Endothelial cells express S100A4, a metastasis-associated protein, but its role in angiogenesis remains to be elucidated. Here we show that knockdown of S100A4 in mouse endothelial MSS31 cells by murine specific small interference RNA (mS100A4 siRNA) markedly suppressed capillary-like tube formation in vitro, in early stage after the treatment, along with down- and up-regulation of some of the pro-angiogenic and anti-angiogenic gene expression, respectively. Of particular note is that intra-tumor administration of the mS100A4 siRNA in a human prostate cancer xenograft significantly reduced tumor vascularity and resulted in the inhibition of tumor growth. These findings show that S100A4 in endothelial cells is involved in tube formation, and suggest its potential as a molecular target for inhibiting tumor angiogenesis, which warrants further development of endothelial S100A4-based strategies for cancer treatment.The online version of this article (doi:10.1007/s10456-013-9372-7) contains supplementary material, which is available to authorized users. S100A4, a member of the S100 family of EF-hand calcium-binding proteins, is involved in the regulation of a variety of biological processes, including tumor progression and metastasis –7. The iIn this study, we used siRNA-mediated depletion and DNA microarray analysis to investigate the function of S100A4 in endothelial cells. Furthermore, the effect of S100A4 knockdown on angiogenesis and tumor growth was examined in a xenograft cancer model. The results suggest that S100A4 in endothelial cells is involved in tube formation and thus siRNA-mediated inhibition of endothelial S100A4 could provide an effective anti-tumor RNAi medicine.We cultured mouse endothelial cells (MSS31) , 24–26, Synthetic 21-nt RNAs were purchased from Ambion/Life Technologies . The murine S100A4 siRNA (mS100A4 siRNA) sequence was 5′-UGA ACA AGA CAG AGC UCA Att-3′ (sense) and 5′-UUG AGC UCU GUC UUG UUC Att-3′ (antisense). AllStars negative control siRNA  was used as a negative control.4 cells/cm2 and cultured in the presence of 50 ng/ml HGF . Capillary formation was assessed after 16 h of Matrigel culture [After 48 h of siRNA transfection using DharmaFECT No.1 , MSS31 cells were seeded on ECM-gel  thin-coated center-well dishes  at a cell density of 4 × 104 cells/cm2 and then counted the number of adhered cells at 30 min, 1 and 3 h.Migration ability was determined using a cell adhesion and a wound healing assay. After 48 h of S100A4 siRNA or negative control siRNA transfection, MSS31 cells was plated on 24-muti-well plate  at a cell density of 4 × 10siRNA-transfected MSS31 cells were grown on 60 mm plates. After the cells reached sub-confluence, the cells were pretreated with mitomycin C (10 μg/ml) for 30 min and then the cells were wounded by scraping the monolayer and grown in medium for 16 h. The width of the wound was measured. Three different locations were visualized and photographed under a phase-contrast inverted microscope . Data was represented as a percent of wound healing.Total RNA was extracted from cells by ISOGEN  and treated with DNase I . Five μg of total RNA was used to produce cDNAs with oligo (dT) 12 primer by superscript III RNA polymerase (Invitrogen/Life Technologies). cDNA was diluted five-fold and used for quantitative PCR. For quantitation, aliquots of 5 μl of cDNA samples were subjected to quantitative PCR in 50 μl reactions using Platinum Quantitative PCR SuperMix-UDG (Invitrogen/Life Technologies) and Assays-on-Demand TaqMan primers/probe sets  specific for target genes (Supplementary Table S1). Reactions were carried out using the ABI PRISM 7700 sequence detection System (Applied biosystems). The reactions were incubated at 50 °C for 2 min, then heated to 95 °C for 2 min followed by 45 cycles of 30 s at 95 °C, 15 s at 60 °C, and 20 s at 72 °C. The GAPDH gene and β-actin gene were used as an internal control.RNA quantity and quality were determined using a Nanodrop ND-1000 spectrophotometer  and an Agilent Bioanalyzer , as per manufacturer’s instructions. Total RNA was amplified and labeled with Cyanine 3 (Cy3) using Agilent Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies) according to the manufacturer’s instructions. Briefly, 100 ng of total RNA was reversed transcribed to double-strand cDNA, using a poly dT-T7 promoter primer. Primer, template RNA and quality-control transcripts of known concentration and quality were first denatured at 65 °C for 10 min, then incubated for 2 h at 40 °C with 5X first strand Buffer, 0.1 M DTT, 10 mM dNTP mix, and AffinityScript RNase Block Mix. The AffinityScript enzyme was then inactivated at 70 °C for 15 min. cDNA products were used as templates for in vitro transcription to generate fluorescent cRNA. cDNA products were mixed with a transcription master mix in the presence of T7 RNA polymerase and Cy3 labeled-CTP and incubated at 40 °C for 2 h. Labeled cRNAs were purified using QIAGEN’s RNeasy mini-spin columns and eluted in 30 μl of nuclease-free water. After amplification and labeling, cRNA quantity and cyanine incorporation were determined using a Nanodrop ND-1000 spectrophotometer and an Agilent Bioanalyzer. For each hybridization, 1.65 μg of Cy3 labeled cRNA was fragmented, and hybridized at 65 °C for 17 h on an Agilent Mouse GE 4x44K v2 Microarray (Design ID: 026655). After washing, microarrays were scanned using an Agilent DNA microarray scanner. Intensity values of each scanned feature were quantified using Agilent feature extraction software version 10.7.3.1, which performs background subtractions. Features that were flagged as having no errors (present flags) were used and features that were not positive, not significant, not uniform, or not above the background level were excluded. Features that were saturated and any population outliers  were also excluded. Normalization was performed using Agilent GeneSpring GX version 11.0.2. . There were 39,429 probes on the Agilent Mouse GE 4x44K v2 Microarray (Design ID: 026655), excluding control probes. The altered transcripts were quantified using the comparative method. A ≥ twofold change in signal intensity was designated as a significant difference in gene expression.Animal experiments were performed in compliance with the guidelines of the Institute for Laboratory Animal Research at the National Cancer Center Research Institute. The mice were maintained under specific pathogen-free conditions with a 12-h light–dark cycle.6 PC-3M-Luc C6 cells in 100 µl sterile Dulbecco’s PBS [2/2, where a and b are the lengths of the long and short axes, respectively. Number of animals was 4 in each group and the experiment was performed twice.Eight- to 11-week-old male athymic nude mice  were used for all experiments. Anesthetized animals were subcutaneously injected with 3 × 10co’s PBS . Nine daDay 9 after the siRNA treatment, signals of AngioSense-IVM-750 were measured using fluorescence molecular tomography . AngioSense-IVM-750 is a 250 kDa macromolecule that freely circulates while staying confined to the intravascular space. Relative value of angiogenesis of mS100A4 siRNA-treated tumor was measured when the negative siRNA control was set to 1.0 and the data was represented as relative angiogenesis. Blood vessels in tumors monitored by Angiosence day 9 after the siRNA treatment, tumor vessels were also visualized using AngioSense-IVM-680  by intravenous administration of 50 μl per mouse. AngioSense-IVM-680 is a near-infrared labeled fluorescent macromolecule that remains localized in the vasculature for extended periods of time and enables imaging of blood vessels and angiogenesis. Fifteen min after the dye administration, the vessel images were acquired by using Olympus OV110 .To prepare the siRNA/atelocollagen complex, equal volumes of atelocollagen  and siRNA solution were combined and mixed by rotation for 20 min at 4 °C. The final concentration of atelocollagen was 0.5 %. Individual mice were injected with 200 μl of atelocollagen containing 50 μg of mS100A4 siRNA or non-silencing control siRNA/atelocollagen by intratumoral injection (negative control siRNA).Caspase-3/7, which plays key effector roles in apoptosis, was determined with the Apo-ONE Homogeneous caspase-3/7 assay (Promega) according to the manufacturer’s instructions. Cells were incubated with the Apo-ONE caspase-3/7 assay reagent for 1.5 h at room temperature, and the fluorescence was then measured at 485Ex/535Em with a Wallac multi-label counter.6 cells) were injected subcutaneously into C57BL/6 mice . Fourteen days after the injection, tumor tissues were removed, embedded in a tissue-freezing medium, and then cut into 5 μm-thick sections. For double-immunostaining of CD31 and S100A4, tissue sections were fixed in acetone for 10 min. The samples were blocked with PBS containing 0.1 % bovine serum albumin and 2 % goat serum and then incubated with a mixture of rat anti-mouse CD31 antibody  and rabbit anti-S100A4 antibody [http://rsb.info.nih.gov/ij), a ratio of S100A4 pixel values to CD31 pixel values was calculated for each image (n = 6) for determination of S100A4/CD31 double-positive areas.B16-BL6 cells  of CD31+ endothelial cells was S100A4-positive. These results suggest that there exist subpopulations of endothelial cells in tumors that might, or might not, be primed for angiogenesis. This prompted us to examine the role of S100A4 in angiogenesis and, to this end, we tested the effect of siRNA-mediated depletion of S100A4 on capillary formation in mouse endothelial MSS31 cells. Specifically, murine S100A4 siRNA (mS100A4 siRNA) completely blocked S100A4 expression in MSS31 cells at both the mRNA and protein levels . Wound healing assay revealed that knockdown of S100A4 inhibited cell migration significantly (P = 0.0003) in Fig. We first examined whether endothelial cells of tumor microvessels express S100A4. For this, we immunostained the microvessels in tumor tissues formed by B16-BL6 melanoma cells that express little S100A4 with anti-CD31 and anti-S100A4 antibody , tumors were treated twice with 50 μg siRNA/atelocollagen complex intratumorally. After the treatment, tumor angiogenesis was measured at day 9 Fig. .Fig. 4Inaqp1) [fgf18) [retn) [map3k5) [thy1) [foxo6) [hs6st1) [mmp3) [cdkn1a) [thbs1) [spry4) [To gain mechanistic insight into the above observations, we compared the gene expression profiles of MSS31 cells treated with either mS100A4 siRNA or control siRNA. Unsupervised hierarchical cluster analysis was performed on 745 angiogenesis-related genes . The analyses showed that significant alterations in angiogenesis-related gene expression occurred in the S100A4 siRNA treatment group, suggesting that S100A4 plays a role in controlling angiogenesis. Genes with significant alterations (twofold or more) in expression level are listed in Table aqp1) , fibrobl [fgf18) , resisti) [retn) , mitogen[map3k5) , thymus ) [thy1) , forkhea [foxo6) , heparan[hs6st1)  and matr) [mmp3) , which a[cdkn1a) , thrombo [thbs1) , and spr [spry4) . Taken tUsing B16BL6 tumor tissues little expressing S100A4, we stained tumor microvessels for CD31 and S100A4 and found that there are subpopulations of endothelial cells in tumors, S100A4-positive and –negative ones. This observation motivated us to examine a possible role of endothelial S100A4 by silencing it. The multiple angiogenesis assay including tube formation, adhesion, and migration analysis of endothelial cells clearly indicated that endothelial S100A4 plays a crucial role in angiogenesis. S100A4-positive endothelial cells in tumors may represent the ones primed for neoangiogenesis. A comparison of the gene expression profiles of siRNA-treated cells with those of untreated cells showed that endothelial S100A4 acts upstream of a variety of angiogenesis-related genes. These findings were confirmed in a xenograft tumor model, where intratumor administration of siRNA distinctly reduced tumor angiogenesis and growth.In the present study, mouse siRNA was delivered in vivo using atelocollagen, a highly purified type I collagen with low immunogenicity. Atelocollagen forms nano-sized particles when mixed with oligonucleotides such as double stranded RNAs and DNAs via electrostatic binding, and is incorporated into cells by endocytosis , 44. In aqp1, map3k5, and fgf18 are highly expressed in tumor-associated blood vessels in several human tumors [cdkn1a, thbs1, and spry4. This means that S100A4 has a dual effect on angiogenesis-related gene expression: it up-regulates angiogenic genes and down-regulates anti-angiogenic ones, thereby determining the fate of endothelial cells in association with tumorigenesis. The biological significance of all these genes has been proven in vitro and in vivo analysis of angiogenesis in previous reports.Microarray analysis further confirmed the molecular mechanism of S100A4-mediated angiogenesis in endothelial cells. Significant changes in angiogenesis-promoting gene expression occurred in S100A4 siRNA-treated endothelial cells. Among the genes exhibiting altered expression levels, n tumors –48. FurtOur sequential observations revealed that mS100A4 knockdown induced apparent cell growth inhibition of MSS31 cells in a later stage after siRNA treatment. It suggests a possible role of S100A4 in regulating endothelial cell growth, which may, in part, account for a drastic inhibition of neoangiogenesis in mS100A4 siRNA-administered tumor. Further analysis is required to know whether S100A4 promotes growth and survival of neovascular endothelial cells.In the human cancer xenograft model used in this study, only host S100A4 was targeted to explore the role of S100A4 in angiogenesis. However, S100A4 in tumor cells is also involved in invasion and metastasis. Therefore, it would be interesting to investigate whether administration of an agent that inhibits both mouse and human S100A4 could suppress the metastasis of xenografted human tumor cells in immuno-compromised mice. If suppression occurs, it would suggest that inhibition of S100A4 could reduce tumor angiogenesis and by inference, tumor metastatic ability, which would have important implications for the development of cancer therapeutics. Furthermore, it would underscore the advantages of S100A4 inhibition over conventional angiogenesis factor/receptor inhibition. Studies are currently under way to develop such agents.In conclusion, the present results show the importance of endothelial S100A4 expression in tumor angiogenesis. As the generation of a new vascular supply is causally involved in the progression of the majority of solid tumors, siRNA-mediated inhibition of endothelial S100A4 could provide an effective anti-tumor RNAi medicine. Thus, the development of inhibitors targeting endothelial S100A4 could represent a promising approach for effective anti-angiogenesis and anti-metastasis therapy.GAPDH and β-actin were used as quality and loading controls. P29 cells were used as a positive control for S100A4 expression [RT-PCR analysis of the expression of S100A4 in B16-BL6 melanoma cells. Total RNA isolated from Lewis lung carcinoma P29 cells and B16-BL6 melanoma cells was subjected to RT-PCR analysis. pression . B16-BL6P = 0.05. Number of animals  in each group was 4 (TIFF 1521 kb)Relative angiogenesis was measured by signals of AngioSense-IVM-750 using FMT. Relative value of angiogenesis of mS100A4 siRNA-treated tumor when the negative siRNA control was set to 1.0. *Short-term effect of mS100A4 siRNA on the growth of xenografted human tumor. Atelocollagen/mS100A4 siRNA or negative control siRNA complex was delivered into the xenografted human tumor, and the tumor volume was assessed 2 days after the siRNA administration. Relative tumor volume of mice 2 days after siRNA treatment is shown. An apparent reduction of tumor volume on day 2 after the mS100A4-knockdown was not evident (TIFF 1521 kb)Unsupervised hierarchical analysis of 745 gene expression profiles and candidate validation. Data were subjected to a hierarchical cluster analysis using a Euclidean distance calculation based on the unweighted pair group method with arithmetic mean. The samples were aligned horizontally: lane S, mS100A4 siRNA-treated; lane A, negative control siRNA-treated; lane R, transfection reagent alone; and lane N, negative control siRNA alone. Red, yellow, and blue color areas represent high, middle, and low expression levels, respectively (TIFF 1521 kb)The gene list from 745 angiogenesis-related genes used for unsupervised hierarchical cluster analysis (TIFF 1521 kb)Below is the link to the electronic supplementary material.


