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The objective of this study was to analyze the expression and prognostic role of the tight junction protein claudin-10 in high-grade serous carcinoma (HGSC). Claudin-10 protein expression by immunohistochemistry was analyzed in 588 HGSC (414 effusions, 174 surgical specimens). Expression in mesotheliomas (n = 97; 47 effusions, 50 surgical specimens) was studied for comparative purposes. CLDN10 mRNA expression by quantitative RT-PCR (qRT-PCR) was analyzed in 40 HGSC effusions. Claudin-10 protein expression was found in 360/588 (61%) HGSC vs. 19/97 (20%) mesotheliomas (p < 0.001), and was higher in HGSC surgical specimens compared to effusions (p < 0.001). qRT-PCR confirmed the presence of CLDN10 mRNA in HGSC effusions. High (> 25%) claudin-10 expression in HGSC effusions was significantly associated with shorter overall survival (OS; p = 0.036) and progression-free survival (PFS; p = 0.045) in univariate analysis, and was an independent prognosticator of OS in multivariate analysis (p = 0.045). In conclusion, claudin-10 protein expression is higher in HGSC compared to mesothelioma, although the diagnostic power of this marker appear to be lesser than other claudin family members. Claudin-10 expression in HGSC effusions is marker of more aggressive disease. [ABSTRACT FROM AUTHOR]

A single transcription factor is known to coordinate expression of a set of metabolites in a biochemical pathway; its use therefore can be a functional strategy in generating plants with desired traits. Triticum aestivum Dof1 (TaDof1) transcription factor is mainly associated with improved nitrogen assimilation in plants. In the current research, the transgenic wheat overexpressing TaDof1 transcription factor, under a constitutive promoter, was developed by Agrobacterium-mediated transformation. The two elite wheat cultivars (Galaxy and Faisalabad-2008) were selected for transformation study. The T0 plants were subjected to screening using selection medium containing herbicide BASTA. PCR results confirmed that only 8 out of 31 plants possessed the complete TaDof1 cassette. A transformation efficiency of 0.46% for Galaxy and 0.08% for Faisalaad-2008 was obtained. The quantitative RT-PCR was performed on T1 plants grown under nitrogen-limiting conditions. A substantial rise in the expression of citrate synthase (CS), isocitrate dehydrogenase (ICDH), phosphoenolpyruvate carboxylase (PEPC), and pyruvate kinase (PK) genes regulated by TaDof1 was observed after 4 weeks of nitrogen stress in T1 plants. The maximum fold increase of 464 was recorded for ICDH. Our findings indicate a cooperative modification of nitrogen and carbon metabolisms since they are intimately linked together. Overexpression of TaDof1 in wheat resulted in a significant increase in various agronomic traits. Furthermore, various physiological and biochemical markers (chlorophyll, protein, and soluble sugar contents) exhibited a profound change in TaDof1 transgenic plants in comparison with wild type plants. The results clearly depict the merits of employing transcription factors in engineering plant metabolisms. [ABSTRACT FROM AUTHOR]

Abstract—Serotonin receptors of subtype 1А (5-HT1A) are involved in the regulation of aggressive behavior. New data show that 5-HT1A receptors are capable of heterodimerization and antagonistic interaction with type 7 serotonin receptors (5-HT7); hence, it is important to study the involvement of 5-НТ7 receptors in aggressive behavior. Specifically, it is necessary to study the patterns of co-expression of 5-НТ1А and 5-НТ7 receptors in the brains of animals that differ in the degree of genetically defined aggression. In this paper, we have studied the expression of serotonin 5-НТ1А and 5-НТ7 receptors in several cerebral structures of rats with genetically defined aggressive behavior or the lack of aggression. We have revealed significant changes in the expression of these receptors both at mRNA and receptor protein levels. Of particular interest is the changes in the levels of the membrane protein of 5-НТ1А and 5-НТ7 receptors in the midbrain and hippocampus of aggressive rats. A decrease in the level of 5-НТ1А receptors in the midbrain is associated with an increase in the level of this receptor in the hippocampus; meanwhile, the level of 5-НТ7 receptor is increased in the midbrain and decreased in the hippocampus of aggressive rats compared to tame animals. These data indicate interaction between 5-НТ7 and 5-НТ1А receptors. In addition, the expression of 5‑НТ7 receptors is increased in the frontal cortex and the mRNA level of the 5-НТ7 receptor gene is decreased in the hypothalamus of aggressive rats compared to tame animals. Therefore, significant changes in the expression of 5-НТ7 receptors in the brain of rats selected for a high level of fear-induced aggression towards humans have been revealed for the first time. Genotype-dependent peculiarities of combined expression of 5-НТ1А and 5-НТ7 receptors have been revealed in the investigated brain structures. Our data suggest that interaction between 5-НТ1А- and 5-НТ7-receptors may be an important factor in the regulation of genetically defined aggression. [ABSTRACT FROM AUTHOR]

Begonia semperflorens (B. semperflorens), belonging to the family Begoniaceae, has now been widely cultivated worldwide and is famous for its ornamental plants with colourful flowers and distinctive leaves. The selection of appropriate internal reference genes is very important to accurately determine target gene expression via quantitative real-time PCR. However, internal reference gene selection has never been conducted in B. semperflorens. In this study, seven candidate reference genes of B. semperflorens, including 18S ribosomal RNA (Bs18S), pentatricopeptide repeat-containing protein (BsPPR), actin-related protein 5 isoform X2 (BsACT), DNAJ homologue subfamily C member 17 (BsDNAJ), glyceraldehyde-3-phosphate dehydrogenase (BsGAPDH), NAD-dependent malic enzyme 59 kDa isoform, mitochondria (BsNAD-ME), and peptidyl-prolyl cis–trans isomerase CYP26-2, chloroplast (BsCYP), which were obtained from our previous studies, were selected. The stabilities of these genes under stress conditions were analysed using geNorm and NormFinder. Validation of target gene expressions, including phenylalanine ammonia-lyase (BsPAL) and respiratory burst oxidase homologue D (BsRBOHD) under biotic and abiotic conditions, phenylalanine ammonia-lyase (BsPAL), anthocyanidin synthase (BsANS), chalcone synthase (BsCHS), and flavanone-3-hydroxylase (BsF3H) under low temperature, using these seven internal reference genes for normalisation further confirmed the stabilities of the selected genes and indicated the need for reference gene selection for normalising gene expressions in B. semperflorens. Of the seven candidate reference genes, the combination of BsACT, BsDNAJ, and BsNAD-ME was the ideal reference gene set for normalising gene expression in samples under biotic conditions. BsCYP combined with BsACT or BsGAPDH was the best reference gene pair under abiotic conditions. BsACT and BsPPR could be combined to normalise gene expression under low temperature. Our results will benefit future studies on gene expression in plants of Begoniaceae. [ABSTRACT FROM AUTHOR]

Fiber length is one of the most important fiber quality traits in Upland cotton (Gossypium hirsutum L.), the most important fiber crop, and its improvement has been impeded in part by a lack of knowledge regarding its genetic basis. Introgressed backcross inbred lines (BILs) or near isogenic lines (NILs) differing in fiber length in the same genetic background, developed through advanced backcrossing between Upland cotton and extra-long staple cotton (G. barbadense L.), provide an important genomic resource for studying the molecular genetic basis of fiber length. In the present study, a long-fiber group and a short-fiber group, each with five BILs of Upland cotton, were selected from a BIL population between G. hirsutum and G. barbadense. Through a microarray-based comparative transcriptome analysis of developing fibers at 10 days postanthesis from the two groups, 1478 differentially expressed genes (DEGs) were identified. A total of 166 DEGs were then mapped to regions of fiber length quantitative trait loci (QTL), including 12 QTL hotspots and 2 QTL identified previously in the BIL population from which the two sets of BILs were selected. Several candidate genes possibly underlying the genetic control of fiber length differences between G. barbadense and G. hirsutum, including GhACX and GhKIF, were identified in this study. These results provide a list of positional candidate genes for the fine-scale mapping and map-based cloning of fiber length QTL, which will facilitate targeted gene transfer from G. barbadense to Upland cotton to further improve fiber quality. [ABSTRACT FROM AUTHOR]

