Cross-Omic Transcription Factor Analysis: An Insight on Transcription Factor Accessibility and Expression Correlation.

It is well known how sequencing technologies propelled cellular biology research in recent years, providing incredible insight into the basic mechanisms of cells. Single-cell RNA sequencing is at the front in this field, with single-cell ATAC sequencing supporting it and becoming more popular. In this regard, multi-modal technologies play a crucial role, allowing the possibility to simultaneously perform the mentioned sequencing modalities on the same cells. Yet, there still needs to be a clear and dedicated way to analyze these multi-modal data. One of the current methods is to calculate the Gene Activity Matrix (GAM), which summarizes the accessibility of the genes at the genomic level, to have a more direct link with the transcriptomic data. However, this concept is not well defined, and it is unclear how various accessible regions impact the expression of the genes. Moreover, the transcription process is highly regulated by the transcription factors that bind to the different DNA regions. Therefore, this work presents a continuation of the meta-analysis of Genomic-Annotated Gene Activity Matrix (GAGAM) contributions, aiming to investigate the correlation between the TF expression and motif information in the different functional genomic regions to understand the different Transcription Factors (TFs) dynamics involved in different cell types.

Neuronal Spike Shapes (NSS): A straightforward approach to investigate heterogeneity in neuronal excitability states.

The mammalian brain exhibits a remarkable diversity of neurons, contributing to its intricate architecture and functional complexity. The analysis of multimodal single-cell datasets enables the investigation of cell types and states heterogeneity. In this study, we introduce the Neuronal Spike Shapes (NSS), a straightforward approach for the exploration of excitability states of neurons based on their Action Potential (AP) waveforms. The NSS method describes the AP waveform based on a triangular representation complemented by a set of derived electrophysiological (EP) features. To support this hypothesis, we validate the proposed approach on two datasets of murine cortical neurons, focusing it on GABAergic neurons. The validation process involves a combination of NSS-based clustering analysis, features exploration, Differential Expression (DE), and Gene Ontology (GO) enrichment analysis. Results show that the NSS-based analysis captures neuronal excitability states that possess biological relevance independently of cell subtype. In particular, Neuronal Spike Shapes (NSS) captures, among others, a well-characterized fast-spiking excitability state, supported by both electrophysiological and transcriptomic validation. Gene Ontology Enrichment Analysis reveals voltage-gated potassium (K+) channels as specific markers of the identified NSS partitions. This finding strongly corroborates the biological relevance of NSS partitions as excitability states, as the expression of voltage-gated K+ channels regulates the hyperpolarization phase of the AP, being directly implicated in the regulation of neuronal excitability.

GAGAM v1.2: An Improvement on Peak Labeling and Genomic Annotated Gene Activity Matrix Construction.

Single-cell Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq) is rapidly becoming a powerful technology for assessing the epigenetic landscape of thousands of cells. However, the sparsity of the resulting data poses significant challenges to their interpretability and informativeness. Different computational methods are available, proposing ways to generate significant features from accessibility data and process them to obtain meaningful results. Foremost among them is the peak calling, which interprets the raw scATAC-seq data generating the peaks as features. However, scATAC-seq data are not trivially comparable with single-cell RNA sequencing (scRNA-seq) data, an increasingly pressing challenge since the necessity of multimodal experiments integration. For this reason, this study wants to improve the concept of the Gene Activity Matrix (GAM), which links the accessibility data to the genes, by proposing an improved version of the Genomic-Annotated Gene Activity Matrix (GAGAM) concept. Specifically, this paper presents GAGAM v1.2, a new and better version of GAGAM v1.0. GAGAM aims to label the peaks and link them to the genes through functional annotation of the whole genome. Using genes as features in scATAC-seq datasets makes different datasets comparable and allows linking gene accessibility and expression. This link is crucial for gene regulation understanding and fundamental for the increasing impact of multi-omics data. Results confirm that our method performs better than the previous GAMs and shows a preliminary comparison with scRNA-seq data.

Identification of miRNAs Potentially Involved in Bronchiolitis Obliterans Syndrome: A Computational Study.

