Inferring microbial co-occurrence networks from amplicon data: a systematic evaluation.

Microbes commonly organize into communities consisting of hundreds of species involved in complex interactions with each other. 16S ribosomal RNA (16S rRNA) amplicon profiling provides snapshots that reveal the phylogenies and abundance profiles of these microbial communities. These snapshots, when collected from multiple samples, can reveal the co-occurrence of microbes, providing a glimpse into the network of associations in these communities. However, the inference of networks from 16S data involves numerous steps, each requiring specific tools and parameter choices. Moreover, the extent to which these steps affect the final network is still unclear. In this study, we perform a meticulous analysis of each step of a pipeline that can convert 16S sequencing data into a network of microbial associations. Through this process, we map how different choices of algorithms and parameters affect the co-occurrence network and identify the steps that contribute substantially to the variance. We further determine the tools and parameters that generate robust co-occurrence networks and develop consensus network algorithms based on benchmarks with mock and synthetic data sets. The Microbial Co-occurrence Network Explorer, or MiCoNE (available at https://github.com/segrelab/MiCoNE) follows these default tools and parameters and can help explore the outcome of these combinations of choices on the inferred networks. We envisage that this pipeline could be used for integrating multiple data sets and generating comparative analyses and consensus networks that can guide our understanding of microbial community assembly in different biomes. IMPORTANCE Mapping the interrelationships between different species in a microbial community is important for understanding and controlling their structure and function. The surge in the high-throughput sequencing of microbial communities has led to the creation of thousands of data sets containing information about microbial abundances. These abundances can be transformed into co-occurrence networks, providing a glimpse into the associations within microbiomes. However, processing these data sets to obtain co-occurrence information relies on several complex steps, each of which involves numerous choices of tools and corresponding parameters. These multiple options pose questions about the robustness and uniqueness of the inferred networks. In this study, we address this workflow and provide a systematic analysis of how these choices of tools affect the final network and guidelines on appropriate tool selection for a particular data set. We also develop a consensus network algorithm that helps generate more robust co-occurrence networks based on benchmark synthetic data sets.

EZH2 inhibitor GSK343 inhibits sepsis-induced intestinal disorders.

Enhancer of zeste homolog 2 (EZH2) is positively associated with poor clinical outcomes in a number of aggressive tumors. Recent studies have demonstrated that inhibition of EZH2 also suppressed the inflammatory response during sepsis. The present study aimed to investigate whether an inhibitor of EZH2, GSK343, could protect the intestine against sepsis-induced injury in vivo. Mice underwent cecal ligation and perforation (CLP) to induce sepsis and were assigned into three groups: Sham, CLP and CLP + GSK343. For GSK343 treatment, the septic mice were intravenously injected with GSK343 at 6 h post-CLP. The results indicated that EZH2 was highly expressed while tight junction (TJ) proteins ZO-1, occludin and claudin-1 expression was reduced in the intestinal tissue of mice subjected to CLP compared with the sham group. CLP operation also caused intestinal pathological injury and the production of inflammatory cytokines including TNF-α, IL-1ß and IL-6 in both serum and intestinal tissues. Meanwhile, CLP induced cell apoptosis of intestinal tissue based on the increased number of apoptotic cells, reduced expression of Bcl-2 and higher expression of caspase-3 and Bax. However, the presence of GSK343 partially rescued intestinal pathological injury, reduced the level of inflammatory cytokines, repressed cell apoptosis and promoted TJ protein expression. Finally, the decreased number of Paneth cells caused by CLP operation was reversed by GSK343 treatment. In conclusion, the results of the present study demonstrated that GSK343 could protect the intestine against sepsis-induced injury in vivo. Inhibition of EZH2 may provide a therapeutic approach for intestinal dysfunction during sepsis.

A network based approach to drug repositioning identifies plausible candidates for breast cancer and prostate cancer.

