text:
[Back to Issue 10 ToC] [Back to Journal Contents] [Back to Biochemistry (Moscow) Home page]
[Download Reprint (PDF)] [Download Supplement]
Synthesis in Escherichia coli and Characterization of Human Recombinant Erythropoietin with Additional Heparin-Binding Domain
A. S. Karyagina1,2,3,a*, T. M. Grunina1, M. S. Poponova1, P. A. Orlova1, V. N. Manskikh1,3, A. V. Demidenko1, N. V. Strukova1, M. S. Manukhina1, K. E. Nikitin1, A. M. Lyaschuk1, Z. M. Galushkina1, S. A. Cherepushkin4, N. B. Polyakov1,5, A. I. Solovyev1, V. G. Zhukhovitsky1, D. A. Tretyak6, I. S. Boksha1,7, A. V. Gromov1,b*, and V. G. Lunin1,2
1Gamaleya National Research Center of Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098 Moscow, Russia
2All-Russia Research Institute of Agricultural Biotechnology, 127550 Moscow, Russia
3Belozersky Institute of Physical and Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia
4State Research Institute of Genetics and Selection of Industrial Microorganisms, Kurchatov Institute National Research Centre, 117545 Moscow, Russia
5Vernadsky Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences, 119334 Moscow, Russia
6Moscow Technological University (Lomonosov Institute of Fine Chemical Technologies), 119571 Moscow, Russia
7Research Center of Mental Health, 115522 Moscow, Russia
Received May 18, 2018
Recombinant human erythropoietin (EPO) with additional N-terminal heparin-binding protein domain (HBD) from bone morphogenetic protein 2 was synthesized in Escherichia coli cells. A procedure for HBD-EPO purification and refolding was developed for obtaining highly-purified HBD-EPO. The structure of recombinant HBD-EPO was close to that of the native EPO protein. HBD-EPO contained two disulfide bonds, as shown by MALDI-TOF mass spectrometry. The protein demonstrated in vitro biological activity in the proliferation of human erythroleukemia TF-1 cell test and in vivo activity in animal models. HBD-EPO increased the number of reticulocytes in the blood after subcutaneous injection and displayed local angiogenic activity after subcutaneous implantation of demineralized bone matrix (DBM) discs with immobilized HBD-EPO. We developed a quantitative sandwich ELISA method for measuring HBD-EPO concentration in solution using rabbit polyclonal serum and commercial monoclonal anti-EPO antibodies. Pharmacokinetic properties of HBD-EPO were typical for bacterially produced EPO. Under physiological conditions, HBD-EPO can reversibly bind to DBM, which is often used as an osteoplastic material for treatment of bone pathologies. The data on HBD-EPO binding to DBM and local angiogenic activity of this protein give hope for successful application of HBD-EPO immobilized on DBM in experiments on bone regeneration.
KEY WORDS: erythropoietin, Escherichia coli, heparin-binding domain
Abbreviations: BCA, 2,2′-bicinchoninic acid; BMP-2, bone morphogenetic protein 2; DBM, demineralized bone matrix; DTT, dithiothreitol; EPO, erythropoietin; HBD, heparin-binding domain; 6His, six-histidine tag; s-tag, 15-amino acid oligopeptide from bovine pancreas ribonuclease A.
Erythropoietin (EPO) is a hormone (cytokine) that initiates differentiation of erythrocyte precursors in bone marrow [1]. Apart from its role in erythropoiesis, EPO is involved in wound healing, neuroregeneration, and other biological processes, such as bone tissue remodeling that requires both angiogenic and osteogenic activities of EPO [2-4]. The effect of recombinant eukaryotic EPO on bone tissue regeneration in mammals has been demonstrated in various experimental models [5-12], in which EPO has been administered systemically or locally. However, there are reasons to believe that local administration of EPO as a component of implanted material is preferable in the treatment of bone pathologies. Using the model of bone tissue regeneration in rabbit long bones, Omlor et al. [12] demonstrated more than two-fold increase in the volume of newly formed bone tissue at three different time points (up to 12 weeks) after surgery upon local EPO administration, as compared to a single systemic parenteral introduction of the same amount of EPO. Multiple systemic parenteral introduction of large doses of EPO may result in impaired hemostasis (increased hematocrit and blood viscosity) and become a risk factor in patients [13]. Therefore, high therapeutic efficacy of EPO administered locally as a component of implanted material makes EPO a promising alternative agent in the treatment of bone pathologies.
Recombinant human EPO used in all the above cited works, as well as in clinical studies, was synthesized in cultured eukaryotic cells (Chinese hamster ovary cells) [14]. Produced molecular forms of EPO have identical amino acid sequence (165 a.a.) but differ in glycosylation and pharmacokinetic profiles: epoetin alpha, epoetin beta, darbepoetin alpha (hyperglycosylated analog of human recombinant epoetin alpha with the elimination half-life after intravenous administration about three times longer than of epoetin alpha and native EPO) [15]. The use of different EPO forms explains the differences in the treatment efficacy and results of model experiments. Besides, common problems in the synthesis of glycosylated recombinant proteins in eukaryotic systems are low yield and synthesis of proteins with different glycosylation profiles requiring their further fractionation.
An alternative approach to the biosynthesis of human recombinant EPO is heterologous prokaryotic expression system in Escherichia coli cells [16-20]. Using this system, EPO could be produced in high amounts in a more standardized state and at much less expense. However, bacterially produced EPO is not glycosylated, which makes it low soluble and prone to aggregation. Compared to the glycosylated forms, non-glycosylated proteins have lower molecular masses favoring their more rapid elimination from the circulation. The problem of low solubility can be solved by introducing amino acid substitutions at the sites of glycosylation [19] or by modifying EPO with additional protein domains that could be later cleaved off by proteolytic hydrolysis [17, 20]. The molecular mass of recombinant protein can be increased by various methods including the widely used procedure of PEGylation [16]. Since the synthesis of eukaryotic proteins in bacterial cells can be accompanied by formation of random disulfide bonds, formation of two correct S–S bonds typical for the native EPO requires additional refolding stage. The protein yield after purification is determined by the portion of correctly refolded molecules [20]. Nevertheless, recombinant EPO produced in E. coli cells displays biological activity [16-20]; hence, development of new procedures for recombinant EPO application, in particular, for regeneration of damaged bone tissue, is a promising approach in clinical practice.
In order to the recombinant EPO exert a long-term action after introduction to the bone defect, its release from the implanted material should be very slow. When using glycosaminoglycan-containing demineralized bone matrix (DBM) as a carrier, the recombinant protein could be retained in the matrix if it contains heparin-binding domain (HBD), an amino acid sequence that forms complexes with heparin and other negatively charged polysaccharides via ionic interactions. HBDs are present in many proteins, e.g., bone morphogenetic protein 2 (BMP-2). Due to the presence of HBD, BMP-2 can be purified on heparin-Sepharose [21] or immobilized on DBM [22]. We suggested that introduction of HBD into the molecule of recombinant EPO will promote EPO retention at the site of implantation if DBM is used as a carrier.
The goal of this work was to produce highly purified HBD-EPO preparations with correctly formed disulfide bonds. The recombinant HBD-EPO should be capable of binding with DBM and display in vitro and in vivo activities, so that it can be used in experiments on regeneration of bone tissue defects.
Escherichia coli M15 [pREP4] (F– Φ80ΔlacM15, thi, lac–, mtl–, recA+, KmR) strain and plasmid vector pQE6 (Qiagen, USA) were used.
HBD-EPO cloning. The synthetic gene encoding HBD from Danio rerio BMP-2 fused to human EPO and flanked with the NcoI and Kpn2I restriction endonuclease sites at the 5′- and 3′-ends, respectively, was synthesized by Eurogen (Russia). Codon composition of the HBD-EPO gene was optimized using the JCat software (http://www.jcat.de/); the secondary structure of the transcribed RNA was corrected using the DINAMelt web server (http://mfold.rna.albany.edu/?q=DINAMelt/Two-state-folding). The synthesized gene was cloned into pQE6 vector by the NcoI and Kpn2I sites to generate the pL610 plasmid that was used to transform E. coli M-15 [pREP4] cells.
Calculated molecular mass and other properties of HBD-EPO. The characteristics of HBD-EPO were theoretically calculated using the https://web.expasy.org/protparam/ resource. The calculated molecular mass of HBD-EPO is 20520.51 Da; theoretical isoelectric point (pI) is 9.62. The absorbance of HBD-EPO solution in water (1 mg/ml) at 280 nm is 1.107 (protein with two disulfide bonds) or 1.095 (reduced protein).
Cultivation of transformed E. coli cells. Transformed E. coli cells were grown in LB medium containing appropriate antibiotics on a shaker at 180 rpm at 37°C to OD600 of 1.0-1.2. Protein synthesis was induced with 0.5 mM IPTG, and the cells were incubated for another 6 h at 180 rpm at 30°C. The cells were collected by centrifugation for 30 min at 5000g at 10°C and stored at –20°C.
Disintegration of E. coli cells and isolation of inclusion bodies. Frozen bacterial biomass (1 g) was thawed and suspended at a 1 : 10 ratio (w/v) in the lysing buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF)) containing 100 μg/ml lysozyme. The cell suspension was incubated for 20 min at room temperature, and the cells were disintegrated on ice by sonication with a Sonics Vibracell sonicator (Sonics, USA) (40% amplitude; 2.5 min; 3-s pulses with 2-s intervals). The resulting homogenate was centrifuged for 30 min at 20,000g at 10°C. The pellet containing inclusion bodies was washed twice with the lysing buffer and once with 20 mM Tris-HCl (pH 8.0).
Column chromatography and electrophoresis. Column chromatography on WorkBeads 40 S (Bio-Works, Sweden) and heparin-Sepharose CL-6B (Amersham Pharmacia Biotech, Sweden) was performed with Äkta Start chromatography system (GE Healthcare Life Sciences, USA). Protein concentration was determined from the protein solution absorbance at 280 nm using calculated extinction coefficients and with BCA Protein Assay (AppliChem, Germany).
Preparation of protein samples, electrophoresis in polyacrylamide gel (PAGE), gel staining, and gel densitometry were performed as described by Grunina et al. [20]. Protein molecular weight markers (14-97 kDa) were from Bio-Rad (USA).
