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Structural comparison reveals remote homology that often fails to be detected by sequence comparison. The DALI web server (http://ekhidna2.biocenter.helsinki.fi/dali) is a platform for structural analysis that provides database searches and interactive visualization, including structural alignments annotated with secondary structure, protein families and sequence logos, and 3D structure superimposition supported by color‐coded sequence and structure conservation. Here, we are using DALI to mine the AlphaFold Database version 1, which increased the structural coverage of protein families by 20%. We found 100 remote homologous relationships hitherto unreported in the current reference database for protein domains, Pfam 35.0. In particular, we linked 35 domains of unknown function (DUFs) to the previously characterized families, generating a functional hypothesis that can be explored downstream in structural biology studies. Other findings include gene fusions, tandem duplications, and adjustments to domain boundaries. The evidence for homology can be browsed interactively through live examples on DALI's website. [ABSTRACT FROM AUTHOR]

The facility of next‐generation sequencing has led to an explosion of gene catalogs for novel genomes, transcriptomes and metagenomes, which are functionally uncharacterized. Computational inference has emerged as a necessary substitute for first‐hand experimental evidence. PANNZER (Protein ANNotation with Z‐scoRE) is a high‐throughput functional annotation web server that stands out among similar publically accessible web servers in supporting submission of up to 100,000 protein sequences at once and providing both Gene Ontology (GO) annotations and free text description predictions. Here, we demonstrate the use of PANNZER and discuss future plans and challenges. We present two case studies to illustrate problems related to data quality and method evaluation. Some commonly used evaluation metrics and evaluation datasets promote methods that favor unspecific and broad functional classes over more informative and specific classes. We argue that this can bias the development of automated function prediction methods. The PANNZER web server and source code are available at http://ekhidna2.biocenter.helsinki.fi/sanspanz/. [ABSTRACT FROM AUTHOR]

Automated protein annotation using the Gene Ontology (GO) plays an important role in the biosciences. Evaluation has always been considered central to developing novel annotation methods, but little attention has been paid to the evaluation metrics themselves. Evaluation metrics define how well an annotation method performs and allows for them to be ranked against one another. Unfortunately, most of these metrics were adopted from the machine learning literature without establishing whether they were appropriate for GO annotations. We propose a novel approach for comparing GO evaluation metrics called Artificial Dilution Series (ADS). Our approach uses existing annotation data to generate a series of annotation sets with different levels of correctness (referred to as their signal level). We calculate the evaluation metric being tested for each annotation set in the series, allowing us to identify whether it can separate different signal levels. Finally, we contrast these results with several false positive annotation sets, which are designed to expose systematic weaknesses in GO assessment. We compared 37 evaluation metrics for GO annotation using ADS and identified drastic differences between metrics. We show that some metrics struggle to differentiate between different signal levels, while others give erroneously high scores to the false positive data sets. Based on our findings, we provide guidelines on which evaluation metrics perform well with the Gene Ontology and propose improvements to several well-known evaluation metrics. In general, we argue that evaluation metrics should be tested for their performance and we provide software for this purpose (https://bitbucket.org/plyusnin/ads/). ADS is applicable to other areas of science where the evaluation of prediction results is non-trivial. In the biosciences, predictive methods are becoming increasingly necessary as novel sequences are generated at an ever-increasing rate. The volume of sequence data necessitates Automated Function Prediction (AFP) as manual curation is often impossible. Unfortunately, selecting the best AFP method is complicated by researchers using different evaluation metrics. Furthermore, many commonly-used metrics can give misleading results. We argue that the use of poor metrics in AFP evaluation is a result of the lack of methods to benchmark the metrics themselves. We propose an approach called Artificial Dilution Series (ADS). ADS uses existing data sets to generate multiple artificial AFP results, where each result has a controlled error rate. We use ADS to understand whether different metrics can distinguish between results with known quantities of error. Our results highlight dramatic differences in performance between evaluation metrics. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) genome is unstable and different types of instabilities, such as chromosomal instability (CIN) and microsatellite instability (MSI) are thought to reflect distinct cancer initiating mechanisms. Although 85% of sporadic CRC reveal CIN, 15% reveal mismatch repair (MMR) malfunction and MSI, the hallmarks of Lynch syndrome with inherited heterozygous germline mutations in MMR genes. Our study was designed to comprehensively follow genome-wide expression changes and their implications during colon tumorigenesis. We conducted a long-term feeding experiment in the mouse to address expression changes arising in histologically normal colonic mucosa as putative cancer preceding events, and the effect of inherited predisposition (Mlh1+/-) and Western-style diet (WD) on those. During the 21-month experiment, carcinomas developed mainly in WD-fed mice and were evenly distributed between genotypes. Unexpectedly, the heterozygote (B6.129-Mlh1tm1Rak) mice did not show MSI in their CRCs. Instead, both wildtype and heterozygote CRC mice showed a distinct mRNA expression profile and shortage of several chromosomal segregation gene-specific transcripts (Mlh1, Bub1, Mis18a, Tpx2, Rad9a, Pms2, Cenpe, Ncapd3, Odf2 and Dclre1b) in their colon mucosa, as well as an increased mitotic activity and abundant numbers of unbalanced/atypical mitoses in tumours. Our genome-wide expression profiling experiment demonstrates that cancer preceding changes are already seen in histologically normal colon mucosa and that decreased expressions of Mlh1 and other chromosomal segregation genes may form a field-defect in mucosa, which trigger MMR-proficient, chromosomally unstable CRC. [ABSTRACT FROM AUTHOR]