Quantitative real-time PCR (qRT-PCR) is a standard method to measure gene expression in function exploring. Accurate and reproducible data of qRT-PCR requires appropriate reference genes, which are stably expressed under different experimental conditions. However, no housekeeping genes were validated as internal controls for qRT-PCR in Sinonovacula constricta. In this study, we classified the transcriptome data of two tissues for Vibrio infection and Cd2+ stress into ten clusters based on the gene expression patterns. Among them, cluster 5 had the most stable gene expression patterns regardless of tissues and treatments as the database for candidate reference genes. A total of 55 orthologs of classical housekeeping genes in the clam transcriptome were annotated. Combined the expression profiles and housekeeping genes in S. constricta, we chose eight candidate reference genes and validated their expression in Vibrio-infected samples and different tissues by qRT-PCR. Their expression stability was analyzed by three different algorithms geNorm, NormFinder and BestKeeper. Although the rank of the eight candidate reference genes is different in different treatments using different software, RS9 could be the best reference genes for normalization of qRT-PCR expression data in S. constricta under various treatments considering the above analysis. Meanwhile, the ranking of genes based on the CV values of transcriptomic data was similar to the validation results. This study provides for the first time a list of suitable reference genes for S. constricta and a valuable resource for further studies of clam immune defense systems. [ABSTRACT FROM AUTHOR]

The ascomycete fungus Beauveria bassiana is a natural pathogen of hundreds of insect species and is commercially produced as an environmentally friendly mycoinsecticide. Many genes involved in fungal insecticide infection have been identified but few have been further explored. In this study, we constructed three transcriptomes of B. bassiana at 24, 48 and 72 h post infection of insect pests (BbI) or control (BbC). There were 3148, 3613 and 4922 genes differentially expressed at 24, 48 and 72 h post BbI/BbC infection, respectively. A large number of genes and pathways involved in infection were identified. To further analyze those genes, expression patterns across different infection stages (0, 12, 24, 36, 48, 60, 72 and 84 h) were studied using quantitative RT-PCR. This analysis showed that the infection-related genes could be divided into four patterns: highly expressed throughout the whole infection process (thioredoxin 1); highly expressed during early stages of infection but lowly expressed after the insect death (adhesin protein Mad1); lowly expressed during early infection but highly expressed after insect death (cation transporter, OpS13); or lowly expressed across the entire infection process (catalase protein). The data provide novel insights into the insect-pathogen interaction and help to uncover the molecular mechanisms involved in fungal infection of insect pests. [ABSTRACT FROM AUTHOR]

Papaya sticky disease, caused by Papaya meleira virus (PMeV), results in complete crop loss in papaya orchards, because infected fruits are unacceptable for consumption. Little is known about sticky disease epidemiology, and the best available strategy for disease management is to rogue infected plants. Identification of alternative hosts for this virus is important for developing control strategies. In Mexico, papayas and watermelons are often cultivated in close proximity or overlapping cycles, which may favor virus interspecies transmission by putative insect vectors of PMeV. Watermelon seedlings (Citrullus lanatus Thunb.) were inoculated with latex from papayas infected with a Mexican variant of PMeV (PMeV-Mx), and infection was confirmed by reverse transcription-PCR (RT-PCR) and quantitative RT-PCR (RT-qPCR). Amplicons (491 bp) of the RNA-dependent RNA polymerase (RdRp) gene of PMeV-Mx were detected in axillary leaves of all inoculated seedlings seven days post-inoculation (dpi). Circa 10 to 12 dpi, necrotic lesions were observed on the edges of the leaves. Absolute quantification of PMeV-Mx-RNA by RT-qPCR showed an increase in viral load over time (from 0.51 to 586.15 pg/μL), three to 14 dpi. Molecular and experimental evidence demonstrate is also a host for PMeV-Mx and may be an alternative host. [ABSTRACT FROM AUTHOR]

Flowering, an important agronomic trait for seed plants, represents the end of vegetative growth and begins to reproduce. In regulating the floral transition, FLOWERING LOCUS T (FT) encoding a mobile floral signal protein that belongs to the phosphatidylethanolamine-binding protein (PEBP) family acts as critical role. Here, an FT-like gene, DcFT, was isolated and cloned from European Daucus carota L. cultivar 'Nantes-H06' (GenBank accession number KY768910), and an alignment of the DcFT protein and other FT-homolog proteins showed that it shared 88.00% similarity with CsFT from Camellia sinensis. Phylogenetic tree analysis indicated that DcFT had the closest relationship with GpFT ( Gypsophila paniculata). Quantitative RT-PCR was analyzed to show that the expression pattern of DcFT in inflorescences sharply increased after 10DAA and then slowly increased reaching the maximum at 30DAA. Subcellular localization made clear that the DcFT protein was located in the nucleus and cytoplasm. Broadly speaking, DcFT is an FT-like homologous gene in carrot regulating the floral transition and could be a candidate gene for accelerating the process in carrot breeding. [ABSTRACT FROM AUTHOR]

Recent progress in plant hormone and macronutrient research is largely associated with characterising the molecular mechanisms of their actions. Quantitative reverse transcription-PCR is a powerful technique that enables elucidation of regulatory relationships through quantifying transcript levels, but its reliability requires precise normalization. We compared eight Arabidopsis thaliana housekeeping genes for expression stability after treatment with cytokinins, auxins, ethylene and after macronutrient level manipulation. The expression stability of housekeeping genes in cytokinin series was studied in wide spectrum of plants treated with different adenine- and phenylurea-type cytokinins as well as in plants with controlled overexpression of agrobacterial isopentenyltransferase. NormFinder and geNorm software were used to predict a unique set of genes as best for particular hormone groups. Generally, 18S rRNA, GAPC, and ACT were confirmed as less stable; and UBQ10, UBC, and EF- 1α exhibited increased stability. Based on geNorm pairwise variation analysis, we confirmed accurate normalization required at least three reference genes. [ABSTRACT FROM AUTHOR]

This study aimed to assess the expression of S100A4, Twist and E-cadherin (mRNA and protein) in urothelial bladder cancer, investigate the correlation between them and evaluate their association with the clinicopathological features of the disease. The study included 54 patients diagnosed as urothelial bladder cancer of different stages and grades. The expression levels of S100A4, Twist and E-cadherin (mRNA and protein) in tissue samples were determined by quantitative RT-PCR and immunohistochemistry. The expression of S100A4 and Twist was significantly upregulated while E- cadherin was significantly downregulated in urothelial bladder cancer tissues compared to the adjacent surrounding normal bladder tissues at both mRNA and protein levels (p < 0.001). Expression levels of S100A4 and Twist were significantly higher in recurrent tumor than in non-recurrent tumors (p < 0.001) while the expression level of E-cadherin was significantly lower in recurrent tumors than in non-recurrent tumors at both mRNA and protein levels (p < 0.001). There was a significant positive correlation between S100A4 and Twist expressions (r = 0.875, p < 0.001) while significant negative correlations were found between E- cadherin and S100A4 expressions(r=- 0.803, p < 0.001) and between E-cadherin and Twist (r = −0.809, p < 0.001). Up-regulation of S100A4 and Twist and down-regulation of E-cadherin in urothelial bladder cancer tissues compared to adjacent normal tissues were observed. There was a significant negative correlation between S100A4 and E- cadherin and between E- cadherin and Twist expression. However, there was a significant positive correlation between S100A4 and Twist expressions. Furthermore, the alterations in the gene expression were associated with disease stage and grade. [ABSTRACT FROM AUTHOR]