BACKGROUND: The high cost and the long time required to bring drugs into commerce is driving efforts to repurpose FDA approved drugs-to find new uses for which they weren't intended, and to thereby reduce the overall cost of commercialization, and shorten the lag between drug discovery and availability. We report on the development, testing and application of a promising new approach to repositioning. METHODS: Our approach is based on mining a human functional linkage network for inversely correlated modules of drug and disease gene targets. The method takes account of multiple information sources, including gene mutation, gene expression, and functional connectivity and proximity of within module genes. RESULTS: The method was used to identify candidates for treating breast and prostate cancer. We found that (i) the recall rate for FDA approved drugs for breast (prostate) cancer is 20/20 (10/11), while the rates for drugs in clinical trials were 131/154 and 82/106; (ii) the ROC/AUC performance substantially exceeds that of comparable methods; (iii) preliminary in vitro studies indicate that 5/5 candidates have therapeutic indices superior to that of Doxorubicin in MCF7 and SUM149 cancer cell lines. We briefly discuss the biological plausibility of the candidates at a molecular level in the context of the biological processes that they mediate. CONCLUSIONS: Our method appears to offer promise for the identification of multi-targeted drug candidates that can correct aberrant cellular functions. In particular the computational performance exceeded that of other CMap-based methods, and in vitro experiments indicate that 5/5 candidates have therapeutic indices superior to that of Doxorubicin in MCF7 and SUM149 cancer cell lines. The approach has the potential to provide a more efficient drug discovery pipeline.

Visualization of Metabolic Interaction Networks in Microbial Communities Using VisANT 5.0.

The complexity of metabolic networks in microbial communities poses an unresolved visualization and interpretation challenge. We address this challenge in the newly expanded version of a software tool for the analysis of biological networks, VisANT 5.0. We focus in particular on facilitating the visual exploration of metabolic interaction between microbes in a community, e.g. as predicted by COMETS (Computation of Microbial Ecosystems in Time and Space), a dynamic stoichiometric modeling framework. Using VisANT's unique metagraph implementation, we show how one can use VisANT 5.0 to explore different time-dependent ecosystem-level metabolic networks. In particular, we analyze the metabolic interaction network between two bacteria previously shown to display an obligate cross-feeding interdependency. In addition, we illustrate how a putative minimal gut microbiome community could be represented in our framework, making it possible to highlight interactions across multiple coexisting species. We envisage that the "symbiotic layout" of VisANT can be employed as a general tool for the analysis of metabolism in complex microbial communities as well as heterogeneous human tissues. VisANT is freely available at: http://visant.bu.edu and COMETS at http://comets.bu.edu.

COMBREX-DB: an experiment centered database of protein function: knowledge, predictions and knowledge gaps.

The COMBREX database (COMBREX-DB; combrex.bu.edu) is an online repository of information related to (i) experimentally determined protein function, (ii) predicted protein function, (iii) relationships among proteins of unknown function and various types of experimental data, including molecular function, protein structure, and associated phenotypes. The database was created as part of the novel COMBREX (COMputational BRidges to EXperiments) effort aimed at accelerating the rate of gene function validation. It currently holds information on ∼ 3.3 million known and predicted proteins from over 1000 completely sequenced bacterial and archaeal genomes. The database also contains a prototype recommendation system for helping users identify those proteins whose experimental determination of function would be most informative for predicting function for other proteins within protein families. The emphasis on documenting experimental evidence for function predictions, and the prioritization of uncharacterized proteins for experimental testing distinguish COMBREX from other publicly available microbial genomics resources. This article describes updates to COMBREX-DB since an initial description in the 2011 NAR Database Issue.

Mutated Pathways as a Guide to Adjuvant Therapy Treatments for Breast Cancer.

Adjuvant therapy following breast cancer surgery generally consists of either a course of chemotherapy, if the cancer lacks hormone receptors, or a course of hormonal therapy, otherwise. Here, we report a correlation between adjuvant strategy and mutated pathway patterns. In particular, we find that for breast cancer patients, pathways enriched in nonsynonymous mutations in the chemotherapy group are distinct from those of the hormonal therapy group. We apply a recently developed method that identifies collaborative pathway groups for hormone and chemotherapy patients. A collaborative group of pathways is one in which each member is altered in the same-generally large-number of samples. In particular, we find the following: (i) a chemotherapy group consisting of three pathways and a hormone therapy group consisting of 20, the members of the two groups being mutually exclusive; (ii) each group is highly enriched in breast cancer drivers; and (iii) the pathway groups are correlates of subtype-based therapeutic recommendations. These results suggest that patient profiling using these pathway groups can potentially enable the development of personalized treatment plans that may be more accurate and specific than those currently available.