The pathogenesis of Bronchiolitis Obliterans Syndrome (BOS), the main clinical phenotype of chronic lung allograft dysfunction, is poorly understood. Recent studies suggest that epigenetic regulation of microRNAs might play a role in its development. In this paper we present the application of a complex computational pipeline to perform enrichment analysis of miRNAs in pathways applied to the study of BOS. The analysis considered the full set of miRNAs annotated in miRBase (version 21), and applied a sequence of filtering approaches and statistical analyses to reduce this set and to score the candidate miRNAs according to their potential involvement in BOS development. Dysregulation of two of the selected candidate miRNAs-miR-34a and miR-21 -was clearly shown in in-situ hybridization (ISH) on five explanted human BOS lungs and on a rat model of acute and chronic lung rejection, thus definitely identifying miR-34a and miR-21 as pathogenic factors in BOS and confirming the effectiveness of the computational pipeline.

Common integration sites of published datasets identified using a graph-based framework.

With next-generation sequencing, the genomic data available for the characterization of integration sites (IS) has dramatically increased. At present, in a single experiment, several thousand viral integration genome targets can be investigated to define genomic hot spots. In a previous article, we renovated a formal CIS analysis based on a rigid fixed window demarcation into a more stretchy definition grounded on graphs. Here, we present a selection of supporting data related to the graph-based framework (GBF) from our previous article, in which a collection of common integration sites (CIS) was identified on six published datasets. In this work, we will focus on two datasets, ISRTCGD and ISHIV, which have been previously discussed. Moreover, we show in more detail the workflow design that originates the datasets.

CyTRANSFINDER: a Cytoscape 3.3 plugin for three-component (TF, gene, miRNA) signal transduction pathway construction.

BACKGROUND: Biological research increasingly relies on network models to study complex phenomena. Signal Transduction Pathways are molecular circuits that model how cells receive, process, and respond to information from the environment providing snapshots of the overall cell dynamics. Most of the attempts to reconstruct signal transduction pathways are limited to single regulator networks including only genes/proteins. However, networks involving a single type of regulator and neglecting transcriptional and post-transcriptional regulations mediated by transcription factors and microRNAs, respectively, may not fully reveal the complex regulatory mechanisms of a cell. We observed a lack of computational instruments supporting explorative analysis on this type of three-component signal transduction pathways. RESULTS: We have developed CyTRANSFINDER, a new Cytoscape plugin able to infer three-component signal transduction pathways based on user defined regulatory patterns and including miRNAs, TFs and genes. Since CyTRANSFINDER has been designed to support exploratory analysis, it does not rely on expression data. To show the potential of the plugin we have applied it in a study of two miRNAs that are particularly relevant in human melanoma progression, miR-146a and miR-214. CONCLUSIONS: CyTRANSFINDER supports the reconstruction of small signal transduction pathways among groups of genes. Results obtained from its use in a real case study have been analyzed and validated through both literature data and preliminary wet-lab experiments, showing the potential of this tool when performing exploratory analysis.

ReNE: a cytoscape plugin for regulatory network enhancement.

One of the biggest challenges in the study of biological regulatory mechanisms is the integration, americanmodeling, and analysis of the complex interactions which take place in biological networks. Despite post transcriptional regulatory elements (i.e., miRNAs) are widely investigated in current research, their usage and visualization in biological networks is very limited. Regulatory networks are commonly limited to gene entities. To integrate networks with post transcriptional regulatory data, researchers are therefore forced to manually resort to specific third party databases. In this context, we introduce ReNE, a Cytoscape 3.x plugin designed to automatically enrich a standard gene-based regulatory network with more detailed transcriptional, post transcriptional, and translational data, resulting in an enhanced network that more precisely models the actual biological regulatory mechanisms. ReNE can automatically import a network layout from the Reactome or KEGG repositories, or work with custom pathways described using a standard OWL/XML data format that the Cytoscape import procedure accepts. Moreover, ReNE allows researchers to merge multiple pathways coming from different sources. The merged network structure is normalized to guarantee a consistent and uniform description of the network nodes and edges and to enrich all integrated data with additional annotations retrieved from genome-wide databases like NCBI, thus producing a pathway fully manageable through the Cytoscape environment. The normalized network is then analyzed to include missing transcription factors, miRNAs, and proteins. The resulting enhanced network is still a fully functional Cytoscape network where each regulatory element (transcription factor, miRNA, gene, protein) and regulatory mechanism (up-regulation/down-regulation) is clearly visually identifiable, thus enabling a better visual understanding of its role and the effect in the network behavior. The enhanced network produced by ReNE is exportable in multiple formats for further analysis via third party applications. ReNE can be freely installed from the Cytoscape App Store (http://apps.cytoscape.org/apps/rene) and the full source code is freely available for download through a SVN repository accessible at http://www.sysbio.polito.it/tools_svn/BioInformatics/Rene/releases/. ReNE enhances a network by only integrating data from public repositories, without any inference or prediction. The reliability of the introduced interactions only depends on the reliability of the source data, which is out of control of ReNe developers.

