GATA4 controls regionalization of tissue immunity and commensal-driven immunopathology.

There is growing recognition that regionalization of bacterial colonization and immunity along the intestinal tract has an important role in health and disease. Yet, the mechanisms underlying intestinal regionalization and its dysregulation in disease are not well understood. This study found that regional epithelial expression of the transcription factor GATA4 controls bacterial colonization and inflammatory tissue immunity in the proximal small intestine by regulating retinol metabolism and luminal IgA. Furthermore, in mice without jejunal GATA4 expression, the commensal segmented filamentous bacteria promoted pathogenic inflammatory immune responses that disrupted barrier function and increased mortality upon Citrobacter rodentium infection. In celiac disease patients, low GATA4 expression was associated with metabolic alterations, mucosal Actinobacillus, and increased IL-17 immunity. Taken together, these results reveal broad impacts of GATA4-regulated intestinal regionalization on bacterial colonization and tissue immunity, highlighting an elaborate interdependence of intestinal metabolism, immunity, and microbiota in homeostasis and disease.

Permissive hypercapnia and hypercapnic hypoxia inhibit signaling pathways of neuronal apoptosis in ischemic/hypoxic rats.

INTRODUCTION: In the present study, we aimed to test the hypothesis that hypercapnia, independently and/or in combination with hypoxia, can activate signaling pathways related to the inhibition of proapoptotic (caspase-dependent and caspase-independent) factors and the induction of antiapoptotic factors in facilitating adaptation to hypoxia/ischemia. MATERIALS AND METHODS: Following exposure to permissive hypercapnia and/or normobaric hypoxia, the degree of apoptosis was evaluated in experimental ischemia models in vivo and in vitro. The percentages of caspase-3, apoptosis-inducing factor (AIF), Bax, and Bcl-2 in astrocytes and neurons derived from male Wistar rats were also calculated. In vitro, cells were subjected to various types of respiratory exposure (hypoxia and/or hypercapnia for 24 or 12 h) as well as further sublethal chemical hypoxia. The percentages of these molecules in nerve cells in the ischemic penumbra of the brain after photothrombotic injury were also calculated. RESULTS: The degree of apoptosis was found to decrease in ischemic penumbra, mostly due to the hypercapnic component. It was also discovered that the levels of caspase-3, AIF, and Bax decreased in this region, whereas the Bcl-2 levels increased following exposure to hypercapnia and hypercapnic hypoxia. CONCLUSIONS: This integrative assessment of the rate of apoptosis/necrosis in astrocyte and neuron cultures shows that the combination of hypercapnia and hypoxia resulted in the maximum neuroprotective effect. The levels of apoptosis mediators in astrocyte and neuron cultures were calculated after modeling chemical hypoxia in vitro. These results show that the exposure models where permissive hypercapnia and normobaric hypoxia were combined also had the most pronounced inhibitory effects on apoptotic signaling pathways.

Blood-Brain Barrier Breakdown in Stress and Neurodegeneration: Biochemical Mechanisms and New Models for Translational Research.

Blood-brain barrier (BBB) is a structural and functional element of the neurovascular unit (NVU), which includes cells of neuronal, glial, and endothelial nature. The main functions of NVU include maintenance of the control of metabolism and chemical homeostasis in the brain tissue, ensuring adequate blood flow in active regions, regulation of neuroplasticity processes, which is realized through intercellular interactions under normal conditions, under stress, in neurodegeneration, neuroinfection, and neurodevelopmental diseases. Current versions of the BBB and NVU models, static and dynamic, have significantly expanded research capabilities, but a number of issues remain unresolved, in particular, personification of the models for a patient. In addition, application of both static and dynamic models has an important problem associated with the difficulty in reproducing pathophysiological mechanisms responsible for the damage of the structural and functional integrity of the barrier in the diseases of the central nervous system. More knowledge on the cellular and molecular mechanisms of BBB and NVU damage in pathology is required to solve this problem. This review discusses current state of the cellular and molecular mechanisms that control BBB permeability, pathobiochemical mechanisms and manifestations of BBB breakdown in stress and neurodegenerative diseases, as well as the problems and prospects of creating in vitro BBB and NVU models for translational studies in neurology and neuropharmacology. Deciphering BBB (patho)physiology will open up new opportunities for further development in the related areas of medicine such as regenerative medicine, neuropharmacology, and neurorehabilitation.

