Micro RNAs (miRNAs) are a class of small, non-coding RNA species that play critical roles throughout cellular development and regulation. miRNA expression patterns taken from various tissue types often point to the cellular lineage of an individual tissue type, thereby being a more invariant hallmark of tissue type. Recent work has shown that these miRNA expression patterns can be used to classify tumor cells, and that this classification can be more accurate than the classification achieved by using messenger RNA gene expression patterns. One aspect of miRNA biogenesis that makes them particularly attractive as a biomarker is the fact that they are maintained in a protected state in serum and plasma, thus allowing the detection of miRNA expression patterns directly from serum. This study is focused on the evaluation of miRNA expression patterns in human serum for five types of human cancer, prostate, colon, ovarian, breast and lung, using a pan-human microRNA, high density microarray. This microarray platform enables the simultaneous analysis of all human microRNAs by either fluorescent or electrochemical signals, and can be easily redesigned to include newly identified miRNAs. We show that sufficient miRNAs are present in one milliliter of serum to detect miRNA expression patterns, without the need for amplification techniques. In addition, we are able to use these expression patterns to correctly discriminate between normal and cancer patient samples.

Most microbes in the biosphere remain unculturable. Whole genome shotgun (WGS) sequencing of environmental DNA (metagenomics) can be used to study the genetic and metabolic properties of natural microbial communities. However, in communities of high complexity, metagenomics fails to link specific microbes to specific ecological functions. To overcome this limitation, we developed a method to target microbial subpopulations by labeling DNA through stable isotope probing (SIP), followed by WGS sequencing. Metagenome analysis of microbes from Lake Washington in Seattle that oxidize single-carbon (C1) compounds shows specific sequence enrichments in response to different C1 substrates, revealing the ecological roles of individual phylotypes. We also demonstrate the utility of our approach by extracting a nearly complete genome of a novel methylotroph, Methylotenera mobilis, reconstructing its metabolism and conducting genome-wide analyses. This high-resolution, targeted metagenomics approach may be applicable to a wide variety of ecosystems.

Ligands of the NKG2D receptor, which activates NK cells and costimulates effector T cells, are inducibly expressed under harmful conditions, such as malignancies and microbial infections. Moreover, aberrant expression in autoimmune disease lesions may contribute to disease progression. Among these ligands are the closely related human MHC class I-related chains (MIC) A and B, which appear to be regulated by cellular stress. Analyses of MIC gene 5′-end flanking regions in epithelial tumor cells defined minimal core promoters that directed near maximum heat shock- or oxidative stress-induced transcriptional activation. Considerably larger fully functional promoters were required for maximum proliferation-associated activation. These activities were dependent on core promoter sequences that included heat shock elements, which inducibly bound heat shock factor 1, TATA-like elements, and constitutively occupied Sp1 and inverted CCAAT box factor sites. By contrast, MIC gene activation by CMV infection was largely independent of these and upstream promoter sequences, and expression of viral immediate early gene (IE1 or IE2) products was sufficient for induction of transcription and surface protein expression. Altogether, these results reveal distinct modes of activation of the genes for the MIC ligands of NKG2D and provide a molecular framework for analyses of gene regulation under different cellular insult conditions.

In the face of concerns over an influenza pandemic, identification of virulent influenza A virus isolates must be obtained quickly for effective responses. Rapid subtype identification, however, is difficult even in well-equipped virology laboratories or is unobtainable in the field under more austere conditions. Here we describe a genome assay and microarray design that can be used to rapidly identify influenza A virus hemagglutinin subtypes 1 through 15 and neuraminidase subtypes 1 through 9. Also described is an array-based enzymatic assay that can be used to sequence portions of both genes or any other sequence of interest.

An addressable electrode array was used for the production of acid at sufficient concentration to allow deprotection of the dimethoxytrityl (DMT) protecting group from an overlaying substrate bound to a porous reaction layer. Containment of the generated acid to an active electrode of 100 micron diameter was achieved by the presence of an organic base. This procedure was then used for the production of a DNA array, in which synthesis was directed by the electrochemical removal of the DMT group during synthesis. The product array was found to have a detection sensitivity to as low as 0.5 pM DNA in a complex background sample.

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Through their metabolic activities, microbial populations mediate the impact of high gradient regions on ecological function and productivity of the highly dynamic Columbia River coastal margin (CRCM). A 2226-probe oligonucleotide DNA microarray was developed to investigate expression patterns for microbial genes involved in nitrogen and carbon metabolism in the CRCM. Initial experiments with the environmental microarrays were directed toward validation of the platform and yielded high reproducibility in multiple tests. Bioinformatic and experimental validation also indicated that >85% of the microarray probes were specific for their corresponding target genes and for a few homologs within the same microbial family. The validated probe set was used to query gene expression responses by microbial assemblages to environmental variability. Sixty-four samples from the river, estuary, plume, and adjacent ocean were collected in different seasons and analyzed to correlate the measured variability in chemical, physical and biological water parameters to differences in global gene expression profiles. The method produced robust seasonal profiles corresponding to pre-freshet spring (April) and late summer (August). Overall relative gene expression was high in both seasons and was consistent with high microbial abundance measured by total RNA, heterotrophic bacterial production, and chlorophyll a. Both seasonal patterns involved large numbers of genes that were highly expressed relative to background, yet each produced very different gene expression profiles. April patterns revealed high differential gene expression in the coastal margin samples (estuary, plume and adjacent ocean) relative to freshwater, while little differential gene expression was observed along the river-to-ocean transition in August. Microbial gene expression profiles appeared to relate, in part, to seasonal differences in nutrient availability and potential resource competition. Furthermore, our results suggest that highly-active particle-attached microbiota in the Columbia River water column may perform dissimilatory nitrate reduction (both dentrification and DNRA) within anoxic particle microniches.

Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluinza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.