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Integrating structure and experimental data annotations with computational modeling framework for predicting micro-nanoplastics toxicities.

The wide use of plastic materials leads to increased emissions of micro-nanoplastics (MNPs) into the environment, raising significant concerns about their impact on human health. Traditional experimental approaches for assessing MNPs toxicity are costly, time-consuming, and there are no experimental protocols that are universally acceptable. Computational modeling using machine learning (ML) approaches provides an efficient alternative to MNP toxicity assessment. However, most modeling studies of MNPs are limited due to the lack of high-quality data and there are few previous modeling studies considering complex structures of MNPs for model training. To address this challenge, we constructed three MNP datasets with popular toxicity endpoints from various resources and used nanostructure annotation techniques to create virtual MNPs (vMNPs) for all MNP structures. The MNP structures were digitalized from annotated vMNPs, and geometrical descriptors were calculated using the Delaunay Tessellation approach. Moreover, important experimental information, such as concentrations and cell lines, were transformed into extra training variables. Partial least squares regression (PLSR) models were built using both experimental and geometrical descriptors and validated through a leave-one-out cross validation procedure. The resulting models showed reasonable performance in predicting toxicity potentials of MNPs for the three endpoints in the present datasets. Moreover, an additional library of vMNPs with their predicted properties and bioactivities was constructed, directing further research of new MNPs. This study provides three novel ML models for MNPs by integrating geometrical and experimental descriptors, which have the potential to assess new MNPs for their toxicity. The modeling strategy developed in this study can be easily expanded to model other MNP toxicity endpoints and create promising new models for MNP toxicity assessments.

Data annotation↗

The expanding role of microarrays in the investigation of macrophage responses to pathogens.

In the last few years, microarray technology has emerged as the method of choice for large-scale gene expression studies. It provides an efficient and rapid method to investigate the entire transcriptome of a cell. No research field has benefited more from microarray technology than the study of the exquisite interplay between pathogens and hosts. Numerous microarray studies have now been published in this field, which have provided insights into the mechanisms of host defence and the tactics employed by pathogens to circumvent these protection strategies. These studies have led to a more comprehensive understanding of the host immune response and identified new avenues of research for potential control strategies against pathogens. In the past, research has concentrated on human and mouse microarrays to investigate host-pathogen interactions, regardless of the host species. This trend is changing with the ever-expanding sequence resources now available for many pathogen and host species, including livestock animals. The use of species-specific microarrays has furthered our understanding of host-pathogen interactions for particular organisms and aided in the annotation of unknown genes. Macrophages play a central role in the host's innate and adaptive immune responses to pathogens. These cells are in the first line of defence and interact with a wide range of pathogens; many of which have evolved strategies to circumvent the macrophage defence mechanisms and survive within these cells. In this report, we review the wealth of studies using microarray technology to investigate the response of macrophages to pathogens. These studies illustrate how microarray technology has expanded our understanding of the dialogue between macrophage and pathogen and provide examples of the benefits and pitfalls of using this technique. Furthermore, we discuss the resources available to use microarray analysis to study the immune response of a non-human, non-rodent species, the cow.

Animals↗

Identification of a common secondary mutation in the Neurospora crassa knockout collection conferring a cell fusion-defective phenotype.

Gene-deletion mutants represent a powerful tool to study gene function. The filamentous fungus Neurospora crassa is a well-established model organism, and features a comprehensive gene knockout strain collection. While these mutant strains have been used in numerous studies, resulting in the functional annotation of many Neurospora genes, direct confirmation of gene-phenotype relationships is often lacking, which is particularly relevant given the possibility of background mutations, sample contamination, and/or strain mislabeling. Indeed, spontaneous mutations resulting in phenotypes resembling many cell fusion mutants have long been known to occur at relatively high frequency in N. crassa, and these secondary mutations are common in the Neurospora deletion collection. The identity of these mutations, however, is largely unknown. Here, we report that the Δada-3 strain from the N. crassa knockout collection, which exhibits a cell fusion defect, harbors a secondary mutation responsible for this phenotype. Through whole-genome sequencing and genetic analyses, we found a ~30-Kb deletion in this strain affecting a known cell fusion-related gene, so/ham-1, and show that it is the absence of this gene—and not of ada-3—that underlies its cell fusion defect. We additionally found three other knockout strains harboring the same deletion, suggesting that this mutation may be common in the collection and could have impacted previous studies. Our findings provide a cautionary note and highlight the importance of proper functional validation of strains from mutant collections. We discuss our results in the context of the spread of cell fusion-defective cheater variants in N. crassa cultures.

