natural products have traditionally been used as herbal medicines to prevent and/or treat bone disorders including osteoporosis. Praeruptorin A is isolated from the dry root extract of 2+ levels were also measured.The effect of praeruptorin A on the differentiation of bone marrow–derived macrophages into osteoclasts was examined by phenotype assay and confirmed by real-time PCR and immunoblotting. The involvement of NFATc1 in the anti-osteoclastogenic action of praeruptorin A was evaluated by its lentiviral ectopic expression. Intracellular Ca2+ oscillation without any changes in the phosphorylation of PLCγ.Praeruptorin A inhibited the RANKL-stimulated osteoclast differentiation accompanied by inhibition of p38 and Akt signaling, which could be the reason for praeruptorin A-downregulated expression levels of c-Fos and NFATc1, transcription factors that regulate osteoclast-specific genes, as well as osteoclast fusion-related molecules. The anti-osteoclastogenic effect of praeruptorin A was rescued by overexpression of NFATc1. Praeruptorin A strongly prevented the RANKL-induced Ca2+ oscillation.Praeruptorin A could exhibit its anti-osteoclastogenic activity by inhibiting p38/Akt-c-Fos-NFATc1 signaling and PLCγ-independent Ca Bone fracture is a public health problem because it occurs easily in patients with bone-related disorders including osteoporosis. As the elderly population is rapidly increasing, the medical costs of hospitalization caused by fractures have been become a serious social issue Bone homeostasis depends on the balance between osteoblastic bone formation and osteoclastic bone resorption, but an imbalance caused by an increased number of osteoclasts or overactivation can lead to impaired bone structure and low bone mass, which are common characteristics in patients with bone disorders Clinically, osteoclast-targeting bisphosphonates have been widely used to treat patients with osteoporosis and/or prevent osteoporotic fracture. The orally available bisphosphonates principally inhibit the activation of osteoclasts by binding to hydroxyapatite Peucedanum praeruptorum Dunn, which has been used as an herbal medicine with several pharmacological activities Praeruptorin A  is isolaPraeruptorin A was purchased from Stanford Chemicals (CA). Mouse soluble receptor activator of nuclear factor-κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) were purchased from R&D Systems (MN). Penicillin, streptomycin, cell culture medium, and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies (NY). The CCK-8 assay kit was from Dojindo Molecular Technologies (ML). Antibodies against c-Fos, NFATc1 and actin were from Santa Cruz Biotechnology (CA). Antibodies against p-p38, p38, p-JNK, JNK, p-ERK, ERK, Akt, p-Akt (Ser473), PLCγ and p-PLCγ were obtained from Cell Signaling Technology (MA).4 cells/well in a 96-well plate or 3×105 cells/well in a 6-well plate) were cultured in the presence of M-CSF (30 ng/ml) and RANKL (10 ng/ml) for 4 days.This study was carried out in strict accordance with the recommendations in the Standard Protocol for Animal Study of Korea Research Institute of Chemical Technology . The protocol (ID No. 7D-M1) was approved by the Institutional Animal Care and Use Committee of KRICT (IACUC-KRICT). All efforts were made to minimize suffering. In detail, after cervical dislocation, bone marrow cells were obtained from 5-wk-old male ICR mice  by flushing femurs and tibias with α-MEM supplemented with antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin). Bone marrow cells were cultured for 1 day on a culture dish in α-MEM supplemented with 10% FBS and M-CSF (10 ng/ml). Non-adherent bone marrow cells were plated on a Petri dish and cultured for 3 days in the presence of M-CSF (30 ng/ml). After non-adherent cells were washed out, adherent cells were used as bone marrow-derived macrophages (BMMs). For osteoclastogenesis, BMMs (1×10p-nitrophenyl phosphate (Sigma-Aldrich) at 37°C for 5min. Reaction mixtures were transferred into a new plate containing an equal volume of 0.1 N NaOH, and optical density values were determined at 405 nm in a Wallac EnVision microplate reader .Cells were fixed with 3.7% formaldehyde for 5 min, permeabilized with 0.1% Triton X-100 for 5 min, and stained with the Leukocyte Acid Phosphatase Kit 387-A . TRAP-positive multinuclear cells with three or more nuclei were counted as osteoclasts. To measure TRAP activity, cells were treated with TRAP buffer  including 3 mM 4 cells/well on a 96-well plate in triplicate. After treatment with M-CSF (30 ng/ml) and praeruptorin A, cells were cultured for 3 days. Then, cell viability was measured with the CCK-8 kit according to the manufacturer’s protocol.BMMs were plated at a density of 1 × 10g for 15 min. After protein quantification of the supernatants by the BCA protein assay , proteins were denatured, separated on SDS-PAGE gels, and transferred onto PVDF membranes . After incubation with antibody, the membranes were developed using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) and visualized with the LAS-3000 luminescent image analyzer . ImageJ software-based quantification of the detected bands was performed, and the relative, normalized ratio between the density of phosphorylated form and that of the protein itself or actin was presented in each figure.Western blot analysis was performed as described previously 4 cells/well on a 48-well plate in triplicate for 1 day. Plasmids containing NFAT-luc (100 ng/well), pcDNA3.1-RANK (100 ng/well), and pRL-Renilla (20 ng/well) were transfected into HEK293T cells with Lipofectamine 2000  according to the manufacturer’s protocol. After 6 h, transfected HEK293T cells were treated with hRANKL (50 ng/mL) and praeruptorin A for 2 days. The luciferase activity was detected in cell extracts with the