Staphylococcus aureus (S. aureus) immediately faces innate immune reactions such as the activated complement system. Here, a novel innate immune evasion strategy of S. aureus is described. The staphylococcal proteins surface immunoglobulin-binding protein (Sbi) and extracellular fibrinogen-binding protein (Efb) bind C3/C3b simultaneously with plasminogen. Bound plasminogen is converted by bacterial activator staphylokinase or by host-specific urokinase-type plasminogen activator to plasmin, which in turn leads to degradation of complement C3 and C3b. Efb and to a lesser extend Sbi enhance plasmin cleavage of C3/C3b, an effect which is explained by a conformational change in C3/C3b induced by Sbi and Efb. Furthermore, bound plasmin also degrades C3a, which exerts anaphylatoxic and antimicrobial activities. Thus, S. aureus Sbi and Efb comprise platforms to recruit plasmin(ogen) together with C3 and its activation product C3b for efficient degradation of these complement components in the local microbial environment and to protect S. aureus from host innate immune reactions.Upon host infection, the human pathogenic microbe  Staphylococcus aureus, a persisting human pathogen, can cause a variety of diseases including skin infections, pneumonia, endocarditis, and sepsis S. aureus is immediately recognized and targeted by innate immune responses, such as the complement system. Activation of complement by foreign surfaces , by antibodies  or by mannan  causes microbial opsonization, leukocyte recruitment, and cell lysis. All three pathways lead to the cleavage of C3 and subsequent formation of anaphylatoxin C3a and opsonin C3b. C3a attracts and activates granulocytes; whereas C3b attaches covalently to the bacterial surface, amplifies complement activation, and thereby labels cells for phagocytosis. Furthermore, C3b deposition leads to inflammatory reactions and formation of the pore-forming terminal complement complex S. aureus evades the complement system by targeting C3 and the activity of C3 convertases S. aureus: staphylococcal immunoglobulin-binding protein (Sbi), extracellular fibrinogen-binding molecule (Efb), and the Efb homologue protein Ehp S. aureus binds the human plasma protein plasminogen (PLG) to its surface and expresses a plasminogen activator, staphylokinase (SAK), that converts plasminogen to the serine protease plasmin (PL) S. aureus binds plasminogen to target the host C3 molecule for complement evasion. Plasmin controls several processes such as fibrinolysis, wound healing and tissue remodeling. Thus pathogenic microbes often exploit the proteolytic activity of plasmin to degrade components of the extracellular matrix (ECM), as well as fibrinogen for dissemination in the host Previous work demonstrated that S. aureus secreted SAK. Bound plasmin subsequently cleaves C3 and C3b that are simultaneously bound by Sbi and Efb. This proteolytic process are enhanced by the conformational changes Efb and presumably Sbi induce in C3 and C3b. In addition, plasmin was shown to degrade C3a that harbors chemoattractant and antimicrobial activities. Thus, recruitment and activation of plasminogen by S. aureus is highly coordinated to maximize complement inhibition at the level of C3.This report demonstrates that plasminogen, which is bound by the staphylococcal proteins Sbi and Efb, is converted to plasmin by S. aureus strain Newman was cultured on blood agar (Invitrogen) or in Luria broth at 37°C.E.coli pET200/D-TOPO expression system (Invitrogen) and Efb forw: CACCTCTTTATTAACATTAGCGGCAATAG and Efb rev: TTATTTAACTAATCCTTGTTTTAATACA, or Efb-C forw: CACCACTGATGCAACTATTAAAA and Efb-C rev: TTATTTAACTAATCCTTGTTTTAATACA primers for amplification. Sbi 1–2 and SAK were cloned into E.coli pET101/D-TOPO expression vector (Invitrogen) using Sbi 1–2 forw: CACCATGACAACTCAAAACAACTACGTAAC and Sbi 1–2 rev: ATTTTGACGTTCTTTAGCTTTAGAAGATTGTACTG, or SAK forw: CACCATGCTCAAAAGAGGTTTATTATTTTTAAC and SAK rev: TTTCTTTTCTATAATAACCTTTGTAATTAAGTTG primers. Expression and purification was performed according the user's manual. Affinity chromatography with Ni-NTA-Agarose  was used as previously described B. burgdorferi was expressed and purified as previously described TM Desalt Spin Columns (Pierce).Recombinant Sbi 1–4 (subsequently referred to as Sbi) and Sbi 3–4 were expressed as previously described b) or C3 (CompTech) overnight at 4°C. Bound PLGb was detected with Streptavidin-Peroxidase , C3 with anti-C3-Fab-HRP , and to assess IgG-binding, rabbit anti-goat-HRP  was applied. All blots were developed using the ECL-substrate (AppliChem).Staphylococcal proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking with 1% BSA, 4% milk powder, 0.1% Tween20 in PBS for 1 h at RT, the membranes were incubated with 1 µg/ml biotin-coupled plasminogen  for 1 h at RT. The reaction was developed with TMB (KEM EM TEC), stopped by addition of 0.25 M H2SO4 and measured at 450 nm.Bacterial proteins were immobilized (equimolar) onto a microtiter plate  blocked with 0.4% gelatin in DPBS for 2 h at 37°C, PLGFor dose-dependent binding of PLG to Sbi 3–4/Efb-C, 5 µg/ml Sbi 3–4 or Efb-C were immobilized to a microtiter plate, blocked with Blocking Solution I (AppliChem) for 2 h at 37°C and 25–800 nM PLG were added for 2 h at 37°C. Bound proteins were detected with anti-plasminogen  in Cross Down Buffer (AppliChem) and anti-goat HRP (1∶2000). In competition assays Sbi 3–4 or Efb-C were immobilized and incubated with concentrations of C3 (200 nM) and plasminogen (200 for Efb-C and 400 nM for Sbi 3–4) that saturate binding sites. C3 binding was detected using polyclonal anti-C3-antibody  and plasminogen binding with polyclonal plasminogen-antiserum.et alBorrelia burgdorferi CRASP-5 protein was included as a positive control and HSA  as a negative control.Combined ELISA-Western blot analysis was performed according to Haupt Binding of plasminogen to Sbi 3–4 or Efb-C was analyzed by surface plasmon resonance  at 25°C in 150 mM PBS. Sbi 3–4 or Efb-C was immobilized on a CMD 500 M sensor chip (Xantec) by standard amino coupling chemistry following the manufacturer's protocol. Plasminogen (400 nM) was injected at a constant flow rate of 5 µl/min.Plasmin activation was determined by hydrolysis of the PL-specific substrate S-2251. Therefore Sbi, Efb, CRASP-5, or HSA were immobilized (equimolar) onto a microtiter plate. After blocking with 0.4% gelatin, 0.2 µM plasminogen was incubated at 4°C overnight. Following washing with PBS +0.05% Tween20, uPa  or recombinant SAK (1 µg/ml) were added together with the chromogenic substrate S-2251 dissolved in reaction buffer . PL activity was recorded at 4 h intervals at 405 nm .S. aureus (3×104) in 10 mM Tris-HCL with 5 mM Glucose (pH 7.4) with or without 5 µg PLG and/or 1 µg SAK at 37°C for 2 h with agitation. Bacteria were diluted 1∶5, 20 µl were plated onto a LB plate in triplicate, and incubated overnight at 37°C. Additionally the supernatant was separated by SDS-PAGE and analyzed by Western blotting with polyclonal anti-C3a antibody .C3a (1 µM) (CompTech) was incubated with Plasminogen (0.2 µM) together with either 0.04 µM C3 or 0.04 µM C3b were incubated with immobilized Sbi, Efb, CRASP-5, or HSA overnight at 4°C. After extensive washing 0.5 µM SAK or 0.06 µM uPa were added for 3 h at 37°C. Samples were reduced with Roti-Load for 5 min at 95°C, separated by SDS-PAGE and transferred onto a nitrocellulose membrane. C3/C3b and their cleavage products were detected with HRP-conjugated anti-C3-Fab antiserum (1∶1000).0.04 µM C3 or C3b with either 0.15 µM plasminogen plus 0.04 µM uPa or 0.2 µM PLG plus 0.5 µM SAK were incubated in the absence or presence of Sbi, Efb, CRASP-5, or HSA (each 1 µM) for 3 h at 37°C. Additionally the same assays were performed using 0.2 or 2 µM of the fragments Sbi 1–2, Sbi 3–4, and Efb-C. Samples were analyzed as described above.C3 (0.04 µM) was immobilized to a microtiter plate at room temperature, after blocking with 0.4 % gelatin, 0.15 µM plasminogen with 0.04 µM uPa or 0.2 µM plasminogen with 0.5 µM SAK incubated for 3 h at 37°C. Sbi 3–4, Efb-C, CRASP-5, or HSA (each1 µM) were added in the presence or absence of plasmin. After extensive washing bound C3 was detected using C3a antiserum supplemented with 15 µg/ml aprotinin (to inhibit plasmin) and HRP-coupled antiserum at 4°C.S. aureus was heat inactivated for 15 min at 72°C and incubated with 5% serum in HEPES buffer  for 20 min at 37°C. Then bacteria were incubated with Sbi 1–2, Sbi 3–4, Efb-C, or HSA (each 2 µM) in the absence or presence of 0.15 µM plasmin  for 2 h at 37°C. Supernatants were reduced with Roti-Load, separated by SDS-PAGE and analyzed by Western blotting using anti-C3-HRP. Bacteria surface-bound C3b was measured by flow cytometry with FITC-labeled anti-C3-Fab (Protos Immunoresearch).Significant differences between two groups were analyzed by the unpaired Student's t-test. Values of *p<0.05, **p<0.01, ***p<0.001 were considered as statistically significant.b) and binding was assayed by ELISA. Plasminogen bound to both staphylococcal proteins Sbi and Efb. Plasminogen binding to Sbi and Efb was similar to the previously identified plasminogen ligand CRASP-5 of Borrelia burgdorferiSbi and Efb are C3 and C3b binding proteins. In order to investigate whether Sbi and Efb also recruit plasminogen, binding of plasminogen to recombinant Sbi and Efb was tested. The accuracy of recombinant Sbi and Efb was confirmed as both molecules showed binding to C3 and in addition Sbi