Expression analysis Research Papers - Academia.edu (original) (raw)

Histone deacetylases (HDAC) are responsible for the transcriptional control of genes through chromatin remodeling and control tumor suppressor genes. In several tumors, their expression has been linked to clinicopathological factors and patient survival. This study investigates HDACs 1, 2, 3, and 7 expressions in hepatocellular carcinoma (HCC) and their correlation with clinical data and patient survival. Tissue microarrays of 170 surgically resected primary HCCs and adjacent uninvolved tissue were evaluated immunohistochemically for the expression of HDACs 1, 2, 3, 7, and Ki-67 and were analyzed with respect to clinicopathological data and patient survival. HDACs 1, 2, 3, and Ki-67 were expressed significantly higher in cancer cells compared to normal tissue (HDAC1: p=0.034, HDACs 2 and 3 and Ki-67: p<0.001), while HDAC7 expression did not differ between HCC and noncancerous liver tissue. In tumor tissue HDACs 1-3 expression levels showed high concordance with each other, Ki-67 and tumor grade (p<0.001). High HDAC2 expression was associated with poor survival in low-grade and early-stage tumors (p<0.05). The expression of the HDACs 1, 2, and 3 (but not HDAC7) isoenzymes correlates with clinicopathological factors, and HDAC2 expression has an impact on patient survival.

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- Immunohistochemistry, HIstone Deacetylase, Hepatocellular Carcinoma, Chromatin remodeling

SPG7 is a newly identified gene involved in an autosomal recessive form of hereditary spastic paraplegia (HSP), a genetically heterogeneous group of neurodegenerative disorders. This gene encodes a protein characterized as a nuclear-encoded mitochondrial metalloprotease. The present report describes the genomic structure of the SPG7 gene. It is organized into 17 exons ranging from 78 to 242 bp and spans approximately 52 kb within three overlapping cosmids. The exon/intron boundaries and all splice junctions are consistent with the published consensus sequences for donor and acceptor sites. The provided genomic structure of SPG7 should facilitate the screening for mutations in this gene in patients with HSP and other related mitochondrial disease syndromes. SPG7 has been mapped to chromosome 16q24.3, a region of frequent loss of heterozygosity (LOH) seen in sporadic breast and prostate cancer. We have performed single-strand conformation polymorphism analysis of ten exons of this gene in a number of sporadic breast cancer samples showing LOH at 16q24.3. No mutations were detected; only single nucleotide polymorphisms were observed in exon 11, intron 7, intron 10 and intron 12. An expression analysis study has revealed the differential expression of SPG7 mRNA in various tissues and at different developmental stages.

- by Grant Sutherland
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- Genetics, Human Genetics, Complementary and Alternative Medicine, Breast Cancer

Microarray based transcription profiling is now a consolidated methodology and has widespread use in areas such as pharmacogenomics, diagnostics and drug target identification. Large-scale microarray studies are also becoming crucial to a new way of conceiving experimental biology. A main issue in microarray transcription profiling is data analysis and mining. When microarrays became a methodology of general use, considerable effort was made to produce algorithms and methods for the identification of differentially expressed genes. More recently, the focus has switched to algorithms and database development for microarray data mining. Furthermore, the evolution of microarray technology is allowing researchers to grasp the regulative nature of transcription, integrating basic expression analysis with mRNA characteristics, i.e. exon-based arrays, and with DNA characteristics, i.e. comparative genomic hybridization, single nucleotide polymorphism, tiling and promoter structure. In this article, we will review approaches used to detect differentially expressed genes and to link differential expression to specific biological functions.

- by Ashik Nazrul
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- Data Analysis, Microarray Data Analysis, Quality Control, Software

Gametophytic apomictic plants form nonreduced embryo sacs that generate clonal embryos by parthenogenesis, in the absence of both meiosis and eggcell fertilization. Here we report the sequence and expression analysis of a lorelei-like Paspalum notatum gene, n20gap-1, which encodes a GPI-anchored protein previously associated with apomixis in this species. Phylogeny trees showed that n20gap-1 was evolutionary related to the Arabidopsis thaliana lorelei genes At4g26466 and At5g56170. The lorelei At4g26466 disruption was shown to be detrimental to sperm cell release in arabidopsis. RFLP (Restriction Fragment Length Polymorphism) analysis revealed the occurrence of several homologous sequences in the Paspalum notatum genome, exhibiting polymorphisms genetically linked to apomixis. Real-time PCR showed that lorelei-family genes present a minor activity peak at pre-meiosis and a major one at anthesis. The apomictic genotype analyzed showed a significantly increased activity at pre-meiosis, post-meiosis and anthesis with respect to a sexual genotype. In situ hybridization assays revealed expression in integuments, nucellus and the egg-cell apparatus. Several n20gap-1 alleles differing mainly at the 3 0 UTR sequence were identified. Allelespecific real-time PCR experiments showed that allele 28 was significantly induced in reproductive tissues of the apomictic genotype with respect to the sexual genotype at anthesis. Our results indicate that P. notatum lorelei-like genes are differentially expressed in representative sexual (Q4188) and apomictic (Q4117) genotypes, and might play a role in the final stages of the apomixis developmental cascade. However, the association of n20gap-1 expression with the trait should be confirmed in significant number of sexual and apomictic genotypes.