Background: Competitive gene set analysis is a standard exploratory tool for gene expression data. Permutation-based competitive gene set analysis methods are preferable to parametric ones because the latter make strong statistical assumptions which are not always met. For permutation-based methods, we permute samples, as opposed to genes, as doing so preserves the inter-gene correlation structure. Unfortunately, up until now, sample permutation-based methods have required a minimum of six replicates per sample group. Results: We propose a new permutation-based competitive gene set analysis method for multi-group gene expression data with as few as three replicates per group. The method is based on advanced sample permutation technique that utilizes all groups within a data set for pairwise comparisons. We present a comprehensive evaluation of different permutation techniques, using multiple data sets and contrast the performance of our method, mGSZm, with other state of the art methods. We show that mGSZm is robust, and that, despite only using less than six replicates, we are able to consistently identify a high proportion of the top ranked gene sets from the analysis of a substantially larger data set. Further, we highlight other methods where performance is highly variable and appears dependent on the underlying data set being analyzed. Conclusions: Our results demonstrate that robust gene set analysis of multi-group gene expression data is permissible with as few as three replicates. In doing so, we have extended the applicability of such approaches to resource constrained experiments where additional data generation is prohibitively difficult or expensive. An R package implementing the proposed method and supplementary materials are available from the website http://ekhidna.biocenter.helsinki.fi/downloads/pashupati/mGSZm.html. [ABSTRACT FROM AUTHOR]

Motivation: The last decade has seen a remarkable growth in protein databases. This growth comes at a price: a growing number of submitted protein sequences lack functional annotation. Approximately 32% of sequences submitted to the most comprehensive protein database UniProtKB are labelled as 'Unknown protein' or alike. Also the functionally annotated parts are reported to contain 30-40% of errors. Here, we introduce a high-throughput tool for more reliable functional annotation called Protein ANNotation with Z-score (PANNZER). PANNZER predicts Gene Ontology (GO) classes and free text descriptions about protein functionality. PANNZER uses weighted k-nearest neighbour methods with statistical testing to maximize the reliability of a functional annotation. Results: Our results in free text description line prediction show that we outperformed all competing methods with a clear margin. In GO prediction we show clear improvement to our older method that performed well in CAFA 2011 challenge. Availability and implementation: The PANNZER program was developed using the Python programming language (Version 2.6). The stand-alone installation of the PANNZER requires MySQL database for data storage and the BLAST (BLASTALL v.2.2.21) tools for the sequence similarity search. The tutorial, evaluation test sets and results are available on the PANNZER web site. [ABSTRACT FROM AUTHOR]

Motivation: Gene set analysis is the analysis of a set of genes that collectively contribute to a biological process. Most popular gene set analysis methods are based on empirical P-value that requires large number of permutations. Despite numerous gene set analysis methods developed in the past decade, the most popular methods still suffer from serious limitations.Results: We present a gene set analysis method (mGSZ) based on Gene Set Z-scoring function (GSZ) and asymptotic P-values. Asymptotic P-value calculation requires fewer permutations, and thus speeds up the gene set analysis process. We compare the GSZ-scoring function with seven popular gene set scoring functions and show that GSZ stands out as the best scoring function. In addition, we show improved performance of the GSA method when the max-mean statistics is replaced by the GSZ scoring function. We demonstrate the importance of both gene and sample permutations by showing the consequences in the absence of one or the other. A comparison of asymptotic and empirical methods of P-value estimation demonstrates a clear advantage of asymptotic P-value over empirical P-value. We show that mGSZ outperforms the state-of-the-art methods based on two different evaluations. We compared mGSZ results with permutation and rotation tests and show that rotation does not improve our asymptotic P-values. We also propose well-known asymptotic distribution models for three of the compared methods.Availability and implementation: mGSZ is available as R package from cran.r-project.org.Contact: pashupati.mishra@helsinki.fiSupplementary information: Available at http://ekhidna.biocenter.helsinki.fi/downloads/pashupati/mGSZ.html [ABSTRACT FROM AUTHOR]

