High-density Lipoprotein (HDL) attenuates endothelial cell apoptosis induced by different cell-death stimuli such as oxidation or growth factor deprivation. HDL is the main plasma carrier of the bioactive lipid sphingosine 1-phosphate (S1P), which it is a signaling molecule that promotes cell survival in response to several apoptotic stimuli. In HDL, S1P is bound to Apolipoprotein M (ApoM), a Lipocalin that is only present in around 5% of the HDL particles. The goal of this study is to characterize ApoM-bound S1P role in endothelial apoptosis protection and the signaling pathways involved.Human umbilical vein endothelial cells (HUVEC) cultures were switched to serum/grow factor deprivation medium to induce apoptosis and the effect caused by the addition of ApoM and S1P analyzed.+ApoM or recombinant ApoM-bound S1P promoted cell viability and blocked apoptosis, whereas HDL-ApoM had no protective effect. Remarkably, S1P exerted a more potent anti-apoptotic effect when carried by ApoM as compared to albumin, or when added as free molecule. Mechanistically, cooperation between S1P1 and S1P3 was required for the HDL/ApoM/S1P-mediated anti-apoptotic ability. Furthermore, AKT and ERK phosphorylation was also necessary to achieve the anti-apoptotic effect of the HDL/ApoM/S1P complex.The addition of HDLAltogether, our results indicate that ApoM and S1P are key elements of the anti-apoptotic activity of HDL and promote optimal endothelial function.The online version of this article (doi:10.1186/s12944-017-0429-2) contains supplementary material, which is available to authorized users. ApoM-bound S1P and ApoM-containing HDL are anti-apoptotic.HDL/ApoM/S1P complex signals through S1P1 and S1P3.ApoM-bound S1P anti-apoptotic effect is more potent than albumin-bound S1P.Apolipoprotein M (ApoM) is a member of the Lipocalin family and its structure is defined by an eight-stranded antiparallel β-barrel enclosing a hydrophobic binding pocket, where different ligands bind, e.g. retinol , oxidizeSphingolipids have multiple key physiological functions that are important for the regulation of cell growth and survival. Ceramide and sphingosine are inducers of growth arrest and apoptosis and many stress stimuli increase the cellular levels of these compounds. In contrast, S1P is associated with suppression of apoptosis , 9.Five The integrity of endothelial cells lining the vessels is crucial for vascular homeostasis and endothelial cell-death triggers vascular leakage and promotes inflammation in adjacent tissues . Additio+ApoM) and ApoM-lacking HDL (HDL-ApoM) to study regulation of apoptosis in human endothelial cells. Moreover, we have elucidated whether the anti-apoptotic properties of S1P are carrier dependent by comparing the anti-apoptotic effects of albumin-bound S1P, ApoM-bound S1P and S1P as a free molecule.HDL particles are potently anti-atherogenic and reduce endothelial cell apoptosis , 16. Cho2 incubator. The culture medium was replaced every 2 days, and cells were subcultured at 90–95% confluence. Cells were studied between passages 2–8.Human Umbilical Vein Endothelial Cells (HUVEC) were obtained from Gibco, grown in 1% gelatin pre-coated plates in M200 medium containing 1% penicillin and streptomycin and low serum growth supplement (LSGS)  at 37 °C in a humidified 5% COLSGS contains fetal bovine serum (FBS), human epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), heparin and hydrocortisone. Removal of all these components was used to induce apoptosis in HUVEC. This treatment will be referred as serum/GF deprivation. For that, cells were washed twice with M200 medium without LSGS. The absence of S1P in M200 medium without LSGS was verified by mass spectrometry as it was previously described in , 20.E. coli, purified from inclusion bodies and refolded as described in Ahnström et al. [Recombinant soluble human ApoM  was expressed in m et al. . ApoM lom et al. .+ApoM and HDL-ApoM were isolated by immunoaffinity chromatography with M23 and M58 monoclonal antibodies against ApoM.HDL was isolated from human plasma obtained from the Blood Bank at Växjö Hospital, Sweden, as described in Ruiz et al. . Briefly+ApoM and ~0.008 μM/mg protein in HDL-ApoM.S1P levels in HDL preparations were quantified by mass spectrometry as it was previously described , 20. S1PSample protein concentration was quantified using BCA protein assay kit (Pierce) according manufacturer’s instructions.Electrophoresis was done in 4–15% gradient pre-casted SDS-gels (Bio-Rad) under reducing conditions. Western blotting was done after separation in a Trans-Blot Turbo transfer system (Bio-Rad). An Antibody against phospho-ERK1 (T202/Y204) / phospho-ERK2 (T185/Y187) ERK1/2 was from R&D systems; antibodies against total ERK (#9102), pSer473 AKT (D9E), total AKT (C67E7) were from Cell Signaling and an antibody against GAPDH was from Santa Cruz Biotechnology (#20357).+ and 7-ADD−.Cells were detached with TrypLE Express (Gibco), washed and resuspended in Annexin V binding buffer (BD Bioscience). Then, cells were stained with PE Annexin V and 7-ADD according manufacturer’s instructions (BD Bioscience) and analyzed in a Cytomics FC500 (Beckman Coulter) flow cytometer. Data were analyzed with FlowJo X v.10.0 7r2. Early apoptotic cells were defined by Annexin VCaspase-3 activity was measured using a colorimetric assay kit according to manufacturer’s instructions (Abcam). Briefly, cell lysates  were incubated in the presence of N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide  and the release of pNA was measured using a plate reader (TECAN Infinite F200) at 405 nm.Cell viability was evaluated by the MTT assay following manufacturer’s instructions (Roche). Briefly, viable cells are defined by their ability to reduce MTT -2,5-diphenyltetrazolium bromide) to formazan, which is a measure of an active metabolism. The conversion was quantified using a plate reader (TECAN Infinite F200) at 570 nm and optical density value was utilized as an indicator of cell viability.GAPDH), Hs00173499_m1 (S1P1), AJ39RQ5 (S1P2), Hs00245464_s1 (S1P3), Hs02330084_s1 (S1P4) and Hs00928195_s1 (S1P5) according manufacturer’s instructions. Samples were measured as quadruplicates. The relative expression of each gene was calculated according to the ΔΔCT method [Total cellular RNA was isolated using RNeasy Kit according to the manufacturer’s instructions (Qiagen) and quantified using a NanoDrop spectrophotometer . qPCR were performed with a CFX384 C1000 thermal cycler (Bio-Rad) using the Super Scrip III Platinum One Step qRT-PCR kit (Invitrogen) and TaqMan probes (Applied Biosystems): 4326317E  was purchased from Avanti Polar Lipids and Sigma; bovine fatty acid free albumin was from Sigma; W146, CAY10444 and ML-031 were from Cayman Chemical; SEW2871 and CYM5541 were from Tocris Bioscience; LY294002, U0126 and PD98059 were from R&D systems.p < 0.05 was defined as threshold for significant changes. Student t-test and Mann-Whitney U-test were used for two-sample comparisons and ANOVA was used when assaying for multiple comparisons. The particular tests used for post hoc analyses depended on homoscedasticity, and are stated in the figure legends.Statistical analyses were performed with SigmaPlot 11.0 software (Systat Software Inc.). A value of +ApoM and HDL-ApoM. Then, HUVEC were serum/GF deprived in the presence of HDL+ApoM or HDL-ApoM for 18 h and the amount of apoptotic cells measured by flow cytometry. HDL+ApoM reduced the percentage of apoptotic cells, whereas HDL-ApoM did not confer any protection against serum/GF deprivation .Since anti-apoptotic and pro-survival effects of S1P were carrier dependent, we investigated if differences can be due to particular activation of S1PR. To study this, we performed Caspase-3 assays as in Fig. Thus, we concluded that the anti-apoptotic effect of S1P in serum/GF deprived endothelial cells was carrier dependent, ApoM-bound S1P being the most powerful of all three carriers. Furthermore, anti-apoptotic activity of S1P was mediated by S1P1 and S1P3 with independence of which S1P carrier was used.+ApoM and albumin-S1P, but not by HDL-ApoM [It has been shown that the anti-apoptotic activities of S1P and HDL are mediated by PI3K/AKT and ERK1/2 signaling pathways , 16, 28.HDL-ApoM . To linkNext, we determined whether S1PR activation mediated the phosphorylation of AKT and ERK by S1P. The selective S1P1 antagonist W146 dramatically reduced the phosphorylation of AKT and ERK by ApoM-bound S1P and albumin-bound S1P. However, when S1P was added as a free molecule, W146 decreased ERK phosphorylation but surprisingly not AKT phosphorylation. In contrast, CAY10444 reduced the phosphorylation of AKT and ERK mediated by free S1P, ApoM-bound S1P and albumin-S1P Fig. .Fig. 7S1In conclusion, S1P anti-apoptotic effect on serum/GF deprived endothelial cells went via S1P1 and S1P3 and required the phosphorylation of AKT and ERK1/2  to inhibit apoptosis on endothelial cells . Indeed,poptosis , but int+ApoM and albumin-S1P, but not HDL-ApoM, phosphorylate AKT and ERK [AKT and ERK1/2 phosphorylation mediate HDL and S1P cytoprotective actions , 27, 28. and ERK , 48. Thiper se in endothelial cells. However, ApoM over-expression promotes apoptosis in the human hepatoma derived cell line HepG2 [Retinol binding protein (RBP) is another member of the Lipocalin family and transports retinol in plasma. Interestingly, apo-RBP is pro-apoptotic, whereas holo-RBP is anti-apoptotic . We did ne HepG2 . Moreovene HepG2 , 52. Intne HepG2 . Thus, aTaken together, our results demonstrate that the HDL/ApoM/S1P-complex plays an essential role in vascular biology and protects endothelial cells from apoptosis. This is especially relevant in pathologies where endothelial cell apoptosis is altered such as in thrombosis and atherosclerosis.