Chromatography on WorkBeads 40 S. The washed inclusion bodies (0.25 g) were dissolved in 8 M urea in 20 mM Tris-HCl (pH 8.0) at a 1 : 10 (w/v) ratio. The solution was incubated for 3 h and then centrifuged at 9000g for 30 min. The supernatant was loaded at a flow rate of 1 ml/min on a column with 10 ml of WorkBeads 40 S equilibrated with 8 M urea in 20 mM Tris-HCl (pH 8.0). The column was washed at 2 ml/min to remove non-bound proteins, and HBD-EPO was eluted at the same flow rate with 0-1.0 M linear gradient of NaCl concentration in the same buffer (total volume, 100 ml). Fractions (5 ml) with the maximal absorbance at 280 nm were combined and analyzed by electrophoresis. Protein concentration in the collected fractions was estimated from the absorbance at 280 nm and by the BCA method (both methods produced convergent results).
HBD-EPO refolding. Protein solution after chromatography on WorkBeads 40 S was diluted with 20 mM Tris-HCl (pH 8.0) containing 6 M guanidine hydrochloride and 0.1 M dithiothreitol (DTT) to a final protein concentration of ~0.1 mg/ml and incubated for 24 h at 4°C. The denatured protein was dialyzed against 10 volumes of 20 mM Tris-HCl (pH 8.0) containing 1 M guanidine hydrochloride for 24 h with three buffer changes and centrifuged for 30 min at 9000g. The supernatant was diluted with the refolding buffer (20 mM Tris-HCl, pH 8.5, containing 1 M L-arginine) at a 1 : 1 ratio to the final protein concentration of ~0.05 mg/ml and incubated for 24 h at 4°C. The refolded protein was dialyzed against 10 volumes of 20 mM Tris-HCl (pH 6.8) containing 4 M urea for 24 h and then centrifuged for 30 min at 9000g.
Affinity chromatography on heparin-Sepharose CL-6B. Protein solution after refolding was loaded at a flow rate of 1 ml/min on a column with 10 ml of heparin-Sepharose CL-6B equilibrated with 4 M urea in 20 mM Tris-HCl (pH 6.8). The column was washed with the same buffer, and the protein was eluted with 0-1.0 M linear gradient of NaCl concentration in the same buffer at the same flow rate. Four fractions (6.5 ml each) were collected; the first three fractions contained monomeric HBD-EPO, while the last fraction contained protein aggregates (seen in PAGE). Fractions containing monomeric HBD-EPO were combined (total volume, 19.5 ml) and dialyzed against 10 volumes of 20 mM ammonium acetate (pH 4.5) for 24 h at 4°C with three buffer changes. For dialysis, we used 25 μm-thick Zellu Trans/ROTH 3,5 E657.1 cellulose dialysis membrane with a 3500-Da cut-off. Protein concentration and total protein amount after dialysis was estimated from the absorbance at 280 nm and by the BCA method (both methods produced convergent results). The absorbance of the combined fraction (20 ml) was 0.181 OU, which corresponded to the protein concentration of 0.165 mg/ml (taking into account theoretical extinction coefficient for the oxidized protein). Purified protein preparations were lyophilized and used in further studies.
HBD-EPO hydrolysis with trypsin for mass-spectrometry. Proteins fractionated by PAGE were digested in the gel as described by Shevchenko et al. [23] in the presence or absence of DTT and iodoacetamide [20].
MALDI mass spectrometry. Peptides were analyzed with a time-of-flight MALDI Ultraflextreme mass spectrometer (Bruker Daltonics, Germany) using dihydroxybenzoic acid as a matrix. Positively charged ions were detected in the reflectron mode at 20.12 kV on the ion source IS1, 17.82 kV – on IS2, 7.47 kV – on lenses, 21.07 kV – on reflectron Ref1, and 10.80 kV – on Ref2.
The ions were detected within the m/z range of 700-5000 Th. Peaks corresponding to trypsin and keratin autolytic fragments were used as internal standards and excluded from the list of detected masses.
The spectra were analyzed with the Flex Analysis 3.4 software (Bruker Daltonics) and Proteinscape 4.1 program suit (Bruker Daltonics) using the following parameters: accuracy – 100 ppm; possible posttranslational modifications – oxidation of methionine, histidine, and tryptophan residues; cysteine modification with acrylamide. When analyzing the primary sequence, we also considered the possibility of cysteine modification with iodoacetamide. Identification of peptides permitted no more than six nonhydrolyzed trypsin sites per protein molecule.
In vitro biological activity assay. Biological activity of EPO preparations was assayed in the in vitro proliferation test in human erythroleukemia cell line TF-1 (ATCC CRL-2003) as suggested by Grunina et al. [20].
Biological activity of BMP-2 preparations was estimated in vitro from the increase in the activity of alkaline phosphatase synthesized by mouse C2C12 myoblasts in response to protein addition as described in [24, 25].
Estimation of HBD-EPO biological activity in vivo. Biological activity of EPO preparations was assayed in CBA mice [26] at the Olfarm Ltd. (Russia) laboratory. Normocythemic females (8 week-old) were injected subcutaneously with 0.5 ml of an equimolar mixture of heparin (molar mass, 8000-25,000 g/mol; BioChemica AppliChem, Germany) with tested EPO preparation at a concentration of 360, 180, or 90 μg/ml. Control mice were injected with physiological saline or Epostim (recombinant epoetin beta produced in eukaryotic cells; 100,000 IU/mg; Farmapark Ltd., Russia) in a concentration of 80, 40, or 20 IU/ml. Blood was collected 96 h after injection and analyzed for the reticulocyte count. Cells with a characteristic reticular network were counted in blood smears under a light microscope after supravital staining of nucleic acids in cell nuclei. The activity of HBD-EPO was estimated relatively to the control using the calibration curve.
Polyclonal anti-EPO serum. Polyclonal anti-EPO serum was obtained by immunization of chinchilla rabbits (body weight of 2.5-3.0 kg) with purified 6His-MBP-EPO and s-tag-EPO proteins [20] by the following scheme: first session – 5 injections 200 μl each (total of 150 μg 6His-MBP-EPO in physiological solution (1 : 1); complete Freund adjuvant) in the top of the shoulder and four zones of axillary and inguinal lymphatic nodes; 2-week break; second session – 5 injections 200 μl each (total of 150 μg 6His-MBP-EPO in physiological solution (1 : 1); incomplete Freund adjuvant) in the top of the shoulder and four zones of axillary and inguinal lymphatic nodes; 2-week break; third session – injection of 1 ml (150 μg) of s-tag-EPO in physiological solution in the thigh muscle; 1-week break; fourth session – injection of 1 ml (150 μg) of s-tag-EPO in physiological solution in another thigh muscle; 1-week break. The serum titer was determined by ELISA using HBD-EPO, s-tag-EPO, and Epostim in a concentration of 5 μg/ml as antigens and was estimated as no less than 1 : 2500 for each protein.
HBD-EPO immunoassay. Six different clones of commercially available monoclonal antibodies (Bialexa LTD, Russia) were tested for their interaction with HBD-EPO and Epostim by immunoblotting according to a standard procedure recommended by Amersham GE Healthcare. After electrophoresis, Epostim and HBD-EPO were transferred onto Hybond-C Extra nitrocellulose membrane using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, USA) at 2 mA/cm2; the membrane was blocked with 0.5% casein, cut into strips, and incubated for 1 h with EP1, EP2, EP3, EP5, EP8, and EP25 antibodies (Bialexa LTD) at a 1 : 1000 dilution and then for 1 h with rabbit anti-mouse immunoglobulins conjugated with horseradish peroxidase (1 : 10,000 dilution; IMTEK, Russia). The membranes were stained by the peroxidase reaction using hydrogen peroxide and diaminobenzidine.
Erythropoietin-EIA-Best kit for sandwich ELISA (Vektor-Best-Evropa, Russia) was used as recommended by the manufacturer, except that EPO concentration was determined in phosphate buffer instead of patient's serum.
Sandwich ELISA for HBD-EPO quantitative assay in solution. The concentrations of HBD-EPO and Epostim were determined by indirect sandwich ELISA using EP8 monoclonal anti-EPO antibody (Bialexa LTD) as a capturing agent, rabbit anti-EPO serum as a detecting agent, and 1 mg/ml bovine serum albumin in phosphate buffer containing 0.1% Tween 20 as a blocking agent.
Pharmacokinetic studies were performed as described by Wang et al. [16] with slight modifications in six Wistar female rats (body weight of 270-280 g). The rats were divided into two groups (3 animals per group). Group 1 was injected with HBD-EPO; group 2 was injected with Epostim. The rats were injected subcutaneously in the top of the shoulder with equimolar mixture of a protein with heparin (molar mass, 8000-25,000 g/mol; BioChemica AppliChem, Germany) at a dose of 100 μg/kg body weight. Blood (200 μl) was collected from the tail vein into tubes with EDTA K3 0.5, 2, 5, 10, 24, 48, and 72 h after injection. After centrifugation at 400g for 15 min at 4°C, the plasma was collected and centrifuged at 2000g at room temperature for 5 min. The plasma was stored at –70°C, EPO concentration was determined using the developed ELISA test.
HBD-EPO binding to DBM. To obtain DBM membranes, diaphyses of bovine femoral bones were cut with band saw into 1- to 3-mm thick slices, that were then delipidated, decalcified, and deproteinized to remove non-collagen protein as described in [27] with modifications [22]. Disks (4 mm in diameter) were cut out, weighed, and standardized according to their mass. Each experiment used disks with maximally similar masses that depended on the amount of the applied protein.
To immobilize HBD-EPO, each disk was placed in 100 μl solution containing 1 to 10 μg HBD-EPO in 25 mM phosphate buffer (pH 5.5) and incubated with shaking for 3 h. The disks were then washed 3 times for 20 min with 1 ml of the same buffer and lyophilized. The amount of bound HBD-EPO was calculated from the content of HBD-EPO in the washing solution (i.e., not bound to the disk) measured by ELISA. To determine the amount of bound protein directly, each disk was incubated in 1 ml of 6 M guanidine hydrochloride at room temperature overnight on a shaker, and then the resulting wash out was dialyzed against 100 volumes of 25 mM phosphate buffer (pH 5.5) for 24 h. The amount of HBD-EPO was determined by the developed ELISA method. We found that under the conditions used, virtually all HBD-EPO bound to the DBM. After this, the disks were frozen, lyophilized, and sterilized by irradiation (adsorbed dose, 20 ± 5 kGy). For simultaneous immobilization of HBD-EPO and BMP-2 of DBM disks, the incubation solution contained 1 to 20 μg s-tag-BMP-2 [25] in addition to HBD-EPO. The amount of bound s-tag-BMP-2 was determined using the earlier developed ELISA test system [28].