Ralstonia solanacearum, a soil-borne bacterium causes bacterial wilt, is a lethal disease of eggplant ( Solanum melongena L.). However, the first line of defense mechanism of R. solanacearum infection remains unclear. The present study focused on the role of induced HO, defense-related enzymes of ascorbate-glutathione pathway variations in resistant and susceptible cultivars of eggplant under biotic stress. Fifteen cultivars of eggplant were screened for bacterial wilt resistance, and the concentration of antioxidant enzymes were estimated upon infection with R. solanacearum. A quantitative real-time PCR was also carried out to study the expression of defense genes. The concentration of HO in the pathogen inoculated seedlings was two folds higher at 12 h after pathogen inoculation compared to control. Antioxidant enzymes of ascorbate-glutathione pathway were rapidly increased in resistant cultivars followed by susceptible and highly susceptible cultivars upon pathogen inoculation. The enzyme activity of ascorbate-glutathione pathway correlates by amplification of their defense genes along with pathogenesis-related protein-1a (PR-1a). The expressions of defense genes increased 2.5−3.5 folds in resistant eggplant cultivars after pathogen inoculation. The biochemical and molecular markers provided an insight to understand the first line of defense responses in eggplant cultivars upon inoculation with the pathogen. [ABSTRACT FROM AUTHOR]

The anthocyanin biosynthetic gene, flavanone 3-hydroxylase ( F3H), was first isolated and characterized from Ascocenda orchid. The complete cDNA sequence of the F3H was isolated from the petals of Ascocenda and designated as AcF3H. The open reading frame of AcF3H was 1113 bp long and encoded 370 amino acid residues. The calculated molecular weight of AcF3H was 41.22 kDa and the predicted isoelectric point (pI) was 5.10. The deduced amino acid sequence of the AcF3H had high homology to the F3H from Bromheadia finlaysoniana (89 %). The analysis of AcF3H expression in Ascocenda was performed with quantitative RT-PCR, and the AcF3H transcript accumulated in the early stage of flower development and declined in the partially opened to fully opened flowers. The expression of AcF3H was also found in all parts of the orchid flower. The recombinant AcF3H enzyme was expressed in Escherichia coli and characterized in terms of the optimal pH and temperature for its activity, which were 8.0 and 35 °C respectively. The temperature stability of the AcF3H enzyme was in the range of 20-45 °C and the activity decreased at 50 °C. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a food-borne pathogen of humans and other animals. The striking ability to survive several stresses usually used for food preservation makes L. monocytogenes one of the biggest concerns to the food industry. This ubiquity can be partly explained by the ability of the organism to grow and persist at very low temperatures, a consequence of its ability to accumulate cryoprotective compound called osmolytes. A quantitative RT-PCR assay was used to measure mRNA transcript accumulation for the stress response genes opuCA and betL (encoding carnitine and betaine transporters, respectively) and the housekeeping gene 16S rRNA. Assays were conducted on mid-exponential phase L. monocytogenes cells exposed to conditions reflecting cold and freezing stress, conditions usually used to preserve foods. We showed that expression of the two cold-adapted genes encoded the transporters of the cryoprotectants carnitine and betaine in ATCC 19115 and the food-isolated L. monocytogenes S1 is induced after cold and freezing stress exposure. Furthermore, transcriptional analysis of the genes encoding opuCA and betL revealed that each transporter is induced to different degrees upon cold shock of L. monocytogenes ATCC 19115 and S1. Our results confirm an increase in carnitine uptake at low temperatures more than in betaine after cold-shocked temperature compared to the non-stress control treatment. It was concluded the use of carnitine and betaine as cryoprotectants is essential for rapid induction of the tested stress response under conditions typically encountered during food preservation. [ABSTRACT FROM AUTHOR]

Quantitative real-time PCR (qPCR) is a sensitive method used to investigate relevant changes in gene expression during somatic embryogenesis. Understanding its regulatory network might be helpful to the process of induction of embryos and facilitate the development of efficient plant regeneration procedures. In this study, a set of 12 genes was selected and their stability was assessed in different tissues of somatic embryogenesis-related cultures of Coffea arabica. Analyses of gene expression stability were performed using the RefFinder tool that integrates the geNorm, NormFinder, BestKeeper and Delta-Ct algorithms. Among the all candidate reference genes studied, APRT/ EF1a, UBQ/ ACT, ACT/ 24S, RPL39/ 24S, PP2A/ RPL39, PP2A/ AP47, emerged as the most stable for normalization of qPCR analyses of embryogenic cell suspensions, non-embryogenic calli, embryogenic calli, combined embryogenic and non-embryogenic calli, somatic embryos and plantlet, respectively. A combination of two genes, 24S and PP2A, was identified as most suitable reference genes across all samples for the C. arabica tissues studied. The commonly employed reference gene encoding glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) was found to be inappropriate as a reference gene for embryogenic tissues of C. arabica. In addition, Baby boom ( BBM) gene expression was investigated to confirm the validity of the selected reference genes, the transcript levels of gene were overestimated when unsuitable reference genes were used for normalization. The results shown herein will permit a more precise and reliable normalization of qPCR in experiments involving somatic embryogenesis of C. arabica. [ABSTRACT FROM AUTHOR]

Abstract Plants are exposed to continual changes in the environment. The daily alternation between light and darkness results in massive recurring changes in the carbon budget, and leads to widespread changes in transcript levels. These diurnal changes are superimposed on slower changes in the environment. Quantitative molecular information about the numbers of ribosomes, of transcripts for 35 enzymes in central metabolism and their loading into polysomes is used to estimate translation rates in Arabidopsis rosettes, and explore the consequences for important sub‐processes in plant growth. Translation rates for individual enzyme are compared with their abundance in the rosette to predict which enzymes are subject to rapid turnover every day, and which are synthesized at rates that would allow only slow adjustments to sustained changes of the environment, or resemble those needed to support the observed rate of growth. Global translation rates are used to estimate the energy costs of protein synthesis and relate them to the plant carbon budget, in particular the rates of starch degradation and respiration at night.

FLO/LEAFY (LFY) genes in the FLO_LFY superfamily are important factors regulating plant transition from inflorescence to floral meristems. In this study, we cloned a full-length LFY cDNA from leaves of carrot (Daucus carota L.). The sequenced gene, designated as DcLFY (GenBank accession number KU297945), comprised 1368 with a 1176-bp open reading frame. The deduced encoded protein, which contained 391 amino acid residues and possessed typical N- and C-domains, shared 67.82 % similarity with LFY of Ziziphus jujube. Quantitative real-time PCR analysis revealed that the relative expression of DcLFY in carrot was the highest in inflorescences and gradually decreased with the growth of the inflorescence during flowering. Expression of green fluorescent protein-fused DcLFY in onion (Allium cepa L.) epidermal cells was localized in nuclei. These results demonstrate that DcLFY is a putative FLO/LFY homolog related to the flowering pathway. Taken together, our data suggest that DcLFY is a crucial transcription factor in floral initiation and floral development in carrot. [ABSTRACT FROM AUTHOR]

Flowers of the complex orchid hybrid Burrageara 'Stefan Isler Lava Flow' had been shown previously to react sensitively to ethylene. Via Agrobacterium tumefaciens, the mutant ethylene receptor ETHYLENE RESPONSE 1 ( etr1- 1) from Arabidopsis thaliana under the control of the flower-specific promoter FLOWER BINDING PROTEIN 1 ( fbp1) from Petunia hybrida was transferred to Burrageara. One single-copy event was analyzed in this study aiming to investigate the expression of the fbp1::etr1- 1 transgene in different plant and flower organs by quantitative RT-PCR and the reaction of flowers and inflorescences to ethylene. It was shown that the heterologous promoter led to high expression levels in the perianth of the orchid flowers compared to low levels in leaves and roots. The expression shift to the first whorl (sepals) described here corresponds to extended expression of endogenous B class MADS box homeotic genes in orchids in general. The transgenic plants grew and developed similar to the wild-type plants, except for slightly faster rooting in vitro and smaller flowers. Flower longevity was improved by 7 days in 10 ppm ethylene. Moreover, bud drop starting at day 5 of incubation of inflorescences in 10 ppm ethylene in the wild-type was efficiently prevented for at least 19 days in the fbp1::etr1- 1 transgenic plants. The function of the tissue-specific promoter fbp1 and the mutant receptor etr1- 1 was shown for the first time in a monocotyledonous plant. [ABSTRACT FROM AUTHOR]

