ViLoN-a multi-layer network approach to data integration demonstrated for patient stratification.

With more and more data being collected, modern network representations exploit the complementary nature of different data sources as well as similarities across patients. We here introduce the Variation of information fused Layers of Networks algorithm (ViLoN), a novel network-based approach for the integration of multiple molecular profiles. As a key innovation, it directly incorporates prior functional knowledge (KEGG, GO). In the constructed network of patients, patients are represented by networks of pathways, comprising genes that are linked by common functions and joint regulation in the disease. Patient stratification remains a key challenge both in the clinic and for research on disease mechanisms and treatments. We thus validated ViLoN for patient stratification on multiple data type combinations (gene expression, methylation, copy number), showing substantial improvements and consistently competitive performance for all. Notably, the incorporation of prior functional knowledge was critical for good results in the smaller cohorts (rectum adenocarcinoma: 90, esophageal carcinoma: 180), where alternative methods failed.

A comprehensive rat transcriptome built from large scale RNA-seq-based annotation.

The rat is an important model organism in biomedical research for studying human disease mechanisms and treatments, but its annotated transcriptome is far from complete. We constructed a Rat Transcriptome Re-annotation named RTR using RNA-seq data from 320 samples in 11 different organs generated by the SEQC consortium. Totally, there are 52 807 genes and 114 152 transcripts in RTR. Transcribed regions and exons in RTR account for ∼42% and ∼6.5% of the genome, respectively. Of all 73 074 newly annotated transcripts in RTR, 34 213 were annotated as high confident coding transcripts and 24 728 as high confident long noncoding transcripts. Different tissues rather than different stages have a significant influence on the expression patterns of transcripts. We also found that 11 715 genes and 15 852 transcripts were expressed in all 11 tissues and that 849 house-keeping genes expressed different isoforms among tissues. This comprehensive transcriptome is freely available at http://www.unimd.org/rtr/. Our new rat transcriptome provides essential reference for genetics and gene expression studies in rat disease and toxicity models.

Loss of the ribosomal RNA methyltransferase NSUN5 impairs global protein synthesis and normal growth.

Modifications of ribosomal RNA expand the nucleotide repertoire and thereby contribute to ribosome heterogeneity and translational regulation of gene expression. One particular m5C modification of 25S ribosomal RNA, which is introduced by Rcm1p, was previously shown to modulate stress responses and lifespan in yeast and other small organisms. Here, we report that NSUN5 is the functional orthologue of Rcm1p, introducing m5C3782 into human and m5C3438 into mouse 28S ribosomal RNA. Haploinsufficiency of the NSUN5 gene in fibroblasts from William Beuren syndrome patients causes partial loss of this modification. The N-terminal domain of NSUN5 is required for targeting to nucleoli, while two evolutionary highly conserved cysteines mediate catalysis. Phenotypic consequences of NSUN5 deficiency in mammalian cells include decreased proliferation and size, which can be attributed to a reduction in total protein synthesis by altered ribosomes. Strikingly, Nsun5 knockout in mice causes decreased body weight and lean mass without alterations in food intake, as well as a trend towards reduced protein synthesis in several tissues. Together, our findings emphasize the importance of single RNA modifications for ribosome function and normal cellular and organismal physiology.

Molecular Mechanisms of Fetal Tendon Regeneration Versus Adult Fibrous Repair.

Tendinopathies are painful, disabling conditions that afflict 25% of the adult human population. Filling an unmet need for realistic large-animal models, we here present an ovine model of tendon injury for the comparative study of adult scarring repair and fetal regeneration. Complete regeneration of the fetal tendon within 28 days is demonstrated, while adult tendon defects remained macroscopically and histologically evident five months post-injury. In addition to a comprehensive histological assessment, proteome analyses of secretomes were performed. Confirming histological data, a specific and pronounced inflammation accompanied by activation of neutrophils in adult tendon defects was observed, corroborated by the significant up-regulation of pro-inflammatory factors, neutrophil attracting chemokines, the release of potentially tissue-damaging antimicrobial and extracellular matrix-degrading enzymes, and a response to oxidative stress. In contrast, secreted proteins of injured fetal tendons included proteins initiating the resolution of inflammation or promoting functional extracellular matrix production. These results demonstrate the power and relevance of our novel ovine fetal tendon regeneration model, which thus promises to accelerate research in the field. First insights from the model already support our molecular understanding of successful fetal tendon healing processes and may guide improved therapeutic strategies.

