Oligonucleotide Microarray Hybridization Research Articles

Simultaneous Detection of Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus by Oligonucleotide Microarray

Background: Traditional laboratory methods to detect pathogenic bacteria are time consuming and laborious. Therefore, it is essential to use powerful and reliable molecular methods for quick and simultaneous detection of microbial pathogens. Objectives: The current study aimed to evaluate the capability and efficiency of 23S rDNA sequence for rapid and simultaneous detection of four important food-borne pathogens by an oligonucleotide microarray technique. Materials and Methods: The 23S rDNA sequences of Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus were obtained from GenBank databases and used to design the oligonucleotide probes and primers by Vector NTI software. Oligonucleotide probes were placed on a nylon membrane and hybridization was performed between probes and 23S rDNA digoxigenin-labeled polymerase chain reaction (PCR) products. Hybridization signals were visualized by NBT/BCIP color development. Results: Positive hybridization color was produced for Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus. The oligonucleotide microarray detected all bacterial strains in a single reaction in less than five hours. The sensitivity of the performed microarray assay was 103 cfu/mL for each species of pathogen. No cross reaction was found between the tested bacterial species. Conclusions: The obtained results indicated that amplification of 23S rDNA gene followed by oligonucleotide microarray hybridization is a rapid and reliable method to identify and discriminate foodborne pathogens tested under the study.

Gene expression analysis of uterine smooth muscle cells exposed to bisphenol A

To evaluate the effect of bisphenol A (BPA) on gene expression changes in uterine myometrium, uterine smooth muscle cells were incubated with 2.28 ng/mL BPA for 48 h. Total RNA was extracted and quantified spectrophotometrically. Gene expression changes were analyzed using oligonucleotide microarray hybridization comparing the treated and control groups (1.5-fold change, p<0.05). Data indicated that 560 genes were upregulated and 231 were downregulated in response to BPA exposure. The biological processes positively associated with gene upregulation included the regulation of cell differentiation, cellular metabolic processes, cell proliferation and smooth muscle contraction. In contrast, genes involved in mitosis, lipid metabolism, regulation of muscle cell differentiation and cell adhesion were significantly downregulated. In the KEGG pathway analysis, MAPK signaling pathway, insulin signaling pathway and extracellular matrix (ECM)-mediated interactions were positively associated with gene upregulation.

Multiplex testing of potentially ‘actionable’ alterations in small cell lung cancer (SCLC).

7592 Background: Therapy and outcomes of SCLC patients have not changed for many years. Recently, driver mutations have been identified in many cancers and targeting these mutations can provide clinical benefit. Recent data from whole genome sequencing suggest that driver mutations may exist in SCLCs. We conducted multiplex testing of SCLCs to identify mutations potentially predictive of response to novel and established therapeutics. Methods: We identified tumor tissues of 30 SCLC patients collected as part of two different institutional review board approved studies. Using the Sequenom MassArray system and a multiplexed panel, we analyzed tumor DNA for 214 mutations in 26 genes associated with the pathogenesis of lung cancer. Gene copy number analysis utilized a 180K oligonucleotide microarray and comparative genomic hybridization assay. Results: The median age at diagnosis of patients was 63 years; 66 % of were males; 23% were African-Americans; 57% patients had extensive stage SCLC at diagnosis. Point...

Gene expression profile of endometrial carcinoma cells exposed to di-(2-ethylhexyl) phthalate

