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Oleacein (OLE), a rare natural compound found in unfiltered extra virgin olive oil, has been shown to have anti-inflammatory and anti-obesity properties. However, little is known regarding the mechanisms by which OLE influences metabolic processes linked to disease targets, particularly in the context of lipid metabolism. In the present study, we conducted whole-genome DNA microarray analyses in adipocytes differentiated from human adipose-derived stem cells (hASCs) and diabetic hASCs (d-hASCs) to examine the effects of OLE on modulating metabolic pathways. We found that OLE significantly inhibited lipid formation in adipocytes differentiated from both sources. In addition, microarray analysis demonstrated that OLE treatment could significantly downregulate lipid-metabolism-related genes and modulate glucose metabolism in both adipocyte groups. Transcription factor enrichment and protein–protein interaction (PPI) analyses identified potential regulatory gene targets. We also found that OLE treatment enhanced the anti-inflammatory properties in adipocytes. Our study findings suggest that OLE exhibits potential benefits in improving lipid and glucose metabolism, thus holding promise for its application in the management of metabolic disorders. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a pathogen that can affect multiple host species. Evidence of transmission between humans and animals and among different animal species has been reported in recent years. In this study, we investigated 284 free‐living red deer (Cervus elaphus) in the Central Italian Alps to assess the prevalence and molecular characteristics of S. aureus in nasal and intestinal samples in relation to host features and environmental factors. A prevalence of 90%, 26.2% and 10.7% of S. aureus was detected in nasal rectal swabs and faeces, respectively. Calves had a higher probability of being S. aureus intestinal carriers than adults, especially in females when considering faecal samples. Clonal complex (CC) 425 was the most prevalent lineage (61.5%). This is a lineage known to be widespread in both domestic and free‐living animals. It was followed by CC2671 (15.4%) and CC350 (6.4%). A high rate of the phage‐borne virulence factor lukM/lukF‐P83 was detected in CC425 and CC350. Further lineages, which are known to occur in both humans and animals, were detected sporadically in red deer faeces only, that is, CC7, CC9, CC121 and CC707, harbouring the genes of the penicillinase operon and a gene for macrolide resistance (CC9 and CC121). Methicillin resistance genes mecA and mecC were not found. Our results suggest that free‐living red deer may be reservoir for S. aureus in Alpine habitats. [ABSTRACT FROM AUTHOR]

In this article, we propose a new method for estimating the number of equal components m0 of two m-dimensional population means when m is large. The proposed method can be used to estimate the number of equally expressed or differentially expressed genes in DNA microarray studies. It can also be applied in the step of estimating m0 in adaptive false discovery rate controlling procedures. Simulation results show that the bias of the moment estimator is very small for both normal and non normal data. It has higher precision than existing methods in most cases. It has more evident advantage under non normal data. [ABSTRACT FROM AUTHOR]

Background: IgG4-related disease (IgG4-RD) is characterized by elevated serum IgG4 and tissue infiltration by IgG4-positive plasma cells. The pathogenesis of this disease is not clear. Transcriptome analysis was performed to identify genes over- and under-expressed in patients with IgG4-RD. Method: DNA microarray analysis was performed using RNA from peripheral blood mononuclear cells of two patients with IgG4-RD and four healthy individuals. Genes showing a greater than threefold change in expression in IgG4-RD patients following steroid therapy were identified. Four genes related to innate immunity such as transcobalamin I (TCN1), secretory leukocyte peptidase inhibitor (SLPI), bactericidal/permeability-increasing protein (BPI) and lactotransferrin (LTF) were assessed by real-time PCR in 15 IgG4-RD patients and 13 healthy individuals. Result: DNA microarray analysis identified 30 genes showing a greater than threefold change in expression in IgG4-RD patients following steroid therapy. Real-time RT-PCR showed that the levels of mRNAs encoding TCNI and SLPI, except for BPI and LTF, were significantly lower in patients with IgG4-RD than in healthy people. The levels of all four mRNAs in patients with IgG4-RD were significantly increased after steroid treatment. Conclusion: These results indicate that reduction in expression of innate immunity-related genes may participate in the pathogenesis of IgG4-RD that steroid treatment may rectify impaired innate immunity as well as acquired immunity. [ABSTRACT FROM AUTHOR]

In recent years, perinatal stem cells, such as human amniotic epithelial cells (hAECs), have attracted increasing interest as a novel tool of stem cell-based high-throughput drug screening. In the present study, we investigated the bioactivities of squalene (SQ) derived from ethanol extract (99.5%) of a microalgae Aurantiochytrium Sp. (EEA-SQ) in hAECs using whole-genome DNA microarray analysis. Tissue enrichment analysis showed that the brain was the most significantly enriched tissue by the differentially expressed genes (DEGs) between EEA-SQ-treated and control hAECs. Further gene set enrichment analysis and tissue-specific functional analysis revealed biological functions related to nervous system development, neurogenesis, and neurotransmitter modulation. Several adipose tissue-specific genes and functions were also enriched. Gene-disease association analysis showed nervous system-, metabolic-, and immune-related diseases were enriched. Altogether, our study suggests the potential health benefits of microalgae-derived SQ and we would further encourage investigation in EEA-SQ and its derivatives as potential therapeutics for nervous system- and metabolism-related diseases.

