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We report a one-pot plasma electrolytic oxidation (PEO) strategy for forming a multi-element oxide layer on the titanium surface using complex electrolytes containing Na2HPO4, Ca(OH)2, (NH2)2CO, Na2SiO3, CuSO4, and KOH compounds. For even better bone implant ingrowth, PEO coatings were additionally loaded with bone morphogenetic protein-2 (BMP-2). The samples were tested in vivoin a mouse craniotomy model. Tests for bactericidal and fungicidal activity were carried out using clinically isolated multi-drug-resistant Escherichia coli(E. coli) K261, E. coliU20, methicillin-resistant Staphylococcus aureus(S. aureus) CSA154 bacterial strains, and Neurospora crassa(N. crassa) and Candida albicans(C. albicans) D2528/20 fungi. The PEO-Cu coating effectively inactivated both Gram-positive and Gram-negative bacteria at low concentrations of Cu2+ions: minimal bactericidal concentration for E. coliand N. crassa(99.9999%) and minimal inhibitory concentration (99.0%) for S. aureuswere 5 ppm. For all studied bacterial and fungal strains, PEO-Cu coating completely prevented the formation of bacterial and fungal biofilms. PEO and PEO-Cu coatings demonstrated bone remodeling and moderate osteoconductivity in vivo, while BMP-2 significantly enhanced osteoconduction and osteogenesis. The obtained results are encouraging and indicate that Ti-based materials with PEO coatings loaded with BMP-2 can be widely used in customized medicine as implants for orthopedics and cranio-maxillofacial surgery.

Growth factor incorporation in biomedical constructs for their local delivery enables specific pharmacological effects such as the induction of cell growth and differentiation. This has enabled a promising way to improve the tissue regeneration process. However, it remains challenging to identify an appropriate approach that provides effective growth factor loading into biomedical constructs with their following release kinetics in a prolonged manner. In the present work, we performed a systematic study, which explores the optimal strategy of growth factor incorporation into sub-micrometric-sized CaCO3core–shell particles (CSPs) and hollow silica particles (SiPs). These carriers were immobilized onto the surface of the polymer scaffolds based on polyhydroxybutyrate (PHB) with and without reduced graphene oxide (rGO) in its structure to examine the functionality of incorporated growth factors. Bone morphogenetic protein-2 (BMP-2) and ErythroPOietin (EPO) as growth factor models were included into CSPs and SiPs using different entrapping strategies, namely, physical adsorption, coprecipitation technique, and freezing-induced loading method. It was shown that the loading efficiency, release characteristics, and bioactivity of incorporated growth factors strongly depend on the chosen strategy of their incorporation into delivery systems. Overall, we demonstrated that the combination of scaffolds with drug delivery systems containing growth factors has great potential in the field of tissue regeneration compared with individual scaffolds.

Electrical conduction and oxygen diffusion mobility in the bixbyite (Ia3̅) and rhombohedral (R3̅) polymorphs of the Ln6MoO12−Δ(Ln = Er, Tm, Yb; Δ = δ, δ1, δ2; δ1 > δ2) heavy lanthanide molybdates, belonging to new, previously unexplored classes of potential mixed (ionic–electronic) conductors, have been studied in the range of 200–900 °C. The oxygen self-diffusion coefficient in bixbyite (Ia3̅) Yb6MoO12−δphase estimated by the temperature-programmed heteroexchange with C18O2was shown to be much higher than that for rhombohedral (R3̅) RI (with large oxygen deficiency) and (R3̅) RII (with small oxygen deficiency) Ln6MoO12−Δ(Ln = Tm, Yb; Δ = δ1; δ1 > δ2) oxides. According to the activation energy for total conduction in ambient air, 0.99, 0.93, and 1.01 eV in Er6MoO12−δ, Tm6MoO12−δ,and Yb6MoO12−δbixbyites, respectively, oxygen ion conductivity prevails in the range ∼200–500 °C. Oxygen mobility data for the rhombohedral Ln6MoO12−Δ(Ln = Er, Tm, Yb; Δ = δ1, δ2) phases RI and RII indicate that the oxygen in these phases exhibits mobility at much higher temperatures, such as those above 600–700 °C. Accordingly, below 600–700 °C they have predominantly electronic conductivity. As shown by total conductivity study of Ln6MoO12−δ(Ln = Er, Tm, Yb) bixbyites (Ia3̅) and rhombohedral phases Ln6MoO12−Δ(Ln = Er, Tm, Yb; Δ = δ1, δ2) (R3̅) in dry and wet air, the proton conductivity contribution exists only in Ln6MoO12−δ(Ln = Er, Tm, Yb) bixbyites up to 450–600 °C and decreases with a decreasing of the lanthanide ionic radius. The obtained data on the mobility of oxygen and the presence of proton contribution in bixbyites in the 300–600 °C temperature range make it possible to confirm unequivocally that Ln6MoO12−δ(Ln = Er, Tm, Yb) bixbyites are mixed electron–proton conductors at these temperatures.