Copper (Cu) and cadmium (Cd) are the most common heavy metals that are easily detected in aquatic environments on a global scale. In this paper, we investigated the messenger RNA (mRNA) and protein levels of HSPs (HSP60, HSP70, and HSP90) in the liver of the common carp exposed to Cu, Cd, and a combination of both metals by real-time quantitative PCR and Western blot. The results indicated that in each exposure group, the mRNA levels of HSP60, HSP70, and HSP90 were increased significantly compared to the corresponding controls after 96 h of exposure ( P < 0.05). A significant increase was observed in the HSP70 protein level in the high-dose Cu group and all of the Cd groups. Significant increases were also observed in the protein levels of HSP60 and HSP90 in the high combination group and the low combination group, respectively. These results indicated that the dynamics of HSP expression observed in the common carp support the role of HSPs as biochemical markers in response to environmental pollution and provided valuable insights into the adaptive mechanisms used by the common carp to adapt to the challenges of stressful environments. [ABSTRACT FROM AUTHOR]

It is assumed that changes in the number of copies that belong to the basic mechanisms that control the expression of genes are important for malignization. Therefore, the characterization of these genes and the precise assessment of the number of copies are important for understanding the molecular basis of tumor emergence and progression in the human organism, as well as for the identification of predictive markers of malignization. In the present study, the relative number of copies of 19 loci ( BAX, GSTP1, CASP3, CASP8, HIF1A, OCT4, C-MYC, SOX2, BCL2, CASP8/FADD, NANOG, P53, CASP9, IL-10, NFKB1, HV2, and ACTB) in cancerous and conventionally healthy tissues from 25 residents of southern Russia with a histologically confirmed diagnosis of adenocarcinoma (stages G1-G2 and G3) or signet cell gastric cancer were determined by quantitative real-time PCR. Changes in the number of copies of the gene were shown to be specific to particular histological types of cancer, as well as to depend on the stage of tumor cell differentiation. The data suggest that the number of copies of changes in the BAX, CASP3, CASP8, OCT4, C-MYC, SOX2, BCL2, NANOG, CASP9, NFKB1, HV2, ACTB, MKI67, IL-10, GSTP1, and P53 genes play an important role in the malignization of gastric tissue. [ABSTRACT FROM AUTHOR]

Grapevine ( Vitis vinifera L.) was genetically modified with a construct containing a cDNA insert encoding the stilbene synthase gene ( Vst1) from grapevine, under the control of the cauliflower mosaic virus 35S promoter in order to test the potential of over-production of resveratrol to protect plants from fungal attack. Southern blot hybridization and quantitative real-time PCR analysis demonstrated the presence and integration of one copy of exogenous DNA sequences in two grapevine-modified lines. Relative expression of the Vst1 gene in different modified lines was confirmed by using gene-specific quantitative real-time PCR. Compared to the control, the concentration of trans-resveratrol quantified by HPLC was up to 7.5 fold higher in the modified plants. The necrotic lesion size of leaves of intact modified plants inoculated by Botrytis cinerea B05.10 strain was consistently smaller and significantly different ( p ≤ 0.05) than in control plants, showing that modified grapevine plants were more resistant to the pathogen than the control plants. [ABSTRACT FROM AUTHOR]

Cotton productivity is affected by water deficit, and little is known about the molecular basis of drought tolerance in cotton. In this study, microarray analysis was conducted to identify drought-responsive genes in the third topmost leaves of the field-grown drought-tolerant cotton ( Gossypium hirsutum L.) cultivar Acala 1517-99 under drought stress conditions. Water stress was imposed by withholding irrigation for 9 days in the early squaring stage, which resulted in 10-15 % reduction in plant growth compared to the well-watered plants. A total of 110 drought-responsive genes (0.5 % of the total genes) were identified, 79 % (88 genes) of which were drought-repressed and 21 % (22 genes) were drought-induced. The drought-induced genes were grouped into six functional categories including stress-related (ten genes, nine of which encode heat shock proteins), metabolism (three genes) and one gene each for transcription factor, proline biosynthesis and cellular transport. The drought-repressed genes were classified into 14 functional categories, comprising metabolism (20 genes), cellular transport (12 genes), stress-related (12 genes), regulation of gene expression (nine genes), transcription factor (four genes), signal transduction (seven genes) and two genes each for biosynthesis of secondary compounds, cell wall, fatty acids/lipids and chlorophyll, and protein degradation. Most of the genes have been reported in other plants as drought-tolerant/responsive. The responsiveness of 19 selected drought-responsive genes was validated by quantitative RT-PCR. Furthermore, primers were developed and assayed for all the drought-responsive genes to develop single-strand conformation polymorphic markers, many of which were found to be correlated with drought tolerance. This report represents the first study on integration of a transcriptome analysis to develop molecular markers that are associated with drought tolerance in cotton. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are short non-coding RNAs that exert a critical influence on tumorigenesis through post-transcriptional modification and are considered to be potential biomarkers for the diagnosis or prognosis of various cancers. Although several miRNAs have been proposed as relevant biomarkers for non-small cell lung cancer (NSCLC), detailed working mechanisms and validated prognostic significance of these miRNAs remain controversial. In this study, we evaluated expression levels of miRNA-126 (miR-126) and miR-200c in 72 NSCLCs and 30 benign lung tissues by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and analyzed the correlation of miRNA expression with a variety of clinicopathological factors and patient survival. Compared with the benign control group, miR-126 expression was significantly downregulated in NSCLCs ( p < 0.001), while miR-200c expression was significantly upregulated in NSCLCs ( p < 0.001). The expression of miR-126 was significantly higher in NSCLCs with a tumor size of ≤3 cm than in those with a tumor size of >3 cm ( p = 0.026). There were no other significant associations between miRNA expression and clinicopathological features. In univariate survival analysis for all NSCLC patients, high miR-200c expression ( p = 0.037), large tumor size ( p = 0.026), and lymphovascular invasion ( p = 0.012) were significantly correlated with worse overall survival. High miR-126 expression was significantly associated with favorable prognosis only in patients with adenocarcinoma ( p = 0.033). In multivariate analysis, miR-200c and tumor size remained as independent prognostic factors. Our results suggest that miR-126 might play tumor-suppressive and miR-200c an oncogenic role, and these miR's are potential prognostic biomarkers for NSCLC. [ABSTRACT FROM AUTHOR]

WW-domain-containing oxidoreductase ( WWOX) is the tumour suppressor gene from the common fragile site FRA16D, whose altered expression has been observed in tumours of various origins. Its suppressive role and influence on basic cellular processes such as proliferation and apoptosis have been confirmed in many in vitro and in vivo studies. Moreover, its protein is thought to take part in the regulation of tissue morphogenesis and cell differentiation. However, its role in colon cancer formation remains unclear. The aim of this study was to characterize the influence of WWOX on the process of colon cancerogenesis, the basic features of the cancer cell and its expression profiles. Multiple biological tests, microarray experiments and quantitative reverse transcriptase (RT)-PCR were performed on two colon cancer cell lines, HT29 and SW480, which differ in morphology, expression of differentiation markers, migratory characteristics and metastasis potential and which represent negative (HT29) and low (SW480) WWOX expression levels. The cell lines were subjected to retroviral transfection, inducting WWOX overexpression. WWOX was found to have diverse effects on proliferation, apoptosis and the adhesion potential of modified cell lines. Our observations suggest that in the HT29 colon cancer cell line, increased expression of WWOX may result in the transition of cancer cells into a more normal colon epithelium phenotype, while in SW480, WWOX demonstrated well-known tumour suppressor properties. Our results also suggest that WWOX does not behave as classical tumour suppressor gene, and its influence on cell functioning is more global and complicated. [ABSTRACT FROM AUTHOR]