N-glycan modulate the biological functions and physicochemical properties of antibodies. By remodeling N-glycan to obtain a homogeneous glycoform or chemically modified glycan, antibody characteristics can be controlled or modified. Such remodeling can be achieved by transglycosylation reactions using a mutant of endoglycosidase from Streptococcus pyogenes (Endo-S) and glycan oxazoline. In this study, we generated improved mutants of Endo-S by introducing additional mutations to the D233Q mutant. Notably, Endo-S D233Q/Q303L, D233Q/E350Q, and several other mutations resulted in transglycosylation efficiencies exceeding 90%, with a single-digit donor-to-substrate ratio of five, and D233Q/Y402F/D405A and several other mutations resulted in slightly reduced transglycosylation efficiencies accompanied by no detectable hydrolysis activity for 48 h. We further demonstrated that the combined use of mutants of Endo-S with Endo-M or Endo-CC, endoglycosidases from Mucor hiemalis and Coprinopsis cinerea, enables one-pot transglycosylation from sialoglycopeptide to antibodies. This novel reaction enables glycosylation remodeling of antibodies, without the chemical synthesis of oxazoline in advance or in situ.The fine structures of Fc  N-linked glycan in the Fc region. In an effort to improve the efficacy and safety of recombinant monoclonal antibody therapeutics, various expression systems have been genetically engineered to produce antibodies with desired glycoforms [Antibodies are major components of humoral immunity that protect the host from infection by specifically binding to pathogens. IgG, the most common antibody found in the circulation, contributes to host defense via several mechanisms, including complement-dependent cytotoxicity –5 and anycoforms –12. Thesycoforms .In vitro chemoenzymatic remodeling is a promising method that enables high uniformity and precise control over the glycoform by the transfer of oligosaccharide oxazolines using endo-β-N-acetylglucosaminidase (ENGase) [Streptococcus pyogenes that selectively hydrolyzes the chitobiose core of the N-linked complex-type glycan of IgG, has been engineered [(ENGase) ,15. Endogineered –18 and ugineered –21. HoweMucor hiemalis [Coprinopsis cinerea [In this paper, we describe improved mutants of Endo-S that enable efficient transglycosylation using a reduced concentration of oxazoline; the mutants were obtained by site-directed mutagenesis based on structural information and methods adapted from studies of various ENGases ,22–26. Fhiemalis  or Endo- cinerea . AuxiliaHuang and coworkers have previously identified the Endo-S D233Q and E235A mutants by targeting residues responsible for promoting oxazolinium ion formation by a substrate-assisted mechanism . AdditioWhile none of the single mutants of Endo-S displayed improvements over the D233Q mutant, a number of multiple mutants exhibited a reduced hydrolysis efficiency, while retaining a comparable or improved transglycosylation efficiency . The Q30i.e., an Endo-S mutant that preferentially targets Fc N-glycan and an Endo-M mutant that targets SGP, but not core-fucosylated Fc N-glycan. We hypothesized that the active intermediate required for transglycosylation catalyzed by the Endo-S mutant could be prepared in situ by an auxiliary endoglycosidase  . While tBased on an analysis of the SGP concentration dependency, greater than approximately 100 eq of SGP is required to achieve 90% transglycosylation with the Endo-S D233Q mutant and Endo-M N175Q mutant . Using tTo test whether Endo-M N175Q could be replaced with another ENGase, Endo-S D233Q was used in combination with variants of Endo-CC at 37°C, the optimal temperature for the latter enzyme. While the reaction with wild-type Endo-CC did not generate a detectable amount of transglycosylation product, that with the Endo-CC N180H mutant showed 57% transglycosylation efficiency at 8 h, but this was followed by significant hydrolysis of the product . LowerinStreptococcus pyogenes, with improved transglycosylation activity compared to that of the previously reported D233Q mutant. Several notable double- and triple-mutants with high transglycosylation activity and low hydrolysis activity were identified. Notably, Endo-S D233Q/Q303L, D233Q/E350Q, and D233Q/D405A demonstrated transglycosylation efficiencies reaching over 90% at a substantially reduced donor-to-acceptor ratio of 5 molar eq . This is lower than the 20 molar eq used in the recently reported highly efficient D184M mutant of Endo-S2 from Streptococcus pyogenes serotype M49 [In this study, we generated mutants of Endo-S, an endoglycosidase from type M49 . While si.e., Y402 is in the proximity of the branched mannose residue, H122 and E350 are likely in contact with the α1–3 arm of the substrate glycan, and D405, R406, and Y348 are buried in the interior with respect to the two residues. Only the F187A mutation, which resulted in a very minor improvement, was identified in the proximity of the α1–6 arm. This area was not thoroughly explored, but it is possible that the interaction with the α1–6 arm is not as extensive as interactions with other regions of the glycan.In the process of generating efficient mutants, various sites that contributed to the reduced hydrolysis activity were identified, including D279 and Q303 in the proximity of catalytic residues . Most ofin situ [Importantly, we showed that the glycan moiety could be transferred from SGP to the antibody by the combination of ENGase mutants. Initially, the use of wild-type Endo-S and Endo-M yielded unsuccessful results due to the hydrolysis of the product and active intermediate, respectively. However, use of the Endo-S D233Q/E350 mutant with the Endo-M N175Q mutant to minimize hydrolysis greatly improved the maximum transglycosylation efficiency to 97%. Although the detailed process remains unclear, the active intermediate generated by the Endo-M mutant may have been transferred to the Endo-S mutant for subsequent transglycosylation of the antibody. In contrast to the recently reported chemical approach in which sialoglycan hydrolyzed by wild-type Endo-M was chemically converted to SG-oxazoline in situ  Fig 8A)in situ [in situ  did not The applicability of the Endo-CC mutant in place of the Endo-M mutant suggests that this type of reaction can be carried out with different combinations of ENGases and their substrates . AdditioUnexpectedly, the Endo-M N175Q mutant, without the help of Endo-S, showed approximately 5% transglycosylation efficiency towards core-fucosylated IgG . This maEnzymatic remodeling of glycan has extensive research uses, but is limited for large-scale applications. In this study, over 120 mutants of Endo-S were screened with the aim of obtaining an efficient enzyme with improved transglycosylation activity and reduced hydrolysis activity. By introducing additional mutations into the D233Q mutant , we obtaStreptococcus pyogenes was subcloned into the pGEX-4T-3 vector  for the expression of glutathione-S-transferase (GST)-fused Endo-S. Mutations were introduced using the QuikChange II Site-Directed Mutagenesis Kit . The plasmids containing mutated Endo-S genes were transformed into Escherichia coli BL21(DE3). The transformants were cultured in 50 mL of Luria Broth supplemented with 100 μg/mL ampicillin, and overexpression was induced by the addition of 0.1 mM isopropyl-β-d-1-thio-galactopyranoside. After 20 h of culture at 16°C, the cells were harvested by centrifugation. The cell pellets were resuspended in PBS  containing 0.5 mg/mL lysozyme  and 40 U of DNaseI  and then sonicated. The supernatant of the cell lysate was affinity-purified with Glutathione Sepharose 4B , buffer exchanged to PBS, and concentrated to approximately 2 to 7 mg/mL by Amicon Ultrafiltration . For the large-scale purification of selected Endo-S variants, 1 L of culture was used and affinity-purified materials were gel-filtered in PBS with HiLoad 26/600 Superdex 200 prep grade , instead of diafiltration, to remove 10 mM glutathione used for elution from Glutathione Sepharose 4B.The synthesized DNA fragment encoding Endo-S from A solution of trastuzumab  was incubated with Endo-S (500 μg) at 37°C in a total volume of 4.39 mL of 50 mM phosphate buffer (pH 6.0) for 4 h. The mixture was immediately subjected to affinity chromatography with HiTrap rProteinA HP , and hydroxyapatite chromatography with CHT Type I , and buffer-exchanged to 50 mM Tris-HCl buffer (pH 7.4), yielding 85.7 mg of deglycosylated trastuzumab. LC-MS results for the heavy chain of  GlcNAc-trastuzumab, M = 49506.0 Da; found (m/z), 49505.5 (deconvolution data).The assay for the hydrolysis activity of each Endo-S mutant (10 μg) was performed at 30°C with commercial trastuzumab (500 μg) as a substrate in Tris-HCl buffer . The transglycosylation activity of each Endo-S mutant (16 μg) was assayed at 30°C with deglycosylated trastuzumab (800 μg) and SG-oxazoline  53.5 μg. The peaA solution of deglycosylated trastuzumab (1 mg) and SGP derived from egg yolk  were incubated with an Endo-S mutant (20 μg) and/or Endo-M mutant  at 28°C in Tris-HCl buffer . The SGP concentration dependency was additionally tested at 50, 100, 200, and 400 eq for the reaction with Endo-S D233Q in concert with Endo-M N175Q. The assay using SG-Asn [2HPO4/NaH2PO4 pH 6.0, Nacalai Tesque, Kyoto, Japan) supplemented with 5 mM EDTA , yielding 8.0 mg of 11.82 mg/mL SG-trastuzumab in 680 μL. LC-MS results for the heavy chain of SG-trastuzumab, M = 51508.7 Da; found (m/z), 51508.0 (deconvolution data). Similarly, a reaction using SG-Asn  was carried out at slightly reduced volume of 570 μL, yielding 8.0 mg of 10.31 mg/mL SG-trastuzumab in 780 μL. LC-MS: calculated for the heavy chain of SG-trastuzumab, M = 51508.7 Da; found (m/z), 51508.0 (deconvolution data).A solution of deglycosylated trastuzumab (10 mg) and SGP  was incubated with the Endo-S D233Q mutant (200 μg) and Endo-M N175Q mutant (0.05 U) at 28°C in Tris-HCl buffer  for 4.5 h. The mixture was immediately subjected to affinity chromatography with HiTrap rProteinA FF  and hydroxyapatite chromatography with CHT Type I (Bio-Rad), and buffer-exchanged to sodium phosphate buffer . The final concentration and volume are indicated.(DOCX)Click here for additional data file.S2 TableThe hydrolysis and transglycosylation efficiencies of Endo-S D233Q and other mutants that displayed reduced hydrolysis or enhanced transglycosylation efficiency at any time point compared to the Endo-S D233Q mutant.(DOCX)Click here for additional data file.S3 TableThe one-pot transglycosylation efficiency by Endo-S D233Q and Endo-M N175Q using various concentrations of SGP as a donor substrate.(DOCX)Click here for additional data file.S1 Fig(A) The electropherogram of the Endo-S D233Q-catalyzed deglycosylation reaction at 1 h obtained using Experion Automated Electrophoresis Station (Bio-Rad). (B) Comparison of the heavy chain peak at various time points of the deglycosylation reaction demonstrates that the relative amount of glycosylated and deglycosylated heavy chains can be approximately quantified.(TIF)Click here for additional data file.