Applications that classify DNA microarray expression data are helpful for diagnosing cancer. Many attempts have been made to analyze these data; however, new methods are needed to obtain better results. In this study, a Complex Network (CN) classifier was exploited to implement the classification task. An algorithm was used to initialize the structure, which allowed input variables to be selected over layered connections and different activation functions for different nodes. Then, a hybrid method integrated the Genetic Programming and the Particle Swarm Optimization algorithms was used to identify an optimal structure with the parameters encoded in the classifier. The single CN classifier and an ensemble of CN classifiers were tested on four bench data sets. To ensure diversity of the ensemble classifiers, we constructed a base classifier using different feature sets, i.e., Pearson's correlation, Spearman's correlation, euclidean distance, Cosine coefficient, and the Fisher-ratio. The experimental results suggest that a single classifier can be used to obtain state-of-the-art results and the ensemble yielded better results. [ABSTRACT FROM AUTHOR]

Although high-risk human papillomaviruses (HPVs) are the major risk factors for cervical cancer they have been associated with several other cancers, such as head and neck and oral cancers. Since integration of low-risk HPV11 DNA has been demonstrated in esophageal tumor genomes, this study compared the effects of low-risk HPV11E6 and high-risk HPV18E6 on cellular gene expression. The HPV11E6 and HPV18E6 genes were cloned into an adenoviral vector and expressed in human keratinocytes (HaCaT) in order to investigate early events and to eliminate possible artifacts introduced by selective survival of fast growing cells in stable transfection experiments. HPV11E6 had very little effect on p21 and p53 gene expression, while HPV18E6 resulted in a marked reduction in both these proteins. Both HPV11E6 and HPV18E6 enabled growth of colonies in soft agar, but the level of colony formation was higher in HPV18E6 infected cells. DNA microarray analysis identified significantly differentially regulated genes involved in the cellular transformation signaling pathways. These findings suggest that HPV11E6 and HPV18E6 are important in initiating cellular transformation via deregulation of signaling pathways such as PI3K/AKT and pathways that are directly involved in DNA damage repair, cell survival, and cell proliferation. This study shows that the low-risk HPV11E6 may have similar effects as the high-risk HPV18E6 during the initial stages of infection, but at a much reduced level.

Although high-risk human papillomaviruses (HPVs) are the major risk factors for cervical cancer they have been associated with several other cancers, such as head and neck and oral cancers. Since integration of low-risk HPV11 DNA has been demonstrated in esophageal tumor genomes, this study compared the effects of low-risk HPV11E6 and high-risk HPV18E6 on cellular gene expression. The HPV11E6 and HPV18E6 genes were cloned into an adenoviral vector and expressed in human keratinocytes (HaCaT) in order to investigate early events and to eliminate possible artifacts introduced by selective survival of fast growing cells in stable transfection experiments. HPV11E6 had very little effect on p21 and p53 gene expression, while HPV18E6 resulted in a marked reduction in both these proteins. Both HPV11E6 and HPV18E6 enabled growth of colonies in soft agar, but the level of colony formation was higher in HPV18E6 infected cells. DNA microarray analysis identified significantly differentially regulated genes involved in the cellular transformation signaling pathways. These findings suggest that HPV11E6 and HPV18E6 are important in initiating cellular transformation via deregulation of signaling pathways such as PI3K/AKT and pathways that are directly involved in DNA damage repair, cell survival, and cell proliferation. This study shows that the low-risk HPV11E6 may have similar effects as the high-risk HPV18E6 during the initial stages of infection, but at a much reduced level. [ABSTRACT FROM AUTHOR]

Background: The aim of this study was to apply a new method for se¬lecting a few genes, out of thousands, as plausible markers of a disease.Methods: Hierarchical clustering technique was used along with Support Vector Machine (SVM) and Naïve Bayes (NB) classifiers to select marker-genes of three types of breast cancer. In this method, at each step, one sub¬ject is left out and the algorithm iteratively selects some clusters of genes from the remainder of subjects and selects a representative gene from each cluster. Then, classifiers are constructed based on these genes and the accu¬racy of each classifier to predict the class of left-out subject is recorded. The classifier with higher precision is considered superior.Results: Combining classification techniques with clustering method re¬sulted in fewer genes with high degree of statistical precision. Although all classifiers selected a few genes from pre-determined highly ranked genes, the precision did not decrease. SVM precision was 100% with 22 genes instead of 50 genes while the NB resulted in higher precision of 97.95% in this case. When 20 highly ranked genes selected to be fed to the algorithm, same precision was obtained using 6 and 5 genes with SVM and NB clas¬sifiers respectively.Conclusion: Using hybrid method could be effective in choosing fewer number of plausible marker genes so that the classification precision of these markers is increased. In addition, this method enables detecting new plausible markers that their association to disease under study is not bio¬logically proved.

Induced pluripotent stem cells (iPSCs) obtained by the ectopic expression of defined transcription factors have tremendous promise and therapeutic potential for regenerative medicine. Many studies have highlighted important differences between iPSCs and embryonic stem cells (ESCs). In this work, we used meta-analysis to compare the global transcriptional profiles of human iPSCs from various cellular origins and induced by different methods. The induction strategy affected the quality of iPSCs in terms of transcriptional signatures. The iPSCs generated by non-integrating methods were closer to ESCs in terms of transcriptional distance than iPSCs generated by integrating methods. Several pathways that could be potentially useful for studying the molecular mechanisms underlying transcription factor-mediated reprogramming leading to pluripotency were also identified. These pathways were mostly associated with the maintenance of ESC pluripotency and cancer regulation. Numerous genes that are up-regulated during the induction of reprogramming also have an important role in the success of human preimplantation embryonic development. Our results indicate that hiPSCs maintain their pluripotency through mechanisms similar to those of hESCs.

