Coincident sequence-specific RNA degradation of linked transgenes in the plant genome

The expression of transgenes in plant genomes can be inhibited by either transcriptional gene silencing or posttranscriptional gene silencing (PTGS). Overexpression of the chalcone synthase-A (CHS-A) transgene triggers PTGS of CHS-A and thus results in loss of flower pigmentation in petunia. We previously demonstrated that epigenetic inactivation of CHS-A transgene transcription leads to a reversion of the PTGS phenotype. Although neomycin phosphotransferase II (nptII), a marker gene co-introduced into the genome with the CHS-A transgene, is not normally silenced in petunia, even when CHS-A is silenced, here we found that nptII was silenced in a petunia line in which CHS-A PTGS was induced, but not in the revertant plants that had no PTGS of CHS-A. Transcriptional activity, accumulation of short interfering RNAs, and restoration of mRNA level after infection with viruses that had suppressor proteins of gene silencing indicated that the mechanism for nptII silencing was posttranscriptional. Read-through transcripts of the CHS-A gene toward the nptII gene were detected. Deep-sequencing analysis revealed a striking difference between the predominant size class of small RNAs produced from the read-through transcripts (22 nt) and that from the CHS-A RNAs (21 nt). These results implicate the involvement of read-through transcription and distinct phases of RNA degradation in the coincident PTGS of linked transgenes and provide new insights into the destabilization of transgene expression.

Epigenetic inactivation of chalcone synthase-A transgene transcription in petunia leads to a reversion of the post-transcriptional gene silencing phenotype

Petunia plants that exhibit a white-flowering phenotype as a consequence of chalcone synthase transgene-induced silencing occasionally give rise to revertant branches that produce flowers with wild-type pigmentation. Transcription run-on assays confirmed that the production of white flowers is caused by post-transcriptional gene silencing (PTGS), and indicated that transgene transcription is repressed in the revertant plants, providing evidence that induction of PTGS depends on the transcription rate. Transcriptional repression of the transgene was associated with cytosine methylation at CpG, CpNpG and CpNpN sites, and the expression was restored by treatment with either 5-azacytidine or trichostatin A. These results demonstrate that epigenetic changes occurred in the PTGS line, and these changes interfere with the initiation of transgene transcription, leading to a reversion of the PTGS phenotype.

Transcript and metabolite profiling for the evaluation of tobacco tree and poplar as feedstock for the bio-based industry

The global demand for food, feed, energy and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Role of intratumoural heterogeneity in cancer drug resistance : molecular and clinical perspectives

Drug resistance continues to be a major barrier to the delivery of curative therapies in cancer. Historically, drug resistance has been associated with over-expression of drug transporters, changes in drug kinetics or amplification of drug targets. However, the emergence of resistance in patients treated with new-targeted therapies has provided new insight into the complexities underlying cancer drug resistance. Recent data now implicate intratumoural heterogeneity as a major driver of drug resistance. Single cell sequencing studies that identified multiple genetically distinct variants within human tumours clearly demonstrate the heterogeneous nature of human tumours. The major contributors to intratumoural heterogeneity are (i) genetic variation, (ii) stochastic processes, (iii) the microenvironment and (iv) cell and tissue plasticity. Each of these factors impacts on drug sensitivity. To deliver curative therapies to patients, modification of current therapeutic strategies to include methods that estimate intratumoural heterogeneity and plasticity will be essential.

A framework for identifying associations in digital evidence using metadata

Digital forensics concerns the analysis of electronic artifacts to reconstruct events such as cyber crimes. This research produced a framework to support forensic analyses by identifying associations in digital evidence using metadata. It showed that metadata based associations can help uncover the inherent relationships between heterogeneous digital artifacts thereby aiding reconstruction of past events by identifying artifact dependencies and time sequencing. It also showed that metadata association based analysis is amenable to automation by virtue of the ubiquitous nature of metadata across forensic disk images, files, system and application logs and network packet captures. The results prove that metadata based associations can be used to extract meaningful relationships between digital artifacts, thus potentially benefiting real-life forensics investigations.