Osteoplastic materials PLA/PCL/HA and PHB/HA and scaffolds with a highly porous structure based on them with potential applications in regenerative medicine have been obtained by solvent casting with thermopressing and salt leaching for PLA-based samples and solid-state mixing with subsequent thermopressing and salt leaching for PHB-based samples. The scaffolds were characterized by SEM-EDX, DSC, FTIR spectroscopy, mechanical tests in compression, measurement of the contact angle, in vitrostudies, including loading by recombinant BMP-2 and EPO and their release kinetics, and in vivostudies on a model of regeneration of critical-sized cranial defects in mice. Biomimetic scaffolds with micropores sizes ranged from 300 to 500 μm and volume porosity of 70% imitate trabecular bone's structure and have increased hydrophilicity to achieve osteoconductive properties. Mechanical characteristics correspond to native trabecular bone. Elastic modulus – key mechanical characteristics of bone implants – showed the values of 0.15 ± 0.04 and 0.18 ± 0.08 GPa for PLA/PCL/HA and PHB/HA scaffolds, respectively. Both materials have high biocompatibility and can be used together with recombinant proteins BMP-2 and EPO. Introduction of BMP-2 leads to induction of new bone formation, introduction of EPO results in increased angiogenesis in the implantation area. The obtained scaffolds with recombinant proteins can be used as bone implants for reconstruction of defects of lightly or non-loaded bones.

Rozrolimupab, a recombinant mixture of 25 fully human RhD-specific monoclonal antibodies, represents a new class of recombinant human antibody mixtures. In a phase 1 or 2 dose escalation study, RhD+ patients (61 subjects) with primary immune thrombocytopenia received a single intravenous dose of rozrolimupab ranging from 75 to 300 μg/kg. The primary outcome was the occurrence of adverse events. The principal secondary outcome was the effect on platelet levels 7 days after the treatment. The most common adverse events were headache and pyrexia, mostly mild, and reported in 20% and 13% of the patients, respectively, without dose relationship. Rozrolimupab caused an expected transient reduction of hemoglobin concentration in the majority of the patients. At the dose of 300 μg/kg platelet responses, defined as platelet count ≥ 30 × 109/L and an increase in platelet count by > 20 × 109/L from baseline were observed after 72 hours and persisted for at least 7 days in 8 of 13 patients (62%). Platelet responses were observed within 24 hours in 23% of patients and lasted for a median of 14 days. Rozrolimupab was well tolerated and elicited rapid platelet responses in patients with immune thrombocytopenia and may be a useful alternative to plasma-derived products. This trial is registered at www.clinicaltrials.gov as #NCT00718692.

Rozrolimupab, a recombinant mixture of 25 fully human RhD-specific monoclonal antibodies, represents a new class of recombinant human antibody mixtures. In a phase 1 or 2 dose escalation study, RhD+patients (61 subjects) with primary immune thrombocytopenia received a single intravenous dose of rozrolimupab ranging from 75 to 300 μg/kg. The primary outcome was the occurrence of adverse events. The principal secondary outcome was the effect on platelet levels 7 days after the treatment. The most common adverse events were headache and pyrexia, mostly mild, and reported in 20% and 13% of the patients, respectively, without dose relationship. Rozrolimupab caused an expected transient reduction of hemoglobin concentration in the majority of the patients. At the dose of 300 μg/kg platelet responses, defined as platelet count ≥ 30 × 109/L and an increase in platelet count by > 20 × 109/L from baseline were observed after 72 hours and persisted for at least 7 days in 8 of 13 patients (62%). Platelet responses were observed within 24 hours in 23% of patients and lasted for a median of 14 days. Rozrolimupab was well tolerated and elicited rapid platelet responses in patients with immune thrombocytopenia and may be a useful alternative to plasma-derived products. This trial is registered at www.clinicaltrials.govas #NCT00718692.