The transmembrane glycoprotein CD26 or dipeptidyl peptidase IV (DPPIV) is a multifunctional protein. In immune system, CD26 plays a role in T-cell function and is also involved in thymic maturation and emigration patterns. In preclinical studies, treatment with DPPIV inhibitors reduces insulitis and delays or even reverses the new -onset of type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. However, the specific mechanisms involved in these effects remain unknown. The aim of the present study was to investigate how DPPIV inhibition modifies the expression of genes in the thymus of NOD mice by microarray analysis. Changes in the gene expression of β-cell autoantigens and Aire in thymic epithelial cells (TECs) were also evaluated by using qRT-PCR. A DPPIV inhibitor, MK626, was orally administered in the diet for 4 and 6 weeks starting at 6–8 weeks of age. Thymic glands from treated and control mice were obtained for each study checkpoint. Thymus transcriptome analysis revealed that 58 genes were significantly over-expressed in MK626-treated mice after 6 weeks of treatment. Changes in gene expression in the thymus were confined mainly to the immune system, including innate immunity, chemotaxis, antigen presentation and immunoregulation. Most of the genes are implicated in central tolerance mechanisms through several pathways. No differences were observed in the expression of Aire and β-cell autoantigens in TECs. In the current study, we demonstrate that treatment with the DPPIV inhibitor MK626 in NOD mice alters the expression of the immune response-related genes in the thymus, especially those related to immunological central tolerance, and may contribute to the prevention of T1D. [ABSTRACT FROM AUTHOR]

There is growing evidence that the distribution of adipose tissue in the body is of importance in the development of metabolic complications of obesity, such as diabetes, hypertension, and hyperlipidemia. The aim of this study was to identify differentially expressed genes in subcutaneous and omental human adipose tissue in obese men, using a subtractive hybridization strategy. From the obtained set of differentially expressed transcripts, we also aimed to identify genes that have a sex-specific pattern of expression in omental or subcutaneous adipose tissue. Representational difference analysis (RDA) was performed on cDNA from subcutaneous and omental fat tissue from a man with extreme abdominal obesity. Forty-four putatively differentially expressed genes were identified. The obtained RDA products were spotted onto glass slides to screen for differential expression in other obese patients by using a microarray hybridization procedure. Five genes were confirmed to be differentially expressed in subcutaneous or omental adipose tissue from male or female obese patients. One gene was detected only in males and was found to be upregulated in subcutaneous tissue.The findings extend previous knowledge that different fat depots have differential gene expression and indicate that sex differences exist in adipose gene expression patterns. Linder, K., P. Arner, A. Flores-Morales, P. Tollet-Egnell, and G. Norstedt. Differentially expressed genes in visceral or subcutaneous adipose tissue of obese men and women. J. Lipid Res. 2004. 45: 148–154.

Protein malnutrition promotes hepatic steatosis, decreases insulin-like growth factor (IGF)-I production and retards growth. To identify new molecules involved in such changes, we conducted DNA microarray analysis on liver samples from rats fed an isoenergetic low-protein diet for 8 h. We identified the fibroblast growth factor 21 gene (Fgf21) as one of the most strongly up-regulated genes under conditions of acute protein malnutrition (P<0·05, false-discovery rate<0·001). In addition, amino acid deprivation increased Fgf21 mRNA levels in rat liver-derived RL-34 cells (P<0·01). These results suggested that amino acid limitation directly increases Fgf21 expression. FGF21 is a polypeptide hormone that regulates glucose and lipid metabolism. FGF21 also promotes a growth hormone-resistance state and suppresses IGF-I in transgenic mice. Therefore, to determine further whether Fgf21 up-regulation causes hepatic steatosis and growth retardation after IGF-I decrease in protein malnutrition, we fed an isoenergetic low-protein diet to Fgf21-knockout (KO) mice. Fgf21-KO did not rescue growth retardation and reduced plasma IGF-I concentration in these mice. Fgf21-KO mice showed greater epididymal white adipose tissue weight and increased hepatic TAG and cholesterol levels under protein malnutrition conditions (P<0·05). Overall, the results showed that protein deprivation directly increased Fgf21 expression. However, growth retardation and decreased IGF-I were not mediated by increased FGF21 expression in protein malnutrition. Furthermore, FGF21 up-regulation rather appears to have a protective effect against obesity and hepatic steatosis in protein-malnourished animals. [ABSTRACT FROM AUTHOR]

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 27- or 38-amino acid neuropeptide, which belongs to the vasoactive intestinal polypeptide/glucagon/secretin family. PACAP and its three receptor subtypes are expressed in neural tissues, with PACAP known to exert pleiotropic effects on the nervous system. This review provides an overview of current knowledge regarding the neuroprotective effects, mechanisms of action, and therapeutic potential of PACAP in response to ischemic brain injuries. [ABSTRACT FROM AUTHOR]

Study Design. A histological, biological, and immunohistochemical study of human lumbar ligamentum flavum. Objective. To analyze changes in the hypertrophied ligamentum flavum and clarify their etiology. Summary of Background Data. Hypertrophy of the ligamentum flavum has been considered a major contributor to the development of lumbar spinal canal stenosis (LSCS). Although previous studies have reported some factors related to ligamentum flavum hypertrophy, its etiology is still unclear. Methods. Ligamentum flavum samples were collected from 20 patients with LSCS (LSCS group) and 10 patients with lumbar disc herniation (LDH group) as a control. The thickness of the ligamentum flavum was measured histologically. The amounts of elastic fibers and proteoglycans were assessed by Elastica-Masson staining and alcian blue staining, respectively. Gene and protein expressions related to fibrosis, inflammation, and chondrogenesis were analyzed by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. The total genes of the 2 groups were compared by DNA microarray analysis. Results. The ligamentum flavum was significantly thicker in the LSCS group, which had a smaller amount of elastic fibers and a larger amount of proteoglycans. The gene expression related to fibrosis was significantly higher in the LSCS group; however, the immunoreactivities of collagen types I and III were weaker on the dorsal side of the ligamentum flavum in the LSCS group. The gene expression related to chondrogenesis and proteoglycan synthesis was significantly higher in the LSCS group. There was no significant difference in the gene expression related to inflammation between the 2 groups. Conclusion. Synthesis of the collagenous fibers and degradation of the elastic and collagenous fibers are both accelerated in the ligamentum flavum of patient with LSCS, which may be the reason for hypertrophy of the tissue. In addition, chondrogenesis and proteoglycan synthesis may have critical roles in the pathogenesis of the ligamentum flavum hypertrophy. [ABSTRACT FROM AUTHOR]