Dual-Luciferase Reporter Assay System  and Wallac EnVision microplate reader (PerkinElmer). Luciferase activity assessed in triplicates was normalized to the average renilla luciferase activity.Luciferase reporter plasmid, NFATc1-luc vector was purchased from Clontech (CA) and pRL-Renilla control vector was purchased from Promega (WI). The full-length human RANK cDNA was amplified from human leukocyte cDNA from Clontech (CA) and cloned into the HindIII-EcoRI site of pcDNA3.1 (Invitrogen). Recombinant human RANKL (hRNAKL) was purchased from R&D Systems (CA). For measurement of luciferase activity, human embryonic kidney HEK293T cells were plated at a density of 5×10−ΔΔCT method t-test with GAPDH-normalized 2−ΔΔCT values; differences were considered significant at p<0.05.Real-time PCR was performed as described previously Retrovirus preparation and infection were conducted as described previously 5 cells on the cover glass and cultured in the presence of RANKL (10 ng/ml) and M-CSF (30 ng/ml) for 24 h. Cells were incubated with vehicle (DMSO) or praeruptorin A (10 µM) for 2 h, treated with Fura-2, AM (5 µM), and then placed in the perfusion chamber to allow continuous perfusion of bath solution  until each experiment was complete. Fluorescent dyes inside the cells were excited at wavelengths of 340 nm and 380 nm sequentially and the emitted fluorescence was passed through 510 nm cut-off filter. Each image was collected with a CCD camera and analyzed with MetaFluor software. All results were digitized to the mean of the ratio (340 nm/380 nm).Isolated BMMs were seeded at a density of 1×10t-test. A value of p<0.05 was considered significant.All quantitative values are presented as mean ± SD. Statistical differences were analyzed using Student’s The effect of praeruptorin A on RANKL-induced osteoclastogenesis was evaluated in BMMs. Pre-treatment with praeruptorin A before RANKL treatment strongly prevented the RANKL-induced formation of TRAP-positive multinucleated cells in a dose-dependent manner . ConsistAnti-osteoclastogenic activity of praeruptorin A was confirmed by staining actin ring of mature osteoclasts . This reAdditionally, the inhibitory effect of praeruptorin A on the expression of transcription factors such as c-Fos and NFATc1 was evaluated by Western blot analysis. As shown in Additionally, the inhibitory effect of praeruptorin A on the activation of NFATc1 was revealed by the NFATc1 luciferase activity assay ; RANKL sThe anti-osteoclastogenic activity of praeruptorin A was confirmed by evaluating the mRNA expression levels of osteoclast-specific genes. Consistent with the previous data, praeruptorin A significantly attenuated the RANKL-induced mRNA expressions of TRAP and NFATc1 . Since NThe anti-osteoclastogenic action of praeruptorin A could result from its potential to block p38 and/or Akt signaling pathways that subsequently affect the expression and/or activity of c-Fos and the most distal transcription factor, NFATc1. To clarify this hypothesis, we investigated whether ectopic expression of the constitutively active form of NFATc1 (CA-NFATc1) could rescue the praeruptorin A-inhibited formation of TRAP-positive multinucleated osteoclasts. Based on GFP signaling, both the control GFP and CA-NFATc1-GFP plasmid were infected well, and the overexpression of NFATc1 was confirmed by Western blot analysis . Consist2+ channel blocking activity of praeruptorin A 2+ oscillation. As shown in 2+ oscillation was triggered, but not with M-CSF alone. However, additional treatment with praeruptorin A for 2 h before measuring Ca2+ oscillation completely inhibited the RANKL-induced Ca2+ oscillation.Since several studies have reported the Ca2+ oscillation by blocking the RANKL-induced phosphorylation of PLCγ. In BMMs, the RANKL-induced phosphorylation of PLCγ was not changed by praeruptorin A.We further examined the possibility that praeruptorin A could inhibit the RANKL-induced CaOsteoclasts are functionally essential for sustaining bone health. However, the overactivation of osteoclasts and/or their increased number can lead to diseases characterized to bone loss, which is a risk factor for fracture. In this study, praeruptorin A attenuated the RANKL-induced osteoclastogenesis in a dose-dependent manner without any cytotoxicity upto 10 µM. Anti-osteoclastogenic activity of praeruptorin A was independent on age, strain and sex of mouse , and impRANKL is the most essential cytokine for osteoclast differentiation (or osteoclastogenesis) Here, praeruptorin A attenuated the RANKL-induced phosphorylation of p38 without affecting JNK and ERK. A pharmacological inhibition experiment using the p38 inhibitor SB203580 revealed direct involvement of p38 in the RANKL-induced osteoclast differentiation Moreover, praeruptorin A also attenuated the RANKL-induced phosphorylation of Akt. Akt has been known to play a critical role in the survival of osteoclasts rather than in osteoclast differentiation through the phosphoinositide 3-kinase (PI3K) kinase signaling pathway 2+ signaling pathway, which is crucial for the activation of NFATc1 Thus, the anti-osteoclastogenic action of praeruptorin A could be due to its potential to inhibit both p38 and Akt signaling pathways that consequently downregulate the expression and/or activity of c-Fos and NFATc1. In particular, NFATc1, which is mainly regulated by c-Fos during osteoclastogenesis, plays a role as the most distal transcription factor required for regulating the expression of osteoclast-specific genes including TRAP, OSCAR, DC-STAMP, cathepsin K and c-Src These results suggested that the downregulation of NFATc1 could be the outcome of the anti-osteoclastogenic action of praeruptorin A by inhibiting p38 and Akt signaling pathways. The hypothesis was proved by the ectopic expression of the constitutively