Using a new method based on a combination of bisulfite reaction, the repair enzyme uracil-DNA glycosylase, and synthetic oligodeoxyribonucleotides, the methylation site of DNA-methyltransferase NlaX (M·NlaX) from Neisseria lactamica was established to be the inner cytosine in the double-stranded pentanucleotide recognition sequence 5′-CCNGG-3′ (where N = any nucleoside). 5-Methylcytosine (m5C) type modification by M·NlaX was confirmed by the use of oligonucleotide substrates that contain 5-fluoro-2′-deoxycytidine.

Using a new method based on a combination of bisulfite reaction, the repair enzyme uracil-DNA glycosylase, and synthetic oligodeoxyribonucleotides, the methylation site of DNA-methyltransferase NlaX (M·NlaX) from Neisseria lactamica was established to be the inner cytosine in the double-stranded pentanucleotide recognition sequence 5′-CCNGG-3′ (where N = any nucleoside). 5-Methylcytosine (m5C) type modification by M·NlaX was confirmed by the use of oligonucleotide substrates that contain 5-fluoro-2′-deoxycytidine.

The functional groups of the DNA methylation site that are involved in the DNA interaction with methyltransferase SsoII at the recognition stage were identified. The contacts in the enzyme–substrate complex were analyzed in the presence of S-adenosyl-L-homocysteine using the interference footprinting assay with formic acid, hydrazine, dimethyl sulfate, or N-ethyl-N-nitrosourea as a modifying reagent. It was shown that the replacement of the central A · T by the G · C pair in the methylation site did not affect enzyme–DNA interaction, whereas the use of a substrate with one strand methylated (monomethylated substrate) instead of the unmethylated substrate dramatically changes the DNA contacts. The binding constants of unmethylated and monomethylated substrates with methyltransferase SsoII in the presence of S-adenosyl-L-homocysteine were calculated.

A target sequence-specific DNA binding region of the restriction endonuclease Sso II was identified by photocross-linking with an oligodeoxynucleotide duplex which was substituted with 5-iododeoxy-uridine (5-IdU) at the central position of the Sso II recognition site (CCNGG). For this purpose the Sso II-DNA complex was irradiated with a helium/cadmium laser (325 nm). The cross-linking yield obtained was approximately 50%. In the presence of excess unmodified oligodeoxynucleotide or with oligode-oxynucleotides substituted with 5-IdU elsewhere, no cross-linking was observed, indicating the specificity of the cross-linking reaction. The cross-linked Sso II-oligodeoxynucleotide complex was digested with chymotrypsin, a cross-linked peptide-oligodeoxy-nucleotide complex isolated and the site of cross-linking identified by Edman sequencing to be Trp61. In line with this identification is the finding that the W61A variant cannot be cross-linked with the IdU-substituted oligodeoxynucleotide, shows a decrease in affinity towards DNA and is inactive in cleavage. It is concluded that the region around Trp61 is involved in specific binding of Sso II to its DNA substrate.

Abstract: Specific features of immune-endocrine interrelationships and of pituitary-adrenal system functioning are studied in NZB mice with hereditary autoimmune pathology. It is established that the development of disease is accompanied by a weakened response to stress, reduced blood corticosteroid level, and decreased adrenal reactivity to exogenous adrenocorticotropic hormone. Moreover, a loss of sensitivity to interleukin-2 is observed. These facts provide evidence of disturbed interaction between the immune and endocrine systems as well as of inhibition of pituitary-adrenal system activity as the autoimmune disease develops.

Specific and non-specific interactions SsoII restriction endonuclease (R·SsoII) were probed by the method of covalent attachment to modified DNA containing an active monosubstituted pyrophosphate internucleotide bond instead of a phosphodiester one. R·SsoII with six N-terminal His residues was shown to be cross-linked to duplexes with this type of modification, either containing or not the recognition sequence. Competition experiments with covalent attachment of R·SsoII to activated DNAs demonstrated the similar affinity of the enzyme to cognate and non-cognate DNAs in the absence of cofactor, Mg2+ ions.