To evaluation of the contribution of DEHP (di-(2-ethylhexyl) phthalate) to the pathophysiology of endometriosis, ECC-1 (human endometrial cancer) cell lines were subjected to DEHP for 48 h with 50 μM DEHP and total RNA was extracted. Quantified spectro-photometrically, and RNA quality was verified via agarose-gel electrophoresis. Gene expression changes were analyzed using oligonucleotide microarray hybridization by comparing the treated and control groups (two-fold change, P<0.05). Three independent samples were analyzed. Data indicated that 522 genes were up-regulated and 379 were down-regulated. These genes were classified according to the GO terms of biological processes and the KEGG pathway. Among biologic processes, regulation of cell death, lipid metabolism, gluconeogenesis, response to hormone stimulus, and regulation of cell differentiation were positively associated with up-regulated genes. Mitosis, meiosis, DNA damage check point, anatomical structure development, reproductive development, and regulation of protein ubiquitination were significantly associated with down-regulated genes. In the KEGG pathway analysis, the MAPK, VEGF, p53, Erbb, mTOR, and phosphatidylinositol signaling pathways were positively associated with up-regulated genes. The cell cycle, oocyte meiosis, spliceosome, and pyrimidine metabolism were positively associated with downregulated genes. In conclusion, After DEHP treatment, some genes associated with cell growth, cell differentiation, meiosis, migration, apoptosis, and the pathophysiology of endometriosis were up regulated.

Detection of disease resistance and susceptibility alleles in pigs using oligonucleotide microarray hybridization

A multiplex DNA microarray chip aimed at the identification of allelic polymorphisms was developed for simultaneous detection of swine disease resistance genes underlying malignant hyperthermia (RYR), postweaning diarrhea, edema disease (FUT1), neonatal diarrhea (MUC4), and influenza (MX1). The on-chip detection was performed with fragmented polymerase chain reaction (PCR)-amplified products. Particular emphasis was placed on the reduction of the number of PCR reactions required. The targets were biotin labeled during the PCR reaction, and the arrays were detected using a colorimetric methodology. Target recognition was provided by specific capture probes designed for each susceptible or resistant allelic variant. Sequencing was chosen as the gold standard to assess chip accuracy. All genotypes retrieved from the microarray (476) fit with sequencing data despite the fact that each pig was heterozygote for at least 1 target gene.

Abstract 3998: The role of serum amyloid A1 (SAA1) in nasopharyngeal carcinoma (NPC)

Abstract Background and aims: Nasopharyngeal carcinoma (NPC) is a cancer which occurs in high frequency in Southern China. The identification of diagnostic and prognostic markers will be highly beneficial for early stage NPC detection. Previous microcell-mediated chromosome transfer (MMCT) studies showed that the transfer of an intact human chromosome 11 suppresses the in vivo tumor growth of a NPC cell line (HONE1) in nude mouse tumorigenicity assays. We aim to identify candidate genes which are associated with tumor suppression in NPC. Methods: Differential expression analysis of 19K genes was performed in oligonucleotide microarray hybridization for the chromosome 11 microcell hybrids (MCHs)/ tumor segregants (TSs) pairs to hunt for some other unknown NPC genes. The gene expression of the candidate gene was examined in the NPC cell lines and tumor tissues. Functional analysis of restoration of the candidate gene expression was studied. Results: Using oligonucleotide microarray analysis, Serum Amyloid A 1 (SAA1), mapping close to 11p15.1, was identified as showing consistent down-regulated expression in the TSs, as compared to their parental tumor-suppressing MCHs. Gene expression and protein analyses show that SAA1 was not expressed in the NPC HONE1 recipient cells, tumor segregants, and other NPC cell lines; SAA1 was exclusively expressed in the non-tumorigenic MCHs. The mechanism of SAA1 gene inactivation in these NPC cell lines was attributed to hypermethylation. The clinical relevance of SAA1 in NPC was examined by RT-PCR; 54.8% (23/42) of NPC specimens showed either down-regulation or loss of SAA1 gene expression. After transfection of SAA1 gene into HONE1 cells, a dramatic reduction of colony formation ability was observed. Conclusions: These findings suggest that SAA1 is a candidate tumor suppressor gene in NPC. Acknowledgments Grant support: Research Grants Council of the Hong Kong Special Administrative Region, People's Republic of China grant HKU772309 (H.L. Lung) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3998. doi:1538-7445.AM2012-3998

A microbial oasis in the hypersaline Atacama subsurface discovered by a life detector chip: implications for the search for life on Mars.