active form of NFATc1; it significantly rescued the anti-osteoclastogenic action of praeruptorin A. The rescue of defected osteoclastogenesis by overexpression of NFATc1 has been reported in several studies Furthermore, the overexpression of c-Fos did not significantly rescue the effect of praeruptorin A on osteoclast differentiation , but the2+ mobilization 2+ mobilization affects the activation of NFATc1 required for regulating osteoclast-specific genes 2+ channel blocking activity of praeruptorin A 2+ oscillation via PLCγ could also downregulate the activity of NFATc1 during osteoclast differentiation. Interestingly, the RANKL-triggered Ca2+ oscillation was inhibited by praeruptorin A, but the RANKL-induced phosphorylation of PLCγ was not changed by praeruptorin A. These data suggest that the anti-osteoclastogenic activity of praeruptorin A involves inhibition of PLCγ-independent Ca2+ oscillation.In osteoclast differentiation, RANKL also triggers the activation of PLCγ, which subsequently leads to Ca2+ oscillation that consequently affect the expression and/or activity of the osteoclast-specific transcription factors, c-Fos and NFATc1. Also, NF-κB signaling was shown to be partly involved in the anti-osteoclastogenic action of praeruptorin A. In a further study, the binding molecules (or target proteins) of praeruptorin A might be identified, and the mechanism how praeruptorin A inhibits the fusion of preosteoclasts and the pit formation of mature osteoclasts might be elucidated.This is the first report of the anti-osteoclastogenic activity of praeruptorin A and its mode of action; praeruptorin A could inhibit the RANKL-induced osteoclast differentiation by inhibiting p38 and Akt signaling pathways and PLCγ-independent CaFigure S1Effect of praeruptorin A on cell spreading during RANKL-induced osteoclast differentiation. BMMs (1×104 cells/well) were seeded in a 96-well plate, treated with the vehicle (0.1% DMSO) or praeruptorin A (10 µM) for 2 h in the presence of M-CSF (30 ng/ml), and incubated with RANKL (10 ng/ml) for 1 and 3 days. Then, cells were fixed, permeabilized, washed, and incubated with 10 µg/ml Hoechst 33342.(TIF)Click here for additional data file.Figure S2Effect of praeruptorin A on RANKL-induced osteoclast differentiation for the indicated periods. BMMs were cultured with praeruptorin A (10 µM) for various times periods (indicated the black arrow) in the presence of M-CSF and RANKL. After TRAP staining, TRAP-positive multinuclear cells  were counted *, P<0.05; **, P<0.01; ***P<0.001.(TIF)Click here for additional data file.Figure S3Effect of praeruptorin A on the formation of actin rings during osteoclast differentiation. BMMs (1×104 cells/well) were seeded in a 96-well plate, treated with the vehicle (0.1% DMSO) or praeruptorin A (10 µM) for 2 h in the presence of M-CSF (30 ng/ml), and incubated with RANKL (10 ng/ml) for 4 days. Then, cells were fixed, permeabilized, washed, and stained with Hoechst 33342 and phalloidin-FITC for nucleus and actin rings, respectively.(TIF)Click here for additional data file.Figure S4P<0.05; **, P<0.01; ***P<0.001.(A) Effect of age, strain or sex on anti-osteoclastogenic action of praeruptorin A. BMMs were isolated from mice  and cultured with praeruptorin A in the presence of M-CSF and RANKL for 4 days. Osteoclast differentiation was visualized by TRAP staining. (B) Effect of age, strain or sex on anti-osteoclastogenic action of praeruptorin A. BMMs were isolated from mice  and cultured with praeruptorin A in the presence of M-CSF and RANKL for 4 days. After TRAP staining, TRAP-positive multinuclear cells  were counted. TRAP activity and cell viability were also evaluated. *, (TIF)Click here for additional data file.Figure S5Anti-resorptive activity of praeruptorin A. (A) After co-culturing BMMs with osteoblasts for 7 days, multinucleated osteoclasts were replated on BioCoat Osteologic MultiTest slides and after 2 h incubation, cells were further incubated with praeruptorin A and RANKL for 6 h. Then, cells were stained for TRAP (upper images). (B) TRAP-positive multinucleated cells were counted. (C) After removing cells, the resorption pits (indicated by asterisks in bottom images) were observed under a light microscope. The relative resorbing areas were evaluated using the ImageJ program. ***P<0.001.(TIF)Click here for additional data file.Figure S6Effect of c-Fos on anti-osteoclastogenic action of praeruptorin A. BMMs were infected with retroviruses harboring the control GFP or c-Fos-GFP vectors. Transduced BMMs were cultured with RANKL (10 ng/ml) and M-CSF (30 ng/ml) in the presence of praeruptorin A (10 µM) or the vehicle (0.1% DMSO). (A) After incubation for 2 days, GFP expression was visualized under a fluorescence microscope (left images). After 2 additional days, mature TRAP-positive multinucleated osteoclasts were visualized by TRAP staining (middle and right images). TRAP-positive cells (nuclear number >3) were counted as osteoclasts (B), and TRAP activity was measured at 405 nm (C). *, P<0.05; **, P<0.01; ***P<0.001.(TIF)Click here for additional data file.Figure S7Effect of praeruptorin A on RANKL-induced activation of NF-κB signaling pathway. BMMs were treated with praeruptorin A (10 µM) for 30 min, stimulated with RANKL (10 ng/ml) for the indicated time. The expression levels of molecules in cytoplasmic or nuclear protein fractions were evaluated by Western blot analysis. Actin and lamin B1 were used for the loading control of cytosolic and nuclear proteins, respectively. Densitometric analysis was performed using ImageJ software and the relative, normalized ratios of IκBα/actin, p-IκBα/actin, cytosolic p65/actin or nuclear p65/lamin B1 were presented.(TIF)Click here for additional data file.File S1Materials and Methods.(DOC)Click here for additional data file.


