RESULTS: The dengue fever mouse model was established by intraperitoneal inoculation of dengue virus, New Guinea C strain at 2 × 106 PFU. Daily oral administration of 1000 mg/kg freeze-dried C. papaya leaf juice (FCPLJ) was done starting from day 1 to day 3 post infection. The RNA was extracted from liver tissues harvested on day 4 post infection. The expression levels of 84 genes related to mouse endothelial cell biology were determined by qRT-PCR technique. Dengue virus infection upregulated 15 genes and downregulated two genes in the liver of AG129 mice. The FCPLJ treatment upregulated monocyte chemoattractant protein 1 and downregulated intercellular adhesion molecule 1, integrin beta 3 and fibronectin 1 genes during dengue virus infection. The data showed the potential effect of FCPLJ treatment on the expression profile of endothelial cell biology related genes in the liver of dengue virus infected-AG129 mice. Further proteomic studies are needed to determine the functional roles of the genes affected by FCPLJ treatment.
METHODS: Data from The Cancer Genome Atlas and the Gene Expression Omnibus database were analyzed to assess ETBR expression. For survival analysis, glioblastoma samples from 25 Swedish patients were immunostained for ETBR, and the findings were correlated with clinical history. The druggability of ETBR was assessed by protein-protein interaction network analysis. ERAs were analyzed for toxicity in in vitro assays with GBM and breast cancer cells.
RESULTS: By bioinformatics analysis, ETBR was found to be upregulated in glioblastoma patients, and its expression levels were correlated with reduced survival. ETBR interacts with key proteins involved in cancer pathogenesis, suggesting it as a druggable target. In vitro viability assays showed that ERAs may hold promise to treat glioblastoma and breast cancer.
CONCLUSIONS: ETBR is overexpressed in glioblastoma and other cancers and may be a prognostic marker in glioblastoma. ERAs may be useful for treating cancer patients.
MATERIAL AND METHODS: Using an Oragene® RNA kit, the total RNA was purified from the saliva of 10 patients with chronic periodontitis and 10 patients without chronic periodontitis. The quantity and quality of the total RNA was determined, and a measure of gene expression via cDNA was undertaken using the Affymetrix microarray system. The microarray profiling result was further validated by real-time quantitative polymerase chain reaction.
RESULTS: Spectrophotometric analysis showed the total RNA purified from each participant ranged from 0.92 μg/500 μL to 62.85 μg/500 μL. There was great variability in the quantity of total RNA obtained from the 2 groups in the study with a mean of 10.21 ± 12.71 μg/500 μL for the periodontitis group and 15.97 ± 23.47 μg/500 μL for the control group. Further the RNA purity (based on the A260 /A280 ratio) for the majority of participants (9 periodontitis and 6 controls) were within the acceptable limits for downstream analysis (2.0 ± 0.1). The study samples, showed 2 distinct bands at 23S (3800 bp) and 16S (1500 bp) characteristic of bacterial rRNA. Preliminary microarray analysis was performed for 4 samples (P2, P6, H5 and H9). The percentage of genes present in each of the 4 samples was not consistent with about 1.8%-18.7% of genes being detected. Quantitative real-time polymerase chain reaction confirmed that the total RNA purified from each sample was mainly bacterial RNA (Uni 16S) with minimal human mRNA.
CONCLUSION: This study showed that minimal amounts of human RNA were able to be isolated from the saliva of patients with periodontitis as well as controls. Further work is required to enhance the extraction process of human mRNA from saliva if the salivary transcriptome is to be used in determining individual patient susceptibility.
FINDINGS: We optimized the assembly of a Hevea bark transcriptome based on 16 Gb Illumina PE RNA-Seq reads using the Oases assembler across a range of k-mer sizes. We then assessed assembly quality based on transcript N50 length and transcript mapping statistics in relation to (a) known Hevea cDNAs with complete open reading frames, (b) a set of core eukaryotic genes and (c) Hevea genome scaffolds. This was followed by a systematic transcript mapping process where sub-assemblies from a series of incremental amounts of bark transcripts were aligned to transcripts from the entire bark transcriptome assembly. The exercise served to relate read amounts to the degree of transcript mapping level, the latter being an indicator of the coverage of gene transcripts expressed in the sample. As read amounts or datasize increased toward 16 Gb, the number of transcripts mapped to the entire bark assembly approached saturation. A colour matrix was subsequently generated to illustrate sequencing depth requirement in relation to the degree of coverage of total sample transcripts.
CONCLUSIONS: We devised a procedure, the "transcript mapping saturation test", to estimate the amount of RNA-Seq reads needed for deep coverage of transcriptomes. For Hevea de novo assembly, we propose generating between 5-8 Gb reads, whereby around 90% transcript coverage could be achieved with optimized k-mers and transcript N50 length. The principle behind this methodology may also be applied to other non-model plants, or with reads from other second generation sequencing platforms.
METHODOLOGY/PRINCIPAL FINDINGS: We profiled gene expression changes in the airway wall using a large animal model of physical injury comprising bronchial brush biopsy in anaesthetised sheep. The experimental design featured sequential studies in the same animals over the course of a week and yielded data relating to the response at 6 hours, and 1, 3 and 7 days after injury. Notable features of the transcriptional response included the early and sustained preponderance of down-regulated genes associated with angiogenesis and immune cell activation, selection and differentiation. Later features of the response included the up-regulation of cell cycle genes at d1 and d3, and the latter pronounced up-regulation of extracellular matrix-related genes at d3 and d7.
CONCLUSIONS/SIGNIFICANCE: It is possible to follow the airway wall response to physical injury in the same animal over the course of time. Transcriptional changes featured coordinate expression of functionally related genes in a reproducible manner both within and between animals. This characterisation will provide a foundation against which to assess the perturbations that accompany airway disease pathologies of comparative relevance.
METHODS: RNA was isolated from peripheral whole blood samples (2 x 10 ml) collected from NPC patients/controls (EDTA vacutainer). Gene expression patterns from 99 samples (66 NPC; 33 controls) were assessed using the Affymetrix array. We also collected expression data from 447 patients with other cancers (201 patients) and non-cancer conditions (246 patients). Multivariate logistic regression analysis was used to obtain biomarker signatures differentiating NPC samples from controls and other diseases. Differences were also analysed within a subset (n=28) of a pre-intervention case cohort of patients whom we followed post-treatment.
RESULTS: A blood-based gene expression signature composed of three genes - LDLRAP1, PHF20, and LUC7L3 - is able to differentiate NPC from various other diseases and from unaffected controls with significant accuracy (area under the receiver operating characteristic curve of over 0.90). By subdividing our NPC cohort according to the degree of patient response to treatment we have been able to identify a blood gene signature that may be able to guide the selection of treatment.
CONCLUSION: We have identified a blood-based gene signature that accurately distinguished NPC patients from controls and from patients with other diseases. The genes in the signature, LDLRAP1, PHF20, and LUC7L3, are known to be involved in carcinoma of the head and neck, tumour-associated antigens, and/or cellular signalling. We have also identified blood-based biomarkers that are (potentially) able to predict those patients who are more likely to respond to treatment for NPC. These findings have significant clinical implications for optimizing NPC therapy.