Interplay between viruses and bacterial microbiota in cancer development.

During the last few decades we have become accustomed to the idea that viruses can cause tumors. It is much less considered and discussed, however, that most people infected with oncoviruses will never develop cancer. Therefore, the genetic and environmental factors that tip the scales from clearance of viral infection to development of cancer are currently an area of active investigation. Microbiota has recently emerged as a potentially critical factor that would affect this balance by increasing or decreasing the ability of viral infection to promote carcinogenesis. In this review, we provide a model of microbiome contribution to the development of oncogenic viral infections and viral associated cancers, give examples of this process in human tumors, and describe the challenges that prevent progress in the field as well as their potential solutions.

Blood-Brain Barrier and Neurovascular Unit In Vitro Models for Studying Mitochondria-Driven Molecular Mechanisms of Neurodegeneration.

Pathophysiology of chronic neurodegeneration is mainly based on complex mechanisms related to aberrant signal transduction, excitation/inhibition imbalance, excitotoxicity, synaptic dysfunction, oxidative stress, proteotoxicity and protein misfolding, local insulin resistance and metabolic dysfunction, excessive cell death, development of glia-supported neuroinflammation, and failure of neurogenesis. These mechanisms tightly associate with dramatic alterations in the structure and activity of the neurovascular unit (NVU) and the blood-brain barrier (BBB). NVU is an ensemble of brain cells (brain microvessel endothelial cells (BMECs), astrocytes, pericytes, neurons, and microglia) serving for the adjustment of cell-to-cell interactions, metabolic coupling, local microcirculation, and neuronal excitability to the actual needs of the brain. The part of the NVU known as a BBB controls selective access of endogenous and exogenous molecules to the brain tissue and efflux of metabolites to the blood, thereby providing maintenance of brain chemical homeostasis critical for efficient signal transduction and brain plasticity. In Alzheimer's disease, mitochondria are the target organelles for amyloid-induced neurodegeneration and alterations in NVU metabolic coupling or BBB breakdown. In this review we discuss understandings on mitochondria-driven NVU and BBB dysfunction, and how it might be studied in current and prospective NVU/BBB in vitro models for finding new approaches for the efficient pharmacotherapy of Alzheimer's disease.

CVID enteropathy is characterized by exceeding low mucosal IgA levels and interferon-driven inflammation possibly related to the presence of a pathobiont.

Common variable immunodeficiency (CVID), the most common symptomatic primary antibody deficiency, is accompanied in some patients by a duodenal inflammation and malabsorption syndrome known as CVID enteropathy (E-CVID).The goal of this study was to investigate the immunological abnormalities in CVID patients that lead to enteropathy as well as the contribution of intestinal microbiota to this process.We found that, in contrast to noE-CVID patients (without enteropathy), E-CVID patients have exceedingly low levels of IgA in duodenal tissues. In addition, using transkingdom network analysis of the duodenal microbiome, we identified Acinetobacter baumannii as a candidate pathobiont in E-CVID. Finally, we found that E-CVID patients exhibit a pronounced activation of immune genes and down-regulation of epithelial lipid metabolism genes. We conclude that in the virtual absence of mucosal IgA, pathobionts such as A. baumannii, may induce inflammation that re-directs intestinal molecular pathways from lipid metabolism to immune processes responsible for enteropathy.

The inhibitory effect of LPS on the expression of GPR81 lactate receptor in blood-brain barrier model in vitro.

BACKGROUND: Lipopolysaccharide (LPS) is one of the main constituents of the cell wall of gram-negative bacteria. As an endotoxin, LPS induces neuroinflammation, which is associated with the blood-brain barrier impairment. Lactate is a metabolite with some significant physiological functions within the neurovascular unit/blood-brain barrier (BBB). Accumulation of extracellular and cerebrospinal fluid lactate is a specific feature of bacterial meningitis. However, the role of lactate production, transport, and sensing by lactate receptors GPR81 in the pathogenesis of bacterial neuroinflammation is still unknown. METHODS: In this study, we analyzed effects of LPS on the expression of GPR81 and MCT-1 and proliferation of cerebral endothelial cells in the BBB model in vitro. We used molecular profiling methods to measure the expression of GPR81, MCT-1, IL-1ß, and Ki67 in the cerebral endothelium after treatment with different concentrations of LPS followed by measuring the level of extracellular lactate, transendothelial electric resistance, and permeability of the endothelial cell layer. RESULTS: Our findings showed that exposure to LPS results in neuroinflammatory changes associated with decreased expression of GPR81 and MCT-1 in endothelial cells, as well as overproduction of IL-1ß and elevation of lactate concentrations in the extracellular space in a dose-dependent manner. LPS treatment reduced JAM tight junction protein expression in cerebral endothelial cells and altered BBB structural integrity in vitro. CONCLUSION: The impairment of lactate reception and transport might contribute to the alterations of BBB structural and functional integrity caused by LPS-mediated neuroinflammation.