- by Juan Ortiz
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- Genetics, Polymorphism, Reproduction, Plant Biology

Drought can negatively impact pod production despite the fact that cacao production usually occurs in tropical areas having high rainfall. Polyamines (PAs) have been associated with the response of plants to drought in addition to their roles in responses to many other stresses. The constitutive and drought inducible expression patterns of genes encoding enzymes involved in PA biosynthesis were determined: an ornithine decarboxylase (TcODC ), an arginine decarboxylase (TcADC ), an S-adenosylmethionine decarboxylase (TcSAMDC ), a spermidine synthase (TcSPDS ), and a spermine synthase (TcSPMS ). Expression analysis using quantitative real-time reverse transcription-PCR (QPCR) results showed that the PA biosynthesis genes were expressed in all plant tissues examined. Constitutive expression of PA biosynthesis genes was generally highest in mature leaves and open flowers. Expression of TcODC, TcADC, and TcSAMDC was induced with the onset of drought and correlated with changes in stomatal conductance, photosynthesis, photosystem II efficiency, leaf water potential and altered emission of blue-green fluorescence from cacao leaves. Induction of TcSAMDC in leaves was most closely correlated with changes in water potential. The earliest measured responses to drought were enhanced expression of TcADC and TcSAMDC in roots along with decreases in stomatal conductance, photosynthesis, and photosystem II efficiency. Elevated levels of putrescine, spermidine, and spermine were detected in cacao leaves 13 days after the onset of drought. Expression of all five PA associated transcripts was enhanced (1.5e3-fold) in response to treatment with abscisic acid. TcODC and TcADC, were also responsive to mechanical wounding, infection by Phytophthora megakarya (a causal agent of black pod disease in cacao), the necrosis-and ethylene-inducing protein (Nep1) of Fusarium oxysporum, and flower abscission. TcSAMDC expression was responsive to all stresses except flower abscission. TcODC, although constitutively expressed at much lower levels than TcADC, TcSAMDC, TcSPDS, and TcSPMS, was highly inducible by the fungal protein Nep1 (135-fold) and the cacao pathogen Phytophthora megakarya (671-fold). The full length cDNA for ODC was cloned and characterized. Among the genes studied, TcODC, TcADC, and TcSAMDC were most sensitive to induction by drought in addition to other abiotic and biotic stresses. TcODC, TcADC, and TcSAMDC may share signal transduction pathways and/or the stress induced signal induction pathways may converge at these three genes leading to similar although not identical patterns of expression. It is possible altering PA levels in cacao will result in enhanced tolerance to multiple stresses including drought and disease as has been demonstrated in other crops. Published by Elsevier Masson SAS.

- by Bryan Bailey and +1
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- Plant Biology, Signal Transduction, Polyamines, Plant Physiology

The human NK gene complex localized on chromosome 12p12.3-p13.2 codes for several lectin-like receptor genes expressed by NK cells as well as by other hematopoietic cells. In this study, by using the expressed sequence tag database we identified a novel receptor gene, designated as killer cell lectin-like receptor, subfamily F, member 1 (KLRF1), encoding a putative type II transmembrane glycoprotein. The KLRF1 gene has been localized on the high-resolution physical map of chromosome 12p. The genomic structure of the KLRF1 gene and the existence of one spliced variant are also described. KLRF1 was expressed at the mRNA level in peripheral blood leukocytes, activated NK cells, monocytes and NK and myeloid cell lines. The presence of two immunoreceptor tyrosine-based inhibitory-like motifs within the cytoplasmic tail of KLRF1 suggests an inhibitory role in NK cell and monocyte activity.

- by P. Roda-Navarro
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- Immunology, Sequence alignment, Natural Killer cells, Lectins
- by vaijayanti tamhane
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- Genetics, Gene expression, Phylogeny, Plant diseases

A phylogenetic analysis of plant FtsH-like proteins was performed using protein sequences from the GENEBANK database and five groups of plant FtsH-like proteins were identified by neighbor-joining analysis. Prediction of the subcellular location of the proteins suggested that two (FtsH-m1 & FtsH-m2) were mitochondrial and three (FtsH-p1, FtsH-p2, FtsH-p3) were plastid targeting. The phylogenetic profile of plant FtsH-like proteins was used to search sugarcane expressed sequence tag (EST) clusters in the SUCEST database. Initially, 153 clusters presenting homology with FtsH-like proteins were recovered, of which 23 were confirmed by a BLAST search in the GENEBANK database and by comparison of their hidropathy index with that of previously described FtsH-like proteins. Sugarcane presented EST clusters in all phylogenetic groups. In silico expression analysis showed that the groups are differentially expressed in sugarcane tissues, with FtsH-p2 and FtsH-m1 presenting increased levels of expression.

- by Phellippe Marbach
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- Genetics, Biology, Molecular Biology and genetics, Phylogenetic analysis

Hemileia vastatrix is a biotrophic fungus, causing coffee leaf rust in all coffee growing countries, leading to serious social and economic problems. Gene expression studies may have a key role unravelling the transcriptomics of this pathogen during interaction with the plant host. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is currently the golden standard for gene expression analysis, although an accurate normalisation is essential for adequate conclusions. Reference genes are often used for this purpose, but the stability of their expression levels requires validation under experimental conditions. Moreover, pathogenic fungi undergo important biomass variations along their infection process in planta, which raises the need for an adequate method to further normalise the proportion of fungal cDNA in the total plant and fungus cDNA pool. In this work, the expression profiles of seven reference genes [glyceraldehyde-3-phosphate dehydrogenase (GADPH), elongation factor (EF-1), Beta tubulin (β-tubulin), cytochrome c oxidase subunit III (Cyt III), cytochrome b (Cyt b), Hv00099, and 40S ribosomal protein (40S_Rib)] were analysed across 28 samples, obtained in vitro (germinated uredospores and appressoria) and in planta (post-penetration fungal growth phases). Gene stability was assessed using the statistical algorithms incorporated in geNorm and NormFinder tools. Cyt b, 40S_Rib, and Hv00099 were the most stable genes for the in vitro dataset, while 40S_Rib, GADPH, and Cyt III were the most stable in planta. For the combined datasets (in vitro and in planta), 40S_Rib, GADPH, and Hv00099 were selected as the most stable. Subsequent expression analysis for a gene encoding an alpha subunit of a heterotrimeric G-protein showed that the reference genes selected for the combined dataset do not differ significantly from those selected specifically for the in vitro and in planta datasets. Our study provides tools for correct validation of reference genes in obligate biotrophic plant pathogens, as well as the basis for RT-qPCR studies in H. vastatrix.► Reference genes were selected for Hemileia vastatrix in planta RT-qPCR studies. ► Gene stability was assessed using geNorm and NormFinder programmes. ► 40S_Rib, GADPH, and Hv00099 were the most stable reference genes. ► An extra correction step enabled normalisation of fungal biomass in planta.