Pituitary surgery generates pituitary tissue for histology, immunohistochemistry, and molecular biological research. In the last decade, the pathogenesis of pituitary adenomas has been extensively studied in humans, and to a lesser degree in dogs, and tumor oncogenesis has been studied in knock-out mice, often by means of quantitative reversed-transcriptase PCR (RT-qPCR). A precondition of such analyses is that so-called reference genes are stably expressed regardless of changes in disease status or treatment. In this study, the expression of six frequently used reference genes, namely, tata box binding protein ( tbp), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide ( ywhaz), hydroxymethylbilane synthase ( hmbs), beta-2-microglobulin ( b2m), succinate dehydrogenase complex subunit A ( sdha), and glyceraldehyde 3 phosphate dehydrogenase 1 ( gapdh), was studied in pituitary tissue (normal and adenoma) from three species (humans, mice, and dogs). The stability of expression of these reference genes differed between species and between healthy and diseased tissue within one species. Quantitative analysis based on a single reference gene that is assumed to be stably expressed might lead to wrong conclusions. This cross-species analysis clearly emphasizes the need to evaluate the expression stability of reference genes as a standard and integral aspect of study design and data analysis, in order to improve the validity of the conclusions drawn on the basis of quantitative molecular analyses. [ABSTRACT FROM AUTHOR]

The expression of the OmpF porin gene in Yersinia pseudotuberculosis in response to antibiotics of two different classes (kanamycin and nalidixic acid) was analyzed using a quantitative PCR and a fluorescence reporter system. Both antibiotics downregulated the expression of the ompF gene. The nalidixic acid significantly reduced the ompF expression, while kanamycin, for which porins are considered to be an alternative transport route, only slightly reduced the ompF level. [ABSTRACT FROM AUTHOR]

Heterobasidion annosum is widely known as a major root and butt rot pathogen of conifer trees, but little information is available on its interaction with the roots of herbaceous angiosperm plants. We investigated the infection biology of H. annosum during challenge with the angiosperm model Arabidopsis and monitored the host response after exposure to different hormone elicitors, chemicals (chitin, glucan and chitosan) and fungal species that represent diverse basidiomycete life strategies [e.g., pathogen ( H. annosum), saprotroph ( Stereum sanguinolentum) and mutualist ( Lactarius rufus)]. The results revealed that the tree pathogen ( H. annosum) and the saprotroph ( S. sanguinolentum) could infect the Col-8 (Columbia) ecotype of Arabidopsis in laboratory inoculation experiments. Germinated H. annosum spores had appressorium-like penetration structures attached to the surface of the Arabidopsis roots. Subsequent invasive fungal growth led to the disintegration of the vascular region of the root tissues. Progression of root rot symptoms in Arabidopsis was similar to the infection development that was previously documented in Scots pine seedlings. Scots pine PsDef1 and Arabidopsis DEFLs (AT5G44973.1) and PDF1.2 were induced at the initial stage of the infection. However, differences in the expression patterns of the defensin gene homologs from the two plant groups were observed under various conditions, suggesting functional differences in their regulation. The potential use of the H. annosum-Arabidopsis pathosystem as a model for studying forest tree diseases is discussed. [ABSTRACT FROM AUTHOR]

The activating KIT D816V mutation plays a central role in the pathogenesis, diagnosis, and targeted treatment of systemic mastocytosis (SM). For improved and reliable identification of KIT D816V, we have developed an allele-specific quantitative real-time PCR (RQ-PCR) with an enhanced sensitivity of 0.01-0.1 %, which was superior to denaturing high-performance liquid chromatography (0.5-1 %) or conventional sequencing (10-20 %). Overall, KIT D816 mutations were identified in 146/147 (99 %) of patients (D816V, n = 142; D816H, n = 2; D816Y, n = 2) with SM, including indolent SM (ISM, n = 63, 43 %), smoldering SM ( n = 8, 5 %), SM with associated hematological non-mast cell lineage disease (SM-AHNMD, n = 16, 11 %), and aggressive SM/mast cell leukemia ± AHNMD (ASM/MCL, n = 60, 41 %). If positive in BM, the KIT D816V mutation was found in PB of all patients with advanced SM (SM-AHNMD, ASM, and MCL) and in 46 % (23/50) of patients with ISM. There was a strong correlation between the KIT D816V expressed allele burden ( KIT D816V EAB) with results obtained from DNA by genomic allele-specific PCR and also with disease activity (e.g., serum tryptase level), disease subtype (e.g., indolent vs. advanced SM) and survival. In terms of monitoring of residual disease, qualitative and quantitative assessment of KIT D816V and KIT D816V EAB was successfully used for sequential analysis after chemotherapy or allogeneic stem cell transplantation. We therefore conclude that RQ-PCR assays for KIT D816V are useful complimentary tools for diagnosis, disease monitoring, and evaluation of prognosis in patients with SM. [ABSTRACT FROM AUTHOR]

Gene expression analysis on messenger RNA (mRNA) purified from formalin-fixed, paraffin-embedded tissue is increasingly used for research purposes. Tissue heterogeneity may question specificity and interpretation of results from mRNA isolated from a whole slide section, and thresholds for minimal tumor content in the paraffin block or macrodissection are used to avoid contamination from non-neoplastic tissue. The aim was to test if mRNA from tissue surrounding breast cancer affected quantification of estrogen receptor α ( ESR1), progesterone receptor ( PGR) and human epidermal growth factor receptor 2 ( ERBB2), by comparing gene expression from whole slide and tumor-enriched sections, and correlating gene expression from whole slide sections with corresponding immunohistochemistry. Gene expression, based on mRNA extracted from a training set (36 paraffin blocks) and two validation sets (133 + 1,083 blocks), were determined by quantitative reverse transcription polymerase chain reaction for all samples, as well as by microarray for 133 validation samples. In the training set, agreement between high vs. low mRNA expression from whole slide and tumor-enriched sections was absolute for ESR1 and ERBB2, and 83 % for PGR. Overall agreements, when comparing mRNA expression to immunohistochemistry, were 100 % ( ERBB2), 89 % ( ESR1) and 83 % ( PGR), which was confirmed in the validation sets. Percentage of tumor in the sections did not influence the results. In conclusion, reliable quantification of ESR1, PGR and ERBB2 mRNA expression can be obtained from a whole slide section, and correlates well with immunohistochemistry. Prior removal of surrounding tissue was found to be unnecessary even with minimal tumor content in the section. [ABSTRACT FROM AUTHOR]

Jasmonates (JAs) play a role in the responses to environmental stress and during growth processes, including fruit/seed development. To better understand the molecular basis of the developmental control exerted by JAs in fruit and seed, methyl jasmonate (MJ, 0.80 mM) was applied to peach fruit ( Prunus persica var. laevis Gray) at an early (S1) developmental stage and under field conditions. Mesocarp and seed were sampled at time intervals until ripening; at harvest, MJ-treated fruit were less ripe than controls as assessed by a nondestructive device called a DA-meter. Real-time reverse-transcription polymerase chain reaction analyses revealed that JA-related gene expression ( AOS1 and JAZs) was affected early (24 h) after treatment, whereas peaks in transcript accumulation of mesocarp ( CYCD3, RD22, SP, Aux/IAA) and seed ( PRP, SSADH, PRU, LEA) developmental marker genes were shifted in accord with a developmental slowing down. At ripening (S4), in the mesocarp the upregulation of the ethylene biosynthetic genes ACO1 and ACS1 and of the softening-related genes PG and EXP2 was dramatically counteracted by MJ. Ethylene signaling ( ETR1, ETR2) was also affected. Because JAs cross-talk with other hormones, the transcript amounts of major hormone-related genes such as GH3, IAA- AH, NCED, and GA2ox were evaluated and showed changes that further support the hypothesis of delay of the developmental program. [ABSTRACT FROM AUTHOR]