MALDI TOF/TOF-Based Approach for the Identification of d- Amino Acids in Biologically Active Peptides and Proteins.

Several biologically active peptides contain a d- amino acid in a well-defined position, which is position 2 in all peptide epimers isolated to date from vertebrates and also some from invertebrates. The detection of such D- residues by standard analytical techniques is challenging. In tandem mass spectrometric (MS) analysis, although fragment masses are the same for all stereoisomers, peak intensities are known to depend on chirality. Here, we observe that the effect of a d- amino acid in the second N-terminal position on the fragmentation pattern in matrix assisted laser desorption time-of-flight spectrometry (MALDI-TOF/TOF MS) strongly depends on the peptide sequence. Stereosensitive fragmentation (SF) is correlated to a neighborhood effect, but the d- residue also exerts an overall effect influencing distant bonds. In a fingerprint analysis, multiple peaks can thus serve to identify the chirality of a sample in short time and potentially high throughput. Problematic variations between individual spots could be successfully suppressed by cospotting deuterated analogues of the epimers. By identifying the [d-Leu2] isomer of the predicted peptide GH-2 (gene derived bombininH) in skin secretions of the toad Bombina orientalis, we demonstrated the analytical power of SF-MALDI-TOF/TOF measurements. In conclusion, SF-MALDI-TOF/TOF MS combines high sensitivity, versatility, and the ability to complement other methods.

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments.

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.

Bacterial protein signals are associated with Crohn's disease.

OBJECTIVE: No Crohn's disease (CD) molecular maker has advanced to clinical use, and independent lines of evidence support a central role of the gut microbial community in CD. Here we explore the feasibility of extracting bacterial protein signals relevant to CD, by interrogating myriads of intestinal bacterial proteomes from a small number of patients and healthy controls. DESIGN: We first developed and validated a workflow-including extraction of microbial communities, two-dimensional difference gel electrophoresis (2D-DIGE), and LC-MS/MS-to discover protein signals from CD-associated gut microbial communities. Then we used selected reaction monitoring (SRM) to confirm a set of candidates. In parallel, we used 16S rRNA gene sequencing for an integrated analysis of gut ecosystem structure and functions. RESULTS: Our 2D-DIGE-based discovery approach revealed an imbalance of intestinal bacterial functions in CD. Many proteins, largely derived from Bacteroides species, were over-represented, while under-represented proteins were mostly from Firmicutes and some Prevotella members. Most overabundant proteins could be confirmed using SRM. They correspond to functions allowing opportunistic pathogens to colonise the mucus layers, breach the host barriers and invade the mucosae, which could still be aggravated by decreased host-derived pancreatic zymogen granule membrane protein GP2 in CD patients. Moreover, although the abundance of most protein groups reflected that of related bacterial populations, we found a specific independent regulation of bacteria-derived cell envelope proteins. CONCLUSIONS: This study provides the first evidence that quantifiable bacterial protein signals are associated with CD, which can have a profound impact on future molecular diagnosis.

Atypical RNA polymerase subunits required for RNA-directed DNA methylation.

RNA-directed DNA methylation, one of several RNA interference-mediated pathways in the nucleus, has been documented in plants and in human cells. Despite progress in identifying the DNA methyltransferases, histone-modifying enzymes and RNA interference proteins needed for RNA-directed DNA methylation, the mechanism remains incompletely understood. We screened for mutants defective in RNA-directed DNA methylation and silencing of a transgene promoter in Arabidopsis thaliana and identified three drd complementation groups. DRD1 is a SNF2-like protein required for RNA-directed de novo methylation. We report here that DRD2 and DRD3 correspond to the second-largest subunit and largest subunit, respectively, of a fourth class of DNA-dependent RNA polymerase (polymerase IV) that is unique to plants. DRD3 is a functionally diversified homolog of NRPD1a or SDE4, identified in a separate screen for mutants defective in post-transcriptional gene silencing. The identical DNA methylation patterns observed in all three drd mutants suggest that DRD proteins cooperate to create a substrate for RNA-directed de novo methylation.