A novel DNA microarray analysis targeting key functional genes involved in most nitrogen cycling reactions was developed to comprehensively analyze microbial populations associated with the nitrogen cycle. The developed microarray contained 876 oligonucleotide probes based on the nucleotide sequences of the nif, amo, hao/ hzo, nap, nar, nirK, nirS, nrf, cnor, qnor and nos genes. An analytical method combining detection by the designed microarray with whole community genome amplification was then applied to monitor the nitrogen cycling microorganisms in river water and wastewater treatment sludge samples. The developed method revealed that nitrogen cycling microorganisms in river water appeared to become less diverse in response to input of effluent from municipal wastewater treatment plants. Additionally, the nitrogen cycling community associated with anaerobic ammonium oxidation and partial nitrification reactors could be reasonably analyzed by the developed method. However, the results obtained for two activated sludge samples from municipal wastewater treatment plants with almost equivalent wastewater treatment performance differed greatly from each other. These results suggested that the developed method is useful for comprehensive analysis of nitrogen cycling microorganisms, although its applicability to complex samples with abundant untargeted populations should be further examined. [ABSTRACT FROM AUTHOR]

The most effective reprogramming methods, somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs), are widely used in biological research and regenerative medicine, yet the mechanism that reprograms somatic cells to totipotency remains unclear and thus reprogramming efficiency is still low. Microarray technology has been employed in analyzing the transcriptomes changes during iPS reprogramming. Unfortunately, it is difficult to obtain enough DNA from SCNT reconstructed embryos to take advantage of this technology. In this study, we aimed to identify critical genes from the transcriptional profile for iPS reprogramming and compared expression levels of these genes in SCNT reprogramming. By integrating gene expression information from microarray databases and published studies comparing somatic cells with either miPSCs or mouse embryonic stem cells (ESCs), we obtained two lists of co-upregulated genes. The gene ontology (GO) enriched analysis of these two lists demonstrated that the reprogramming process is associated with numerous biological processes. Specifically, we selected 32 genes related to heterochromatin, embryonic development, and cell cycle from our co-upregulated gene datasets and examined the gene expression level in iPSCs and SCNT embryos by qPCR. The results revealed that some reprogramming related genes in iPSCs were also expressed in SCNT reprogramming. We established the network of gene interactions that occur with genes differentially expressed in iPS and SCNT reprogramming and then performed GO analysis on the genes in the network. The network genes function in chromatin organization, heterochromatin, transcriptional regulation, and cell cycle. Further researches to improve reprogramming efficiency, especially in SCNT, will focus on functional studies of these selected genes. [ABSTRACT FROM AUTHOR]

We have previously shown that whey protein hydrolysate (WPH) causes a greater increase in muscle protein synthesis than does a mixture of amino acids that is identical in amino acid composition. The present study was conducted to investigate the effect of WPH on gene expression. Male Sprague–Dawley rats subjected to a 2 h swimming exercise were administered either a carbohydrate–amino acid diet or a carbohydrate–WPH diet immediately after exercise. At 1 h after exercise, epitrochlearis muscle mRNA was sampled and subjected to DNA microarray analysis. We found that ingestion of WPH altered 189 genes after considering the false discovery rate. Among the up-regulated genes, eight Gene Ontology (GO) terms were enriched, which included key elements such as Cd24, Ccl2, Ccl7 and Cxcl1 involved in muscle repair after exercise. In contrast, nine GO terms were enriched in gene sets that were down-regulated by the ingestion of WPH, and these GO terms fell into two clusters, ‘regulation of ATPase activity’ and ‘immune response’. Furthermore, we found that WPH activated two upstream proteins, extracellular signal-regulated kinase 1/2 (ERK1/2) and hypoxia-inducible factor-1α (HIF-1α), which might act as key factors for regulating gene expression. These results suggest that ingestion of WPH, compared with ingestion of a mixture of amino acids with an identical amino acid composition, induces greater changes in the post-exercise gene expression profile via activation of the proteins ERK1/2 and HIF-1α. [ABSTRACT FROM PUBLISHER]

Background: The aim of this study was to apply a new method for selecting a few genes, out of thousands, as plausible markers of a disease. Methods: Hierarchical clustering technique was used along with Support Vector Machine (SVM) and Naïve Bayes (NB) classifiers to select marker-genes of three types of breast cancer. In this method, at each step, one subject is left out and the algorithm iteratively selects some clusters of genes from the remainder of subjects and selects a representative gene from each cluster. Then, classifiers are constructed based on these genes and the accuracy of each classifier to predict the class of left- out subject is recorded. The classifier with higher precision is considered superior. Results: Combining classification techniques with clustering method resulted in fewer genes with high degree of statistical precision. Although all classifiers selected a few genes from pre-determined highly ranked genes, the precision did not decrease. SVM precision was 100% with 22 genes instead of 50 genes while the NB resulted in higher precision of 97.95% in this case. When 20 highly ranked genes selected to be fed to the algorithm, same precision was obtained using 6 and 5 genes with SVM and NB classifiers respectively. Conclusion: Using hybrid method could be effective in choosing fewer number of plausible marker genes so that the classification precision of these markers is increased. In addition, this method enables detecting new plausible markers that their association to disease under study is not biologically proved. [ABSTRACT FROM AUTHOR]