Evaluation and integration of cancer gene classifiers: identification and ranking of plausible drivers.

The number of mutated genes in cancer cells is far larger than the number of mutations that drive cancer. The difficulty this creates for identifying relevant alterations has stimulated the development of various computational approaches to distinguishing drivers from bystanders. We develop and apply an ensemble classifier (EC) machine learning method, which integrates 10 classifiers that are publically available, and apply it to breast and ovarian cancer. In particular we find the following: (1) Using both standard and non-standard metrics, EC almost always outperforms single method classifiers, often by wide margins. (2) Of the 50 highest ranked genes for breast (ovarian) cancer, 34 (30) are associated with other cancers in either the OMIM, CGC or NCG database (P < 10(-22)). (3) Another 10, for both breast and ovarian cancer, have been identified by GWAS studies. (4) Several of the remaining genes--including a protein kinase that regulates the Fra-1 transcription factor which is overexpressed in ER negative breast cancer cells; and Fyn, which is overexpressed in pancreatic and prostate cancer, among others--are biologically plausible. Biological implications are briefly discussed. Source codes and detailed results are available at http://www.visantnet.org/misi/driver_integration.zip.

Using VisANT to Analyze Networks.

VisANT is a Web-based workbench for the integrative analysis of biological networks with unique features such as exploratory navigation of interaction network and multi-scale visualization and inference with integrated hierarchical knowledge. It provides functionalities for convenient construction, visualization, and analysis of molecular and higher order networks based on functional (e.g., expression profiles, phylogenetic profiles) and physical (e.g., yeast two-hybrid, chromatin-immunoprecipitation and drug target) relations from either the Predictome database or user-defined data sets. Analysis capabilities include network structure analysis, overrepresentation analysis, expression enrichment analysis etc. Additionally, network can be saved, accessed, and shared online. VisANT is able to develop and display meta-networks for meta-nodes that are structural complexes or pathways or any kind of subnetworks. Further, VisANT supports a growing number of standard exchange formats and database referencing standards, e.g., PSI-MI, KGML, BioPAX, SBML(in progress) Multiple species are supported to the extent that interactions or associations are available (i.e., public datasets or Predictome database).

Identification of collaborative driver pathways in breast cancer.

BACKGROUND: An important challenge in cancer biology is to computationally screen mutations in cancer cells, separating those that might drive cancer initiation and progression, from the much larger number of bystanders. Since mutations are large in number and diverse in type, the frequency of any particular mutation pattern across a set of samples is low. This makes statistical distinctions and reproducibility across different populations difficult to establish. RESULTS: In this paper we develop a novel method that promises to partially ameliorate these problems. The basic idea is although mutations are highly heterogeneous and vary from one sample to another, the processes that are disrupted when cells undergo transformation tend to be invariant across a population for a particular cancer or cancer subtype. Specifically, we focus on finding mutated pathway-groups that are invariant across samples of breast cancer subtypes. The identification of informative pathway-groups consists of two steps. The first is identification of pathways significantly enriched in genes containing non-synonymous mutations; the second uses pathways so identified to find groups that are functionally related in the largest number of samples. An application to 4 subtypes of breast cancer identified pathway-groups that can highly explicate a particular subtype and rich in processes associated with transformation. CONCLUSIONS: In contrast to previous methods that identify pathways across a set of samples without any further validation, we show that mutated pathway-groups can be found in each breast cancer subtype and that such groups are invariant across the majority of samples. The algorithm is available at http://www.visantnet.org/misi/MUDPAC.zip.

Functional characterization of breast cancer using pathway profiles.