Overexpression of the transcription factor RAP2.6 leads to enhanced callose deposition in syncytia and enhanced resistance against the beet cyst nematode Heterodera schachtii in Arabidopsis roots.

BACKGROUND: Cyst nematodes invade the roots of their host plants as second stage juveniles and induce a syncytium which is their source of nutrients throughout their life. A transcriptome analysis of syncytia induced by the beet cyst nematode Heterodera schachtii in Arabidopsis roots has shown that gene expression in the syncytium is different from that of the root with thousands of genes upregulated or downregulated. Among the downregulated genes are many which code for defense-related proteins. One gene which is strongly downregulated codes for the ethylene response transcription factor RAP2.6. The genome of Arabidopsis contains 122 ERF transcription factor genes which are involved in a variety of developmental and stress responses. RESULTS: Expression of RAP2.6 was studied with RT-PCR and a promoter::GUS line. During normal growth conditions the gene was expressed especially in roots and stems. It was inducible by Pseudomonas syringae but downregulated in syncytia from a very early time point on. Overexpression of the gene enhanced the resistance against H. schachtii which was seen by a lower number of nematodes developing on these plants as well as smaller syncytia and smaller female nematodes. A T-DNA mutant had a reduced RAP2.6 transcript level but this did not further increase the susceptibility against H. schachtii. Neither overexpression lines nor mutants had an effect on P. syringae. Overexpression of RAP2.6 led to an elevated expression of JA-responsive genes during early time points after infection by H. schachtii. Syncytia developing on overexpression lines showed enhanced deposition of callose. CONCLUSIONS: Our results showed that H. schachtii infection is accompanied by a downregulation of RAP2.6. It seems likely that the nematodes use effectors to actively downregulate the expression of this and other defense-related genes to avoid resistance responses of the host plant. Enhanced resistance of RAP2.6 overexpression lines seemed to be due to enhanced callose deposition at syncytia which might interfere with nutrient import into syncytia.

Mycoparasitism related targets of Tmk1 indicate stimulating regulatory functions of this MAP kinase in Trichoderma atroviride.

Mycoparasitism is a key feature of Trichoderma (Hypocreales, Ascomycota) biocontrol agents. Recent studies of intracellular signal transduction pathways of the potent mycoparasite Trichoderma atroviride revealed the involvement of Tmk1, a mitogen-activated protein kinase (MAPK), in triggering the mycoparasitic response. We previously showed that mutants missing Tmk1 exhibit reduced mycoparasitic activity against several plant pathogenic fungi. In this study, we identified the most robustly regulated targets that were governed by Tmk1 during mycoparasitism using transcriptome and proteome profiling. Tmk1 mainly exerts a stimulating function for T. atroviride during its mycoparasitic interaction with the fungal plant pathogen Rhizoctonia solani, as reflected by 89% of strongly differently responding genes in the ∆tmk1 mutant compared to the wild type. Specifically, 54% of these genes showed strong downregulation in the response with a deletion of the tmk1 gene, whereas in the wild type the same genes were strongly upregulated during the interaction with the fungal host. These included the gene encoding the mycoparasitism-related proteinase Prb1; genes involved in signal transduction pathways such as a candidate coding for a conserved 14-3-3 protein, and a gene coding for Tmk2, the T. atroviride cell-wall integrity MAP kinase; genes encoding a specific siderophore synthetase, and multiple FAD-dependent oxidoreductases and aminotransferases. Due to the phosphorylating activity of Tmk1, different (phospho-)proteomics approaches were applied and identified proteins associated with cellular metabolism, energy production, protein synthesis and fate, and cell organization. Members of FAD- and NAD/NADP-binding-domain proteins, vesicular trafficking of molecules between cellular organelles, fungal translational, as well as protein folding apparatus were among others found to be phosphorylated by Tmk1 during mycoparasitism. Outstanding downregulation in the response of the ∆tmk1 mutant to the fungal host compared to the wild type at both the transcriptome and the proteome levels was observed for nitrilase, indicating that its defense and detoxification functions might be greatly dependent on Tmk1 during T. atroviride mycoparasitism. An intersection network analysis between the identified transcripts and proteins revealed a strong involvement of Tmk1 in molecular functions with GTPase and oxidoreductase activity. These data suggest that during T. atroviride mycoparasitism this MAPK mainly governs processes regulating cell responses to extracellular signals and those involved in reactive oxygen stress.