- by Helena Azinheira
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- Microbiology, Plant Biology, Gene expression, Plant diseases
- by Eric Miska
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- Pharmacology, Biochemistry, Bioinformatics, Evolutionary Biology

recent advances in high-throughput cDna sequencing (rna-seq) can reveal new genes and splice variants and quantify expression genome-wide in a single assay. the volume and complexity of data from rna-seq experiments necessitate scalable, fast and mathematically principled analysis software. topHat and cufflinks are free, open-source software tools for gene discovery and comprehensive expression analysis of high-throughput mrna sequencing (rna-seq) data. together, they allow biologists to identify new genes and new splice variants of known ones, as well as compare gene and transcript expression under two or more conditions. this protocol describes in detail how to use topHat and cufflinks to perform such analyses. It also covers several accessory tools and utilities that aid in managing data, including cummerbund, a tool for visualizing rna-seq analysis results. although the procedure assumes basic informatics skills, these tools assume little to no background with rna-seq analysis and are meant for novices and experts alike. the protocol begins with raw sequencing reads and produces a transcriptome assembly, lists of differentially expressed and regulated genes and transcripts, and publication-quality visualizations of analysis results. the protocol's execution time depends on the volume of transcriptome sequencing data and available computing resources but takes less than 1 d of computer time for typical experiments and ~1 h of hands-on time.

- by Geo Pertea
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- Genomics, Open Source and Free Software Studies, Nature, Biological Sciences
- by Tarek Kapiel
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- Biology, Pseudomonas Fluorescens, Molecular cloning, Expression analysis

Extraction of discriminative features from salient facial patches plays a vital role in effective facial expression recognition. The accurate detection of facial landmarks improves the localization of the salient patches on face images. This paper proposes a novel framework for expression recognition by using appearance features of selected facial patches. A few prominent facial patches, depending on the position of facial landmarks, are extracted which are active during emotion elicitation. These active patches are further processed to obtain the salient patches which contain discriminative features for classification of each pair of expressions, thereby selecting different facial patches as salient for different pair of expression classes. One-against-one classification method is adopted using these features. In addition, an automated learning-free facial landmark detection technique has been proposed, which achieves similar performances as that of other state-of-art landmark detection methods, yet requires significantly less execution time. The proposed method is found to perform well consistently in different resolutions, hence, providing a solution for expression recognition in low resolution images. Experiments on CK+ and JAFFE facial expression databases show the effectiveness of the proposed system.

- by S L Happy
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- Engineering, Human Computer Interaction, Computer Animation, Face Recognition

A copy DNA encoding the plastid-located isoform of the fructose-l,6-bisphosphatase (cp-FBPase) has been cloned from potato (Solanum tuberosum L.). Sequence analysis reveals a high degree of homology to cp-FBPases from wheat, spinach, and Arabidopsis. Analysis of RNA blots shows that the expression of the cp-FBPase is limited to green tissue such as leaf and stem, and is absent from photosynthetically inactive tissue such as roots, tubers and stolons. This provides additional evidence that hexoses or hexose phosphates are imported into amyloplasts of heterotrophic tissues. Incubation of detached leaves of potato in darkness in a sucrosecontaining medium leads to massive accumulation of both starch and transcripts encoding starch biosynthetic enzymes. However, no transcripts encoding the cp-FBPase are detectable under these conditions.

- by Jens Kossmann
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- Plant Biology, Sequence Analysis, Potato, Enzyme

Tight control over circulating juvenile hormone (JH) levels is of prime importance in an insect's life cycle. Consequently, enzymes involved in JH metabolism, especially juvenile hormone esterases (JHEs), play major roles during metamorphosis and reproduction. In the highly eusocial Hymenoptera, JH has been co-opted into additional functions, primarily in the development of the queen and worker castes and in age-related behavioral development of workers. Within a set of 21 carboxylesterases predicted in the honey bee genome we identified one gene (Amjhe-like) that contained the main functional motifs of insect JHEs. Its transcript levels during larval development showed a maximum at the switch from feeding to spinning behavior, coinciding with a JH titer minimum. In adult workers, the highest levels were observed in nurse bees, where a low JH titer is required to prevent the switch to foraging. Functional assays showed that Amjhe-like expression is induced by JH-III and suppressed by 20-hydroxyecdysone. RNAi-mediated silencing of Amjhe-like gene function resulted in a six-fold increase in the JH titer in adult worker bees. The temporal profile of Amjhe-like expression in larval and adult workers, the pattern of hormonal regulation and the knockdown phenotype are consistent with the function of this gene as an authentic JHE.

- by Adriana Márcia
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- Physiology, RNA interference, Bees, Apis mellifera

Mutations in the PCCA or PCCB genes, encoding both subunits of propionyl-CoA carboxylase, result in propionic acidemia, a life-threatening inborn error of metabolism with autosomal recessive inheritance. To date, 41 mutations in the PCCA gene and 54 in the PCCB gene have been reported, most of them single base substitutions causing amino acid replacements, and a variety of small insertions and deletions and splicing defects. A greater heterogeneity is observed in the PCCA gene, specially in Caucasians, with no prevalent mutations, while in the Japanese population three mutations account for more than half of the alleles studied. For the PCCB gene a limited number of mutations is responsible for the majority of the alleles characterized in both Caucasian and Oriental populations. These two populations show a diVerent mutational spectrum, only sharing some involving CpG dinucleotides probably as recurrent mutational events. Functional characterization of the mutant missense alleles has been accomplished using diVerent prokaryotic and eukaryotic systems, and the structural consequences have been analyzed in the available crystal models. For the PCCA gene, the main molecular eVect of the expressed mutations is related to protein instability, except two mutations in the active site predictably aVecting ATP binding. In the PCCB gene the majority of the analyzed mutations are predicted to alter the active site conformation resulting in diminished activity. A few carboxy-terminal PCCB mutations aVect the interaction between subunits and the assembly with PCCA to form a functional PCC oligomer. The amount of normal transcripts resulting from some PCCA and PCCB splicing mutations has also been analyzed. Overall, the data generated from the expression analysis reveal potential genotype-phenotype correlations for this clinically heterogeneous disorder.  2004 Elsevier Inc. All rights reserved.