An extended gene protein/products Boolean network model including post-transcriptional regulation.

BACKGROUND: Networks Biology allows the study of complex interactions between biological systems using formal, well structured, and computationally friendly models. Several different network models can be created, depending on the type of interactions that need to be investigated. Gene Regulatory Networks (GRN) are an effective model commonly used to study the complex regulatory mechanisms of a cell. Unfortunately, given their intrinsic complexity and non discrete nature, the computational study of realistic-sized complex GRNs requires some abstractions. Boolean Networks (BNs), for example, are a reliable model that can be used to represent networks where the possible state of a node is a boolean value (0 or 1). Despite this strong simplification, BNs have been used to study both structural and dynamic properties of real as well as randomly generated GRNs. RESULTS: In this paper we show how it is possible to include the post-transcriptional regulation mechanism (a key process mediated by small non-coding RNA molecules like the miRNAs) into the BN model of a GRN. The enhanced BN model is implemented in a software toolkit (EBNT) that allows to analyze boolean GRNs from both a structural and a dynamic point of view. The open-source toolkit is compatible with available visualization tools like Cytoscape and allows to run detailed analysis of the network topology as well as of its attractors, trajectories, and state-space. In the paper, a small GRN built around the mTOR gene is used to demonstrate the main capabilities of the toolkit. CONCLUSIONS: The extended model proposed in this paper opens new opportunities in the study of gene regulation. Several of the successful researches done with the support of BN to understand high-level characteristics of regulatory networks, can now be improved to better understand the role of post-transcriptional regulation for example as a network-wide noise-reduction or stabilization mechanisms.

Reducing the complexity of complex gene coexpression networks by coupling multiweighted labeling with topological analysis.

Undirected gene coexpression networks obtained from experimental expression data coupled with efficient computational procedures are increasingly used to identify potentially relevant biological information (e.g., biomarkers) for a particular disease. However, coexpression networks built from experimental expression data are in general large highly connected networks with an elevated number of false-positive interactions (nodes and edges). In order to infer relevant information, the network must be properly filtered and its complexity reduced. Given the complexity and the multivariate nature of the information contained in the network, this requires the development and application of efficient feature selection algorithms to be able to exploit the topological characteristics of the network to identify relevant nodes and edges. This paper proposes an efficient multivariate filtering designed to analyze the topological properties of a coexpression network in order to identify potential relevant genes for a given disease. The algorithm has been tested on three datasets for three well known and studied diseases: acute myeloid leukemia, breast cancer, and diffuse large B-cell lymphoma. Results have been validated resorting to bibliographic data automatically mined using the ProteinQuest literature mining tool.

A systematic analysis of a mi-RNA inter-pathway regulatory motif.