Neurospora crassa↗

Dendritic mRNAs encode diversified functionalities in hippocampal pyramidal neurons.

BACKGROUND: Targeted transport of messenger RNA and local protein synthesis near the synapse are important for synaptic plasticity. In order to gain an overview of the composition of the dendritic mRNA pool, we dissected out stratum radiatum (dendritic lamina) from rat hippocampal CA1 region and compared its mRNA content with that of stratum pyramidale (cell body layer) using a set of cDNA microarrays. RNAs that have over-representation in the dendritic fraction were annotated and sorted into function groups. RESULTS: We have identified 154 dendritic mRNA candidates, which can be arranged into the categories of receptors and channels, signaling molecules, cytoskeleton and adhesion molecules, and factors that are involved in membrane trafficking, in protein synthesis, in posttranslational protein modification, and in protein degradation. Previously known dendritic mRNAs such as MAP2, calmodulin, and G protein gamma subunit were identified from our screening, as were mRNAs that encode proteins known to be important for synaptic plasticity and memory, such as spinophilin, Pumilio, eEF1A, and MHC class I molecules. Furthermore, mRNAs coding for ribosomal proteins were also found in dendrites. CONCLUSION: Our results suggest that neurons transport a variety of mRNAs to dendrites, not only those directly involved in modulating synaptic plasticity, but also others that play more common roles in cellular metabolism.

Animals↗

IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains.

IMGT, the international ImMunoGeneTics database (http://imgt.cines.fr) is a high quality integrated information system specializing in immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of human and other vertebrates. IMGT provides a common access to expertly annotated data on the genome, proteome, genetics and structure of the IG and TR, based on the IMGT Scientific chart and IMGT-ONTOLOGY. The IMGT unique numbering defined for the IG and TR variable regions and domains of all jawed vertebrates has allowed a redefinition of the limits of the framework (FR-IMGT) and complementarity determining regions (CDR-IMGT), leading, for the first time, to a standardized description of mutations, allelic polymorphisms, 2D representations (Colliers de Perles) and 3D structures, whatever the antigen receptor, the chain type, or the species. The IMGT numbering has been extended to the V-like domain and is, therefore, highly valuable for comparative analysis and evolution studies of proteins belonging to the IG superfamily.

Amino Acid Sequence↗

Image analysis for automatic segmentation of cytoplasms and classification of Rac1 activation.

BACKGROUND: Rac1 is a GTP-binding molecule involved in a wide range of cellular processes. Using digital image analysis, agonist-induced translocation of green fluorescent protein (GFP) Rac1 to the cellular membrane can be estimated quantitatively for individual cells. METHODS: A fully automatic image analysis method for cell segmentation, feature extraction, and classification of cells according to their activation, i.e., GFP-Rac1 translocation and ruffle formation at stimuli, is described. Based on training data produced by visual annotation of four image series, a statistical classifier was created. RESULTS: The results of the automatic classification were compared with results from visual inspection of the same time sequences. The automatic classification differed from the visual classification at about the same level as visual classifications performed by two different skilled professionals differed from each other. Classification of a second image set, consisting of seven image series with different concentrations of agonist, showed that the classifier could detect an increased proportion of activated cells at increased agonist concentration. CONCLUSIONS: Intracellular activities, such as ruffle formation, can be quantified by fully automatic image analysis, with an accuracy comparable to that achieved by visual inspection. This analysis can be done at a speed of hundreds of cells per second and without the subjectivity introduced by manual judgments.

Animals↗

Characterization of adhesion threads of Deinococcus geothermalis as type IV pili.