LMNA. LMNA mutations can lead to laminopathies such as lipodystrophies, premature aging syndromes (progeria) and muscular dystrophies. Here, we identified a novel heterozygous LMNA p.R388P de novo mutation in a patient with a non-previously described severe phenotype comprising congenital muscular dystrophy (L-CMD) and lipodystrophy. In culture, the patient’s skin fibroblasts entered prematurely into senescence, and some nuclei showed a lamina honeycomb pattern. C2C12 myoblasts were transfected with a construct carrying the patient’s mutation; R388P-lamin A (LA) predominantly accumulated within the nucleoplasm and was depleted at the nuclear periphery, altering the anchorage of the inner nuclear membrane protein emerin and the nucleoplasmic protein LAP2-alpha. The mutant LA triggered a frequent and severe nuclear dysmorphy that occurred independently of prelamin A processing, as well as increased histone H3K9 acetylation. Nuclear dysmorphy was not significantly improved when transfected cells were treated with drugs disrupting microtubules or actin filaments or modifying the global histone acetylation pattern. Therefore, releasing any force exerted at the nuclear envelope by the cytoskeleton or chromatin did not rescue nuclear shape, in contrast to what was previously shown in Hutchinson-Gilford progeria due to other LMNA mutations. Our results point to the specific cytotoxic effect of the R388P-lamin A mutant, which is clinically related to a rare and severe multisystemic laminopathy phenotype.A-type lamins, the intermediate filament proteins participating in nuclear structure and function, are encoded by  LMNA gene encodes A-type lamins, intermediate filament proteins that are regulated during development and cell differentiation. The two major isoforms in mammals are lamins A and C (LA/C). Prelamin A ) and lamin C  are produced by alternative splicing. Mature lamin A  is generated after posttranslational modifications of prelamin A, a process which includes farnesylation and carboxymethylation of Cysteine 661 followed by proteolysis of the last 18 a.a. by the metalloproteinase Zmpste24 [The  C LC, 57 a.a. areLMNA gene have been identified as causal for laminopathies, which encompass tissue-specific dystrophies affecting cardiac, cartilage, bone, skin, peripheral nerves, skeletal muscles and/or adipose tissues. Laminopathies include muscular dystrophies and/or cardiomyopathies, lipodystrophies and syndromes of premature ageing such as Hutchinson-Gilford progeria syndrome (HGPS) [In the last 15 years, ~400 mutations in the e (HGPS) , 4.LMNA inhibition or in HGPS, the frequency of nuclear dysmorphy can be reduced by relieving the forces exerted on the nuclei by microtubules, using drugs such as nocodazole or an N-acetyl transferase 10 inhibitor [Wild-type lamin A helps regulate the normal ovoid nuclear shape within cells. Accordingly, the extinction of lamin A/C by siRNA or the expression of laminopathy-associated mutant lamins A/C induces abnormal nuclear shapes , 6. For nhibitor , and alsnhibitor . In addinhibitor , ameliornhibitor , 21.LMNA p.R388P de novo mutation. In contrast to the previous studies, our study revealed that nuclear dysmorphy associated with the R388P lamin A mutation responsible for congenital muscular dystrophy L-CMD and lipodystrophy relies on distinct mechanisms.We describe here a patient with a severe phenotype, congenital muscular dystrophy (L-CMD) and lipodystrophy, in whom we detected a novel heterozygous LMNA were amplified by PCR with specific primers in the proband and her relatives for direct sequencing as previously described [Clinical, biological and molecular studies of the patient and her relatives  were performed after full informed consent according to legal procedures. Genomic DNA was extracted from peripheral-blood leukocytes using a commercial kit . The entire coding region and splice junctions of escribed . Direct ™ and pyruvate (Gibco), and 10% foetal calf serum for experimental use between passages 6 and 14. We are authorised to store and use our registered and anonymised collection of human samples in the INSERM Unit number 938 (Centre de Recherche Saint-Antoine) (anonymised collection registered under number DC-2008-529). We obtained specific authorisation for the study “Physiopathological modelling of insulin resistance and/or lipodystrophic syndromes” from the ethics committee "Comité de Protection des Personnes Ile-de-France V" . In accordance, a written consent was obtained from the patient, her legal representative, and controls subjects for their participation in this study and its potential publications.Subcutaneous fibroblasts obtained via skin biopsy from the patient (aged 16) and from two healthy individuals (her 43-year-old mother and an unrelated 18-year-old man) were cultured in low-glucose DMEM containing GlutaMAXWhole cell extracts of fibroblasts from controls and proband were analysed by western blot using antibodies directed against lamins  and2. Senescence-associated- (SA) β-galactosidase  activity was estimated by quantifying the percentage of positive blue cells by direct observation of the samples under light microscope (n = 900–1100 cells in each case).Fibroblasts adherent to coverslips were fixed for 12 min at 22°C with 3% formaldehyde, then incubated overnight at 37°C in 1 mg/mL X-gal, 40 mM citric acid, 126 mM sodium phosphate pH 6.0, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 150 mM NaCl, and 2 mM MgCl™ (Gibco) and 10% foetal bovine serum. For cell transfection, lipofectamine™ 2000 (Life technologies) was used according to the manufacturer's instructions. Cells were analysed 24 h after transfection.C2C12 cells were obtained from American Type Culture Collection and grown in DMEM containing high glucose and GlutaMAXBamH1 and EcoR1 sites of pCMV-Tag 2A (Stratagene). The construct encoding GFP fusion of WT prelamin A was previously described [5’ GAG GAG AGG CTA CCA CTG TCC CCC AGC 3’ and reverse 5’ GCT GGG GGA CAG TGG TAG CCT CTC CTC 3’.Constructs encoding FLAG fusions of WT or p.L647R prelamin A (a.a. 1–664) and mature lamin A (a.a. 1–646) were generated by PCR amplification of the relevant regions of cDNA using as templates pSVK3-prelamin A and pSVK3-mature-lamin A ; amplifiescribed . VectorsSix hours after transfection, cells were incubated in the presence of 10 μM mevinolin (Sigma-Aldrich) or in 1% DMSO for 18 h. Twenty-four hours after transfection, cells were incubated with a cytoskeleton destabilising drug: nocodazole (Sigma-Aldrich) at 5 or 10 μM for 3 h, or cytochalasin D (Sigma-Aldrich) at 1 μM or 2 μM for 3 h. Alternatively, cells were incubated in the presence of either trichostatin A  at 100 nM or anacardic acid at 100 μM for 16 h.For immunofluorescence (IF), cells were fixed with a 3% paraformaldehyde solution (PFA), then processed as previously described . IF was 2; a form factor of 1 representing a perfect circle).Nuclear dysmorphy was estimated from FLAG IF images, either directly by visual observation or indirectly when specified using Image J software to calculate the nuclear circularity , or medians with third and first quartiles. Comparisons between samples were performed with the Mann-Whitney test, the Kruskal-Wallis test with or without the pairwise comparisons of groups or the two-way ANOVA test coupled with the Bonferroni post-tests, as indicated.2 at age 11, 60th percentile). Glucose tolerance abnormalities, hyperinsulinemia and oligomenorrhea improved with dietary management while lipodystrophy persisted. Serum triglycerides were normal. A c.1163G>C heterozygous change in LMNA exon 7, predicting a p.R388P substitution, was identified in the patient  but not in her parents or her healthy sister. This de novo mutation was absent in more than 150 unrelated control subjects. Sequence alignment of the mutated region across various species revealed that residue p.R388 is highly conserved during evolution  level was elevated (up to 1951 UI/L), electromyography was myopathic, brain magnetic resonance imaging and Fukutin-related protein gene (FKRP) sequencing were normal and a muscle biopsy showed dystrophic changes . The course of disease was steadily progressive, leading to loss of independent ambulation outdoors at 5 years, joint contractures  from age 6.5 years that initially coexisted with knee hyperextension (genu recurvatum) requiring casts and/or braces and, subsequently, total loss of ambulation and generalised joint contractures  from 11 years. Dorsal hyperlordosis was associated with restrictive respiratory insufficiency that appeared at 9 years and was slowly progressive  = 49% of the predicted value and moderate polysomnography abnormalities at 18 years). Holter studies revealed symptomatic supraventricular hyperexcitability at age 10 years and mixed (supra- and ventricular) symptomatic hyperexcitability from 12 years with ventricular doublets and extrasystoles, which were improved by treatment with bisoprolol. Systolic function was normal. No conduction block was observed on 24-h holter-electrocardiogram, regularly performed every 6 months. In addition, a lipodystrophic morphotype developed progressively from early puberty (age 10), with a striking accumulation of adipose tissue in the cervicofacial and subclavicular regions, and a subcutaneous lipoatrophy of distal upper and lower limbs, while BMI remained in the normal range  , the incTo study the consequences of the L-CMD-related p.R388P mutation in myoblasts, C2C12 cells were transfected with pCMV plasmids encoding WT or R388P FLAG-tagged prelamin A . TransfeIn transfected cells, both WT and R388P preLA proteins were incompletely processed as assessed by western blot comparing bands stained by anti-FLAG, anti-LA/C antibodies (which recognises both endogenous and ectopic lamins) and anti-prelamin A specific antibodies . The mixThis suggested that the p.R388P mutation might alter the binding of LA to specific partners at the nuclear periphery or within the nucleoplasm. We focused on two partners that bind LA predominantly within the nucleoplasm LAP2α,  or at thTherefore, expression of R388P-LA triggers a significant depletion of LA interaction with both LAP2α and emerin specifically at the intranuclear periphery.Nuclear shape was assessed on cells stained for DNA and FLAG-LA by observation  and by mIn conclusion, nuclear dysmorphy induced by R388P lamins A in myoblasts, does not rely on defective prelamin A processing.Considering the hypothetical models proposed to generate dysmorphic nuclei see , we inveDepolymerisation of microtubules with nocodazole did not modify significantly the frequency of dysmorphic nuclei in myoblasts expressing R388P-LA . ConversUsing antibodies directed against H3K9ac or H3K27ac, we observed an increased histone acetylation upon R388P-LA overexpression in C2C12 myoblasts, suggesting that the mutation could alter some chromatin epigenetic marks (A and B in LMNA mutations (~23) previously reported as responsible for L-CMD are located all along the different regions of A-type lamins  [th amino acid of lamin A was previously shown to be pathogenic, and was associated with L-CMD  –33, wherd C-ter) . Only on .Xenopus  located at the end of coil 2B and at the C-ter of the rod domain that are highly conserved between species from human to Xenopus . Interesf nuclei , 40. In f nuclei . We thusLMNA mutations as responsible for several diseases, extensive work has been produced to characterise