Estrogen and the endometrium: lessons learned from gene expression profiling in rodents and human

To date, research into the biological processes and molecular mechanisms associated with endometrial receptivity and embryo implantation has been a focus of attention, whereas the complex events that occur in the human endometrium during the menstrual and proliferative phase under the influence of estrogen have received little attention. The objective of this review is to provide an update of our current understanding of the actions of estrogen on both human and rodent endometrium, with special emphasis on the regulation of uterine growth and cell proliferation, and the value of global gene expression analysis, in increasing understanding of these processes.

Tumor-suppressor gene promoter hypermethylation in saliva of head and neck cancer patients

Head and neck squamous cell carcinoma (HNSCC) accounts for a bulk of the oral and laryngeal cancers, the majority (70%) of which are associated with smoking and excessive drinking, major known risk factors for the development of HNSCC. In contrast to reports that suggest an inverse relationship between smoking and global DNA CpG methylation, hypermethylation of promoters of a number of genes was detected in saliva collected from patients with HNSCC. Using a sensitive methylation-specific polymerase chain reaction (MSP) assay to determine specific methylation events in the promoters of RASSF1A, DAPK1, and p16 genes, we demonstrate that we can detect tumor presence with an overall accuracy of 81% in the DNA isolated from saliva of patients with HNSCC (n = 143) when compared with the DNA isolated from the saliva of healthy nonsmoker controls (n = 31). The specificity for this MSP panel was 87% and the sensitivity was 80%(with a Fisher exact test P < .0001). In addition, the test panel performed extremely well in the detection of the early stages of HNSCCs, with a sensitivity of 94% and a specificity of 87%, and a high. concordance value of 0.8, indicating an excellent overall agreement between the presence of HNSCC and a positive MSP panel result. In conclusion, we demonstrate that the promoter methylation of RASSF1A, DAPK1, and p16 MSP panel is useful in detecting hypermethylation events in a noninvasive manner in patients with HNSCC.

miRNAs in head and neck cancer revisited

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cause of cancer mortality in the world and the 5th most commonly occurring cancer. Tobacco smoking, alcohol consumption and human papilloma virus (HPV) infections have been associated with the occurrence of HNSCC. Despite advances that have been made in HNSCC treatment, smoking-associated HNSCC patients still exhibit a poor 5 year survival rate (30-50 %) and a concomitant poor quality of life. The major clinical challenge to date lies in the early detection of dysplastic lesions,which can progress to malignancy. In addition, there are currently no tools available to monitor HNSCC patients for early stages of local recurrences or distant metastases. In the recent past, micro-RNAs (miRNA) have been assessed for their role in cancer initiation and progression, including HNSCC. It is now well-established that deregulation of these single stranded, small non-coding, 19-25 nt RNAs can e.g. enhance the expression of oncogenes or subdue the expression of tumor suppressor genes. The aims of this review are three-fold: first to retrieve from the literature miRNAs that have specifically been associated with HNSCC, second to group these miRNAs into those regulating tumor initiation, progression and metastasis, and third to discern miRNAs related to smoking-associated HNSCC versus HPV-associated HNSCC development. This review gives an overview on the miRNAs regulating the development of head and neck cancers. The ultimate establishment of miRNA expression profiles that are HNSCC specific, and miRNAs that orchestrate altered gene and protein expression levels in HNSCC, could pave the way for a better understanding of the mechanism underlying its pathogenesis and the development of novel, targeted therapies.

A novel saliva-based microRNA biomarker panel to detect head and neck cancers

Background MicroRNAs (miRNAs) are known to play an important role in cancer development by post-transcriptionally affecting the expression of critical genes. The aims of this study were two-fold: (i) to develop a robust method to isolate miRNAs from small volumes of saliva and (ii) to develop a panel of saliva-based diagnostic biomarkers for the detection of head and neck squamous cell carcinoma (HNSCC). Methods Five differentially expressed miRNAs were selected from miScript™ miRNA microarray data generated using saliva from five HNSCC patients and five healthy controls. Their differential expression was subsequently confirmed by RT-qPCR using saliva samples from healthy controls (n = 56) and HNSCC patients (n = 56). These samples were divided into two different cohorts, i.e., a first confirmatory cohort (n = 21) and a second independent validation cohort (n = 35), to narrow down the miRNA diagnostic panel to three miRNAs: miR-9, miR-134 and miR-191. This diagnostic panel was independently validated using HNSCC miRNA expression data from The Cancer Genome Atlas (TCGA), encompassing 334 tumours and 39 adjacent normal tissues. Receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic capacity of the panel. Results On average 60 ng/μL miRNA was isolated from 200 μL of saliva. Overall a good correlation was observed between the microarray data and the RT-qPCR data. We found that miR-9 (P <0.0001), miR-134 (P <0.0001) and miR-191 (P <0.001) were differentially expressed between saliva from HNSCC patients and healthy controls, and that these miRNAs provided a good discriminative capacity with area under the curve (AUC) values of 0.85 (P <0.0001), 0.74 (P < 0.001) and 0.98 (P < 0.0001), respectively. In addition, we found that the salivary miRNA data showed a good correlation with the TCGA miRNA data, thereby providing an independent validation. Conclusions We show that we have developed a reliable method to isolate miRNAs from small volumes of saliva, and that the saliva-derived miRNAs miR-9, miR-134 and miR-191 may serve as novel biomarkers to reliably detect HNSCC. © 2014 International Society for Cellular Oncology.