Deinococcus geothermalis E50051 forms tenuous biofilms on paper machine surfaces. Field emission electron microscopy analysis revealed peritrichous appendages which mediated cell-to-surface and cell-to-cell interactions but were absent in planktonically grown cells. The major protein component of the extracellular extract of D. geothermalis had an N-terminal sequence similar to the fimbrial protein pilin annotated in the D. geothermalis DSM 11300 draft sequence. It also showed similarity to the type IV pilin sequence of D. radiodurans and several gram-negative pathogenic bacteria. Other proteins in the extract had N-terminal sequences identical to D. geothermalis proteins with conservative motifs for serine proteases, metallophosphoesterases, and proteins whose function is unknown. Periodic acid-Schiff staining for carbohydrates indicated that these extracellular proteins may be glycosylated. A further confirmation for the presence of glycoconjugates on the cell surface was obtained by confocal laser scanning imaging of living D. geothermalis cells stained with Amaranthus caudatus lectin, which specifically binds to galactose residues. The results indicate that the thread-like appendages of D. geothermalis E50051 are glycosylated type IV pili, bacterial attachment organelles which have thus far not been described for the genus Deinococcus.

Bacterial Adhesion↗

Cluster analysis of protein array results via similarity of Gene Ontology annotation.

BACKGROUND: With the advent of high-throughput proteomic experiments such as arrays of purified proteins comes the need to analyse sets of proteins as an ensemble, as opposed to the traditional one-protein-at-a-time approach. Although there are several publicly available tools that facilitate the analysis of protein sets, they do not display integrated results in an easily-interpreted image or do not allow the user to specify the proteins to be analysed. RESULTS: We developed a novel computational approach to analyse the annotation of sets of molecules. As proof of principle, we analysed two sets of proteins identified in published protein array screens. The distance between any two proteins was measured as the graph similarity between their Gene Ontology (GO) annotations. These distances were then clustered to highlight subsets of proteins sharing related GO annotation. In the first set of proteins found to bind small molecule inhibitors of rapamycin, we identified three subsets containing four or five proteins each that may help to elucidate how rapamycin affects cell growth whereas the original authors chose only one novel protein from the array results for further study. In a set of phosphoinositide-binding proteins, we identified subsets of proteins associated with different intracellular structures that were not highlighted by the analysis performed in the original publication. CONCLUSION: By determining the distances between annotations, our methodology reveals trends and enrichment of proteins of particular functions within high-throughput datasets at a higher sensitivity than perusal of end-point annotations. In an era of increasingly complex datasets, such tools will help in the formulation of new, testable hypotheses from high-throughput experimental data.

Algorithms↗

cDNA-AFLP analysis of seed germination in Arabidopsis thaliana identifies transposons and new genomic sequences.

A cDNA-AFLP experiment was designed to identify and clone nucleotide sequences induced during seed germination in Arabidopsis thaliana. Sequences corresponding to known genes involved in processes important for germination, such as mitochondrial biogenesis, protein synthesis and cell cycle progression, were isolated. Other sequences correspond to Arabidopsis BAC clones in regions where genes have not been annotated. Notably, a number of the sequences cloned did not correspond to available sequences in the databases from the Arabidopsis genome, but instead present significant similarity with DNA from other organisms, for example fish species; among them, some may encode transposons. A number of the sequences isolated showed no significant similarity with any sequences in the public databases. Oligonucleotides derived from these new sequences were used to amplify genomic DNA of Arabidopsis. Expression analysis of representative sequences is presented. This work suggests that, during germination, there may be a massive transposon mobilization that may be useful in the annotation of new genome sequences and identification of regulatory mechanisms.

Amino Acid Motifs↗

Genomewide transcriptional changes associated with genetic alterations and nutritional supplementation affecting tryptophan metabolism in Bacillus subtilis.