The aspartic protease cathepsin-D (cath-D) is overexpressed by human epithelial breast cancer cells and is closely correlated with poor prognosis in breast cancer. The adipocyte is one of the most prominent cell types in the tumor-microenvironment of breast cancer, and clinical studies have shown that obesity increases the incidence of breast cancer. Here, we provide the first evidence that cath-D expression is up-regulated in adipose tissue from obese human beings, as well as in adipocytes from the obese C57BI6/J mouse. Cath-D expression is also increased during human and mouse adipocyte differentiation. We show that cath-D silencing in 3T3-F442A murine preadipocytes leads to lipid-depleted cells after adipogenesis induction, and inhibits of the expression of PPARγ, HSL and aP2 adipocyte differentiation markers. Altogether, our findings demonstrate the key role of cath-D in the control of adipogenesis, and suggest that cath-D may be a novel target in obesity. The consumption of foods containing high levels of fat and carbohydrates is a major cause of obesity, resulting in the formation of excessive white adipose tissue. This increase in adipose tissue mass results from a combination of hypertrophy of existing adipocytes (hypertrophic adipocytes), and adipogenic differentiation of precursor cells (adipocyte hyperplasia). Recently, clinical studies have shown that obesity is a major risk factor for cancer Interestingly, proteases have also been recently shown to affect the biology of the adipocyte. The metalloproteinases Here, we investigated the expression of cath-D in adipocytes from obese subjects, and its role in the control of adipogenesis. We show, for the first time, that cath-D expression is up-regulated in mouse and human obese adipose tissues, as well as during mouse and human adipogenesis. We also demonstrate that cath-D silencing inhibits the adipogenic process, indicating the crucial positive role of cath-D in adipogenesis.Because of the recently established relationship between obesity and cancer incidence Cath-D mRNA expression was investigated in intra-abdominal visceral adipose tissue (VAT) from lean and obese human subjects , panel aIn order to find out whether this up-regulation of cath-D was a general characteristic of adipocytes from obese subjects, we next analysed cath-D mRNA levels in adipocytes isolated from C57BI6/J mice fed either a High Fat Diet (HFD) or a Normal Diet (ND) . HFD-fedSince we were not aware of any report establishing that cath-D protein is expressed in adipocyte cells, we analysed cath-D expression in well-established mouse adipocyte cell lines (3T3-F442A and 3T3-L1), and compared it to that in mouse fibroblasts (NIH-3T3). Cath-D is synthesized as a 52-kDa precursor that is rapidly converted in endosomes as an active 48-kDa single-chain intermediate, and then in the lysosomes into the fully active mature protease, composed of a 34 kDa heavy chain and a 14 kDa light chain. As per se and not by insulin.Obesity is characterized by the increased intracellular accumulation of lipids, a characteristic of adipocyte differentiation that is significantly correlated with adipocyte differentiation. Since cath-D expression was up-regulated in obese tissues, we next investigated whether cath-D plays any role in adipogenesis. We first investigated the regulation of cath-D expression during the course of differentiation of the 3T3-F442A preadipocyte cell line, a valuable model of adipogenesis Although 3T3-F442A and 3T3-L1 cells are valuable experimental models, these preadipocytes have some distinct attributes compared with human cells in primary culture beyond the obvious species differences. We therefore next investigated the regulation of cath-D expression during adipogenesis in human preadipocytes purified from abdominal subcutaneous adipose tissue Since cath-D was up-regulated in tissues from obese individuals, and as its expression increased during the adipocytic process, we went on to investigate whether preadipocytes require cath-D expression to differentiate into mature adipocytes. Cath-D expression was stably silenced in 3T3-F442A preadipocytes with cath-D shRNA1 and shRNA2 generating the D10 and A4 clones, respectively . ControlTo evaluate the consequences of cath-D silencing on adipocyte differentiation, we analysed the cellular lipid levels in adipocytes which had or had not been silenced for cath-D at day 7 of differentiation . Indeed,Our results demonstrate, for the first time, that cath-D expression is up-regulated in abdominal visceral and subcutaneous tissues from obese human subjects. A previous study observed cath-D mRNA expression in abdominal subcutaneous adipose tissue Our data show that cath-D expression increases gradually as 3T3-F442A and 3T3-L1 cells differentiate into mature adipocytes. Interestingly, our findings also reveal that fully-differentiated 3T3-F442A adipocytes secrete pro-cath-D, suggesting a potential new function as an adipokine. A recent study analysing the secretome of adipocytes reported that cath-D is a secretory protein induced by insulin Our report highlights that cath-D silencing by shRNAs in 3T3-F442A preadipocytes leads to lipid-depleted cells, and to a reduction of the expression of PPARγ, HSL and aP2 adipocyte markers of differentiation, indicating the essential role of cath-D in the differentiation process of preadipocytes. Given that cath-D protein is required for adipocyte differentiation and as it is abundantly expressed in fully-differentiated adipocytes, we propose that cath-D may be involved in the onset of obesity. Moreover, murine cath-D deficiency led to the accumulation of cholesteryl esters in the brain, suggesting that cath-D may play a key role in lipid metabolism Taken together, our findings reveal that cath-D is up-regulated in tissues from obese humans and mice, and that it is a novel key regulator of adipogenesis. We propose that targeting cath-D in obesity could reduce the number of hypertrophic adipocytes and decrease adipocyte hyperplasia. In the future, we intend to investigate the mechanism(s) by which cath-D controls adipogenesis. Since experimental studies have demonstrated that adipocytes play a supportive role in breast growth All subjects gave their informed written consent to participate to the study.http://www.wma.net/e/policy/b3.htm) and