The common marmoset ( Callithrix jacchus) is increasingly being used as a non-human primate animal model in biomedical research. To perform accurate quantitative analysis of gene expression by quantitative reverse transcription polymerase chain reaction, reliable reference genes should be selected. In this study, we evaluated the expressions of 11 widely used reference genes: ACTB, ATP5F1, B2M, GAPDH, HPRT1, PGK1, PPIA, RN18S1, RPLP0, TBP and UBC in 12 tissues and five brain areas of healthy common marmosets. NormFinder and geNorm indicated that the most suitable reference genes for cross-sectional studies of the 17 tissues were RN18S1 and RPLP0. Conversely, ACTB and PPIA were the most suitable for analyzing brain samples; however, the expression of PGK1 fluctuated among brain areas. These results indicate that suitable reference genes differ between the tissues examined. This study provides fundamental information for gene expression studies of the common marmoset and highlights the importance of validating reference genes before quantification of target mRNAs. [ABSTRACT FROM AUTHOR]

Although the survival of lung cancer patients has improved significantly due to the development of early detective tools, lung cancer remains a leading cause of cancer-related death after curative surgery. So it is extremely important for cancer patients to predict early metastasis, especially pleural dissemination, the most frequent type of recurrence in patients after surgery. Based on a retrospective study of 86 curatively resected lung cancer patients (training set), we determined a cutoff value of NSE mRNA using receiver-operating characteristic curve. Then, we prospectively used this cutoff value to validate the risk of pleural recurrence in a new cohort of 81 lung cancer patients (validation set) between April 2009 and June 2010 by real-time reverse transcriptase-polymerase chain reaction. During the median 27 months of postoperative surveillance, 16 of the 81 patients died, and 9 of the 16 developed pleural metastases. Multivariate analysis with the Cox proportional hazards model showed that positive NSE mRNA was a significant independent risk factor with both overall survival and pleural recurrence-free survival (both P < 0.0001) as end points which were significantly worse in patients with positive NSE mRNA ( P < 0.0001). These results indicate that quantitative NSE mRNA in pleural fluid is a reliable prognostic indicator of pleural recurrence in the clinical setting. [ABSTRACT FROM AUTHOR]

Fungal proteases, such as subtilisin-like serine protease (Pr1) and trypsin-like serine protease (Pr2), play an important role in penetration through the host cuticle in entomopathogenic fungi, including Isaria farinosa. In the present study, one gene of I. farinosa subtilisin-like protease ( Ifa- pr1A), showing high homology to a Beauveria bassiana cuticle-degrading protease, was amplified by rapid amplification of cDNA ends PCR. The resulting full-length cDNA displayed an open reading frame (ORF) of 960 bp, encoding a protein of 319 amino acids. The Ifa-Pr1A protein was expressed in Escherichia coli to verify its protease activity. The recombinant Ifa-Pr1A protein exhibited high enzymatic activity according to the enzyme assay using a synthetic substrate. The expression profiles of Ifa- pr1A and Ifa- pr1H (another subtilisin-like protease gene from I. Farinosa) were analyzed at different induction times by adding cuticle material to the media. Quantitative reverse transcriptase PCR (RT-PCR) analysis revealed that Ifa- pr1A transcripts increased 58,800-fold at 12 h post-induction, whereas Ifa- pr1H transcripts peaked at 12 h with only an 11-fold increase, indicating that Ifa- pr1A may be a major gene of cuticle-degrading proteases in I. farinosa. Furthermore, Ifa- pr1A gene expression under in vivo conditions showed a clear upward trend during the early stages of the infection process. These results suggest that Pr1A cloned from I. farinosa is a potential virulence factor for the development of engineered biopesticides. [ABSTRACT FROM AUTHOR]

Fungal laccases ( p-diphenol:oxygen oxidoreductase; EC 1.10.3.2) are multi-copper-containing oxidases that catalyse the oxidation of a great variety of phenolic compounds and aromatic amines through simultaneous reduction of molecular oxygen to water. Fungi generally produce several laccase isoenzymes encoded by complex multi-gene families. The Pleurotus ostreatus genome encodes 11 putative laccase coding genes, and only six different laccase isoenzymes have been isolated and characterised so far. Laccase expression was found to be regulated by culture conditions and developmental stages even if the redundancy of these genes still raises the question about their respective functions in vivo. In this context, laccase transcript profiling analysis has been used to unravel the physiological role played by the different isoforms produced by P. ostreatus. Even if reported results depict a complex picture of the transcriptional responses exhibited by the analysed laccase genes, they were allowed to speculate on the isoform role in vivo. Among the produced laccases, LACC10 (POXC) seems to play a major role during vegetative growth, since its transcription is downregulated when the fungus starts the fructification process. Furthermore, a new tessera has been added to the puzzling mosaic of the heterodimeric laccase LACC2 (POXA3). LACC2 small subunit seems to play an additional physiological role during fructification, beside that of LACC2 complex activation/stabilisation. [ABSTRACT FROM AUTHOR]

The molecular mechanisms underlying gravitropic bending of shoots are poorly understood and how genes related with this growing progress is still unclear. To identify genes related to asymmetric growth in the creeping shoots of chrysanthemum, suppression subtractive hybridization was used to visualize differential gene expression in the upper and lower halves of creeping shoots of ground-cover chrysanthemum under gravistimulation. Sequencing of 43 selected clones produced 41 unigenes (40 singletons and 1 unigenes), which were classifiable into 9 functional categories. A notable frequency of genes involve in cell wall biosynthesis up-regulated during gravistimulation in the upper side or lower side were found, such as beta tubulin (TUB), subtilisin-like protease (SBT), Glutathione S-transferase (GST), and expensing-like protein (EXP), lipid transfer proteins (LTPs), glycine-rich protein (GRP) and membrane proteins. Our findings also highlighted the function of some metal transporter during asymmetric growth, including the boron transporter (BT) and ZIP transporter (ZT), which were thought primarily for maintaining the integrity of cell walls and played important roles in cellulose biosynthesis. CmTUB (beta tubulin) was cloned, and the expression profile and phylogeny was examined, because the cytoskeleton of plant cells involved in the plant gravitropic bending growth is well known. [ABSTRACT FROM AUTHOR]

Although circulating microRNAs (miRNAs) were frequently detected in sera of cancer patients, there is still a lack of analysis of the dynamic changes of miRNAs expression in sera of pre- and post-operative lung carcinoma patients. Thus, we conducted quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to examine the expression of four miRNAs (miR-21, miR-205, miR-30d, and miR-24) in the sera of a set of 82 pre-operative lung carcinoma patients and paired 10 days post-operative patients, as well as in 50 normal volunteers. We showed that, compared to that in normal volunteers, the expression of miR-21, miR-205, miR-30d, and miR-24 was increased in lung cancer sera samples, as well as in sera of early stage lung cancer patients according to their clinical-pathological characteristics. The area under roc curves (AUCs) for levels of miR-21, miR-205, miR-30d, and miR-24 in sera were significantly higher than those for Carcinoma embryonic antigen (CEA) ( P < 0.05), whereas the AUC for combination of serum levels of miRNA with serum CEA showed no significant difference from that for serum levels of miRNAs only ( P > 0.05). The expression levels of miR-21 and miR-24 were significantly decreased in post-operative sera compared with levels in paired pre-operative sera ( P = 0.0004 and <0.0001, respectively). In addition, high expressions of miR-21 and miR-30d in pre-operative sera were independently correlated with shorter overall survival in lung cancer patients (log-rank test: P = 0.0498, 0.0019). In summary, our results suggest that miR-21, miR-205, miR-30d, and miR-24 may serve as potential novel non-invasive biomarkers for diagnosis of lung cancer. In addition, miR-21 and miR-24 serum levels were lower in post-operative samples than those in pre-operative samples, suggesting they can potentially be used as biomarkers for disease recurrence after surgery operation. [ABSTRACT FROM AUTHOR]