DNA microarrays comprising approximately 95% of the Bacillus subtilis annotated protein coding ORFs were deployed to generate a series of snapshots of genomewide transcriptional changes that occur when cells are grown under various conditions that are expected to increase or decrease transcription of the trp operon segment of the aromatic supraoperon. Comparisons of global expression patterns were made between cells grown in the presence of indole acrylic acid, a specific inhibitor of tRNA(Trp) charging; cells deficient in expression of the mtrB gene, which encodes the tryptophan-activated negative regulatory protein, TRAP; WT cells grown in the presence or absence of two or three of the aromatic amino acids; and cells harboring a tryptophanyl tRNA synthetase mutation conferring temperature-sensitive tryptophan-dependent growth. Our findings validate expected responses of the tryptophan biosynthetic genes and presumed regulatory interrelationships between genes in the different aromatic amino acid pathways and the histidine biosynthetic pathway. Using a combination of supervised and unsupervised statistical methods we identified approximately 100 genes whose expression profiles were closely correlated with those of the genes in the trp operon. This finding suggests that expression of these genes is influenced directly or indirectly by regulatory events that affect or are a consequence of altered tryptophan metabolism.

Bacillus subtilis↗

The Database of Quantitative Cellular Signaling: management and analysis of chemical kinetic models of signaling networks.

MOTIVATION: Analysis of cellular signaling interactions is expected to pose an enormous informatics challenge, perhaps even larger than analyzing the genome. The complex networks arising from signaling processes are traditionally represented as block diagrams. A key step in the evolution toward a more quantitative understanding of signaling is to explicitly specify the kinetics of all chemical reaction steps in a pathway. Technical advances in proteomics and high-throughput protein interaction assays promise a flood of such quantitative data. While annotations, molecular information and pathway connectivity have been compiled in several databases, and there are several proposals for general cell model description languages, there is currently little experience with databases of chemical kinetics and reaction level models of signaling networks. RESULTS: The Database of Quantitative Cellular Signaling is a repository of models of signaling pathways. It is intended both to serve the growing field of chemical-reaction level simulation of signaling networks, and to anticipate issues in large-scale data management for signaling chemistry. AVAILABILITY: The Database of Quantitative Cellular Signaling is available at http://doqcs.ncbs.res.in. Links to the signaling model simulator, GENESIS/Kinetikit are at http://www.ncbs.res.in/~bhalla/kkit/index.html and are also provided from within the database. The database source code is available under the GNU Public License.

Abstracting and Indexing↗

Rhodopseudomonas palustris regulons detected by cross-species analysis of alphaproteobacterial genomes.

Rhodopseudomonas palustris, an alpha-proteobacterium, carries out three of the chemical reactions that support life on this planet: the conversion of sunlight to chemical-potential energy; the absorption of carbon dioxide, which it converts to cellular material; and the fixation of atmospheric nitrogen into ammonia. Insight into the transcription-regulatory network that coordinates these processes is fundamental to understanding the biology of this versatile bacterium. With this goal in mind, we predicted regulatory signals genomewide, using a two-step phylogenetic-footprinting and clustering process that we had developed previously. In the first step, 4,963 putative transcription factor binding sites, upstream of 2,044 genes and operons, were identified using cross-species Gibbs sampling. Bayesian motif clustering was then employed to group the cross-species motifs into regulons. We have identified 101 putative regulons in R. palustris, including 8 that are of particular interest: a photosynthetic regulon, a flagellar regulon, an organic hydroperoxide resistance regulon, the LexA regulon, and four regulons related to nitrogen metabolism (FixK2, NnrR, NtrC, and sigma54). In some cases, clustering allowed us to assign functions to proteins that previously had been annotated with only putative functions; we have identified RPA0828 as the organic hydroperoxide resistance regulator and RPA1026 as a cell cycle methylase. In addition to predicting regulons, we identified a novel inverted repeat that likely forms a highly conserved stem-loop and that occurs downstream of over 100 genes.

Alphaproteobacteria↗

MeMo: a hybrid SQL/XML approach to metabolomic data management for functional genomics.