bound to IgG . ImmobilTo determine the Sbi and Efb domains responsible for interaction with plasminogen, plasminogen binding to the structurally related domains Sbi 3–4 and Efb-C in Sbi and Efb, respectively, was assayed. Plasminogen added to the immobilized domain fragments bound to both Sbi 3–4  and Efb-To determine whether plasminogen and C3 bind simultaneously to Sbi and Efb, each plasma protein was added to immobilized Sbi 3–4 or Efb-C using concentrations of plasminogen (400 nM for Sbi 3–4 and 200 nM for EfbC) which saturate all binding sites. Plasminogen and C3 binding were measured in parallel. C3 binding to Sbi 3–4 and Efb-C, as well as plasminogen binding to Sbi 3–4 remained unchanged. Plasminogen binding to Efb-C was slightly reduced in the presence of C3 . The datTo exert proteolytic activity, plasminogen needs conversion to plasmin by an activator. To proof whether plasminogen bound to Sbi or Efb is converted to plasmin. Plasminogen was bound to immobilized Sbi or Efb and treated with the human activator uPa. Plasmin generation was followed by cleavage of a plasmin-specific chromogenic substrate. Plasminogen-bound Sbi or Efb was activated by uPa to plasmin, as seen by the increased conversion of the substrate S-2251. Similarly, plasminogen bound to CRASP-5 was activated to the protease plasmin .SAK is known to activate plasminogen via a non-proteolytic mechanism. To determine whether SAK is also able to activate Sbi- or Efb-bound plasminogen SAK was used in similar experiments instead of uPa. Also SAK activated plasminogen bound to Sbi or Efb, and the resulting plasmin cleaved the chromogenic substrate . In summAfter plasmin(ogen) was shown to form complexes with C3 and Sbi or Efb, the plasmin activity was assayed to cleave complexed C3. Plasminogen was bound together with C3 to immobilized Sbi or Efb and activated by the addition of SAK. C3 degradation was investigated by separating the reaction mixture by SDS-PAGE and Western blot analysis using anti-C3 Fab'-fragments. Multiple C3-degradation products with mobilities of 115, 87, 68, 40, and 27 kDa were generated indicating that Sbi- or Efb-bound plasminogen was activated by SAK to plasmin which subsequently cleaved bound C3 . In a siUpon binding, Efb changes the structural conformation of C3, leading to an increased susceptibility of C3 to degradation by trypsin S. aureus was incubated with human serum to allow complement-mediated C3b deposition. The C3b-coated bacteria were then washed, incubated with plasmin plus Sbi 3–4 or Efb-C, and C3b deposition was analyzed by flow cytometry. Plasmin substantially reduced the C3b opsonization of S. aureus by about 37%. In the presence of Sbi 3–4, C3b deposition was further decreased to 50%; and in the presence of Efb-C, to 38%  85% of S. aureus survived. Plasminogen added without SAK resulted in 50% survival of the bacteria, and addition of SAK without plasminogen had no effect on the bacteria  The importance of the complement system in defense against H. influenzaeB. burgdoferiC. albicansPlasmin cleaves C3b at several sites and generates C3c and C3d S. aureus strains express the sak gene and produce the non-proteolytic staphylokinase Complexed with C3 and Sbi or Efb, plasminogen remains accessible for the staphylococcal activator SAK and the human activator uPa to be converted to plasmin. More than 67% of S. aureus are decreased. These results are in accordance with those of Amara et al, who observed that while plasmin generates C3a, the C3a amount decreased when plasmin concentrations increased S. aureus helps the pathogen inactivate host antimicrobial activities and obviously represents a further survival strategy.Plasmin has also been described as a complement activator, because it cleaves C3 to C3a and C3b S. aureus is remarkably effective, using several plasmin activation pathways and expressing at least five plasminogen binding proteins. Among these proteins Sbi and Efb act like an amplifier of plasmin-mediated C3 cleavage to reduce local complement activity.The powerful inhibitory effect of acquired plasmin(ogen) against several immune defense mechanisms apparently explains why many pathogenic microbes attach human plasminogen to their surface and activate plasminogen to plasmin.


The aim of this study was to assess the effects of resveratrol on hydrogen peroxide  and intracellular Ca2+ properties were determined using whole-cell patch-clamp techniques and dual-excitation fluorescence photomultiplier system (IonOptix), respectively, in rabbit ventricular myocytes.INa.L in myocytes both in the absence and presence of H2O2 (300 µM) in a concentration dependent manner. Ranolazine (3–9 µM) and tetrodotoxin , INa.L inhibitors, decreased INa.L in cardiomyocytes in the presence of 300 µM H2O2. H2O2 (300 µM) increased the reverse INCX and this increase was significantly attenuated by either 20 µM resveratrol or 4 µM ranolazine or 4 µM TTX. In addition, 10 µM resveratrol and 2 µM TTX significantly depressed the increase by 150 µM H2O2 of the diastolic intracellular Ca2+ fura-2 fluorescence intensity (FFI), fura-fluorescence intensity change (△FFI), maximal velocity of intracellular Ca2+ transient rise and decay. As expected, 2 µM TTX had no effect on ICa.L.Resveratrol  decreased 2O2-induced augmentation of INa.L.and may contribute to the reduction of ischemia-induced lethal arrhythmias.Resveratrol protects the cardiomyocytes by inhibiting the H INa.L) has been recognized as an important factor contributing to the abnormal repolarization in ischemic and failured hearts INa.L plays an important role in determining the action potential duration (APD) + concentration ([Na+]i) INa.L in rat ventricular myocytes + inflow during hypoxia increased [Na+]i which in turn rose the intracellular Ca2+ concentration ([Ca2+]i) via the Na+-Ca2+ exchanger (NCX) resulting in a Na+-dependent intracellular Ca2+ overload induced by INa.L2+]i caused cardiac arrhythmias and irreversible cell damage INa.L caused arrhythmic activity and contractile dysfunction INa.L is considered to be a new potential target for therapeutic intervention in patients with myocardial ischaemia and heart failure Despite intensive research has been conducted in recent years, cardiac arrhythmias remain a serious problem. Late sodium current , a polyphenol in various vegetables and fruits, is abundant in grapes. The root extracts of Polygonum cuspidatum, a constituent of Chinese and Japanese folk medicine, is also a good source of resveratrol INa.L. Previous investigation showed that 50 µM of resveratrol reduced INa.L in a recombinant expression system with the R1623Q LQT3 mutation INa.L in ventricular myocytes with increased H2O2 has not been reported. Therefore, this study was designed to address the impact of resveratrol on the Na+-dependent Ca2+ overload induced by H2O2-induced increase in INa.L in ventricular myocytes, with the intention to shed some light on its potential clinical application in the future.To further clarify the pharmacological mechanisms and the scope of application of the agent, it is critical to determine the effect of resveratrol on  −1 i.v.) and xylazine (7.5 mg kg−1 i.m.). Hearts were excised rapidly and perfused retrogradely on a Langendorff apparatus for 5 min with a Ca2+-free Tyrode’s solution containing (in mM): NaCl 135, KCl 5.4, MgCl2 1, NaH2PO4 0.33, HEPES 10 and glucose 10 , and then a Tyrode’s solution containing enzyme  and bovine serum albumin  for 40–50 min. The perfusate was finally switched to KB solution containing (in mM): KOH 70, taurine 20, glutamic acid 50, KCl 40, KH2PO4 20, MgCl2 3, EGTA 0.5, HEPES 10, and glucose 10 (pH 7.4), for 5 min. All perfusates were bubbled with 100% O2 and maintained at 37°C. The left ventricles were then cut into small chunks and gently agitated in KB solution. The cells were filtered through nylon mesh and stored in KB solution at 25°C. The use of animals in this investigation was approved by the Institutional Animal Care and Use Committee of Wuhan University of Science and Technology and conformed to the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health  and the Guide for the Care and Use of Laboratory Animals of Hubei Province, China.Adult New Zealand white rabbits (body weight 1.7–2 kg) of either sex were heparinized (2000 U) and anesthetized with ketamine (30 mg kg2 (control) and were then exposed to Tyrode's solution containing 300 µM H2O2 for 7 min. Next, isolated cells were perfused with Tyrode's solution containing both 300 µM H2O2 and one of the following, resveratrol  for 10 min, 4 µM tetrodotoxin (TTX) for 10 min, or 4 µM ranolazine for 5 min.Isolated cells were perfused with Tyrode's solution saturated oxygenated with 100% OINa.L, the intracellular pipette solution contained (mM): CsCl 120, CaCl2 1, MgCl2 5, Na2ATP 5, TEACl 10, EGTA 11, HEPES 10 (pH 7.3). The extracellular solution contained (mM): NaCl 135, CsCl 70, CaCl2 1,MgCl2 1, CdCl2 0.05, glucose 5, HEPES 5 (pH 7.4). In addition, 1 µM nicardipine was used to block the L-type Ca2+ channels.To record +-Ca2+ exchanger current (INCX), the intracellular pipette solution contained (mM): NaCl 20, CaCl2 10, aspartic acid 50, MgCl2 3, EGTA 20, HEPES 10, MgATP 5 and CsOH 120 (pH 7.3). The bath solution contained (mM): NaCl 140,CaCl2 2, MgCl2 2, HEPES 5, and glucose 10 (pH 7.4). In addition, 20 µmol L−1 ouabain, 1 mmol L−1 BaCl2, 2 mmol L−1 CsCl and 1 µmol L−1 nicardipine were used to block the Na+-K+ pump, K+ channels and L-Ca2+ channels, respectively. INCX