- by Lourdes Desviat
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- Molecular Genetics, Mutation, Inborn errors of metabolism, Phenotype

DREB transcription factors play key roles in plant stress signalling transduction pathway, they can specifically bind to DRE/CRT element (G/ACCGAC) and activate the expression of many stress inducible genes. Here, a novel rice DREB transcription factor, OsDREB1F, was cloned and characterised via subtractive suppression hybridisation (SSH) from upland rice. Expression analysis revealed that OsDREB1F gene was induced by salt, drought, cold stresses, and also ABA application, but not by pathogen, wound, and H2O2. Subcellular localization results indicated that OsDREB1F localizes in nucleus. Yeast activity assay demonstrated that OsDREB1F gene encodes a transcription activator, and can specifically bind to DRE/CRT but not to ABRE element. Transgenic plants harbouring OsDREB1F gene led to enhanced tolerance to salt, drought, and low temperature in both rice and Arabidopsis. The further characterisation of OsDREB1F-overexpressing Arabidopsis showed that, besides activating the expression of COR genes which contain DRE/CRT element in their upstream promoter regions, the expression of rd29B and RAB18 genes were also activated, suggested that OsDREB1F may also participate in ABA-dependent pathway.

- by QIUYUN WANG
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- Genetics, Plant Biology, Abiotic Stress, Plant Molecular Biology

A simple and efficient protocol for the Agrobacterium-mediated transformation of an agronomically useful abiotic sensitive popular indica rice cv. ADT 43 has been developed. Initiation of calli were best achieved from the leaf bases of 4 days old rice seedlings on LS medium supplemented with 2.5mg/L 2,4-D and 1.0mg/L thiamine-HCl. Rice calli immersed in Agrobacterium suspension (strain EHA 105, OD(600)=0.8) were co-cultured on LS30-AsPC medium for 2 days at 25±2°C in the dark. Based on GUS expression analysis, 10min co-cultivation time with 100μM acetosyringone was found optimum for the delivery of gus gene. Calli were proved to be very sensitive to Agrobacterium infection and we found that the level of necrotic response can be minimized after co-cultivation with 30% LS, 10g/L PVP, 10% coconut water and 250mg/L timentin which improved the final transformation efficiency to 9.33%. Molecular and genetic analysis of transgenic plants reveals the integration, expression and inheritance of transgene in the progeny (T(1)) of these plants. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants (T(0) and T(1)).

- by Manikandan Ramesh
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- Plant Biology, Plant Science, Rhizobium, Plant Growth Regulators

Agrobacterium tumefaciens mediated in planta transformation protocol was developed for castor, Ricinus communis. Two-day-old seedlings were infected with Agrobacterium strain EHA105/pBinBt8 harboring cry1AcF and established in the greenhouse. Screening the T1 generation seedlings on 300 mg L -1 kanamycin identified the putative transformants. Molecular and expression analysis confirmed the transgenic nature and identified high-expressing plants. Western blot analysis confirmed the co-integration of the nptII gene in the selected transgenic plants. Bioassay against Spodoptera litura corroborated with high expression and identified five promising effective lines. Analysis of the T2 generation plants proved the stability of the transgene indicating the feasibility of the method.

- by Kalpana Reddy
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- Insect Resistance, Crop, Western blot, Expression analysis

DNA plasmids of Escherichia coli are common vectors for recombinant protein and metabolite production and have potential therapeutic applications as genetic vaccines and therapeutics. However, plasmid maintenance imposes a metabolic burden on the host cells, resulting in reduced growth rate and cell density. In 2 L batch fermentation, DH5␣ cells carrying a 7.3 kb NS3 plasmid had a lower specific growth rate than the non-plasmid-bearing host (0.64 h −1 versus 0.87 h −1 ). In this work, global transcriptional analysis was combined with proteomics studies to evaluate the effect of plasmid maintenance on gene expression. Global transcriptional expression analysis of plasmid-bearing cells over host showed a general trend of downregulated biosynthetic/energy metabolism genes, differentially expressed transport genes and upregulated heat shock proteins. In the central metabolic pathways, most glycolytic genes were downregulated, while less expression difference was found in the pentose phosphate pathway. Expression ratios of 19 proteins identified from proteomics studies were consistent with these observations. Our findings suggest that plasmid maintenance alone perturbs global gene regulation, and leads to significant changes in central metabolic pathways in the host. This work contributes to our understanding of plasmid metabolic load at the gene expression level and could potentially aid in future metabolic engineering efforts.

- by Robin Philp
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- Genetics, Gene regulation, Metabolic Engineering, Gene expression

Amplified segments of the long arm of chromosome 12 are frequently observed in human sarcomas. In most cases there are separate amplified regions around the MDM2 and CDK4 genes. Here we show recurrent amplification of a third region... more

- by O. Myklebost
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- Transcription Factors, Oncogene, Sarcoma, Transcription Factor

Winter rye (Secale cereale L.) is known to be recalcitrant to tissue culture response (TCR). Moreover, the mechanisms controlling TCR are poorly recognized. In the present study, a Genetically Directed Differential Subtraction Chain (GDDSC) strategy was used to isolate genomic regions associated with TCR. Two pairs of bulks, R-NR and E > 90–E < 25, were prepared, and for each pair, the bulk was used both as a tester and as a driver. After eight rounds of subtraction, 45 unique GDDSC products were obtained. To verify the connection between GDDSCs and TCR two approaches were applied: Real-Time RT-PCR analysis and genetic mapping. The expression profiles of four out of six investigated products agrees with the phenotype and subtraction direction. Two from the developed GDDSC-SCAR markers showed polymorphism in lines L9 and L318, the parental components of a mapping population. The polymorphic SCAR GDDSC markers were mapped on the rye chromosome 4R (SCAR-GDDSC NR 440BamHI) and 6R (SCAR-GDDSC E < 25 340BamHI). The marker E < 25_340B9 was localized in the border region of QTL for embryogenic callus production.