approved by the Ethical commitee of the Rangueil hospital  with permit number 07/858/03/07. Mice were handled in accordance with National Institute of Medical Research (INSERM) principles and guidelines. C57Bl6/J female mice were obtained from Charles River laboratory . Philippe Valet, with the license number # 31–147 given by the French government, performed the experiments on the animals. This study was approved by the ethics committee of Midi-Pyrénées (France) with the permit number MP/01/01/01/2010.Our protocol is using adipose tissues from plastic surgery considered as “waste”, and we thus do not need a specific agreement. However the study was performed accordingly to the declaration of Helsinki as revised in 2000 (Cell lines were cultured in DMEM (Invitrogen) supplemented with 10% fetal calf serum (FCS). Differentiation was induced by incubating confluent 3T3-F442A cells in differentiation medium (DMEM supplemented with 10% FCS and 50 nM insulin) as described Total RNA was extracted using the Reasy minikit  according to the manufacturer's instructions. Reverse transcription of total RNA was performed at 37°C using Moloney murine leukemia virus reverse transcriptase enzyme  and random hexanucleotide primers . Quantitative PCR was carried out by real-time PCR using a LightCycler and the DNA double-strand-specific SYBR green I dye for detection . Results for mouse RNA were normalized to RS9 levels. For human RNA, analysis of the 18 S ribosomal RNA was performed in parallel using the ribosomal RNA control Taqman Assay Kit (Applied Biosystem) to normalize gene expression.The sequences of the primers were:5′CGGCCCGGGAGCTGTTGACG3′, reverse 5′CTGCTTGCGGACCCTAATGTGACG3′),mouse RS9 ,mouse aP2 5′CTGAAGGCTCTGAGTTGGTCAA3′, reverse 5′GGCTTACTGGGCACAGATACCT3′),mouse HSL ,mouse PPARγ .human cath-D  using Nucleofector Technology (Amaxa biosystems) according to the manufacturer's instructions and 40 clones resistant to Blasticidin (4 µg/ml) were isolated. Clone D10 transfected with anti-cath-D shRNA1 and clone A4 transfected with anti-cath-D shRNA2 were the best clones selected for cath-D silencing.5′GGTTCCATGTAAGTCTGACCATCAAGAGTGGTCAGACTTACATGGACCC3′sh1 5′GACCAGTCAAAGGCAAGAGGTTCAAGAGACCTCTTGCCTTTGACTGGTC3′sh2 Adipocytes were washed with phosphate-buffered saline (pH 7.4) and then fixed with Antigenfix . Cells were stained with Oil Red O dye (saturated Oil Red O dye in six parts of isopropanol and four parts of water), an indicator of cell lipid content, and then exhaustively rinsed with water. Spectrophotometric quantification of the stain was performed by dissolving the stained oil droplets in the cell in isopropanol and measuring their absorbance at 540 nm.2) undergoing cosmetic abdominal lipectomy. No clinical data from these patients were available. Human VAT adipose tissue samples were obtained from 27 morbidly (grade-III) obese subjects  before they underwent bariatric surgery.Human adipose tissue was collected according to the guidelines of the Ethical Committee of Toulouse-Rangueil and Nancy J. d'Arc Hospitals and with the full ethical approval of the Ethical Committee of Toulouse-Rangueil and Nancy J. d'Arc Hospitals. All subjects gave their informed written consent to participate in the study. Human abdominal visceral (VAT) adipose tissue and human subcutaneous adipose tissue (SAT) samples were obtained from 9 healthy volunteer patients  with the approval of the locals Ethic Committee. All subjects gave their informed written consent to participate in the study. Adipose tissue fragments were immediately used for collagenase digestion, as previously described For Mice were handled in accordance with National Institute of Medical Research (INSERM) principles and guidelines. C57Bl6/J female mice were obtained from Charles River laboratory . Mice were housed conventionally in an animal room at constant temperature (20–22°C) and humidity (50–60%), and with a 12 h light–dark cycle. All the mice had free access to food and water throughout the experiment. The C57Bl6/J mice were assigned to a normal diet (ND) or high-fat diet (HFD) . The energy contents of the diets were as follows: 20% protein, 70% carbohydrate, and 10% fat for the ND; and 20% protein, 35% carbohydrate, and 45% fat for the HFD. The main source of fat in the HFD was lard (20 g/100g of food). C57Bl6/J mice  were fed an ND or HFD for 20 weeks. A 20 weeks period of HFD is used to mimic the slow development of obesity in humans associated with the settlement of insulin resistance and low grade inflammation. All the mice were sacrificed at 30 weeks of age.Mouse intra-abdominal adipose tissues were dissected immediately after sacrifice, minced in 5 ml of Dulbecco's modified Eagle's medium  supplemented with 1 mg/ml collagenase (SIGMA) and 1% BSA for 30 min at 37°C while shaking. Digestion was followed by filtration through a 150 µm screen, and the floating adipocytes were separated from the medium containing the stroma-vascular fraction (SVF). Adipocytes were washed twice in DMEM, and then processed for RNA extraction using the RNeasy mini kit .2, 1 mM EGTA, 100 mM NaF, 10 mM NaPPI, 500 µm Na-Vanadate, 1 mM PMSF, 10 µM Aprotinine, and a protease inhibitor cocktail). After shaking gently for 20 min at 4°C, cell extracts were obtained by centrifugation in a microfuge at 13,000 rpm for 15 min at 4°C. Equal amounts of protein (100 µg) from cell extracts, quantitated by the Bradford assay, were separated on a 7% gel by SDS-PAGE. Proteins were electro-transferred to a PVDF membrane, and then incubated with 1 µg/ml anti-mouse cath-D (Santa Cruz Biotechnology), 1 µg/ml anti-human cath-D (BD Biosciences), 1 µg/ml anti-α tubulin (Lab Vision Corporation), 1 µg/ml anti-β actin (Sigma), 1 µg/ml ERK2 (Santa Cruz Biotechnology), or 0.4 µg/ml anti-HSL (Santa Cruz Biotechnology). Proteins were visualized with horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin (ECL Amersham) or horseradish peroxidase-conjugated rabbit anti-goat immunoglobulin (ECL Amersham) followed by the Renaissance chemiluminescence system (Perkin Life Sciences).Cells were lysed in lysis buffer , and adipocytes differentiated for 7 days (D7) from the 3T3-F442A and 3T3-L1 preadipocytic cell lines, and NIH-3T3 mouse fibroblasts. α-tubulin was used as a loading control.(TIF)Click here for additional data file.


