Allele resolution of HLA-A using oligonucleotide probes in a two-stage typing strategy

High-resolution polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) typing methods for HLA-A identification have been established. The four systems, which operate independently of each other, are intended for use as secondary typing systems following HLA-A identification with a medium-resolution PCR-SSOP technique. The systems, all using digoxigenin-labelled probes, are based on group specific amplifications for resolution of: i) HLA-A*29 & -A*33; ii) HLA-A*24 & -A*30; and iii) HLA-A*26, -A*25, -A*11, -A*34, -A*66 and -A*68 alleles, respectively. The fourth system, for the detection of HLA-A*02 alleles, is a modification of a previously reported PCR-SSOP subtyping system. The methods have been applied to individuals from the local bone marrow registry and HLA-A allele frequencies for the Northern Ireland population have been established.

Line-profile tomography of exoplanet transits - II. A gas-giant planet transiting a rapidly rotating A5 star

Most of our knowledge of extrasolar planets rests on precise radial-velocity measurements, either for direct detection or for confirmation of the planetary origin of photometric transit signals. This has limited our exploration of the parameter space of exoplanet hosts to solar- and later-type, sharp-lined stars. Here we extend the realm of stars with known planetary companions to include hot, fast-rotating stars. Planet-like transits have previously been reported in the light curve obtained by the SuperWASP survey of the A5 star HD15082 (WASP-33 V = 8.3, v sini = 86 km s-1). Here we report further photometry and time-series spectroscopy through three separate transits, which we use to confirm the existence of a gas-giant planet with an orbital period of 1.22d in orbit around HD15082. From the photometry and the properties of the planet signal travelling through the spectral line profiles during the transit, we directly derive the size of the planet, the inclination and obliquity of its orbital plane and its retrograde orbital motion relative to the spin of the star. This kind of analysis opens the way to studying the formation of planets around a whole new class of young, early-type stars, hence under different physical conditions and generally in an earlier stage of formation than in sharp-lined late-type stars. The reflex orbital motion of the star caused by the transiting planet is small, yielding an upper mass limit of 4.1MJupiter on the planet. We also find evidence of a third body of substellar mass in the system, which may explain the unusual orbit of the transiting planet. In HD 15082, the stellar line profiles also show evidence of non-radial pulsations, clearly distinct from the planetary transit signal. This raises the intriguing possibility that tides raised by the close-in planet may excite or amplify the pulsations in such stars.

Validation of Speciation Techniques: A Study of Chlorozincate(II) Ionic Liquids

The speciation of chlorozincate(II) ionic liquids, prepared by mixing 1-octyl-3-methylirnidazolium chloride, [C(8)mim]Cl, and zinc(II) chloride in various molar ratios, chi zncl(2), was investigated using Raman spectroscopy and differential scanning calorimetry; the Gutmann acceptor number, which is a quantitative measure of Lewis acidity, was also determined as a function of the composition. These results were combined with literature data to define the anionic speciation; in the neat liquid phase, the existence of cl(-), [ZnCl4](2-), [Zn2Cl6](2-), [Zn3Cl8](2-), and [Zn4Cl10](2-) anions was confirmed. From two chlorozincate(H) ionic liquids with [C(2)mim](+) cations (chi zncl(2) = 0.33 and chi zncl(2) = 0.50), crystals have been obtained, revealing the structures of [C(2)mim)(2)[ZnCl4] and [C(2)mim](2)[Zn2Cl6] forming three-dimensional hydrogen-bond networks. The compound [C(2)mim](2){Zn4Cl10} was crystallized from the chi zncl(1) = 0.75 composition, showing an open-framework structure, with the first example of zinc in a trigonal-bipyramidal chloride coordination. Reinvestigation of the electrospray ionization mass spectrometry of these systems demonstrated that it is an unreliable technique to study liquid-phase speciation.

OXIDATION OF RUTHENIUM(II) TRIS(2,2'-BIPYRIDINE) IONS BY THALLIC IONS IN NITRIC-ACID, MEDIATED BY RUTHENIUM DIOXIDE HYDRATE - AN EXAMPLE OF REVERSIBLE HETEROGENEOUS REDOX CATALYSIS

The kinetics of the oxidation of Ru(bpy)32+ to Ru(bpy)33+ by T13+ ions, catalyzed by a dispersion of RuO2-xH2O in 3 mol dm-3 HNO3, are reported as a function of [Ru(bpy)32+], [Tl3+], [Tl+], [RuO2.xH2O], and temperature. The kinetics of Ru(bpy)32+ oxidation fit an electrochemical model of redox catalysis involving electron transfer between the two electrochemically reversible redox couples, i.e. Ru(bpy)33+/Ru(bpy)32+ and Tl3+/Tl+, mediated by the dispersion of microelectrode particles of RuO2.xH2O. In this model, the rate of reaction is assumed to be controlled by the diffusion of Ru(bpy)32+ toward, and Ru(bpy)33+ away from, the catalyst particles. The Arrhenius activation energy for the catalyzed reaction is 25.9 +/- 0.7 kJ mol-1, and the changes in enthalpy and entropy for the reaction are 36 +/- 2 kJ mol-1 and 127 +/- 6 J mol-1 K-1, respectively. This work describes a rare example of reversible heterogeneous redox catalysis.