The MyD88+ phenotype is an adverse prognostic factor in epithelial ovarian cancer

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.

Transcriptome analyses of amoebic gill disease-affected atlantic salmon (Salmo salar) tissues reveal localized host gene suppression

The transcriptome response of Atlantic salmon (Salmo salar) displaying advanced stages of amoebic gill disease (AGD) was investigated. Naïve smolt were challenged with AGD for 19 days, at which time all fish were euthanized and their severity of infection quantified through histopathological scoring. Gene expression profiles were compared between heavily infected and naïve individuals using a 17 K Atlantic salmon cDNA microarray with real-time quantitative RT-PCR (qPCR) verification. Expression profiles were examined in the gill, anterior kidney, and liver. Twenty-seven transcripts were significantly differentially expressed within the gill; 20 of these transcripts were down-regulated in the AGD-affected individuals compared with naïve individuals. In contrast, only nine transcripts were significantly differentially expressed within the anterior kidney and five within the liver. Again the majority of these transcripts were down-regulated within the diseased individuals. A down-regulation of transcripts involved in apoptosis (procathepsin L, cathepsin H precursor, and cystatin B) was observed in AGD-affected Atlantic salmon. Four transcripts encoding genes with antioxidant properties also were down-regulated in AGD-affected gill tissue according to qPCR analysis. The most up-regulated transcript within the gill was an unknown expressed sequence tag (EST) whose expression was 218-fold (± SE 66) higher within the AGD affected gill tissue. Our results suggest that Atlantic salmon experiencing advanced stages of AGD demonstrate general down-regulation of gene expression, which is most pronounced within the gill. We propose that this general gene suppression is parasite-mediated, thus allowing the parasite to withstand or ameliorate the host response. © 2008 Springer Science+Business Media, LLC.

Clustering and labeling a web scale document collection using Wikipedia clusters

Clustering is an important technique in organising and categorising web scale documents. The main challenges faced in clustering the billions of documents available on the web are the processing power required and the sheer size of the datasets available. More importantly, it is nigh impossible to generate the labels for a general web document collection containing billions of documents and a vast taxonomy of topics. However, document clusters are most commonly evaluated by comparison to a ground truth set of labels for documents. This paper presents a clustering and labeling solution where the Wikipedia is clustered and hundreds of millions of web documents in ClueWeb12 are mapped on to those clusters. This solution is based on the assumption that the Wikipedia contains such a wide range of diverse topics that it represents a small scale web. We found that it was possible to perform the web scale document clustering and labeling process on one desktop computer under a couple of days for the Wikipedia clustering solution containing about 1000 clusters. It takes longer to execute a solution with finer granularity clusters such as 10,000 or 50,000. These results were evaluated using a set of external data.

An analysis of DNA methylation in human adipose tissue reveals differential modification of obesity genes before and after gastric bypass and weight loss

Background Environmental factors can influence obesity by epigenetic mechanisms. Adipose tissue plays a key role in obesity-related metabolic dysfunction, and gastric bypass provides a model to investigate obesity and weight loss in humans. Results Here, we investigate DNA methylation in adipose tissue from obese women before and after gastric bypass and significant weight loss. In total, 485,577 CpG sites were profiled in matched, before and after weight loss, subcutaneous and omental adipose tissue. A paired analysis revealed significant differential methylation in omental and subcutaneous adipose tissue. A greater proportion of CpGs are hypermethylated before weight loss and increased methylation is observed in the 3′ untranslated region and gene bodies relative to promoter regions. Differential methylation is found within genes associated with obesity, epigenetic regulation and development, such as CETP, FOXP2, HDAC4, DNMT3B, KCNQ1 and HOX clusters. We identify robust correlations between changes in methylation and clinical trait, including associations between fasting glucose and HDAC4, SLC37A3 and DENND1C in subcutaneous adipose. Genes investigated with differential promoter methylation all show significantly different levels of mRNA before and after gastric bypass. Conclusions This is the first study reporting global DNA methylation profiling of adipose tissue before and after gastric bypass and associated weight loss. It provides a strong basis for future work and offers additional evidence for the role of DNA methylation of adipose tissue in obesity.