We introduce a doubly stochastic marked point process model for supervised classification problems. Regardless of the number of classes or the dimension of the feature space, the model requires only 2–3 parameters for the covariance function. The classification criterion involves a permanental ratio for which an approximation using a polynomial-time cyclic expansion is proposed. The approximation is effective even if the feature region occupied by one class is a patchwork interlaced with regions occupied by other classes. An application to DNA microarray analysis indicates that the cyclic approximation is effective even for high-dimensional data. It can employ feature variables in an efficient way to reduce the prediction error significantly. This is critical when the true classification relies on nonreducible high-dimensional features. [ABSTRACT FROM PUBLISHER]

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Cytochrome P450(CYP)s are known to show a sexual dimorphic expression in rat livers. However, the comprehensive analysis for the sex-dependent gene expressions of drug metabolizing enzymes except for CYPs, transporters and nuclear receptors in rat livers and kidneys has not been investigated yet. The purpose of the present study was to identify the novel drug metabolizing and pharmacokinetics (DMPK)-related gene(s) which show the sex difference in the mRNA expressions in rat livers and kidneys. Total RNAs were prepared from livers and kidneys in both male and female rats (Crl:CD(SD) and Crlj:WI). A DNA microarray analysis using a "GeneSQUARE Multiple Assay DNA Microarray Drug Metabolism Gene Expression for Rat" was performed. DMPK-related genes which showed sex differences in the mRNA expression were identified in rat livers or kidneys. Especially, the female dominant expressions of UDP glucuronosyltransferase (UGT) s were seen in rat livers and kidneys. The sex difference of UGT expressions in rats might be one of the causal factors of the sex difference of the biological response to UGT substrates. [ABSTRACT FROM AUTHOR]

An early prognostic indicator which warns of progressive joint destruction of rheumatoid arthritis (RA) was explored using a novel suspension-array technique in moderate (Steinbrocker stage I and II) and severe (Steinbrocker stage IV) RA patients. DNA microarray analysis of peripheral blood lymphocytes showed significant increase of interleukin (IL)-2 receptor α-chain (CD25) gene expression, a regulatory T cell (Treg) surface marker in severe RA patients. In contrast, suspension array, a comprehensive bead-based enzyme-linked immunosorbent assay (ELISA), revealed decreased production of IL-10 and increased production of interferon (IFN)-γ in sera in the incipient stage of the aggressive disease process. Both in moderate and in severe RA patients, the IFN-γ/IL-10 ratio indicated deterioration of the disease with universal validity. Fluorescence-activated cell sorting (FACS) and reverse-transcription polymerase chain reaction (RT-PCR) analysis showed extant CD4+CD25+ regulatory T cells in severe RA patients, however Foxp3, a regulatory T cell-specific transcription factor, gene expression was absent, while glucocorticoid-induced tumor necrosis factor (TNF) receptor family-related protein (GITR), which transmits a signal that abrogates regulatory T cell functions, was elevated. In the current study, we showed the validity of suspension-array analysis for enabling more complete understanding of RA, and showed that IFN-γ/IL-10 ratio can be a prognostic tool for early lesion and more aggressive RA. [ABSTRACT FROM AUTHOR]

Objective and design: To investigate whether di-(2-ethylhexyl) phthalate (DEHP) affects the production of inflammatory cytokines by human macrophages. Materials and methods: Differentiated macrophage-like THP-1 cells were exposed to 200 μM DEHP for 3 h, followed by incubation in the presence or absence of opsonized zymosan A, and the concentrations of TNF-α, IL-1β, IL-8, and IL-6 in the culture media were determined by ELISA. DNA microarray and quantitative real-time RT-PCR analyses were performed to identify genes that showed changes in expression in response to DEHP. Results: DEHP treatment increased the concentrations of TNF-α, IL-1β, IL-8, and IL-6 in the media, regardless of whether the cells phagocytosed zymosan. DNA microarray analysis showed that DEHP increased the levels of expression of IL-8, CXCL1, CXCL2, CXCL3, CXCL6, CCL3, MMP3, MMP10, MMP14, and CSF2 mRNA, and real-time RT-PCR showed that DEHP significantly enhanced the levels of expression of IL-8, CXCL1, CXCL2, CXCL3, CXCL6, CCL3, MMP10, CSF2, TNF-α, IL-1β, and IL-6 mRNA in THP-1 cells. DEHP significantly induced translocation of p65 NF-κB into the nucleus. Conclusion: DEHP enhances the production of inflammatory cytokines and chemokines by macrophages, and exacerbates their inflammatory response. [ABSTRACT FROM AUTHOR]

The article focuses on a study that investigates the important properties of a high-malic acid-producing strains of Saccharomyces cerevisiae isolated from sake liquors. It mentions the efforts developed to maintain the diversification and quality of sake liquor while characterizing the malic-acid profile and gene profile of high malic acid-producing sake yeast strains. Finding of the study claimed that stress response has been upregulated while thiamine synthesis were downregulated.