the associated cellular phenotypes. However, only few studies have been performed in the context of L-CMD [Since the discovery of of L-CMD , 42, 43.of L-CMD , 45, 46.LMNA-/- fibroblasts [To our knowledge, our study is the first to precisely characterise the phenotypes of myoblasts expressing a mutant lamin A responsible for L-CMD. Beside nuclear dysmorphies, previously associated with other laminopathies including Hutchinson-Gilford progeria, our study reveals several specific defects induced by the R388P-LA: i) R388P-LA accumulates in the nucleoplasm and is more soluble than WT-LA (this study), whereas progerin, which is constitutively farnesylated, forms a stiff lamina at the NE , 48; ii)roblasts  but not roblasts , but it LMNA p.R388P mutation, responsible for a complex and severe multisystemic laminopathy phenotype, on the organisation of the nuclear compartment in myoblasts. The difficulty in reversing the abnormal cellular nuclear phenotypes using drugs validated in other laminopathy contexts highlights a need to develop combinatorial approaches or to consider the depletion of that mutant lamin A for therapeutic purposes.In conclusion, our study highlights the multiple deleterious impacts of the S1 FigXenopus laevis lamin A, respectively.The position of the mutation described in this study is shown in bold. The corresponding Swiss Prot accession numbers are P02545, P48679 and P48678 for human, rat and mouse lamin A/C, respectively, and P13648 and P11048 for chicken and (PDF)Click here for additional data file.S2 FigA) Nuclear circularity  of whole cell populations expressing either WT FLAG-LA or R388P FLAG-LA. ** p = 0.002 for n = 6 independent experiments (Mann Whitney test) B) Percentage (mean ± s.e.m.) of dysmorphic nuclei among cells expressing either WT FLAG-LA or R388P FLAG-LA. The criteria of dysmorphy rely on the circularity value (inferior to 0.74) or on the visual observation, as indicated. ** p = 0.002 for n = 6 independent experiments (Mann Whitney test). C) Nuclear circularity (mean ± s.e.m.) of dysmorphic nuclei as revealed by visual observation of cell populations expressing WT FLAG-LA or R388P FLAG-LA. ** p = 0.002 for n = 6 independent experiments (Mann Whitney test). D) Size of nuclei (mean expressed in μm2 ± s.e.m.) with either normal shape (Norm.) or with dysmorphy (Dysm.) as assessed by visual observation of cell populations expressing either WT FLAG-LA or R388P FLAG-LA. * p = 0.01 for n = 6 independent experiments .C2C12 cells overexpressing WT (WT) or mutant (R388P) FLAG-LA were fixed and labelled with anti-FLAG antibodies and analysed by immunofluorescence under confocal microscopy. (PDF)Click here for additional data file.S3 FigA) C2C12 cells overexpressing WT or R388P FLAG-LA were fixed and labelled with mouse anti-FLAG (green) and rabbit anti-H3K9ac (red) antibodies before observation under confocal microscopy. Arrows in A) indicate the absence of H3K9ac signal in cells overexpressing WT lamins A. Scale bar, 20 μm. B) The graph illustrates the H3K9ac median immunofluorescence signal intensity observed per nucleus of cells processed as in A) that express either WT or R388P-LA. Signals are normalised to the signal measured in untransfected cells for 3 independent experiments . Boxes show first and third quartiles, bars are put according to Tukey method for n > 125 nuclei per condition, *** p < 0.001 (Mann Whitney test).(PDF)Click here for additional data file.S4 FigThis model postulates electrostatic attraction of lamin A dimers via distinct positively charged patches  and negatively charged patches . The position of the head, coil 1, coil 2 and C-ter regions and of the Ig-fold domain of A-type lamins are identified. Numbers refer to the amino acid sequence. The localisation of the p.R388P mutation is highlighted within one positively charged patch close to the end of coil 2.(PDF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


To revise the American Joint Committee on Cancer TNM staging system for colorectal cancer (CRC) based on a nomogram analysis of Surveillance, Epidemiology, and End Results (SEER) database, and to prove the rationality of enhancing T stage’s weighting in our previously proposed T-plus staging system.Total 115,377 non-metastatic CRC patients from SEER were randomly grouped as training and testing set by ratio 1:1. The Nomo-staging system was established via three nomograms based on 1-year, 2-year and 3-year disease specific survival (DSS) Logistic regression analysis of the training set. The predictive value of Nomo-staging system for the testing set was evaluated by concordance index (c-index), likelihood ratio (L.R.) and Akaike information criteria (AIC) for 1-year, 2-year, 3-year overall survival (OS) and DSS. Kaplan–Meier survival curve was used to valuate discrimination and gradient monotonicity. And an external validation was performed on database from the Second Affiliated Hospital of Zhejiang University (SAHZU).Patients with T1-2 N1 and T1N2a were classified into stage II while T4 N0 patients were classified into stage III in Nomo-staging system. Kaplan–Meier survival curves of OS and DSS in testing set showed Nomo-staging system performed better in discrimination and gradient monotonicity, and the external validation in SAHZU database also showed distinctly better discrimination. The Nomo-staging system showed higher value in L.R. and c-index, and lower value in AIC when predicting OS and DSS in testing set.The Nomo-staging system showed better performance in prognosis prediction and the weight of lymph nodes status in prognosis prediction should be cautiously reconsidered.The online version of this article (10.1186/s12885-017-3796-1) contains supplementary material, which is available to authorized users. However, patients with early T stage who are N+ can have better outcomes than high T stages N− (negative lymph nodes) patients . (TIFF 2963 kb)


MYCN-non-amplified SK-N-AS NB xenografts. Human SK-N-AS cells, which are poorly differentiated and express PlGF and VEGF-A, were implanted subcutaneously in athymic nude mice. Treatment was done by intratumoral injection of replication-incompetent adenoviruses (Ad) expressing PlGF- or VEGF-specific short hairpin (sh)RNA, or soluble (s)VEGF receptor 2 (VEGFR2). The effect on tumor growth and angiogenesis was analyzed. High PlGF expression levels were observed in human advanced-stage NBs. Down-regulating PlGF significantly reduced NB growth in established NB xenografts by reducing cancer cell proliferation but did not suppress angiogenesis. In contrast, blocking VEGF by administration of Ad(sh)VEGF and Ad(s)VEGFR2 reduced tumor growth associated with decreased tumor vasculature. These findings suggest that PlGF and VEGF-A modulate MYCN-non-amplified NB tumors by different mechanisms and support a role for PlGF in NB biology.Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and is a rapidly growing, highly-vascularized cancer. NBs frequently express angiogenic factors and high tumor angiogenesis has been associated with poor outcomes. Placental growth factor (PlGF) is an angiogenic protein belonging to the vascular endothelial growth factor (VEGF) family and is up-regulated mainly in pathologic conditions. Recently, PlGF was identified as a member of a gene expression signature characterizing highly malignant NB stem cells drawing attention as a potential therapeutic target in NB. In the present study, we sought to investigate the expression of PlGF in NB patients and the effect of PlGF inhibition on high-risk  MYCN oncogene, which is observed in about 20% of neuroblastoma cases, also play a role in determining the tumor phenotype and predicting the outcome. Together with other prognostic factors, they have been used to categorize patients in four categories, very low risk, low risk, intermediate risk, and high risk [Neuroblastoma (NB) arises from primitive neuroepithelial cells of the neural crest and is the most frequently occurring solid tumor in children. Although some NB patients have a good response to treatment, a subset of NB patients is poorly responsive to current therapeutic schemes . Consequigh risk ,3.Additionally, several reports on NB have shown the important dependency of NB on angiogenesis, demonstrating that high vascularity is characteristic for the progressed tumor stages and poor outcome in human NB ,5. High-MYCN up-regulates VEGF-A in NB cells [MYCN-amplified NBs induced a higher angiogenic response compared to the non-amplified ones [MYCN-amplified NB xenografts suggest that established MYCN-amplified NB tumors are relatively VEGF-independent, and display the ability to up-regulate alternative pro-angiogenic mechanisms, including the PlGF pathway, that may stabilize vasculature and reduce the efficacy of an antiangiogenic therapy [Increased expression of VEGF-A, a central mediator of tumor angiogenesis, was found in advanced-stage NBs (stages 3 and 4) compared with low-stage tumors  . It has NB cells  and MYCNied ones . However therapy .Recently, PlGF-2 was identified as gene belonging to a gene expression signature elevated in highly malignant NB stem cell lines . PlGF isMYCN-nonamplified NB.In the present study, we analyzed the expression PlGF in NB patients. We also aimed to define the potential anti-tumor and antiangiogenic effects of an anti-PlGF treatment in comparison to anti-VEGF treatments in a xenograft model of We first examined serum PlGF levels of 37 children with NB and evaluated PlGF mRNA and protein expression in NB biopsies by quantitative real-time reverse transcriptase–polymerase chain reaction (qRT-PCR) and Western blotting. Patient characteristics, including tumor staging and location, are detailed in p < 0.01; p < 0.003; p = 0.03) and IV (p < 0.001) compared to sera from control patients (PlGF (PlGF-1 and PlGF-2) mRNA expression was significantly up-regulated in biopsies of stage I–IV NBs vs. control biopsies  and Ad(sh)VEGF (p < 0.025), respectively, as shown in PlGF and VEGF-A-specific shRNA-expressing plasmids were then constructed to express shRNAs under the control of a human U6 promoter. The shRNA oligonucleotides containing the 19-nucleotide PlGF or VEGF-specific targeting sequences were cloned into RNAi-Ready pSIREN-plasmid, generating PlGF and VEGF-specific shRNA-expressing plasmids p(sh)PlGF and p(sh)VEGF-A A. We theTaken together, these in vitro data show robust adenoviral infection of SK-N-AS cells and efficient shRNA target gene suppression as well as efficient secretion of (s)VEGFR2 from Ads-transduced NB cells.p < 0.032; To assess the role of PlGF on SK-N-AS tumor growth, we used a xenograft model in which SK-N-AS cells were injected subcutaneously into mice. Ad(sh)PlGF, Ad(sh)VEGF, or Ad(s)VEGFR2 were injected into established tumors on day 10 and the effects on tumor weight were compared. AdRFP and PBS were used as controls. At the beginning of treatment on day 10 after tumor cell inoculation, mice developed human tumors of comparable size as measured using a caliper. On day 22, at which time the animals were sacrificed, the mean tumor mass of the Ad(sh)PlGF treated group (2045 ± 1654 mg) was significantly reduced compared to PBS treated control (5738 ± 853 mg) and AdRFP (4791 ± 1266 mg) control mice (p < 0.034) along with the tumor suppression following Ad(sh)PlGF treatment as compared to controls  and cancer cell (human) tissue PlGF mRNA levels significantly decreased in NB xenografts (controls B. No