Uncovering effects of antibiotics on the host and microbiota using transkingdom gene networks.

OBJECTIVE: Despite widespread use of antibiotics for the treatment of life-threatening infections and for research on the role of commensal microbiota, our understanding of their effects on the host is still very limited. DESIGN: Using a popular mouse model of microbiota depletion by a cocktail of antibiotics, we analysed the effects of antibiotics by combining intestinal transcriptome together with metagenomic analysis of the gut microbiota. In order to identify specific microbes and microbial genes that influence the host phenotype in antibiotic-treated mice, we developed and applied analysis of the transkingdom network. RESULTS: We found that most antibiotic-induced alterations in the gut can be explained by three factors: depletion of the microbiota; direct effects of antibiotics on host tissues and the effects of remaining antibiotic-resistant microbes. Normal microbiota depletion mostly led to downregulation of different aspects of immunity. The two other factors (antibiotic direct effects on host tissues and antibiotic-resistant microbes) primarily inhibited mitochondrial gene expression and amounts of active mitochondria, increasing epithelial cell death. By reconstructing and analysing the transkingdom network, we discovered that these toxic effects were mediated by virulence/quorum sensing in antibiotic-resistant bacteria, a finding further validated using in vitro experiments. CONCLUSIONS: In addition to revealing mechanisms of antibiotic-induced alterations, this study also describes a new bioinformatics approach that predicts microbial components that regulate host functions and establishes a comprehensive resource on what, why and how antibiotics affect the gut in a widely used mouse model of microbiota depletion by antibiotics.

Establishment of neurogenic microenvironment in the neurovascular unit: the connexin 43 story.

Connexins (Cx) play an important role in the coordination of intercellular communication, and autocrine and paracrine regulation of cells within the neurovascular unit (NVU). Gap junctional mechanisms control proliferation and differentiation processes underlying neurogenesis and angiogenesis in the brain. Cx43 possesses some unique properties [the ability to form either intercellular channels permeable for regulatory molecules and ions or hemichannels open to the extracellular space to provide release of cell metabolites; functional coupling with nicotinamide adenine dinucleotide (NAD+)-consuming and NAD+-dependent enzymatic processes] which may be of great importance for the fate of the stem cells. Dynamic changes in Cx43 expression are associated with different stages of brain cells development either at embryonic or adult periods of ontogenesis. This review summarizes recent data on Cx43-controlled neurogenesis in the context of NVU development and functioning. Understanding the molecular mechanisms of gap junctional intercellular communication will support translational studies focused on the development of regeneration-based approaches for the therapy of central nervous system pathology.

Bridging immunity and lipid metabolism by gut microbiota.

The human gut is a unique organ in which hundreds of different microbial species find their habitat and in which different host physiologic functions, such as digestion, nutrition, and immunity, coexist. Although all these players were studied separately for decades, recently, there has been an explosion of studies demonstrating the essential role for interactions between these components in gut function. Furthermore, new systems biology methods provide essential tools to study this complex system as a whole and to identify key elements that define the crosstalk between the gut microbiota, immunity, and metabolism. This review is devoted to several human diseases resulting from the disruption in this crosstalk, including immunodeficiency-associated and environmental enteropathies, celiac disease, inflammatory bowel disease, and obesity. We describe findings in experimental models of these diseases and in germ-free animals that help us understand the mechanisms and test new therapeutic strategies. We also discuss current challenges that the field is facing and propose that a new generation of antibiotics, prebiotics, and probiotics coupled with novel, systems biology-driven diagnostics will provide the basis for future personalized therapy.

Transkingdom Network Analysis (TkNA): a systems framework for inferring causal factors underlying host-microbiota and other multi-omic interactions.