was measured as the Ni2+ sensitive current that could be blocked by NiCl2 5.0 mmol L−1.To record Na2+ fluorescence measurement, the bath solution contained (mM): NaCl 131, KCl 4, CaCl2 1, MgCl2 1, glucose 10, and HEPES 10 (pH 7.4).Krebs-Henseleit bicarbonate (KHB) buffer for intracellular CaICa.L), the intracellular pipette solution contained (mM): CsCl 80, CsOH 60, aspartic acid 40, CaCl2 0.65, HEPES 5, EGTA 10, MgATP 5 and disodium creatine phosphate 5 (pH 7.2). The bath solution contained (mM): NaCl 135, KCl 5.4, MgCl2 0.5, CaCl2 1.8, NaH2PO4 0.33, HEPES 10, glucose 10 (pH 7.4).To record L-type calcium current . All other chemicals were purchased from Sigma. Stock solutions of drugs were prepared in water. Each of the stocks was diluted to the required concentrations in the external recording solution immediately before use.H−1. INa.L, INCX and ICa.L were recorded in voltage clamp mode by using whole-cell patch-clamp techniques in rabbit ventricular myocytes. Patch electrodes were pulled with a two-stage puller . Their resistances were in the range of 1–1.5 MΩ. Capacitance and series resistances were adjusted to obtain minimal contribution of the capacitive transients. A 60% to 80% compensation of the series resistance was usually achieved without ringing. Currents were obtained with an EPC 9 amplifier  and a Multiclamp 700B amplifier , filtered at 2 kHz, digitized at 10 kHz, and stored on a computer hard disk for further analysis.Experiments were performed at room temperature (22–24°C). Rabbit ventricular myocytes were placed into a recording chamber that was bathed with normal extracellular solution, in the absence and presence of drug (s), at a rate of 2 ml min2+ level were inferred from the ratio of the fura-fluorescence intensity (FFI) at both wavelengths. Intracellular Ca2+ fluorescence measurements were assessed using the following indices: diastolic intracellular Ca2+ level (diastolic FFI) (340/380 ratio), electrically stimulated rise in intracellular Ca2+ (△FFI) (340/380 ratio), maximal velocity of Ca2+ rise and Ca2+ decay (340/380 ratio).Myocytes were loaded with fura-2-AM (0.5 µM) for 10 min at 25°C, and fluorescence measurements were recorded with a dual excitation fluorescence photomultiplier system (Ionoptix). Myocytes were imaged through an Olympus IX-70 Fluor 40 × oil objective. The cells were field stimulated with a suprathreshold (150%) voltage and at a frequency of 0.5 Hz, 3-ms duration, using a pair of platinum wires placed on opposite sides of the chamber connected to a FHC stimulator . The polarity of the stimulatory electrodes was reversed frequently to avoid possible build up of electrolyte by-products. Cells were exposed to light emitted by a 75-W lamp and passed through either a 340- or 380-nm filter (bandwidths were ±15 nm) while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after first illuminating the cells at 340 nm for 0.5 s then at 380 nm for the duration of the recording protocol (333 Hz sampling rate). The 360 excitation scan was repeated at the end of the protocol, and qualitative changes in intracellular CaINa.L were tested at 200 ms in depolarization testing pulse to eliminate the influence of transient sodium current (INa.T). Statistical significance between two groups and multiple groups were evaluated by Student’s t-test and one-way analysis of variance (ANOVA), respectively. All values were expressed as mean ± SD, and the number of cells (n) in each group was given. P<0.05 was considered to be statistically significant.Whole-cell recordings were analyzed using clampfit 9.0  and PulseFit . Figures were plotted by Origin . All amplitudes of INa.L, the current was recorded first in the absence and then in the presence of 4 µM TTX with 300 ms voltage steps from a holding potential (HP) of −120 to −20 mV. The values of current recorded before (control condition) and after application of TTX were −0.400±0.050 and −0.154±0.038 pA pF−1 , respectively, indicating that this TTX-sensitive current recorded was INa.L. When INa.L was recorded under normal condition using depolarizing pulses with a duration of 300 ms applied at 0.25 Hz from a HP of −120 mV in 10 mV increments between −70 and −20 mV, administration of 10, 20, 40 and 80 µM resveratrol resulted in decreased amplitudes of INa.L in a concentration dependent manner  increased the amplitudes of INa.L but 10, 20, 40 and 80 µM resveratrol decreased the amplitudes of INa.L in the continued presence of H2O2  induced by 300 µM H2O2 with an IC50 of 26.192 µM. Ranolazine  attenuated the increased INa.L in the presence of 300 µM H2O2 in a concentration dependent manner  reversed the increased INa.L caused by 300 µM H2O2. The values of INa.L under the control conditions, after application of 300 µM H2O2 and 4 µM TTX were −0.323±0.087, −0.878±0.071 and −0.258± −0.045 pA pF−1 , respectively.Currents were recorded using depolarizing pulses with a duration of 300 ms at a rate of 0.25 Hz from a HP of −120 mV, in 10 mV increments between −70 and −20 mV. Administration of resveratrol at concentrations of 10, 20, 40 and 80 µM resulted in a decrease in the amplitudes of  to H2O2 . H2O2  before returning to −40 mV. The current-time relationship was constructed from the declining slope of the ramp pulse (2+-sensitive (NCX) current obtained by subtracting the data in the trace d, h or l from the data in the trace a, e, i, b, f, j, c, g or k in the panel 4A, 4D or 4G. INCX measured at voltage levels of +50 mV and −100 mV, respectively, as the Ni2+-sensitive current by subtracting the current recorded in the presence from that in the absence of 5 mM NiCl2.Electrogenic mp pulse . Figure INCX was recorded after 7 minutes of exposure of H2O2. The mean current density of the inward INCX had little change, while the reverse INCX increased significantly , fura-fluorescence intensity change (△FFI), maximal velocity of Ca2+ rise and Ca2+ decay were all enhanced by 150 µM H2O2  TTX is relatively a selective INa.L blocker. Using depolarizing pulses with a duration of 300 ms applied at 0.5 Hz from a HP of −80 mV, in 10 mV increments between −40 and +40 mV, ICa.L was recorded in the absence  and ultimately causing cell damage INa.L by H2O2 and hypoxia INa and INa.L in the R1623Q LQT3 mutation in a recombinant expression system INa.L and reverse INCX under proarrhythmic conditions (H2O2) in rabbit ventricular myocytes has not been investigated yet. The data from this study addressed the impact of resveratrol on the Na+-dependent Ca2+ overload.The mechanisms underlying the genesis of ischemia- and reperfusion-induced arrhythmias are notoriously complex and controversial. There has been an interest in the concept that oxygen free radicals play a role in the pathogenesis of myocardial ischemia and infarction. It has been reported that a burst of HINa.L was increased by H2O2  2+ levels. Ranolazine is currently approved as an antianginal agent that reduces the Na+-dependent Ca2+ overload via inhibition of the INa.L and thus improves diastolic tone and oxygen handling during myocardial ischemia INa.L is an important contributing factor to intracellular Ca2+ overload in the pathogenesis of myocardial ischemia and infarction. In rabbit ventricular myocytes, low concentrations of TTX (1.5–4.0 µM) did not alter the L-type Ca2+ current  attenuated the increase in the reverse INCX by H2O2. Thus, we concluded that the effect of resveratrol to inhibit the increased reverse INCX caused by H2O2 was from its inhibition of INa.L.Recently, it has been reported that reverse ficantly . Differe2O2 significantly increased the amplitude of calcium transients and diastolic calcium concentration in the ventricular cell which could be reversed by TTX (2 µM). The intracellular Ca2+ overload caused by ROS was due to an increase in [Na+]i followed with an increase in Ca2+ influx via the reverse mode of the NCX 2+ into the cell will cause intracellular Ca2+ overload 2+ channel with an IC50 value of 55±2 µM INa.L and restrained Ca2+ overload induced by H2O2 but not affected L-type Ca2+ channels  also restrained the increased calcium transients amplitude and the diastolic calcium concentration induced by H2O2 (150 µM). Therefore the effects of resveratrol on the Na+-dependent Ca2+ overload induced by enhanced INa.L were similar to 2 µM TTX, suggesting that the reduction of Ca2+ overload by resveratrol may have similar mechanism to TTX, i.e., inhibition of INa.L. Indeed, resveratrol has also been suggested to inhibit the ryanodine receptor-induced intracellular Ca2+ increase 2+]i. The results in this study indicated that resveratrol reduced both INa.L and reverse INCX which was responsible for the reversal of intracellular Ca2+ overload in the presence of H2O2. Resveratrol may inhibit both the ryanodine receptor-induced intracellular Ca2+ overload and INa.L-induced increase in reverse INCX to attenuate the intracellular Ca2+ overload. Further research will be needed to clarify the contribution of the two pathways by resveratrol in the absence and presence of H2O2.In this study, 150 µM Hchannels , supportINa.L is an important target for resveratrol to prevent or treat ventricular arrhythmias. INa.L increased by H2O2 induces intracellular Ca2+ overload  through the increase in the reverse INCX. The inhibitive effect of resveratrol on H2O2-induced INa.L may reduce the concentration of [Na+]i, lower [Ca2+]i by attenuating reverse NCX to eliminate Ca+ overload, and ultimately inhibit the electrical abnormalities.