The quality of the bound proteins and the ratio between them were estimated by PAGE. DBM disks with immobilized proteins were incubated with 50 μl Laemmli's sample buffer containing 0.2 M DTT, 2 mM EDTA, 125 mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, and 0.015% Bromophenol Blue for 5 min at 95°C and then centrifuged for 5 min at 16,000g. The supernatant was fractionated by PAGE.
HBD-EPO angiogenic activity. The angiogenic activity of HBD-EPO was assayed in six Wistar female rats (body weight of 270-280 g). The rats were divided into two groups (3 animals per group). The animals were anesthetized intravenously with Zoletil 100 (15 mg/kg body weight) and intraperitoneally with Rometar (5 mg/kg body weight). In each rat, two DBM disks with the same immobilized protein were implanted subcutaneously through 1-cm cuts in the temples symmetrically on both sides of the head. The first group was implanted with disks loaded with 1 μg HBD-EPO; the second group (control) was implanted with disks with immobilized 1 μg Epostim. To immobilize HBD-EPO and Epostim, the disks were placed in 1.5-ml plastic tubes containing 1 μg of protein in 20 μl of 25 mM phosphate buffer (pH 5.5), incubated overnight at 10°C, and used for implantation without further lyophilization.
The animals were euthanized with carbon dioxide 5 days later, and the disks with surrounding tissues were removed. One of the two disks from the same animal was fixed for 24 h in freshly prepared 10% formalin buffered solution (pH 7.4) and used for preparation of paraffin slides for hematoxylin-eosin and Heidenhain's AZAN trichrome staining [29] with further histological analysis and histomorphometry. The second disk was used for determining the residual protein. For this, the disk was incubated in 1 ml of 6 M guanidine hydrochloride in 25 mM phosphate buffer at 4°C overnight on a shaker and then dialyzed against 100 volumes of 25 mM phosphate buffer (pH 5.5) at 4°C for 24 h. The amount of the residual protein was determined using the developed ELISA test system.
To create EPO variants capable of retention on DBM, we needed to find small protein domain that could be introduced into EPO molecule to provide immobilization of the resulting chimeric protein on DBM. The most obvious candidate was heparin-binding domain (HBD) of BMP-2. The function of this domain is a long-term retention of BMP-2 in the bone tissue. HBD is located in the N-terminal part of BMP-2 molecule and contains a large number of positively charged amino acid residues. Since we planned to use the created recombinant protein in experiments on the bone tissue regeneration, in particular, in combination with BMP-2, it was undesirable to fuse EPO with amino acid sequence identical to the fragment of human BMP-2 because it could hinder its immunological detection in experiments on the binding and kinetics of EPO elution from DBM. We compared BMP-2 sequences from various vertebrate species (data not shown) and chose HBD from the zebra fish Danio rerio that is maximally distant from the human HBD (Fig. 1).
Fig. 1. Amino acid sequence of HBD-EPO. EPO sequence is shown in bold; HBD (D. rerio) is shown in italic; linker is underlined; cysteine residues in EPO are shown in bold and underlined. Amino acid sequence of HBD from Homo sapiens is shown in italic directly under the HBD sequence of HBD-EPO.
We developed a synthetic gene encoding for the chimeric HBD-EPO protein consisting of the HBD from D. rerio and human EPO connected with a serine/glycine linker.
The obtained genetic construct was used for creation of bacterial strain for the production of recombinant HBD-EPO in quantities sufficient for performing experiments described below in this article. We also developed the procedure for HBD-EPO purification by column chromatography.
The results of electrophoretic analysis of protein fractions at different stages of HBD-EPO purification are shown in Fig. 2.
Fig. 2. Electrophoresis in 12% polyacrylamide gel of protein preparations obtained during HBD-EPO purification from E. coli M-15 [pREP4, pL610] producer strain: 1, 2) extract of E. coli cells before (1) and after (2) induction of protein synthesis with IPTG; 3) soluble proteins from E. coli cells after the protein synthesis induction with IPTG; 4) washed inclusion bodies; 5, 6) HBD-EPO-containing fractions eluted from WorkBeads 40 S in the sample buffer with (5) and without (6) DTT; 7, 8) HBD-EPO fractions after refolding eluted from heparin-Sepharose CL-6B in the sample buffer with (7) and without (8) DTT; M, protein molecular weight markers, 14-97 kDa (Bio-Rad, USA). The presence (+) or absence (–) of DTT in the sample buffer is shown under the lanes.
Comparison of electrophoregrams of bacterial cell extracts before and after the protein synthesis induction with IPTG (Fig. 2, lanes 1 and 2) revealed a protein band corresponding to the recombinant HBD-EPO with apparent molecular mass of 24 kDa, which was higher than its calculated molecular mass (20.5 kDa). This protein comprised ~50 % of total cell protein and was synthesized mostly as insoluble inclusion bodies (Fig. 2, lanes 3 and 4). After column chromatography on WorkBeads 40 S, fractions with the maximal absorbance at 280 nm were combined. The total volume of the combined protein fractions was 10 ml; protein concentration was 2.15 mg/ml. Electrophoretic analysis in the presence of DTT showed that the isolated protein was homogenous; however, multiple bands presumably corresponding to differently folded monomeric and oligomeric forms of HBD-EPO were observed in electrophoregrams in the absence of DTT (Fig. 2, lanes 5 and 6).
Protein refolding was performed as described by Wang et al. [16] with slight modifications; HBD-EPO was then subjected to chromatography on heparin-Sepharose CL-6B. The first three eluted fractions (total volume, 20 ml) contained almost homogenous HBD-EPO monomer, as judged by electrophoresis in the presence and absence of DTT. However, in the absence of DTT, the refolded protein had a higher electrophoretic mobility with apparent molecular mass of ~23 kDa (Fig. 2, lanes 7 and 8). Similar increase in the electrophoretic mobility of the refolded protein in the absence of DTT was observed for bacterially produced non-glycosylated 6His-s-tag-EPO [20].
The total amount of purified protein was ~3.2 mg. The data on the HBD-EPO purification efficiency and yield at different purification stages are shown in the table.
Yield of recombinant HBD-EPO at different purification stages
To study the structure of refolded HBD-EPO after chromatography on WorkBeads 40 S, the protein band was cut from the gel (Fig. 2, lane 7) and digested with trypsin with subsequent addition of DTT and to the gel (see "Materials and Methods" for details). The results of MALDI mass spectrometry of the tryptic peptides are shown in Fig. 3, a-c; peptide sequences and their modifications are presented in Table S1 (see Supplement to this paper on the site of the journal http://protein.bio.msu.ru/biokhimiya and Springer site Link.springer.com). Analysis of mass spectra revealed a number of peaks corresponding to the protonated molecular ions of cysteine-containing peptides: T[7-30] + IAA (m/z 2607.3409), T[13-34] + IAA (m/z 2299.1836), T7 + 2IAA (m/z 2803.2123) containing two cysteine residues that form one of the disulfide bridges in the native protein, T[164-182] + IAA (m/z 2275.3167), T[164-186] + IAA (m/z 2704.375), and T26 + IAA (m/z 969.4701). Cysteine residues in these peptides were S-carbamidomethylated. The spectra also had three peaks corresponding to cysteine-containing peptides T[7-30] + IAA + oxH (m/z 2623.3226), T[10-30] + IAA + oxH (m/z 2211.0515), and T[10-34] + IAA + oxH (m/z 2708.3472) containing oxidized histidine residues in addition to iodoacetamide-modified cysteines. The coverage of HBD-EPO sequence with tryptic peptides (Fig. 3d) was 94.6%. The absence of some peptides in the spectra could be explained by their low molecular mass (<700 Da), which was below the detection limit.
Fig. 3. MALDI mass spectra of peptides obtained by tryptic digestion of reduced HBD-EPO (a-c) and diagram showing the coverage of HBD-EPO sequence with tryptic peptides (d). a, b) MALDI mass spectra of tryptic peptides obtained by digestion of the protein band with apparent molecular mass of ~24 kDa (Fig. 2, lane 7) with m/z 700-3300 (a), 910-990 (b), and 2180-2270 Th (c). Before digestion in the gel, the protein was treated with DTT, and cysteine residues were reduced with iodoacetamide. d) Identified peptides are shown in rectangles with peptide name inside (T with peptide number); corresponding amino acid sequences are shown above; IAA, modification with iodoacetamide; oxH, oxidize histidine; gray rectangles, cysteine-containing peptides.
To estimate the efficiency of HBD-EPO refolding, the corresponding protein band was cut out of the gel (Fig. 2, lane 8) and subjected to trypsinolysis in the absence of DTT and iodoacetamide. The samples were loaded on the gel in the DTT-free sample buffer. MALDI mass spectra of the obtained peptides are shown in Fig. 4, a and b; the sequences of these peptides and their modifications are presented in Table S2 (Supplement). The mass spectra lack the peaks corresponding to peptides containing S-carbamidomethyl cysteine (Fig. 3, a and b) but contain peaks confirming the presence of disulfide bonds in the studied protein. Thus, the peak T7 S–S (Fig. 4a) with m/z 2687.1215 confirms the presence of disulfide bond between Cys79 and Cys83. The presence of the second disulfide bridge between Cys57 and Cys211 is corroborated by the peaks T5 S–S and T6 S–S with m/z 1615.6938 and 2542.306, respectively. Therefore, mass spectrometry analysis confirms the presence of two disulfide bridges in refolded HBD-EPO. The absence in mass spectra of peaks corresponding to molecular ions of cysteine-containing peptides with no post-translational modification with iodoacetamide or peptides with modifications such as cysteinyl-S-β-propionamide [30] formed by cysteine reaction with unpolymerized acrylamide monomers indicates that HBD-EPO folded correctly.
Fig. 4. MALDI mass spectra of peptides obtained by tryptic digestion of reduced HBD-EPO (a, b) and diagram showing the coverage of HBD-EPO sequence with tryptic peptides (c). a, b) MALDI mass spectra of tryptic peptides obtained by digestion of the protein band with apparent molecular mass of ~23 kDa (Fig. 2, lane 8) with m/z 970-5000 Th (a) (with two detailed inserts) and m/z 900-1330 Th (b). Before digestion in the gel, the protein was not treated with DTT or iodoacetamide. Dashed lines, disulfide bonds between identified peptides; for other designations, see Fig. 3c.
Activity of purified HBD-EPO was tested in vitro in TF-1 cell line (Fig. 5).