Silicone Breast Implant Surface Texture Impacts Gene Expression in Periprosthetic Fibrous Capsules.

BACKGROUND: Silicone breast implants with smooth outer shells are associated with higher rates of capsular contracture, whereas textured implants have been linked to the development of breast implant-associated anaplastic large cell lymphoma. By assessing the gene expression profile of fibrous capsules formed in response to smooth and textured implants, insight into the development of breast implant-associated abnormalities can be gained. METHODS: Miniature smooth or textured silicone implants were surgically inserted into female rats ( n = 10) and harvested for the surrounding capsules at postoperative week 6. RNA sequencing and quantitative polymerase chain reaction were performed to identify genes differentially expressed between smooth and textured capsules. For clinical correlation, the expression of candidate genes was assayed in implant capsules harvested from human patients with and without capsular contracture. RESULTS: Of 18,555 differentially expressed transcripts identified, three candidate genes were selected: matrix metalloproteinase-3 ( MMP3 ), troponin-T3 ( TNNT3 ), and neuregulin-1 ( NRG1 ). In textured capsules, relative gene expression and immunostaining of MMP3 and TNNT3 was up-regulated, whereas NRG1 was down-regulated compared to smooth capsules [mean relative fold change, 8.79 ( P = 0.0059), 4.81 ( P = 0.0056), and 0.40 ( P < 0.0001), respectively]. Immunostaining of human specimens with capsular contracture revealed similar gene expression patterns to those of animal-derived smooth capsules. CONCLUSIONS: An expression pattern of low MMP3 /low TNNT3 /high NRG1 is specifically associated with smooth implant capsules and human implant capsules with capsular contracture. The authors' clinically relevant breast implant rat model provides a strong foundation to further explore the molecular genetics of implant texture and its effect on breast implant-associated abnormalities. CLINICAL RELEVANCE STATEMENT: The authors have demonstrated that there are distinct gene expression profiles in response to smooth versus textured breast implants. Since surface texture may be linked to implant-related pathology, further molecular analysis of periprosthetic capsules may yield strategies to mitigate implant-related complications.

GMI1, a structural-maintenance-of-chromosomes-hinge domain-containing protein, is involved in somatic homologous recombination in Arabidopsis.

DNA double-strand breaks (DSBs) pose one of the most severe threats to genome integrity, potentially leading to cell death. After detection of a DSB, the DNA damage and repair response is initiated and the DSB is repaired by non-homologous end joining and/or homologous recombination. Many components of these processes are still unknown in Arabidopsis thaliana. In this work, we characterized γ-irradiation and mitomycin C induced 1 (GMI1), a member of the SMC-hinge domain-containing protein family. RT-PCR analysis and promoter-GUS fusion studies showed that γ-irradiation, the radio-mimetic drug bleocin, and the DNA cross-linking agent mitomycin C strongly enhance GMI1 expression particularly in meristematic tissues. The induction of GMI1 by γ-irradiation depends on the signalling kinase Ataxia telangiectasia-mutated (ATM) but not on ATM and Rad3-related (ATR). Epistasis analysis of single and double mutants demonstrated that ATM acts upstream of GMI1 while the atr gmi1-2 double mutant was more sensitive than the respective single mutants. Comet assay revealed a reduced rate of DNA double-strand break repair in gmi1 mutants during the early recovery phase after exposure to bleocin. Moreover, the rate of homologous recombination of a reporter construct was strongly reduced in gmi1 mutant plants upon exposure to bleocin or mitomycin C. GMI1 is the first member of its protein family known to be involved in DNA repair.

