Association of the FCRL3 gene with rheumatoid arthritis: a further example of population specificity?

Association of a functional promoter polymorphism mapping to the Fc receptor-like 3 (FCRL3) gene has recently been reported and replicated with rheumatoid arthritis (RA) in Japanese populations. The aim of this study was to investigate association of the FCRL3 gene with RA in UK subjects. DNA was available from 1065 patients with RA and 2073 population controls from the UK. Four single nucleotide polymorphism (SNP) markers (FCRL3-169*C/T (fclr3_3, rs7528684), fclr3_4 (rs11264799), fclr3_5 (rs945635), fclr3_6 (rs3761959)) all previously associated with RA in a Japanese population were genotyped in 761 RA samples and 484 controls. In the remaining samples, only the putative disease causal polymorphism, FCRL3-169*C/T, was tested. Genotyping was performed using either the Sequenom MassArray iPlex platform or a 5' Allelic discrimination assay (Taqman, ABI). Extensive linkage disequilibrium was present across the promoter SNPs genotyped (r² values = 0.60-0.98). Allele frequencies did not differ between RA cases and controls either for the putative disease causal polymorphism (odds ratio FCRL3-169*C allele = 0.97 (0.87-1.07), p = 0.51) or for the other SNPs tested. Similarly, no association was detected with RA using haplotype analysis or when stratification by shared epitope carriage or by presence of rheumatoid factor was undertaken. This study was powered to detect an effect size of 1.24 or greater for the FCRL3-169*C/T functional promoter polymorphism but no evidence for association was detected, suggesting that this gene will not have a substantial effect in determining susceptibility to RA in populations of Northern European descent.     

Yields of damage to C4' deoxyribose and to pyrimidines in pUC18 by the direct effect of ionizing radiation

Our mechanistic understanding of damage formation in DNA by the direct effect relies heavily on what is known of free radical intermediates studied by EPR spectroscopy. Bridging this information to stable product formation requires methods with comparable sensitivities, a criterion met by the (32)P-post-labeling assay developed by Weinfeld and Soderlind, [Weinfeld,M. and Soderlind,K.-J.M. (1991) (32)P-Postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and phosphoglycolate termini. Biochemistry, 30, 1091-1097] which when applied to the indirect effect, detected phosphoglycolate (pg) and thymine glycol (Tg). Here we applied this assay to the direct effect, measuring product yields in pUC18 films with hydration levels (Γ) of 2.5, 16 or 23 waters per nucleotide and X-irradiated at either 4 K or room temperature (RT). The yields of pg [G(pg)] for Γ ≈ 2.5 were 2.8 ± 0.2 nmol/J (RT) and 0.2 ± 0.3 nmol/J (4 K), which is evidence that the C4' radical contributes little to the total deoxyribose damage via the direct effect. The yield of detectable base damage [G(B*)] at Γ ≈ 2.5 was found to be 30.2 ± 1.0 nmol/J (RT) and 12.9 ± 0.7 nmol/J (4 K). While the base damage called B*, could be due to either oxidation or reduction, we argue that two reduction products, 5,6-dihydrouracil and 5,6-dihydrothymine, are the most likely candidates.     

Apomixis and reticulate evolution in the Asplenium monanthes fern complex

Background and Aims

Asexual reproduction is a prominent evolutionary process within land plant lineages and especially in ferns. Up to 10 % of the approx. 10 000 fern species are assumed to be obligate asexuals. In the Asplenium monanthes species complex, previous studies identified two triploid, apomictic species. The purpose of this study was to elucidate the phylogenetic relationships in the A. monanthes complex and to investigate the occurrence and evolution of apomixis within this group.

Methods

DNA sequences of three plastid markers and one nuclear single copy gene were used for phylogenetic analyses. Reproductive modes were assessed by examining gametophytic and sporophyte development, while polyploidy was inferred from spore measurements.

