High-throughput Microarray Analysis Research Articles

Simple and sensitive microRNA labeling by terminal deoxynucleotidyl transferase

MicroRNAs (miRNAs) constitute a critically important class of non-translated, small RNAs, which post-transcriptionally regulate gene expression via one of the multiple mechanisms. To profile miRNA expression, microarrays have been extensively applied to the high-throughput detection of miRNAs. Here, we described a novel 3'-end miRNA-labeling method for microarray detection by terminal-deoxynucleotidyl transferase (TdT). TdT can catalyze the formation of polynucleotides at RNA receptor molecule with deoxycytidine triphosphate (dCTP). Using this activity, miRNA was successfully labeled by adding fluorescence dCTPs to its 3'-end. This labeling method was very simple and sensitive. The TdT-labeling method can detect as little as 0.04 fmol of synthetic small RNA, and produce precise and accurate measurements that span a linear dynamic range from 0.04 to 5 fmol of synthetic small RNA. The high consistency of miRNA expression data between our TdT method and real-time polymerase chain reaction analysis indicated the reliability and accuracy of the TdT method. Taken together, these results emphasize the immense potential application of the TdT-labeling method for sensitive and high-throughput microarray analysis of miRNA expression.

Identification of the role of C/EBP in neurite regeneration following microarray analysis of a L. stagnalisCNS injury model

BackgroundNeuronal regeneration in the adult mammalian central nervous system (CNS) is severely compromised due to the presence of extrinsic inhibitory signals and a reduced intrinsic regenerative capacity. In contrast, the CNS of adult Lymnaea stagnalis (L. stagnalis), a freshwater pond snail, is capable of spontaneous regeneration following neuronal injury. Thus, L. stagnalis has served as an animal model to study the cellular mechanisms underlying neuronal regeneration. However, the usage of this model has been limited due to insufficient molecular tools. We have recently conducted a partial neuronal transcriptome sequencing project and reported over 10,000 EST sequences which allowed us to develop and perform a large-scale high throughput microarray analysis.ResultsTo identify genes that are involved in the robust regenerative capacity observed in L. stagnalis, we designed the first gene chip covering ~15, 000 L. stagnalis CNS EST sequences. We conducted microarray analysis to compare the gene expression profiles of sham-operated (control) and crush-operated (regenerative model) central ganglia of adult L. stagnalis. The expression levels of 348 genes were found to be significantly altered (p < 0.05) following nerve injury. From this pool, 67 sequences showed a greater than 2-fold change: 42 of which were up-regulated and 25 down-regulated. Our qPCR analysis confirmed that CCAAT enhancer binding protein (C/EBP) was up-regulated following nerve injury in a time-dependent manner. In order to test the role of C/EBP in regeneration, C/EBP siRNA was applied following axotomy of cultured Lymnaea PeA neurons. Knockdown of C/EBP following axotomy prevented extension of the distal, proximal and intact neurites. In vivo knockdown of C/EBP postponed recovery of locomotory activity following nerve crush. Taken together, our data suggest both somatic and local effects of C/EBP are involved in neuronal regeneration.ConclusionsThis is the first high-throughput microarray study in L. stagnalis, a model of axonal regeneration following CNS injury. We reported that 348 genes were regulated following central nerve injury in adult L. stagnalis and provided the first evidence for the involvement of local C/EBP in neuronal regeneration. Our study demonstrates the usefulness of the large-scale gene profiling approach in this invertebrate model to study the molecular mechanisms underlying the intrinsic regenerative capacity of adult CNS neurons.

Automatic Spot Identification for High Throughput Microarray Analysis

High throughput microarray analysis has great potential in scientific research, disease diagnosis, and drug discovery. A major hurdle toward high throughput microarray analysis is the time and effort needed to accurately locate gene spots in microarray images. An automatic microarray image processor will allow accurate and efficient determination of spot locations and sizes so that gene expression information can be reliably extracted in a high throughput manner. Current microarray image processing tools require intensive manual operations in addition to the input of grid parameters to correctly and accurately identify gene spots. This work developed a method, herein called auto-spot, to automate the spot identification process. Through a series of correlation and convolution operations, as well as pixel manipulations, this method makes spot identification an automatic and accurate process. Testing with real microarray images has demonstrated that this method is capable of automatically extracting subgrids from microarray images and determining spot locations and sizes within each subgrid, regardless of variations in array patterns and background noises. With this method, we are one step closer to the goal of high throughput microarray analysis.