We present Transkingdom Network Analysis (TkNA), a unique causal-inference analytical framework that offers a holistic view of biological systems by integrating data from multiple cohorts and diverse omics types. TkNA helps to decipher key players and mechanisms governing host-microbiota (or any multi-omic data) interactions in specific conditions or diseases. TkNA reconstructs a network that represents a statistical model capturing the complex relationships between different omics in the biological system. It identifies robust and reproducible patterns of fold change direction and correlation sign across several cohorts to select differential features and their per-group correlations. The framework then uses causality-sensitive metrics, statistical thresholds and topological criteria to determine the final edges forming the transkingdom network. With the subsequent network's topological features, TkNA identifies nodes controlling a given subnetwork or governing communication between kingdoms and/or subnetworks. The computational time for the millions of correlations necessary for network reconstruction in TkNA typically takes only a few minutes, varying with the study design. Unlike most other multi-omics approaches that find only associations, TkNA focuses on establishing causality while accounting for the complex structure of multi-omic data. It achieves this without requiring huge sample sizes. Moreover, the TkNA protocol is user friendly, requiring minimal installation and basic familiarity with Unix. Researchers can access the TkNA software at https://github.com/CAnBioNet/TkNA/ .

Intestinal IL-22RA1 signaling regulates intrinsic and systemic lipid and glucose metabolism to alleviate obesity-associated disorders.

IL-22 is critical for ameliorating obesity-induced metabolic disorders. However, it is unknown where IL-22 acts to mediate these outcomes. Here we examine the importance of tissue-specific IL-22RA1 signaling in mediating long-term high fat diet (HFD) driven metabolic disorders. To do so, we generated intestinal epithelium-, liver-, and white adipose tissue (WAT)-specific Il22ra1 knockout and littermate control mice. Intestinal epithelium- and liver-specific IL-22RA1 signaling upregulated systemic glucose metabolism. Intestinal IL-22RA1 signaling also mediated liver and WAT metabolism in a microbiota-dependent manner. We identified an association between Oscillibacter and elevated WAT inflammation, likely induced by Mmp12 expressing macrophages. Mechanistically, transcription of intestinal lipid metabolism genes is regulated by IL-22 and potentially IL-22-induced IL-18. Lastly, we show that Paneth cell-specific IL-22RA1 signaling, in part, mediates systemic glucose metabolism after HFD. Overall, these results elucidate a key role of intestinal epithelium-specific IL-22RA1 signaling in regulating intestinal metabolism and alleviating systemic obesity-associated disorders.

The C-SHIFT Algorithm for Normalizing Covariances.

Omics technologies are powerful tools for analyzing patterns in gene expression data for thousands of genes. Due to a number of systematic variations in experiments, the raw gene expression data is often obfuscated by undesirable technical noises. Various normalization techniques were designed in an attempt to remove these non-biological errors prior to any statistical analysis. One of the reasons for normalizing data is the need for recovering the covariance matrix used in gene network analysis. In this paper, we introduce a novel normalization technique, called the covariance shift (C-SHIFT) method. This normalization algorithm uses optimization techniques together with the blessing of dimensionality philosophy and energy minimization hypothesis for covariance matrix recovery under additive noise (in biology, known as the bias). Thus, it is perfectly suited for the analysis of logarithmic gene expression data. Numerical experiments on synthetic data demonstrate the method's advantage over the classical normalization techniques. Namely, the comparison is made with Rank, Quantile, cyclic LOESS (locally estimated scatterplot smoothing), and MAD (median absolute deviation) normalization methods. We also evaluate the performance of C-SHIFT algorithm on real biological data.

Transkingdom Network Analysis (TkNA): a systems framework for inferring causal factors underlying host-microbiota and other multi-omic interactions.