Key Results

Asplenium monanthes and A. resiliens are confirmed to be apomictic. Asplenium palmeri, A. hallbergii and specimens that are morphologically similar to A. heterochroum are also found to be apomictic. Apomixis is confined to two main clades of taxa related to A. monanthes and A. resiliens, respectively, and is associated with reticulate evolution. Two apomictic A. monanthes lineages, and two putative diploid sexual progenitor species are identified in the A. monanthes clade.

Conclusions

Multiple origins of apomixis are inferred, in both alloploid and autoploid forms, within the A. resiliens and A. monanthes clades.     

Phase I trial of tremelimumab in combination with short-term androgen deprivation in patients with PSA-recurrent prostate cancer

CTLA-4 blockade has demonstrated antitumor efficacy in human clinical trials. The antitumor mechanism is presumably mediated in part by the expansion of tumor-specific T cells. Androgen deprivation, the cornerstone of treatment for patients with metastatic prostate cancer, has been shown to elicit prostate tissue apoptosis and lymphocytic inflammation. We hypothesized that treatment with androgen deprivation, followed by an anti-CTLA-4 antibody, could augment a tumor-specific immune response elicited by androgen deprivation. We report here the results of a phase I trial evaluating a humanized monoclonal antibody targeting CTLA-4, CP-675,206 (tremelimumab), in combination with androgen deprivation using an antiandrogen. Eligible patients were those with PSA-recurrent prostate cancer after primary surgery and/or radiation therapy, not previously treated with androgen deprivation, and without radiographic evidence of metastatic disease. Subjects were treated in two cycles, 3 months apart, in which they received bicalutamide 150 mg daily days 1-28 and tremelimumab on day 29. The primary endpoint of the trial was safety. Secondary endpoints included measures of PSA kinetics and identification of a maximum tolerated dose. Eleven patients were enrolled and completed at least 1 year of follow-up. Dose-limiting toxicities included grade 3 diarrhea and skin rash. No favorable changes in PSA doubling time were observed in a period shortly after completing treatment; however, three patients experienced a prolongation in PSA doubling time detectable several months after completing treatment. The identification of delayed, prolonged favorable changes in serum PSA suggests that future studies could explore this combination in studies evaluating time to disease progression.     

Identification of candidate SNPs for drug induced toxicity from differentially expressed genes in associated tissues

The growing collection of publicly available high-throughput data provides an invaluable resource for generating preliminary in silico data in support of novel hypotheses. In this study we used a cross-dataset meta-analysis strategy to identify novel candidate genes and genetic variations relevant to paclitaxel/carboplatin-induced myelosuppression and neuropathy. We identified genes affected by drug exposure and present in tissues associated with toxicity. From ten top-ranked genes 42 non-synonymous single nucleotide polymorphisms (SNPs) were identified in silico and genotyped in 94 cancer patients treated with carboplatin/paclitaxel. We observed variations in 11 SNPs, of which seven were present in a sufficient frequency for statistical evaluation. Of these seven SNPs, three were present in ABCA1 and ATM, and showed significant or borderline significant association with either myelosuppression or neuropathy. The strikingly high number of associations between genotype and clinically observed toxicity provides support for our data-driven computations strategy to identify biomarkers for drug toxicity.     

Activation of the Arabidopsis thaliana mitogen-activated protein kinase MPK11 by the flagellin-derived elicitor peptide, flg22

Mitogen-activated protein kinases (MAPK) mediate cellular signal transduction during stress responses, as well as diverse growth and developmental processes in eukaryotes. Pathogen infection or treatments with conserved pathogen-associated molecular patterns (PAMPs) such as the bacterial flagellin-derived flg22 peptide are known to activate three Arabidopsis thaliana MAPK: MPK3, MPK4, and MPK6. Several stresses, including flg22 treatment, are known to increase MPK11 expression but activation of MPK11 has not been shown. Here, we show that MPK11 activity can, indeed, be increased through flg22 elicitation. A small-scale microarray for profiling defense-related genes revealed that cinnamyl alcohol dehyrogenase 5 requires MPK11 for full flg22-induced expression. An mpk11 mutant showed increased flg22-mediated growth inhibition but no altered susceptibility to Pseudomonas syringae, Botrytis cinerea, or Alternaria brassicicola. In mpk3, mpk6, or mpk4 backgrounds, MPK11 is required for embryo or seed development or general viability. Although this developmental deficiency in double mutants and the lack of or only subtle mpk11 phenotypes suggest functional MAPK redundancies, comparison with the paralogous MPK4 reveals distinct functions. Taken together, future investigations of MAPK roles in stress signaling should include MPK11 as a fourth PAMP-activated MAPK.     

