Duplication of 7q34 is specific to juvenile pilocytic astrocytomas and a hallmark of cerebellar and optic pathway tumours

BACKGROUND: Juvenile pilocytic astrocytomas (JPA), a subgroup of low-grade astrocytomas (LGA), are common, heterogeneous and poorly understood subset of brain tumours in children. Chromosomal 7q34 duplication leading to fusion genes formed between KIAA1549 and BRAF and subsequent constitutive activation of BRAF was recently identified in a proportion of LGA, and may be involved in their pathogenesis. Our aim was to investigate additional chromosomal unbalances in LGA and whether incidence of 7q34 duplication is associated with tumour type or location. METHODS AND RESULTS: Using Illumina-Human-Hap300-Duo and 610-Quad high-resolution-SNP-based arrays and quantitative PCR on genes of interest, we investigated 84 paediatric LGA. We demonstrate that 7q34 duplication is specific to sporadic JPA (35 of 53-66%) and does not occur in other LGA subtypes (0 of 27) or NFI-associated-JPA (0 of 4). We also establish that it is site specific as it occurs in the majority of cerebellar JPA (24 of 30-80%) followed by brainstem, hypothalamic/optic pathway JPA (10 of 16-62.5%) and is rare in hemispheric JPA (1 of 7-14%). The MAP-kinase pathway, assessed through ERK phosphorylation, was active in all tumours regardless of 7q34 duplication. Gain of function studies performed on hTERT-immortalised astrocytes show that overexpression of wild-type BRAF does not increase cell proliferation or baseline MAPK signalling even if it sensitises cells to EGFR stimulation. CONCLUSIONS AND INTERPRETATION: Our results suggest that variants of JPA might arise from a unique site-restricted progenitor cell where 7q34 duplication, a hallmark of this tumour-type in association to MAPK-kinase pathway activation, potentially plays a site-specific role in their pathogenesis. Importantly, gain of function abnormalities in components of MAP-Kinase signalling are potentially present in all JPA making this tumour amenable to therapeutic targeting of this pathway. British Journal of Cancer (2009) 101, 722-733. doi: 10.1038/sj.bjc.6605179 www.bjcancer.com Published online 14 July 2009 (C) 2009 Cancer Research U

Multi-Modal Proteomic Analysis of Retinal Protein Expression Alterations in a Rat Model of Diabetic Retinopathy

As a leading cause of adult blindness, diabetic retinopathy is a prevalent and profound complication of diabetes. We have previously reported duration-dependent changes in retinal vascular permeability, apoptosis, and mRNA expression with diabetes in a rat model system. The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies.A multi-modal proteomic approach of antibody (Luminex)-, electrophoresis (DIGE)-, and LC-MS (iTRAQ)-based quantitation methods was used to maximize coverage of the retinal proteome. Transcriptomic profiling through microarray analysis was included to identify additional targets and assess potential regulation of protein expression changes at the mRNA level. The proteomic approaches proved complementary, with limited overlap in proteomic coverage. Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts. In an independent experiment, insulin replacement therapy normalized the expression of some proteins (Dbi, Anxa5) while other proteins (Cp, Cryba3, Lgals3, Stat3) were only partially normalized and Fgf2 and Crybb2 expression remained elevated.These results expand the understanding of the changes in retinal protein expression occurring with diabetes and their responsiveness to normalization of blood glucose through insulin therapy. These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies

Levels of DNA cytosine methylation in the Drosophila genome

Insects provide an accessible system to study the contribution of DNA methylation to complex epigenetic phenotypes created to regulate gene expression, chromatin states, imprinting and dosage compensation. The members of genus Drosophila have been used as a model system to study aspects of biology like development, behaviour and genetics. Despite the popularity of Drosophila melanogaster as a genetic and epigenetic model organism, DNA methylation studies are limited due to low levels of genomic 5-methylcytosine. Our study employs a sensitive liquid chromatography-mass spectrometry (LCMS) based method to quantify the levels of 5-methylcytosine from the genomic DNA in different members of the genus Drosophila. Our results reveal that, despite being phylogenetically related, there is a marked variation in the levels of 5-methylcytosine between the genomes of the members of genus Drosophila. Also, there is a change in the genomic levels of 5-methylcytosine through each life cycle stage of holometabolous development in D. melanogaster