Metaphyseal anchored short-stem hip implants were designed to improve load transmission and preserve femoral bone stock. Until now, only few outcome data have been available and migration studies are one of the few ways of obtaining data that are predictive of implant survival. We therefore evaluated a metaphyseal anchored short-stem hip implant by Ein Bild Roentgen Analyse femoral component analysis (EBRA-FCA). First, the EBRA-FCA method was validated for the short-stem hip implant. Then 80 of the first 100 consecutive implants were evaluated after at least 2 years. Clinical assessment was performed using the WOMAC and the UCLA score. After 2.7 (2.0–4.2), years none of the implants had been revised and by that time the stems had subsided by a mean of 0.7 mm (SD 1.8) (95% CI: 0.3–1.1). Of the 80 implants, 78 were stable after 2 years, with 74 being primary stable and 4 showing secondary stabilization after initial subsidence. Continuous migration was seen in only 2 patients. The clinical outcome showed good results with a mean WOMAC of 11 (SD 13) and a mean UCLA score of 7.3 (SD 2.0). [OK?] The metaphyseal anchored short-stem hip implant showed good functional results and a high degree of stability after 2 years. The outcome is comparable to that of clinically proven conventional hip implants and if the results are confirmed by long-term studies, short-stem hip arthroplasty might be an alternative for young patients requiring hip replacement. The number of young patients with total hip arthroplasty is steadily increasing. However, it is well known that young patients face a higher risk of implant failure , which mShort-stem hip implants are shorter than standard stems and the first designs, the Mayo and the Pipino short stem, were already introduced several years ago . Due to Initial stability and early migration of implants have been shown to predict long-term survival . SeveralThe study cohort comprised the first 100 consecutively implanted short-stem hip arthroplasties at our orthopedic department (from May 2006 through November 2008). From this cohort, 82 implants (74 patients) could be evaluated. 10 patients were living abroad or had moved, and 8 refused to come to the clinic for reasons not related to the implant. The final study cohort comprised 43 males and 31 females, with a mean age of 55 (SD 12) years and a mean BMI of 27 (SD 4). Reasons for hip replacement were osteoarthritis (n = 34), dysplasia (n = 24), avascular necrosis of the femoral head (AVN) (n = 20), posttraumatic osteoarthritis (n = 1), and rheumatoid arthritis (n = 3). The mean follow-up time was 2.7 (2.0–4.2) years.The study protocol was approved by the Ethics Committee of the university (No. 054-10). All patients were informed about the study and signed an informed consent form.All patients received the same short-stem hip implant . The implant size ranges from 0 to 7, available as a monoblock (neck-shaft angle (CCD) 130°, 135°) or as a modular implant with cone adapters . Monoblock implants were used as follows: 130° (n = 3) and 135° (n = 3). Modular implants were used as follows:130°/0° (n = 27), 130°/7.5° ante (n = 4), 130°/7.5° retro (n = 2), 135°/0° (n = 32), 135°/7.5° ante (n = 8), 135°/7.5° retro (n = 1), 140°/0° (n = 1), and 140°/7.5° retro (n = 1).For the acetabular component, either a threaded cup or a press-fit cup was used . Surgery was performed by 3 senior surgeons  with a minimally invasive anterolateral Harding approach in supine position. All implants were templated before surgery and evaluated intraoperatively by fluoroscopy. Weight bearing was allowed to half the body weight for the first 2 weeks, followed by a rapid increase to full weight bearing.All patients had an AP radiograph postoperatively, and they were usually followed up at 3, 6, and 12 months, and then by annual assessment. Patients included in this study were required to have their implant longer than 24 months and to have at least 3 radiographs accepted by the EBRA-FCA software: 1 postoperatively, 1 within 2 years, and 1 at the final follow-up.Clinical assessment at the last follow-up included the Western Ontario and McMaster Universities arthritis index (WOMAC), and the UCLA activity score. Radiographic evaluation included measurement of the CCD angle and the cup inclination. The size of the femoral cavity was evaluated, according to Short-stem implants have a curved shape, which made it necessary for the author of the EBRA method to adapt the software and place the central axis as a tangent to the medial side of the implant . Due to Validation was performed similarly to A worst-case study was performed to evaluate possible rotational deviations of the femur that might occur during radiographic acquisition. According to recent studies, the evaluation was carried out at –5° and +20° of flexion, and at –10° and +50° of rotation . At 5 diTo determine the measurement accuracy of an implant migration, a controlled implant subsidence was simulated. The implant was shifted down the femur from 0 to 6 mm. This was done in 1-mm steps with an integrated vernier calliper  firmly fixed at the implant and at the distal femur, allowing a precision of 0.05 mm. A series of radiographs from every single shift was taken at the 5 different time points. Any difference between the adjusted position and the measured position was taken to represent a measurement error.Migration analysis for the in vitro validation and for the implanted prosthesis was performed by the modified EBRA-FCA software . The magnitude of stem migration is presented as mean (SD) with 95% confidence interval (CI).To account for bilateral hips, we used a linear mixed model with a fixed intercept and a random effect for each patient, assuming a compound-symmetry covariance pattern for the repeated implantations . The choice of the covariance pattern was supported by the Akaike information criterion. Statistical analysis was performed with SPSS 19.0. Graphs were created with SigmaPlot 8.02 .At the last postoperative follow-up (mean 2.7 (2.0–4.2) years), none of the stems or acetabular cups had been revised. At the last follow-up, the mean WOMAC was 11 (SD 13) (CI: 8–14), with 2 (SD 1) in the stiffness section, 2 (SD 3) in the pain section, and 8 (SD 10) in the function section. The activity level was rated on the UCLA scale, with a mean score of 7 (SD 2) (CI: 6.9–7.8).The median size of the acetabular component was 54 (48–58) mm for men and 48 (44–52) mm for women. The median size of the stem was 4 (1–6) for men and 1 (0–4) for women. Radiographic evaluation showed a mean cup inclination of 45° (SD 6) and a mean CCD angle of 137° (SD 6). The mean endosteal diameter at the midpoint of the lesser trochanter (point 1) was 28 mm (SD 3.8) (range: 19.2–40), and 12 mm (SD 2.1) (range: 8.2–18) 10 cm below (point 2). The endosteal distances with respect to the implant size are given in Rotation error. Compared to the neutral position, a flexion of 20° gave a maximum error of –1.5 mm with a mean of –1.5 mm (SD 0.1) (CI: –1.5 to –1.4). An extension of 5° gave a maximum error of +0.9 mm with a mean of +0.8 mm (SD 0.1) (CI: 0.8–0.9). External rotation was conducted at different angles, with 50°, 30°, and 20° being rejected by the software due to detected inaccuracy. The maximum accepted external rotation was 10°, which gave a maximum error of +0.2 mm with a mean of +0.1 mm (SD 0.1) (CI: 0–0.2). Internal rotation with 10° gave a maximum error of –0.2 mm with a mean of –0.1 mm (SD 0.1) (CI: –0.2 to 0) (.2 to 0) .Measurement precision. Evaluation of the 5 controlled-migration series, running from the neutral position to 6 mm, resulted in a measured subsidence between 5.9 to 6.3 mm, indicating an error ranging from –0.1 mm to +0.3 mm with a mean of 0.0 mm (SD 0.2) (CI: –0.2 to 0.2). Analysis of each single step (1 mm) for all series gave an error ranging from –0.5 mm to +0.4 mm with a mean of 0.1 mm (SD 0.2) (CI: 0.0 to 0.1) ( to 0.1) .From the 82 implants, 2 series of radiographs were rejected by the EBRA-FCA software. The remaining 80 implants (73 patients) were analyzed with a mean of 4.5 (SD 1.8) (range 3–9) radiographs for each implant. The numbers of radiographs at defined time points were accepted and measured by EBRA-FCA as follows: n = 80 postoperatively, n = 80 at 3 months, n = 71 at 1 year, n = 68 at 2 years, n = 23 at 3 years, and n = 4 at 4 years.In the total study cohort, the mean vertical migration of the short-stem implants was –0.7 mm (SD 1.8) (CI: –1.1 to –0.3) after a mean of 2.7 (2.0–4.2) years. The implants were also assigned to 4 different patterns of migration (A–D) . AccordiFrom the 4 implants with pattern B, 3 migrated within the first 2 months and then stabilized. For the fourth implant, no definite time point within the first 2 years could be determined, as no sufficient radiographs were available in between. However, the implant did not migrate further after 2 years and showed complete osseous integration.Of the 6 migrated stems, 4 in 3 patients) were implanted due to AVN—1 due to osteoarthritis and 1 due to dysplasia. Both patients with continuous stem migration were diagnosed with AVN, 1 receiving high-dose steroid therapy due to kidney transplant rejection and 1 with severe chronic alcohol abuse . From th patientsAlthough only long-term results in hip arthroplasty should be considered valid, initial evaluation is necessary to identify undesirable results . Early mEBRA-FCA is a reliable and accurate method , but it After 2.7 years, the mean subsidence was –0.7 mm, which is clearly below the reported critical threshold of –1.5 mm for the first 2 years . This inAs it is not only the average migration that is important, we assigned the implants to 4 different patterns of migration (A–D) . 74 out 6 stems migrated more than –1.5 mm within the first 2 years, and 2 of them showed a continuous migration (pattern A) and were at risk of early failure. The remaining 4 implants showed an initial migration but then stabilized (pattern B).A retrospective EBRA-FCA study with a 10-year follow-up showed that initial migration with secondary stabilization can succeed, and is not necessarily followed by implant failure . SimilarOnly limited data are available regarding the cause of initial migration. Functional results in our patients were good, with a mean WOMAC of 11. Similarly, good results were reported for the CFP short-stem implant with a HHS of 93 after 2 years and a HHS of 96 after 7 years . For theOur study has some limitations. Although EBRA-FCA is an accurate method, it mainly addresses the initial stability and does not take account of all loosening mechanisms, e.g. failure due to wear. Furthermore, the study was performed retrospectively, thus preoperative WOMAC had not been obtained and some patients could not be followed up. Still, we were able to analyze 80 of the first 100 implants, which is comparable to previous migration studies with an evaluation rate of 66–85% .In summary, the early evaluation of the metaphyseally anchored short-stem implant revealed good clinical results and showed good stability comparable to that of clinically proven cementless THA stems. Migration was mainly seen in patients with avascular necrosis of the hip and with a possible mismatch between stem and femur size. After 2 years, 78 of 80 implants were stable and—if those results are confirmed by long-term studies—short-stem arthroplasty might be an alternative for young patients requiring hip replacement.