Characterization and improvement of RNA-Seq precision in quantitative transcript expression profiling.

MOTIVATION: Measurement precision determines the power of any analysis to reliably identify significant signals, such as in screens for differential expression, independent of whether the experimental design incorporates replicates or not. With the compilation of large-scale RNA-Seq datasets with technical replicate samples, however, we can now, for the first time, perform a systematic analysis of the precision of expression level estimates from massively parallel sequencing technology. This then allows considerations for its improvement by computational or experimental means. RESULTS: We report on a comprehensive study of target identification and measurement precision, including their dependence on transcript expression levels, read depth and other parameters. In particular, an impressive recall of 84% of the estimated true transcript population could be achieved with 331 million 50 bp reads, with diminishing returns from longer read lengths and even less gains from increased sequencing depths. Most of the measurement power (75%) is spent on only 7% of the known transcriptome, however, making less strongly expressed transcripts harder to measure. Consequently, <30% of all transcripts could be quantified reliably with a relative error<20%. Based on established tools, we then introduce a new approach for mapping and analysing sequencing reads that yields substantially improved performance in gene expression profiling, increasing the number of transcripts that can reliably be quantified to over 40%. Extrapolations to higher sequencing depths highlight the need for efficient complementary steps. In discussion we outline possible experimental and computational strategies for further improvements in quantification precision. CONTACT: rnaseq10@boku.ac.at

RNA-directed DNA methylation and plant development require an IWR1-type transcription factor.

RNA-directed DNA methylation (RdDM) in plants requires two RNA polymerase (Pol) II-related RNA polymerases, namely Pol IV and Pol V. A genetic screen designed to reveal factors that are important for RdDM in a developmental context in Arabidopsis identified DEFECTIVE IN MERISTEM SILENCING 4 (DMS4). Unlike other mutants defective in RdDM, dms4 mutants have a pleiotropic developmental phenotype. The DMS4 protein is similar to yeast IWR1 (interacts with RNA polymerase II), a conserved putative transcription factor that interacts with Pol II subunits. The DMS4 complementary DNA partly complements the K1 killer toxin hypersensitivity of a yeast iwr1 mutant, suggesting some functional conservation. In the transgenic system studied, mutations in DMS4 directly or indirectly affect Pol IV-dependent secondary short interfering RNAs, Pol V-mediated RdDM, Pol V-dependent synthesis of intergenic non-coding RNA and expression of many Pol II-driven genes. These data suggest that DMS4 might be a regulatory factor for several RNA polymerases, thus explaining its diverse roles in the plant.

Expression Analyses in the Rachis Hint towards Major Cell Wall Modifications in Grape Clusters Showing Berry Shrivel Symptoms.

Berry shrivel (BS) is one of the prominent and still unresolved ripening physiological disorders in grapevine. The causes of BS are unclear, and previous studies focused on the berry metabolism or histological studies, including cell viability staining in the rachis and berries of BS clusters. Herein, we studied the transcriptional modulation induced by BS in the rachis of pre-symptomatic and symptomatic clusters with a custom-made microarray qPCR in relation to a previous RNASeq study of BS berries. Gene set analysis of transcript expression in symptomatic rachis tissue determined suppression of cell wall biosynthesis, which could also be confirmed already in pre-symptomatic BS rachis by CESA8 qPCR analyses, while in BS berries, a high number of SWITCH genes were suppressed at veraison. Additionally, genes associated with the cell wall were differently affected by BS in berries. A high percentage of hydrolytic enzymes were induced in BS grapes in rachis and berries, while other groups such as, e.g., xyloglucan endotransglucosylase/hydrolase, were suppressed in BS rachis. In conclusion, we propose that modulated cell wall biosynthesis and cell wall assembly in pre-symptomatic BS rachis have potential consequences for cell wall strength and lead to a forced degradation of cell walls in symptomatic grape clusters. The similarity to sugar starvation transcriptional profiles provides a link to BS berries, which are low in sugar accumulation. However, further studies remain necessary to investigate the temporal and spatial coordination in both tissues.

An analysis of single amino acid repeats as use case for application specific background models.