BACKGROUND: The genome sequencing projects have shown our limited knowledge regarding gene function, e.g. S. cerevisiae has 5-6,000 genes of which nearly 1,000 have an uncertain function. Their gross influence on the behaviour of the cell can be observed using large-scale metabolomic studies. The metabolomic data produced need to be structured and annotated in a machine-usable form to facilitate the exploration of the hidden links between the genes and their functions. DESCRIPTION: MeMo is a formal model for representing metabolomic data and the associated metadata. Two predominant platforms (SQL and XML) are used to encode the model. MeMo has been implemented as a relational database using a hybrid approach combining the advantages of the two technologies. It represents a practical solution for handling the sheer volume and complexity of the metabolomic data effectively and efficiently. The MeMo model and the associated software are available at http://dbkgroup.org/memo/. CONCLUSION: The maturity of relational database technology is used to support efficient data processing. The scalability and self-descriptiveness of XML are used to simplify the relational schema and facilitate the extensibility of the model necessitated by the creation of new experimental techniques. Special consideration is given to data integration issues as part of the systems biology agenda. MeMo has been physically integrated and cross-linked to related metabolomic and genomic databases. Semantic integration with other relevant databases has been supported through ontological annotation. Compatibility with other data formats is supported by automatic conversion.

Computer Simulation↗

Gene expression signature of estrogen receptor alpha status in breast cancer.

BACKGROUND: Estrogens are known to regulate the proliferation of breast cancer cells and to modify their phenotypic properties. Identification of estrogen-regulated genes in human breast tumors is an essential step toward understanding the molecular mechanisms of estrogen action in cancer. To this end we generated and compared the Serial Analysis of Gene Expression (SAGE) profiles of 26 human breast carcinomas based on their estrogen receptor alpha (ER) status. Thus, producing a breast cancer SAGE database of almost 2.5 million tags, representing over 50,000 transcripts. RESULTS: We identified 520 transcripts differentially expressed between ERalpha-positive (+) and ERalpha-negative (-) primary breast tumors (Fold change >or= 2; p < 0.05). Furthermore, we identified 220 high-affinity Estrogen Responsive Elements (EREs) distributed on the promoter regions of 163 out of the 473 up-modulated genes in ERalpha (+) breast tumors. In brief, we observed predominantly up-regulation of cell growth related genes, DNA binding and transcription factor activity related genes based on Gene Ontology (GO) biological functional annotation. GO terms over-representation analysis showed a statistically significant enrichment of various transcript families including: metal ion binding related transcripts (p = 0.011), calcium ion binding related transcripts (p = 0.033) and steroid hormone receptor activity related transcripts (p = 0.031). SAGE data associated with ERalpha status was compared with reported information from breast cancer DNA microarrays studies. A significant proportion of ERalpha associated gene expression changes was validated by this cross-platform comparison. However, our SAGE study also identified novel sets of genes as highly expressed in ERalpha (+) invasive breast tumors not previously reported. These observations were further validated in an independent set of human breast tumors by means of real time RT-PCR. CONCLUSION: The integration of the breast cancer comparative transcriptome analysis based on ERalpha status coupled to the genome-wide identification of high-affinity EREs and GO over-representation analysis, provide useful information for validation and discovery of signaling networks related to estrogen response in this malignancy.

Biomarkers, Tumor↗

Functional genomics approaches in arthritis.

The post-genomic era of functional genomics and target validation will allow us to narrow the bridge between clinically correlative data and causative data for complex diseases, such as arthritis, for which the etiological agent remains elusive. The availability of human and other annotated genome sequences, and parallel developments of new technologies that allow analysis of minute amounts of human and animal cells (peripheral blood cells and infiltrating cells) and tissues (synovium and cartilage) under different pathophysiological conditions, has facilitated high-throughput gene mining approaches that can generate vast amounts of clinically correlative data. Characterizing some of the correlative/causative genes will require reverting to the hypothesis-driven, low throughput method of complementary experimental biology using genomic approaches as a tool. This will include in silico gene expression arrays, genome-wide scans, comparative genomics using various animal models (such as rodents and zebrafish), bioinformatics and a team of well trained translational scientists and physicians. For the first time, the "genomic tools" will allow us to analyze small amounts of surgical samples (such as needle biopsies) and clinical samples in the context of the whole genome. Preliminary genomic analysis in osteoarthritis has already resurrected the debate on the semantic issues in the definition of inflammation. Further analyses will not only facilitate the development of unbiased hypotheses at the molecular level, but also assist us in the identification and characterization of novel targets and disease markers for pharmacological intervention, gene therapy, and diagnosis.