The Cortical and Striatal Gene Expression Profile of 100 Hz Electroacupuncture Treatment in 6-Hydroxydopamine-Induced Parkinson's Disease Model

Electroacupuncture (EA), especially high-frequency EA, has frequently been used as an alternative therapy for Parkinson disease (PD) and is reportedly effective for alleviating motor symptoms in patients and PD models. However, the molecular mechanism underlying its effectiveness is not completely understood. To implement a full-scale search for the targets of 100 Hz EA, we selected rat models treated with 6-hydroxydopamine into the unilateral MFB, which mimic end-stage PD. High-throughput microarray analysis was then used to uncover the regulated targets in the cortex and striatum after 4-week EA treatment. In the differentially regulated transcripts, the proportion of recovered expression profiles in the genes, the functional categories of targets in different profiles, and the affected pathways were analyzed. Our results suggested that the recovery of homeostasis in the transcript network and many regulated functional clusters in the cortex and striatum after EA treatment may contribute to the behavioral improvement of PD rats.

Computational Analysis on the Role of GPM6A in Human Thyroid Cancer

Thyroid cancer is one of the major cancers around the world. In this study the whole thyroid genome is systematically scanned in order to hit those molecular targets which are highly associated with thyroid cancer. To achieve this goal bioinformatics methodologies are combined. These include the high throughput microarray analysis combined with Serial analysis of gene expression. The results obtained revealed Glycoprotein M6A (GPM6A) as a novel associated gene marker. It belongs to the glycoprotein’s family which plays a major role in cell migration and also known as the major contributors in tumor formation. Moreover the biological pathway of GPM6A is not yet been defined. In this study by assessing the whole biological mechanism the pathway is also inferred .This new drug target will help the biologists in finding the early diagnosis and better treatment for the thyroid cancer patients.

Neuroendocrine Mechanisms of Acupuncture

Neuroendocrine Mechanisms of Acupuncture

Unraveling the ischemic brain transcriptome in a permanent middle cerebral artery occlusion mouse model by DNA microarray analysis

SUMMARYBrain ischemia, also termed cerebral ischemia, is a condition in which there is insufficient blood flow to the brain to meet metabolic demand, leading to tissue death (cerebral infarction) due to poor oxygen supply (cerebral hypoxia). Our group is interested in the protective effects of neuropeptides for alleviating brain ischemia, as well as the underlying mechanisms of their action. The present study was initiated to investigate molecular responses at the level of gene expression in ischemic brain tissue. To achieve this, we used a mouse permanent middle cerebral artery occlusion (PMCAO) model in combination with high-throughput DNA microarray analysis on an Agilent microarray platform. Briefly, the right (ipsilateral) and left (contralateral) hemispheres of PMCAO model mice were dissected at two time points, 6 and 24 hours post-ischemia. Total RNA from the ischemic (ipsilateral) hemisphere was subjected to DNA microarray analysis on a mouse whole genome 4x44K DNA chip using a dye-swap approach. Functional categorization using the gene ontology (GO, MGD/AMIGO) of numerous changed genes revealed expression pattern changes in the major categories of cellular process, biological regulation, regulation of biological process, metabolic process and response to stimulus. Reverse-transcriptase PCR (RT-PCR) analysis on randomly selected highly up- or downregulated genes validated, in general, the microarray data. Using two time points for this analysis, major and minor trends in gene expression and/or functions were observed in relation to early- and late-response genes and differentially regulated genes that were further classified into specific pathways or disease states. We also examined the expression of these genes in the contralateral hemisphere, which suggested the presence of bilateral effects and/or differential regulation. This study provides the first ischemia-related transcriptome analysis of the mouse brain, laying a strong foundation for studies designed to elucidate the mechanisms regulating ischemia and to explore the neuroprotective effects of agents such as target neuropeptides.