We have utilized mice in which SIRT2 has been reduced or ablated to further explore the function of SIRT2 and to assess whether SIRT2 loss has a beneficial impact on disease progression in the R6/2 mouse model of HD. Surprisingly we found that reduction or loss of SIRT2 had no effect on the acetylation of α-tubulin or H4K16 or on cholesterol biosynthesis in the brains of wild type mice. Equally, genetic reduction or ablation of SIRT2 had no effect on HD progression as assessed by a battery of physiological and behavioural tests. Furthermore, we observed no change in aggregate load or levels of soluble mutant huntingtin transprotein. Intriguingly, neither the constitutive genetic loss nor acute pharmacological inhibition of SIRT2 affected the expression of cholesterol biosynthesis enzymes in the context of HD. Therefore, we conclude that SIRT2 inhibition does not modify disease progression in the R6/2 mouse model of HD and SIRT2 inhibition should not be prioritised as a therapeutic option for HD.Huntington's disease (HD) is a devastating neurodegenerative disorder for which there are no disease-modifying treatments. The molecular pathogenesis of HD is complex and many mechanisms and cellular processes have been proposed as potential sites of therapeutic intervention. However, prior to embarking on drug development initiatives, it is essential that therapeutic targets can be validated in mammalian models of HD. Previous studies in invertebrate and cell culture HD models have suggested that inhibition of SIRT2 could have beneficial consequences on disease progression. SIRT2 is a NAD HTT), which translates into an expanded polyglutamine (polyQ) tract in the N-terminus of the huntingtin protein (HTT) Huntington's Disease (HD) is a devastating, autosomal dominant, neurodegenerative disorder, with a mean age of onset of 40 years + (nicotinamide adenine dinucleotide) dependent and proceeds through the formation of an oxocarbenium-like transition species and the release of nicotinamide (NAM) Histone deacetylase (HDAC) inhibition has recently emerged as an attractive therapeutic intervention for many complex diseases, including HD. There are altogether 18 mammalian HDACs, divided into four classes depending on their sequence homology to yeast enzymes. HDACs 1 through 11 are all zinc dependent enzymes, share the same reaction mechanism and belong to classes I, II and IV SIRT2 is the only member of the sirtuin family residing primarily in the cytoplasm, where it has been shown to deacetylate α-tubulin Sirt2 to 50% of normal levels prevented photoreceptor neuron degeneration in a HTT exon 1 D. melanogaster HD model, but did not rescue lethality D. melanogaster, C. elegans and primary striatal cell models of HD Evidence for a protective role of SIRT2 inhibition in HD came from two important studies. Firstly, genetic knock-down of Sirt2 knock-out (Sirt2KO) mice have been crossed to the R6/2 mouse model of HD and disease progression was assessed with physiological, behavioural and molecular readouts. The R6/2 mouse has an early onset and rapid phenotype progression and at late stage disease expresses HD-related phenotypes that are extremely similar to those that develop in the genetically precise HdhQ150 knock-in HD model Sirt2KO mice or in mice acutely dosed with a SIRT2 inhibitor. We find no evidence for the therapeutic potential of SIRT2 modulation in a mammalian model of HD and suggest that SIRT2 inhibition should not be prioritised as a therapeutic strategy for HD.Based on previous studies in worm, fly and cell culture HD models, we might expect that loss of SIRT2 would decrease aggregate load and cholesterol levels and modify HD progression in a mouse model of HD Sirt2 knock-out (Sirt2KO) mice were generated by the targeted insertion of a puromycin resistance gene into exon 11 of the Sirt2 locus. The insertion was sequenced and BLAST analysis confirmed that in addition to vector backbone sequences, the mutation introduced a puromycin resistance gene countersense to the Sirt2 gene  with primers binding upstream of the insertion in Sirt2KO, Sirt2HET (Sirt2 heterozygous) and wild type (WT) mice at 4 weeks of age. Sirt2HET and Sirt2KO mice were found to express 80% and 60% of Sirt2 mRNA levels as compared to WT respectively  40 of α-tubulin and K16 of histone 4 (H4) he brain . To assehe brain .Sirt2HET and Sirt2KO mice could be detected in cortices from 9 week old mice, or brain stem and liver from mice at 4 weeks of age , Sirt2HET (WT HET) (n = 21), Sirt2KO (WT KO) (n = 16), R6/2 (n = 18), Sirt2HETxR6/2 (R6/2 HET) (n = 19) and Sirt2KOxR6/2 (R6/2 KO) (n = 15) mice were monitored in a phenotyping study from 4 to 14 weeks of age and sacrificed at 15 weeks of age. The expression pattern of SIRT2 throughout the brain was consistent across this time frame  = 10.655, p = 0.002) and gained weight at a significantly slower rate  = 61.278, p<0.0005). Interestingly, Sirt2HET and Sirt2KO mice had significantly increased weight  = 4.668, p = 0.012)  = 3.152, p = 0.047). This phenomenon showed a trend to become significant with time  = 2.131, p = 0.051). However, the Sirt2 mutation had no overall effect on R6/2 weight loss  = 0.087, p = 0.917) or on the decrease in weight of R6/2 mice over time  = 1.902, p = 0.082), suggesting that if the Sirt2 mutation modifies weight, it does so independently of HD progression.Mice were weighed weekly from 4 until 14 weeks of age. R6/2 mice weighed significantly less than WT mice overall (F= 0.012) , startin = 66.79, p<0.0005). This deficit appeared by 11 weeks of age  = 54.462, p<0.0005) and deteriorated over time  = 78.857, p<0.0005) (Sirt2HET) or absent (Sirt2KO) overall  = 0.654, p = 0.522) or with time  = 0.355, p = 0.916) and there was no significant effect of the modulation of SIRT2 levels on the grip strength deterioration observed in R6/2 mice either overall  = 0.116, p = 0.891), or with time  = 0.9, p = 0.5) (Grip strength was measured in mice at 4 weeks of age and bi-weekly from 7 to 13 weeks of age. R6/2 mice had reduced grip strength compared to WT mice (F<0.0005) . Grip stp = 0.5) . These f = 2.128, p = 0.148)  = 23.792, p<0.0005)  = 0.23, p = 0.795) or with time  = 1.118, p = 0.351) and down-regulation or ablation of SIRT2 had no effect on the rotarod performance of R6/2 mice  = 0.634, p = 532) or its deterioration over time  = 0.376, p = 0.865) (Rotarod performance is a robust measure of motor coordination and was assessed at 4 weeks of age and bi-weekly from 8 to 14 weeks . In line<0.0005) . Sirt2HE= 0.865) . Therefo = 17.197, p<0.0005 at 9 weeks and F = 268.291, p<0.0005 at 15 weeks of age)  = 0.734, p = 0.486 at 9 weeks and F = 1.107, p = 0.335 at 15 weeks).As expected, R6/2 brains weighed significantly less than those of WT mice (F of age) . Geneticp-values are presented in Sirt2KO Genotype, Time*Sirt2KO, R6/2*Sirt2KO). These data imply that depletion of SIRT2 has no effect on the progressive hypoactivity of R6/2 mice.Spontaneous motor activity was measured bi-weekly from 5 to 13 weeks of age. Data were analysed with General Linear Model repeated measures ANOVA and Although the genetic ablation of SIRT2 had no discernible disease modifying effect on HD related behavioural and physiological phenotypes in R6/2 mice, it remained possible that SIRT2 depletion elicited molecular changes. Mutant HTT aggregates are formed in HD patients and all known mouse models and correlate with disease progression As has been observed previously, the aggregate load in R6/2 mice was highest in the cortex and hippocampus and much lower in the brain stem  [26]. AsThe results obtained from the Seprion ELISA and TR-FRET experiments were confirmed by an MSD assay . ImportaSirt2 genotype alone had no significant effect on the expression of any of the cholesterogenic enzymes. Surprisingly, however, the Sirt2 genotype significantly modified the effect of the R6/2 genotype on the expression of Hmgcr  = 6.03, p = 0.006), Dhcr7  = 5.14, p = 0.011) and Sqle  = 3.38, p = 0.045). Close inspection of the data revealed that the statistical significance arises from a SIRT2-related increase in the WT, and decrease in the R6/2 groups, which would be inconsistent with the proposed biological mechanism. All of the tested cholesterogenic enzymes are targets of SREBP-2, therefore, if SIRT2 ablation was causing a decrease in the nuclear shift of SREBP-2, KO mice should display lowered expression for all seven enzymes in both R6/2 and WT mice. As this was not the case, the observed statistical differences are more likely a result of biological variability than a consequence of SIRT2 depletion.SIRT2 genetic ablation had no effect on the expression of cholesterogenic enzymes in WT mice . However50 for SIRT2 inhibition, as measured at Cerep (assay 2582) is 560 nM and [S]-35 was found to inhibit SIRT1 with a similar IC50 (assay 2581). LC/MS/MS analysis of brain tissue from mice dosed with [S]-35 revealed that at 4 h post dosing, [S]-35 was still present in both WT and R6/2 mice at concentrations above the SIRT2 IC50  or high (3 mg/kg) dose of the dual SIRT1/SIRT2 inhibitor, [S]-35 RT2 IC50 .in vivo.In line with previous reports and data from this study, expression of all cholesterogenic enzymes was diminished in R6/2 vehicle treated mice as compared to WT vehicle treated mice  [40]. HoPrevious studies in invertebrate and cell culture models of HD have indicated that SIRT2 inhibition alters HD-related phenotypes, particularly by modulating the expression of cholesterol biosynthesis enzymes in vitro and in cell culture experiments Hdac6KO mice Sirt2KO mice and those of wild type mice. It is possible that SIRT2 is not a bona fide tubulin deacetylase in vivo. Consistent with this, previous studies could not detect changes in the levels of acetylated tubulin in mouse embryonic fibroblasts derived from Sirt2KO mice Hdac6KOxSirt2KO mice are viable and do not display any overt phenotypes up to 4 weeks of age, arguing for non-redundant roles for HDAC6 and SIRT2 (unpublished observations). Therefore, it is possible that HDAC6, and not SIRT2, is the major tubulin deacetylase in the brain and that previous reports suggesting that SIRT2 is a tubulin deacetylase do not translate to the mammalian system.SIRT2 has been shown to deacetylate tubulin Sirt2KO mice. This could indicate that SIRT2 is only important for cell cycle regulation under specific conditions, as has been suggested by some, but not all, previous studies Sirt2KO mice.Although cytoplasmic in nature, SIRT2 has been shown to regulate cell cycle progression through deacetylation of H4K16 D. melanogaster models of HD and Parkinson's disease (PD) Sirt2HET or Sirt2KO mice could lead to activation of the AMPK pathway, which in turn has been shown to affect multiple downstream events, including hypothalamic inhibition of POMC, activation of NPY/AgRP and an increase in food intake A partial rescue of toxicity after genetic knock-down or pharmacological inhibition of SIRT2 has been observed in The lack of an effect of SIRT2 loss on aggregate load or the levels of soluble mHTT was very interesting. The pharmacological inhibition of SIRT2 was found to reduce mHTT inclusion number in primary striatal neurons, but over-expression of a catalytically dead SIRT2, had little effect on mHTT inclusion number in the same model Sirt2KO mice. Equally, reduction and depletion of SIRT2 did not affect the expression of cholesterol biosynthesis enzymes at late stage disease in R6/2 mice. These data indicate that SIRT2 does not play a role in cholesterol synthesis in the mouse. As previous work linking SIRT2 to cholesterol synthesis was performed with inhibitors, the observed effects on the cholesterol pathway could be the result of off-target effects. Although changes in the expression of Fdft1, Hgmcs1 and Hmgcr were also detected after over-expression of a catalytically dead SIRT2, viral transduction and over-expression of a protein can also evoke compensatory mechanisms or aberrant interactions within cells A role for SIRT2 in the modulation of cholesterol content was proposed after acute inhibition of SIRT2 ameliorated mHTT toxicity in worm, fly and cell culture HD models HdhQ150 knock-in models suggests that the rate limiting step in disease onset and progression in these mice is likely to be the formation of an N-terminal mHTT fragment HdhQ150 knock-in phenotypes, and as the time and resources required to perform these experiments are considerable, priorities should be directed toward the validation of more convincing therapeutic targets.Previously published reports on the role of SIRT2 as a modifier of neurodegenerative disease clearly provided a rationale for conducting a phenotypic study in a mouse model of HD. Our finding that depletion of SIRT2 does not modify HD progression in R6/2 mice was surprising and the relevance of this model could be questioned. Although R6/2 mice express an N-terminal fragment of mHTT and thus lack the context of full length mutant protein, the possible involvement of SIRT2 in cholesterol biosynthesis originated from studies in cells expressing an N-terminal fragment of mHTT Given the devastating nature of HD and the lack of disease modifying treatments, considerable effort has been invested in the search for, and validation of, therapeutic strategies. Pan HDAC and sirtuin inhibitors such as SAHA and NAM have been shown to ameliorate disease phenotype in HD mice but can be associated with significant toxic effects. In order to divorce the toxic and beneficial effects of HDAC inhibition, considerable efforts have been made to determine which HDAC(s) and sirtuin(s) are key modifiers of disease progression. Genetic knock-down or knock-out of HDAC3, 5, 6, 7 and 9 has shown no benefit in R6/2 mice  mice were on C57BL/6/129Ola background and were backcrossed to CBF three times.Hemizygous R6/2 mice were maintained by backcrossing R6/2 males to CBAxC57BL/6 F1 (CBF) females . At 4 weeks of age, mice were weaned into cages of 5 or 6 animals. Each cage contained at least one representative of each genotype when available. Animals were housed under 12 h light/12 h dark cycle, with unlimited access to water and chow . Cages were environmentally enriched as described Sirt2 mice were genotyped with all 3 primers ×30 and 68°C for 10 min. The PCR product was purified with the Qiaquick Gel Extraction kit according to manufacturer's instructions and cloned into TOP10 bacteria using the Invitrogen TOPO TA cloning kit according to manufacturer's instructions. Plasmid DNA was isolated with Qiaprep Spin Miniprep Kit according to manufacturer's instructions and eluted in ultra-pure water. Sequencing reactions were carried out in a 6.25 µL volume containing 200–500 ng DNA, 0.25 µL BigDye v3.1 (Applied Biosystems), 1.25 µL sequencing buffer, 0.25 µL primer (80 ng/µL) under cycling conditions of: 96°C for 2 min, ×30. PCR products for sequencing were precipitated with 26 µL sequencing precipitation solution (120 mM C2H3O2Na in 95% absolute ethanol), incubated for 10 min at room temperature and centrifuged at 3,000× g for 20 min. The supernatant