Recently, acetate-free citrate containing dialysate (A(−)D) was developed. We have already reported about the significant effect of A(−)D on metabolic acidosis, anemia, and malnutrition in maintenance hemodialysis (MHD) patients. In this study, we compared the effect of A(−)D and acetate containing dialysate (A(+)D) on serum calcium and intact-parathyroid hormone (int-PTH) levels.3-, total (t)-calcium, ionized (i)-calcium, and int-PTH were evaluated at the beginning and the end of each session. Cross over study: A total of 29 patients with MHD were treated with A(+)D for 4 months, switched to A(−)D for next 4 months, and returned to A(+)D for the final 4 months.Single session study: Seventeen patients were treated with A(+)D in one session and also treated with A(−)D in another session. Serum levels of pH, HCOIn single session study, serum i-calcium and t-calcium levels significantly increased, and int-PTH levels decreased after HD with A(+)D, whereas HD with A(−)D did not affect iCa and int-PTH. In cross over study, if all patients were analyzed, there was no significant difference in serum int-PTH or bone alkaline phosphatase (BAP) levels during each study period. In contrast, in the patients with low int-PTH (<60 pg/mL), serum levels of int-PTH and BAP were significantly increased during the A(−)D, without significant changes in serum t-calcium or i-calcium levels.A(−)D containing citrate could affect calcium and PTH levels, and, in 4 month period of crossover study, increased int-PTH levels pararelled with increasing BAP levels, exclusively in MHD patients with low int-PTH levels. We recently demonstrated in a cross-over study that HD with acetate-free citrate dialysate (A(−)D) could contribute to improved clinical status, including improved hemodynamic conditions during HD [3- concentration affected PTH and osteocalcin levels and bone resorption surfaces in 21 MHD patients [Newly developed acetate-free citrate containing dialysate D iuring HD -5. Furthuring HD . From thpatients . The purIn this study, for clarifying the effect of A(−)D on calcium and int-PTH metabolism, we evaluated the changes of total (t)-calcium, i-calcium, and int-PTH in a single HD session and as part of previous cross-over study.Seventeen patients (for the single session study) and 29 patients (for the cross-over study), who had received MHD for more than one year and who were in stable clinical condition, participated in the present study. Patients with chronic inflammatory diseases, malignant tumors, or severe hepatic or respiratory diseases were excluded from the study. Written informed consent was obtained from all patients. All subjects gave their informed consent in accordance with the requirements of the institutional committee on human research, and the committee approved the study protocol (Hyogo College of Medicine No. 436). The study was registered with the University Hospital Medical Information Network (UMIN) Clinical Trial Registry (UMIN number: 000000773).The patients included in cross-over study were part of an earlier clinical trial designed to evaluate the effect of A(−)D on dialysis efficiency, nutritional status, inflammatory conditions, and response to ESA, as previously reported .®, Ajinomoto Pharmaceuticals Co., Ltd., Tokyo, Japan) in one session and with A(−)D in another session, without changing other dialysis conditions . Blood samples were taken at the beginning and the end of each session. We measured pH and serum i-calcium, t-calcium, HCO3-, and int-PTH levels. The blood levels of pH, HCO3−, t-calcium, and i-calcium were measured immediately after sampling from patients. The remaining blood samples were stored at −30°C until the BAP and int-PTH were measured.Seventeen MHD patients were treated with acetate-containing dialysate (A(+)D, AK-soritaTwenty-nine MHD patients were treated with A(+)D for 4 months (first A(+)D period), were switched to A(−)D for the next 4 months (A(−)D period) and were returned to A(+)D for the final 4 months (second A(+)D period). Other parameters of HD therapy, including the dialysis membrane used, blood flow rate, dialysate flow rate, and anticoagulant agent (drug type and dosage), except for dialysate, was unchanged during the entire study period. Standard unfractioned heparin (not citrate) was administered as an anticoagulant, and the schedule of administration was not altered during the study for each patient.3-, int-PTH, and bone-specific alkaline phosphatase (BAP) at the same time. Serum levels of BAP and int-PTH were measured by chemiluminescence enzyme immunoassay. The blood levels of pH, HCO3−, t-calcium, and i-calcium were measured immediately after sampling from patients. The remaining blood samples were stored at −30°C until the BAP and int-PTH were measured.At the beginning of the study, 25 patients were treated using a phosphate binder , 13 patients were treated using an active vitamin D compound , and 2 patients were treated using calcimimetics. There were no significant differences in the doses of phosphate binder, active vitamin D compound, and calcimimetics during the study period. Blood samples were collected before and after the HD session at the beginning of the study, at each switch of dialysate and at the end of the study. We measured serum pH values and levels of t-calcium, i-calcium, HCOAll values are presented as the mean ± SD. Differences between pre- and post-HD levels were analyzed using the paired Student’s t-test. The Mann–Whitney test was used for the analysis of the changes of several