Conversion of a molecular classifier obtained by gene expression profiling into a classifier based on real-time PCR: a prognosis predictor for gliomas

<p>Abstract</p> <p>Background</p> <p>The advent of gene expression profiling was expected to dramatically improve cancer diagnosis. However, despite intensive efforts and several successful examples, the development of profile-based diagnostic systems remains a difficult task. In the present work, we established a method to convert molecular classifiers based on adaptor-tagged competitive PCR (ATAC-PCR) (with a data format that is similar to that of microarrays) into classifiers based on real-time PCR.</p> <p>Methods</p> <p>Previously, we constructed a prognosis predictor for glioma using gene expression data obtained by ATAC-PCR, a high-throughput reverse-transcription PCR technique. The analysis of gene expression data obtained by ATAC-PCR is similar to the analysis of data from two-colour microarrays. The prognosis predictor was a linear classifier based on the first principal component (PC1) score, a weighted summation of the expression values of 58 genes. In the present study, we employed the delta-delta Ct method for measurement by real-time PCR. The predictor was converted to a Ct value-based predictor using linear regression.</p> <p>Results</p> <p>We selected <it>UBL5 </it>as the reference gene from the group of genes with expression patterns that were most similar to the median expression level from the previous profiling study. The number of diagnostic genes was reduced to 27 without affecting the performance of the prognosis predictor. PC1 scores calculated from the data obtained by real-time PCR showed a high linear correlation (r = 0.94) with those obtained by ATAC-PCR. The correlation for individual gene expression patterns (r = 0.43 to 0.91) was smaller than for PC1 scores, suggesting that errors of measurement were likely cancelled out during the weighted summation of the expression values. The classification of a test set (n = 36) by the new predictor was more accurate than histopathological diagnosis (log rank p-values, 0.023 and 0.137, respectively) for predicting prognosis.</p> <p>Conclusion</p> <p>We successfully converted a molecular classifier obtained by ATAC-PCR into a Ct value-based predictor. Our conversion procedure should also be applicable to linear classifiers obtained from microarray data. Because errors in measurement are likely to be cancelled out during the calculation, the conversion of individual gene expression is not an appropriate procedure. The predictor for gliomas is still in the preliminary stages of development and needs analytical clinical validation and clinical utility studies.</p

Pyelonephritis in slaughter pigs and sows: Morphological characterization and aspects of pathogenesis and aetiology

<p>Abstract</p> <p>Background</p> <p>Pyelonephritis is a serious disease in pig production that needs to be further studied. The purpose of this study was to describe the morphology, investigate the pathogenesis, and evaluate the aetiological role of <it>Escherichia coli </it>in pyelonephritis in slaughtered pigs by concurrent bacteriological, gross and histopathological examinations.</p> <p>Methods</p> <p>From Danish abattoirs, kidneys and corresponding lymph nodes from 22 slaughtered finishing pigs and 26 slaughtered sows with pyelonephritis were collected and evaluated by bacteriology and pathology. Based on gross lesions, each kidney (lesion) was grouped as acute, chronic, chronic active, or normal and their histological inflammatory stage was determined as normal (0), acute (1), sub-acute (2), chronic active (3), or chronic (4). Immunohistochemical identification of neutrophils, macrophages, T-lymphocytes, B-lymphocytes, plasma cells, <it>E. coli </it>and Tamm-Horsfall protein (THP) in renal sections was performed. The number of <it>E. coli </it>and the proportion of immunohistochemically visualized leukocytes out of the total number of infiltrating leukocytes were scored semi-quantitatively.</p> <p>Results</p> <p>Lesions in finishing pigs and sows were similar. Macroscopically, multiple unevenly distributed foci of inflammation mostly affecting the renal poles were observed. Histologically, tubulointerstitial infiltration with neutrophils and mononuclear cells and tubular destruction was the main findings. The significant highest scores of L1 antigen⁺neutrophils were in inflammatory stage 1 while the significant highest scores of CD79αcy⁺B-lymphocytes, IgG⁺and IgA⁺plasma cells were in stage 3 or 4. Neutrophils were the dominant leukocytes in stage 1 while CD3ε⁺T-lymphocytes dominated in stage 2, 3 and 4. Interstitially THP was seen in 82% and 98% of kidneys with pyelonephritis from finishing pigs and sows, respectively. <it>E. coli </it>was demonstrated in monoculture and/or identified by immunohistochemistry in relation to inflammation in four kidneys from finishing pigs and in 34 kidneys from sows.</p> <p>Conclusions</p> <p><it>E. coli </it>played a significant role in the aetiology of pyelonephritis. Neutrophils were involved in the first line of defence. CD3ε⁺T-lymphocytes were involved in both the acute and chronic inflammatory response while a humoral immune response was most pronounced in later inflammatory stages. The observed renal lesions correspond with an ascending bacterial infection with presence of intra-renal reflux.</p