The values for the column rec in rows ic SimGIC2, ic2 SimGIC2, AJacc E, and ic2 Smin1 are incorrect. Here we show RC (Rank Correlation) and FP (False Positive) results for the best performing methods. [Extracted from the article]

WRKY transcription factors ( TFs) have been mainly associated with plant defense, but recent studies have suggested additional roles in the regulation of other physiological processes. Here, we explored the possible contribution of two related group III WRKY TFs, WRKY70 and WRKY54, to osmotic stress tolerance. These TFs are positive regulators of plant defense, and co-operate as negative regulators of salicylic acid ( SA) biosynthesis and senescence., We employed single and double mutants of wrky54 and wrky70, as well as a WRKY70 overexpressor line, to explore the role of these TFs in osmotic stress (polyethylene glycol) responses. Their effect on gene expression was characterized by microarrays and verified by quantitative PCR. Stomatal phenotypes were assessed by water retention and stomatal conductance measurements., The wrky54wrky70 double mutants exhibited clearly enhanced tolerance to osmotic stress. However, gene expression analysis showed reduced induction of osmotic stress-responsive genes in addition to reduced accumulation of the osmoprotectant proline. By contrast, the enhanced tolerance was correlated with improved water retention and enhanced stomatal closure., These findings demonstrate that WRKY70 and WRKY54 co-operate as negative regulators of stomatal closure and, consequently, osmotic stress tolerance in Arabidopsis, suggesting that they have an important role, not only in plant defense, but also in abiotic stress signaling. [ABSTRACT FROM AUTHOR]

Soft rot disease is economically one of the most devastating bacterial diseases affecting plants worldwide. In this study, we present novel insights into the phylogeny and virulence of the soft rot model Pectobacterium sp. SCC3193, which was isolated from a diseased potato stem in Finland in the early 1980s. Genomic approaches, including proteome and genome comparisons of all sequenced soft rot bacteria, revealed that SCC3193, previously included in the species Pectobacterium carotovorum, can now be more accurately classified as Pectobacterium wasabiae. Together with the recently revised phylogeny of a few P. carotovorum strains and an increasing number of studies on P. wasabiae, our work indicates that P. wasabiae has been unnoticed but present in potato fields worldwide. A combination of genomic approaches and in planta experiments identified features that separate SCC3193 and other P. wasabiae strains from the rest of soft rot bacteria, such as the absence of a type III secretion system that contributes to virulence of other soft rot species. Experimentally established virulence determinants include the putative transcriptional regulator SirB, two partially redundant type VI secretion systems and two horizontally acquired clusters (Vic1 and Vic2), which contain predicted virulence genes. Genome comparison also revealed other interesting traits that may be related to life in planta or other specific environmental conditions. These traits include a predicted benzoic acid/salicylic acid carboxyl methyltransferase of eukaryotic origin. The novelties found in this work indicate that soft rot bacteria have a reservoir of unknown traits that may be utilized in the poorly understood latent stage in planta. The genomic approaches and the comparison of the model strain SCC3193 to other sequenced Pectobacterium strains, including the type strain of P. wasabiae, provides a solid basis for further investigation of the virulence, distribution and phylogeny of soft rot bacteria and, potentially, other bacteria as well. [ABSTRACT FROM AUTHOR]

Background: High throughput methods of the genome era produce vast amounts of data in the form of gene lists. These lists are large and difficult to interpret without advanced computational or bioinformatic tools. Most existing methods analyse a gene list as a single entity although it is comprised of multiple gene groups associated with separate biological functions. Therefore it is imperative to define and visualize gene groups with unique functionality within gene lists. Results: In order to analyse the functional heterogeneity within a gene list, we have developed a method that clusters genes to groups with homogenous functionalities. The method uses Nonnegative Matrix Factorization (NMF) to create several clustering results with varying numbers of clusters. The obtained clustering results are combined into a simple graphical presentation showing the functional groups over-represented in the analyzed gene list. We demonstrate its performance on two data sets and show results that improve upon existing methods. The comparison also shows that our method creates a more simplified view that aids in discovery of biological themes within the list and discards less informative classes from the results. Conclusion: The presented method and associated software are useful for the identification and interpretation of biological functions associated with gene lists and are especially useful for the analysis of large lists. [ABSTRACT FROM AUTHOR]