The proteomic profiles of primary needles from Cr2-resistant and cr2-susceptible Pinus monticola seedlings were analysed post Cronartium ribicola inoculation by 2-DE. One hundred-and-five protein spots exhibiting significant differential expression were identified using LC-MS/MS. Functional classification showed that the most numerous proteins are involved in defence signalling, oxidative burst, metabolic pathways, and other physiological processes. Our results revealed that differential expression of proteins in response to C. ribicola inoculation was genotype- and infection-stage dependent. Responsive proteins in resistant seedlings with incompatible white pine blister rust (WPBR) interaction included such well-characterized proteins as heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, and intermediate factors functioning in the signal transduction pathways triggered by well-known plant R genes, as well as new candidates in plant defence like sugar epimerase, GTP-binding proteins, and chloroplastic ribonucleoproteins. Fewer proteins were regulated in susceptible seedlings; most of them were in common with resistant seedlings and related to photosynthesis among others. Quantitative RT-PCR analysis confirmed HSP- and ROS-related genes played an important role in host defence in response to C. ribicola infection. To the best of our knowledge, this is the first comparative proteomics study on WPBR interactions at the early stages of host defence, which provides a reference proteomic profile for other five-needle pines as well as resistance candidates for further understanding of host resistance in the WPBR pathosystem. [ABSTRACT FROM AUTHOR]

Purpose: We investigated whether miRNA expression profiles can distinguish and predict outcome of non-small-cell lung carcinoma (NSCLC) patients with different histological subtypes. Methods: High-throughput microarray was used to measure miRNA expression levels in six NSCLC samples. Subsequently, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify findings in an independent set of 54 squamous-cell lung carcinomas (SCC), 51 lung adenocarcinomas (AD), and paired adjacent non-neoplastic lung tissue. Results: We showed that, compared to adjacent non-neoplastic lung tissues, the expressions of miR-125a-5p and let-7e were decreased in AD and SCC samples, while increased expressions of miR-93, miR-205, and miR-221 were observed in SCC samples. In addition, miR-205 expression was significantly higher in SCC patients with lymph node metastasis. Lower let-7e expression was associated with lymph node metastasis, >3 cm tumor size, and differentiation of the NSCLC AD subtype. High levels of miR-100 expression also correlated with the AD subtype in current smokers. Moreover, induction of miR-93 and miR-205 expressions and reduction of let-7e were strongly associated with shorter overall survival in SCC patients, whereas AD patient survival was only associated with reduced let-7e. Conclusions: We identified differential expression profiles of miRNAs in AD and SCC. More importantly, in addition to morphology and immunocytochemistry approaches, we report that miR-93, miR-205, miR-221, and let-7e may represent novel biomarkers for differential diagnosis and prognosis of certain NSCLC subtypes or be new targets of histology-specific treatments. Furthermore, our results suggest a strong correlation between high expression of miR-100 and AD patients with history of heavy smoking. [ABSTRACT FROM AUTHOR]

The homozygous T-DNA mutants of AtCAL1 ( Rat1) and AtCAL2 ( Rat2) were obtained. The double mutant of Rat2/Rat1RNAi was constructed which showed obvious late-flowering phenotype from others. The expression of various flowering-related genes was studied among mutants and wild-type plants by quantitative RT-PCR. The double mutant plants showed the shortest root length compared with T-DNA insertion mutants and wild type plants under red light, blue light, and white light. The double mutants showed hypersensitivity to NaCl and ABA. However, these mutants had no effect on stomatal closure by ABA. [ABSTRACT FROM AUTHOR]

Diverse cellular and environmental stresses can activate the heat shock response, an evolutionarily conserved mechanism to protect proteins from denaturation. Stressors activate heat shock transcription factor 1 (HSF1), which binds to heat shock elements in the genes for heat shock proteins, leading to rapid induction of these important molecular chaperones. Both heat and noise stress are known to activate the heat shock response in the cochlea and protect it from subsequent noise trauma. However, the contribution of HSF1 to induction of heat shock proteins following noise trauma has not been investigated at the molecular level. We evaluated the role of HSF1 in the cochlea following noise stress by examining induction of heat shock proteins in Hsf1 control and Hsf1 mice. Heat stress rapidly induced expression of Hsp25, Hsp47, Hsp70.1, Hsp70.3, Hsp84, Hsp86, and Hsp110 in the cochleae of wild-type and Hsf1 mice, but not in Hsf1 mice, confirming the essential role of HSF1 in mediating the heat shock response. Exposure to broadband noise (2-20 kHz) at 106 dB SPL for 2 h produced partial hearing loss. Maximal induction of heat shock proteins occurred 4 h after the noise. In comparison to heat stress, noise stress resulted in lower induced levels of Hsp25, Hsp70.1, Hsp70.3, Hsp86, and Hsp110 in Hsf1 mice. Induction of these heat shock proteins was attenuated, but not completely eliminated, in Hsf1 mice. These same noise exposure conditions induced genes for several immediate early transcription factors and maximum induction occurred earlier than for heat shock proteins. Thus, additional signaling pathways and transcriptional regulators that are activated by noise probably contribute to induction of heat shock proteins in the cochlea. [ABSTRACT FROM AUTHOR]

Over a period of two years, more than 5,000 citrus trees were tested for the presence of Citrus tristeza virus (CTV) on the island of Crete, resulting in thirty eight positives. Comparisons of the relative transcript levels of CTV p23, coat protein (CP), polymerase (POL) and an intergenic (POL/p33) region using quantitative RTPCR, revealed consistent differences in abundance for each of these RNAs among flowers, stems, young fruits and leaves of infected orange trees. CTV p23 RNAs accumulated at highest levels, reaching a maximum in the flowers and the lowest levels in the leaves, while POL RNAs consistently accumulated at low levels in all tissues tested. A PCR-amplified dig-labelled CTV p23 DNA probe was applied to stem and leaf prints, and to crude and total RNA leaf extracts, using non isotopic hybridization. This technique, when applied to stem or leaf prints, and particularly to total RNA, unequivocally provided strong signals with minimal backgrounds. Moreover, an antiserum with high sensitivity and specificity of CTV detection as shown by DAS and immunoprint ELISA was produced against bacterially-expressed CTV CP. In the former method, stems and flowers contained higher levels of CTV CP when compared to leaf extracts. Taking into account Cretan geography and the importance of citrus to the island, systematic surveys for CTV eradication, sustainable control measurements and epidemiological studies need to be undertaken. The observations, materials and methods presented here may assist all three tasks at local and national level. [ABSTRACT FROM AUTHOR]

Continuous dopaminergic treatment is considered to prevent or delay the occurrence of dyskinesia in patients with Parkinson's disease (PD). Rotigotine is a non-ergolinic D > D > D dopamine-receptor agonist for the treatment of PD using a transdermal delivery system providing stable plasma levels. We aimed to investigate the differential influence on gene expression of pulsatile l-DOPA or rotigotine versus a continuous rotigotine treatment. The gene expression profile within the nigro-striatal system of unilateral 6-hydroxydopamine-lesioned rats was assessed in order to differentiate potential changes in gene expression following the various treatment using Affymetrix microarrays and quantitative RT-PCR. The expression of 15 genes in the substantia nigra and of 11 genes in the striatum was altered under pulsatile treatments inducing dyskinetic motor response, but was unchanged under continuous rotigotine treatment that did not cause dyskinetic motor response. The route of administration of a dopaminergic drug is important for the induction or prevention of motor abnormalities and adaptive gene expressions. The decline of neurotrophin-3 expression under pulsatile administration was considered of particular importance. [ABSTRACT FROM AUTHOR]

Objective: Previous reports have shown that the gene promoter region of retinoic acid receptor β (RARβ) was hypermethylated in cervical carcinoma, implying the inhibition of gene transcription. The aim of this study was to investigate the association of cervical cancer development with the RARβ gene expression at the mRNA and protein level to assess the impact of RARβ as a marker for early detection of the cancer. Methods: We collected 126 cases of formalin fixed and paraffin embedded cervical tissue specimens as well as 37 cases of fresh tissue samples from women with cervicitis, cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC). The RARβ mRNA and protein expression was detected by quantitative RT-PCR and immunohistochemistry, respectively. Results: (1) The mRNA expression of RARβ in CIN and cervical cancer was markedly decreased compared to cervicitis with a statistically very significant difference, but no difference was found between CIN and cervical cancer. (2) RARβ protein was normally expressed in the epithelial cells of cervicitis and partially lost in a few cases, but with the development of cervical lesion pathogenesis and cancer, a significant loss of protein expression was detected in CIN (38%) and CSCC (57%) compared to cervicitis ( P < 0.01). Conclusion: The downregulation of RARβ transcription or loss of protein expression is an important indicator of cervical cancer and its precursur lesions. The detection of RARβ expression coupled with aberrant methylation of the gene may become a biomarker for the early prognosis or diagnosis of the cancer. [ABSTRACT FROM AUTHOR]