- by Monika Rakoczy-Trojanowska
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- Genetics, Polymorphism, Plant Biology, Real-time RT-PCR

Muscle biopsy is required to provide a definitive diagnosis in many neuromuscular disorders. It can be performed through an open or needle technique under local anesthesia. The major limitations of the needle biopsy technique are the sample size, which is smaller than that obtained with open biopsy, and the impossibility of direct visualization of the sampling site. However, needle biopsy is a less invasive procedure than open biopsy and is particularly indicated for diagnosis of neuromuscular disease in infancy and childhood. The biopsied muscle should be one affected by the disease but not be too weak or too atrophic. Usually, in case of proximal muscle involvement, the quadriceps and the biceps are biopsied, while under suspicion of mitochondrial disorder, the deltoid is preferred. The samples must be immediately frozen or fixed after excision to prevent loss of enzymatic reactivity, DNA depletion or RNA degradation. A battery of stainings is performed on muscle sections from every frozen muscle biopsy arriving in the pathology laboratory. Histological, histochemical, and histoenzymatic stainings are performed to evaluate fiber atrophy, morphological, and structural changes and metabolic disorders. Moreover, immunohistochemistry and Western blotting analysis may be used for expression analysis of muscle proteins to obtain a specific diagnosis. There are myopathies that do not need muscle biopsy since a genetic test performed on a blood sample is enough for definitive diagnosis. Muscle biopsy is a useful technique which can make an enormous contribution in the field of neuromuscular disorders but should be considered and interpreted together with the patient's family and clinical history.

- by Rosanna Cardani and +1
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- Neurology, Immunohistochemistry, Biopsy, Genetic Testing

A total of 210 Salmonella isolates, representing 64 different serovars, were isolated from imported seafood samples, and 55/210 isolates were found to be resistant to at least one antibiotic. Class 1 integrons from three multidrug-resistant Salmonella enterica strains (Salmonella enterica serovars Newport [strain 62], Typhimurium var. Copenhagen [strain 629], and Lansing [strain 803], originating from Hong Kong, the Philippines, and Taiwan, respectively) were characterized. Southern hybridization of plasmids isolated from these strains, using a class 1 integron probe, showed that trimethoprim-sulfamethoxazole and streptomycin resistance genes were located on a megaplasmid in strain 629. Our study indicates that imported seafood could be a reservoir for Salmonella isolates resistant to multiple antibiotics.

- by Bruno Oliveira
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- Genetics, Microbiology, Parasitology, Immunology

Restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA) were investigated to detect intraspecific variation among the Italian cultivated and wild Olea europaea L. populations. RFLPs were visualized by Southern hybridization of Eco RI and Hind III restricted total cellular DNA using two mitochondrial genes (cox3 and atpA ) as probes. The mtDNA RFLP data gathered in our analysis allow the identification of three distinct olive tree mitotypes; each characterized by specific hybridization patterns. The result on genetic variability is also confirmed by the unweighted pair group method with arithmetic averages dendrogram (UPGMA) that combines the Italian thirty-seven different sources of Olea europaea L. into three major clusters of cytoplasmic relatedness. Therefore, at least as many maternal lineages are involved in the evolution and origin of the Italian olive tree mitochondrial genomes, with the cultivar Cerasòla showing the greatest diversity with the cox3 probe and two restriction enzymes. We, thus, report the identification of three novel diagnostic probe/enzyme combinations that can be used as markers of cytoplasmic diversity in the Euromediterranean O. europaea L. population. Further characterization of the polymorphic cox3 locus in the mtDNA of the cultivar Cerasòla by sequencing and expression analysis at the level of transcription and RNA editing pattern suggests a correlation between a duplication event of the cox3 locus and the male sterile phenotype of this cultivar. The mtDNA RFLPs provide, thus, a sensitive and reliable method to assess intraspecific cytoplasmic diversity within the economically important olive tree cultivars in Italy. Our results also provide new insights into the maternal lineages involved in the evolution of the Euromediterranean olive trees. #

- by Teresa Regina
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- Polymorphism, Plant Biology, Plant Science, Gene expression

Background: Even though the process of potato tuber starch biosynthesis is well understood, mechanisms regulating biosynthesis are still unclear. Transcriptome analysis provides valuable information as to how genes are regulated. Therefore, this work aimed at investigating transcriptional regulation of starch biosynthetic genes in leaves and tubers of potato plants under various conditions. More specifically we looked at gene expression diurnally in leaves and tubers, during tuber induction and in tubers growing at different velocities. To determine velocity of potato tuber growth a new method based on X-ray Computed Tomography (X-ray CT) was established. Results: Comparative transcriptome analysis between leaves and tubers revealed striking similarities with the same genes being differentially expressed in both tissues. In tubers, oscillation of granule bound starch synthase (GBSS) expression) was observed which could be linked to sucrose supply from source leaves. X-ray CT was used to determine time-dependent changes in tuber volume and the growth velocity was calculated. Although there is not a linear correlation between growth velocity and expression of starch biosynthetic genes, there are significant differences between growing and non-growing tubers. Co-expression analysis was used to identify transcription factors positively correlating with starch biosynthetic genes possibly regulating starch biosynthesis.

- by Uwe Sonnewald
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- Transcription Regulation, Starch, Gene expression, Potato

Software-based image analysis is a crucial step in the biological interpretation of two-dimensional gel electrophoresis experiments. Recent significant advances in image processing methods combined with powerful computing hardware have enabled the routine analysis of large experiments. We cover the process starting with the imaging of 2-D gels, quantitation of spots, creation of expression profiles to statistical expression analysis followed by the presentation of results. Challenges for analysis software as well as good practices are highlighted. We emphasize image warping and related methods that are able to overcome the difficulties that are due to varying migration positions of spots between gels. Spot detection, quantitation, normalization, and the creation of expression profiles are described in detail. The recent development of consensus spot patterns and complete expression profiles enables one to take full advantage of statistical methods for expression analysis that are we...