Fig. 5. Dependence of the optical density (measured in the plate well) on the sample protein concentration in the in vitro assay of HBD-EPO (open circles) and Epostim (closed circles, control) activity in TF-1 cells. The data are shown as mean ± standard deviation (n = 3).
EC50 values for the control epoetin beta produced in eukaryotic cells (Epostim; Farmapark, Russia) and HBD-EPO produced in procaryotes were 2.3 and 614 ng/ml, respectively (average from three plates), i.e., the specific activity of HBD-EPO was less than 0.5% of the specific activity of Epostim and ~4% of the specific activity of bacterially expressed EPO obtained in [20]. Such a low activity of HBD-EPO could be explained by the absence of glycosylation and, probably, by the presence of additional positively charged N-terminal domain (Fig. 1) that can destabilize protein globule or hinder its interaction with cell receptors. We introduced the N-terminal HBD to provide protein retention at the site of bone damage. Despite low activity of recombinant HBD-EPO, we still believed that it would produce required effects upon local application, because an increase in the local HBD-EPO concentration will compensate its low activity, while HBD-EPO immobilization on heparin-containing substrate will promote protein stabilization.
To estimate the principal possibility of HBD-EPO application in experiments on bone tissue regeneration, we had to characterize its activity in vivo. We failed to find published data on the bacterial expression and in vivo characterization of EPO variants, so to estimate the in vivo activity of HBD-EPO, we used the test commonly used for EPO expressed in eukaryotic cells.
Biological activity of HBD-EPO was tested in vivo in 8-week-old CBA mice by counting the number of reticulocytes in blood after injection of the tested preparations using Epostim as a control [26]. Both HBD-EPO and Epostim were injected subcutaneously as equimolar mixtures with heparin (see "Materials and Methods"). The activity of HBD-EPO was 174 IU/mg, which is approximately 100 times lower than the activity of Epostim. Such a low in vivo activity of HBD-EPO might be related to its fast elimination from the circulation. To perform pharmacokinetic studies, we had to develop an adequate system for measuring HBD-EPO concentration in a solution.
The concentrations of HBD-EPO and other EPO variants were measured using commercially available sandwich ELISA kit (Vektor-Best-Evropa, Russia). This kit is advertised as a tool for measuring the concentration of native EPO in patient's blood serum. However, when we used this kit for determining Epostim concentration, the obtained values were approximately two times lower that the actual values; while the determined concentrations for bacterially expressed HBD-EPO were approximately an order of magnitude lower than the values determined spectrophotometrically.
The ability of commercially available monoclonal antibodies EP1, EP2, EP3, EP5, EP8, and EP25 to interact with HBD-EPO and other recombinant EPO variants [20] was studied by immunoblotting. Out of six antibody clones tested, only EP8 interacted with all bacterially produced recombinant EPOs and Epostim (data not shown).
We also raised immune sera interacting with all EPO variants, both produced in prokaryotes and eukaryotic cells, by immunization of rabbits with purified 6His-MBP-EPO and s-tag-EPO [20], and then chose the serum with the maximal titer (1 : 2500).
Using EP8 antibody and obtained anti-EPO rabbit serum, we developed sandwich ELISA test system for quantitative assay of HBD-EPO and epoetin beta (Epostin) in a solution. Monoclonal EP8 antibody was adsorbed on the plate at a concentration of 1 µg/ml and used for capturing the antigen, while the rabbit serum was used as detecting agent (dilution of 1 : 2000); goat anti-rabbit immunoglobulins conjugated with horseradish peroxidase (IMTEK, Russia) were used at dilution of 1 : 10,000. The developed test system was able to detect EPO proteins in a concentration range from 1 to 100 ng/ml and was used in further experiments.
The results of pharmacokinetic studies in rats are shown in Fig. 6. HBD-EPO was completely eliminated from the rat circulation within 10 h after its subcutaneous injection, while the concentration of Epostim in the samples of blood plasma decreased negligible within 48 h after injection.
Fig. 6. Dependence of the HBD-EPO (diamonds, dashed line) and Epostim (squares, solid line) concentrations in rat blood plasma on the time elapsed from the subcutaneous administration of the protein preparations in a dose of 100 μg/kg body weight. The data are shown as mean ± standard deviation (n = 3).
HBD-EPO was injected subcutaneously, because this method of administration increases the elimination half-life of the protein. Indeed, according to the published data, the elimination half-life of Epostim after its intravenous injection in patients is 5-6 h, whereas subcutaneous injection increases it to 16-24 h [31] or even more (as in our experiments).
To study the pharmacokinetics of the tested recombinant proteins and to measure their in vivo activity, we used a mixture of HBD-EPO or Epostim (control) with heparin to screen the positive charge of HBD due to its complex formation with heparin and, probably, to slow down protein release into the blood. The complex of heparin with HBD-EPO is non-covalent (experimentally determined Kd of the BMP-2 complex with heparin is ~20 nM [32]). The concentration of HBD-EPO in its equimolar mixture with heparin used in the experiments was 160 μg/ml, which corresponds to 18 μM, i.e., to the value that is three orders of magnitude higher than the supposed Kd (20 nM). This means that in the injected mixture, all protein was in a complex with heparin. However, considering low rate of protein release to the blood and, therefore, its strong dilution, and heparin redistribution between other heparin-binding proteins, dissociation of the complex in the blood should prevail, so that the protein will be easily eliminated from the organism.
The observed pharmacokinetics of nonglycosylated HBD-EPO and glycosylated Epostim almost completely reproduced the data for EPO variants synthesized in bacterial and eukaryotic cells presented in Wang et al. [16]. Both rh-ng-EPO synthesized in E. coli cells, which has been used in study of Wang et al. [16], and HBD-EPO obtained in our work were almost completely eliminated from the circulation within 10 h, while EPO synthesized in Chinese hamster ovary cells (Sunshine Pharmaceutical Company, China) was found in the blood for longer periods of time, although the rate of its elimination was higher than for Epostim. This means that HBD-EPO combination with heparin for subcutaneous injection does not noticeably increase the elimination half-life of HBD-EPO.
It should be noted that both control and experimental groups of animals displayed the symptoms of impaired hemostasis: blood samples withdrawn after the injection of EPO preparations were very viscous and exhibited signs of hemolysis. In the experimental group, the major signs of hemostasis impairment were observed already at the first hours of the experiment, while in the control group, these symptoms were delayed. We did not perform general and biochemical blood assays; nevertheless, the observed hemostasis impairments suggested pronounced side effects upon systemic administration of both EPO preparations in a dose of 100 μg/kg body weight.
The in vivo and in vitro biological activity of HBD-EPO indicates the functional activity of the obtained protein preparation. We assume that the presence of HBD in HBD-EPO will provide its efficacy upon local administration in small doses due to high local concentrations achieved by HBD-EPO immobilization on DBM. Fast elimination of small amounts of nonglycosylated low-molecular-weight protein through its slow release from DBM into the blood could prevent the development of negative responses that would be induced by large doses of the same preparation.
For further experiments, we developed a method for HBD-EPO immobilization on DBM. DBM disks were incubated with varying amounts of HBD-EPO, washed, and lyophilized as described in "Materials and Methods". The amounts of non-bound and bound protein were estimated by ELISA. As a result of these experiments, we determined the DBM capacity for the HBD-EPO and s-tag-BMP-2 (at least, 4 mg/g DBM). By using the developed method and by loading the proteins in amounts below the matrix capacity, it was possible to immobilize any required quantity of HBD-EPO, s-tag-BMP-2, or both proteins at a desired ratio.
The quantity of the immobilized proteins and the ratio between them were estimated electrophoretically. Figure 7 shows the results of fractionation in polyacrylamide gel of HBD-EPO, s-tag-BMP-2, and samples eluted from disks by heating them in the sample buffer (see "Materials and Methods").
Fig. 7. Electrophoresis in 12% polyacrylamide gel of HBD-EPO and s-tag-BMP-2 preparations and proteins eluted from DBM disks: 1) HBD-EPO; 2) s-tag-BMP-2; 3) proteins eluted from DBM disk loaded with 10 μg of HBD-EPO; 4) proteins eluted from DBM disk loaded with 10 μg HBD-EPO and 20 μg BMP-2; M, molecular weight markers.
The disks were loaded with 10 µg HBD-EPO or with 10 μg HBD-EPO and 20 μg s-tag-BMP-2. Proteins eluted from the disks (Fig. 5, lanes 3 and 4) had the same electrophoretic mobility as loaded proteins (Fig. 5, lanes 1 and 2). No protein degradation was observed, and the ratio between the proteins (1 : 2) was kept. Lanes 3 and 4 also contained protein bands with higher molecular masses that corresponded to proteins extracted from DBM by the loading buffer as described in "Materials and Methods".
To verify the possibility of application of DBM disks with immobilized HBD-EPO in experiments on bone regeneration, it was important (i) to elucidate local effects of disk implantation and (ii) to estimate protein release from the disks within the period of observation under in vivo conditions when the disk is washed by bodily fluids.
Two identical DBM disks were implanted subcutaneously in rats into the temporal areas (see "Materials and Methods") on the opposite sides of the head. Rats of the experimental group received disks loaded with 1 μg of HBD-EPO; the control group received disks loaded with 1 μg of Epostim. After 5 days, the disks were removed. One of the two disks with surrounding tissue fragment was used for histochemical analysis; the second disk was used for measuring residual protein. Disks loaded with 1 μg of HBD-EPO were covered with a connective tissue capsule 1.0-1.5 cm in diameter penetrated with a dense network of blood vessels. Capsule exsection was accompanied with strong bleeding. The disks themselves were of intense red color. Disks loaded with 1 μg of Epostim were covered with less pronounced connective tissue capsule 1 cm in diameter and had lighter coloring. Figure 8 (a, e) shows the disks after their removal on day 5 after implantation and incubation in 6 M guanidine hydrochloride (for extraction of residual protein). The amount of residual protein in the disks loaded with 1 μg of HBD-EPO was 0.9 ± 0.06 μg, as determined with our test system. However, we failed to measure the amount of residual protein in disks loaded with 1 μg of Epostim, which might indicate that Epostim did not bind to DBM and was completely eluted from the disk within 5 days. On the contrary, only 10% of HBD-EPO was eluted from the disks within 5 days after subcutaneous implantation, which means that it was eluted gradually. If the same elution rate preserves (although it usually slows down with time), HBD-EPO will be completely eluted from the disk in 45 days. Such prolonged and gradual elution of factors from the implants is essential for the prolongation of their action and maximal efficacy, in particular, in the bone tissue repair. The maximal activity of BMP-2 requires its slow release from the implant at least for two weeks [33].