BACKGROUND: The molecular characteristics of human diseases are often represented by a list of genes termed "signature genes". A significant challenge facing this approach is that of reproducibility: signatures developed on a set of patients may fail to perform well on different sets of patients. As diseases are resulted from perturbed cellular functions, irrespective of the particular genes that contribute to the function, it may be more appropriate to characterize diseases based on these perturbed cellular functions. METHODS: We proposed a profile-based approach to characterize a disease using a binary vector whose elements indicate whether a given function is perturbed based on the enrichment analysis of expression data between normal and tumor tissues. Using breast cancer and its four primary clinically relevant subtypes as examples, this approach is evaluated based on the reproducibility, accuracy and resolution of the resulting pathway profiles. RESULTS: Pathway profiles for breast cancer and its subtypes are constructed based on data obtained from microarray and RNA-Seq data sets provided by The Cancer Genome Atlas (TCGA), and an additional microarray data set provided by The European Genome-phenome Archive (EGA). An average reproducibility of 68% is achieved between different data sets (TCGA microarray vs. EGA microarray data) and 67% average reproducibility is achieved between different technologies (TCGA microarray vs. TCGA RNA-Seq data). Among the enriched pathways, 74% of them are known to be associated with breast cancer or other cancers. About 40% of the identified pathways are enriched in all four subtypes, with 4, 2, 4, and 7 pathways enriched only in luminal A, luminal B, triple-negative, and HER2+ subtypes, respectively. Comparison of profiles between subtypes, as well as other diseases, shows that luminal A and luminal B subtypes are more similar to the HER2+ subtype than to the triple-negative subtype, and subtypes of breast cancer are more likely to be closer to each other than to other diseases. CONCLUSIONS: Our results demonstrate that pathway profiles can successfully characterize both common and distinct functional characteristics of four subtypes of breast cancer and other related diseases, with acceptable reproducibility, high accuracy and reasonable resolution.

VisANT 4.0: Integrative network platform to connect genes, drugs, diseases and therapies.

With the rapid accumulation of our knowledge on diseases, disease-related genes and drug targets, network-based analysis plays an increasingly important role in systems biology, systems pharmacology and translational science. The new release of VisANT aims to provide new functions to facilitate the convenient network analysis of diseases, therapies, genes and drugs. With improved understanding of the mechanisms of complex diseases and drug actions through network analysis, novel drug methods (e.g., drug repositioning, multi-target drug and combination therapy) can be designed. More specifically, the new update includes (i) integrated search and navigation of disease and drug hierarchies; (ii) integrated disease-gene, therapy-drug and drug-target association to aid the network construction and filtering; (iii) annotation of genes/drugs using disease/therapy information; (iv) prediction of associated diseases/therapies for a given set of genes/drugs using enrichment analysis; (v) network transformation to support construction of versatile network of drugs, genes, diseases and therapies; (vi) enhanced user interface using docking windows to allow easy customization of node and edge properties with build-in legend node to distinguish different node type. VisANT is freely available at: http://visant.bu.edu.

Analysis strategy of protein-protein interaction networks.

Protein interactions, as well as the networks they formed, play a key role in many cellular processes and the distortion of the protein interacting interfaces may lead to the development of many diseases. In this chapter, we will briefly introduce the background knowledge of the protein-protein interaction, followed by the detailed explanation of varied analysis-from basic to advanced, as well as related tools and databases. VisANT (http://visant.bu.edu)-a free Web-based software platform for the integrative visualization, mining, analysis, and modeling of the biological networks-will be used as a main tool for all examples used in this section.

Using functional signatures to identify repositioned drugs for breast, myelogenous leukemia and prostate cancer.

The cost and time to develop a drug continues to be a major barrier to widespread distribution of medication. Although the genomic revolution appears to have had little impact on this problem, and might even have exacerbated it because of the flood of additional and usually ineffective leads, the emergence of high throughput resources promises the possibility of rapid, reliable and systematic identification of approved drugs for originally unintended uses. In this paper we develop and apply a method for identifying such repositioned drug candidates against breast cancer, myelogenous leukemia and prostate cancer by looking for inverse correlations between the most perturbed gene expression levels in human cancer tissue and the most perturbed expression levels induced by bioactive compounds. The method uses variable gene signatures to identify bioactive compounds that modulate a given disease. This is in contrast to previous methods that use small and fixed signatures. This strategy is based on the observation that diseases stem from failed/modified cellular functions, irrespective of the particular genes that contribute to the function, i.e., this strategy targets the functional signatures for a given cancer. This function-based strategy broadens the search space for the effective drugs with an impressive hit rate. Among the 79, 94 and 88 candidate drugs for breast cancer, myelogenous leukemia and prostate cancer, 32%, 13% and 17% respectively are either FDA-approved/in-clinical-trial drugs, or drugs with suggestive literature evidences, with an FDR of 0.01. These findings indicate that the method presented here could lead to a substantial increase in efficiency in drug discovery and development, and has potential application for the personalized medicine.