Analysis of substrate specificity and cyclin Y binding of PCTAIRE-1 kinase

PCTAIRE-1 (cyclin-dependent kinase [CDK] 16) is a highly conserved serine/threonine kinase that belongs to the CDK family of protein kinases. Little is known regarding PCTAIRE-1 regulation and function and no robust assay exists to assess PCTAIRE-1 activity mainly due to a lack of information regarding its preferred consensus motif and the lack of bona fide substrates. We used positional scanning peptide library technology and identified the substrate-specificity requirements of PCTAIRE-1 and subsequently elaborated a peptide substrate termed PCTAIRE-tide. Recombinant PCTAIRE-1 displayed vastly improved enzyme kinetics on PCTAIRE-tide compared to a widely used generic CDK substrate peptide. PCTAIRE-tide also greatly improved detection of endogenous PCTAIRE-1 activity. Similar to other CDKs, PCTAIRE-1 requires a proline residue immediately C-terminal to the phosphoacceptor site (+1) for optimal activity. PCTAIRE-1 has a unique preference for a basic residue at +4, but not at +3 position (a key characteristic for CDKs). We also demonstrate that PCTAIRE-1 binds to a novel cyclin family member, cyclin Y, which increased PCTAIRE-1 activity towards PCTAIRE-tide >100-fold. We hypothesised that cyclin Y binds and activates PCTAIRE-1 in a way similar to which cyclin A2 binds and activates CDK2. Point mutants of cyclin Y predicted to disrupt PCTAIRE-1-cyclin Y binding severely prevented complex formation and activation of PCTAIRE-1. We have identified PCTAIRE-tide as a powerful tool to study the regulation of PCTAIRE-1. Our understanding of the molecular interaction between PCTAIRE-1 and cyclin Y further facilitates future investigation of the functions of PCTAIRE-1 kinase.     

Cross-talk between insulin and Wnt signaling in preadipocytes: role of Wnt co-receptor low density lipoprotein receptor-related protein-5 (LRP5)

Disturbed Wnt signaling has been implicated in numerous diseases, including type 2 diabetes and the metabolic syndrome. In the present study, we have investigated cross-talk between insulin and Wnt signaling pathways using preadipocytes with and without knockdown of the Wnt co-receptors LRP5 and LRP6 and with and without knock-out of insulin and IGF-1 receptors. We find that Wnt stimulation leads to phosphorylation of insulin signaling key mediators, including Akt, GSK3β, and ERK1/2, although with a lower fold stimulation and slower time course than observed for insulin. These Wnt effects are insulin/IGF-1 receptor-dependent and are lost in insulin/IGF-1 receptor double knock-out cells. Conversely, in LRP5 knockdown preadipocytes, insulin-induced phosphorylation of IRS1, Akt, GSK3β, and ERK1/2 is highly reduced. This effect is specific to insulin, as compared with IGF-1, stimulation and appears to be due to an inducible interaction between LRP5 and the insulin receptor as demonstrated by co-immunoprecipitation. These data demonstrate that Wnt and insulin signaling pathways exhibit cross-talk at multiple levels. Wnt induces phosphorylation of Akt, ERK1/2, and GSK3β, and this is dependent on insulin/IGF-1 receptors. Insulin signaling also involves the Wnt co-receptor LRP5, which has a positive effect on insulin signaling. Thus, altered Wnt and LRP5 activity can serve as modifiers of insulin action and insulin resistance in the pathophysiology of diabetes and metabolic syndrome.