Fig. 8. DBM disks removed 5 days after subcutaneous implantation and tissue samples from the implantation areas. a) Disk was loaded with HBD-EPO; b-d) tissue samples after subcutaneous implantation of the disk with HBD-EPO. e) Disk was loaded with Epostim; f-h) tissue samples after subcutaneous implantation of the disk with Epostim. (b, c) and (f, g) General view of tissues from the implantation area (magnification, 25×): 1) dermis; 2) muscle layer (striated cutaneous muscle); 3) adventitia (layer of loose connective tissue); thick white arrows, connective tissue borders. d, h) Peripheral area with vessels (4); magnification, 200×; corresponding areas in panels (c) and (g) are shown with yellow rectangles. b, f) Hematoxylin–eosin staining; c, d, g, h) Heidenhain's AZAN staining.
Analysis of histological preparations showed the first stages of connective tissue capsule formation including moderate inflammatory response of the exudative type with the granulation tissue formation. No specific manifestations of cell response to the implant were observed (Fig. 8, b-d, f-h). The general appearance of the preparations was virtually the same in the control and experimental groups: epidermis and its appendages, dermis, hypodermis, and cutaneous muscle were intact; the implant as a fragment of decellularized trabecular bone without signs of its resorption by osteoclasts or perifocal osteogenesis was located in the adventitia; the spaces between the trabeculae contained fibrin and neutrophils. Active inflammation with the formation of immature granulation tissue from fibroblast-like cells and macrophages with moderate number of thin-walled vessels and moderate infiltration of neutrophils including cells with karyorrhexis and karyolysis was observed at the implant periphery. Staining with hematoxylin and eosin (Fig. 8, b and f) showed that preparations from the experimental group were more vascularized that preparations from the control group. Preparations from the experimental group contained areas with well-expressed development of capillary-like thin-walled vessels that showed extravasation of erythrocytes into surrounding connective tissue. To estimate the extent of vascularization, we used Heidenhain's AZAN staining to identify vessels by contrasting (red or yellowish) erythrocytes [34] (Fig. 8, c, d, g and h) for further histomorphometry (estimation of vessel number and area). The average number of vessels in the periphery of samples from the experimental group (251 ± 27) slightly exceeded the average number of vessels in the control group (204 ± 20). The average vessel diameter in the control group was 15 ± 4 vs. 30 ± 6 μm in the experimental group. The portion of capillaries over 30 μm in diameter in the experimental group was 47%, while in the control group, such capillaries were absent. The average capillary area in the control group was 177 ± 6 vs. 707 ± 10 μm2 in the experimental group (i.e., 4 times higher). The difference in the extent of vascularization in the control and experimental samples is clearly seen in Fig. 8, d and g.
Therefore, HBD-EPO immobilized on DBM disks caused much more pronounced vascularization than Epostim. HBD-EPO administration resulted in a slight increase (by 23%) in the number of vessels with significant (4-fold) elevation in the vessel area in the peripheral zone of the implants. This difference can be explained by the fact that immediately after disk implantation, both preparations induced angiogenesis to the same extent. However, Epostim was completely and rapidly eluted from the implantation area, whereas HBD-EPO was retained on DBM due to presence of HBD in its molecule. HBD-EPO release into surrounding tissues was gradual and lasted for an extended period of time, so that HBD-EPO could stimulate growth of blood vessels. The ability of EPO to cause tissue vascularization by directly acting on endothelial cells is one of its most characterized biological functions [35, 36]. It should be noted that in our experiments, HBD-EPO exhibited angiogenic properties when administered at a sufficiently low dose (1 μg). When used at the same dose, Epostim, which has a significantly higher in vivo activity upon subcutaneous injection but does not bind to DBM, induced local vascularization to a considerably lesser extent than HBD-EPO.
The efficiency of HBD-EPO binding to DBM is similar to that of s-tag-BMP-2 that displays high osteoinductive activity in animal models when immobilized on DBM [22, 37-41]. Besides its action on cell cultures, HBD-EPO induces erythropoiesis and local angiogenesis in vivo. It should be noted that the HBD-EPO in vitro activity (measured in the proliferation tests in human TF-1 erythroleukemia cells) and in vivo activity (estimated from the increase in the reticulocyte count in the blood) observed after HBD-EPO subcutaneous injection were two orders of magnitude lower than for the commercial preparation Epostim. This difference could result from the presence of heparin-binding domain that destabilizes the protein and decreases its ability to bind to the EPO receptor (EPOR). The reduced in vivo activity might be caused by rapid elimination of HBD-EPO from the circulation, which in turn, might result from the lower (~1.5 times) molecular mass of the protein due to the absence of its glycosylation (as compared to Epostim). On the contrary, local angiogenic activity observed after subcutaneous implantation of DBM with immobilized tested proteins was higher for HBD-EPO, which might be related to the retention of HBD-EPO (and not Epostim). This pronounced local angiogenic activity of HBD-EPO gives hope for successful use of this preparation in the development of new highly efficient materials for local application with the purpose of bone regeneration. In our further studies, we plan to characterize osteogenic properties of HBD-EPO and to develop new variants of recombinant EPO with improved in vitro and in vivo activities using the methods of protein engineering by creating different combination of protein domains and replacing domain–domain linkers with more rigid sequences that would reduce negative influence of these domains at each other in the resulting multidomain proteins [42].
This work was supported by the Russian Science Foundation (project 16-15-00133).
A. S. Karyagina, A. V. Gromov, and V. G. Lunin proposed research plan and conducted general research management, T. M. Grunina, Z. M. Galushkina, D. A. Tretyak, and I. S. Boksha were engaged in the preparation and purification of proteins, M. S. Poponova and A. V. Demidenko obtained antibodies and performed immunological testing, P. A. Orlova and V. N. Manskikh performed histological studies, N. V. Strukova and M. S. Manukhina worked with laboratory animals, K. E. Nikitin obtained DBM, A. M. Lyaschuk cloned genes, S. A. Cherepushkin tested protein preparations in vitro, N. B. Polyakov, A. I. Solovyev, and V. G. Zhukhovitsky performed mass-spectrometry, A. S. Karyagina wrote this article.
We thank D. A. Grumov and A. A. Pereborova for help with mass-spectrometry analysis and discussion of the obtained results, N. A. Lavrova for performing immunoblotting, and L. A. Soboleva for growing bacterial biomass for protein purification.
Authors declare no conflict of interests neither in financial nor in any other area.
All procedures with animals were conducted in accordance with the ethical standards of the Gamaleya National Research Center of Epidemiology and Microbiology (Rules for Using Animals in Experiments) and legal requirements adopted by the Russian Federation and international organizations (EU Directive 2010/63/EU and Appendix A of Convention ETS 123).
1.Krantz, S. B. (1991) Erythropoietin, Blood, 77, 419-434.
2.Shiozawa, Y., Jung, Y., Ziegler, A. M., Pedersen, E. A., Wang, J., Wang, Z., Song, J., Wang, J., Lee, C. H., Sud, S., Pienta, K. J., Krebsbach, P. H., and Taichman, R. S. (2010) Erythropoietin couples hematopoiesis with bone formation, PLoS One, 5, e10853.
3.Wu, C., Giaccia, A. J., and Rankin, E. B. (2014) Osteoblasts: a novel source of erythropoietin, Curr. Osteoporos. Rep., 4, 428-432.
4.Li, C., Shi, C., Kim, J., Chen, Y., Ni, S., Jiang, L., Zheng, C., Li, D., Hou, J., Taichman, R. S., and Sun, H. (2015) Erythropoietin promotes bone formation through EphrinB2/EphB4 signaling, J. Dent. Res., 94, 455-463.
5.Holstein, J. H., Menger, M. D., Scheuer, C., Meier, C., Culemann, U., Wirbel, R. J., Garcia, P., and Pohlemann, T. (2007) Erythropoietin (EPO): EPO-receptor signaling improves early endochondral ossification and mechanical strength in fracture healing, Life Sci., 80, 893-900.
6.Holstein, J. H., Orth, M., Scheuer, C., Tami, A., Becker, S. C., Garcia, P., Histing, T., Morsdorf, P., Klein, M., Pohlemann, T., and Menger, M. D. (2011) Erythropoietin stimulates bone formation, cell proliferation, and angiogenesis in a femoral segmental defect model in mice, Bone, 49, 1037-1045.
7.Garcia, P., Speidel, V., Scheuer, C., Laschke, M. W., Holstein, J. H., Histing, T., Pohlemann, T., and Menger, M. D. (2011) Low dose erythropoietin stimulates bone healing in mice, J. Orthop. Res., 29, 165-172.
8.Rolfing, J. H. D., Bendtsen, M., Jensen, J., Stiehler, M., Foldager, C. B., Hellfritzsch, M. B., and Bunger, C. (2012) Erythropoietin augments bone formation in a rabbit posterolateral spinal fusion model, J. Orthop. Res., 30, 1083-1088.
9.Sun, H., Jung, Y., Shiozawa, Y., Taichman, R. S., and Krebsbach, P. H. (2012) Erythropoietin modulates the structure of bone morphogenetic protein 2-engineered cranial bone, Tissue Eng. Part A, 18, 2095-20105.
10.Rolfing, J. H., Jensen, J., Jensen, J. N., Greve, A. S., Lysdahl, H., Chen, M., Rejnmark, L., and Bunger, C. (2014) A single topical dose of erythropoietin applied on a collagen carrier enhances calvarial bone healing in pigs, Acta Orthop., 85, 201-209.
11.Patel, J. J., Modes, J. E., Flanagan, C. L., and Krebsbach, P. H. (2015) Dual delivery of EPO and BMP2 from a novel modular poly-ε-caprolactone construct to increase the bone formation in prefabricated bone flaps, Tissue Eng. Part C Methods, 21, 889-897.
12.Omlor, G. W., Kleinschmidt, K., Gantz, S., Speicher, A., Guehring, T., and Richter, W. (2016) Increased bone formation in a rabbit long-bone defect model after single local and single systemic application of erythropoietin, Acta Orthop., 87, 425-431.
13.Sinyukhin, V. N., Stetsyuk, E. A., and Lovchinsky, E. V. (1994) Pharmakinetics of human recombinant erythropoietin, Terapevt. Arkhiv, 66, 60-62.
