Transcriptome analysis of a parasitic clade V nematode:comparative analysis of potential molecular anthelmintic targets in Cylicostephanus goldi

Clade V nematodes comprise several parasitic species that include the cyathostomins, primary helminth pathogens of horses. Next generation transcriptome datasets are available for eight parasitic clade V nematodes, although no equine parasites are included in this group. Here, we report next generation transcriptome sequencing analysis for the common cyathostomin species, Cylicostephanus goldi. A cDNA library was generated from RNA extracted from 17 C. goldi male and female adult parasites. Following sequencing using a 454 GS FLX pyrosequencer, a total of 475,215 sequencing reads were generated, which were assembled into 26,910 contigs. Using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases, 27% of the transcriptome was annotated. Further in-depth analysis was carried out by comparing the C. goldi dataset with the next generation transcriptomes and genomes of other clade V nematodes, with the Oesophagostomum dentatum transcriptome and the Haemonchus contortus genome showing the highest levels of sequence identity with the cyathostomin dataset (45%). The C. goldi transcriptome was mined for genes associated with anthelmintic mode of action and/or resistance. Sequences encoding proteins previously associated with the three major anthelmintic classes used in horses were identified, with the exception of the P-glycoprotein group. Targeted resequencing of the glutamate gated chloride channel α4 subunit (glc-3), one of the primary targets of the macrocyclic lactone anthelmintics, was performed for several cyathostomin species. We believe this study reports the first transcriptome dataset for an equine helminth parasite, providing the opportunity for in-depth analysis of these important parasites at the molecular level. Sequences encoding enzymes involved in key processes and genes associated with levamisole/pyrantel and macrocyclic lactone resistance, in particular the glutamate gated chloride channels, were identified. This novel data will inform cyathostomin biology and anthelmintic resistance studies in future.

Glycemic targets in the second and third trimester of pregnancy for women with type 1 diabetes

OBJECTIVE: To assess the relationship between second and third trimester glycemic control and adverse outcomes in pregnant women with type 1 diabetes, as uncertainty exists about optimum glycemic targets.

RESEARCH DESIGN AND METHODS: Pregnancy outcomes were assessed prospectively in 725 women with type 1 diabetes from the Diabetes and Pre-eclampsia Intervention Trial. HbA1c (A1C) values at 26 and 34 weeks' gestation were categorized into five groups, the lowest, <6.0% (42 mmol/mol), being the reference. Average pre- and postprandial results from an eight-point capillary glucose profile the previous day were categorized into five groups, the lowest (preprandial <5.0 mmol/L and postprandial <6.0 mmol/L) being the reference.

RESULTS: An A1C of 6.0-6.4% (42-47 mmol/mol) at 26 weeks' gestation was associated with a significantly increased risk of large for gestational age (LGA) (odds ratio 1.7 [95% CI 1.0-3.0]) and an A1C of 6.5-6.9% (48-52 mmol/mol) with a significantly increased risk of preterm delivery (odds ratio 2.5 [95% CI 1.3-4.8]), pre-eclampsia (4.3 [1.7-10.8]), need for a neonatal glucose infusion (2.9 [1.5-5.6]), and a composite adverse outcome (3.2 [1.3-8.0]). These risks increased progressively with increasing A1C. Results were similar at 34 weeks' gestation. Glucose data showed less consistent trends, although the risk of a composite adverse outcome increased with preprandial glucose levels between 6.0 and 6.9 mmol/L at 34 weeks (3.3 [1.3-8.0]).

CONCLUSIONS: LGA increased significantly with an A1C ≥6.0 (42 mmol/mol) at 26 and 34 weeks' gestation and with other adverse outcomes with an A1C ≥6.5% (48 mmol/mol). The data suggest that there is clinical utility in regular measurement of A1C during pregnancy.