The Atacama Desert has long been considered a good Mars analogue for testing instrumentation for planetary exploration, but very few data (if any) have been reported about the geomicrobiology of its salt-rich subsurface. We performed a Mars analogue drilling campaign next to the Salar Grande (Atacama, Chile) in July 2009, and several cores and powder samples from up to 5 m deep were analyzed in situ with LDChip300 (a Life Detector Chip containing 300 antibodies). Here, we show the discovery of a hypersaline subsurface microbial habitat associated with halite-, nitrate-, and perchlorate-containing salts at 2 m deep. LDChip300 detected bacteria, archaea, and other biological material (DNA, exopolysaccharides, some peptides) from the analysis of less than 0.5 g of ground core sample. The results were supported by oligonucleotide microarray hybridization in the field and finally confirmed by molecular phylogenetic analysis and direct visualization of microbial cells bound to halite crystals in the laboratory. Geochemical analyses revealed a habitat with abundant hygroscopic salts like halite (up to 260 g kg(-1)) and perchlorate (41.13 μg g(-1) maximum), which allow deliquescence events at low relative humidity. Thin liquid water films would permit microbes to proliferate by using detected organic acids like acetate (19.14 μg g(-1)) or formate (76.06 μg g(-1)) as electron donors, and sulfate (15875 μg g(-1)), nitrate (13490 μg g(-1)), or perchlorate as acceptors. Our results correlate with the discovery of similar hygroscopic salts and possible deliquescence processes on Mars, and open new search strategies for subsurface martian biota. The performance demonstrated by our LDChip300 validates this technology for planetary exploration, particularly for the search for life on Mars.

The Global Spread of Rotavirus G10 Strains: Detection in Ghanaian Children Hospitalized with Diarrhea

From October 2003 through September 2004, a total of 289 stool samples were collected from children <5 years of age who had severe diarrhea at admission to or when visiting the emergency department at the Navrongo War Memorial Hospital in rural Ghana during a study on rotavirus disease burden. Rotavirus antigen was detected in 115 stool samples (39.8%) tested for rotavirus. Four rotavirus-positive samples were found to bear G10P[6] specificity by reverse-transcription polymerase chain reaction, polymerase chain reaction-enzyme-linked immunosorbent assay, and oligonucleotide microarray hybridization. Two of these strains further exhibited serotype G10 specificity by neutralization and subgroup II specificity by enzyme immunoassay and possessed long electropheretic patterns by polyacrylamide gel electrophoresis. Their VP7 genes shared a much closer nucleotide identity with other African human G10 strains (>97%) than with human G10 strain from Asia or South America (<86%) or animal strains (<85%). The VP8* genes of the Ghanaian G10 strains exhibited >94% identity to that of human P[6] virus strains and belonged to the P[6] lineage 1a. The deduced VP7 amino acid sequence showed that the Ghanaian strains were more closely related to human G10 strains than to animal G10 strains. The possession of the typical human subgroup II specificity and the P[6] specificity (frequently found in Ghana and the rest of Africa) and the marked similarity in the VP7 antigenic sites suggest that these G10 strains may have evolved through genetic reassortment between bovine and human strains.

Detection and genotyping of porcine circovirus in naturally infected pigs by oligo-microarray

A rapid and reliable method for the identification of porcine circovirus (PCV) genotypes based on oligonucleotide microarray hybridization has been developed. The genotype-specific oligonucleotides (22-30 mer) immobilized on the surface of glass slides were selected to bind to the multiple target sites within the replication gene that are conserved among individual PCV genotypes. Cy5-labeled DNA targets were amplified in a PCR with primers common to both genotypes. The identification of PCV genotype was based on hybridization with several individual genotype-specific oligonucleotides. This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hybridization. The utility and feasibility of oligonucleotide microarray hybridization was evaluated by testing standard and 87 clinical isolates. Analysis of the specimens showed that this microarray-based method is capable of unambiguous identification of both genotypes and fivefold more sensitive than gel electrophoresis. Our results indicated that the oligonucleotide array is useful for the identification and discrimination of PCV from clinical isolates and specimens in a clinical laboratory.