BACKGROUND: Sequence analysis aims to identify biologically relevant signals against a backdrop of functionally meaningless variation. Increasingly, it is recognized that the quality of the background model directly affects the performance of analyses. State-of-the-art approaches rely on classical sequence models that are adapted to the studied dataset. Although performing well in the analysis of globular protein domains, these models break down in regions of stronger compositional bias or low complexity. While these regions are typically filtered, there is increasing anecdotal evidence of functional roles. This motivates an exploration of more complex sequence models and application-specific approaches for the investigation of biased regions. RESULTS: Traditional Markov-chains and application-specific regression models are compared using the example of predicting runs of single amino acids, a particularly simple class of biased regions. Cross-fold validation experiments reveal that the alternative regression models capture the multi-variate trends well, despite their low dimensionality and in contrast even to higher-order Markov-predictors. We show how the significance of unusual observations can be computed for such empirical models. The power of a dedicated model in the detection of biologically interesting signals is then demonstrated in an analysis identifying the unexpected enrichment of contiguous leucine-repeats in signal-peptides. Considering different reference sets, we show how the question examined actually defines what constitutes the 'background'. Results can thus be highly sensitive to the choice of appropriate model training sets. Conversely, the choice of reference data determines the questions that can be investigated in an analysis. CONCLUSIONS: Using a specific case of studying biased regions as an example, we have demonstrated that the construction of application-specific background models is both necessary and feasible in a challenging sequence analysis situation.

The impact of quantitative optimization of hybridization conditions on gene expression analysis.

BACKGROUND: With the growing availability of entire genome sequences, an increasing number of scientists can exploit oligonucleotide microarrays for genome-scale expression studies. While probe-design is a major research area, relatively little work has been reported on the optimization of microarray protocols. RESULTS: As shown in this study, suboptimal conditions can have considerable impact on biologically relevant observations. For example, deviation from the optimal temperature by one degree Celsius lead to a loss of up to 44% of differentially expressed genes identified. While genes from thousands of Gene Ontology categories were affected, transcription factors and other low-copy-number regulators were disproportionately lost. Calibrated protocols are thus required in order to take full advantage of the large dynamic range of microarrays.For an objective optimization of protocols we introduce an approach that maximizes the amount of information obtained per experiment. A comparison of two typical samples is sufficient for this calibration. We can ensure, however, that optimization results are independent of the samples and the specific measures used for calibration. Both simulations and spike-in experiments confirmed an unbiased determination of generally optimal experimental conditions. CONCLUSIONS: Well calibrated hybridization conditions are thus easily achieved and necessary for the efficient detection of differential expression. They are essential for the sensitive pro filing of low-copy-number molecules. This is particularly critical for studies of transcription factor expression, or the inference and study of regulatory networks.

A systematic comparison of spectral-domain optical coherence tomography and fundus autofluorescence in patients with geographic atrophy.