Functional genomics to identify therapeutic prophylactic targets

Infectious diseases are a leading cause of global human mortality. The use of antimicrobials remains the most common strategy for treatment. However, the isolation of pathogens resistant to virtually all antimicrobials makes it urgent to develop effective therapeutics based on new targets. Here we review a new drug discovery paradigm focusing on identifying and targeting host factors important for infection as well as pathogen determinants involved in disease progression. We summarize innovative strategies which by combining bioinformatics with transcriptomics and chemical genetics have already identified host factors essential for pathogen entry, survival and replication. We describe how the discovery of RNA interference which allows loss-of-function studies has facilitated functional genomic studies in human cells. It is expected that these studies will identify targets to be used as host-directed drug therapy which, together with antimicrobials targeting microbial virulence factors, will efficiently eliminate the invading pathogen.

Abstract 1905: Defining a therapeutic classification of breast cancer by actionable targets

Breast cancer remains a frequent cause of female cancer death despite the great strides in elucidation of biological subtypes and their reported clinical and prognostic significance. We have defined a general cohort of breast cancers in terms of putative actionable targets, involving growth and proliferative factors, the cell cycle, and apoptotic pathways, both as single biomarkers across a general cohort and within intrinsic molecular subtypes.

We identified 293 patients treated with adjuvant chemotherapy. Additional hormonal therapy and trastuzumab was administered depending on hormonal and HER2 status respectively. We performed immunohistochemistry for ER, PR, HER2, MM1, CK5/6, p53, TOP2A, EGFR, IGF1R, PTEN, p-mTOR and e-cadherin. The cohort was classified into luminal (62%) and non-luminal (38%) tumors as well as luminal A (27%), luminal B HER2 negative (22%) and positive (12%), HER2 enriched (14%) and triple negative (25%). Patients with luminal tumors and co-overexpression of TOP2A or IGF1R loss displayed worse overall survival (p=0.0251 and p=0.0008 respectively). Non-luminal tumors had much greater heterogeneous expression profiles with no individual markers of prognostic significance. Non-luminal tumors were characterised by EGFR and TOP2A overexpression, IGF1R, PTEN and p-mTOR negativity and extreme p53 expression.

Our results indicate that only a minority of intrinsic subtype tumors purely express single novel actionable targets. This lack of pure biomarker expression is particular prevalent in the triple negative subgroup and may allude to the mechanism of targeted therapy inaction and myriad disappointing trial results. Utilising a combinatorial biomarker approach may enhance studies of targeted therapies providing additional information during design and patient selection while also helping decipher negative trial results.

In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets of aberrant promoter hypermethylation in prostate cancer

BACKGROUND: Aberrant DNA methylation has been implicated as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many cellular processes including apoptosis. Limited data is available on the methylation profile of apoptotic genes in prostate cancer (CaP). The aim of this study was to profile methylation of apoptotic-related genes in CaP using denaturing high performance liquid chromatography (DHPLC).

METHODS: Based on an in silico selection process, 13 genes were screened for methylation in CaP cell lines using DHPLC. Quantitative methylation specific PCR was employed to determine methylation levels in prostate tissue specimens (n = 135), representing tumor, histologically benign prostate, high-grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. Gene expression was measured by QRT-PCR in cell lines and tissue specimens.

RESULTS: The promoters of BIK, BNIP3, cFLIP, TMS1, DCR1, DCR2, and CDKN2A appeared fully or partially methylated in a number of malignant cell lines. This is the first report of aberrant methylation of BIK, BNIP3, and cFLIP in CaP. Quantitative methylation analysis in prostate tissues identified 5 genes (BNIP3, CDKN2A, DCR1, DCR2 and TMS1) which were frequently methylated in tumors but were unmethylated in 100% of benign tissues. Furthermore, 69% of tumors were methylated in at least one of the five-gene panel. In the case of all genes, except BNIP3, promoter hypermethylation was associated with concurrent downregulation of gene expression.

CONCLUSION: Future examination of this "CaP apoptotic methylation signature" in a larger cohort of patients is justified to further evaluate its value as a diagnostic and prognostic marker.