Seeking gene candidates responsible for developmental origins of health and disease

Human epidemiological evidence has led scientists to theorize that undernutrition during gestation is an important early origin of adult diseases. Animal models have successfully demonstrated that maternal diet could contribute to some adult diseases. Undernutrition is perceived harmful in pregnant women, whereas calorie restriction is a strategy proven to extend healthy and maximum lifespan in adult. This diagrammatically opposite effect of nutritional condition might provide us with hints to search for genes underlying health conditions. Here, we have initiated a study examining the effect of undernutrition on maternal and fetal livers, utilizing high-throughput DNA microarray analysis for screening genome-wide changes in their transcriptomes. Briefly, pregnant mice were exposed to food deprivation (FD) on gestation day (GD) 17, and cesarean section was performed on GD18. Control mice were supplied with chow ad libitum until sacrifice. Total RNA extracted from mother and fetal livers for each control and treatment (FD) was analyzed with an Agilent mouse whole genome DNA chip. A total of 3058 and 3126 up- (>1.5-fold) and down- (<0.75-fold) regulated genes, and 1475 and 1225 up- (>1.5-fold) and down- (<0.75-fold) regulated genes showed differential expression at the mRNA level, in the maternal and fetal livers, respectively. Interestingly, 103 genes up-regulated in the mother were down-regulated in the fetus, whereas 108 down-regulated maternal genes were up-regulated in the fetus; these 211 genes are potential candidates related to longevity or health. The role of some of these genes, in context of the proposed mechanisms for developmental origins of health and disease is discussed.

Effects of dietary plant-derived phytonutrients on the genome-wide profiles and coccidiosis resistance in the broiler chickens

BackgroundThe present study was conducted to investigate the effects of dietary plant-derived phytonutrients, carvacrol, cinnamaldehyde and Capsicum oleoresin, on the translational regulation of genes associated with immunology, physiology and metabolism using high-throughput microarray analysis and in vivo disease challenge model of avian coccidiosis.MethodsIn this study, we used nutrigenomics technology to investigate the molecular and genetic mechanisms of dietary modulation of host innate immunity and metabolism by three phytonutrients. To validate their immunomodulatory effects in a disease model, young broiler chickens fed a standard diet supplemented with three phytochemicals (carvacrol, cinnamaldehyde, and Capsicum oleoresin) from one day post-hatch were orally challenged with E. acervulina. The body weight gain and fecal oocyst production were used to evaluate coccidiosis disease parameters.ResultsAnalysis of global gene expression profiles of intestinal tissues from phytonutrient-fed birds indicated that Capsicum oleoresin induced the most gene changes compared to the control group where many of these genes were associated with those of metabolism and immunity. The most reliable network induced by dietary cinnamaldehyde treatment was related with the functions of antigen presentation, humoral immune response, and inflammatory disease. Furthermore, dietary supplementation with these phytonutrients significantly protected broiler chickens against live coccidiosis challenge infection based on body weight and parasite fecundity.ConclusionsThe results of this study provide clear evidence to support the idea that plant-derived phytochemicals possess immune-enhancing properties in chickens and these new findings create a new possibility to develop effective drug-free alternative strategies for disease control for poultry infectious diseases.

An evaluation of microRNA signature and response of patients with gastric cancer to cisplatin and fluorouracil.

4062 Background: In previous miRNA microarray studies in gastric cancer, adjacent non-tumor tissue was used as the control as opposed to tissue from healthy volunteers. Also, most of previous studies attempting to identify miRNA predictors of clinical chemoresistance in cancer have examined only individual miRNAs. Very few high-throughput microarray analyses have been performed to identify miRNA expression signatures predictive of chemotherapy resistance in metastatic solid tumor patients. In order to identify miRNA signatures for gastric cancer and for predicting clinical resistance to cisplatin/fluorouracil (CF) chemotherapy, a comprehensive miRNA microarray analysis was performed using endoscopic biopsy samples. Methods: Endoscopic biopsy samples were collected prior to chemotherapy from 90 gastric cancer patients treated with CF and from 34 healthy volunteers. At the time of disease progression, post-treatment samples were collected from 8 clinical responders. miRNA expression was determined using a custom-designed Agilent microarray. In order to identify a miRNA signature for chemotherapy resistance, we correlated miRNA expression levels with the time to progression (TTP) after CF. Results: A signature distinguishing gastric cancer from normal stomach epithelium was identified. Fifty-eight miRNAs were significantly correlated with TTP of 82 cancer patients (P<0.05). Prominent among upregulated chemosensitive miRNAs were miRNAs associated with apoptosis. When this 58-miRNA predictor was applied to a separate set of pre- and post-treatment tumor samples from the 8 clinical responders, all of 8 pre-treatment samples were correctly predicted as low-risk, whereas samples from the post-treatment, chemoresistant state were enriched for the 58 miRNA signature, suggesting that selection for the expression of these miRNAs occurred as chemoresistance developed. Conclusions: This study identified a miRNA expression signature that distinguishes gastric cancer from normal stomach epithelium from healthy volunteers, and a chemoreresistance miRNA expression signature that is correlated with TTP after CF, although limited by the small sample size of the validation set.