sigp < 0.003) following Ad(sh)VEGF treatment (p < 0.001), while Ad(sh)PlGF treatment did not affect VEGF-A levels , while cancer cell VEGFR1 and VEGFR2 were not significantly different from controls. No significant differences were observed for VEGFR1 and VEGFR2 mRNA levels following Ad(sh)PlGF treatment (Host VEGFR1 and VEGFR2 mRNA levels were significantly down-regulated following Ad(sh)VEGF and Ad(s)VEGFR2 treatment in SK-N-AS xenografts PlGF group also appeared as extensively hemorrhagic tumors. In contrast, Ad(sh)VEGF and Ad(s)VEGFR2 groups were rather firm, with only limited areas of hemorrhage A. In Fig+ proliferating NB cells in SK-N-AS xenografts were significantly reduced in the PlGF-shRNA group by 55% as compared to controls PlGF shrinks the tumor. Accompanying the reduction in tumor growth, Ki67p < 0.022)  were detected by staining with an anti-CD34 monoclonal antibody. In untreated and AdRFP control tumors, mean overall EM density was 9.0 and 9.2 microvessels per 40× field visually (range 5–15 and 3–19), respectively. In Ad(sh)PlGF-treated tumors the mean overall EM density was 10.8 microvessels per 40× field visually (range 6–14), while in Ad(s)VEGFR2- and Ad(sh)VEGF-treated tumors the mean overall EM density was 4.2 and 4.9 microvessels per 40× field visually (range 1–9 in both groups), respectively. Thus, a significantly lower microvessel density was only encountered in the Ad(s)VEGFR2 and Ad(sh)VEGF groups compared to control groups (< 0.022) A,B. ThesMYCN-unamplified SK-N-AS NBs.Taken together, PlGF and VEGF-blockade resulted in a heterogeneous histological appearance of SK-N-AS tumors. PlGF-blockade reduced tumor cell proliferation but did not affect endothelial microvessel density, while VEGF-blockade reduced microvessel density and induced some degree of vessel normalization in MYCN upregulates VEGF-A in NB cells [MYCN-amplified NBs compared to the non-amplified ones [MYCN-amplified stage 4 NB [The very poor prognosis of advanced NB and the modest therapeutic achievements underline the need for designing alternative therapeutic strategies, such as adding an anti-angiogenesis treatment. Angiogenesis is a hallmark of tumor pathogenesis and contributes to the progression of cancer and is a logical target for new and established treatment strategies . It has NB cells . This fiied ones . Additioage 4 NB . Howeverage 4 NB . In addiage 4 NB  and the age 4 NB ,26. Consage 4 NB .Thus, it is not surprising that clinical results of VEGF inhibitors have only demonstrated limited responses in NB ,28. In gMYCN-amplified NB xenografts [An attractive alternative for overcoming these problems is the modulation of PlGF/VEGFR1 signaling, since PlGF expression, unlike that of VEGF, is undetectable in most organs in healthy conditions, but is highly up-regulated in tumor conditions . Thus, inografts .MYCN-non-amplified NB. Intriguingly, we demonstrated that PlGF inhibition reduced the proliferation of tumor cells in established MYCN-non-amplified NB xenografts resulting in suppression of tumor growth, but unexpectedly did not alter the tumor vasculature. In contrast, VEGF blockade affected the tumor vasculature but did not affect tumor cell proliferation. These results suggest that both PlGF and VEGF are critical to NB growth in established MYCN-non-amplified NB and that they act via different mechanisms. Interestingly, PlGF mRNA levels were significantly up-regulated in all tumor stages, being most pronounced in stage II tumors, a pattern opposite to that of PlGF protein and serum levels, which were highest in advanced stages III and IV. This discrepancy can be explained, at least in part, by post-transcriptional regulation of PlGF mRNA under pathological conditions. In support of this, up-regulation of PlGF protein without a concomitant increase in PlGF mRNA was shown in a retinopathy mouse model [In this study, we demonstrate that PlGF is up-regulated in NB tissue and serum of patients and provide experimental evidence for a cancer promoting activity of PlGF in se model . Anotherse model . In thisse model . Thus, PPlGF transmits its signal through FLT-1 and affects a wide range of different cell types and induces various biological effects, such as vessel growth and maturation . In tumoFurthermore, experimental models using human melanoma and sarcoma xenografts in mice showed elevated murine but not human PlGF levels following anti-VEGF treatment, suggesting that PlGF up-regulation by VEGF blockade is at least in part a host response . Partly The study has been approved by the Ethics Committee of the Medical University of Vienna . Biopsies and serum samples were obtained from 37 patients with NB, following a written informed consent given by the parents of the children. Patients were scheduled for surgical intervention and had routine medical programs before surgery. As controls, adrenal biopsies and serum were obtained from seven children diagnosed with congenital ureteropelvic junction obstruction who underwent Anderson-Hynes pyeloplasty, following a written informed consent. All biopsies were analyzed by histological screening according to standard procedures. Tumor and adrenal biopsies, as well as serum of patients and controls, were analyzed using qRT-PCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA) as described below.MYCN single copy, chromosomal status of 1p36: loss of heterozygosity (LOH), derived from a six year old girl with a poorly-differentiated NB) [2. Cell lines were tested for authenticity by using STR-PCR .Human NB cell line, SK-N-AS (ated NB)  and humaBamHI and EcoRI restriction site overhangs were designed. The target sequences of the used shRNAs were: hu/muPlGF: 5′-CCAAGCCAGATTCTCTTGA-3′ and hu/muVEGF: 5′-CCTCCGAAACCATGAACTT-3′ The sequences of the used shRNAs oligonucleotides were: hu/muPlgf-sh-s: 5′-GATCCGCCAAGCCAGATTCTCTTGATTCAAGAGATCAAGAGAATCTGGCTTGGTTTTTTACGCGTG-3′, hu/muPlgf-sh-as: 5′-AATTCACGCGTAAAAAACCAAGCCAGATTCTCTTGATCTCTTGAATCAAGAGAATCTGGCTTGGCG-3′; hu/muVEGF-sh-s: 5′-GATCCGCCTCCGAAACCATGAACTTTTCAAGAGAAAGTTCATGGTTTCGGAGGTTTTTTACGCGTG-3′, hu/muVEGF-sh-as: 5′-AATTCACGCGTAAAAAACCTCCGAAACCATGAACTTTCTCTTGAAAAGTTCATGGTTTCGGAGGCG-3′. shPlGF and shVEGF genes were first subcloned into RNAi-Ready pSIREN shuttle vector  using BamHI-EcoRI, generating pSIREN-shPlGF and pSIREN-shVEGF. The shRNA sequences were screened for functionality in mouse S3T3 fibroblasts (ATCC) and SK-N-AS cells. The shRNA expression cassette from the newly-constructed pSIREN-shPlGF and pSIREN-shVEGF shuttle vectors was then ligated into the replication-deficient Ad5 genome-based 32kb BD Adeno-X Viral DNA  using Pi-SceI/I-CeuI. The resulting Adeno-X-shPlGF and -VEGF product was then transformed into Escherichia coli for expansion and purified using NucleoBond Plasmid Midi Kit (Clontech). HEK293 cells were then transfected with PacI-digested Adeno-X constructs using Lipofectamine (Life Technologies). The viruses were propagated in HEK293 cells and purified using the Adeno-X Virus Purification kit (Clontech) according to the manufacturers’ protocol. The adenoviral titers were determined using the Adeno-X Rapid Titer kit (Clontech). The function of viruses was evaluated in SK-N-AS cells. SK-N-AS cells were infected with a multiplicity of 100 pfu/cell in six-well plates and the down-regulation of the target genes was determined by qRT-PCR and Western blotting.To generate functional recombinant adenovirus constructs for silencing of human and mouse PlGF or VEGF-A, shRNAs with EcoRI and BamHI linkers. The 2289 bp construct was gel purified with Qiaex II  and recombinant adenovirus Ad(s)VEGFR2 was constructed as described [The soluble murine VEGFR2 open reading frame was purchased from Invivogen  and amplified with forward (5′-GGAATTCCACCATGGAGAGCAAGGCGCTGC-3′) and reverse (5′-CGGGATCCCGCTAGGCACCTTCTATTATGA-3′) primers incorporating escribed . Adeno-XNB cells and tissues were processed for qRT-PCR as described . The priLCDA Version 3.5.3  was used for PCR data analysis. Relative quantification of the signals was performed by normalizing the signals of the different genes to β2-microglobulin as described .NB cells and tissues were processed for Western blotting as described . In brieImmunoassay (ELISA) for human PlGF  was performed using 100 µL of cell culture supernatant according to manufacturer’s protocol. The absorbance was detected using a microplate reader  at 450 nm.4 events was conducted on a FACScan flow cytometer (BD Biosciences) with an argon laser tuned to 488 nm.SK-N-AS cells were seeded in 10 cm plates and allowed to adhere before infection with Ad(s)VEGFR2 and one portion of the cells were left untreated. Cells were washed with PBS and then fixed with Cytofix fixation buffer  for 30 min at 37 °C, washed, and then permeabilized with Perm buffer III (BD Biosciences) and stained with VEGFR2 antibody (LifeSpan BioSciences) to detect intracellular VEGFR2 expression. The analysis of 10n = 4. Mice were anesthetized PlGF, Ad(sh)VEGF, or Ad(s)VEGFR2 viruses in 100 µL PBS intratumorally. Control animals received injections of 109 AdRFP in PBS or 100 µL PBS only. Treatment was repeated twice at five day intervals. Tumor mass was determined on termination. All mice were sacrificed on day 22, and tumors were isolated and weighed. One portion of the tissue was processed for paraffin embedding, and the remainder was processed for qRT-PCR.The experiments performed in this study were approved by the Institutional Animal Care and Use Committee at the Medical University of Vienna. Pathogen free immune-deficient male athymic nu/nu (nude) mice , five weeks of age were weighed, coded, and randomly assigned to experimental groups of Cells were harvested from AdRFP and Ad(s)VEGFR2 infected SK-N-AS cultures. Cytospins were fixed in acetone for 8 min at 4 °C, and sequentially incubated with primary goat anti-mouse VEGFR2/FLK-1 antibody (LifeSpan BioSciences), a biotinylated donkey anti-goat IgG secondary antibody (LifeSpan BioSciences), and horseradish peroxidase (HRP)-conjugated streptavidin   developed with 3,3′-diaminobenzidine , counterstained with hemalaun, dehydrated, and mounted in DPX .2O2 to block endogenous peroxidase activity and antigens were stained with hematoxylin and eosin (H and E), or subjected to immunohistochemical analysis for Ki-67 antibody  to evaluate the density of proliferating cells [Paraffin-embedded tumor sections were rehydrated, incubated in 5% Hng cells , and antp values of <0.05 were considered to indicate statistical significance. Statistical tests were performed with the use of Statistical Package for the Social Sciences (SPSS) software .Differences between groups were studied using analysis of variance (ANOVA) followed by post hoc tests . All statistical tests were two-sided. Data are expressed as means ± the standard deviation (SD). MYCN-non-amplified NB. PlGF inhibition by virally-expressed shPlGF reduced tumor growth in a MYCN-non-amplified NB xenograft model by affecting proliferation of tumor cells without remodeling of the vascular network. In contrast, blockade of VEGF-A by using virally-expressed shVEGF-A or sVEGFR2, which antagonizes VEGFR2 activity, reduced tumor growth associated with vessel normalization and decreased hemorrhage. A multiple target approach based on the combination of PlGF blockade with anti-tumor cell activity and anti-vascular therapies could be expected to improve the therapeutic effects of anti-angiogenic strategies against NB.In this study, we show that PlGF is up-regulated in NB tissue and serum of NB patients and that PlGF acts as a positive regulator of NB growth. Moreover, we identified PlGF as a potential therapeutic target in high-risk