Quantitative imaging of concentrated suspensions under flow

We review recent advances in imaging the flow of concentrated suspensions, focussing on the use of confocal microscopy to obtain time-resolved information on the single-particle level in these systems. After motivating the need for quantitative (confocal) imaging in suspension rheology, we briefly describe the particles, sample environments, microscopy tools and analysis algorithms needed to perform this kind of experiments. The second part of the review focusses on microscopic aspects of the flow of concentrated model hard-sphere-like suspensions, and the relation to non-linear rheological phenomena such as yielding, shear localization, wall slip and shear-induced ordering. Both Brownian and non-Brownian systems will be described. We show how quantitative imaging can improve our understanding of the connection between microscopic dynamics and bulk flow.Comment: Review on imaging hard-sphere suspensions, incl summary of methodology. Submitted for special volume 'High Solid Dispersions' ed. M. Cloitre, Vol. xx of 'Advances and Polymer Science' (Springer, Berlin, 2009); 22 pages, 16 fig

Regulated Nuclear Trafficking of rpL10A Mediated by NIK1 Represents a Defense Strategy of Plant Cells against Virus

The NSP-interacting kinase (NIK) receptor-mediated defense pathway has been identified recently as a virulence target of the geminivirus nuclear shuttle protein (NSP). However, the NIK1–NSP interaction does not fit into the elicitor–receptor model of resistance, and hence the molecular mechanism that links this antiviral response to receptor activation remains obscure. Here, we identified a ribosomal protein, rpL10A, as a specific partner and substrate of NIK1 that functions as an immediate downstream effector of NIK1-mediated response. Phosphorylation of cytosolic rpL10A by NIK1 redirects the protein to the nucleus where it may act to modulate viral infection. While ectopic expression of normal NIK1 or a hyperactive NIK1 mutant promotes the accumulation of phosphorylated rpL10A within the nuclei, an inactive NIK1 mutant fails to redirect the protein to the nuclei of co-transfected cells. Likewise, a mutant rpL10A defective for NIK1 phosphorylation is not redirected to the nucleus. Furthermore, loss of rpL10A function enhances susceptibility to geminivirus infection, resembling the phenotype of nik1 null alleles. We also provide evidence that geminivirus infection directly interferes with NIK1-mediated nuclear relocalization of rpL10A as a counterdefensive measure. However, the NIK1-mediated defense signaling neither activates RNA silencing nor promotes a hypersensitive response but inhibits plant growth and development. Although the virulence function of the particular geminivirus NSP studied here overcomes this layer of defense in Arabidopsis, the NIK1-mediated signaling response may be involved in restricting the host range of other viruses

The Arabidopsis Resistance-Like Gene SNC1 Is Activated by Mutations in SRFR1 and Contributes to Resistance to the Bacterial Effector AvrRps4