Technological advances have generated tremendous amounts of high-throughput omics data. Integrating data from multiple cohorts and diverse omics types from new and previously published studies can offer a holistic view of a biological system and aid in deciphering its critical players and key mechanisms. In this protocol, we describe how to use Transkingdom Network Analysis (TkNA), a unique causal-inference analytical framework that can perform meta-analysis of cohorts and detect master regulators among measured parameters that govern pathological or physiological responses of host-microbiota (or any multi-omic data) interactions in a particular condition or disease. TkNA first reconstructs the network that represents a statistical model capturing the complex relationships between the different omics of the biological system. Here, it selects differential features and their per-group correlations by identifying robust and reproducible patterns of fold change direction and sign of correlation across several cohorts. Next, a causality-sensitive metric, statistical thresholds, and a set of topological criteria are used to select the final edges that form the transkingdom network. The second part of the analysis involves interrogating the network. Using the network's local and global topology metrics, it detects nodes that are responsible for control of given subnetwork or control of communication between kingdoms and/or subnetworks. The underlying basis of the TkNA approach involves fundamental principles including laws of causality, graph theory and information theory. Hence, TkNA can be used for causal inference via network analysis of any host and/or microbiota multi-omics data. This quick and easy-to-run protocol requires very basic familiarity with the Unix command-line environment.

Early transcriptome changes associated with western diet induced NASH in Ldlr-/- mice points to activation of hepatic macrophages and an acute phase response.

Background: Nonalcoholic fatty liver disease (NAFLD) is a global health problem. Identifying early gene indicators contributing to the onset and progression of NAFLD has the potential to develop novel targets for early therapeutic intervention. We report on the early and late transcriptomic signatures of western diet (WD)-induced nonalcoholic steatohepatitis (NASH) in female and male Ldlr-/- mice, with time-points at 1 week and 40 weeks on the WD. Control Ldlr-/- mice were maintained on a low-fat diet (LFD) for 1 and 40 weeks. Methods: The approach included quantitation of anthropometric and hepatic histology markers of disease as well as the hepatic transcriptome. Results: Only mice fed the WD for 40 weeks revealed evidence of NASH, i.e., hepatic steatosis and fibrosis. RNASeq transcriptome analysis, however, revealed multiple cell-specific changes in gene expression after 1 week that persisted to 40 weeks on the WD. These early markers of disease include induction of acute phase response (Saa1-2, Orm2), fibrosis (Col1A1, Col1A2, TGFß) and NASH associated macrophage (NAM, i.e., Trem2 high, Mmp12 low). We also noted the induction of transcripts associated with metabolic syndrome, including Mmp12, Trem2, Gpnmb, Lgals3 and Lpl. Finally, 1 week of WD feeding was sufficient to significantly induce TNFα, a cytokine involved in both hepatic and systemic inflammation. Conclusion: This study revealed early onset changes in the hepatic transcriptome that develop well before any anthropometric or histological evidence of NALFD or NASH and pointed to cell-specific targeting for the prevention of disease progression.

Multi-omic network analysis identified betacellulin as a novel target of omega-3 fatty acid attenuation of western diet-induced nonalcoholic steatohepatitis.

Clinical and preclinical studies established that supplementing diets with ω3 polyunsaturated fatty acids (PUFA) can reduce hepatic dysfunction in nonalcoholic steatohepatitis (NASH) but molecular underpinnings of this action were elusive. Herein, we used multi-omic network analysis that unveiled critical molecular pathways involved in ω3 PUFA effects in a preclinical mouse model of western diet induced NASH. Since NASH is a precursor of liver cancer, we also performed meta-analysis of human liver cancer transcriptomes that uncovered betacellulin as a key EGFR-binding protein upregulated in liver cancer and downregulated by ω3 PUFAs in animals and humans with NASH. We then confirmed that betacellulin acts by promoting proliferation of quiescent hepatic stellate cells, inducing transforming growth factor-ß2 and increasing collagen production. When used in combination with TLR2/4 agonists, betacellulin upregulated integrins in macrophages thereby potentiating inflammation and fibrosis. Taken together, our results suggest that suppression of betacellulin is one of the key mechanisms associated with anti-inflammatory and anti-fibrotic effects of ω3 PUFA on NASH.

Reproducibility of developmental neuroplasticity in in vitro brain tissue models.