The WWOX gene is a tumour suppressor gene affected in various types of malignancies. Numerous studies showed either loss or reduction of the WWOX expression in variety of tumours, including breast, ovary, liver, stomach and pancreas. Recent study demonstrated that breast cancer patients exhibiting higher WWOX expression showed significantly longer disease-free survival in contrast to the group with lower relative WWOX level. This work was undertaken to show whether similar phenomena take place in colon tumours and cell lines. To assess the correlation of WWOX gene expression with prognosis and cancer recurrence in 99 colorectal cancer patients, we performed qRT-PCR analysis. We also performed analysis of WWOX promoter methylation status using MethylScreen method and analysis of loss of heterozygosity (LOH) status at two WWOX-related loci, previously shown to be frequently deleted in various types of tumours. A significantly better disease-free survival was observed among patients with tumours exhibiting high level of WWOX (hazard ratio = 0.39; p = 0.0452; Mantel-Cox log-rank test), but in multivariate analysis it was not an independent prognostic factor. We also found that although in colorectal cancer WWOX expression varies among patients and correlates with DFS, the exact mode of decrease in this type of tumour was not found. We failed to find the evidence of LOH in WWOX region, or hypermethylation in promoter regions of this gene. Although we provide the evidence for tumour-suppressive role of WWOX gene expression in colon, we were unable to identify the molecular mechanism responsible for this. [ABSTRACT FROM AUTHOR]

iso-Migrastatin (iso-MGS) has been actively pursued recently as an outstanding candidate of antimetastasis agents. Having characterized the iso-MGS biosynthetic gene cluster from its native producer Streptomyces platensis NRRL 18993, we have recently succeeded in producing iso-MGS in five selected heterologous Streptomyces hosts, albeit the low titers failed to meet expectations and cast doubt on the utility of this novel technique for large-scale production. To further explore and capitalize on the production capacity of these hosts, a thorough investigation of these five engineered strains with three fermentation media for iso-MGS production was undertaken. Streptomyces albus J1074 and Streptomyces lividans K4-114 were found to be preferred heterologous hosts, and subsequent analysis of carbon and nitrogen sources revealed that sucrose and yeast extract were ideal for iso-MGS production. After the initial optimization, the titers of iso-MGS in all five hosts were considerably improved by 3-18-fold in the optimized R2YE medium. Furthermore, the iso-MGS titer of S. albus J1074 (pBS11001) was significantly improved to 186.7 mg/L by a hybrid medium strategy. Addition of NaHCO to the latter finally afforded an optimized iso-MGS titer of 213.8 mg/L, about 5-fold higher than the originally reported system. With S. albus J1074 (pBS11001) as a model host, the expression of iso-MGS gene cluster in four different media was systematically studied via the quantitative RT-PCR technology. The resultant comparison revealed the correlation of gene expression and iso-MGS production for the first time; synchronous expression of the whole gene cluster was crucial for optimal iso-MGS production. These results reveal new insights into the iso-MGS biosynthetic machinery in heterologous hosts and provide the primary data to realize large-scale production of iso-MGS for further preclinical studies. [ABSTRACT FROM AUTHOR]

Noroviruses (NoV) are the most common cause of acute nonbacterial gastroenteritis in the United States, and human hands play an important role in their transmission. Little is known about the efficacy of hand hygiene agents against these highly infectious pathogens. We investigated the activity of seven commercially available hand hygiene products against human noroviruses by in vivo fingerpad tests. The in vivo activity of alcohol-based handrubs ranged from 0.10 to 3.74 log reduction and was not solely dependent on alcohol concentration. A handrub (VF481) based on 70% ethanol and a blend of other skin care ingredients reduced Norwalk virus (NV) by 3.74 log in 15 s and provided significantly greater NV reduction than all the other products tested ( P < 0.001). Furthermore, VF481 was the most effective product tested against the NoV genogroup II strains Snow Mountain virus (GII.2) and a GII.4 strain. These results demonstrate that alcohol by itself is not effective against NoV, but effective formulation of alcohol-based handrubs can achieve significant reduction of norovirus RNA on fingers. [ABSTRACT FROM AUTHOR]

Background: The last decade has seen significant progress in understanding the molecular biology of pancreatic adenocarcinoma. There is now an urgent need to translate these molecular techniques to clinical practice in order to improve diagnosis and prediction of response to treatment. The objectives of this study are to utilise poly(A) RT-PCR to measure expression levels of diagnostic Indicator genes, selected from microarray studies, of RNA from intraoperatively sampled pancreatic ductal juice and to correlate these expression levels with those in matched pancreatic tissue resection samples.Methods: Intraoperative sampling of pancreatic juice and collection of matched tissue samples was undertaken in patients undergoing pancreaticoduodenectomy for suspected tumour. RNA was isolated and poly(A) PCR and real-time PCR used to measure expression levels of 30 genes. Spearman's rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue.Results: Of the 30 Indicator genes measured, just one, ANXA1, showed a significant correlation of expression level between pancreatic juice and tissue samples, whereas three genes, IGFBP3 (P0.035), PSCA (P0.001) and SPINK1 (P0.05), showed significantly different expression between cancerous and benign pancreatic tissue samples.Conclusions: These results demonstrate that RNA analysis of pancreatic juice is feasible using the poly(A) cDNA technique, that correlation of gene expression exists between pancreatic juice and tissue for very few genes and that gene expression profiling can distinguish between benign and malignant pancreatic tissue. This indicates possible use of the technique for measurement of Indicator genes in pancreatic tissue for diagnosis of pancreatic cancer from very small tissue samples. [ABSTRACT FROM AUTHOR]

vailable data suggest that voltage-dependent anion selective channel (VDAC) constitutes an important component of a cellular regulatory mechanism based on the intracellular reduction/oxidation (redox) state. Here, using quantitative RT-PCR, we demonstrated that depletion of VDAC1 (termed here VDAC) in Saccharomyces cerevisiae cells distinctly affected levels of mRNAs encoding nuclear proteins sensitive to changes of the intracellular redox state including the nuclear transcription factors important for adaptation to the redox state and proteins involved in communication between mitochondria and the nucleus. We also revealed that the changes of the studied protein transcript levels generally correlated with changes of the intracellular redox state although VDAC appears also to affect mRNA levels by a mechanism not based on changes of the intracellular redox states. Thus, VDAC seems to be an important element of the intracellular signaling network. [ABSTRACT FROM AUTHOR]

Preliminary findings of various types of globin expressed in the respiratory bronchiolar and alveolar epithelium prompted us to compare the expression of erythropoietin (Epo) and its receptor (EpoR) in normal (healthy) human lung tissues with that in malignant lung tissues. The expression of Epo and EpoR was examined at the transcriptional and protein levels in normal and malignant lung tissues by reverse transcription-PCR, western blot, and immunohistochemical analyses. EpoR mRNA, but not Epo mRNA, was detected in all samples. In normal tissues, EpoR was detected in the mesothelium, chondrocytes, alveolar cells, vascular endothelial cells, smooth muscle fibers, macrophages, and neutrophils, while in malignant foci, the cancer cells of five malignant types showed various intensities of EpoR immunoreactivity. The pattern of staining of EpoR protein was generally stronger in the malignant tissues than in the normal samples. Phosphorylation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK-ERK1/2) was frequently seen in malignant cells, but not in the normal tissues, with the exception of macrophages. Based on the expression of Epo and EpoR mRNA with the EpoR in almost all cell components in normal tissues, we suggest that the normal lung may produce various types of globin through the autocrine and/or paracrine role of Epo. When the Epo signal is upregulated by hypoxic stress, the normal cells appear to transform into malignant cells and proliferate through activated MAPK signaling. [ABSTRACT FROM AUTHOR]