Over the past several years, we have been interested in understanding the mechanisms by which mammary carcinomas acquire hormone independence. We demonstrated that carcinoma associated fibroblasts participate in the ligand-independent activation of progesterone receptors inducing tumor growth. In this study, we used DNA microarrays to compare the gene expression profiles of tumors from the MPA mouse breast cancer model, one hormone-dependent (C4-HD) and one hormone-independent (C4-HI), using whole tumor samples or laser-captured purified stromal and epithelial cells obtained from the same tumors. The expression of selected genes was validated by immunohistochemistry and immunofluorescence assays. We identified 413 genes specifically expressed in tumor stroma. Eighty-five percent of these genes were upregulated, whereas the remaining 15% were downregulated in C4-HI relative to their expression in the C4-HD tumor stroma. Several matrix metallopeptidases were overexpressed in the C4-HI tumor microenvironment. On the other hand, 1100 genes were specifically expressed in the tumor parenchyma. Among them, the 29% were upregulated, whereas the remaining 71% were downregulated in C4-HI relative to C4-HD tumor epithelium. Steap, Pdgfc, Runx2, Cxcl9, and Sdf2 were among the genes with high expression in the C4-HI tumor parenchyma. Interestingly, Fgf2 was one of the few genes upregulated by MPA in C4-HD tumors, confirming its pivotal role in regulating tumor growth in this model. In conclusion, we demonstrate herein a gene expression profile that distinguishes both the epithelial and the stromal cells in mammary tumors with different hormone dependence, supporting the hypothesis that the tumor-associated stroma may contribute to hormone-independent tumor growth. [ABSTRACT FROM AUTHOR]

Proteolytic degradation by secreted proteases into the culture medium is one of the significant problems to be solved in heterologous protein production by filamentous fungi including Aspergillus oryzae. Double ( tppA, and pepE) and quintuple ( tppA, pepE, nptB, dppIV, and dppV) disruption of protease genes enhanced human lysozyme (HLY) and bovine chymosin (CHY) production by A. oryzae. In this study, we used a quintuple protease gene disruptant and performed successive rounds of disruption for five additional protease genes ( alpA, pepA, AopepAa, AopepAd, and cpI), which were previously investigated by DNA microarray analyses for their expression. Gene disruption was performed by pyrG marker recycling with a highly efficient gene-targeting background (∆ ligD) as previously reported. As a result, the maximum yields of recombinant CHY and HLY produced by a decuple protease gene disruptant were approximately 30% and 35%, respectively, higher than those produced by a quintuple protease gene disruptant. Thus, we successfully constructed a decuple protease gene disruptant possessing highly improved capability of heterologous protein production. This is the first report on decuple protease gene disruption that improved the levels of heterologous protein production by the filamentous fungus A. oryzae. [ABSTRACT FROM AUTHOR]

Theiler's virus-induced demyelination represents an important animal model to study the chronic-progressive form of multiple sclerosis (MS). The aim of the present study was to identify specific genes and pathways in the deep cervical lymph node (cLN) and spleen of experimentally infected SJL-mice, using DNA microarrays. Analyses identified 387 genes in the deep cLN and only 6 genes in the spleen of infected animals. The lymph node presented 27.4% of genes with fold changes ±1.5 at 14 days post infection (dpi) and a reduced transcription at later time points. K-means clustering analyses resulted in five clusters. Accordingly, functional annotation revealed that the B-cell immune response pathway was the most up-regulated cluster at the early phase. Additionally, an increase of CD68- and lysozyme-positive cells in the deep cLN was observed by immunohistochemistry. Polioencephalitis was most intense at 14 dpi, and the spinal cord demyelinating leukomyelitis started at 42 dpi. In summary, early gene expression is indicative of virus-trigged immune responses in the central nervous system (CNS)-draining lymph node. The decreased gene transcription in the deep cLN during the chronic phase and the low number of spleen genes supports the hypothesis of a compartmentalized inflammation within the CNS, as described in progressive MS. [ABSTRACT FROM AUTHOR]

Despite its importance in plant growth and development, the auxin biosynthetic pathway has remained elusive. In this study, we analyzed hormone series transcriptome data from AtGenExpress in Arabidopsis and found that aminoethoxyvinylglycine (AVG) had the strongest anti-auxin activity. We also identified other effective compounds such as l-amino-oxyphenylpropionic acid (AOPP) through additional screening. These inhibitors shared characteristics in that they inhibited pyridoxal enzymes and/or aminotransferases. They reduced endogenous IAA levels in both monocots and dicots. l-AOPP inhibited root development of Arabidopsis in main root elongation, gravitropism, root skewing and root hair formation. This inhibition was generally recovered after exogenous IAA treatment, and the recovery was almost completely to the level of non-inhibited seedlings. The compounds inhibited conversion from tryptophan to indole-3-pyruvic acid in enzyme extracts from Arabidopsis and wheat. Our data collectively suggest that the inhibitors directly blocked auxin biosynthesis, and that the major target site was tryptophan aminotransferase. This enzyme probably makes up one of the major biosynthesis pathways conserved among higher plants. Each inhibitor, however, demonstrated a different action spectrum in shoot and root of rice and tomato, indicating diversity in biosynthesis pathways between organs and species. Our results provide novel insights into auxin biosynthesis and action, and uncover structural characteristics of auxin biosynthesis inhibitors. [ABSTRACT FROM PUBLISHER]

Abstract: Background: Enterocytes of the jejunum express several genes related to digestion/absorption of nutrients and ions when these cells rapidly differentiate from crypt to villus cells. However, it is unknown whether the distribution of extensive gene expression along the villus–crypt axis of the jejunum is altered during differentiation. Methods: We investigated the changes in jejunal gene expression during differentiation from crypt to villus cells in rats using DNA microarray analysis on cryostat sections of the villus–crypt columns. Results: During differentiation, the expression of many genes related to cell growth rapidly decreased, while expression of genes related to digestion and absorption of nutrients and ions increased. Expression of a subset of genes related to the digestion and absorption of starch and sucrose was highest at the middle of the villi, whereas expression of genes related to dietary fat absorption was highest at the top of the villi. Several transcriptional factors such as Pdx1, Foxa2 and Thra were expressed in the crypt, whereas Klf15 was highly expressed during the crypt–villus transition. Expression of Klf4 and Pparg was highest at the top of the villi. Conclusions: Subsets of genes related to the digestion and absorption of starch/sucrose and dietary fat as well as their transcriptional factors/co-factors are expressed in the specific locations along the crypt–villus axis. General Significance: The jejunum may absorb nutrients effectively by simultaneously expressing subsets of genes along the villus-crypt axis. [Copyright &y& Elsevier]