A platform for functional prediction and comparative analyses of transcription factors of legumes and beyond

Sequence-specific DNA-binding transcription factors (TFs) are key molecular switches that control many of the biological processes in a cell or organism. Recent completion of the genomic sequences of soybean (Glycine max), Locus japonicus and Medicago truncatula has enabled us to carry out large-scale data mining to identify and annotate regulatory TFs for functional and comparative genomics of the TF repertoires of these three major legumes in themselves or with those of other species. The identified TFs of each legume were grouped in 61 TF families, and these TFs all together were integrated into a so-called LegumeTFDB (http://legumetfdb.psc.riken.jp). Detailed annotations of TF genes can be accessed at LegumeTFDB. Moreover, we included hyperlinks to expression data of a number of soybean TF genes, generated by high-throughput microarray analyses to facilitate functional prediction. LegumeTFDB enables the search for all types of known abiotic stress responsive motifs as well as cis-motifs provided by the PLACE database in promoter region of each of the TFs. Integration of expression analysis, comparative sequence analysis and cis-motif and GO annotations provided through this study would aid in systematic functional prediction of the legume TFs. LegumeTFDB provides an important user-friendly public resource for comparative genomics and understanding of transcriptional regulation in three important legumes and beyond.

Microarray Analyses of Shrimp Immune Responses

Shrimp aquaculture is one of the major foodproducing industries in the world. However, it is being impacted by several problems including diseases, antibiotic use, and environmental factors. The extent of the effects of these problems in the immune system of the shrimp at the molecular level is just beginning to be understood. Here, we review the gene expression profile of shrimp in response to some of these problems using the high-throughput microarray analysis, including white spot syndrome virus, yellow head virus, Vibrio spp., peptidoglycan, oxytetracycline, oxolinic acid, salinity, and temperature.

2136 TRANSFORMING GROWTH FACTOR-β (TGF-β])-INDUCED DNA METHYLTRANSFERASE (DNMT) IS ASSOCIATED WITH WORSE CLINICAL OUTCOME AFTER RADICAL PROSTATECTOMY