The investigation of Sso II DNA-methyltransferase (M.Sso II) interaction with the intergenic region of Sso II restriction-modification system was carried out. Seven guanine residues protected by M. Sso II from methylation with dimethylsulfate and thus probably involved in enzyme-DNA recognition were identified. Six of them are located symmetrically within the 15 bp inverted repeat inside the Sso II promoter region. The crosslinking of Sso II methyltransferase with DNA duplexes containing 5-bromo-2'-deoxyuridine (br5dU) instead of thymidine was performed. The crosslinked products were obtained in all cases, thus proving that tested thymines were in proximity with enzyme. The ability to produce the crosslinked products in one case was 2-5-fold higher than in other ones. This allowed us to imply that thymine residue in this position of the inverted repeat could be in contact with M. Sso II. Based on the experimental data, two symmetrical 4 bp clusters (GGAC), which could be involved in the interaction with M. Sso II in the DNA-protein complex, were identified. The model of M. Sso II interaction with its own promoter region was proposed.

Without Abstract:

Specific and non‐specific interactions SsoII restriction endonuclease (R·SsoII) were probed by the method of covalent attachment to modified DNA containing an active monosubstituted pyrophosphate internucleotide bond instead of a phosphodiester one. R·SsoII with six N‐terminal His residues was shown to be cross‐linked to duplexes with this type of modification, either containing or not the recognition sequence. Competition experiments with covalent attachment of R·SsoII to activated DNAs demonstrated the similar affinity of the enzyme to cognate and non‐cognate DNAs in the absence of cofactor, Mg2+ions.

The investigation of SsoII DNA‐methyltransferase (M.SsoII) interaction with the intergenic region of SsoII restriction‐modification system was carried out. Seven guanine residues protected by M.SsoII from methylation with dimethylsulfate and thus probably involved in enzyme–DNA recognition were identified. Six of them are located symmetrically within the 15 bp inverted repeat inside the SsoII promoter region. The crosslinking of SsoII methyltransferase with DNA duplexes containing 5‐bromo‐2′‐deoxyuridine (br5dU) instead of thymidine was performed. The crosslinked products were obtained in all cases, thus proving that tested thymines were in proximity with enzyme. The ability to produce the crosslinked products in one case was 2–5‐fold higher than in other ones. This allowed us to imply that thymine residue in this position of the inverted repeat could be in contact with M.SsoII. Based on the experimental data, two symmetrical 4 bp clusters (GGAC), which could be involved in the interaction with M.SsoII in the DNA–protein complex, were identified. The model of M.SsoII interaction with its own promoter region was proposed.

The genes encoding SsoI and SsoII restriction endonuclease (ENase) and methyltransferase (MTase) are located on the small plasmids P6 and P4, respectively, of Shigella sonneistrain 47. Functions provided by plasmids P5, P7 and P9, which include colicinogenicity and immunity to colicin E1, resistance to streptomycin (Sm), and conjugative DNA transfer, respectively, have also been identified. The genes of the SsoII restriction-modification (R-M) system have been cloned into Escherichia coliexpressing the 35-kDa (ENase) and 43-kDa (MTase) products. A restriction map of the P4 plasmid DNA was determined, and the approximate location of the genes encoding SsoII ENase and MTase (ssoIIRand ssoIIM) on that have been established. SsoI is an isoschisomer of EcoRI and SsoII cleaves the 5′-/CCNGG/recognition sequence producing 5′-protruding 5-nt long cohesive ends.

The misfolding of α-synuclein (αS) to a cross-β-sheet amyloid structure is associated with pathological conditions in Parkinson's and other neurodegenerative diseases. Using pulse electron paramagnetic resonance spectroscopy combined with a cross-labeling strategy involving four double mutants, we were able to determine the intramolecular distance between the extremal β-strands. The distance of 4.5 ± 0.5 nm is in good agreement with the dimensions of a protofilament reported by other low-resolution techniques, such as x-ray scattering and atomic force microscopy.