Abstract: We have recently demonstrated that upregulation of the innate immune system plays a key role in KRV-induced autoimmune diabetes in the BBDR rat, but the nature of this proinflammatory reaction has not yet been addressed. Using a DNA microarray approach, we identified 569 genes upregulated in pancreatic lymph nodes following virus infection. Among the most highly activated are IL-1 pathways, IFN-γ-induced chemokines, and genes associated with interferon production and signaling. Ex vivo and in vitro studies indicate that KRV upregulates proinflammatory cytokines and chemokines in B lymphocytes and Flt-3L-induced plasmacytoid DCs (pDCs). Finally, in contrast to KRV, infection of BBDR rats with the non-diabetogenic KRV homologue H-1 parvovirus fails to induce a robust proinflammatory response in pancreatic lymph nodes. Our findings provide new insights into KRV-induced innate immune pathways that may play a role in early mechanisms leading to islet inflammation and diabetes. [Copyright &y& Elsevier]

Aspergillus oryzae has received attention as a host for heterologous protein production. However, A. oryzae has 134 protease genes, which is recognized to be one of the major reasons for the proteolytic degradation of heterologously produced proteins. We previously reported that double disruption of the protease genes ( tppA and pepE) improved heterologous protein (human lysozyme) production by A. oryzae. In this study, we performed successive round of five protease genes ( tppA, pepE, nptB, dppIV, and dppV) disruption in A. oryzae by pyrG marker recycling with highly efficient gene-targeting background (Δ ligD). The multiple disruption of protease genes were confirmed by Southern blot analysis. Furthermore, the quintuple protease gene disruptants showed the maximum production level of bovine chymosin (CHY) that was 34% higher than those of the double protease gene disruptant (Δ tppA Δ pepE). Consequently, we successfully constructed a multiple protease gene disruptant bearing enhanced levels of CHY productivity. We presented the first evidence that the quintuple disruption of the protease genes improved the production level of a heterologous protein by A. oryzae. [ABSTRACT FROM AUTHOR]

Biologists rely on morphology, function and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three ‘cell lineage trajectories’, which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ cells and induced pluripotent cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast and fibroblast cell lines are mapped near the far end of the trajectories. We suggest that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation. [ABSTRACT FROM PUBLISHER]

Expression of the epithelial cell adhesion molecule EpCAM is upregulated in a variety of carcinomas. This antigen is therefore explored in tumour diagnosis, and clinical trials have been initiated to examine EpCAM-based therapies. Notably, the possible intracellular effects and signalling pathways triggered by EpCAM-specific antibodies are unknown. Here, we show treatment of the mouse lung carcinoma cell line A2C12, of the human lung carcinoma cell line A549 and the human colorectal cell line Caco-2 with the monoclonal EpCAM antibody G8.8 to cause dose dependently an increase in cell proliferation, as determined by the MTS and the 5'-bromo-2'-deoxyuridine (BrdU) labelling assay. Furthermore, a genome-wide approach identified networks of regulated genes, most notably cell cycle regulators, upon treatment with an EpCAM-specific antibody. Indeed, changes in the expression of cell cycle regulators agreed well with the BrdU labelling data, and an analysis of differentially expressed genes revealed the processes with the strongest over-representation of modulated genes, for example, cell cycle, cell death, cellular growth and proliferation, and cancer. These data suggest that EpCAM is involved in signal transduction triggering several intracellular signalling pathways. Knowing EpCAM signalling pathways might lead to a reassessment of EpCAM-based therapies. [ABSTRACT FROM AUTHOR]

Many different methods exist for pattern detection in gene expression data. In contrast to classical methods, biclustering has the ability to cluster a group of genes together with a group of conditions (replicates, set of patients or drug compounds). However, since the problem is NP-complex, most algorithms use heuristic search functions and therefore might converge towards local maxima. By using the results of biclustering on discrete data as a starting point for a local search function on continuous data, our algorithm avoids the problem of heuristic initialization. Similar to OPSM, our algorithm aims to detect biclusters whose rows and columns can be ordered such that row values are growing across the bicluster's columns and vice-versa. Results have been generated on the yeast genome (Saccharomyces cerevisiae), a human cancer dataset and random data. Results on the yeast genome showed that 89% of the one hundred biggest non-overlapping biclusters were enriched with Gene Ontology annotations. A comparison with OPSM and ISA demonstrated a better efficiency when using gene and condition orders. We present results on random and real datasets that show the ability of our algorithm to capture statistically significant and biologically relevant biclusters. [ABSTRACT FROM PUBLISHER]