How to sequence and annotate insect mitochondrial genomes for systematic and comparative genomics research

Over the past decade the mitochondrial (mt) genome has become the most widely used genomic resource available for systematic entomology. While the availability of other types of ‘–omics’ data – in particular transcriptomes – is increasing rapidly, mt genomes are still vastly cheaper to sequence and are far less demanding of high quality templates. Furthermore, almost all other ‘–omics’ approaches also sequence the mt genome, and so it can form a bridge between legacy and contemporary datasets. Mitochondrial genomes have now been sequenced for all insect orders, and in many instances representatives of each major lineage within orders (suborders, series or superfamilies depending on the group). They have also been applied to systematic questions at all taxonomic scales from resolving interordinal relationships (e.g. Cameron et al., 2009; Wan et al., 2012; Wang et al., 2012), through many intraordinal (e.g. Dowton et al., 2009; Timmermans et al., 2010; Zhao et al. 2013a) and family-level studies (e.g. Nelson et al., 2012; Zhao et al., 2013b) to population/biogeographic studies (e.g. Ma et al., 2012). Methodological issues around the use of mt genomes in insect phylogenetic analyses and the empirical results found to date have recently been reviewed by Cameron (2014), yet the technical aspects of sequencing and annotating mt genomes were not covered. Most papers which generate new mt genome report their methods in a simplified form which can be difficult to replicate without specific knowledge of the field. Published studies utilize a sufficiently wide range of approaches, usually without justification for the one chosen, that confusion about commonly used jargon such as ‘long PCR’ and ‘primer walking’ could be a serious barrier to entry. Furthermore, sequenced mt genomes have been annotated (gene locations defined) to wildly varying standards and improving data quality through consistent annotation procedures will benefit all downstream users of these datasets. The aims of this review are therefore to: 1. Describe in detail the various sequencing methods used on insect mt genomes; 2. Explore the strengths/weakness of different approaches; 3. Outline the procedures and software used for insect mt genome annotation, and; 4. Highlight quality control steps used for new annotations, and to improve the re-annotation of previously sequenced mt genomes used in systematic or comparative research.

Analysis of albumin-associated peptides and proteins from ovarian cancer patients

Albumin binds low–molecular-weight molecules, including proteins and peptides, which then acquire its longer half-life, thereby protecting the bound species from kidney clearance. We developed an experimental method to isolate albumin in its native state and to then identify [mass spectrometry (MS) sequencing] the corresponding bound low–molecular-weight molecules. We used this method to analyze pooled sera from a human disease study set (high-risk persons without cancer, n= 40; stage I ovarian cancer, n = 30; stage III ovarian cancer, n = 40) to demonstrate the feasibility of this approach as a discovery method. Methods Albumin was isolated by solid-phase affinity capture under native binding and washing conditions. Captured albumin-associated proteins and peptides were separated by gel electrophoresis and subjected to iterative MS sequencing by microcapillary reversed-phase tandem MS. Selected albumin-bound protein fragments were confirmed in human sera by Western blotting and immunocompetition. Results In total, 1208 individual protein sequences were predicted from all 3 pools. The predicted sequences were largely fragments derived from proteins with diverse biological functions. More than one third of these fragments were identified by multiple peptide sequences, and more than one half of the identified species were in vivo cleavage products of parent proteins. An estimated 700 serum peptides or proteins were predicted that had not been reported in previous serum databases. Several proteolytic fragments of larger molecules that may be cancer-related were confirmed immunologically in blood by Western blotting and peptide immunocompetition. BRCA2, a 390-kDa low-abundance nuclear protein linked to cancer susceptibility, was represented in sera as a series of specific fragments bound to albumin. Conclusion Carrier-protein harvesting provides a rich source of candidate peptides and proteins with potential diverse tissue and cellular origins that may reflect important disease-related information.

Clinicopathological relevance of BRAF mutations in human cancer

BRAF represents one of the most frequently mutated protein kinase genes in human tumours. The mutation is commonly tested in pathology practice. BRAF mutation is seen in melanoma, papillary thyroid carcinoma (including papillary thyroid carcinoma arising from ovarian teratoma), ovarian serous tumours, colorectal carcinoma, gliomas, hepatobiliary carcinomas and hairy cell leukaemia. In these cancers, various genetic aberrations of the BRAF proto-oncogene, such as different point mutations and chromosomal rearrangements, have been reported. The most common mutation, BRAF V600E, can be detected by DNA sequencing and immunohistochemistry on formalin fixed, paraffin embedded tumour tissue. Detection of BRAF V600E mutation has the potential for clinical use as a diagnostic and prognostic marker. In addition, a great deal of research effort has been spent in strategies inhibiting its activity. Indeed, recent clinical trials involving BRAF selective inhibitors exhibited promising response rates in metastatic melanoma patients. Clinical trials are underway for other cancers. However, cutaneous side effects of treatment have been reported and therapeutic response to cancer is short-lived due to the emergence of several resistance mechanisms. In this review, we give an update on the clinical pathological relevance of BRAF mutation in cancer. It is hoped that the review will enhance the direction of future research and assist in more effective use of the knowledge of BRAF mutation in clinical practice.