Abstract: The development of autoimmune hemolytic anemia in NZB mice aged 2–3, 6–7, and 10–12 months is associated with decreased corticosterone and increased corticosteroid-binding globulin concentrations in the blood. An increase in the pool of free glucocorticoids induced by dexamethasone decelerates the autoimmune processes. (NZBDBZ/2) F1 mice, which never develop the disease, are characterized by a high adrenal activity and a low blood level of corticosteroid-binding globulin. High level of free glucocorticoids is believed to prevent autoimmune reactions.

Gisslinger: AOP Orphan Pharmaceuticals AG, Novartis, Celgene, Baxalta: Consultancy, Honoraria, Research Funding. Klade:AOP Orphan: Employment. Kyrcz-Krzemien:AOP Orphan Pharmaceuticals AG, Novartis, BMS, Medac: Honoraria. Warzocha:BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Grohmann-Izay:AOP Orphan Pharmaceuticals AG: Employment. Hodisch:AOP Orphan Pharmaceuticals: Employment. Widmann:AOP Orphan Pharmaceuticals: Employment. Kralovics:Qiagen: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Research Funding. Schwarz:AOP Orphan Pharmaceuticals: Consultancy. Kiladjian:Novartis: Research Funding; AOP Orphan: Research Funding.

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No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Study objectives

Background. The increase of life expectancy in patients after 65 years with MM is the main aim of treatment. Lack of carrying out aggressive anti-multiple myeloma therapy increase influence of different factors on OS.

No relevant conflicts of interest to declare.

Objectives and background:Normal karyotype (NK) in AML patients accounts for nearly 45% of all cases and were assigned into intermediate risk group. The identification of new molecular markers in this group is the focus of most of researches. The application of the next-generation sequence techniques led to detect molecular markers with valuable prognostic significance. F.e., identification of DNMT3Amutations has gained the tremendous attention in recent times, because of its essential role in cell development, high frequency in AML patients and association of poor clinical outcome. Objects: to analyse character and frequency of DNMT3Amutations in AML patients; to study their associations with clinical and laboratory parameters and other molecular markers; to investigate their prognostic value.

The incidence of chronic myeloid leukemia (CML), reported from some population based registries, varies significantly. CML is known as age-dependent disease, so population age structure may strongly influent on the data. For international comparisons several systems for age-standardization are using in epidemiological studies. We conducted our retrospective study to reveal differences in CML incidence rates on the basis of calculation – crude or age-adjusted according to different population standards in St. Petersburg and Leningrad region.In 2005 the database of Ph- and/or bcr-abl- positive CML patients (pts) was conducted in St. Petersburg and Leningrad region. Since then the data from all newly diagnosed CML patients were included prospectively on population basis. The database was updated at least bi-annually. The data were obtained from hematologists, as general practitioners and private physicians are not licensed to treat oncohematological disorders. The data were double checked from the list of Imatinib distribution (the only drug reimbursed for first line treatment). To calculate crude CML incidence rate we use the data of the general census of the population in Russia in 2010 (the whole population of our region is 6596434 with population in age 15 and above 5821133). For age-adjusted CML incidence rate we use three of currently existing standards: The Segi (“World”), The Scandinavian (“European”) and the WHO standard (based on world average population between 2000–2025).There are 258 (242 in chronic, 9 in accelerated and 7 in blastic phases) CML adult (15 years and above) pts, registered during 2006–2011. The median age is 53 years (48,5 and 55,5 years for men and women respectively). Sokal score was evaluable in 209 pts. It is low in 37%, intermediate in 35% and high in 28% pts. The crude CML incidence rate is slightly higher in men than in women with ratio 1,2:1. Mean annual crude CML incidence rate was 0,65 per 100 000 whole population of Saint Petersburg and Leningrad region, but it was 0,74 in adult population (15 years old and above). Mean annual CML incidence rates in the same age groups were slightly higher in all three standardized systems: 0,94 in Segi, 0,84 in Scandinavian and 0,88 in WHO standard populations. CML incidence rates in all age groups are presented in the table 1. CML incidence rate was lowest in young pts. It was unexpectedly very low in senior pts.CML incidence rates nearly for all age groups were slightly higher in St. Petersburg than in the Leningrad region. The majority of pts (98%) were treated with Imatinib (93% first or second line) or other tyrosine kinase inhibitors (5% first line-in international clinical trials, 18% after Imatinib failure or intolerance). Stem cell transplantation was performed only in 8/258 (3%) pts. Only 25235 (7,5%) evaluable pts progressed from chronic to advanced phases. Only 29/258 (11%) pts dead mostly due to CML (21 CML related deaths were reported). Estimated 5 years overall survival is 91,5%. Mean annual overall CML pts death rate was 1,9% (mean annual death rate between 2006–2010 in whole population of our region was 1,6%). Mean pts accumulated very fast - annual CML prevalence increasing rate between 2005–2011 was more than 14% (Picture 1).CML incidence both crude and age-adjusted in our population based registry is nearly the same in young and middle age, but much lower in senior (65 years and above) pts groups in comparison with published data from other registries which probably represents peculiarities of health system rather than real incidence. In the tyrosine kinase inhibitors era CML patients death rate is very low (nearly the same as in whole population) and CML pts is accumulated very fast in our region.No relevant conflicts of interest to declare.