AppNL-G-F/NL-G-F and 3xTg-AD-H mouse models. Genes commonly altered in AppNL-G-F/NL-G-F and human AD cortices correlated with the inflammatory response or immunological disease. Among them, expression of AD-related genes  was increased in the AppNL-G-F/NL-G-F cortex as Aβ amyloidosis progressed with exacerbated gliosis, while genes commonly altered in the 3xTg-AD-H and human AD cortices correlated with neurological disease. The AppNL-G-F/NL-G-F cortex also had altered expression of genes  defined as risk factors for AD by genome-wide association study or identified as genetic nodes in late-onset AD. These results suggest a strong correlation between cortical Aβ amyloidosis and the neuroinflammatory response and provide a better understanding of the involvement of gender effects in the development of AD.Alzheimer’s disease (AD) is the most common form of dementia, characterized by accumulation of amyloid β (Aβ) and neurofibrillary tangles. Oxidative stress and inflammation are considered to play an important role in the development and progression of AD. However, the extent to which these events contribute to the Aβ pathologies remains unclear. We performed inter-species comparative gene expression profiling between AD patient brains and the  Alzheimer’s disease (AD) is the most common form of dementia, and amyloid β (Aβ) plaques and neurofibrillary tangles (NFTs) are the classical hallmarks of this disease2. Currently, a growing body of evidence supports the concept that oxidative stress and inflammation may also play an important role in the development and progression of AD pathologies. Data from clinical studies revealed systemic immune-related changes in AD brains6. However, whether those events occur during the later stages of disease or contribute to the Aβ pathologies remains unclear.Dementia affects over 47 million people throughout the world, and this number is likely to increase to more than 131 million by 20509. To more accurately reproduce AD pathologies, App knock-in mouse models that carry pathogenic mutation(s) in App and/or Psen1 genes have been established. These mouse models show age-dependent amyloidosis, with activated astrocytes and microglia surrounding Aβ plaques, synaptic dysfunction and deficits in behavioural and cognition assays; revealing that amyloidosis triggered by pathological modifications in APP processing is sufficient to induce cognitive impairment11.To better understand the molecular mechanisms of AD pathologies, different animal models have been established. Transgenic mouse models overexpress genetically modified Aβ precursor protein (APP), presenilin (PSEN) and/or the microtubule-associated protein tau (MAPT), to induce accumulation of Aβ or neuronal dysfunction. However, these transgenic mouse models develop AD-like pathologies at different ages and to different extents due to expression levels of AD-related proteins that are dependent on promoters used in transgene constructs, as well as copy number of transgenes and inserted regions16, raising the question of how Aβ accumulation in the brain cortex is involved in pathophysiological alterations observed in AD.Most studies in mouse models have focused on the effect of AD pathologies in the hippocampal area. However, cortical areas also play an important role in the maintenance of brain integrity; novel imaging technologies show Aβ depositions and morphological alterations in the cortex of AD patientsAppNL-G-F/NL-G-F and 3xTg-AD (3xTg-AD-H) mouse models to determine differential gene expression profiles to understand how expression changes contribute to the progression of AD pathologies. AppNL-G-F/NL-G-F mice carrying the homozygous mutant App gene encoding the humanised Aβ sequence  with three pathogenic mutations, namely Swedish (KM595/596NL), Beyreuther/Iberian (I641F), and Arctic (E618G)10, progressively exhibit Aβ accumulation starting at 4 to 6 months of age, dense distributions of microglia and astrocytes from 9 months of age, and behavioural symptoms from 8 to 12 months of age11. The 3xTg-AD-H mice that overexpress two mutated human transgenes, Swedish APP (KM670/671NL) and MAPT (P301L) driven by the exogenous neuronal Thy1.2 promoter, with a knock-in mutation of Psen1 (M146V) that promotes formation of Aβ plaques and NFTs, also exhibit behavioural symptoms and Aβ and Tau pathologies before 12 months of age17. It is noteworthy that the Tau pathology that occurs in the 3xTg-AD-H brain is induced by a pathogenic Tau protein encoded by a mutant MAPT (P301L) gene, not as a result of elevated Aβ, therefore this AD mouse model enables us to examine the brain response to Tau pathology.The present study aimed to identify expression profiles of cortical genes in AD patients and AD mouse models, as well as their associated biological functions. We performed inter-species comparative gene expression profiling between AD patient brains and the AppNL-G-F/NL-G-F mice, but not 3xTg-AD-H mice, exhibited an altered expression profile of cortical genes, indicating a strong correlation between cortical Aβ amyloidosis and the neuroinflammatory response, similar to that observed in the human AD cortex.We thus examined the two mouse models, and the gene expression profiles altered by only Aβ, or by Aβ and Tau pathologies, were compared with those obtained from human AD brains. We found that 18. In the present study, we aimed to characterise gene expression profiles in AD cortical regions by re-analysing the microarray data from temporal and frontal cortices of AD patients and controls  > 6.64, a fold change ≥ 1.2 or ≤−1.2)  genes in the 3xTg-AD-H mice were differentially expressed compared with the corresponding controls  > 6.64, a fold change ≥ 1.2 or ≤−1.2)  and microglia  were significantly increased in the human AD temporal cortex and more prominently in the AppNL-G-F/NL-G-F cortex, suggesting gliosis. These trends were barely observed in human AD frontal and 3xTg-AD-H cortices. Expression levels of some oligodendrocyte markers were also significantly increased in human AD temporal cortex, and to a lesser extent in AppNL-G-F/NL-G-F cortex. Most neuronal markers exhibited a trend towards decreased expression in human AD cortices. In particular, the expression levels of RBFOX3 encoding neuronal nuclear antigen (NeuN), a marker for post-mitotic neurons, were 18 to 27% lower than non-AD controls, thus supporting the neuronal loss observed in AD cortices. In contrast, there was no significant reduction in the expression of any neuronal marker in the two AD mouse models, in good agreement with the observation that these AD mouse models do not exhibit neuronal loss in the brains17, indicating that the stages of disease being compared between human and mouse brains is not the same.We compared expression levels of genes encoding specific markers for four major types of brain cells: astrocytes, microglia, neurons and oligodendrocytes Table , in ordeAppNL-G-F/NL-G-F cortex, in accordance with previous reports11, suggesting that neuroinflammation in the AppNL-G-F/NL-G-F cortex, with increased Aβ burden, may represent pathological alterations seen in the human AD temporal cortex.Taken together, these data indicate aggressive gliosis in AD cortices, especially in the AppNL-G-F/NL-G-F and human AD cortices were subjected to biological function analysis using IPA, and were categorised into various biofunctions: inflammatory response (33), immunological disease (34), organismal injury and abnormalities (57), neurological disease (34), inflammatory disease (22), and others , the human leukocyte antigen system (HLA), B2M, LILRB4, CD37, CD9, IL13RA1 and AQP4 , which have direct or indirect connections with APP  in 3xTg-AD-H cortex were categorised into the same group  were commonly upregulated in the AppNL-G-F/NL-G-F mouse and human AD temporal cortices , but these increases were not statistically significant  among the 1372 altered genes in the AD temporal cortex, and 17 (7.2%) out of 236 altered genes in the AD frontal cortex were categorised as AD-related genes according to IPA function annotation, whereas in AD mouse models, a total of 37 out of 280 genes (13%) in the % among tAppNL-G-F/NL-G-F and wild-type mice at 5, 7 and 12 months of age, in order to explore effects of gender and age on expression of the AD-related genes. During these periods, the area of Aβ deposition in the cortex progressively increases, together with behavioural symptoms starting at 8–9 months of age, and these events are more rapidly observed in female mice11. We performed qRT-PCR using RNA from entire cortex, and found that all 10 genes exhibited an age-dependent increase in their expression levels in male and female AppNL-G-F/NL-G-F mice , Tf  and Vim  genes between AppNL-G-F/NL-G-F and wild-type mice, although expression of C4b, Ctss, Gfap, Nef2l2, Phyhd1 , S100b, Tf , Tgfbr2 and Vim  was significantly increased. In AppNL-G-F/ NL-G-F mice, gene expression levels of C4b, Ctss, Gfap, S100b, Tf, Tgfbr2 and Vim genes were greater in females than in males but S100b, Tf and Tgfbr2 showed higher expression in females only at 12 months of age. At 7 months of age, male AppNL-G-F/NL-G-F mice expressed higher levels of Cd74 and Phyhd1 than females, while there was no obvious gender difference in the expression level of Nfe2l2. Expression levels of Vim in female AppNL-G-F/NL-G-F mice were higher than in males at any age, reaching its peak at 7 months of age, then decreasing, while males exhibited a continuous increase during aging. In the human AD brain, expression levels of PHYHD1 in the AD frontal cortex were significantly greater in females than males (ANOVA: P = 0.0097), and VIM expression in the AD temporal cortex was greater in females (ANOVA: P = 0.0697).We next evaluated expression levels of the 10 AD-related genes in the cortices of male and female ice Fig.  and S7. AppNL-G-F/NL-G-F mice, in comparison with wild-type mice  whose expression is reported to be significantly altered in late-onset AD (LOAD) patients and APPK670N/M671L/PSEN1M146V transgenic mice30, in our microarray data from human and mouse brains  were significantly increased in human AD temporal but not frontal cortex, while expression of GRIN2B was significantly decreased in both human AD cortices. In AppNL-G-F/NL-G-F cortex, expression levels of 13 genes  were significantly increased, and that of Frmd6 was significantly decreased. In 3xTg-AD-H cortex, expression levels of two genes (Ab13 and Frmd6) were significantly decreased, and only that of Trem2 was significantly increased.Finally, we evaluated the expression levels of 57 genes defined as risk factors for AD by genome-wide association study (GWAS), together with genes in the immune/microglia module . The AD patient brains exhibited a much larger number of genes with altered expression in temporal cortex than in frontal cortex. Expression levels of 59 genes were commonly altered in the AppNL-G-F/NL-G-F and human AD temporal cortices, and most of these genes (34 genes) were related to inflammatory response or immunological disease. Among them, expression of 10 genes , which are categorised as AD-related by IPA, was increased in the AppNL-G-F/NL-G-F cortex as Aβ amyloidosis progressed with exacerbated