Strategic targeting of the PI3K–NFκB axis in cisplatin-resistant NSCLC

Chemoresistance is a major therapeutic challenge to overcome in NSCLC, in order to improve the current survival rates of <15% at 5 years. We and others have shown increased PI3K signaling in NSCLC to be associated with a more aggressive disease, and a poorer prognosis. In this study, targeted inhibition of three strategic points of the PI3K–NFκB axis was performed with the aim of exploiting vulnerabilities in cisplatin-resistant NSCLC cells. Cisplatin-resistant cell lines were previously generated through prolonged exposure to the drug. Expression of PI3K and NFκB pathway-related genes were compared between cisplatin-resistant cells and their matched parent cells using a gene expression array, qRT-PCR, DNA sequencing, western blot, and immunofluorescence. Targeted inhibition was performed using GDC-0980, a dual PI3K–mTOR inhibitor currently in Phase II clinical trials in NSCLC, and DHMEQ, an inhibitor of NFκB translocation which has been used extensively both in vitro and in vivo. Effects of the two inhibitors were assessed by BrdU proliferation assay and multiparameter viability assay. NFKBIA was shown to be 12-fold overexpressed in cisplatin-resistant cells, with no mutations present in exons 3, 4, or 5 of the gene. Corresponding overexpression of IκBα was also observed. Treatment with DHMEQ (but not GDC-0980) led to significantly enhanced effects on viability and proliferation in cisplatin-resistant cells compared with parent cells. We conclude that NFκB inhibition represents a more promising strategy than PI3K–mTOR inhibition for treatment in the chemoresistance setting in NSCLC.

Correlation between BRAF mutation and the clinicopathological parameters in papillary thyroid carcinoma with particular reference to follicular variant

Mutation of the BRAF gene is common in thyroid cancer. Follicular variant of papillary thyroid carcinoma is a variant of papillary thyroid carcinoma that has created continuous diagnostic controversies among pathologists. The aims of this study are to (1) investigate whether follicular variant of papillary thyroid carcinoma has a different pattern of BRAF mutation than conventional papillary thyroid carcinoma in a large cohort of patients with typical features of follicular variant of papillary thyroid carcinoma and (2) to study the relationship of clinicopathological features of papillary thyroid carcinomas with BRAF mutation. Tissue blocks from 76 patients with diagnostic features of papillary thyroid carcinomas (40 with conventional type and 36 with follicular variant) were included in the study. From these, DNA was extracted and BRAF V600E mutations were detected by polymerase chain reaction followed by restriction enzyme digestion and sequencing of exon 15. Analysis of the data indicated that BRAF V600E mutation is significantly more common in conventional papillary thyroid carcinoma (58% versus 31%, P = .022). Furthermore, the mutation was often noted in female patients (P = .017), in high-stage cancers (P = .034), and in tumors with mild lymphocytic thyroiditis (P = .006). We concluded that follicular variant of papillary thyroid carcinoma differs from conventional papillary thyroid carcinoma in the rate of BRAF mutation. The results of this study add further information indicating that mutations in BRAF play a role in thyroid cancer development and progression.

Assembly and annotation of a non-model gastropod (Nerita melanotragus) transcriptome: a comparison of De novo assemblers