Expression of the NF-kappaB targets BCL2 and BIRC5/Survivin characterizes small B-cell and aggressive B-cell lymphomas, respectively

Nuclear factor kappa B (NF-kappaB) activation has been proposed as a cardinal feature of tumourigenesis, although the precise mechanism, frequency, relevance, and extent of NF-kappaB activation in lymphomas remain to be fully elucidated. In this study, expression profiling and tissue microarray studies of 209 and 323 non-Hodgkin's lymphomas (NHLs) respectively, including the most frequent sub-types of NHL, were employed to generate a hypothesis concerning the most common NF-kappaB targets in NHL. These analyses showed that NF-kappaB activation is a common phenomenon in NHL, resulting in the expression of distinct sets of NF-kappaB target genes, depending on the cell context. BCL2 and BIRC5/Survivin were identified as key NF-kappaB targets and their expression distinguished small and aggressive B-cell lymphomas, respectively. Interestingly, in the vast majority of B-cell lymphomas, the expression of these markers was mutually exclusive. A set of genes was identified whose expression correlates either with BIRC5/Survivin or with BCL2. BIRC5/Survivin expression, in contrast to BCL2, was associated with a signature of cell proliferation (overexpression of cell cycle control, DNA repair, and polymerase genes), which may contribute to the aggressive phenotype and poor prognosis of these lymphomas. Strikingly, mantle cell lymphoma and chronic lymphocytic leukaemia expressed highly elevated levels of BCL2 protein and mRNA, higher than that observed in reactive mantle zone cells or even in follicular lymphomas, where BCL2 expression is deregulated through the t(14;18) translocation. In parallel with this observation, BIRC5/Survivin expression was higher in Burkitt's lymphoma and diffuse large B-cell lymphoma than in non-tumoural germinal centre cells. In vitro studies confirmed that NF-kappaB activation contributes to the expression of both markers. In cell lines representing aggressive lymphomas, NF-kappaB inhibition resulted in a decrease in BIRC5/Survivin expression. Meanwhile, in chronic lymphocytic leukaemia (CLL)-derived lymphocytes, NF-kappaB inhibition resulted in a marked decrease in BCL2 expression.

New androgen receptor genomic targets show an interaction with the ETS1 transcription factor

The androgen receptor (AR) initiates important developmental and oncogenic transcriptional pathways. The AR is known to bind as a homodimer to 15-base pair bipartite palindromic androgen-response elements; however, few direct AR gene targets are known. To identify AR promoter targets, we used chromatin immunoprecipitation with on-chip detection of genomic fragments. We identified 1,532 potential AR-binding sites, including previously known AR gene targets. Many of the new AR target genes show altered expression in prostate cancer. Analysis of sequences underlying AR-binding sites showed that more than 50% of AR-binding sites did not contain the established 15 bp AR-binding element. Unbiased sequence analysis showed 6-bp motifs, which were significantly enriched and were bound directly by the AR in vitro. Binding sequences for the avian erythroblastosis virus E26 homologue (ETS) transcription factor family were also highly enriched, and we uncovered an interaction between the AR and ETS1 at a subset of AR promoter targets.

M-L band x-rays (3-3.5 KeV) from palladium coated targets for isochoric radiative heating of thin foil samples

We describe experiments designed to produce a bright M-L band x-ray source in the 3-3.5 keV region. Palladium targets irradiated with a 10(15) W cm(-2) laser pulse have previously been shown to convert up to similar to 2% of the laser energy into M-L band x-rays with similar pulse duration to that of the incident laser. This x-ray emission is further characterized here, including pulse duration and source size measurements, and a higher conversion efficiency than previously achieved is demonstrated (similar to 4%) using more energetic and longer duration laser pulses (200 ps). The emission near the aluminium K-edge (1.465-1.550 keV) is also reported for similar conditions, along with the successful suppression of such lower band x-rays using a CH coating on the rear side of the target. The possibility of using the source to radiatively heat a thin aluminium foil sample to uniform warm dense matter conditions is discussed.