Comprehensive molecular characterization of gastric adenocarcinoma

Gastric cancer is a leading cause of cancer deaths, but analysis of its molecular and clinical characteristics has been complicated by histological and aetiological heterogeneity. Here we describe a comprehensive molecular evaluation of 295 primary gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) project. We propose a molecular classification dividing gastric cancer into four subtypes: tumours positive for Epstein–Barr virus, which display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and PDCD1LG2 (also knownasPD-L2); microsatellite unstable tumours, which show elevated mutation rates, including mutations of genes encoding targetable oncogenic signalling proteins; genomically stable tumours, which are enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins; and tumours with chromosomal instability, which show marked aneuploidy and focal amplification of receptor tyrosine kinases. Identification of these subtypes provides a roadmap for patient stratification and trials of targeted therapies

Comprehensive Pan-Genomic Characterization of Adrenocortical Carcinoma

SummaryWe describe a comprehensive genomic characterization of adrenocortical carcinoma (ACC). Using this dataset, we expand the catalogue of known ACC driver genes to include PRKAR1A, RPL22, TERF2, CCNE1, and NF1. Genome wide DNA copy-number analysis revealed frequent occurrence of massive DNA loss followed by whole-genome doubling (WGD), which was associated with aggressive clinical course, suggesting WGD is a hallmark of disease progression. Corroborating this hypothesis were increased TERT expression, decreased telomere length, and activation of cell-cycle programs. Integrated subtype analysis identified three ACC subtypes with distinct clinical outcome and molecular alterations which could be captured by a 68-CpG probe DNA-methylation signature, proposing a strategy for clinical stratification of patients based on molecular markers

Rapid and efficient <em>Agrobacterium </em> mediated transformation of early scutellum derived calli of <em>indica</em> rice

20-28Rice is a staple food for humans and its demand in 2035 has been put at 852 million tons. Knowledge on genes and genome architecture helps in better understanding of growth and development mechanisms for crop improvement. Transgenic crops may offer a solution by means of higher yield and resistance to biotic and abiotic stresses. In this context, modification of Agrobacterium mediated transformation protocol for indica rice cultivar is imperative to increase transformation efficiency and reduce duration of transgenic development. Here, we developed an efficient Agrobacterium mediated transformation protocol using early scutellum derived calli of the indica rice cultivar Pusa Sugandh 2. Competency of 3, 4, 5 and 6 day old primary calli was compared with 21- day old secondary calli for Agrobacterium mediated transformation using a modified pCAMBIA 1304 harbouring GFP-GUS fusion gene driven by maize ubiquitin 1 promoter. The highest competency with stable transformation efficiency of 51% was observed for 5-6 day old primary calli. Molecular analysis confirmed stable integration of the transgene. Transgenic lines of Pusa Sugandh 2 were developed within a short period of two months using 5-6 day old primary calli

Silencing of <i>HaAce1</i> gene by host-delivered artificial microRNA disrupts growth and development of <i>Helicoverpa armigera</i>

<div><p>The polyphagous insect-pest, <i>Helicoverpa armigera</i>, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of <i>Bt</i> transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement <i>Bt</i> to tackle insect-pests menace. In this study, we report host-delivered silencing of <i>HaAce1</i> gene, encoding the predominant isoform of <i>H</i>. <i>armigera</i> acetylcholinesterase, by an artificial microRNA, <i>HaAce1</i>-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with <i>HaAce1</i>-amiR1/<i>HaAce1</i>-amiR1* sequences. The recombinant <i>HaAce1</i>-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing <i>HaAce1</i>-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of <i>HaAce1</i> mRNA was 70–80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of <i>HaAce1</i> gene for <i>H</i>. <i>armigera</i> management.</p></div

Designing and construction of pBI::HAR1.

<p>Primer extension PCR was done with a pair of overlapping primers, <i>HAR-</i>F & <i>HAR</i>-R (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194150#pone.0194150.s006" target="_blank">S1 Table</a>) containing <i>Xba</i>I and <i>Sac</i>I recognition sequences at their 5′ ends. <i>Xba</i>I and <i>Sac</i>I digested PCR product was ligated onto pUC19. pBI121 backbone was generated by digestion with <i>Xba</i>I and <i>Sac</i>I that released <i>GUS</i> coding sequence. After sequence confirmation, <i>HaAce1</i>-preamiRNA1 DNA fragment was released from recombinant pUC19 and ligated onto pBI121 backbone to get pBI::HAR1.</p

Microarray analysis of the astrocyte transcriptome in the aging brain: Relationship to Alzheimer's pathology and APOE genotype