Abstract: Objective: The problem of marker selection in DNA microarray analysis has been addressed so far by two basic types of approaches, the so-called filter and wrapper methods. Wrapper methods operate in a recursive fashion where feature (gene) weights are re-evaluated and dynamically changing from iteration to iteration, while in filter methods feature weights remain fixed. Our objective in this study is to show that the application of filter criteria in a recursive fashion, where weights are potentially adjusted from cycle to cycle, produces noticeable improvement on the generalization performance measured on independent test sets. Methods and materials: Toward this direction we explore the behavior of two well known and broadly accepted pattern recognition approaches namely the support vector machines (SVM) and a single linear neuron (LN), properly adapted to the problem of marker selection. Within this context we also show how the kernel ability of SVM could be employed in a practical manner to provide alternative ways to approach the problem of reliable marker selection. Results: We explore how the proposed approaches behave in two application domains (breast cancer and leukemia), achieving comparable or even better results than those reported in the related bibliography. An important advantage of these approaches is their ability to derive stable performance without deteriorating due to the complexity of the application domain. Validation is performed using internal leave one out (ILOO) and 10-fold cross validation as well as independent test set evaluation. Conclusions: Results show that the proposed methodologies achieve remarkable performance and indicate that applying filter criteria in a wrapper fashion (‘wrapper filtering criteria’) provides a useful tool for marker selection. The contribution of this study is threefold. First it provides a methodology to apply filter criteria in a wrapper way (which is a new approach), second it introduces a fundamental pattern recognition component namely the single neuron (which is a linear estimator) and explores its behavior on marker selection and third it demonstrates an approach to exploit the kernel ability of SVMs in a practical and effective manner. [Copyright &y& Elsevier]

Abstract: The long-term cardiac effects of amiodarone resemble many aspects of hypothyroidism. The anti-arrhythmic potential of amiodarone may therefore be the result of a drug-induced, local hypothyroid-like condition. To investigate this controversial issue, we compared gene expression profiles in the hearts of rats treated with amiodarone with those of rats with hypothyroidism. Wistar male rats were assigned to 3 groups (n =6–8): Control, systemic hypothyroidism (Hypothyroidism) and amiodarone treatment (Amiodarone, 150 mg/kg/day, p.o., 4 weeks). Electrocardiogram (ECG) recordings, gene profiling by DNA microarray and Northern blotting were carried out. Amiodarone, like Hypothyroidism, caused significant prolongation of RR and QT intervals in ECGs. Microarray analysis of 8435 genes in the left ventricular myocardium revealed a significant similarity in expression profiles between Hypothyroidism and Amiodarone (R =0.63, p <0.00001). The gene expression profiles of Hypothyroidism and Amiodarone showed closer correlation when top 100 up-regulated and 100 down-regulated genes in Hypothyroidism (total 200 genes) were analyzed (R =0.78, p <0.00001). Northern blots of left ventricular myocardium showed a parallel decrease in mRNAs for myosin heavy chain (MHC)-alpha and a parallel increase for myosin heavy chain (MHC)-beta in Hypothyroidism and Amiodarone. In the liver and pituitary, in contrast, Northern blots showed quite different changes in the transcripts of the representative T3-responsive genes in the Hypothyroidism and Amiodarone. In conclusion, long-term treatment with amiodarone causes cardioselective hypothyroid-like alterations in gene expression profiles. The potent anti-arrhythmic activity of amiodarone may be attributable, in part at least, to this unique transcriptional remodeling. [Copyright &y& Elsevier]

Gene expression is altered in mammalian cells (MCF-7 cells), by exposure to a variety of chemicals that mimic steroid hormones or interact with endocrine receptors or their co-factors. Among those populations chronically exposed to these endocrine disruptive chemicals are persons, and their families, who are employed in agriculture or horticulture, or who use agricultural/horticultural chemicals. Among the chemicals most commonly used, both commercially and in the home, is the herbicide glyphosate. Although glyphosate is commonly considered to be relatively non-toxic, we utilized in vitro DNA microarray analysis of this chemical to evaluate its capacity to alter the expression of a variety of genes in human cells. We selected a group of genes, determined by DNA microarray analysis to be dysregulated, and used quantitative real-time PCR to corroborate their altered states of expression. We discussed the reported function of those genes, with emphasis on altered physiological states that are capable of initiating adverse health effects that might be anticipated if gene expression were significantly altered in either adults or embryos exposed in utero. [ABSTRACT FROM AUTHOR]

Clustering algorithms, such as hierarchical clustering, k-means, and fuzzy k-means, have become important tools for gene expression analysis of microarray data. However, the need of prior knowledge of the number of clusters, k, and the fuzziness parameter, b, limits the usage of fuzzy clustering. In this paper, we use simulated annealing (SA) and fuzzy k-means clustering to determine the optimal parameters, namely the number of clusters, k, and the fuzziness parameter, b. To improve SA, two methods, searching with Tabu List and shrinking the scope of randomization, are applied. Our results show that a nearly-optimal pair of k and b (a near optimal value of k) can be obtained without exploring the entire search space. [ABSTRACT FROM AUTHOR]

Diethylhexylphthalate (DEHP) is a widely distributed phthalate, to which humans are exposed to due to its variety of commercial and manufacturing uses. As a plasticiser, it is found in a wide number of products, and metabolites of DEHP have been detected in urine samples from a high percentage of the people screened for phthalates. We utilised DNA microarray analysis to evaluate DEHP for gene expression disrupting activity using the human cell line MCF-7, and found that DEHP significantly dysregulated approximately 34% of the 2400 genes spotted on the NEN2400 chip we used. The results suggest that DEHP, a known estrogen agonist and probable androgen antagonist, alters the expression of a number of genes, many of which are critical for fetal development. Down-regulation of two genes, FGD1 and PAFAH1B1, related in that both are essential for fetal brain development, was corroborated using quantitative real time PCR. These studies show DEHP to be a highly effective human gene expression-altering chemical, and that, at appropriate concentrations, it has the possibility of altering fetal central nervous system development, resulting in the birth defects lissencephaly and/or faciodigitogenital dysplasia. [ABSTRACT FROM AUTHOR]