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Not AvailablePhysiological and biochemical changes during fruit growth, development and maturity of eleven genotypes of guava fruit were studied at 30, 60, 90, 105, 120 and 127 days after fruit set (DAFS). Fruit weight, length and diameter of guava fruit increased continuously from the initial stage of fruit development till maturity and ripening in all the genotypes. The increases in fruit weight and diameter were comparatively more between 30 to 60 DAFS and 90 to 127 DAFS than 60 to 90 DAFS where the increase in fruit weight and diameter was slow in most of the genotypes. However, RCG-1, RCG-2 and RCG-3 showed the continued rapid increase in fruit weight upto 120 days and thereafter the rate of increase was quite slow. A gradual increase in total soluble solids (TSS) was observed in all the genotypes throughout development and ripening stage of the fruits. However, the amount of total titratable acidity increased steadily in the beginning upto 105 days after fruit set in most of the genotypes except RCG-1, RCG-2 and RCG-3 which showed upto 90 days only and afterwards there was a continuous reduction till ripening. The fruits showed continuous and progressive increase in TSS: acid ratio from 30 DAFS till maturity and ripening in all the genotypes except RCG-11, RCGH-1 and RCGH-7 that showed from 60 DAFS. Based on the present findings, days taken from flowering to harvest maturity (>105 days), TSS (>9.50%) and TSS: acid ratio (>15.00) were some of the parameters for judging the maturity indices of genotypes like RCG-1, RCG-2 and RCG-3. Similarly, days taken from flowering to harvest maturity (>110 days), TSS (>10.50%), TSS: acid ratio (>21.00) and fruit skin colour (whitish green) for RCGH-1 were some of the parameters for judging the maturity indices. Whereas, other genotypes were also exhibited the variation for days taken from flowering to harvest maturity (>120 days), skin colours, TSS (9.20-11.00%) and TSS: acid ratio (13.50-23.50).Not Availabl

Spreading of antibody reactivity to non-thyroid antigens during experimental immunization with human thyroglobulin

Intermolecular spreading of antibody reactivity has been implicated in the evolution of autoimmune disease. In this study, spreading of antibody reactivity to non-thyroid autoantigens after experimental immunization with thyroglobulin (Tg) was investigated. For this purpose, two rabbits were injected with human Tg six times (stages 1–6) every 3 weeks. Animals were also bled before priming. Antisera were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity to several non-thyroid antigens: bovine serum albumin (BSA), native DNA (nDNA), human myosin, human globular (G) and filamentous (F) actin and porcine tubulin. Tg-immunized animals developed the following serological reactivity pattern: (a) high reactivity to myosin from stage 2 onward, (b) significant reactivity to F-actin, remaining high up to stage 6, (c) reactivity to BSA with a peak at stage 3, (d) a small increase of reactivity to G-actin at stage 3 and (e) no increase of reactivity to nDNA and tubulin. The study of affinity-purified anti-Tg antibodies and the use of competitive assays revealed that reactivity to F-actin was not due to cross-reaction with Tg. On the contrary, reactivity to myosin during the first stages of immunization was due to cross-reaction with Tg, while at stage 6 it became myosin-specific. Reactivity to BSA at stage 3 was also due to cross-reaction with Tg. We conclude that at least part of the induced anti-Tg antibodies may result from the expansion of B cell clones producing polyreactive natural autoantibodies, and polyreactivity of anti-Tg antibodies during the first stages of Tg-immunization may be responsible for the intermolecular spreading of antibody response