Astrocytes contribute to a variety of functions in the brain, including homeostasis, synapse formation, plasticity, and metabolism. Astrocyte dysfunction may disrupt their normal role, including neuronal support, thereby contributing to neurodegenerative pathologies, including Alzheimer's disease (AD). To understand the role of astrocytes in the pathogenesis of age-related disorders, we isolated astrocytes by laser capture microdissection, using glial fibrillary acidic protein (GFAP) as a marker, and characterized the astrocyte transcriptome at different Braak neurofibrillary tangle stages in postmortem temporal cortex samples derived from the Medical Research Council Cognitive Function and Ageing Study (MRC CFAS) cohort, using microarray analysis. The largest number of significant, differentially expressed genes were identified when the expression profile of astrocytes from isocortical stages of neurofibrillary tangle pathology (Braak stages V-VI) were compared with entorhinal stages (Braak stages I-II). Dysregulation of genes associated with the actin cytoskeleton, proliferation, apoptosis, and ubiquitin-mediated proteolysis occurred at low Braak stages, while altered regulation of intracellular signaling pathways, including insulin, phosphatidylinositol 3-kinase (PI3K)/Akt, and mitogen-activated protein kinase (MAPK) pathways were primarily associated with high levels of Alzheimer-type pathology, and occurred at lower Braak stages in individuals with the APOEε4 allele. Our findings implicate astrocyte dysfunction in the pathogenesis of neurodegenerative pathology in the aging brain, and provide a basis for future candidate studies based on specific pathways. © 2011 Elsevier Inc

Expression of <i>HaAce1</i>-amiR1 in transgenic tobacco lines and down regulation of <i>HaAce1</i> in <i>H</i>. <i>armigera</i> adults.

<p>(A) Northern blot showing <i>HaAce1</i>-amiR1 expression in the transgenic tobacco lines, 17R1 and 29R1. Small RNAs were hybridized with DIG end labelled Anti-amiR<i>Ace</i> probe of 21 nt. C, pBI121 transformed tobacco (vector control); 29R1 & 17R1, <i>HaAce1</i>-amiR1 expression in the selected two transgenic tobacco line, 29R1 and 17R1, respectively; P, labelled Anti-amiR<i>Ace</i> probe. (B) Relative expression of <i>HaAce1</i> in the <i>H</i>. <i>armigera</i> adults. Vector control, <i>HaAce1</i> transcript abundance in <i>H</i>. <i>armigera</i> adults emerged from vector control tobacco treated group; 17R1 and 29R1, <i>HaAce1</i> transcript abundance in deformed <i>H</i>. <i>armigera</i> adults emerged from 17R1 and 29R1 transgenic tobacco treatment groups, respectively. <i>β</i>-<i>Actin</i> gene of <i>H</i>. <i>armigera</i> was used as an internal control. Real time PCR data was analysed using delta-delta Ct method. One way ANOVA test was used to perform the statistical analysis of the data. *** Extremely significant at P<0.001. The test was performed three times.</p

Mortality and developmental deformity in <i>H</i>. <i>armigera</i>.

<p>Thirty second instar larvae of <i>H</i>. <i>armigera</i> were fed continuously on each transgenic line (Line 17R1 and 29R1) and vector control separately (one larva per plate) till their active feeding stage<b>.</b> (A) Mortality percentage of <i>H</i>. <i>armigera</i> larvae. Vector control, mortality percentage in larvae fed on pBI121 transformed tobacco leaves; 17R1, mortality percentage in larvae fed on 17R1 transgenic tobacco line; 29R1, mortality percentage in larvae fed on 29R1 transgenic tobacco line. (B) Deformity percentage of <i>H</i>. <i>armigera</i> adults. Vector control, percentage of emergence of deformed adults from larvae fed on pBI121 transformed tobacco leaves; 17R1, percentage of emergence of deformed adults from larvae fed on 17R1 transgenic tobacco line; 29R1, percentage of emergence of deformed adults from larvae fed on 29R1 transgenic tobacco line. (C) Phenotype of <i>H</i>. <i>armigera</i> adults. Upper panel: Normal adults developed from vector control fed larvae; Lower panel: Deformed adults developed from transgenic fed larvae. One way ANOVA test was used to perform statistical analysis of the data. *** denotes extremely significant differences at P<0.001. The test was repeated three times.</p

Loss of nuclear TDP-43 in amyotrophic lateral sclerosis (ALS) causes altered expression of splicing machinery and widespread dysregulation of RNA splicing in motor neurones

Loss of nuclear TDP-43 characterizes sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether (1) RNA splicing dysregulation is present in lower motor neurones in ALS and in a motor neurone-like cell model; and (2) TARDBP mutations (mtTARDBP) are associated with aberrant RNA splicing using patient-derived fibroblasts. Affymetrix exon arrays were used to study mRNA expression and splicing in lower motor neurones obtained by laser capture microdissection of autopsy tissue from individuals with sporadic ALS and TDP-43 proteinopathy. Findings were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in NSC34 motor neuronal cells following shRNA-mediated TDP-43 depletion. Exon arrays and immunohistochemistry were used to study mRNA splicing and TDP-43 expression in fibroblasts from patients with mtTARDBP-associated, sporadic and mutant SOD1-associated ALS. We found altered expression of spliceosome components in motor neurones and widespread aberrations of mRNA splicing that specifically affected genes involved in ribonucleotide binding. This was confirmed in TDP-43-depleted NSC34 cells. Fibroblasts with mtTARDBP showed loss of nuclear TDP-43 protein and demonstrated similar changes in splicing and gene expression, which were not present in fibroblasts from patients with sporadic or SOD1-related ALS. Loss of nuclear TDP-43 is associated with RNA processing abnormalities in ALS motor neurones, patient-derived cells with mtTARDBP, and following artificial TDP-43 depletion, suggesting that splicing dysregulation directly contributes to disease pathogenesis. Key functional pathways affected include those central to RNA metabolis