- by Kostas Karpouzis
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- Psychology, Human Computer Interaction, Face Recognition, Facial expression

Abstracts / Current Opinion in Biotechnology 22S (2011) S15-S152 stress. In the present study, six different wheat (Triticum aestivum L.) genotypes including two tolerant sister lines (Ducula-1 and Ducula-4), two standard cultivars widely grown in the Eastern Mediterranean ('Basribey' and 'Golia') and two susceptible cultivars ('Seri-82' and 'Dogankent') were subjected to different waterlogging treatments and their proline and chlorophyll contents were measured by spectrophotometer. About 25 anaerobically induced genes were analyzed using real time polymerase chain reaction (RT-PCR) method. Wheat seedlings were subjected to waterlogging treatments of 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72 h and after each treatment, leaf samples were collected for chlorophyll, carotenoid, proline contents and gene expression analyses. A total of 32 primer pairs obtained from 25 anaerobically induced genes were used for RT-PCR. Total chlorophyll content decreased while proline content increased as waterlogging duration was prolonged. Gene expression differences among varieties under waterlogging and control conditions will be also presented.

- by Che E Zain
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- Engineering, Technology, Biological Sciences, Oryza Sativa

We report the progress of a multi-disciplinary research project on solid-state fermentation (SSF) of the filamentous fungus Aspergillus oryzae. The molecular and physiological aspects of the fungus in submerged fermentation (SmF) and SSF are compared and we observe a number of differences correlated with the different growth conditions. First, the aerial hyphae which occur only in SSFs are mainly responsible for oxygen uptake. Second, SSF is characterised by gradients in temperature, water activity and nutrient concentration, and inside the hyphae different polyols are accumulating. Third, pelleted growth in SmF and mycelial growth in SSF show different gene expression and protein secretion patterns. With this approach we aim to expand our knowledge of mechanisms of fungal growth on solid substrates and to exploit the biotechnological applications. ß

- by Te Biesebeke
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- Engineering, Microbiology, Technology, Biotechnology

In order to determine the suitability of reference or housekeeping genes as internal controls in real-time reverse transcriptase PCR (RT-PCR) assays for quantification of target mRNAs, we studied the levels of expression of four candidate reference genes in maritime pine by real-time RT-PCR. The expression levels obtained for glyceraldehyde-3-phosphate-dehydrogenase, 18S ribosomal RNA, eukaryotic translation initiation factor eIF4AII and ubiquitin in nine stages of embryo development revealed that none of the genes tested proved to be suitable as an internal control. Copy number quantification of the four transcripts showed an average relative variation of seven fold. We propose that the combination of a precise method for RNA quantification, internal controls for monitoring RT reaction and PCR efficiency and a robust external standard curve can guarantee a reliable absolute quantification of mRNA transcripts in real time RT-PCR. This approach may avoid the controversy in the use of housekeeping genes and may assume special significance in tissues undergoing developmental changes.

- by João Maroco
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- Plant Biology, Real-time RT-PCR, Internal Control, real time PCR

Sex steroid hormones are known to play a central role in vertebrate sex determination and differentiation. However, the tissues in which they are produced or received during development, especially around the period of sex determination of the gonads, have rarely been investigated. In this study, we identified the cDNA sequence, including the full-length of the coding region of cholesterol side-chain cleavage enzyme (P450scc), from the leopard gecko; a lizard with temperature-dependent sex determination. Embryonic expression analysis of two steroidogenic enzymes, P450scc and P450 aromatase (P450arom), and four sex steroid hormone receptors, androgen receptor, estrogen receptor α and β, and progesterone receptor, was subsequently conducted. mRNA expression of both steroidogenic enzymes was observed in the brain and gonads prior to the temperature-sensitive period of sex determination. The mRNAs of the four sex steroid hormone receptors were also detected in the brain and gonads at all stages examined. These results suggest the existence of a gonad-independent sex steroid hormone signaling system in the developing leopard gecko brain.

- by Min Kyun Park
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- Physiology, Zoology, Gene expression, Estrogen Receptor

Piriformospora indica (Basidiomycota, Sebacinales) is a root colonizing fungus which is able to increase biomass and yield of crop plants and to induce local and systemic resistance to fungal diseases and tolerance to abiotic stress. A prerequisite for the elucidation of the mode of action of this novel kind of symbiosis is knowledge of the genome organization as well as the development of tools to study and modify gene functions. Here we provide data on the karyotype and genetic transformation strategies. The fungus was shown to possess at least six chromosomes and a genome size of about 15.4-24 Mb. Sequences of the genes encoding the elongation factor 1-a (TEF) and glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) were used for genome size estimation through real-time PCR analysis. Chromosomal location investigated by Southern blot and expression analysis suggested that TEF and GAPDH are single-copy genes with strong and constitutive promoters. A genetic transformation system was established using a fragment of the TEF promoter region for construction of vectors carrying the selectable marker hygromycin B phosphotransferase. Results demonstrate that P. indica can be stably transformed by random genomic integration of foreign DNA and that it posses a relative small genome as compared to other members of the Basidiomycota.

- by Karl-heinz Kogel
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- Genetics, Microbiology, Plant Biology, Genome Size