You have accessJournal of UrologyProstate Cancer: Markers II1 Apr 20102136 TRANSFORMING GROWTH FACTOR-β (TGF-β])-INDUCED DNA METHYLTRANSFERASE (DNMT) IS ASSOCIATED WITH WORSE CLINICAL OUTCOME AFTER RADICAL PROSTATECTOMY Qiang Zhang, Thomas Jang, Lin Chen, Brian Helfand, Smith Norm, Borko Jovanovic, Yinglu Guo, and Chung Lee Qiang ZhangQiang Zhang Chicago, IL More articles by this author , Thomas JangThomas Jang New Brunswick, NJ More articles by this author , Lin ChenLin Chen Chicago, IL More articles by this author , Brian HelfandBrian Helfand Chicago, IL More articles by this author , Smith NormSmith Norm Chicago, IL More articles by this author , Borko JovanovicBorko Jovanovic Chicago, IL More articles by this author , Yinglu GuoYinglu Guo Beijing, People's Republic of China More articles by this author , and Chung LeeChung Lee Chicago, IL More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.2224AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Gradual loss of sensitivity to and over-expression of TGF-β are features associated with advanced prostate cancer. Previously, we reported that TGF-β induced DNMT and corresponding downregulation of TGF-β receptors (TβRs) are associated with aggressive prostate cancer cell lines. However, the association between DNMT expression and clinical outcome following radical prostatectomy has yet to be defined. In this study, we evaluate the prognostic significance of DNMTs expression in men with prostate cancer following prostatectomy. METHODS A total of 287 radical prostatectomy specimens were obtained from Northwestern University's Prostate SPORE tissue bank to determine the association between DNMT expression in radical prostatectomy specimens and clinical outcome. Levels of TGF-β, TβRs, and DNMT were analyzed using a high-throughput tissue microarray analysis. Tumor grade was categorized as follows: Gleason score 4-5, Gleason score 7, and Gleason score 8-10. Disease recurrence was defined as PSA>0.2 ng/ml and based on patient follow-up information from the Prostate SPORE clinical-linked database. Median follow-up was 60 months (range 22 to 82). RESULTS There were 165 specimens had Gleason score 4-6, 55 had Gleason score 7, and 67 had Gleason score 8-10. High levels of DNMT1 expression occurred in 27.9%, 1.8% and 1.3% of Gleason score 8-10, Gleason score 7 and Gleason score 4-6 tumors, respectively. High levels of DNMT 3a expression occurred in 40.6%, 11.9% and 6.7% of of Gleason score 8-10, Gleason score 7 and Gleason score 4-6 tumors, respectively. High levels of DNMT3b expression occurred in 58.7%, 18.3% and 22.8% of Gleason score 8-10, Gleason score 7 and Gleason score 4-6, respectively. TGF-β1 and DNMTs (1, 3a and 3b) directly correlated with increasing Gleason score. There was a significant correlation between the expression of TGF-β1 and DNMT1, DNMT3a and DNMT3b. DNMT1 was inversely correlated to TβRI and TβRII. Kaplan Meier curves show that expression levels of TGF-β1, age, Gleason Score and DNMT1were associated with PSA recurrence. When these significant variables were retained in a COX proportional hazards model, only DNMT1 <3 or 3 was found to be predictive of PSA recurrence (Hazard ratio 4.03, 95%CI 1.0-4.12, p= 0.0004). CONCLUSIONS Tumor derived TGF-β-induced DNMTs mediates promoter hypermethylation of its own receptors, which contributes to insensitivity in growth inhibition by this cytokine, and may independently predict disease recurrence of prostate cancer patients following prostatectomy. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e830 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Qiang Zhang Chicago, IL More articles by this author Thomas Jang New Brunswick, NJ More articles by this author Lin Chen Chicago, IL More articles by this author Brian Helfand Chicago, IL More articles by this author Smith Norm Chicago, IL More articles by this author Borko Jovanovic Chicago, IL More articles by this author Yinglu Guo Beijing, People's Republic of China More articles by this author Chung Lee Chicago, IL More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

The German Research Unit “Neuronal and glial P2 receptors; molecular basis and functional significance”

The German Research Unit “Neuronal and glial P2 receptors; molecular basis and functional significance”

From tumor immunology to cancer immunotherapy: Miles to go

Advances in tumor immunology are supporting the clinical implementation of several immunological approaches to cancer in the clinical setting. However, the alternate success of current immunotherapeutic regimens underscores the fact that the molecular mechanisms underlying immune-mediated tumor rejection are still poorly understood. Given the complexity of the immune system network and the multidimensionality of tumor/host interactions, the comprehension of tumor immunology might greatly benefit from high-throughput microarray analysis, which can portrait the molecular kinetics of immune response on a genome-wide scale, thus accelerating the discovery pace and ultimately catalyzing the development of new hypotheses in cell biology. Although the general principles of microarray-based gene profiling with the application of DNA microarray have rapidly spread in the scientific community, the need for mastering this technique to produce meaningful data and correctly interpret the enormous output of information generated by this technology is critical and represents a tremendous challenge for investigators. To describe the sequence of events conductive to an effective immune recognition and killing of malignant cells, clinicians might use gene profiling as a powerful tool to assess the activation status of the immune system before and during immunotherapy.