Abstract 4432

Rozrolimupab is the first in-class fully human, recombinant antibody mixture for the treatment of ITP. Rozrolimupab comprises 25 unique human antibodies with documented binding capacity to the Rhesus D antigen on red blood cells, and constitutes a promising, modern counter-part to the blood-derived immunoglobulin products currently used in the treatment of ITP. The antibody mixture constituting rozrolimupab is produced by a novel single batch manufacturing strategy.The objectives of the trial were to investigate the safety and efficacy of a single dose of rozrolimupab in RhD positive, non-splenectomized adult patients with primary ITP. The primary endpoint was incidence and severity of Adverse Events (AEs) during the 6-week trial period. Secondary endpoints included identification of the optimal dose of rozrolimupab and percentage of patients responding to treatment at 72 hours, 7 and 15 days.Patients from 27 sites in 11 countries were enrolled in this dose escalation (75 μg/kg – 300 μg/kg), multicentre, open label trial. Inclusion criteria included confirmed presence of thrombocytopenia with two individual pre-dose platelet counts of < 30 × 109/L. The patients received a single iv dose of rozrolimupab, and were followed for 6 weeks with frequent evaluations for safety and efficacy. Response was defined as platelet count ≥ 30 ×109/L and increase in platelet count from baseline by > 20 × 109/L.A total of 61 ITP patients were treated with rozrolimupab: 75 μg/kg (11 patients), 100 μg/kg (10 patients), 125 μg/kg (10 patients), 150 μg/kg (5 patients), 200 μg/kg (6 patients), 250 μg/kg (6 patients) and 300 μg/kg (13 patients). Data from the 300 μg/kg cohort were not available for this abstract, but will be included in the final presentation.Given the small size of this study, the dose cohorts differed in a number of baseline characteristics. 60–90% of patients were females in all dose cohorts except in the 75 μg/kg cohort where 73% were males. Median platelet count before trial entry ranged from 11.5 to 22.3 × 109/L, and median time from first ITP diagnosis ranged from 5 to 68 months. In the individual dose groups, up to 83% of the patients responded at day 7. Up to 50% and 60% of the patients responded at 24 and 72 hours, respectively, and some responses lasted for the duration of the 6 weeks follow up period. Platelet responses also included patients who had baseline platelet counts below 10 ×109/L. A total of 51 AEs considered related to trial drug were reported in 20 patients. The most frequently reported adverse reactions were headache (7 events in 5 patients) and pyrexia (5 events in 4 patients). All but 2 of the adverse reactions (one headache, one extravascular haemolysis) were of mild or moderate intensity. Four of the 51 adverse reactions were categorized as Serious Adverse Events: One mild haemoglobin decrease in the 100 μg/kg cohort, one extravascular haemolysis in the 200 μg/kg cohort (mentioned above) and two mild to moderate, transient increases in D-dimer with no clinical symptoms in the 250 μg/kg cohort. Laboratory data showed a mean maximum decrease in haemoglobin ranging from 1.1 g/dL to 2.7 g/dL in individual cohorts with a slight dose dependency. A drop in haemoglobin of ≥ 3 g/dL was seen in 2 patients, one of 3.1 g/dL in the 100 μg/kg cohort and one of 4.5 g/dL in the 200 μg/kg cohort.Rozrolimupab is well tolerated with a favourable safety profile. In the individual dose groups, up to 83% of patients responded at day 7 including patients who had baseline platelet counts below 10 ×109/L. Final safety and efficacy data from all dose groups in the completed Phase II trial of rozrolimupab in primary ITP patients will be presented.Wiktor-Jedrzejczak: Celgene: Honoraria; Janssen-Cilag: Honoraria; Novartis: Honoraria, Research Funding. Flensburg:Symphogen: Employment. Petersen:Symphogen: Employment.