Ruthenium(II) bis(terpyridine) electron transfer complexes with alkynyl-ferrocenyl bridges: synthesis, structures, and electrochemical and spectroscopic studies

Two novel alkynyl-bridged symmetric bis-tridentate ligands 1,2-bis(1'-[4'-(2,2':6', 2 ''-terpyridinyl)]-ferrocenyl)ethyne (3a; tpy-Fc-C C-Fc-tpy; Fc = ferrocenyl; tpy = terpyridyl) and 1,4-bis(1'-[4'-(2,2':6', 2 ''-terpyridinyl)]ferrocenyl)-1,3-butadiyne (3b; tpy-Fc-C C-C C-Fc-tpy) and their Ru2+ complexes 6a and 6b have been synthesized and characterized by cyclic voltammetry, UV-vis and luminescence spectroscopy, and in the case of 3b by single-crystal X-ray diffraction. Cyclic voltammograms of both compounds, 3a and 3b, display two severely overlapping ferrocene-based oxidative peaks with only one reductive peak. The redox behavior of 6a and 6b is dominated by the Ru2+/Ru3+ redox couple (E-1/2 from 1.33 to 1.34 V), the Fe2+/Fe3+ redox couples (E-1/2 from 0.46 to 0.80 V), and the tpy/tpy(-)/tpy(2-)redox couples (E-1/2 from -1.19 to -1.48 V). The UV-vis spectra of 6a and 6b show absorption bands assigned to the (1)[(d(pi)(Fe))(6)] -> (1)[(d(pi)(Fe))(5)(pi*(Ru)(tpy))(1)] MMLCT transition at similar to 555 nm. Complexes 6a and 6b are luminescent in H2O-CH3CN (4 : 1, v/v) solution at room temperature, and 6b exhibits the strongest luminescence intensity (lambda(em)(max): 710 nm, Phi(em): 2.28 x 10(-4), tau: 358 ns) relative to analogous ferrocene-based bis(terpyridine) Ru(II) complexes reported so far.

Anthelmintic resistance in Northern Ireland (II): Variations in nematode control practices between lowland and upland sheep flocks

A questionnaire to obtain information on nematode control practices and sheep management was sent to over 1000 farmers in Northern Ireland. Replies were received from 305 flock owners, and data from 252 of them were analysed. Farms were divided into lowland and upland areas. Sizes of pasture and stocking rates on lowland and upland farms were 59.5 hectares, 6.99 sheep/hectare and 62.9 hectares and 10.01 sheep/hectare, respectively. Mean drenching rates for lambs and adults were 2.33 and 2.44, respectively, in lowland flocks and 2.73 and 2.71, respectively, in upland flocks. Between 2008 and 2011, the most frequently identified compounds in use were benzimidazoles and moxidectin in lowland flocks, and benzimidazoles and avermectins in upland flocks. Over the same period the most frequently identified commercial formulations were Tramazole (R), Panacur (R) and Allverm (R) (white drench), Levacide (R) (yellow drench), Oramec (R) (clear drench; avermectin), Cydectin (R) (clear drench; moxidectin) and Monepantel (R) (orange drench).

Most respondents (56.35%) treated their lambs at weaning and the most common time to treat ewes was identified to be pre-mating (67.86% of respondents).

The results of the questionnaire survey revealed that lowland annual drench frequency was 233 and 2.44 in lambs and ewes, respectively, although drench frequencies were higher in upland flocks: 2.73 and 2.71 for lambs and ewes, respectively.

Annual drench rotation was practiced by 43.96% of flock owners, but whether this was true rotation or pseudo-rotation (i.e., substitution of one anthelmintic product by another product belonging to the same chemical group of anthelmintics) could not be explicitly determined. (C) 2012 Elsevier B.V. All rights reserved.

The use of erythropoiesis-stimulating agents with ruxolitinib in patients with myelofibrosis in COMFORT-II: an open-label, phase 3 study assessing efficacy and safety of ruxolitinib versus best available therapy in the treatment of myelofibrosis

BACKGROUND: Anemia is considered a negative prognostic risk factor for survival in patients with myelofibrosis. Most patients with myelofibrosis are anemic, and 35-54 % present with anemia at diagnosis. Ruxolitinib, a potent inhibitor of Janus kinase (JAK) 1 and JAK2, was associated with an overall survival benefit and improvements in splenomegaly and patient-reported outcomes in patients with myelofibrosis in the two phase 3 COMFORT studies. Consistent with the ruxolitinib mechanism of action, anemia was a frequently reported adverse event. In clinical practice, anemia is sometimes managed with erythropoiesis-stimulating agents (ESAs). This post hoc analysis evaluated the safety and efficacy of concomitant ruxolitinib and ESA administration in patients enrolled in COMFORT-II, an open-label, phase 3 study comparing the efficacy and safety of ruxolitinib with best available therapy for treatment of myelofibrosis. Patients were randomized (2:1) to receive ruxolitinib 15 or 20 mg twice daily or best available therapy. Spleen volume was assessed by magnetic resonance imaging or computed tomography scan.