parameters during cross-over study. P values of <0.05 were considered statistically significant. Statistical analyses were performed with the Statistical Package for Social Science (SPSS), version 18.0 .Seventeen MHD patients  were enrolled in the single session study (Table 3- significantly increased after HD sessions with both A(−)D and A(+)D, the increase of pH and HCO3- with A(−)D  were significantly larger than A(+)D  D, both i-calcium (from 1.14 ± 0.06 mEq/L to 1.28 ± 0.06) and t-calcium (from 8.8 ± 0.48 mg/dL to 9.6 ± 0.68) significantly increased. On the other hand, after HD with A(−)D, although t-calcium significantly increased (from 8.7 ± 0.48 to 9.9 ± 0.56), there was no significant difference in i-calcium (from 1.14 ± 0.06 to 1.13 ± 0.05) between pre- and post-HD levels with A(−)D D, serum levels of int-PTH were significantly decreased (from 149.9 ± 49.7 to 94.9 ± 42.0 pg/mL). In contrast, there was no significant difference in serum int-PTH between pre- HD (132.2 ± 54.8 pg/mL) and post-HD (154.4 ± 62.9 pg/mL) levels with A(−)D  were enrolled in the cross-over study Table . All pat3- levels during the 3 study periods. According to the pre-dialysis plasma HCO3- levels at the beginning of the study, the patients were stratified into two groups ; HCO3- levels ≥ 20 mEq/L (n=13) and < 20 mEq/L (n=16). In the patients with high plasma HCO3- levels (≥ 20 mEq/L), there were no significant differences in plasma HCO3- levels during the three study periods. Conversely, in the patients with low plasma HCO3- levels (< 20 mEq/L), plasma HCO3- levels were significantly (P < 0.05) increased from 17.8 ± 1.8 to 22 ± 3 mEq/L after switching from A(+)D to A(−)D. After switching back for the second period of A(+)D, they decreased to baseline levels  after the switch from A(+)D to A(−)D period (38 ± 3 to 58 ± 4 pg/mL). Furthermore, they decreased to the baseline levels after switching back to A(+)D period  increased, from 12 ± 1 to 16 ± 2 U/L after switching for A(+)D period to the A(−)D period D or A(+)D. We found significant increase in i-calcium, t-calcium and decrease in int-PTH after HD with A(+)D. In contrast, there was no significant difference in i-calcium and int-PTH levels after HD with A(−)D. These differences could be affected by the post-dialysis iCa levels. Despite the calcium concentrations of A(−)D and A(+)D being the same level (3.0 mEq/L), A(−)D contains a small amount of citrate as a buffer source. Because citrate is an effective chelator of i-calcium, HD with A(−)D containing citrate might decrease serum i-calcium levels. Therefore, Ca chelating effect of citrate as well as alkalizing effect of A(−)D might decrease iCa which affects intPTH.In the cross-over study, we conducted a stratified analysis, according to the target int-PTH levels of 60–180 pg/mL, as recommended by the 2006 guidelines for the management of secondary hyperparathyroidism in chronic dialysis patients by JSDT [Past studies have reported the impact of calcium concentrations in dialysates on bone mineral metabolism and int-PTH levels -5. In thThere were no significant differences in pre-dialysis t-calcium and i-calcium levels during the cross-over study. On the other hand, in the single session study, we found significant increases in i-calcium and t-calcium and decreases in int-PTH levels after HD with A(+)D, while there were no significant differences in i-calcium or int-PTH between pre- and post-HD levels with A(−)D in the single session study. From these results, we presumed that citrate in the dialysate could chelate i-calcium during HD sessions with A(−)D. Also from the results of this study, we concluded that not only the decrease in the calcium concentration of dialysate but also the chelation of i-calcium by citrate in dialysate might affect int-PTH levels in patients undergoing MHD.3- levels: 15–16.8 mmol/L). The amelioration of metabolic acidosis by raising the dialysate base HCO3- concentration was found to the rise in PTH, reduce bone resorption in the patients with low PTH levels, on the other hand, improve bone formation in the patients with high PTH levels. Furthermore, Krieger et al. reported, from an in vitro study, a direct, suppressive effect of metabolic acidosis on osteoblastic-induced collagen synthesis, which decreased by 15% after exposure to an acidic milieu [The bone diseases that accompany CKD are due to alterations in PTH levels and vitamin D levels. However, a substantial quantity of data has been accrued to implicate chronic metabolic acidosis as an additional important factor . In our c milieu . We did It had been reported that metabolic acidosis triggers an increase in bone resorption and a decrease in bone formation in parathyroidectomized rats . FurtherAcetate induces the expression of pro-inflammatory cytokines in monocytes and polymorphonuclear neutrophil leukocytes, which may be responsible for chronic inflammation in HD patients . The remThe primary limitations of this study are the small patient population, the lack of values for pharmacological parameters such as the blood levels for citrate and acetate, and our inability to specify a single type of dialysis membrane, the glucose concentration or the pH in the dialysate.Although further studies are needed, the mechanisms involved in increased int-PTH and BAP levels in patients with low int-PTH levels using A(−)D are associated with the adsorption of i-calcium, improvements in metabolic acidosis and with nutritional status. Our data demonstrate that HD with A(−)D could increase int-PTH and BAP levels in MHD patients, especially in the patients with low PTH levels that could be associated with low turnover bone disease.The authors declare that they have no competing interests.TK and MF participated in the design of the study. MY and AK performed the statistical analysis. YO and YH helped to draft the manuscript. AM and TN conceived of the study, and participated in its design of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/14/18/prepub