Prediction of qualitative outcome of oligonucleotide microarray hybridization by measurement of RNA integrity using the 2100 Bioanalyzer™ capillary electrophoresis system

RNA quality is critical to achieve valid results in microarray experiments and to save resources. The RNA integrity number (RIN) can be measured with minimal sample consumption by microfluidics-based capillary electrophoresis. To determine whether RIN can predict the qualitative outcome of microarray hybridization, we measured RIN in total RNA samples from 484 different experiments by the 2100 Bioanalyzer system and correlated with the percentage of present calls (%pc) of downstream oligonucleotide microarrays. The correlation coefficient for RNA and %pc in all 408 samples for which the bioanalyzer algorithm was able to produce an RIN was 0.475 (p < 0.05), ranging from 0.039 to 0.673 for different tissue- and assay-type subgroups. Multivariate analysis found RIN to be the best predictor of microarray quality as assessed by %pc, outperforming the 28S to 18S ratio. For a %pc threshold of 25% and 35%, we determined optimal cut points for RIN at 7.15 and 8.05, respectively. Using the suggested cut points, RIN can support the final decision whether a certain RNA sample is appropriate for successful microarray hybridization.

Carboplatin-induced gene expression changes in vitroare prognostic of survival in epithelial ovarian cancer

BackgroundWe performed a time-course microarray experiment to define the transcriptional response to carboplatin in vitro, and to correlate this with clinical outcome in epithelial ovarian cancer (EOC). RNA was isolated from carboplatin and control-treated 36M2 ovarian cancer cells at several time points, followed by oligonucleotide microarray hybridization. Carboplatin induced changes in gene expression were assessed at the single gene as well as at the pathway level. Clinical validation was performed in publicly available microarray datasets using disease free and overall survival endpoints.ResultsTime-course and pathway analyses identified 317 genes and 40 pathways (designated time-course and pathway signatures) deregulated following carboplatin exposure. Both types of signatures were validated in two separate platinum-treated ovarian and NSCLC cell lines using published microarray data. Expression of time-course and pathway signature genes distinguished between patients with unfavorable and favorable survival in two independent ovarian cancer datasets. Among the pathways most highly induced by carboplatin in vitro, the NRF2, NF-kB, and cytokine and inflammatory response pathways were also found to be upregulated prior to chemotherapy exposure in poor prognosis tumors.ConclusionDynamic assessment of gene expression following carboplatin exposure in vitro can identify both genes and pathways that are correlated with clinical outcome. The functional relevance of this observation for better understanding the mechanisms of drug resistance in EOC will require further evaluation.

Abstract 559: Elevated Level of sCD40L During Acute Coronary Syndrome Leads to Systemic MIP-2 Release

Purpose: Acute coronary syndromes (ACS) have been associated with sCD40 Ligand (sCD40L) release. Endothial cells are located at the interface between the blood and parenchymal cells and take active part in many physiological and pathologic processes, including inflammation. It has been suggested that CD40L is a key player in the pathogenesis of atherosclerosis and ACS, originating from endothelial cells. When sCD40L is present in human plasma during ACS, it is mainly derived from activated platelets. This may lead to activation of endothelial cells resulting in atherosclerosis and eventually atherothrombosis. Therefore, the aim of the present study was to investigate differential gene expression profiles of endothelial cells after stimulation with sCD40L. Methods: Human endothelial cells (HUVEC) were stimulated with sCD40L for 2 hours. Differential gene expression patterns were evaluated with Oligonucleotide microarray hybridization (UG-U133A, Affymetrix Inc, Santa Clara, CA). Significantly up-regulated gene expression was confirmed and quantitated by Real-time-PCR as well as ELISA. In serum from ACS patients expression of sCD40L was correlated with these up-regulated genes products. Results: Stimulation of HUVEC with sCD40L leads to a profound release of proinflammatory mediators like IL-8, ICAM1, ISGF3 as well as MCP-1 on a transcriptional level. In addition, gene expression profiling revealed a highly significant increase in expression of the macrophage inflammatory protein 2 (MIP-2), also known as GRO-beta or CXCL2. This was quantitated as approximately 30 fold up-regulation by real-time-PCR as well as in ELISA analysis (8 fold at 8 to 24 hours) derived from supernations. In clinical serum samples from ACS patients there was a highly significant correlation between sCD40L and MIP-2 levels (r = 0.88, p &lt; 0.01) Conclusion: Proinflammatory mediators as well as MIP-2 play an important role in the chemoattraction of macrophages, which underlines the key role of CD40L as a link between thrombotic events, e.g ACS, and inflammatory processes, e.g. atherosclerosis. Therefore targeting MIP-2 up-regulation might be a potential approach to prevent the progression of atherosclerotic diseases as well as a new therapeutic approach to treat ACS.