Investigation of post-transcriptional gene regulatory networks associated with autism spectrum disorders by microRNA expression profiling of lymphoblastoid cell lines

BackgroundAutism spectrum disorders (ASD) are neurodevelopmental disorders characterized by abnormalities in reciprocal social interactions and language development and/or usage, and by restricted interests and repetitive behaviors. Differential gene expression of neurologically relevant genes in lymphoblastoid cell lines from monozygotic twins discordant in diagnosis or severity of autism suggested that epigenetic factors such as DNA methylation or microRNAs (miRNAs) may be involved in ASD.MethodsGlobal miRNA expression profiling using lymphoblasts derived from these autistic twins and unaffected sibling controls was therefore performed using high-throughput miRNA microarray analysis. Selected differentially expressed miRNAs were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis, and the putative target genes of two of the confirmed miRNA were validated by knockdown and overexpression of the respective miRNAs.ResultsDifferentially expressed miRNAs were found to target genes highly involved in neurological functions and disorders in addition to genes involved in gastrointestinal diseases, circadian rhythm signaling, as well as steroid hormone metabolism and receptor signaling. Novel network analyses of the putative target genes that were inversely expressed relative to the relevant miRNA in these same samples further revealed an association with ASD and other co-morbid disorders, including muscle and gastrointestinal diseases, as well as with biological functions implicated in ASD, such as memory and synaptic plasticity. Putative gene targets (ID3 and PLK2) of two RT-PCR-confirmed brain-specific miRNAs (hsa-miR-29b and hsa-miR-219-5p) were validated by miRNA overexpression or knockdown assays, respectively. Comparisons of these mRNA and miRNA expression levels between discordant twins and between case-control sib pairs show an inverse relationship, further suggesting that ID3 and PLK2 are in vivo targets of the respective miRNA. Interestingly, the up-regulation of miR-23a and down-regulation of miR-106b in this study reflected miRNA changes previously reported in post-mortem autistic cerebellum by Abu-Elneel et al. in 2008. This finding validates these differentially expressed miRNAs in neurological tissue from a different cohort as well as supports the use of the lymphoblasts as a surrogate to study miRNA expression in ASD.ConclusionsFindings from this study strongly suggest that dysregulation of miRNA expression contributes to the observed alterations in gene expression and, in turn, may lead to the pathophysiological conditions underlying autism.

MiR-21: An Androgen Receptor–Regulated MicroRNA that Promotes Hormone-Dependent and Hormone-Independent Prostate Cancer Growth

Androgen receptor (AR)-mediated oncogenic pathways have not been fully elucidated. In this study, we used high-throughput microarray analysis on two AR-positive prostate cancer (CaP) cell lines to identify 16 AR-responsive microRNAs (miRNA). We focused on miR-21 because of its previously reported oncogenic activity in other cancers. We show androgen-induced AR binding to the defined miR-21 promoter, miPPR-21, suggesting direct transcriptional regulation. Inhibition of miR-21 diminished androgen-induced CaP cell proliferation, providing new evidence that miRNAs can contribute to androgen-driven cell growth. Elevated expression of miR-21 enhanced CaP tumor growth in vivo and, surprisingly, was sufficient for androgen-dependent tumors to overcome castration-mediated growth arrest. Thus, elevated miR-21 expression alone is sufficient to impart castration resistance. Moreover, quantitative reverse transcription-PCR analysis revealed elevated miR-21 expression in CaP when compared with adjacent normal tissue. These results suggest that miR-21 may contribute to CaP pathogenesis.

Nuclear Factor-κB-Mediated Transforming Growth Factor-β-Induced Expression of Vimentin Is an Independent Predictor of Biochemical Recurrence after Radical Prostatectomy

Transforming growth factor-beta (TGF-beta)-mediated epithelial-to-mesenchymal transition (EMT) has been shown to occur in some cancers; however, the pathway remains controversial and varies with different cancers. In addition, the mechanisms by which TGF-beta and the EMT contribute to prostate cancer recurrence are largely unknown. In this study, we elucidated TGF-beta-mediated EMT as a predictor of disease recurrence after therapy for prostate cancer, which has not been reported before. We analyzed TGF-beta-induced EMT using nuclear factor-kappaB (NF-kappaB) as an intermediate mediator in prostate cancer cell lines. A total of 287 radical prostatectomy specimens were evaluated using immunohistochemistry in a high-throughput tissue microarray analysis. Levels of TGF-beta signaling components and EMT-related factors were analyzed using specific antibodies. Results were expressed as the percentage of cancer cells that stained positive for a given antibody and were correlated with disease recurrence rates at a mean of 7 years following radical prostatectomy. In prostate cancer cell lines, TGF-beta-induced EMT was mediated by NF-kappaB signaling. Blockade of NF-kappaB or TGF-beta signaling resulted in abrogation of vimentin expression and inhibition of the invasive capability of these cells. There was high risk of biochemical recurrence associated with tumors that displayed high levels of expression of TGF-beta1, vimentin, and NF-kappaB and low level of cytokeratin 18. This was particularly true for vimentin, which is independent of patients' Gleason score. The detection of NF-kappaB-mediated TGF-beta-induced EMT in primary tumors predicts disease recurrence in prostate cancer patients following radical prostatectomy. The changes in TGF-beta signaling and EMT-related factors provide novel molecular markers that may predict prostate cancer outcomes following treatment.