During the pandemic [Influenza A(H1N1)pdm09] period in 2009-2010, an influenza A (Inf-A) virus with H1N2 subtype (designated as A/Eastern India/N-1289/2009) was detected from a 25 years old male from Mizoram (North-eastern India).To characterize full genome of the H1N2 influenza virus.For initial detection of Influenza viruses, amplification of matrix protein (M) gene of Inf-A and B viruses was carried out by real time RT-PCR. Influenza A positive viruses are then further subtyped with HA and NA gene specific primers. Sequencing and the phylogenetic analysis was performed for the H1N2 strain to understand its origin.The outcome of this full genome study revealed a unique reassortment event where the N-1289 virus acquired it’s HA gene from a 2009 pandemic H1N1 virus with swine origin and the other genes from H3N2-like viruses of human origin.This study provides information on possibility of occurrence of reassortment events during influenza season when infectivity is high and two different subtypes of Inf-A viruses co-circulate in same geographical location. Influenza A viruses belongs to the family Orthomyxoviridae. Orthomyxoviruses have negative-sense single-stranded RNA genomes that are segmented, allowing for reassortment and production of novel viruses. There are two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), that define subtypes and are important for host range, antigenicity, pathogenesis, and diagnostic detection. The H3N2 viruses were demonstrated to have acquired HA, NA, and PB1 genes of human virus origin, PA and PB2 genes of avian virus origin, and the remaining internal genes, NP, M, and NS, of swine virus origin, thus are also referred as the triple reassortant viruses (TR H3N2). H3N2 viIn the present study, we are reporting detection and molecular characterization of a H1N2 strain (designated as A/Eastern India/N-1289/2009); detected from a 25 years old male in North-eastern India who was suffering from fever, and acute respiratory tract infections (ARTIs) and had histories of close contact with a person suffering from influenza A(H1N1)pdm09. Since H1N2 is not common in this region, all gene segments were sequenced to characterize the virus.Nasal and/or throat swabs of hospitalized cases were referred from different hospitals in eastern and north-eastern India during the pandemic period (June 2009-Dec 2010),13. The For sequencing, viral genes were amplified as described earlier,15. NuclPairwise sequence alignments were performed using LALIGN software. Multiple alignments were done with Clustal W program which is available at DDBJ software. Phylogenetic tree was constructed by the neighbor-joining method using thThe nucleotide sequence data were submitted to the GenBank under the accession numbers JQ340003, JQ340004, JQ340005, JQ340006, JQ340007, JQ340008, JQ340009, and JQ340010 for HA, NA, NP, PA, PB1, PB2, Matrix and Non structural genes respectively.During the influenza A(H1N1)pdm09 period in 2009-2010, an Inf-A positive sample (designated as A/Eastern India/N-1289/2009) was detected from a 25 years old male in North-eastern India among 2931 samples screened till date. The sample was initially classified as influenza A(H1)pdm09, though NA gene could not be amplified using N1 specific primers. To rule out mix infection in patient, primers specific for H3 and N2 were also used, H3 did not amplify, however NA gene was amplified using N2 specific primers. Full length sequencing of HA and NA gene segments confirmed the virus to be of H1N2 subtype. For further genetic analysis, the complete coding sequences of all 8 segments were determined.FigureThe phylogeny of the NA gene Figure demonstrFiguresThe non-structural gene also shares the same clade with the current circulating H3N2 strains and also with the vaccine strain A/Brisbane/10/2007 Figure. A singlInfluenza A viruses of the H1N2 subtype were isolated previously from humans in India and JapaCo-circulation of both influenza A(H1N1)pdm09 and seasonal  influenza during 2009 and complete disappearance of seasonal A/H3N2 and A/H1N1 strains in 2010 was observed in eastern India. Such coThe strain A/Eastern India/N-1289/2009 is a classical case of sporadic reassortment event occurring during the 2009-2010 influenza season when infectivity is high and different subtypes co-circulate.Aa: Amino acid; Nt: Nucleotide; RT-PCR: Reverse transcriptase polymerase chain reaction.The authors declare that they have no competing interests.TRM drafted the manuscript and performed phylogenetic analysis. ASA performed screening of clinical samples by RT-PCR. SC provided guidance and has given final approval of the version to be published. MCS conceived the study, provided guidance and edited manuscript. All authors read and approved the final manuscript.Comparison of amino acid (aa) changes at different positions of the internal genes  of A/Eastern India/N-1289/2009 with that of the vaccine strain (A/Brisbane/10/2007).Click here for fileTable indicating closest ancestor strain (name and host origin) and % homology using BLAST analysis.Click here for file