Faecal ribosomal protein L19 is a genetic prognostic factor for survival in colorectal cancer

Ribosomal proteins are encoded by a gene family, members of which are overexpressed in human cancers. Many of them have been found, using oligonucleotide microarray hybridization, to be differentially expressed in the faeces of patients with various stages of col-orectal cancer (CRC). The gene encoding ribosomal protein L19 (RPL19), a prognostic marker for human prostate cancer, is differentially expressed in CRC patients. Measurement of faecal RPL19 mRNA might improve prognostic prediction for CRC patients. Using quantitative real-time reverse transcription PCR, levels of RPL19 mRNA were detected in samples of colonic tissues from 44 CRC patients, in the faeces of 54 CRC patients and 15 controls, and in 11 colonic cell lines. Seven of 24 patients with late-stage CRC (Dukes' stages C and D) expressed over 2-fold more RPL19 in colonic tumour tissues than in corresponding normal tissues (P= 0.038). The mean faecal RPL19 mRNA levels of late-staged patients were higher than those of controls (P= 0.003) and early-staged patients (P= 0.008). Patients with both high serum levels of carcinoembryonic antigen (CEA; >5 ng/mL) and high-faecal RPL19 mRNA (≥0.0069) had higher risk (odds ratio, 8.0; P= 0.015) and lower overall 48-month survival (33.8 ± 13.7%, P= 0.013). Oligonucleotide microarray hybridization analysis of faecal molecules identified gene transcripts differentially present in faeces. In conclusion, faecal RPL19 expression is associated with advanced tumour stages and addictive to serum CEA in predicting prognosis of CRC patients.

Array analysis of epilepsy-associated gangliogliomas reveals expression patterns related to aberrant development of neuronal precursors

Gangliogliomas, the most frequent neoplasms in patients with pharmacoresistant focal epilepsies, are characterized by histological combinations of glial and dysplastic neuronal elements, a highly differentiated phenotype and rare gene mutations. Their molecular basis and relationship to other low-grade brain tumours are not completely understood. Systematic investigations of altered gene expression in gangliogliomas have been hampered by their cellular complexity, the lack of suitable control tissue and of sensitive expression profiling approaches. Here, we have used discrete microdissected ganglioglioma and adjacent control brain tissue obtained from the neurosurgical access to the tumour of identical patients (n = 6) carefully matched for equivalent glial and neuronal elements in an amount sufficient for oligonucleotide microarray hybridization without repetitive amplification. Multivariate statistical analysis identified a rich profile of genes with altered expression in gangliogliomas. Many differentially expressed transcripts related to intra- and intercellular signalling including protein kinase C and its target NELL2 in identical ganglioglioma cell components as determined by real-time quantitative RT-PCR (qRT-PCR) and in situ hybridization. We observed the LIM-domain-binding 2 (LDB2) transcript, critical for brain development during embryogenesis, as one of the strongest reduced mRNAs in gangliogliomas. Subsequent qRT-PCR in dysembryoplastic neuroepithelial tumours (n = 7) revealed partial expression similarities as well as marked differences from gangliogliomas. The demonstrated gene expression profile differentiates gangliogliomas from other low-grade primary brain tumours. shRNA-mediated silencing of LDB2 resulted in substantially aberrant dendritic arborization in cultured developing primary hippocampal neurons. The present data characterize novel molecular mechanisms operating in gangliogliomas that contribute to the development of dysplastic neurons and an aberrant neuronal network.