14.Egrie, J. C., Strickland, T. W., Lane, J., Aoki, K., Cohen, A. M., Smalling, R., Trail, G., Lin, F. K., Browne, J. K., and Hines, D. K. (1986) Characterization and biological effects of recombinant human erythropoietin, Immunobiology, 172, 213-224.
15.Egrie, J. C., Dwyer, E., Browne, J. K., Hitz, A., and Lykos, M. A. (2003) Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin, Exp. Hematol., 31, 290-299.
16.Wang, Y. J., Liu, Y. D., Chen, J., Hao, S. J., Hu, T., Ma, G. H., and Su, Z. G. (2010) Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. coli, Int. J. Pharm., 386, 156-164.
17.Jeong, T. H., Son, Y. J., Ryu, H. B., Koo, B. K., Jeong, S. M., Hoang, P., Do, B. H., Song, J. A., Chong, S. H., Robinson, R. C., and Choe, H. (2014) Soluble expression and partial purification of recombinant human erythropoietin from E. coli, Protein Expr. Purif., 95, 211-218.
18.Boissel, J. P., Lee, W. R., Presnell, S. R., Cohen, F. E., and Bunn, H. F. (1993) Erythropoietin structure–function relationships. Mutant proteins that test a model of tertiary structure, J. Biol. Chem., 268, 5983-5993.
19.Narhi, L. O., Arakawa, T., Aoki, K., Wen, J., Elliott, S., Boone, T., and Cheetham, J. (2001) Asn to Lys mutations at three sites which are N-glycosylated in the mammalian protein decrease the aggregation of Escherichia coli-derived erythropoietin, Protein Eng., 14, 135-140.
20.Grunina, T. M., Demidenko, A. V., Lyaschuk, A. M., Poponova, M. S., Galushkina, Z. M., Soboleva, L. A., Cherepushkin, S. A., Polyakov, N. B., Grumov, D. A., Solovyev, A. I., Zhukhovitsky, V. G., Boksha, I. S., Subbotina, M. E., Gromov, A. V., Lunin, V. G., and Karyagina, A. S. (2017) Recombinant human erythropoietin with additional processable protein domains: purification of protein synthesized in Escherichia coli heterologous expression system, Biochemistry (Moscow), 82, 1285-1294.
21.Karyagina, A. S., Boksha, I. S., Grunina, T. M., Demidenko, A. V., Poponova, M. S., Sergienko, O. V., Lyaschuk, A. M., Galushkina, Z. M., Soboleva, L. A., Osidak, E. O., Semikhin, A. S., Gromov, A. V., and Lunin, V. G. (2016) Optimization of rhBMP-2 active-form production in a heterologous expression system using microbiological and molecular genetic approaches, Mol. Genet. Microbiol. Virol., 31, 208-213.
22.Bartov, M. S., Gromov, A. V., Poponova, M. S., Savina, D. M., Nikitin, K. E., Grunina, T. M., Manskikh, V. N., Gra, O. A., Lunin, V. G., Karyagina, A. S., and Gintsburg, A. L. (2016) Modern approaches to research of new osteogenic biomaterials on the model of regeneration of cranial critical-sized defects in rats, Bull. Exp. Biol. Med., 162, 273-276.
23.Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., and Mann, M. (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes, Nat. Protoc., 1, 2856-2860.
24.Sharapova, N. E., Kotnova, A. P., Galushkina, Z. M., Lavrova, N. V., Poletaeva, N. N., Tukhvatulin, A. E., Semikhin, A. S., Gromov, A. V., Soboleva, L. A., Ershova, A. S., Zaitsev, V. V., Sergeenko, O. V., Lunin, V. G., and Karyagina, A. S. (2010) Production of the recombinant human bone morphogenetic protein-2 in Escherichia coli and testing of its biological activity in vitro and in vivo, Mol. Biol. (Moscow), 44, 1036-1044.
25.Karyagina, A. S., Boksha, I. S., Grunina, T. M., Demidenko, A. V., Poponova, M. S., Sergienko, O. V., Lyashchuk, A. M., Galushkina, Z. M., Soboleva, L. A., Osidak, E. O., Bartov, M. S., Gromov, A. V., and Lunin, V. G. (2017) Two variants of recombinant human bone morphogenetic protein 2 (rhBMP-2) with additional protein domains: synthesis in an Escherichia coli heterologous expression system, Biochemistry (Moscow), 82, 613-624.
26.Ramos, A. S., Schmidt, C. A., Andrade, S. S., Fronza, M., Rafferty, B., and Dalmora, S. L. (2003) Biological evaluation of recombinant human erythropoietin in pharmaceutical products, Braz. J. Med. Biol. Res., 36, 1561-1569.
27.Gromov, A. V., Nikitin, K. E., Karpova, T. A., Zaitsev, V. V., Sidirova, E. I., Andreeva, E. V., Bartov, M. S., Mishina, D. M., Subbotina, M. E., Shevelyagina, N. V., Sergienkov, M. A., Soboleva, L. A., Kotnova, A. P., Sharapova, N. E., Semikhin, A. S., Didenko, L. V., Karyagina, A. S., and Lunin, V. G. (2012) Development of a procedure for obtaining osteoplastic material based on demineralized bone matrix with the maximal content of native factor of bone tissue growth, Biotekhnologiya, 5, 66-75.
28.Osidak, E. O., Osidak, M. S., Sivogrivov, D. E., Portnaya, T. S., Grunina, T. M., Galushkina, Z. M., Lunin, V. G., Karyagina, A. S., and Domogatskii, S. P. (2014) Kinetics of BMP-2 release from collagen carrier: evaluation by enzyme immunoassay in the presence of plasma proteins, Bull. Exp. Biol. Med., 158, 104-108.
29.Heidenhain, M. (1905) Zeitschrift fur wissenschaftliche mikroskopie und fur mikroskopische technik, S. Hirzel-Leipzig, 22, 339.
30.Xing, G., Zhang, J., Chen, Y., and Zhao, Y. (2008) Identification of four novel types of in vitro protein modifications, J. Proteome Res., 7, 4603-4608.
31.Biryukova, L. S., Ushakova, A. I., Kamshilova, N. I., Ose, I. V., Akimov, A. V., Skobeleva, T. F., and Badmaev, A. L. (2011) Estimation of safety and therapeutic efficacy of new preparation stimulating erythropoiesis (epoetin β) in patients subjected to programmed hemodialysis, Nefrol. Dializ, 13, 128-132.
32.Ruppert, R., Hoffmann, E., and Sebald, W. (1996) Human bone morphogenetic protein 2 contains a heparin-binding site, which modifies its biological activity, Eur. J. Biochem., 237, 295-302.
33.Vo, T. N., Kasper, F. K., and Mikos, A. G. (2012) Strategies for controlled delivery of growth factors and cells for bone regeneration, Adv. Drug Deliv. Rev., 64, 1292-1309.
34.Zwingenberger, S., Langanke, R., Vater, C., Lee, G., Niederlohmann, E., Sensenschmidt, M., Jacobi, A., Bernhardt, R., Muders, M., Rammelt, S., Knaack, S., Gelinsky, M., Gunther, K. P., Goodman, S. B., and Stiehler, M. (2016) The effect of SDF-1α on low dose BMP-2 mediated bone regeneration by release from heparinized mineralized collagen type I matrix scaffolds in a murine critical size bone defect model, J. Biomed. Mater. Res. A, 104, 2126-2134.
35.Ribatti, D., Presta, M., Vacca, A., Ria, R., Giuliani, R., Dell'Era, P., Nico, B., Roncali, L., and Dammacco, F. (1999) Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo, Blood, 93, 2627-2636.
36.Kimakova, P., Solar, P., Solarova, Z., Komel, R., and Debeljak, N. (2017) Erythropoietin and its angiogenic activity, Int. J. Mol. Sci., 18, E1519.
37.Gaifullin, N. M., Karyagina, A. S., Gromov, A. V., Terpilovsky, A. A., Malanin, D. A., Demeshchenko, M. V., and Novochadov, V. V. (2016) Morphological properties of osteointegration upon using titanium implants with bioactive coating and recombinant bone morphogenetic protein rhBMP-2, Morfologiya, 149, 77-84.
38.Andreev, A. Yu., Zakharov, V. D., Zaratyants, O. V., Borzenok, S. A., Khubenova, M. Kh., Osidak, E. O., Krasheninnikov, S. V., Karyagina, A. S., and Domogatsky, S. P. (2016) Prospects in the administration of bone tissue growth factor in the content of collagen matrix for cornea strengthening (experimental study), Sovr. Tekhnol. Oftalmol., 4, 11-16.
39.Zakharov, V. D., Andreev, A. Yu., Zaratyants, O. V., Osidak, E. O., Borzenok, S. A., Krasheninnikov, S. V., Karyagina, A. S., and Domogatsky, S. P. (2016) Morphological changes in the rabbit cornea under the action of the bone and cartilage tissue growth factor rhBMP-2 within the intracorneal collagen implant, Klin. Eksp. Morfol., 4, 36-42.
40.Zakharov, V. D., Zaratyants, O. V., Andreev, A. Yu., Osidak, E. O., Borzenok, S. A., Krasheninnikov, S. V., Karyagina, A. S., and Domogatsky, S. P. (2016) Effect of the growth factor rhBMP-2 in the content of collagen matrix on morphological and biomechanical properties of retina, Oftalmokhirurgiya, 4, 20-28.
41.Bartov, M. S., Gromov, A. V., Manskih, V. N., Makarova, E. B., Rubshtein, A. P., Poponova, M. S., Savina, D. M., Savin, K. S., Nikitin, K. E., Grunina, T. M., Boksha, I. S., Orlova, P. A., Krivozubov, M. S., Subbotina, M. E., Lunin, V. G., Karyagina, A. S., and Gintsburg, A. L. (2017) Recombinant human Bone Morphogenetic Protein-2 (rhBMP-2) with additional protein domain synthesized in E. coli: in vivo osteoinductivity in experimental models on small and large laboratory animals, Bull. Exp. Biol. Med., 164, 148-151.
42.Chen, X., Zaro, J. L., and Shen, W. C. (2013) Fusion protein linkers: property, design and functionality, Adv. Drug Deliv. Rev., 65, 1357-1369.