Animals↗

Mining the Giardia lamblia genome for new cyst wall proteins.

The Giardia lamblia cyst wall (CW), which is required for survival outside the host and infection, is a primitive extracellular matrix. Because of the importance of the CW, we queried the Giardia Genome Project Database with the coding sequences of the only two known CW proteins, which are cysteine-rich and contain leucine-rich repeats (LRRs). We identified five new LRR-containing proteins, of which only one (CWP3) is up-regulated during encystation and incorporated into the cyst wall. Sequence comparison with CWP1 and -2 revealed conservation within the LRRs and the 44-amino-acid N-flanking region, although CWP3 is more divergent. Interestingly, all 14 cysteine residues of CWP3 are positionally conserved with CWP1 and -2. During encystation, C-terminal epitope-tagged CWP3 was transported to the wall of water-resistant cysts via the novel regulated secretory pathway in encystation-secretory vesicles (ESVs). Deletion analysis revealed that the four LRRs are each essential to target CWP3 to the ESVs and cyst wall. In a deletion of the most C-terminal region, fewer ESVs were stained in encysting cells, and there was no staining in cysts. In contrast, deletion of the 44 amino acids between the signal sequence and the LRRs or the region just C-terminal to the LRRs only decreased the number of cells with CWP3 targeting to ESVs and cyst wall by approximately 50%. Our studies indicate that virtually every portion of the CWP3 protein is needed for efficient targeting to the regulated secretory pathway and incorporation into the cyst wall. Further, these data demonstrate the power of genomics in combination with rigorous functional analyses to verify annotation.

Amino Acid Sequence↗

Novel candidate targets of beta-catenin/T-cell factor signaling identified by gene expression profiling of ovarian endometrioid adenocarcinomas.

The activity of beta-catenin (beta-cat), a key component of the Wnt signaling pathway, is deregulated in about 40% of ovarian endometrioid adenocarcinomas (OEAs), usually as a result of CTNNB1 gene mutations. The function of beta-cat in neoplastic transformation is dependent on T-cell factor (TCF) transcription factors, but specific genes activated by the interaction of beta-cat with TCFs in OEAs and other cancers with Wnt pathway defects are largely unclear. As a strategy to identify beta-cat/TCF transcriptional targets likely to contribute to OEA pathogenesis, we used oligonucleotide microarrays to compare gene expression in primary OEAs with mutational defects in beta-cat regulation (n = 11) to OEAs with intact regulation of beta-cat activity (n = 17). Both hierarchical clustering and principal component analysis based on global gene expression distinguished beta-cat-defective tumors from those with intact beta-cat regulation. We identified 81 potential beta-cat/TCF targets by selecting genes with at least 2-fold increased expression in beta-cat-defective versus beta-cat regulation-intact tumors and significance in a t test (P < 0.05). Seven of the 81 genes have been previously reported as Wnt/beta-cat pathway targets (i.e., BMP4, CCND1, CD44, FGF9, EPHB3, MMP7, and MSX2). Differential expression of several known and candidate target genes in the OEAs was confirmed. For the candidate target genes CST1 and EDN3, reporter and chromatin immunoprecipitation assays directly implicated beta-cat and TCF in their regulation. Analysis of presumptive regulatory elements in 67 of the 81 candidate genes for which complete genomic sequence data were available revealed an apparent difference in the location and abundance of consensus TCF-binding sites compared with the patterns seen in control genes. Our findings imply that analysis of gene expression profiling data from primary tumor samples annotated with detailed molecular information may be a powerful approach to identify key downstream targets of signaling pathways defective in cancer cells.

Binding Sites↗

Annotating eukaryote genomes.

The Genome Annotation Assessment Project tested current methods of gene identification, including a critical assessment of the accuracy of different methods. Two new databases have provided new resources for gene annotation: these are the InterPro database of protein domains and motifs, and the Gene Ontology database for terms that describe the molecular functions and biological roles of gene products. Efforts in genome annotation are most often based upon advances in computer systems that are specifically designed to deal with the tremendous amounts of data being generated by current sequencing projects. These efforts in analysis are being linked to new ways of visualizing computationally annotated genomes.

Animals↗