Hedgehog signaling is an important component of cell-cell communication during bilaterian development, and abnormal Hedgehog signaling contributes to disease and birth defects. Hedgehog genes are composed of a ligand ("hedge") domain and an autocatalytic intein ("hog") domain. Hedgehog (hh) ligands bind to a conserved set of receptors and activate downstream signal transduction pathways terminating with Gli/Ci transcription factors. We have identified five intein-containing genes in the anthozoan cnidarian Nematostella vectensis, two of which (NvHh1 and NvHh2) contain definitive hedgehog ligand domains, suggesting that to date, cnidarians are the earliest branching metazoan phylum to possess definitive Hh orthologs. Expression analysis of NvHh1 and NvHh2, the receptor NvPatched, and a downstream transcription factor NvGli (a Gli3/ Ci ortholog) indicate that these genes may have conserved roles in planar and trans-epithelial signaling during gut and germline development, while the three remaining intein-containing genes (NvHint1,2,3) are expressed in a cell-type-specific manner in putative neural precursors. Metazoan intein-containing genes that lack a hh ligand domain have previously only been identified within nematodes. However, we have identified intein-containing genes from both Nematostella and in two newly annotated lophotrochozoan genomes. Phylogenetic analyses suggest that while nematode inteins may be derived from an ancestral true hedgehog gene, the newly identified cnidarian and lophotrochozoan inteins may be orthologous, suggesting that both true hedgehog and hint genes may have been present in the cnidarian-bilaterian ancestor. Genomic surveys of N. vectensis suggest that most of the components of both protostome and deuterostome Hh signaling pathways are present in anthozoans and that some appear to have been lost in ecdysozoan lineages. Cnidarians possess many bilaterian cell-cell signaling pathways (Wnt, TGFβ, FGF, and Hh) that appear to act in concert to pattern tissues along the oral-aboral axis of the polyp. Cnidarians represent a diverse group of animals with a predominantly epithelial body plan, and perhaps selective pressures to pattern epithelia resulted in the ontogeny of the hedgehog pathway in the common ancestor of the Cnidaria and Bilateria.

- by David Matus
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- Developmental Biology, Molecular Evolution, Pattern Formation, Cell Signaling

Aspartic proteinases (EC 3.4.23) are widely distributed in the plant kingdom, and a number of cDNAs have been isolated from different plants. Here we report the isolation an expression analysis of a cDNA from Solanum tuberosum L. (cv. Pampeana) named StAsp. The StAsp cDNA clone was obtained using a reverse transcriptase-polymerase chain reaction (RT-PCR) and degenerated primers encoding to plant aspartic proteinases conserved domains. The coding region of the gene is 1494 bp long encoding 497 amino acids of a predicted 54 kDa molecular mass and with a pI of 5.5. The gene shares a high homology with an aspartic proteinase cDNA of tomato, 97% and 94% homology on the level of DNA and protein, respectively. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant specific insert. Northern blot analysis indicated that StAps transcripts are differentially accumulated in potato leaves after Phytophthora infestans infection in two potato cultivars with different degree of field resistance to this pathogen. In the resistant cultivar (Pampeana), induction was higher and more durable than in the susceptible cultivar (Bintje), suggesting that the StAsp level expression are associated with the resistance degree of potato cultivars to P. infestans. Results obtained previously about the induction of StAP proteins in stress conditions and these results suggest that potato aspartic proteinases are components of the plant defense response.

- by Paula Veríssimo and +1
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- Plant Biology, Western blotting, Potato, Plant Physiology

The aims of this study were to isolate and characterize putative fragrance-related cDNAs from the floral cDNA library of Vanda Mimi Palmer, an orchid hybrid that has won several international awards for its sweet fragrance. A total of 1,000,000 pfu were screened by hybridizing cDNA library plaques with fully open flower cDNA probe of Vanda Mimi Palmer representing all mRNAs expressed during daytime. The clones that gave positive signals were in vivo excised and PCR-amplified inserts were subjected to reverse-Northern analysis by hybridizing with cDNA probes of fully open flower of Vanda Mimi Palmer (fragrant orchid) and its bud or fully open flower of Vanda Tan Chay Yan (non-fragrant orchid) separately. The clones up-regulated in fully open flower stage of Vanda Mimi Palmer compared to its bud stage or fully open flower of Vanda Tan Chay Yan were sequenced. Sequence analyses showed the presence of eight putative fragrance-related cDNAs of which two were putative Vanda Mimi Palmer 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase (VMPCMEK) and Vanda Mimi Palmer cytochrome P450 protein (VMPCyP450). These two transcripts were selected for full-length cDNA isolation and expression analysis by real-time RT-PCR. The VMPCMEK transcript encodes a polypeptide of 400 amino acid residues, while the VMPCyP450 encodes 538 amino acid residues. Relative expression analysis of VMPCMEK and VMPCyP450 transcripts by real-time RT-PCR showed up-regulated expressions in floral tissues compared to vegetative tissues, and both were found to be developmentally regulated.

Structural and biochemical analysis of proteins requires access to purified protein material. Modern molecular biology technologies facilitate straightforward molecular cloning and expression analysis of multiple protein constructs in... more

- by Anne Lagendijk
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- Genetics, Membrane Proteins, Development, Biological Sciences

Chromosome region 17q12-23 commonly shows an increase in DNA copy number in breast cancers, suggesting that several oncogenes are located at this site. We performed a high-resolution expression array and comparative genomic hybridization analysis of genes mapped to the entire 17q12-23 region, to identify novel candidate oncogenes. We identified 24 genes that showed significant overexpression in breast cancers with gain of 17q12-23, compared to cancers without gain. These genes included previously identified oncogenes, together with several novel candidate oncogenes. FISH analysis using specific gene probes hybridized to tissue arrays confirmed the underlying amplification of overexpressed genes. This high-resolution analysis of the 17q12-23 region indicates that several established and novel candidate oncogenes, including a Wnt-signaling pathway member, are amplified and overexpressed within individual primary breast cancer samples. We were also able to confirm the presence of two apparently separate and reciprocally amplified groups of genes within this region. Investigation of these genes and their functional interactions will facilitate our understanding of breast oncogenesis and optimal management of this disease.

- by Jane Armes
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- Breast Cancer, Nucleic acid hybridization, Gene Dosage, Genes

We conducted a genetic yeast screen to identify Thermo-tolerance genes (TTOs) in maize kernel cDNA library. During the screening, we identified a maize clone (TTO6) that seemed to confer elevated heat tolerance in comparison to control cells. TTO6 cDNA (GenBank accession no. AY103785) encodes an 11-kDa protein which is 69% similarity to the Arabidopsis GASA4 gene. To further examine heat tolerance in Arabidopsis, we functionally characterized the GASA4 gene and found that heat induced GASA4 expression. Constitutive expression of GASA4 in Arabidopsis led to elevated heat tolerance in transgenic lines. Interestingly, endoplasmic reticulum chaperone expression analysis suggests that GASA4 influences BiP gene expression during heat stress.