Gene set enrichment analysis: performance evaluation and usage guidelines.

A central goal of biology is understanding and describing the molecular basis of plasticity: the sets of genes that are combinatorially selected by exogenous and endogenous environmental changes, and the relations among the genes. The most viable current approach to this problem consists of determining whether sets of genes are connected by some common theme, e.g. genes from the same pathway are overrepresented among those whose differential expression in response to a perturbation is most pronounced. There are many approaches to this problem, and the results they produce show a fair amount of dispersion, but they all fall within a common framework consisting of a few basic components. We critically review these components, suggest best practices for carrying out each step, and propose a voting method for meeting the challenge of assessing different methods on a large number of experimental data sets in the absence of a gold standard.

COMBREX: a project to accelerate the functional annotation of prokaryotic genomes.

COMBREX (http://combrex.bu.edu) is a project to increase the speed of the functional annotation of new bacterial and archaeal genomes. It consists of a database of functional predictions produced by computational biologists and a mechanism for experimental biochemists to bid for the validation of those predictions. Small grants are available to support successful bids.

The BioPAX community standard for pathway data sharing.

Biological Pathway Exchange (BioPAX) is a standard language to represent biological pathways at the molecular and cellular level and to facilitate the exchange of pathway data. The rapid growth of the volume of pathway data has spurred the development of databases and computational tools to aid interpretation; however, use of these data is hampered by the current fragmentation of pathway information across many databases with incompatible formats. BioPAX, which was created through a community process, solves this problem by making pathway data substantially easier to collect, index, interpret and share. BioPAX can represent metabolic and signaling pathways, molecular and genetic interactions and gene regulation networks. Using BioPAX, millions of interactions, organized into thousands of pathways, from many organisms are available from a growing number of databases. This large amount of pathway data in a computable form will support visualization, analysis and biological discovery.

Identification of functional modules that correlate with phenotypic difference: the influence of network topology.

One of the important challenges to post-genomic biology is relating observed phenotypic alterations to the underlying collective alterations in genes. Current inferential methods, however, invariably omit large bodies of information on the relationships between genes. We present a method that takes account of such information - expressed in terms of the topology of a correlation network - and we apply the method in the context of current procedures for gene set enrichment analysis.

Genome-wide prioritization of disease genes and identification of disease-disease associations from an integrated human functional linkage network.

We integrate 16 genomic features to construct an evidence-weighted functional-linkage network comprising 21,657 human genes. The functional-linkage network is used to prioritize candidate genes for 110 diseases, and to reliably disclose hidden associations between disease pairs having dissimilar phenotypes, such as hypercholesterolemia and Alzheimer's disease. Many of these disease-disease associations are supported by epidemiology, but with no previous genetic basis. Such associations can drive novel hypotheses on molecular mechanisms of diseases and therapies.

VisANT 3.5: multi-scale network visualization, analysis and inference based on the gene ontology.

Despite its wide usage in biological databases and applications, the role of the gene ontology (GO) in network analysis is usually limited to functional annotation of genes or gene sets with auxiliary information on correlations ignored. Here, we report on new capabilities of VisANT--an integrative software platform for the visualization, mining, analysis and modeling of the biological networks--which extend the application of GO in network visualization, analysis and inference. The new VisANT functions can be classified into three categories. (i) Visualization: a new tree-based browser allows visualization of GO hierarchies. GO terms can be easily dropped into the network to group genes annotated under the term, thereby integrating the hierarchical ontology with the network. This facilitates multi-scale visualization and analysis. (ii) Flexible annotation schema: in addition to conventional methods for annotating network nodes with the most specific functional descriptions available, VisANT also provides functions to annotate genes at any customized level of abstraction. (iii) Finding over-represented GO terms and expression-enriched GO modules: two new algorithms have been implemented as VisANT plugins. One detects over-represented GO annotations in any given sub-network and the other finds the GO categories that are enriched in a specified phenotype or perturbed dataset. Both algorithms take account of network topology (i.e. correlations between genes based on various sources of evidence). VisANT is freely available at http://visant.bu.edu.