Microparticle-mediated gene delivery for the enhanced expression of a 19-KDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl-terminal fragment of merozoite surface protein 1 (MSP119) is a major component of the invasion-inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP119 is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h1. High levels of gene expression and moderate cytotoxicity in COS-7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z-average hydrodynamic diameters of 1.50-2.10 lm and zeta potentials of 17.8-23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA-mediated microparticles can be employed as potential gene delivery systems to antigen-presenting cells in the prevention of malaria.

The pragmatic approach : the myriad gene patents before the Australian courts

The Full Court of the Federal Court of Australia in D'Arcy v Myriad Genetics [2014] FCAFC 115 recently upheld the validity of Myriad Genetics' Australian BRCA1 gene patent over isolated DNA sequences.

Edge-weighting of gene expression graphs

In recent years, considerable research efforts have been directed to micro-array technologies and their role in providing simultaneous information on expression profiles for thousands of genes. These data, when subjected to clustering and classification procedures, can assist in identifying patterns and providing insight on biological processes. To understand the properties of complex gene expression datasets, graphical representations can be used. Intuitively, the data can be represented in terms of a bipartite graph, with weighted edges corresponding to gene-sample node couples in the dataset. Biologically meaningful subgraphs can be sought, but performance can be influenced both by the search algorithm, and, by the graph-weighting scheme and both merit rigorous investigation. In this paper, we focus on edge-weighting schemes for bipartite graphical representation of gene expression. Two novel methods are presented: the first is based on empirical evidence; the second on a geometric distribution. The schemes are compared for several real datasets, assessing efficiency of performance based on four essential properties: robustness to noise and missing values, discrimination, parameter influence on scheme efficiency and reusability. Recommendations and limitations are briefly discussed. Keywords: Edge-weighting; weighted graphs; gene expression; bi-clustering

The O-specific polysaccharide structure and biosynthetic gene cluster of Yersinia pseudotuberculosis serotype O:11

In the Yersinia pseudotuberculosis serotyping scheme, 21 serotypes are present originating from about 30 different O-factors distributed within the species. With regard to the chemical structures of lipopolysaccharides (LPSs) and the genetic basis of their biosynthesis, a number, but not all, of Y. pseudotuberculosis strains representing different serotypes have been investigated. In order to present an overall picture of the relationship between genetics and structures, we have been working on the genetics and structures of various Y. pseudotuberculosis O-specific polysaccharides (OPSs). Here, we present a structural and genetic analysis of the Y. pseudotuberculosis serotype O:11 OPS. Our results showed that this OPS structure has the same backbone as that of Y. pseudotuberculosis O:1b, but with a 6d-l-Altf side-branch instead of Parf. The 3′ end of the gene cluster is the same as that for O:1b and has the genes for synthesis of the backbone and for processing the completed repeat unit. The 5′ end has genes for synthesis of 6d-l-Altf and its transfer to the repeating unit backbone. The pathway for the synthesis of the 6d-l-Altf appears to be different from that for 6d-l-Altp in Y. enterocolitica O:3. The chemical structure of the O:11 repeating unit is [Figure]

The O-specific polysaccharide structure and gene cluster of serotype O:12 of the Yersinia pseudotuberculosis complex, and the identification of a novel L-quinovose biosynthesis gene

A major virulence factor for Yersinia pseudotuberculosis is lipopolysaccharide, including O-polysaccharide (OPS). Currently, the OPS based serotyping scheme for Y. pseudotuberculosis includes 21 known O-serotypes, with genetic and structural data available for 17 of them. The completion of the OPS structures and genetics of this species will enable the visualization of relationships between O-serotypes and allow for analysis of the evolutionary processes within the species that give rise to new serotypes. Here we present the OPS structure and gene cluster of serotype O:12, thus adding one more to the set of completed serotypes, and show that this serotype is present in both Y. pseudotuberculosis and the newly identified Y. similis species. The O:12 structure is shown to include two rare sugars: 4-C[(R)-1-hydroxyethyl]-3,6-dideoxy-d-xylo-hexose (d-yersiniose) and 6-deoxy-l-glucopyranose (l-quinovose). We have identified a novel putative guanine diphosphate (GDP)-l-fucose 4-epimerase gene and propose a pathway for the synthesis of GDP-l-quinovose, which extends the known GDP-l-fucose pathway.