The Plant-Associated Microbe Gene Ontology (PAMGO) Consortium: community development of new Gene Ontology terms describing biological processes involved in microbe-host interactions

All microbes that form beneficial, neutral, or pathogenic associations with hosts face similar challenges. They must physically adhere to and/or gain entry to host tissues; they must avoid, suppress, or tolerate host defenses; they must acquire nutrients from the host and successfully multiply. Microbes that associate with hosts come from many kingdoms of life and include bacteria, fungi, oomycetes, and nematodes. The increasing numbers of full genome sequences from these diverse microbes provide the opportunity to discover common mechanisms by which the microbes forge and maintain intimate associations with host organisms. However, cross-genome analyses have been hindered by lack of a universal vocabulary for describing biological processes involved in the interplay between microbes and their hosts. The Plant-Associated Microbe Gene Ontology (PAMGO) Consortium has been working for three years as an official interest group of the Gene Ontology (GO) Consortium to develop well-defined GO terms that describe many of the biological processes common to diverse plant- and animal-associated microbes. Creating these terms, over 700 at this time, has required a synthesis of diverse points of view from many research communities. The use of these terms in genome annotation will allow cross-genome searches for genes with common function (without demand for sequence similarity) and also improve the interpretation of data from high-throughput microarray and proteomic analyses. This article, and the more focused mini-reviews that make up this supplement to BMC Microbiology, describe the development and use of these terms.

Internal Controls for Quantitative Polymerase Chain Reaction of Swine Mammary Glands During Pregnancy and Lactation

High-throughput microarray analysis is an efficient means of obtaining a genome-wide view of transcript profiles across physiological states. However, quantitative PCR (qPCR) remains the chosen method for high-precision mRNA abundance analysis. Essential for reliability of qPCR data is normalization using appropriate internal control genes (ICG), which is now, more than ever before, a fundamental step for accurate gene expression profiling. We mined mammary tissue microarray data on >13,000 genes at −34, −14, 0, 7, 14, 21, and 28 d relative to parturition in 27 crossbred primiparous gilts to identify suitable ICG. Initial analysis revealed TBK1, PCSK2, PTBP1, API5, VAPB, QTRT1, TRIM41, TMEM24, PPP2R5B, and AP1S1 as the most stable genes (sample/reference=1±0.2). We also included 9 genes previously identified as ICG in bovine mammary tissue. Gene network analysis of the 19 genes identified AP1S1, API5, MTG1, VAPB, TRIM41, MRPL39, and RPS15A as having no known co-regulation. In addition, UXT and ACTB were added to this list, and mRNA abundance of these 9 genes was measured by qPCR. Expression of all 9 of these genes was decreased markedly during lactation. In a previous study with bovine mammary tissue, mRNA of stably expressed genes decreased during lactation due to a dilution effect brought about by large increases in expression of highly abundant genes. To verify this effect, highly abundant mammary genes such as CSN1S2, SCD, FABP3, and LTF were evaluated by qPCR. The tested ICG had a negative correlation with these genes, demonstrating a dilution effect in the porcine mammary tissue. Gene stability analysis identified API5, VABP, and MRPL39 as the most stable ICG in porcine mammary tissue and indicated that the use of those 3 genes was most appropriate for calculating a normalization factor. Overall, results underscore the importance of proper validation of internal controls for qPCR and highlight the limitations of using absence of time effects as the criteria for selection of appropriate ICG. Further, we showed that use of the same ICG from one organism might not be suitable for qPCR normalization in other species.