The current prevalence of neurodevelopmental, neurodegenerative diseases, stroke and brain injury stimulates studies aimed to identify new molecular targets, to select the drug candidates, to complete the whole set of preclinical and clinical trials, and to implement new drugs into routine neurological practice. Establishment of protocols based on microfluidics, blood-brain barrier- or neurovascular unit-on-chip, and microphysiological systems allowed improving the barrier characteristics and analyzing the regulation of local microcirculation, angiogenesis, and neurogenesis. Reconstruction of key mechanisms of brain development and even some aspects of experience-driven brain plasticity would be helpful in the establishment of brain in vitro models with the highest degree of reliability. Activity, metabolic status and expression pattern of cells within the models can be effectively assessed with the protocols of system biology, cell imaging, and functional cell analysis. The next generation of in vitro models should demonstrate high scalability, 3D or 4D complexity, possibility to be combined with other tissues or cell types within the microphysiological systems, compatibility with bio-inks or extracellular matrix-like materials, achievement of adequate vascularization, patient-specific characteristics, and opportunity to provide high-content screening. In this review, we will focus on currently available and prospective brain tissue in vitro models suitable for experimental and preclinical studies with the special focus on models enabling 4D reconstruction of brain tissue for the assessment of brain development, brain plasticity, and drug kinetics.

Microbiota and adipocyte mitochondrial damage in type 2 diabetes are linked by Mmp12+ macrophages.

Microbiota contribute to the induction of type 2 diabetes by high-fat/high-sugar (HFHS) diet, but which organs/pathways are impacted by microbiota remain unknown. Using multiorgan network and transkingdom analyses, we found that microbiota-dependent impairment of OXPHOS/mitochondria in white adipose tissue (WAT) plays a primary role in regulating systemic glucose metabolism. The follow-up analysis established that Mmp12+ macrophages link microbiota-dependent inflammation and OXPHOS damage in WAT. Moreover, the molecular signature of Mmp12+ macrophages in WAT was associated with insulin resistance in obese patients. Next, we tested the functional effects of MMP12 and found that Mmp12 genetic deficiency or MMP12 inhibition improved glucose metabolism in conventional, but not in germ-free mice. MMP12 treatment induced insulin resistance in adipocytes. TLR2-ligands present in Oscillibacter valericigenes bacteria, which are expanded by HFHS, induce Mmp12 in WAT macrophages in a MYD88-ATF3-dependent manner. Thus, HFHS induces Mmp12+ macrophages and MMP12, representing a microbiota-dependent bridge between inflammation and mitochondrial damage in WAT and causing insulin resistance.

Intestinal microbiota signatures of clinical response and immune-related adverse events in melanoma patients treated with anti-PD-1.

Ample evidence indicates that the gut microbiome is a tumor-extrinsic factor associated with antitumor response to anti-programmed cell death protein-1 (PD-1) therapy, but inconsistencies exist between published microbial signatures associated with clinical outcomes. To resolve this, we evaluated a new melanoma cohort, along with four published datasets. Time-to-event analysis showed that baseline microbiota composition was optimally associated with clinical outcome at approximately 1 year after initiation of treatment. Meta-analysis and other bioinformatic analyses of the combined data show that bacteria associated with favorable response are confined within the Actinobacteria phylum and the Lachnospiraceae/Ruminococcaceae families of Firmicutes. Conversely, Gram-negative bacteria were associated with an inflammatory host intestinal gene signature, increased blood neutrophil-to-lymphocyte ratio, and unfavorable outcome. Two microbial signatures, enriched for Lachnospiraceae spp. and Streptococcaceae spp., were associated with favorable and unfavorable clinical response, respectively, and with distinct immune-related adverse effects. Despite between-cohort heterogeneity, optimized all-minus-one supervised learning algorithms trained on batch-corrected microbiome data consistently predicted outcomes to programmed cell death protein-1 therapy in all cohorts. Gut microbial communities (microbiotypes) with nonuniform geographical distribution were associated with favorable and unfavorable outcomes, contributing to discrepancies between cohorts. Our findings shed new light on the complex interaction between the gut microbiome and response to cancer immunotherapy, providing a roadmap for future studies.

Construct and Compare Gene Coexpression Networks with DAPfinder and DAPview.

BACKGROUND: DAPfinder and DAPview are novel BRB-ArrayTools plug-ins to construct gene coexpression networks and identify significant differences in pairwise gene-gene coexpression between two phenotypes. RESULTS: Each significant difference in gene-gene association represents a Differentially Associated Pair (DAP). Our tools include several choices of filtering methods, gene-gene association metrics, statistical testing methods and multiple comparison adjustments. Network results are easily displayed in Cytoscape. Analyses of glioma experiments and microarray simulations demonstrate the utility of these tools. CONCLUSIONS: DAPfinder is a new friendly-user tool for reconstruction and comparison of biological networks.