Effects of hypothermia on gene expression in zebrafish gills:upregulation in differentiation and function of ionocytes as compensatory responses

Ectothermic vertebrates are different from mammals that are sensitive to hypothermia and have to maintain core temperature for survival. Why and how ectothermic animals survive, grow and reproduce in low temperature have been for a long time a scientifically challenging and important inquiry to biologists. We used a microarray to profile the gill transcriptome in zebrafish (Danio rerio) after exposure to low temperature. Adult zebrafish were acclimated to a low temperature of 12 degrees C for 1 day and up to 30 days, and the gill transcriptome was compared with that of control fish in 28 degrees C by oligonucleotide microarray hybridization. Results showed 11 and 22 transcripts were found to be upregulated, whereas 56 and 70 transcripts were downregulated by low-temperature treatment for 1 day and 30 days, respectively. The gill transcriptome profiles revealed that ionoregulation-related genes were highly upregulated in cold-acclimated zebrafish. This paved the way to investigate the role of ionoregulatory genes in zebrafish gills during cold acclimation. Cold acclimation caused upregulation of genes that are essential for ionocyte specification, differentiation, ionoregulation, acid-base balance and the number of cells expressing these genes increased. For instance, epithelial Ca2+ channel (EcaC; an ionoregulatory protein) mRNA increased in parallel with the level of Ca2+ influx, revealing a functional compensation after long-term acclimation to cold. Phosphohistone H3 and TUNEL staining showed that the cell turnover rate was retarded in cold-acclimated gills. Altogether, these results suggest that gills may sustain their functions by producing mature ionocytes from pre-existing undifferentiated progenitors in low-temperature environments.

Detection of Genetically Modified Canola Using Multiplex PCR Coupled with Oligonucleotide Microarray Hybridization

A rapid method was developed for concurrent screening of transgenic elements in GM canola. This method utilizes a single multiplex PCR coupled with an oligonucleotide DNA array capable of simultaneously detecting the 12 approved GM canola lines in Canada. The assay includes construct-specific elements for identification of approved lines, common elements (e.g., CaMV 35S promoter, Agrobacterium tumefaciens nos terminator, or nptII gene) for screening of approved or unapproved lines, a canola-specific endogenous gene, and endogenous genes from heterologous crops to serve as additional controls. Oligonucleotide probes were validated individually for functionality and specificity by amplification of specific transgene sequences from appropriate GM canola lines corresponding to each probe sequence, and hybridization of amplicons to the array. Each target sequence hybridized to its corresponding oligonucleotide probe and no significant cross-hybridization was observed. The limit of detection was examined for the GM lines GT73, T45, and MS8/RF3, and was determined to be 0.1%, 0.1%, and 0.5%, respectively, well within the European food and feed labeling threshold level of 0.9% for approved GM product. Practically, the method was demonstrated to be effective for the detection of GM canola in several types of animal feed, as well as in commercial canola meal.

Osteopontin Expression in Squamous Cell Cancer of the Esophagus

The purpose of this study was to better understand the role of osteopontin (OPN) in esophageal squamous cell carcinoma (ESCC) by comparing the OPN mRNA level in ESCC tumor tissue and matched normal tissue and by determining the prognostic significance of the gene expression. Initially, by an oligo-nucleotide microarray hybridization technique, OPN expressions were found to consistently elevate at least twofold in three ESCC tissues compared with their adjacent normal ones. Subsequently, the expression of OPN mRNA was detected by real-time QRT-PCR among the 58 fresh surgical ESCC specimens. The clinical information was obtained by chart review. OPN mRNA expression was detectable in 58 of 58 (100%) tumor specimens and 57 of 58 (98.3%) nonmalignant esophageal specimens. OPN expression was higher in tumor tissue than in the matched normal tissue in 54 of 58 (93.1%) individual cases. The overall median mRNA expression level of OPN was approximately 8.8-fold higher in tumor tissues (4.1; range: 0.02-247) compared with matched normal esophageal tissues (0.5; range, 0.0-21.4; p < 0.001). Overexpression of OPN mRNA was significantly associated with clinical stage (p = 0.01). The more severe the clinical stage (from I-II, III, to IV) was the higher frequency of overexpression of OPN mRNA. No significant associations were found between overexpression of OPN mRNA and the patients' survival (p = 0.27). Our findings suggest OPN is associated with esophageal tumorigenesis and progression, but not patients' survival.