RESULTS: Thirteen of 146 ruxolitinib-treated patients had concomitant ESA administration (+ESA). The median exposure to ruxolitinib was 114 weeks in the +ESA group and 111 weeks in the overall ruxolitinib arm; the median ruxolitinib dose intensity was 33 mg/day for each group. Six weeks before the first ESA administration, 10 of the 13 patients had grade 3/4 hemoglobin abnormalities. These had improved to grade 2 in 7 of the 13 patients by 6 weeks after the first ESA administration. The rate of packed red blood cell transfusions per month within 12 weeks before and after first ESA administration remained the same in 1 patient, decreased in 2 patients, and increased in 3 patients; 7 patients remained transfusion independent. Reductions in splenomegaly were observed in 69 % of evaluable patients (9/13) following first ESA administration.

CONCLUSIONS: Concomitant use of an ESA with ruxolitinib was well tolerated and did not affect the efficacy of ruxolitinib. Further investigations evaluating the effects of ESAs to alleviate anemia in ruxolitinib-treated patients are warranted (ClinicalTrials.gov identifier, NCT00934544; July 6, 2009).

Association Between Results of a Gene Expression Signature Assay and Recurrence-Free Interval in Patients With Stage II Colon Cancer in Cancer and Leukemia Group B 9581 (Alliance)

PURPOSE: Conventional staging methods are inadequate to identify patients with stage II colon cancer (CC) who are at high risk of recurrence after surgery with curative intent. ColDx is a gene expression, microarray-based assay shown to be independently prognostic for recurrence-free interval (RFI) and overall survival in CC. The objective of this study was to further validate ColDx using formalin-fixed, paraffin-embedded specimens collected as part of the Alliance phase III trial, C9581.

PATIENTS AND METHODS: C9581 evaluated edrecolomab versus observation in patients with stage II CC and reported no survival benefit. Under an initial case-cohort sampling design, a randomly selected subcohort (RS) comprised 514 patients from 901 eligible patients with available tissue. Forty-nine additional patients with recurrence events were included in the analysis. Final analysis comprised 393 patients: 360 RS (58 events) and 33 non-RS events. Risk status was determined for each patient by ColDx. The Self-Prentice method was used to test the association between the resulting ColDx risk score and RFI adjusting for standard prognostic variables.

RESULTS: Fifty-five percent of patients (216 of 393) were classified as high risk. After adjustment for prognostic variables that included mismatch repair (MMR) deficiency, ColDx high-risk patients exhibited significantly worse RFI (multivariable hazard ratio, 2.13; 95% CI, 1.3 to 3.5; P < .01). Age and MMR status were marginally significant. RFI at 5 years for patients classified as high risk was 82% (95% CI, 79% to 85%), compared with 91% (95% CI, 89% to 93%) for patients classified as low risk.

CONCLUSION: ColDx is associated with RFI in the C9581 subsample in the presence of other prognostic factors, including MMR deficiency. ColDx could be incorporated with the traditional clinical markers of risk to refine patient prognosis.

A Phase II Biomarker-Embedded Study of Lapatinib plus Capecitabine as First-line Therapy in Patients with Advanced or Metastatic Gastric Cancer

An exploratory phase II biomarker-embedded trial (LPT109747; NCT00526669) designed to determine the association of lapatinib-induced fluoropyrimidine gene changes with efficacy of lapatinib plus capecitabine as first-line treatment for advanced gastric cancer or gastroesophageal junction adenocarcinoma independent of tumor HER2 status. Tumor biopsies obtained before and after 7-day lapatinib (1,250 mg) to analyze changes in gene expression, followed by a 14-day course of capecitabine (1,000 mg/m(2) twice daily, 14/21 days) plus lapatinib 1,250 mg daily. Blood samples were acquired for pharmacokinetic analysis. Primary clinical objectives were response rate (RR) and 5-month progression-free survival (PFS). Secondary objectives were overall survival (OS), PFS, time to response, duration of response, toxicity, and identification of associations between lapatinib pharmacokinetics and biomarker endpoints. Primary biomarker objectives were modulation of 5-FU-pathway genes by lapatinib, effects of germline SNPs on treatment outcome, and trough steady-state plasma lapatinib concentrations. Sixty-eight patients were enrolled; (75% gastric cancer, 25% gastroesophageal junction). Twelve patients (17.9%) had confirmed partial response, 31 (46.3%) had stable disease, and 16 (23.9%) had progressive disease. Median PFS and OS were 3.3 and 6.3 months, respectively. Frequent adverse events included diarrhea (45%), decreased appetite (39%), nausea (36%), and fatigue (36%). Lapatinib induced no changes in gene expression from baseline and no significant associations were found for SNPs analyzed. Elevated baseline HER3 mRNA expression was associated with a higher RR (33% vs. 0%; P = 0.008). Lapatinib plus capecitabine was well tolerated, demonstrating modest antitumor activity in patients with advanced gastric cancer. The association of elevated HER3 and RR warrants further investigation as an important player for HER-targeted regimens in combination with capecitabine