neuroinflammation. Only 17 genes were commonly altered in the 3xTg-AD-H and human AD temporal cortices, most of which related to neurological disease.In the present study, we performed inter-species comparative gene expression profiling using cortical RNA prepared from AD patient brains  and two different AD mouse models  with the human AD hippocampus18, some of which are related to neuronal metabolic and synaptic functions, sugesting that the 3xTg-AD-H cortex mimics hippocampal and to lesser extent cortical profiles in AD patient brains. These results support the fact that different AD mouse models represent different features of human AD pathologies7.In human AD, only the temporal cortex exhibited significant upregulation of several marker genes for astrocytes, microglia and oligodendrocytes, and significant downregulation of several neuronal marker genes Table , supportles Fig. . It is nAppNL-G-F/NL-G-F mice exhibit aggressive extracellular Aβ deposition as early as at 5 months of age, and gliosis from 7 to 12 months of age, throughout cortical and hippocampal regions, and memory impairment in an age-dependent manner  were categorized to inflammatory response or immunological disease  or only Tau (MAPTP301L) pathology revealed that immune gene expression correlated tightly with Aβ plaques, whereas synaptic genes correlated negatively with NFTs26.Studies on post-mortem brains have shown that AD pathologies are accompanied by neuroinflammation, probably as a consequence of Aβ amyloidosis or neuronal damage. However, recent neuroimaging and genome-wide association studies further suggest that neuroinflammation is an early event that takes place even before Aβ amyloidosisrks Figs  and 6. I30, in our microarray data from human and mouse brains  were significantly upregulated in the AppNL-G-F/NL-G-F cortex. Among these, overexpression of TYROBP in microglial cells has been reported to alter the expression of the microglia module that is dominated by genes involved in pathogen phagocytosis25. Moreover, Fcer1g and Trem2 have been identified as member of the hub genes  of the immune module in the cortex from transgenic mouse lines with only Aβ pathology but not with only Tau pathology26. Our results clearly indicate that the prominent neuroinflammation observed in the AppNL-G-F/NL-G-F cortex is a result of pure Aβ pathology induced by App knock-in mutations. Only the Trem2 gene was mildly upregulated in 3xTg-AD-H cortex, in agreement with their milder Aβ pathology and inflammatory responses in comparison with AppNL-G-F/NL-G-F cortex  was significantly increased in the AppNL-G-F/NL-G-F and human AD cortices. A recent report showed a significant increase in the copy number of C4 genes in AD patients, compared with healthy controls37, which may contribute to the elevated levels of C4 in cerebrospinal fluid or serum in the AD patients39. Moreover, it has been shown that C4 surrounds Aβ plaques in the cortex of an AD mouse model40. C4b, a cleaved product of C4 by the C1 complex, which can be activated by Aβ41, functions as a C3 convertase with C2b, thus resulting in activation of the complement system, which may in turn inappropriately activate microglia, thereby mediating synapse loss42 or a further inflammatory response43.Expression of several complement component genes 44. It has been shown that CD74 expression increases in microglia, astrocytes and NFT-positive neurons of AD patients46. Moreover, CD74 was reported to interact with APP and suppress production of Aβ48, while CD74 itself is processed by cathepsin S, encoded by CTSS, thus releasing its cytoplasmic domain49, which is essential for the proinflammatory NF- κB activation50. Because only full-length CD74 can interact with APP48, an increased expression of cathepsin S in the AD cortex could deplete the full-length CD74, thereby cancelling the suppression of Aβ production and rather activating NF-κB. Additionally, cathepsin S could be involved in lysosomal processing of APP to produce Aβ51.AppNL-G-F/NL-G-F cortex, gene expression of Ctss as well as Nfe2l2, Tgfbr2 and Gfap was already elevated at 5 months of age compared with wild-type mice 54, which shows decreased levels with age but remains responsive to oestradiol treatment55. This may explain why the female-specific Vim expression in AppNL-G-F/NL-G-F cortex peaked at 7 months of age , in complex manners59. Our results thus suggest that oestrogen together with TGF-β may induce more severe Aβ amyloidosis and changes in gene expression profiles in females.When we compared expression levels of the 10 genes shown in Fig. AppNL-G-F/NL-G-F mouse, a novel AD mouse model with authentic expression of pathogenic Aβ, exhibits a cortical gene expression profile that reproduces changes observed in the human AD brain, in a limited but faithful manner. Results from the present study indicate a strong correlation between cortical Aβ amyloidosis and neuroinflammation, and also provide important clues to better understand the role of gender effects in AD development.In conclusion, the The use of human postmortem brain tissue was approved by the Ethics Committee of the Faculty of Medicine, Kyushu University, Fukuoka, Japan, and was performed in accordance with the ethical standards described in the latest revision of the Declaration of Helsinki. Written informed consent for all subjects was obtained from their families. The handling and killing of all animals was performed in accordance with national prescribed guidelines, and ethical approval for the study was granted by the Animal Experiment Committee of Kyushu University, Fukuoka, Japan.18. All RNA samples were preserved at −80 °C until further use.We previously prepared total RNA from freshly frozen cerebral cortices of frontal and temporal poles at several centimeters thick removed from post-mortem brains donated for the Hisayama study between December 15, 2008 and February 24, 201118 are available from the GEO database (accession number GSE36980). CEL files were imported into the Affymetrix Expression Console  and CHP files were obtained using a Gene Level-RMA-Sketch method. CHP files were input into the Affymetrix Transcriptome Analysis Console (TAC) software and a gene level differential expression analysis was performed according to the user’s guide. One-way between subject ANOVA was performed between AD and non-AD subjects and a list of transcripts was created. Principal Component Analysis (PCA) and hierarchical clustering was performed in the Affymetrix Expression Console and TAC software, respectively, and several samples were found to be outliers, likely owing to biological heterogeneity or technical issues  and frontal cortices (15 AD and 18- non-AD cases)Psen1M146V knock-in mutation and homozygous mutant transgenes for Swedish APPKM670/671NL and MAPTP301L, and control non-Tg mice were previously established18. Heterozygous App+/NL-G-F mice carrying humanised Aβ sequence , Swedish (ML595/596NL), Beyreuther/Iberian (I641F), and Arctic (E618G) mutations, were previously established10. Homozygous AppNL-G-F/NL-G-F and wild-type mice were obtained by crossing, and were maintained as inbred lines. All animals were maintained in a specific pathogen-free room.The homozygous triple-transgenic mouse model of AD (3xTg-AD-H), carrying a homozygous For transcriptomic analyses, mice were anesthetized, transcardially perfused with saline, and brain cortices were quickly dissected, snap-frozen in liquid nitrogen, and preserved at −80 °C until RNA preparation. For immunofluorescence, mice were perfused with saline followed by cold 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). The brains were removed and post-fixed in 4% PFA for 24 hours at 4 °C. Tissue blocks were cryoprotected in 20% sucrose, followed by 30% sucrose, in PBS, and then embedded in FSC 22 frozen section media . The tissue blocks were quickly frozen and stored at −80 °C until further use.AppNL-G-F/NL-G-F and 3xTg-AD-H mice together with the corresponding wild-type or non-Tg control mice, respectively . Total RNA was prepared from frozen cortex using Isogen  according to the manufacturer instructions. RNA concentrations were determined by measuring the UV absorbance spectra, and the total RNA profile was analysed using an Agilent 2100 Bioanalyzer  to determine RNA integrity number (RIN). RNA (100 ng) was used for microarray analysis. The GeneChip WT PLUS Reagent Kit (Affymetrix Japan K.K.) was used to generate amplified and biotinylated sense-strand DNA targets. Manufacturer instructions were followed for hybridisation, washing, and scanning steps with Affymetrix Mouse Gene 2.0ST Array, and CEL files were generated. CEL files were further analysed as described for analysis of human microarray data. The lists of transcript clusters significantly altered  > 6.64, compared with control) were further analysed using Ingenuity Pathway Analysis  software to determine the commonly altered genes between AD patients and each AD mouse model, as well as the relevant biological function categories and network-based interactions. All microarray data were deposited in the GEO database (accession number GSE92926).We performed microarray analyses using cortical RNA prepared from 12-month-old 60. Gapdh was used as an internal control and we verified that Gapdh levels do not change between mutant and control mice . Primer pairs  were prepared using a cryostat and collected as free-floating sections. Sections were blocked in 2 × Block Ace solution  for 2 hours at room temperature, then incubated with a corresponding mix of primary antibodies , and either rabbit anti-GFAP  or anti-IBA1 ) overnight at 4 °C. Corresponding Alexa Fluor-labelled secondary antibodies (Life Technologies Japan) were then added and incubated for 45 minutes at room temperature, followed by 0.05 μg/ml DAPI for 10 min at room temperature, and mounted on slides. All sections were rinsed in 0.3% Triton X-100 in PBS, 3 times for 5 min. The sections were mounted on glass slides and air-dried. The sections were then embedded with VECTASHIELD Mounting Medium . Multiple z-stack images of 15 fields were obtained, tiled, and stacked together using a confocal microscope  with Zen 2012 software (Carl Zeiss Microscopy). The intensity of GFAP or IBA1 immunofluorescence was measured in each digital image using ImageJ 1.51n (NIH) to obtain the GFAP or IBA1 index, which corresponds to one thousandth of the mean intensity per µmP-value < 0.05 was considered statistically significant.Gene-level estimates from microarray data were subjected to one-way between subject ANOVA using Affymetrix TAC software. Statistical analysis was performed using JMP Pro Version 13.2.0 software . A Supplementary InformationSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S6Supplementary Table S7Supplementary Table S8Supplementary Table S9Supplementary Table S10