Gene Expression Profiling in Postmortem Prefrontal Cortex of Major Depressive Disorder

Investigations of the molecular mechanisms underlying major depressive disorder (MDD) have been hampered by the complexity of brain tissue and sensitivity of gene expression profiling approaches. To address these issues, we used discrete microdissections of postmortem dorsolateral prefrontal cortex (DLPFC) (area 9) and an oligonucleotide (60mer) microarray hybridization procedure that increases sensitivity without RNA amplification. Mixed-effects statistical methods were used to rigorously control for medication usage in the subset of medicated depressed subjects. These analyses yielded a rich profile of dysregulated genes. Two of the most highly dysregulated genes of interest were stresscopin, a neuropeptide involved in stress responses, and Forkhead box D3 (FOXD3), a transcription factor. Secondary cell-based analysis demonstrated that stresscopin and FoxD3 are increased in neurons of DLPFC gray matter of MDD subjects. These findings identify abnormal gene expression in a discrete region of MDD subjects and contribute to further elucidation of the molecular alterations of this complex mood disorder.

Frequent decreased expression of candidate tumor suppressor gene, DEC1, and its anchorage‐independent growth properties and impact on global gene expression in esophageal carcinoma

Previous studies showed that expression of the novel candidate tumor suppressor gene, DEC1 (Deleted in Esophageal Cancer 1), is reduced in esophageal carcinoma and suppresses cancer cell growth in vitro and tumor growth in vivo in nude mice. This study shows that DEC1 gene expression was downregulated in 100% of 16 esophageal squamous cell carcinoma (ESCC) cell lines and 52 and 45%, respectively, of esophageal tumor specimens from Hong Kong and a high-risk ESCC region of Henan, China. Using epitope tagging, the DEC1 protein was localized to both the cytoplasm and nucleus of the cell. In 3D Matrigel culture, no significant difference in colony numbers formed was observed for DEC1 stable transfectants, as compared to vector-alone transfectant controls. However, significantly smaller colony sizes were observed for the DEC1 transfectants. In in vitro cell migration, invasion and soft agar assays of DEC1 transfectants, only the soft agar assay showed statistically significant differences in colony numbers with the vector-alone controls, indicating that DEC1 may be involved in anchorage-independent cell growth. In addition, the global gene expression affected by DEC1 in tumor-suppressive stable transfectants was investigated using cDNA oligonucleotide microarray hybridization. Three candidate genes, TFPI-2, GDF15 and DUSP6, were identified through this approach; they are downregulated in tumor segregants of DEC1 stable transfectants, ESCC cell lines and esophageal tumors and have a potential role in tumor growth and progression. These studies show that DEC1 is involved in esophageal cancer development and help elucidate its functional role in tumor development.

A DNA oligonucleotide microarray for detecting human astrovirus serotypes

Human astroviruses have been shown in numerous studies to be an important cause of gastroenteritis in young children worldwide. The present communication addresses their characterization by use of oligonucleotide microarray hybridization. The system developed consists of an RT-PCR using primers of low degeneracy capable of detecting all eight serotypes of human astroviruses. RT-PCR products are then hybridized against a microarray consisting of short oligonucleotide probes 17–18 nucleotides in length. Cy3-labeled ssDNA targets are generated using a Cy3-labeled primer in the RT-PCR. The non-labeled strand is enzymatically digested, and the labeled target is rescued by column purification. This method of generating labeled target uses equimolar concentrations of the amplifying primers and does not compromise assay sensitivity for initial detection of the virus. Hybridization can be performed without the need for additional amplification. Although the amplicon spans a relatively conserved region of the astrovirus genome, the use of short probes enables type distinction despite such limited diversity. Probes differing by as little as a single nucleotide can be used to distinguish isolates. The microarray developed was capable of distinguishing representatives of the eight known serotypes of human astroviruses.