Structural basis for the coiled-coil architecture of human CtIP.

The DNA repair factor CtIP has a critical function in double-strand break (DSB) repair by homologous recombination, promoting the assembly of the repair apparatus at DNA ends and participating in DNA-end resection. However, the molecular mechanisms of CtIP function in DSB repair remain unclear. Here, we present an atomic model for the three-dimensional architecture of human CtIP, derived from a multi-disciplinary approach that includes X-ray crystallography, small-angle X-ray scattering (SAXS) and diffracted X-ray tracking (DXT). Our data show that CtIP adopts an extended dimer-of-dimers structure, in agreement with a role in bridging distant sites on chromosomal DNA during the recombinational repair. The zinc-binding motif in the CtIP N-terminus alters dynamically the coiled-coil structure, with functional implications for the long-range interactions of CtIP with DNA. Our results provide a structural basis for the three-dimensional arrangement of chains in the CtIP tetramer, a key aspect of CtIP function in DNA DSB repair.

Certolizumab pegol in rheumatoid arthritis patients with low to moderate activity: the CERTAIN double-blind, randomised, placebo-controlled trial.

OBJECTIVES: This 52-week, randomised, double-blind phase IIIb study assessed efficacy and safety of certolizumab pegol (CZP) as add-on therapy to non-biologic disease-modifying antirheumatic drugs (DMARDs) in rheumatoid arthritis (RA) patients with low to moderate disease activity, and stopping therapy in patients in sustained remission. METHODS: Patients were randomised 1:1 to CZP (400 mg at weeks 0, 2 and 4, then 200 mg every 2 weeks) or placebo (every 2 weeks) plus current non-biologic DMARDs. At week 24, patients who achieved the primary endpoint of Clinical Disease Activity Index (CDAI) remission at both weeks 20 and 24 stopped study treatment and continued in the study until week 52. RESULTS: Of 194 patients (CZP=96; placebo=98), >90% had moderate disease activity at baseline. Significantly more CZP patients met the primary endpoint than placebo patients (week 20 and 24 CDAI remission rates: 18.8% vs 6.1%; p≤0.05). At week 24, 63.0% vs 29.7% of CZP versus placebo patients (p<0.001) achieved LDA. Disease activity score (ESR) based on 28-joint count and Simplified Disease Activity Index remission rates were also significantly higher with CZP versus placebo (19.8% vs 3.1%; p≤0.01 and 14.6% vs 4.1%; p≤0.05). CZP patients reported improvements in physical function versus placebo (mean Health Assessment Questionnaire-Disability-Index change from baseline: CZP, -0.25 vs placebo, -0.03; p≤0.01). During the period following withdrawal of CZP or placebo, only 3/17 prior CZP patients and 2/6 prior placebo patients maintained CDAI remission until week 52, but CZP reinstitution allowed renewed improvement. Adverse and serious adverse event rates were comparable between CZP and placebo groups. CONCLUSIONS: Addition of CZP to non-biologic DMARDs is an effective treatment in RA patients with predominantly moderate disease activity, allowing low-disease activity or remission to be reached in a majority of the patients. However, the data suggest that CZP cannot be withdrawn in patients achieving remission. TRIAL REGISTRATION NUMBER: NCT00674362.

Single shot tetanus vaccine manufactured by a supercritical fluid encapsulation technology.

Single shot vaccines of tetanus toxoid (TT) were manufactured using the NanoMix process - a low temperature solvent free encapsulation technology using supercritical fluids. The formulations were injected into mice, and compared to multiple injections of a commercially available alum adsorbed TT vaccine. After 5 months the antibody titres were found to be similar for both the alum adsorbed and microparticle formulations, demonstrating for the first time the potential of formulating antigens in PLA microparticles using the supercritical fluid (NanoMix) technique to produce single shot vaccines. The results are likely to be due to the maintenance of toxoid bioactivity and some degree of sustained release of the encapsulated antigens, resulting in repeated stimulation of antigen presenting cells eliminating the need for multiple immunisations. This demonstrates the potential of this supercritical fluid processing technique to reduce the need for booster doses in a vaccine regimen.