Background The sequencing, de novo assembly and annotation of transcriptome datasets generated with next generation sequencing (NGS) has enabled biologists to answer genomic questions in non-model species with unprecedented ease. Reliable and accurate de novo assembly and annotation of transcriptomes, however, is a critically important step for transcriptome assemblies generated from short read sequences. Typical benchmarks for assembly and annotation reliability have been performed with model species. To address the reliability and accuracy of de novo transcriptome assembly in non-model species, we generated an RNAseq dataset for an intertidal gastropod mollusc species, Nerita melanotragus, and compared the assembly produced by four different de novo transcriptome assemblers; Velvet, Oases, Geneious and Trinity, for a number of quality metrics and redundancy. Results Transcriptome sequencing on the Ion Torrent PGM™ produced 1,883,624 raw reads with a mean length of 133 base pairs (bp). Both the Trinity and Oases de novo assemblers produced the best assemblies based on all quality metrics including fewer contigs, increased N50 and average contig length and contigs of greater length. Overall the BLAST and annotation success of our assemblies was not high with only 15-19% of contigs assigned a putative function. Conclusions We believe that any improvement in annotation success of gastropod species will require more gastropod genome sequences, but in particular an increase in mollusc protein sequences in public databases. Overall, this paper demonstrates that reliable and accurate de novo transcriptome assemblies can be generated from short read sequencers with the right assembly algorithms. Keywords: Nerita melanotragus; De novo assembly; Transcriptome; Heat shock protein; Ion torrent

Pituitary tumours : all silent on the epigenetics front

Investigation of the epigenome of sporadic pituitary tumours is providing a more detailed understanding of aberrations that characterise this tumour type. Early studies, in this and other tumour types adopted candidate-gene approaches to characterise CpG island methylation as a mechanism responsible for or associated with gene silencing. However, more recently, investigators have adopted approaches that do not require a priori knowledge of the gene and transcript, as example differential display techniques, and also genome-wide, array-based approaches, to 'uncover' or 'unmask' silenced genes. Furthermore, through use of chromatin immunoprecipitation as a selective enrichment technique; we are now beginning to identify modifications that target the underlying histones themselves and that have roles in gene-silencing events. Collectively, these studies provided convincing evidence that change to the tumour epigenome are not simply epiphenomena but have functional consequences in the context of pituitary tumour evolution. Our ability to perform these types of studies has been and is increasingly reliant upon technological advances in the genomics and epigenomics arena. In this context, other more recent advances and developing technologies, and, in particular, next generation or flow cell re-sequencing techniques offer exciting opportunities for our future studies of this tumour type.

Health education action research (HEAR) framework

As a mandatory assessment technique of the Health Education Senior Syllabus (QSA, 2010), action research provides an opportunity for unique insights into health related knowledge, behaviours, beliefs, attitudes and values. It has the potential to be a very motivating and engaging form of learning and assessment. Despite these benefits, action research can be a daunting prospect for some students and a challenging form of assessment for teachers to conceptualise and construct. Session participants will be provided with a visual framework for understanding, structuring and sequencing action research projects. This framework will present a series of action research phases and map them against the four general objectives and three assessable dimensions of the syllabus (QSA, 2010. Participants are strongly encouraged to bring a copy of the syllabus for reference and use during this session. Reference Queensland Studies Authority. (2010). Health Education Senior Syllabus 2010. Brisbane: QSA.

A population-specific HTR2B stop codon predisposes to severe impulsivity

Impulsivity, describing action without foresight, is an important feature of several psychiatric diseases, suicidality and violent behaviour. The complex origins of impulsivity hinder identification of the genes influencing it and the diseases with which it is associated. Here we perform exon-focused sequencing of impulsive individuals in a founder population, targeting fourteen genes belonging to the serotonin and dopamine domain. A stop codon in HTR2B was identified that is common (minor allele frequency > 1%) but exclusive to Finnish people. Expression of the gene in the human brain was assessed, as well as the molecular functionality of the stop codon, which was associated with psychiatric diseases marked by impulsivity in both population and family-based analyses. Knockout of Htr2b increased impulsive behaviours in mice, indicative of predictive validity. Our study shows the potential for identifying and tracing effects of rare alleles in complex behavioural phenotypes using founder populations, and indicates a role for HTR2B in impulsivity.

Speckle-type POZ protein mutations interrupt tumor suppressor function of speckle-type POZ protein in prostate cancer by affecting androgen receptor degradation

Large scale exome sequencing studies have revealed regions of the genome, which contribute to the castrate resistant prostate cancer (CRPC) phenotype. [1],[2],[3] Such studies have identified mutations in genes, which may have diagnostic/prognostic potential, or which may be targeted therapeutically. Two of these genes include the androgen receptor (AR) and speckle-type POZ protein (SPOP) genes. However, the findings from these exome sequencing studies can only be translated therapeutically once the functional consequences of these mutations have been determined. Here, we highlight the recent study by An et al. [4] which investigated the functional effects of mutations in the SPOP gene that were identified in the aforementioned exome sequencing studies, particularly in the context of SPOP-mediated degradation of the AR.