PURPOSE: To evaluate spectral-domain optical coherence tomography (SD-OCT) in providing reliable and reproducible parameters for grading geographic atrophy (GA) compared with fundus autofluorescence (FAF) images acquired by confocal scanning laser ophthalmoscopy (cSLO). DESIGN: Prospective observational study. PARTICIPANTS: A total of 81 eyes of 42 patients with GA. METHODS: Patients with atrophic age-related macular degeneration (AMD) were enrolled on the basis of total GA lesion size ranging from 0.5 to 7 disc areas and best-corrected visual acuity of at least 20/200. A novel combined cSLO-SD-OCT system (Spectralis HRA-OCT, Heidelberg Engineering, Heidelberg, Germany) was used to grade foveal involvement and to manually measure disease extent at the level of the outer neurosensory layers and retinal pigment epithelium (RPE) at the site of GA lesions. Two readers of the Vienna Reading Center graded all obtained volume stacks (20×20 degrees), and the results were correlated to FAF. MAIN OUTCOME MEASURES: Choroidal signal enhancements and alterations of the RPE, external limiting membrane (ELM), and outer plexiform layer by SD-OCT. These parameters were compared with the lesion measured with severely decreased FAF. RESULTS: Foveal involvement or sparing was definitely identified in 75 of 81 eyes based on SD-OCT by both graders (inter-grader agreement: κ=0.6, P < 0.01). In FAF, inter-grader agreement regarding foveal involvement was lower (48/81 eyes, inter-grader agreement: κ=0.3, P < 0.01). Severely decreased FAF was measured over a mean area of 8.97 mm(2) for grader 1 (G1) and 9.54 mm(2) for grader 2 (G2), consistent with the mean SD-OCT quantification of the sub-RPE choroidal signal enhancement (8.9 mm(2) [G1] -9.4 mm(2) [G2]) and ELM loss with 8.7 mm(2) (G1) -10.2 mm(2) (G2). In contrast, complete morphologic absence of the RPE layer by SD-OCT was significantly smaller than the GA size in FAF (R(2)=0.400). Inter-reader agreement was highest regarding complete choroidal signal enhancement (0.98) and ELM loss (0.98). CONCLUSIONS: Absence of FAF in GA lesions is consistent with morphologic RPE loss or advanced RPE disruption and is associated with alterations of the outer retinal layers as identified by SD-OCT. Lesion size is precisely determinable by SD-OCT, and foveal involvement is more accurate by SD-OCT than by FAF.

Model-based probe set optimization for high-performance microarrays.

A major challenge in microarray design is the selection of highly specific oligonucleotide probes for all targeted genes of interest, while maintaining thermodynamic uniformity at the hybridization temperature. We introduce a novel microarray design framework (Thermodynamic Model-based Oligo Design Optimizer, TherMODO) that for the first time incorporates a number of advanced modelling features: (i) A model of position-dependent labelling effects that is quantitatively derived from experiment. (ii) Multi-state thermodynamic hybridization models of probe binding behaviour, including potential cross-hybridization reactions. (iii) A fast calibrated sequence-similarity-based heuristic for cross-hybridization prediction supporting large-scale designs. (iv) A novel compound score formulation for the integrated assessment of multiple probe design objectives. In contrast to a greedy search for probes meeting parameter thresholds, this approach permits an optimization at the probe set level and facilitates the selection of highly specific probe candidates while maintaining probe set uniformity. (v) Lastly, a flexible target grouping structure allows easy adaptation of the pipeline to a variety of microarray application scenarios. The algorithm and features are discussed and demonstrated on actual design runs. Source code is available on request.

Effects of aneuploidy on genome structure, expression, and interphase organization in Arabidopsis thaliana.

Aneuploidy refers to losses and/or gains of individual chromosomes from the normal chromosome set. The resulting gene dosage imbalance has a noticeable affect on the phenotype, as illustrated by aneuploid syndromes, including Down syndrome in humans, and by human solid tumor cells, which are highly aneuploid. Although the phenotypic manifestations of aneuploidy are usually apparent, information about the underlying alterations in structure, expression, and interphase organization of unbalanced chromosome sets is still sparse. Plants generally tolerate aneuploidy better than animals, and, through colchicine treatment and breeding strategies, it is possible to obtain inbred sibling plants with different numbers of chromosomes. This possibility, combined with the genetic and genomics tools available for Arabidopsis thaliana, provides a powerful means to assess systematically the molecular and cytological consequences of aberrant numbers of specific chromosomes. Here, we report on the generation of Arabidopsis plants in which chromosome 5 is present in triplicate. We compare the global transcript profiles of normal diploids and chromosome 5 trisomics, and assess genome integrity using array comparative genome hybridization. We use live cell imaging to determine the interphase 3D arrangement of transgene-encoded fluorescent tags on chromosome 5 in trisomic and triploid plants. The results indicate that trisomy 5 disrupts gene expression throughout the genome and supports the production and/or retention of truncated copies of chromosome 5. Although trisomy 5 does not grossly distort the interphase arrangement of fluorescent-tagged sites on chromosome 5, it may somewhat enhance associations between transgene alleles. Our analysis reveals the complex genomic changes that can occur in aneuploids and underscores the importance of using multiple experimental approaches to investigate how chromosome numerical changes condition abnormal phenotypes and progressive genome instability.