Nematodes from Swainson's spurfowl Pternistis swainsonii and an Orange River francolin Scleroptila levaillantoides in Free State Province, South Africa, with a description of Tetrameres swainsonii n. sp. (Nematoda: Tetrameridae).

Five Swainson's spurfowl collected in Free State Province, South Africa, were examined for helminth parasites, and the nematodes Acuaria gruveli, Cyrnea parroti, Gongylonema congolense, Subulura dentigera, Subulura suctoria and a new Tetrameres species were recovered. Their respective prevalence was 100, 20, 80, 20, 20 and 20%. These nematodes are all new parasite records for Swainson's spurfowl, and Acuaria gruveli constitutes a new geographical record as well. A single specimen of Cyrnea eurycerca was found in an Orange River francolin, representing a new host and geographical record for this parasite. The new species, for which the name Tetrameres swainsonii is proposed, can be differentiated from its congeners by a combination of the following characters of males: two rows of body spines, a single spicule which is 1152-1392 microm long, and eight pairs of caudal spines arranged in two ventral and two lateral rows of four spines each. The single female has the globular shape typical of the genus.

The assessment of hookworm calreticulin as a potential vaccine for necatoriasis.

A vaccine against the human hookworm Necator americanus is urgently required to reduce hookworm-induced morbidity in endemic areas. In the present study, recombinant hookworm calreticulin, a nominated vaccine candidate, has been tested in mice. Mice given calreticulin had 43-49% fewer worms in their lungs, compared to non-vaccinated controls, following challenge infection with infective hookworm larvae. These levels of protection were achieved in the absence of adjuvant following intraperitoneal administration of three doses of 15 microg antigen. Antigen was also encapsulated in PLG microparticles. Encapsulated calreticulin elicited higher levels of anti-calreticulin IgG1 than free antigen but failed to induce protective immunity. The protection induced by free calreticulin was associated with low levels of serum IgE and moderate lung eosinophilia whilst administration of calreticulin-loaded microparticles was associated with high levels of serum IgE and higher lung eosinophil activity, suggesting that the classical Th2 phenotype may not always be associated with protective immunity, albeit in experimental necatoriasis.

Synthesis and characterisation of polyamine-poly(ethylene glycol) constructs for DNA binding and gene delivery.

Improved non-viral vector systems are needed for efficient delivery of DNA to target cell nuclei in gene therapy. A series of linear polyamine poly(ethylene glycol) (PEG) constructs has been synthesised by reaction of appropriately Boc-protected thermine derivatives with omega-methoxyPEG oxiranylmethyl ethers. Constructs carrying 1-3 MeOPEG units and 0, 2 or 4 N-methyl groups have been prepared by this method. H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NHBoc was prepared efficiently by mono-trifluoroacetylation of thermine, attachment of Boc and removal of the trifluoroacetyl group in one pot. A similar process gave H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NH2. BocMeN(CH2)3NHMe was alkylated by 1,3-dibromopropane to give BocMeN(CH2)3NMe(CH2)3NMe(CH2)3NMeBoc. A cyanoethylation/reduction sequence extended H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NH2 to give H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NBoc(CH2)3NBoc(CH2) 3NH2, which was converted to its mono- and di-MeOPEG550 derivatives. Deprotection gave the linear polyamine MeOPEG constructs. A branched triamine-poly(ethylene glycol) construct was prepared by acylation of (BocHN(CH2)3)2N(CH2)3NH2 with omega-methoxyPEG 550 chloroformate, followed by deprotection. A cyanoethylation/reduction/protection sequence from (H2N(CH2)3)2 N(CH2)3NHBoc gave a protected pentamine. Alkylation with Br(CH2)5CONH(CH2)2NHBoc, deprotection, acylation with MeOPEG chloroformate and deprotection gave a pentamine MeOPEG construct in which the MeOPEG is attached through a linker to the central amine. The linear hexamine construct carrying MeOPEG550 at only one terminus was the most effective DNA-interactive member of the two series in an ethidium displacement assay and was effective in delivering a reporter gene to RIF-1 tumours.