Brain-derived neurotrophic factor (BDNF) regulates neuronal survival, differentiation and plasticity. It has been shown to promote epileptogenesis and transgenic mice with decreased and increased BDNF signaling show opposite alterations in epileptogenesis. However, the mechanisms of BDNF action are largely unknown. We studied the gene expression changes 12 days after kainic acid-induced status epilepticus in transgenic mice overexpressing either the functional BDNF receptor trkB or a dominant-negative truncated trkB. Epileptogenesis produced marked changes in expression of 27 of 1090 genes. Cluster analysis revealed BDNF signalling-mediated regulation of functional gene classes involved in cellular transport, DNA repair and cell death, including kinesin motor kinesin family member 3A involved in cellular transport. Furthermore, the expression of cytoskeletal and extracellular matrix components, such as tissue inhibitor of metalloproteinase 2 was altered, emphasizing the importance of intracellular transport and interplay between neurons and glia during epileptogenesis. Finally, mice overexpressing the dominant-negative trkB, which were previously shown to have reduced epileptogenesis, showed a decrease in mRNAs of several growth-associated genes, including growth-associated protein 43. Our data suggest that BDNF signaling may partly mediate the development of epilepsy and propose that regrowth or repair processes initiated by status epilepticus and promoted by BDNF signaling may not be as advantageous as previously thought. [ABSTRACT FROM AUTHOR]

Background: Conventionally, the first step in analyzing the large and high-dimensional data sets measured by microarrays is visual exploration. Dendrograms of hierarchical clustering, self-organizing maps (SOMs), and multidimensional scaling have been used to visualize similarity relationships of data samples. We address two central properties of the methods: (i) Are the visualizations trustworthy, i.e., if two samples are visualized to be similar, are they really similar? (ii) The metric. The measure of similarity determines the result; we propose using a new learning metrics principle to derive a metric from interrelationships among data sets. Results: The trustworthiness of hierarchical clustering, multidimensional scaling, and the self-organizing map were compared in visualizing similarity relationships among gene expression profiles. The self-organizing map was the best except that hierarchical clustering was the most trustworthy for the most similar profiles. Trustworthiness can be further increased by treating separately those genes for which the visualization is least trustworthy. We then proceed to improve the metric. The distance measure between the expression profiles is adjusted to measure differences relevant to functional classes of the genes. The genes for which the new metric is the most different from the usual correlation metric are listed and visualized with one of the visualization methods, the self-organizing map, computed in the new metric. Conclusions: The conjecture from the methodological results is that the self-organizing map can be recommended to complement the usual hierarchical clustering for visualizing and exploring gene expression data. Discarding the least trustworthy samples and improving the metric still improves it. [ABSTRACT FROM AUTHOR]

Agents acting as noncompetitive N-methyl-d-aspartate (NMDA)/glutamate receptor antagonists induce the expression of several genes in limbic cortical regions, such as the cingulate, retrosplenial, and entorhinal cortices. These include important regulatory genes such as the neurotrophin brain–derived neurotrophic factor (BDNF), its receptor trkB, and c- fos. We applied expression profiling methods to find genes coregulated with BDNF following treatment with the prototypical NMDA/glutamate receptor antagonist MK-801. Expression profiling provides a useful technique for describing the molecular and transcriptional level events that follow various processes. We illustrate the utility of microarrays to find novel ESTs regulated by MK-801. We also used expression profiling with microarrays to characterize the levels of transcription factor cAMP response element modulator (CREM) and inducible cAMP early repressor (ICER) isoforms that are induced by MK-801. These factors may act as the eventual repressors for BDNF expression via competition and heterodimerization with phosphorylated CREB, a transcription factor important for BDNF expression. Finally, we find and confirm the regulation of Erp29, RTN1, and an ABC transporter by antagonism of NMDA/glutamate receptors as potential stress related molecules in brain. The emerging picture generated by using these expression profiling approaches, identifies several of what likely will be many molecules that take part in the complex events that occur during BDNF signaling mediated by blockade of NMDA/glutamate receptors. [ABSTRACT FROM AUTHOR]