was removed, the pellet washed with 100 µL 70% ethanol, centrifuged at 3,000× g for 20 min, cleared of supernatant and re-suspended in 10 µL Hidi-formamide (Applied Biosystems). The sample was then denatured at 96°C for 2 min and analysed with an ABI3730 sequencer. Sequence traces were analysed with the ABI Sequencing Analysis and Vector NTI programmes. The primer sequences used are given in Tail-tip DNA from Dissected brain regions, whole brains or peripheral tissues were snap frozen in liquid nitrogen and stored at −80°C until use.2, 1 mM DTT, 1 mM PMSF, 5 µM TSA, 10 mM nicotinamide, Complete protease inhibitor cocktail (Roche)) with 10 gentle pestle strokes in a Dounce homogeniser. The homogenates were centrifuged at 500× g for 15 min at 4°C. The supernatant (cytoplasmic fraction) was removed and stored at −80°C. The pellet was re-suspended in 1 mL of ice cold sucrose buffer 1 with an additional 10 gentle pestle strokes in the Dounce homogeniser. Two volumes of ice cold sucrose buffer 2 ) were added to the homogenate and the solution was mixed until uniform. Ultracentrifuge tubes were layered with a 0.5 mL cushion of sucrose buffer 2 and the homogenate was gently layered on top. The tubes were appropriately balanced and spun in pre-cooled SW41 rotor tubes at 124,000× g for 1 h at 4°C. Subsequently, the supernatant was removed and the pellet re-suspended in 0.5 mL sucrose buffer 1, transferred to fresh 1.5 mL tubes and spun at 800× g for 15 min at 4°C. The supernatant was removed and the pellet was re-suspended in 0.5 mL buffer 1, spun at 800× g for 15 min at 4°C twice more before being re-suspended in 50 µL of buffer 1. The nuclear fraction was sonicated on ice for 10 s at 80 Hz. The protein concentration of both fractions was determined with the BCA assay (Thermo Scientific) for each sample.Whole brains were homogenised in 2 volumes of ice cold sucrose buffer 1  Triton X-100, 3 mM DTT, 1 mM Orthovanadate, 5 mM NaF, 1 mM PMSF, 5 µM TSA, 10 mM nicotinamide, Complete protease inhibitor cocktail) using a polytron homogenising probe and centrifuged at 800× g for 15 min at 4°C. Protein concentration was measured by BCA assay for each sample.Unless nuclear/cytoplasmic fractionation, acid extraction or Seprion ELISA were performed, tissue was homogenised in 1 volume of ice cold RIPA buffer  NP-40, 0.5% (w/v) Na deoxycholate, 0.1% (w/v) SDS, 50 mM Tris-HCl pH 8.0, 1 mM 2-mercaptoethanol, 10 mM DTT, 1 mM PMSF, 5 µM TSA, 10 mM nicotinamide, Complete protease inhibitor cocktail) or TX buffer  Triton - X100, 1 mM PMSF, 5 µM TSA, 10 mM nicotinamide, Complete protease inhibitor cocktail)  with a polytron homogenising probe. Homogenates were sonicated on ice for 10 s at 80 Hz. Lysates were cleared by centrifugation at 16,200× Samples were diluted with 2× Laemmli buffer  bromophenol blue) and denatured for 5 min at 95°C. Equal amounts of protein were loaded onto an SDS polyacrylamide gel alongside a size reference. Proteins were transferred onto nitrocellulose membrane at 120 V for 90 min by a submerged transfer apparatus in transfer buffer. Membranes were blocked in 5% non-fat dried milk in PBS for at least 1 h. Primary antibodies were applied in 0.02% PBS-Tween 20 (PBST) for 20 min , 1 hour  at room temperature or overnight (SIRT2 (sc-20966); SIRT1; SREBP-2; Acetylated H4K16; Acetylated p53; Acetylated FOXO1) at 4°C. Blots were washed three times for 5 min in 0.2% PBST and incubated with appropriate HRP coupled secondary antibody. For signal detection the enhanced chemi-luminescence (ECL) detection system, hyperfilms and Xenograph developer were used according to the manufacturers' instructions. Signals were quantified using GS-800 densitometer.Antibodies and the dilutions at which they were used are presented in 2O and three times for 10 min in de-staining solution (16.5% (v/v) methanol, 0.5% (v/v) acetic acid). Gels were dried in a vacuum gel drier.Polyacrylamide gels were stained with Coomassie solution (0.25% (w/v) Brilliant blue R-250, 50% (v/v) methanol, 10% (v/v) acetic acid) for 30 min with gentle agitation. Gels were then washed several times in ddHFor SDS-insoluble aggregate detection, 2.5% (w/v) lysates were prepared by homogenising tissue in ice cold RIPA buffer. Aggregate load was measured as previously described Time Resolved – Förster Resonance Energy Transfer (TR-FRET) experiments were performed as previously described 384-well high bind MesoScale Discovery L21XB plates were coated at 4°C over night with 12 µl/well of 30 µg/ml 2B7-antibody for soluble mutant huntingtin detection or 10 µg/ml MW8-antibody for aggregated mutant huntingtin detection. Wells were washed with wash buffer  and blocked with 35 µl Starting Block T20 (#37539 Thermo) per well. After washing, 10 µl tissue homogenate was applied. Plates were incubated for 1 h at room temperature while shaking at 450 rpm. Wells were washed and 10 µl/well detection antibody  were added. Plates were incubated for 1 h at room temperature while shaking at 450 rpm. After final washing steps, 2× R92TC-2 MSD Read Buffer was added and plates were analyzed with a MSD SECTOR Imager 6000 reader.Sirt4, Sirt6 and cholesterol pathway genes were purchased from Primer Design. Primers for analysis of expression of Hdac1-11, Sirt1-3, Sirt5 and Sirt7 are listed in RNA extraction, cDNA synthesis, Taqman RT-qPCR and ΔCt analyses were performed as previously described The [S]-35 SIRT1/SIRT2 inhibitor was synthesized at Cychem. [S]-35 was formulated in the vehicle solution  immediately prior to dosing. 12 week old WT and R6/2 mice (N≥9/genotype) were weighed and given one acute dose of either vehicle, or formulated [S]-35 at 1 mg/kg or 3 mg/kg by oral gavage and dissected either 4 or 8 hours later. Each treatment group contained mice matched for age, sex and CAG repeat number.Blood was collected into EDTA tubes after cardiac puncture and stored on ice before centrifugation for 10 min at 4°C at 16 100× g. The plasma was removed and frozen at −20°C. Brains were removed, rinsed with saline to remove blood, weighed, and immediately frozen at −20°C. Analysis of the pharmacokinetic properties of [S]-35 was performed in 6–7 week old male C57BL/6 mice at Pharmadex and the levels of [S]-35 in the brains of WT and R6/2 mice 4 h and 8 h post dosing was performed at BioFocus.Brain tissue was homogenized in acetonitrile∶water  using an Precellys tissue homogenizer; the tissue-to-solvent ratio was of 1∶3 (w/v). Aliquots (25 µL of plasma or 100 µL tissue homogenate) of study samples, and corresponding controls were dispensed into 96-well plates. One hundred (100) µL of solvent containing internal standard  were added to all samples except matrix double blanks and solvent blanks. Acetonitrile∶water (75∶25 v/v) without internal standard (100 µL) was added to matrix double blanks. Samples were vortexed and centrifuged for 5 min, 100 µL of the supernatants were transferred to a new plate, diluted with 100 µL of acetonitrile∶Milli-Q water (75∶25 v/v) and submitted to LC-MS/MS analysis.Concentrations in mouse plasma and brain were determined using LC-MS/MS assays. UPLC reverse phase separation was performed on a Waters Acquity UPLC chromatography system using a Waters Acquity UPLC BEH C18 column ; the column temperature was maintained at 40°C. The mobile phase consisted of acetonitrile containing 0.01% v/v formic acid (B) and milliQ water containing 0.01% v/v formic acid (A). A gradient elution program (flow rate 0.6 mL/min) was used with initial conditions of 5% B. These conditions were maintained for 0.2 min, followed by a linear increase to 95% B over 1 min, maintained at 95% B for 0.6 min before returning to initial conditions; the total run time was 2 min. An injection volume of 5 µL was used. The entire LC eluent was directly introduced to an electrospray (ESI) source for LC/MS/MS analysis on a Waters TQMS Xevo triple quadrupole mass spectrometer with a source temperature of 150°C and a desolvation temperature of 500°C. Positive ion mode of ESI was used for the analysis. The mass spectrometer ion optics were set in the multiple reaction monitoring (MRM) mode, and the following transitions were monitored: 263.98>218.92 for [S]-35 and 296.05>214.14 for diclofenac . The data was processed using QuanLynx software from Waters. Using this method, the retention time of [S]-35 and the IS were 1.18 and 1.28 min, respectively. The lower limit of assay quantitation (LLOQ) for [S]-35 was 3.8 nM for both plasma and brain.t-test) software. For mouse phenotypic assessment, weight, rotarod and grip strength were analysed with repeated measures ANOVA and at each time point with three-way ANOVA. Brain weight was analysed by three-way ANOVA. Activity was analysed with repeated measures ANOVA at each time point separately. For western blotting, ELISA, TR-FRET and qPCR, group means were compared by Student's t-test, two or three-way ANOVA.Statistical analysis was performed with SPSS  or Microsoft Excel  is highlighted in yellow. The mouse Sirt2 genomic sequence was obtained from NCBI under accession number: NC_000073.5.(DOCX)Click here for additional data file.Figure S2Expression of Hdacs 1-11, Sirt1-7 and SIRT1. (A–B) Taqman qPCR assays measuring the mRNA expression of all known deacetylases in the cortex of 4 week old wild type (WT), Sirt2HET (HET) and Sirt2KO (KO) mice. Expression was normalised to the housekeeping genes Atp5b and Canx and expressed as fold change of WT ± SEM. n≥6/genotype. (C) Representative western blot probed with SIRT1 antibody showing SIRT1 expression between wild type (WT) and Sirt2KO (KO) brain lysates extracted from cortices of 9 week old mice in RIPA buffer. * denotes a non-specific band. Tubulin (Tub) was used as loading control. n≥3/genotype.(TIF)Click here for additional data file.Figure S3SIRT2 expression is comparable between 4 and 15 weeks and not affected by HD progression. (A) Representative western blot showing SIRT2 expression in different brain regions at 4 and 15 weeks of age in wild type mice. The signal for SIRT2 was normalised to total protein loaded (Coomasie stain) and expressed as fold change of rest of brain (Rb) , striatum (S), hippocampus (H), cerebellum (Cb) and brain stem (BS)) ± SEM. n = 4/brain region/time point. (B) Taqman qPCR assay measuring levels of Sirt2 mRNA between wild type (WT) and R6/2 mice at 4 and 15 weeks of age in cortex, striatum and cerebellum. Expression was normalised to the housekeeping genes Atp5b and Canx, and expressed as fold change of WT ± SEM. n≥6/genotype.(TIF)Click here for additional data file.Figure S4Activity and mobility. Activity (single lower beam break) and mobility (consecutive lower beam breaks) were recorded for wild type (WT), Sirt2HET (HET), Sirt2KO (KO), R6/2, Sirt2HETxR6/2 (R6/2 HET) and Sirt2KOxR6/2 (R6/2 KO) mice over the course of 30 min at 5, 7, 9, 11, and 13 weeks of age. Activity parameters measurements were visualised by plotting 5 min moving averages.(TIF)Click here for additional data file.Figure S5Rearing and centre rearing. Rearing (upper beam break) and centre rearing (unsupported upper beam break) were recorded for wild type (WT), Sirt2HET (HET), Sirt2KO (KO), R6/2, Sirt2HETxR6/2 (R6/2 HET) and Sirt2KOxR6/2 (R6/2 KO) mice over the course of 30 min at 5, 7, 9, 11, and 13 weeks of age. Activity parameters measurements were visualised by plotting 5 min moving averages.(TIF)Click here for additional data file.Figure S6Soluble and aggregated mHTT in the cortex, hippocampus, brain stem and cerebellum measured by MSD. Levels of soluble (upper panels) and aggregated (lower panels) mHTT as measured by Mesoscale Discovery (MSD) in (A) cortex, (B) hippocampus, (C) brain stem, and (D) cerebellum at 4, 9 and 15 weeks of age in R6/2, Sirt2HETxR6/2 (R6/2 HET) and Sirt2KOxR6/2 (R6/2 KO) mice. Soluble mHTT was measured with 2B7-MW1 and aggregated mHTT with MW8-MW8 antibody combinations. The signal for each tissue was expressed as fold change of R6/2 at 4 weeks of age. A signal was not observed above background for mice without the R6/2 transgene. n≥4/genotype/tissue/time point (except for R6/2 KO at 4 weeks where n = 3). Error bars represent SEM. *p≤0.05, **p≤0.01 (to the R6/2 at the respective time point).(TIF)Click here for additional data file.Figure S7Pharmacokinetic properties of [S]-35 in mouse brain after acute dosing. Plasma and brain levels of [S]-35 following intravenous (IV), or oral gavage (PO) administration. Male, 6–7 weeks old C57BL/6 mice were dosed with 5 mg/kg (IV) or 10 mg/kg (PO) of [S]-35 and plasma and brains were harvested at various timepoints after compound administration. Concentration of [S]-35 in the analyte was measured by LC/MS/MS. n = 4 per timepoint, mean ± standard deviation shown.(TIF)Click here for additional data file.Table S1Genetic depletion of SIRT2 does not modify R6/2 exploratory behaviour. The p values displayed in Sirt2 genotype, Sex and the duration of the 30 min test (Time) at 5, 7, 9, 11 and 13 weeks of age. Significant p-values are highlighted in blue for p<0.05, orange for p<0.01 and pink for p<0.001.(DOCX)Click here for additional data file.Table S2Summary of primer and probe sequences designed in-house.(DOCX)Click here for additional data file.Table S3Summary of all antibodies used in this study.(DOCX)Click here for additional data file.