- by Dong Ju Lee
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- Biochemistry, Genetics, Plant Biology, Stress
- by Mirna Sweet
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- Pharmacology, Biochemistry, Bioinformatics, Evolutionary Biology

The CDKA gene is linked to the cellular control. This gene was isolated from coconut palm (Cocos nucifera L) and a detailed expression analysis was done during somatic embryogenesis. Analysis of the deduced amino acid sequence showed the most important residues to be conserved. The highest homology was with Picea abies (96% similarity). Expression of the putative CnCDKA gene steadily increased during embryogenic callus formation phase when embryogenic competence is attained. In situ hybridization specified the localization of the transcripts as being mainly in a few cell layers within the meristematic centres in embryogenic calli at 90 day cultures. Analysis of CnCDKA expression at different somatic embryo formation stages showed that the expression was decreased progressively with the lowest expression in germinated somatic embryos.

- by Francisco Paredes
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- Plant Biology, Gene expression, Cell Division, Somatic Embryogenesis

Human leukocyte antigen (HLA) expression is important for the elimination of tumor cells by the immune system and immunotherapy. Activated T cells directed against tumor-associated antigens are fully capable of recognizing and eradicating neoplastic cells. Therefore, HLA expression loss is considered to be a main factor in tumor development. We report for the first time HLA-A and HLA-B allele-specific expression analysis by immunohistochemical staining of fresh tumor tissue and 9 lymph node metastases of 15 patients with head-and-neck squamous cell carcinoma. Heterogeneous HLA expression and HLA expression loss was detected in 13 tumor patients. Approximately 50% of the tumors had allele-specific expression loss, which would have remained undetected using HLA monomorphic and locus-specific antibodies. In the majority of the patients with head-and-neck squamous cell carcinoma, HLA allele-specific expression loss differed between primary lesions and metastases. This is important for the efficacy of immunotherapy in these patients. It can be concluded that it is crucial to study HLA expression at the allele-specific level of primary lesions and metastases. It increases and refines our knowledge of HLA expression loss in tumorgenesis, which will improve the development of specific immunotherapy. Human Immunology 67, 692-699 (2006).

Bu bildiride, ifadenin varlığında sabit kayıtlamaya dayalı 3B yüz tanıma yöntemlerinin iyi başarım sağlamadığı gösterilmiş olup, başarım arttırımı için bölgesel kayıtlama ve tanıma yapılması önerilmiştir. Hızlı hizalama amaçlı, ortalama bölge modelleriyle yinelemeli en yakın nokta (ICP) algoritmasına dayalı kayıtlama yöntemi kullanılmıştır. Bölgelerden elde edilen benzerlik skorları, tümleştirme yöntemleriyle birleştirilerek başarım arttırımı sağlanmıştır. Bu çalışmada, ifade tanıma ve ifade varlığında yüz tanıma için kullanılmak üzere toplanmış yeni bir 3B yüz veri kütüphanesi tanıtılmış ve deneylerde bu veri kütüphanesi kullanılmıştır.

- by Nese Alyuz
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- Face Recognition, Facial expression, Expression analysis
- by Seong Bhoo
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- Plant Biology, Phylogeny, Plant diseases, Host-parasite interactions
- by Mario Ruales
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- Genetics, Geography, Demography, Archaeology

bottles and cartons of commercially pasteurized cow's milk were obtained at random from retail outlets throughout the Czech Republic. During the same period, samples of raw milk and of milk that was subsequently subjected to a minimum of 71.7°C for 15 s in a local pasteurization unit were also obtained from two dairy herds, designated herds A and B, with low and high levels, respectively, of subclinical Mycobacterium avium subsp. paratuberculosis infection, and from one herd, herd C, without infection. Infection in individual cows in each herd was tested by fecal culturing. Milk samples were brought to the Veterinary Research Institute in Brno, Czech Republic, processed, inoculated onto Herrold's egg yolk slants, and incubated for 32 weeks. Colonies were characterized by morphology, Ziehl-Neelsen staining, mycobactin J dependency, and IS900 PCR results. M. avium subsp. paratuberculosis was cultured from 4 of 244 units (1.6%) of commercially pasteurized retail milk. M. avium subsp. paratuberculosis was also cultured from 2 of 100 (2%) cartons of locally pasteurized milk derived from infected herds A and B and from 0 of 100 cartons of milk from uninfected herd C. Raw milk from 1 of 10 (10%) fecal culture-positive cows in herd A and from 13 of 66 (19.7%) fecal culture-positive cows in herd B was culture positive for M. avium subsp. paratuberculosis. These findings confirm that M. avium subsp. paratuberculosis is present in raw milk from subclinically infected dairy cows. The culture of M. avium subsp. paratuberculosis in the Czech Republic from retail milk that had been pasteurized locally or commercially to the required national and European Union standards is in agreement with similar research on milk destined for consumers in the United Kingdom and the United States and shows that humans are being exposed to this chronic enteric pathogen by this route.

- by Roberto Sargo
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- Zoology, Medical Microbiology, European Union, Adolescent

Crenate broomrape (Orobanche crenata) is the major constraint for pea and faba bean production in the Mediterranean region. In this study, a systematic sequencing of expressed sequence tags (ESTs) was chosen to obtain a first global picture of the assembly of genes involved in defence response. A cDNA-library was established by suppression subtractive hybridization in the model legume Medicago truncatula infected by O. crenata in order to identify a large number of host plant ESTs. Eighty-one presumably up-regulated genes were identified and classified in functional categories. EST-annotations showed homologies to a number of well-characterized genes. Most of the proteins encoded by these genes, are already known in defence in M. truncatula, such as genes related to the JA pathway or involved in cell wall modifications. A notable number of the ESTs, however, were derived from novel genes not matching entries of the large-scale M. truncatula sequences collections. Expression analyses by quantitative RT-PCR of 20 genes corresponding to different functional categories showed high expression levels, supporting their involvement in the defence response. r

- by Christina Ingvardsen
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- Genetics, Microbiology, Plant Biology, Plant Molecular Biology
- by Maria Forlenza
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- Immunology, Fish Diseases, Macrophages, Phylogeny