Abstract 527This icon denotes a clinically relevant abstract

A study was made of the interaction between restriction endonucleases recognizing CCNGG (Ssoll and ScrFl) or CCA/TGG (Mval and EcoRll) DNA sequences and a set of synthetic substrates containing 1,3-propanediol, 1,2-dideoxy-D-ribofuranose or 9-[1′-hydroxy-2′-(hydroxymethyl)ethoxy] methylguanine (glG) residues replacing either one of the central nucleosides or dG residues in the recognition site. The non-nucleotide inserts (except for glG) introduced into the recognition site both increase the efficiency of Ssoll and change its specificity. A cleavage at the noncanonical position takes place, in some cases in addition to the correct ones. Noncanonical hydrolysis by Ssoll occurs at the phosphodiester bond adjacent to the point of modification towards the 5′-end. With the guanine base returned (the substrate with glG), the correct cleavage position is restored. ScrFl specifically cleaves all the modified substrates. DNA duplexes with non-nucleotide inserts (except for the glG-containing duplex) are resistant to hydrolysis by Mval and EcoRll. Prompted by the data obtained we discuss the peculiarities of recognition by restriction endonucleases of 5-membered DNA sequences which have completely or partially degenerated central base pairs. It is suggested that Ssoll forms a complex with DNA in an ‘open’ form.

The regulation of the SsoII restriction-modification system from Shigella sonnei was studied in vivo and in vitro. In lacZ fusion experiments, SsoII methyltransferase (M.SsoII) was found to repress its own synthesis but stimulate expression of the cognate restriction endonuclease (ENase). The N-terminal 72 amino acids of M.SsoII, predicted to form a helix-turn-helix (HTH) motif, was found to be responsible for the specific DNA-binding and regulatory function of M.SsoII. Similar HTH motifs are predicted in the N-terminus of a number of 5-methylcytosine methyltransferases, particularly M.EcoRII, M.dcm and M.MspI, of which the ability to regulate autogenously has been proposed. In vitro, the binding of M.SsoII to its target DNA was investigated using a mobility shift assay. M.SsoII forms a specific and stable complex with a 140 bp DNA fragment containing the promoter region of SsoII R-M system. The dissociation constant (Kd) was determined to be 1.5 × 10−8 M. DNaseI footprinting experiments demonstrated that M.SsoII protects a 48–52 bp region immediately upstream of the M.SsoII coding sequence which includes the predicted −10 promoter sequence of M.SsoII and the −10 and −35 sequences of R.SsoII.

Oligonucleotldes containing 2-amlnopurine (2-AP) In place of G or A in the recognition site of EcoRII (CCT/AGG) or Ssoll (CCNGG) restriction endonucleases have been synthesized In order to Investigate the specific interaction of DNA with these enzymes. Physicochemlcal properties (CD spectra and melting behaviour) have shown that DNA duplexes containing 2-aminopurine exist largely in a stable B-IIke form. 2-Aminopurine base paired with cytidine, however, essentially Influences the helix structure. The presence of a 2-APC mismatch strongly reduces the stability of the duplexes in comparison with the natural double strand, indicated by a blphaslc melting behaviour. Ssoil restriction endonuclease recognizes and cleaves the modified substrate with a 2-APT mismatch In the centre of the recognition site, but it does not cleave the duplexes containing 2-amlnopurine in place of inner and outer G, or both. EcoRII restriction endonuclease does not cleave duplexes containing 2-amlnopurine at all. The two-substrate mechanism of EcoRII-DNA interaction, however, allows hydrolysis of the duplex containing 2-amlnopurine In place of adenine In the presence of the canonical substrate.