Muscle atrophy may arise from many factors such as inactivity, malnutrition, and inflammation. In the present study, we investigated the stimulatory effect of nitric oxide (NO) on muscle protein synthesis. Primarily, C2C12 cells were supplied with extra L-arginine (L-Arg) in the culture media. L-Arg supplementation increased the activity of inducible nitric oxide synthase (iNOS), the rate of protein synthesis, and the phosphorylation of mTOR (Thr 2446) and p70S6K (Thr 389). L-NAME, an NOS inhibitor, decreased NO concentrations within cells and abolished the stimulatory effect of L-Arg on protein synthesis and the phosphorylation of mTOR and p70S6K. In contrast, SNP (sodium nitroprusside), an NO donor, increased NO concentrations, enhanced protein synthesis, and upregulated mTOR and p70S6K phosphorylation, regardless of L-NAME treatment. Blocking mTOR with rapamycin abolished the stimulatory effect of both L-Arg and SNP on protein synthesis and p70S6K phosphorylation. These results indicate that L-Arg stimulates protein synthesis via the activation of the mTOR (Thr 2446)/p70S6K signaling pathway in an NO-dependent manner. Nitric oxide (NO) is a free radical that is produced by nitric oxide synthase (NOS) enzymes, which catalyze the conversion of L-arginine (L-Arg) to L-citrulline . NO partmdx mice  [L-Arg, the precursor to NO, is involved in protein phosphorylation cascades and gene expression –7. L-Argstrophy) . Additiostrophy) , 11. Howstrophy) . Therefostrophy) . L-arginstrophy) . L-citrustrophy) . NO is istrophy) . The actstrophy) . The maistrophy) , 19. Thestrophy) . There istrophy) , 22. Howin vitro in an mTORC1-dependent manner [The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, plays a central role as a nutrient and energy sensor in skeletal muscle. The mTOR signaling pathway controls cell growth and metabolic progression by phosphorylating two downstream proteins: the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and the ribosomal p70S6 kinase1 (p70S6K) , 24. As t manner . Therefoin vitro in differentiated mouse C2C12 myoblasts. The protein synthesis rate was estimated with a nonradioactive method by labeling the newly synthesized polypeptides with low concentrations of puromycin and subsequently detecting these proteins with an anti-puromycin antibody [In this study, the effect of L-Arg on myocyte protein synthesis and the involvement of NO were investigated antibody , 29. Then = 6 or 7).Mouse C2C12 myoblasts  were plated and cultured in high-glucose Dulbecco's modified Eagle's medium  with 10% fetal bovine serum  and 1% penicillin/streptomycin . At 80% confluence, the cells were induced to differentiate and form myotubes by culturing in DMEM supplemented with 2% horse serum  for 84 h. Before treatment, the medium was replaced with serum-free DMEM for 12 h. Finally, the C2C12 cells were exposed to the treatments detailed below. Each treatment was performed on 6 or 7 samples (μM of L-Arg), extra L-arginine supplementation , N-nitro-L-arginine methyl ester , and L-Arg supplementation (1 mM) plus L-NAME (10 mM). The treatment doses were selected based on previous studies [μM of sodium nitroprusside  or SNP (1 μM) plus L-NAME (10 mM). At 36 h after treatment, puromycin  was added to the culture media for an additional 30 min, and proteins were extracted from C2C12 cells and used for subsequent analyses.The C2C12 cells were subjected to the following treatments: control  supplementation for 36 h. Thereafter, puromycin was added to the culture medium for an additional 30 min, and proteins were extracted from the C2C12 cells and used for subsequent analyses.The C2C12 cells were treated with 100 nM of rapamycin  for 30 min, followed by L-Arg (1 mM) or SNP . Intracellular NOS activity, including total NOS enzymes (TNOS) and inducible NOS (iNOS), were determined using a commercial kit  according to the manufacturer's instructions. In the reaction system, NOS catalyzes L-arginine to produce NO, which reacts with nucleophilic substances to form nonferrous compounds. The absorbance was determined at 530 nm using a UV-2450 spectrophotometer . The experiment was also performed in the absence of calcium and the presence of a calcium chelator to determine the calcium-independent NOS activity, which was taken to represent iNOS activity.NO concentrations in the media and cells were assessed using a commercial kit . NO is very chemically active and is thus easily converted into NOμg of protein were separated by 7.5% SDS polyacrylamide gel electrophoresis, and the separated proteins were transferred onto polyvinylidene fluoride membranes  at 200 mA for 2 h in western transfer buffer  containing 20% methanol. Membranes were then blocked for 1 h at room temperature and incubated at 4°C overnight with primary antibodies at an appropriate dilution ratio. The following primary antibodies were used: anti-phospho-4E-BP1 (Thr 37/46), anti-4E-BP1, anti-phospho-p70S6K (Thr 389), anti-p70S6K, anti-phospho-mTOR (Ser 2448), anti-mTOR , anti-phospho-mTOR (Ser 2481) and anti-phospho-mTOR (Thr 2446) , anti-mouse puromycin , and anti-β-actin . After washing, the proteins were probed with horseradish peroxidase-linked anti-rabbit or anti-mouse secondary antibodies with gentle agitation for 4 h. The membranes were subsequently exposed to enhanced chemiluminescence plus western blot detection reagents . When two different proteins had the same or similar molecular weight, we used different membranes to separately detect them. In contrast, when two proteins were of different molecular weights or when the phosphorylated and total levels of one protein needed to be analyzed, the same membrane was used again. During this process, the membrane was blocked again and incubated with another antibody after one protein was detected. Finally, the membrane was scanned, and specific bands were quantified using Vilber Fusion FX7 Spectra . The band intensity was normalized to the β-actin band in the same sample. For phosphorylated proteins, when the total protein bands showed significant differences in different treatments, the phosphorylated protein bands were normalized to the total protein bands. In contrast, if the total protein bands remained constant among different groups, both the phosphorylated and total protein bands were normalized to β-actin.The cells were washed briefly with PBS  and collected in 0.2 mL of RIPA (radio immunoprecipitation assay) lysis buffer . Cell debris were removed by centrifugation at 12,000 ×g for 5 min at 4°C, and the supernatants were used for immunoblotting analysis. The BCA Protein Assay Kit  was used to determine protein concentrations. Aliquots containing 18 The protein synthesis rate was detected using a nonradioactive method . The newP < 0.05 and as approaching significance at P < 0.10.The data are expressed as the means ± SEM. The results were analyzed using one-way ANOVA and the Statistical Analysis Systems statistical software package . For the observations of NO, TNOS, and iNOS at 3, 18, and 36 h time points, two-way ANOVA was used to estimate the main effects of L-Arg or L-NAME supplementation and time. Differences between the means were evaluated using Duncan's honestly significant difference tests. Differences were considered as significant at P < 0.05, P < 0.05, Figures P > 0.05) were observed in the levels of phospho-mTOR (Ser 2448 and Ser 2481)  . AdditioP < 0.05) at 18 and 36 h  at 3 h, 18 h, and 36 h, whereas TNOS activity was significantly inhibited  at 18 h (P = 0.093) (P > 0.05) on NO concentrations in either the cells or the cell-free supernatant. Conversely, iNOS activity was significantly increased by L-Arg treatment , regardless of the presence of L-NAME (P > 0.05)  . Comparef L-NAME  at 36 h. > 0.05) .P < 0.05) protein synthesis  in the levels of the total or phosphorylated mTOR (Ser 2448 and Ser 2481)  .P < 0.05) and in the cell-free supernatants , but not TNOS (P > 0.05), activity was suppressed by SNP treatment . Neither SNP nor L-NAME treatment altered the levels of the phosphorylated mTOR (Ser 2448 and Ser 2481)  , and upregulated phosphorylated mTOR (Thr 2446) levels. The stimulatory effect of L-Arg on protein synthesis and p70S6K (Thr 389) and mTOR (Thr 2446) phosphorylation was abolished by the presence of L-NAME, an NOS inhibitor. In contrast, SNP, an NO donor, increased protein synthesis and upregulated both p70S6K and mTOR phosphorylation ; this effect was not altered by L-NAME. Blocking the phosphorylation of p70S6K with rapamycin, however, abolished the stimulatory effect of both L-Arg and SNP on protein synthesis. These results demonstrate that NO is associated with the regulation of muscle protein synthesis via the mTOR/p70S6K pathway. Moreover, except for one mTOR phosphorylation site (Thr 2446), the phosphorylated levels of mTOR (Ser 2448 and Ser 2481) were not altered by the L-Arg, L-NAME, or SNP treatment, suggesting that this specific mTOR site (Thr 2446) is the target site involved in the regulation of protein synthesis by NO.In the present study, we investigated the role of L-Arg on mdx mice [In animal experiments, L-Arg has been demonstrated to enhance protein synthesis in skeletal muscle , 32. Mormdx mice , 9. The mdx mice –35.In the present study, we further investigated the direct role of L-Arg in muscle cell protein synthesis. We confirmed the enhancement of protein synthesis in C2C12 cells following L-Arg supplementation, which was in line with the results of , in whicβL, PRAS40, and DEPTOR, phosphorylates 4E-BP1 and p70S6K and thus stimulates protein synthesis [In vivo, L-Arg treatment was shown to stimulate p70S6K phosphorylation [mTOR complex 1 (mTORC1), consisting of Raptor, Gynthesis . In vivorylation . Under wrylation . In linerylation , who repIn humans, skeletal muscle participates in the overall NO metabolism by serving as a nitrate reservoir . As the To further verify this hypothesis, we evaluated the effect of SNP, an NO donor, on protein synthesis and p70S6K and mTOR phosphorylation. The elevated NO production in cells and in the media, as well as the decreased iNOS activity, indicated that SNP treatment provides sufficient NO independent of NOS. The increased protein synthesis caused by SNP also suggests that NO, rather than L-Arg, is associated with the regulation of protein synthesis in muscle cells. This hypothesis was confirmed by the observation that the negative effect of L-NAME on protein synthesis could be rescued by SNP supplementation. Therefore, the present results demonstrate that NO, rather than L-Arg, was associated with the regulation of protein synthesis in C2C12 cells, which is consistent with the mechanism in intestinal epithelial cells . This reThe SNP-induced upregulation of p70S6K and 4E-BP1 phosphorylation suggests that p70S6K and 4E-BP1 are the target proteins of NO, which is consistent with previous studies , 41. HowNO participates in cellular signal transduction mainly through S-nitrosylation of allosteric and active-site cysteine thiols within proteins , 52, 53.α-induced cachectic muscle loss and in age-related muscle wasting (sarcopenia) [P < 0.01) and 36 h (P = 0.093) but had little influence on iNOS, suggesting that TNOS may be responsible for blocking protein synthesis. In contrast, L-Arg supplementation increased iNOS and TNOS at different time points. Hence, the role of iNOS and nNOS on protein synthesis and the mTOR/p70S6K pathway requires further study.NOS has three isoforms: iNOS, nNOS , and eNOS . In skeletal muscle, nNOS is the major NOS isoform. There is growing evidence that NOS is associated with the development of muscle atrophy. The nNOS/NO system modulates muscle functions such as insulin sensitivity and glucose uptake, muscle contraction, vasodilation, and activation of satellite cells , 55, 56.copenia) . Under pcopenia) . NO may copenia) . L-Citrucopenia) . In the In conclusion, our results demonstrate that L-Arg is associated with the regulation of muscle development via the mTOR (Thr 2446)/p70S6K signaling pathway in an NO-dependent manner . These r