Supplementary Tables S1 and S2 (PDF)


text:
Kremlin stays mum on comments by Blair-era MI6 chief 'regretting helping Putin win power'
The Kremlin spokesman refrains from commenting on an interview Richard Dearlove, the former head of the MI6 had with The Times
© Mikhail Metzel/TASS
MOCSOW, October 1. /TASS/. Kremlin Spokesman Dmitry Peskov has chosen to refrain from commenting on an interview Richard Dearlove, the former head of the MI6 had with The Times. In that discussion, the ex-MI6 boss regretted his agency's assistance in organizing then Prime Minister Tony Blair's attendance to a social event with Vladimir Putin in the run-up to the Russian presidential elections back in 2000, thus helping him come to power.
Putin hopes US authorities will one day realize futility of sanctions against Russia
The Times' Monday issue published an interview with Richard Dearlove, then chief of the Secret Intelligence Service in 1999-2004, who said he regretted that he had helped organize Tony Blair's visit to Russia's St. Petersburg where he attended a premier at the Mariinsky Theatre alongside Vladimir Putin just a few weeks before the presidential polls in Russia. According to Dearlove, Blair's visit helped Putin win power, and improve relations between the two countries, which however soon soured again.
"Frankly speaking, we never knew that it is the MI6 that issues permits for prime ministers to visit any social event. This is news for us," Peskov said on Monday.
"Otherwise, I don't think it is worth any comments," he added.

redpajama_set_name: RedPajamaCommonCrawl


King Salman ordered that Saudi citizens held imprisoned on financial – rather than criminal – cases be freed, provided that their debts do not exceed SAR 1 million ($266,655) and whose insolvency can be proven.
Saudi Arabia's King Salman has launched a $22 billion project in the Riyadh to improve infrastructure, amenities and education in the region, it was announced on Wednesday.
The 1,281 development projects include 15 housing projects in the Riyadh region designed to increase home ownership among Saudi citizens, as well as the world's largest Islamic museum, sports cities, seven medical cities and 16 educational projects.
Additionally, the Saudi government will create a 315,000 square metre environmental area spanning the lakes to the south of Riyadh, as well as airport expansions and the development and upgrading of the area's road network.
The plan also calls for the development of an ambulance centre, a modernisation project for the Riyadh TV renovation project, and the establishment of a specialist mental health hospital.
The projects were announced by Riyadh governor Prince Faisal bin Bandar during the an event at Qasr Al-Hukm.
During the event, he noted that the projects form part of a wider programme of 2,830 projects collectively worth over $90 billion.
At the same event, King Salman ordered that Saudi citizens held imprisoned on financial – rather than criminal – cases be freed, provided that their debts do not exceed SAR 1 million ($266,655) and whose insolvency can be proven.
King Salman previously served as governor of Riyadh for nearly 50 years.


English carmaker Jaguar Land Rover today reported a model hostile for India, which will see the brand dispatch no less than 10 models in 2018-19 (April-March) with an expect to catch a bigger bit of the extravagance vehicle deals pie.
While the organization has not uncovered the model arrangement for the year, we can let you know no less than four models will be from JLR's SVO (Special Vehicle Operations) division.
The invigorated Range Rover Sport SVR and the refreshed Range Rover SVAutobiography will lead the SVO show line-up as they are set to be presented in the primary quarter (April-June) of 2018-19.
The as of late refreshed Range Rover Sport SVR accompanies a 5.0-liter supercharged V8 that now delivers 575hp at 6,000rpm, and 700Nm of torque from 3,500rpm – changes of 24hp and 19Nm, separately.
The SVAutobiography is RR's sumptuous long-wheelbase SUV that was divulged at the LA Motor show a year ago. The SUV is based on Range Rover's more drawn out 3,120mm wheelbase to furnish raise travellers with more legroom (around 1,200mm) and seats that can lean back by 40 degrees. All around, there are three power trains on offer – a 339hp, 4.4-liter SDV8 diesel, a 404hp, 2.0-liter oil electric and a 565hp, supercharged 5.0-liter V8 (like the SVR).
JLR will dispatch the quickest street going Jaguar, the F-Type SVR, in India in the second quarter (July-Sep) of 2018-19. The model packs in a decent blend of execution and solace, as we as of late discovered in our drive. It is fuelled by a 5.0-liter supercharged V8 acquired from the Project 7, making 575hp and 700Nm of pulling power. The model is outfitted with an eight-speed auto gearbox, a four-wheel-drive framework and has high utilization of carbon fibre in its body shell.
Going to the fourth SVO display, JLR is required to bring the as of late revealed new Range Rover SV Coupe to India in the second from last quarter (October-December) of 2018-19. The SUV, which made its open presentation at the 2018 Geneva engine appear, is Land Rover's most costly model situated at the highest point of the range, enabling the organization to better contend with the Lamborghini Urus and Bentley Bentayga. Restricted to only 999 units, the model is fueled by a 575hp, 5.0-liter supercharged V8 petroleum.
In a current collaboration with Autocar India, Mark Stanton, executive, SVO division, stated, aside from offering elevated amounts of execution, the SVO models are outlined remembering regular utilization, rather than being simply track-centred.
Aside from the four models, JLR is additionally anticipated that would present facelifts of the standard Range Rover and Range Rover Sport, both of which got a mid-cycle invigorate a year ago. While the previous gets a revived grille with a gleam dark encompass in a front guard, the last gets new LED headlamps and unpretentious changes to the grille.
The carmaker is additionally taking a gander at its most up to date module half and half in its line-up, the Range Rover PHEV, yet the model is probably not going to come to India until the point that JLR is certain of the strategy for crossovers. The present strategy intensely charges mixtures, which could value RR's half breed relatively out of dispute.


Grace Insurance is a team of passionate insurance brokers with a background in being brokers and underwriters. We are able to assist and direct you on what cover would be more suitable for your unique risk.
Utilising cutting edge technology we ensure we provide our personalised service and the right product for you as our valued client.
We are located in Perth in WA, but we service the whole of Australia.
Nothing gives us greater pleasure than seeing you and your business grow along side ours. After a few years we do not have clients, we have loyal friends in business.
we have a range of options available online for you to choose from.
Choice of $5 million, $10 million and $20 million.
When you call Grace Insurance you will speak to an insurance professional not a robot or call centre.
Professional Indemnity Insurance – What is it? Do you need it?
Cyber Insurance – What is it and why do you need it?


The Fibox range of standard enclosures provides the designer with over 800 choices for packaging electrical and electronics equipment operating in hostile environments. Fibox enclosures comply with EN 60529 (IEC 529) and UL 50 and UL 508. Our enclosures have achieved ingress ratings of IP 67 and NEMA 4X and 6, 6P. Enclosure performance is verified by independent laboratory testing and on-going monitoring of production. With over 700 standard accessories, Fibox enclosures are easy to use and flexible. Standard solutions exist for panel mounting, hinging inner doors, wire and management and air ventilation.
3S Outdoor use to provide a degree of protection against windblown dust, rain and sleet; external mechanisms remain operable while ice laden.
Indoor or outdoor use to provide a degree of protection against the entry of water during temporary submersion at a limited depth; undamaged by the formation of ice on the enclosure.
6P Indoor and outdoor use to provide a degree of protection against the entry of water during prolonged submersion at a limited depth.
Indoor use to provide by oil immersion a degree of protection of the enclosed equipment against the corrosive effects of corrosive liquids and gases.


text:
Amphan may intensify into super cyclone in next 12 hours, says IMD
It is likely to make landfall in Digha, West Bengal, by May 20
May 18, 2020 11:07 am | Updated 11:51 am IST - NEW DELHI
Warning signal number two has been hoisted in Pamban port in Tamil Nadu on Sunday as cyclonic storm Amphan lay centred in Bay of Bengal.
Amphan , now raging in the Bay of Bengal, is likely to intensify into a super cyclone — the severest category — in the next 12 hours, according to an update from the India Meteorological Department (IMD) on Monday.
Also read | How lessons learnt from 1999 supercyclone is helping Odisha even today
Until Sunday evening, the IMD had said it was likely to be a less strong 'extreme cyclonic storm.' Amphan is expected to generate wind speeds up to 230 kmph, gusting to 265 kmph and make landfall in Digha, West Bengal, by May 20 and veer towards Bangladesh.
Last year, an extremely severe cyclone Phailin had ravaged Odisha. Amphan, according to its present track is expected to be strongest in the ocean and weaken as it approaches land
Orissa / cyclones


FAIRFAX, VA – (October 2, 2018) – mLINQS, LLC today announced that Stephen E. Kovalenko has joined the company as its Director of Business Development. In this position, he is now responsible for account management and customer expansion of the company's industry-leading system to manage federal agency employee relocation worldwide. Mr. Kovalenko previously worked for 22 years with FranklinCovey Company as its Enterprise Client Partner for the Departments of the Navy, Air Force, and Homeland Security. "Steve Kovalenko has an unprecedented record of success," said mLINQS President Greg McIntyre. "His consistency for effectively serving the needs of federal agencies and their employees will prove invaluable to the growth of mLINQS. Steve is a most welcome new member of our team." "My intent is to bring an even higher degree of enthusiasm for business development and customer engagement to mLINQS," said Steve Kovalenko. "I firmly believe we can help government agencies to more cost-effectively streamline mission-critical staff relocations, and ease any burden on employees at the same time." In his previous position, Mr. Kovalenko received more than 19 awards for his professionalism, and was a charter member of the FranklinCovey elite $20 million club. He holds a Bachelor of Arts degree in American Studies from Brigham Young University, and also has earned an MBA in International Management from Thunderbird, The American Graduate School of International Management. Mr. Kovalenko's new responsibilities with mLINQS include working closely with the existing client base, while supporting development of new accounts. In addition, he will be deeply involved with evaluating client recommendations for system improvements. This system, moveLINQ, is currently used to support relocation expense management for the employees of over 40 federal agencies, including 11 of the 15 federal executive departments. Further information about mLINQS ® product, services, and management is available by calling 703.425.1122, or visiting www.mlinqs.net.
mLINQS ® , headquartered in Fairfax, Virginia, is a Service-Disabled Veteran-Owned Small Business (SDVOSB) founded in 2003. In 2004, the company released moveLINQ ® , the first and only relocation expense management system designed to meet the unique needs of federal agencies, including FedRAMP and DISA security accreditation. mLINQS product and consulting services, are described in detail on the company website, www.mlinqs.net, and are available on GSA Schedule, including TDRS, TSS, and PSS.


Cycling bottle with nice, secure handle even when you're giving it your all. Ideal for all the tougher challenges and rigours of sport and exercise, thanks to its robust design. Very popular among those who really want to keep in shape.
Qty/pkg: 161 per bag. We also offer other packaging solutions.