BACKGROUND: The continuing discovery of new types and functions of small non-coding RNAs is suggesting the presence of regulatory mechanisms far more complex than the ones currently used to study and design Gene Regulatory Networks. Just focusing on the roles of micro RNAs (miRNAs), they have been found to be part of several intra-pathway regulatory motifs. However, inter-pathway regulatory mechanisms have been often neglected and require further investigation. RESULTS: In this paper we present the result of a systems biology study aimed at analyzing a high-level inter-pathway regulatory motif called Pathway Protection Loop, not previously described, in which miRNAs seem to play a crucial role in the successful behavior and activation of a pathway. Through the automatic analysis of a large set of public available databases, we found statistical evidence that this inter-pathway regulatory motif is very common in several classes of KEGG Homo Sapiens pathways and concurs in creating a complex regulatory network involving several pathways connected by this specific motif. The role of this motif seems also confirmed by a deeper review of other research activities on selected representative pathways. CONCLUSIONS: Although previous studies suggested transcriptional regulation mechanism at the pathway level such as the Pathway Protection Loop, a high-level analysis like the one proposed in this paper is still missing. The understanding of higher-level regulatory motifs could, as instance, lead to new approaches in the identification of therapeutic targets because it could unveil new and "indirect" paths to activate or silence a target pathway. However, a lot of work still needs to be done to better uncover this high-level inter-pathway regulation including enlarging the analysis to other small non-coding RNA molecules.

A combined approach for genome wide protein function annotation/prediction.

BACKGROUND: Today large scale genome sequencing technologies are uncovering an increasing amount of new genes and proteins, which remain uncharacterized. Experimental procedures for protein function prediction are low throughput by nature and thus can't be used to keep up with the rate at which new proteins are discovered. On the other hand, proteins are the prominent stakeholders in almost all biological processes, and therefore the need to precisely know their functions for a better understanding of the underlying biological mechanism is inevitable. The challenge of annotating uncharacterized proteins in functional genomics and biology in general motivates the use of computational techniques well orchestrated to accurately predict their functions. METHODS: We propose a computational flow for the functional annotation of a protein able to assign the most probable functions to a protein by aggregating heterogeneous information. Considered information include: protein motifs, protein sequence similarity, and protein homology data gathered from interacting proteins, combined with data from highly similar non-interacting proteins (hereinafter called Similactors). Moreover, to increase the predictive power of our model we also compute and integrate term specific relationships among functional terms based on Gene Ontology (GO). RESULTS: We tested our method on Saccharomyces Cerevisiae and Homo sapiens species proteins. The aggregation of different structural and functional evidence with GO relationships outperforms, in terms of precision and accuracy of prediction than the other methods reported in literature. The predicted precision and accuracy is 100% for more than half of the input set for both species; overall, we obtained 85.38% precision and 81.95% accuracy for Homo sapiens and 79.73% precision and 80.06% accuracy for Saccharomyces Cerevisiae species proteins.

Building gene expression profile classifiers with a simple and efficient rejection option in R.

BACKGROUND: The collection of gene expression profiles from DNA microarrays and their analysis with pattern recognition algorithms is a powerful technology applied to several biological problems. Common pattern recognition systems classify samples assigning them to a set of known classes. However, in a clinical diagnostics setup, novel and unknown classes (new pathologies) may appear and one must be able to reject those samples that do not fit the trained model. The problem of implementing a rejection option in a multi-class classifier has not been widely addressed in the statistical literature. Gene expression profiles represent a critical case study since they suffer from the curse of dimensionality problem that negatively reflects on the reliability of both traditional rejection models and also more recent approaches such as one-class classifiers. RESULTS: This paper presents a set of empirical decision rules that can be used to implement a rejection option in a set of multi-class classifiers widely used for the analysis of gene expression profiles. In particular, we focus on the classifiers implemented in the R Language and Environment for Statistical Computing (R for short in the remaining of this paper). The main contribution of the proposed rules is their simplicity, which enables an easy integration with available data analysis environments. Since in the definition of a rejection model tuning of the involved parameters is often a complex and delicate task, in this paper we exploit an evolutionary strategy to automate this process. This allows the final user to maximize the rejection accuracy with minimum manual intervention. CONCLUSIONS: This paper shows how the use of simple decision rules can be used to help the use of complex machine learning algorithms in real experimental setups. The proposed approach is almost completely automated and therefore a good candidate for being integrated in data analysis flows in labs where the machine learning expertise required to tune traditional classifiers might not be available.