There is no current treatment alternative for patients with FMF whose disease is resistant to, or do not tolerate colchicine. Since pyrin has an important role in IL-1β regulation we hypothesize that IL-1 inhibition will decrease the number of FMF attacks in these patients.To determine if rilonacept, a fusion protein that binds and neutralizes IL-1, decreases the number of FMF attacks compared to placebo.Subjects were FMF patients ≥4 years of age recruited at 6 U.S. sites, who had at least 1 FMF attack per month despite receiving adequate doses of, or who were intolerant of colchicine. Subjects received two 3-month courses of rilonacept (Arm A) at 2.2 mg/kg (max 160 mg) by weekly SC injection and two 3-month courses of placebo (Arm B). Patients were randomized to 1 of 4 treatment sequences . Escape visits were allowed to permit switching arms (blinding was maintained) for patients with at least 2 attacks within a course. The primary outcome was the difference of FMF attacks between rilonacept and placebo courses with responders defined as subjects with a >40% difference. Results were analyzed by paired t-and signed rank tests.Fourteen subjects were randomized, 8 males and 6 females, mean (±SD) age 24.4±11.8 years , disease duration 17.5±12.6 yrs, with a baseline of 3.1±2.0 attacks per month. Eleven completed the full study and 3 dropped out . Among 12 patients who completed at least 2 treatment courses the mean number of attacks per month on rilonacept was 1.0±1.2 vs. 1.8±0.9 on placebo (P=0.021 by paired t-test and 0.027 by signed rank test). There were 8 responders; all 4 non-responders were adults. There were 2 respiratory infection SAEs, 1 on rilonacept and 1 on placebo. Injections site reactions were significantly more frequent with rilonacept but no differences were seen in other adverse events, including infections.Rilonacept significantly reduced the number of FMF attacks and had an acceptable safety profile. IL-1 inhibition is a treatment option for most  colchicine resistant FMF patients.


Many of the error products commonly formed by Taq polymerase also are removed. Sequencing of the IMAC-processed PCR product gave base-calling accuracy comparable to that obtained with a commercial PCR product purification method. The results show that IMAC matrices  can be used for the purification of PCR products. Due to the generality of the base-specific mechanism of adsorption, IMAC matrices may also be used in the purification of oligonucleotides, cDNA, mRNA and micro RNAs.Immobilized Metal Affinity Chromatography (IMAC) has been used for decades to purify proteins on the basis of amino acid content, especially surface-exposed histidines and “histidine tags” genetically added to recombinant proteins. We and others have extended the use of IMAC to purification of nucleic acids via interactions with the nucleotide bases, especially purines, of single-stranded RNA and DNA. We also have demonstrated the purification of plasmid DNA from contaminating genomic DNA by IMAC capture of selectively-denatured genomic DNA. Here we describe an efficient method of purifying PCR products by specifically removing error products, excess primers, and unincorporated dNTPs from PCR product mixtures using flow-through metal-chelate affinity adsorption. By flowing a PCR product mixture through a Cu Current methods of PCR product purification include electrophoretic separation, enzymatic degradation of single-stranded DNA with Exonuclease I and Shrimp Alkaline Phosphatase, and adsorption on silica matrices. Smith et al. have demonstrated the removal of mismatch sequences from PCR products using mutHLS proteins 2+, Ni2+, and Zn2+ immobilized on chelators such as iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) have been shown to interact with histidine and tryptophan residues 20 tail as an affinity tag for capturing double-stranded PCR products on metal-chelate matrices and also proposed a method of IMAC purification of polyA-tailed mRNA 2+-IDA spin columns, with substantial reduction of erroneous PCR products, unincorporated primers and dNTPs.We have developed a technique for removing PCR error products, unincorporated primers and dNTPs from a PCR product mixture using immobilized metal affinity chromatography (IMAC). IMAC is widely employed for the purification of both native and histidine-tagged recombinant proteins The use of a spin column allows the efficient use of small amounts of adsorbent and sample. The columns can be equilibrated with the appropriate buffer in a few minutes with a few centrifugation steps. With the appropriate equipment, e.g., filter-bottom plates, most steps are automatable.Previously, it was determined that single-stranded DNA binds to IMAC adsorbents whereas double-stranded DNA does not Taq PCR reaction product mixture from a lambda bacteriophage genomic DNA template was applied to each of two 125 µL Cu2+-IDA IMAC spin columns. The pooled flow-through product from two identical columns was loaded onto a 1.2% agarose SB (sodium boric acid) gel stained with SYBR Gold Nucleic Acid Gel Stain  for analysis by electrophoresis. nd and 3rd elutions, 20 µL of eluent was added and centrifuged for 2 min at 1100 xg (no incubation). The primer is eluted from the column in the first two elutions, along with more double-stranded product , and DNA precipitated using 1/10 volume of 3 M potassium acetate, pH 5.0 and 2.5 volumes of absolute ethanol. The pellet was washed with 250 µL of 70% ethanol, air dried and then dissolved in 5 µL of water and loaded into the gel.20 µL of a Taq PCR by insertion of mismatched bases Taq has difficulty extending the DNA chain past a mismatch, resulting in sticky-ended products 2+ column. Similar results as shown for lambda DNA were obtained for removal of PCR error products from products of PCR amplification of a 1.1 kbp D-alanine-D-alanine ligase A gene from E.coli genomic DNA  and 99±0.2% (1.50±0.01 absorbance), respectively. The deoxy pyrimidine triphosphates dCTP and dTTP bound to the column less stringently - 95±2% (0.88±0.02 absorbance) and 94±1% (1.36±0.00 absorbance) bound, respectively. In each case, the concentration of dNTPs was reduced from 100 µM to less than 6 µM, a level that should not inhibit subsequent sequencing or other enzymatic reactions Each dNTP (as 60 µL of a 100 µM solution) was loaded onto triplicate 100 µL CuContaminants such as excess salts, PCR primers, and dNTPs have a detrimental effect on sequencing of PCR products. In the sequencing reaction, primers and nucleotides will compete with the sequencing primer and ddNTPs. This results in additional products which, when analyzed on the sequencing gel, make it difficult or impossible to determine the correct bases, causing a “no-call”, the number of which can be used to judge the relative quality of a PCR product. Phred quality scores also can be used to assess the quality of the sequence, with a Phred quality score of 20 implying a 1-in-100 probability that the base is incorrectly assigned E.coli genomic DNA, this PCR product after purification by IMAC, and this PCR product purified using the QIAquick PCR Purification kit . Four independent PCR reaction samples were sequenced after each treatment, for a total of 12. Each product was sequenced from each end (using primers designated CT and NT), for a total of 24 reactions. Sequencing used BigDyes™ ddNTPs , according to the manufacturer's directions. The average no-calls ‘N’s observed after 10 correctly called consecutive bases in the first 800 bases for unpurified PCR products, QIAquick purified and IMAC purified samples were 38±24, 2±2 and 7±4 respectively using CT primer  performed dideoxynucleotide sequencing reactions on three sets of samples – the products of PCR amplification of a 1.1 kbp D-alanine-D-alanine ligase A encoding region of the T primer  and 73±3T primer . Raw seqT primer . Figure T primer , demonst2+-IDA) is a promising approach to the purification of PCR products.From these results, it is clear that IMAC can remove primers, error products and dNTPs from PCR product mixtures, suggesting that IMAC , Helicase-dependent amplification (HDA) and Multiple displacement amplification (MDA). These results also suggest the application of IMAC in the purification of cDNAs, mRNAs and microRNAs.IMAC purification might also improve the results of PCR product cloning, and sequencing by non-Sanger methods. In addition to purifying double-stranded PCR products, IMAC should be applicable to the purification of double-stranded DNA amplicons generated by other Taq Flexi DNA Polymerase , 1.3 mM magnesium chloride and 2 µM each of the primers, 5′-GGCTTCGGTCCCTTCTGT-3′ and 5′-CACCACCTGTTCAAACTCTGC-3′. The reactions were carried out in a MJ Mini Personal thermal cycler  as follows: 35 cycles of 45 s at 95°C (denaturation), 45 s at 55°C , 45 s at 72°C (elongation), followed by a final elongation step at 72°C for 5 min.A 280 bp region of lambda bacteriophage genomic DNA  was amplified in 50 µL PCR reactions containing 2 pg of lambda genomic DNA template, 1X PCR buffer, 200 µM each dNTP, 1 U GoE.coli genomic DNA was amplified in 50 µL PCR reactions containing 10 ng of the genomic DNA, 1X PCR buffer, 200 µM each dNTP, 1 U GoTaq Flexi DNA Polymerase , 1 mM Magnesium chloride and 1 µM each of the primers, 5′- GGGGCATATGGAAAAACTGCGGGTAGGAATCG -3′ (NT primer) and 5′- GGGGGGATCCGGGCGTTAAAATATTACATTGTGGTT -3′ (CT primer). The reactions were carried out in a MJ Mini Personal thermal cycler  as follows: 30 cycles of 45 s at 95°C (denaturation), 1 min at 66.5°C , 1 min at 72°C (elongation), followed by a final elongation step at 72°C for 5 min.For other experiments, an 1.1 kbp D-alanine-D-alanine ligase A encoding region of the 2O to remove residual ethanol. Then, 0.6 mL of 50 mM CuCl2 was added and the tube was mixed end-over-end on a Cole-Parmer rotor for 15 minutes. Finally, the beads were washed three times with 20 mM HEPES, 250 mM NaCl, pH 7.0 (“Buffer A”) to remove excess unchelated metal ions and to equilibrate the matrix.2 mL of Chelating Sepharose Fast Flow  in 20% ethanol was added to a 2 mL polypropylene tube. The tube was centrifuged in a tabletop microcentrifuge at 11,000 xg for 2 min, the supernatant was decanted, and the beads were washed three times with DI Hca. 90 µm adsorbent particles, 250 µL of vortex-suspended metal-charged matrix slurry  was added to a Micro Bio-Spin Chromatography column . The column was then inserted into a 2 mL polypropylene microcentrifuge tube and centrifuged for 2 min at 1100 xg in a tabletop microcentrifuge, leaving 125 µL of packed wet beads.Using a 1 ml pipette tip with the bottom quarter cut off to accommodate the The receiving microcentrifuge tube was replaced with a fresh one. Then, 20 µL of the PCR mixture was added to the prepared spin column and after allowing 15 min for adsorption, the column was centrifuged for 2 min at 1100 xg. The PCR mixture passes through the column and is depleted of primers, nucleotides and error products, producing purified double-stranded product. Some double-stranded product is trapped in the interstitial space of the column and can be recovered if desired by washing the column with non-eluting buffer A.Figure S12+-IMAC purification of PCR product mixture from amplifying a region of E.coli genomic DNA. Lane 1: normal loading (0.5 µL) of unpurified PCR product; Lane 2: overloading (4 µL) of unpurified PCR product; Lane 3: normal loading (0.5 µL) of purified PCR product, flow-through after direct application of PCR product mixture to Cu2+-IMAC column; Lane 4: overloading(4 µL) of purified PCR product Lanes 5-7: 4 µL of consecutive 20 µL column washes with 250 mM NaCl, 20 mM HEPES, pH 7.0; Lane 8: 4 µL of first elution with 20 µL 500 mM imidazole in 250 mM NaCl, 20 mM HEPES, pH 7.0; Lanes 9-11: 4 µL of first, second and third elutions, respectively with 20 µL 500 mM imidazole in 250 mM NaCl, 20 mM HEPES, pH 7.0. Lanes 9-11 were concentrated 10-fold by ethanol precipitation to enhance sensitivity.Cu(1.42 MB TIF)Click here for additional data file.Figure S2Raw sequence trace using CT primer.(5.44 MB PDF)Click here for additional data file.Figure S3Raw sequence trace using NT primer.(5.62 MB PDF)Click here for additional data file.Figure S4ClustalW alignment of sequence data generated using NT and CT Primers.(0.08 MB DOC)Click here for additional data file.Figure S5Average Phred score (using CT primer) versus base position. IMAC purified (red) and QIAquick purified (green) samples have better quality scores than unpurified (blue) samples.(0.36 MB PDF)Click here for additional data file.Table S1Number of no-calls using CT primer.(0.03 MB DOC)Click here for additional data file.Table S2Number of no-calls using NT primer.(0.03 MB DOC)Click here for additional data file.