text:
Quebec Police Set Up Special Task Force to Deal With Latest Mob War
Experts say power struggles within the Sicilian Mafia and against rival factions like the Calabrian Ndrangheta are likely to end with more violence and police can do little to stop it.
by Nick Rose
May 16 2019, 3:42pm
Scene of the Sheraton hotel shooting in Laval | Image via CP.
Police in Montreal's largest suburb are working with provincial and federal law enforcement in an aggressive crackdown on organized crime following a number of high-profile murders.
On Monday, Laval police chief Pierre Brochet announced the creation of Project Repercussion and a specialized task force made up of agents from Laval's Service de police (SPL), Sûreté du Québec (SQ), and the RCMP, to work together in the wake of a recent rash of very public shootings. However, organized crime experts told VICE that this new task force will do little to get to the root of the problem.
The project's name, as well of the police chief's tone, left little doubt as to the ultimate goal of the operation.
"We will not hesitate to double down on our efforts and maintain the security of our citizens at any cost," Brochet said in a statement, adding, "The fact that an individual opened fire inside of a hotel with total disregard for repercussions. It's unacceptable!"
Brochet was referring to the death of 49-year-old Salvatore Scoppa, who was shot multiple times in the lobby of a Sheraton Hotel in Laval on May 4, in close proximity to children and family members.
Scoppa, who had well-documented ties with organized crime in Montreal, was pronounced dead on arrival at hospital. The brazenness of the attack prompted the creation of the new task force that police say will be "omnipresent in businesses frequented by individuals linked to organized crime, notably bars, restaurants, and other licensed establishments."
Police also promised more officers "on the ground," an increase in "interventions to suppress any act of violence (possession of weapons, intimidation, etc.)," and information gathering relative to organized crime.
The announcement was made after a string of shootings that took place in Montreal's suburbs.
On May 10, less than a week after Scoppa's death, an unidentified 25-year-old man was killed after being gunned down at a restaurant in Brossard, on Montreal's South Shore. Police found a gun and a burning car nearby shortly thereafter.
On May 12, Éric Chabot, a 42-year-old private investigator and father of five with no criminal record was gunned down near his home in Terrebonne, just north of Laval. While Terrebonne police suspected a link with organized crime and handed the investigation over to provincial investigators, no such connection has been established as of yet.
Coincidentally, Terrebonne is also where Salvatorre Scoppa survived an earlier attempt on his life after being shot outside of a restaurant in 2017. Scoppa's fate was also a topic of conversation between Leonardo Rizzuto (son of late Montreal mob boss Vito Rizzutto), Stefano Sollecito (Vito's rumoured interim successor), and Montreal street gang leader Gregory Wooley in 2015, obtained during the SQ's massive Magot-Mastiff operation, which deployed more than 200 officers and led to the arrests of 43 individuals suspected of ties with organized crime.
These conversations were surreptitiously recorded by police inside the office of Loris Cavaliere (a Montreal lawyer who later pleaded guilty to gangsterism in 2017) and revealed Rizzuto, Sollcito, and Wooley discussing a plot to kill Scoppa, who apparently posed a threat to drug trafficking operations, before being dissuaded by Leonardo Rizzutto.
Salvatore Scoppa was the brother of equally notorious Andrew Scoppa, once named "the head of a Mafia clan in Montreal" by an SQ officer during a bail hearing in 2017.
For John Westlake, a retired 34-year veteran of both Montreal and RCMP organized crime units, who knew both Scoppa brothers personally, the creation of the Repercussion task force is of no surprise and little consolation.
"Now they're calling for a 'War against the Mafia'!" Westlake told VICE. "Did you read that in the papers? Give me a break! They've been doing a war on the Mafia for a hundred fucking years—nobody wins. If you look at the history of the Mafia, they all take care of their own and they kill who they don't want in the picture, and that's how it goes. But the hotel thing could have been a lot more serious, with all those kids running around."
Westlake said that Salvatore Scoppa's murder was so extreme that it forced police to react aggressively, but that Repercussion will probably have a more soothing effect on public opinion than on controlling mob violence.
"The last time something serious like this happened was with the Hells, when the kid got blown up near the car in the street," Westlake said, referring to the death of 11-year-old Daniel Desrochers during the early years of the Quebec biker war. "And then the government wakes up and says, 'OK, let's put a bit of heat.' [...] But the killing still goes on."
Antonio Nicaso agrees. Nicaso is an expert on organized crime who has written extensively about Montreal's organized crime factions, as depicted in his book Business or Blood: Mafia Boss Vito Rizzuto's Last War, which was later adapted into the TV show Bad Blood.
"There is only a reaction when we see atrocity and when we see an increase in violence," Nicaso said. "Now, because Scoppa was killed in front of many people, like in a Western movie, it forces a kind of reaction.
"After 9/11, they moved all of the resources and money away from organized crime against terrorists in the name of national security. That is the main issue. We lost track with intelligence. Building and rebuilding task forces doesn't make any sense to me."
While neither Westlake or Nicaso would speculate as to who could be behind the murder, they both said that his death marks a shift in the delicate balance of power of organized crime in Quebec, which has been marred by internal violence within the Sicilian Mafia as well as the resurgence of the rival Ontario-based Ndragheta.
"He was part of a power struggle," Nicaso said, recalling the chaos that followed Vito Rizzuto's imprisonment and death. "Scoppa was one of the most important players in this conflict and this is another indication that there will be more violence until they settle and fill the void left by [Vito] Rizzuto. We should expect nothing good. There will be more murders. There will be more violence."
In fact, for Nicaso, the very nature of Scoppa's murder is a testament to the volatility and brutal absurdity of Quebec's seemingly interminable mob war.
"It shows that they have no respect for the victims or the victims' families," he said. "Killing Scoppa in front of many, many people is an indication that there is an increase in violence and that there are no rules. There is nothing. There is only violence to eliminate violence."
Scoppa's funeral, which was scheduled to take place in Montreal's Little Italy on Monday was cancelled, with police citing possible "dangers." No arrests have been made in connection to any of the three shootings that preceded the announcement of Project Repercussion.
Follow Nick Rose on Twitter.
Sicilian Mafia
Tony Magi
pierre brochet
project repercussion
calabrian ndrangheta


Hi there, Are you looking for Free Twitch Accounts of 2019? Video Game Lovers! Gaming is ruling the Internet these days. YouTube, Instagram, and Facebook, everywhere Gaming Clips, Videos, and Memes are trending. YouTube Streaming service is on fire these days. Even Facebook has also launched its own Streaming Service sometime before. So in this article, Geeks10 is going to explain step by step methods to access Prime Twitch Accounts easily.
Twitch Prime Accounts: But Twitch has been thrashing the internet in Gaming Industry ever since Gaming got its hype. Twitch is the home of Gaming and the Gamers. The world's biggest platform for live Video Game's streaming and broadcasts is Twitch. Nearly 140 million unique monthly users visit this amazing website and spend hours of their lives watching live gaming streams with a platform who has 2-million-plus creators that stream at least 8 hours per day, one of our favorites PUBG, Apex Legends, Fortnite, Warcraft and Dota 2.
Anyone in this world can become a Twitch Streamer. Gaming Consoles like PS4 and Xbox One, already have built-in functions to stream through Twitch but before Streaming, you must have an account on Twitch, so here is the guide to create an account on Twitch Account.
1 How to create an account on Twitch?
How to create an account on Twitch?
Follow these instructions as per written.
Go to the Twitch Website.
At the top of the page, click on the button "Sign Up".
Enter your desired username, password, your birthdate, and your e-mail address, where you can get your account confirmation mail.
Click on the "Sign Up" button to create an account. You will be asked to visit your mailbox and do verify your account.
If you are a PC Gamer or if you want to stream games and grow your own Twitch Channel from other kinds of console boxes such as the Nintendo Switch, all you need, is good and decent kind of hardware, streaming software like OBS Studio, and just create an account on Twitch.
Install a broadcasting software on your PC. A common solution for OBS Studio, which is widely used and it is available on Windows, Mac, and Linux, and there is one more named as, XSplit, and is built for Windows. OBS is a free and open source but requires to be set up a bit complicated but it is one of the best, while XSplit is highly intuitive but requires a paid subscription for some of its better features.
Log in to your account on Twitch.tv.
Select the option Dashboard from the drop-down menu at the top-right corner of the screen.
Search for Game and then select it, whatever you want to play under the Playing tab.
Enter a title for your Stream.
Click on OBS Studio's icon and select Run as Administrator (It is necessary for Screen/Game Capture).
Go to Settings menu and Select Broadcast Settings.
Select Twitch as your Streaming Service and click Optimize on the bottom-left of the menu.
Go back to your Twitch dashboard and select Stream Key.
Copy and Paste that code into the Stream Key box in the Broadcast Settings menu. Select OK.
On the main OBS screen, right-click on the Sources box and select Add > Game Capture.
Right-click the Sources box again to add any additional feeds. You can add images, stickers and text to customize your layout, use Monitor Capture if you want to show anything on your display or select Video Capture to use your webcam.
Select Preview Stream and Edit Scene to test your stream layout and make changes as your desires. For example, you might want to add any kind of stickers or clips to your gameplay, you might like to add a small box with live streaming of yourself using a webcam.
Click on Start Streaming on the OBS main screen. Now you are streaming live and gonna rock the Twitch World!!
If you are a PS4 or Xbox One Gamer, you can directly broadcast from your console box without worrying about any external hardware or software. To stream on Twitch through PS4, just press the DualShock 4's Share button, select Broadcast Gameplay and choose Twitch as the streaming service. Once you logged in, you can go live with the just a push of a button.
Streaming to Twitch on Xbox One is just simple as it was for PS4. First, download the free Twitch app from the Xbox Store before you start. Once you have the app downloaded and have logged in, just fire up whichever game you would like to play and stream. Then, open up the Twitch and select the Broadcast option from the main menu. Just remember that you can also stream on Microsoft's Mixer platform directly from your Xbox One by just pressing the home button and selecting the Broadcast icon.
Both of these consoles will let you chat with your viewers from your headset, as well as it will show you your face via the PlayStation's Camera or Microsoft's Kinect. You won't get to customize the layout of your stream the way you can do it on PC, but console streaming has a wide watching around the globe and you can easily build your audience.
We wish you to have great success on Twitch. In case, If you have faced any kind of issues while creating an account on Twitch or Setting up OBS Studio, feel free to submit your queries in the comment section below, we will surely reply to you.
Next article7 Shocking Reasons Why Mars is better than Earth?