The SUPPRESSOR OF rps4-RLD1 (SRFR1) gene was identified based on enhanced AvrRps4-triggered resistance in the naturally susceptible Arabidopsis accession RLD. No other phenotypic effects were recorded, and the extent of SRFR1 involvement in regulating effector-triggered immunity was unknown. Here we show that mutations in SRFR1 in the accession Columbia-0 (Col-0) lead to severe stunting and constitutive expression of the defense gene PR1. These phenotypes were temperature-dependent. A cross between srfr1-1 (RLD background) and srfr1-4 (Col-0) showed that stunting was caused by a recessive locus in Col-0. Mapping and targeted crosses identified the Col-0-specific resistance gene SNC1 as the locus that causes stunting. SRFR1 was proposed to function as a transcriptional repressor, and SNC1 is indeed overexpressed in srfr1-4. Interestingly, co-regulated genes in the SNC1 cluster are also upregulated in the srfr1-4 snc1-11 double mutant, indicating that the overexpression of SNC1 is not a secondary effect of constitutive defense activation. In addition, a Col-0 RPS4 mutant showed full susceptibility to bacteria expressing avrRps4 at 24°C but not at 22°C, while RLD susceptibility was not temperature-dependent. The rps4-2 snc1-11 double mutant showed increased, but not full, susceptibility at 22°C, indicating that additional cross-talk between resistance pathways may exist. Intriguingly, when transiently expressed in Nicotiana benthamiana, SRFR1, RPS4 and SNC1 are in a common protein complex in a cytoplasmic microsomal compartment. Our results highlight SRFR1 as a convergence point in at least a subset of TIR-NBS-LRR protein-mediated immunity in Arabidopsis. Based on the cross-talk evident from our results, they also suggest that reports of constitutive resistance phenotypes in Col-0 need to consider the possible involvement of SNC1

Genome-Wide Transcript Profiling of Endosperm without Paternal Contribution Identifies Parent-of-Origin–Dependent Regulation of AGAMOUS-LIKE36

Seed development in angiosperms is dependent on the interplay among different transcriptional programs operating in the embryo, the endosperm, and the maternally-derived seed coat. In angiosperms, the embryo and the endosperm are products of double fertilization during which the two pollen sperm cells fuse with the egg cell and the central cell of the female gametophyte. In Arabidopsis, analyses of mutants in the cell-cycle regulator CYCLIN DEPENDENT KINASE A;1 (CKDA;1) have revealed the importance of a paternal genome for the effective development of the endosperm and ultimately the seed. Here we have exploited cdka;1 fertilization as a novel tool for the identification of seed regulators and factors involved in parent-of-origin–specific regulation during seed development. We have generated genome-wide transcription profiles of cdka;1 fertilized seeds and identified approximately 600 genes that are downregulated in the absence of a paternal genome. Among those, AGAMOUS-LIKE (AGL) genes encoding Type-I MADS-box transcription factors were significantly overrepresented. Here, AGL36 was chosen for an in-depth study and shown to be imprinted. We demonstrate that AGL36 parent-of-origin–dependent expression is controlled by the activity of METHYLTRANSFERASE1 (MET1) maintenance DNA methyltransferase and DEMETER (DME) DNA glycosylase. Interestingly, our data also show that the active maternal allele of AGL36 is regulated throughout endosperm development by components of the FIS Polycomb Repressive Complex 2 (PRC2), revealing a new type of dual epigenetic regulation in seeds

A genome-wide genetic map of NB-LRR disease resistance loci in potato

Like all plants, potato has evolved a surveillance system consisting of a large array of genes encoding for immune receptors that confer resistance to pathogens and pests. The majority of these so-called resistance or R proteins belong to the super-family that harbour a nucleotide binding and a leucine-rich-repeat domain (NB-LRR). Here, sequence information of the conserved NB domain was used to investigate the genome-wide genetic distribution of the NB-LRR resistance gene loci in potato. We analysed the sequences of 288 unique BAC clones selected using filter hybridisation screening of a BAC library of the diploid potato clone RH89-039-16 (S. tuberosum ssp. tuberosum) and a physical map of this BAC library. This resulted in the identification of 738 partial and full-length NB-LRR sequences. Based on homology of these sequences with known resistance genes, 280 and 448 sequences were classified as TIR-NB-LRR (TNL) and CC-NB-LRR (CNL) sequences, respectively. Genetic mapping revealed the presence of 15 TNL and 32 CNL loci. Thirty-six are novel, while three TNL loci and eight CNL loci are syntenic with previously identified functional resistance genes. The genetic map was complemented with 68 universal CAPS markers and 82 disease resistance trait loci described in literature, providing an excellent template for genetic studies and applied research in potato