Publications by authors named "Christopher C Overall"

28 Publications

  • Page 1 of 1

Mesenchymal stromal cells reprogram monocytes and macrophages with processing bodies.

Stem Cells 2021 Jan 9;39(1):115-128. Epub 2020 Nov 9.

Marcus Center for Cellular Cures, Duke University, Durham, North Carolina, USA.

Mesenchymal stromal cells (MSCs) are widely used in clinical trials because of their ability to modulate inflammation. The success of MSCs has been variable over 25 years, most likely due to an incomplete understanding of their mechanism. After MSCs are injected, they traffic to the lungs and other tissues where they are rapidly cleared. Despite being cleared, MSCs suppress the inflammatory response in the long term. Using human cord tissue-derived MSCs (hCT-MSCs), we demonstrated that hCT-MSCs directly interact and reprogram monocytes and macrophages. After engaging hCT-MSCs, monocytes and macrophages engulfed cytoplasmic components of live hCT-MSCs, then downregulated gene programs for antigen presentation and costimulation, and functionally suppressed the activation of helper T cells. We determined that low-density lipoprotein receptor-related proteins on monocytes and macrophages mediated the engulfment of hCT-MSCs. Since a large amount of cellular information can be packaged in cytoplasmic RNA processing bodies (p-bodies), we generated p-body deficient hCT-MSCs and confirmed that they failed to reprogram monocytes and macrophages in vitro and in vivo. hCT-MSCs suppressed an inflammatory response caused by a nasal lipopolysaccharide challenge. Although both control and p-body deficient hCT-MSCs were engulfed by infiltrating lung monocytes and macrophages, p-body deficient hCT-MSCs failed to suppress inflammation and downregulate MHC-II. Overall, we identified a novel mechanism by which hCT-MSCs indirectly suppressed a T-cell response by directly interacting and reprogramming monocytes and macrophages via p-bodies. The results of this study suggest a novel mechanism for how MSCs can reprogram the inflammatory response and have long-term effects to suppress inflammation.
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http://dx.doi.org/10.1002/stem.3292DOI Listing
January 2021

Experimental autoimmune encephalomyelitis is associated with changes of the microbiota composition in the gastrointestinal tract.

Sci Rep 2020 09 16;10(1):15183. Epub 2020 Sep 16.

Center for Brain Immunology and Glia, University of Virginia, Charlottesville, VA, 22908, USA.

The gut microbiome is known to be sensitive to changes in the immune system, especially during autoimmune diseases such as Multiple Sclerosis (MS). Our study examines the changes to the gut microbiome that occur during experimental autoimmune encephalomyelitis (EAE), an animal model for MS. We collected fecal samples at key stages of EAE progression and quantified microbial abundances with 16S V3-V4 amplicon sequencing. Our analysis of the data suggests that the abundance of commensal Lactobacillaceae decreases during EAE while other commensal populations belonging to the Clostridiaceae, Ruminococcaceae, and Peptostreptococcaceae families expand. Community analysis with microbial co-occurrence networks points to these three expanding taxa as potential mediators of gut microbiome dysbiosis. We also employed PICRUSt2 to impute MetaCyc Enzyme Consortium (EC) pathway abundances from the original microbial abundance data. From this analysis, we found that a number of imputed EC pathways responsible for the production of immunomodulatory compounds appear to be enriched in mice undergoing EAE. Our analysis and interpretation of results provides a detailed picture of the changes to the gut microbiome that are occurring throughout the course of EAE disease progression and helps to evaluate EAE as a viable model for gut dysbiosis in MS patients.
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http://dx.doi.org/10.1038/s41598-020-72197-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494894PMC
September 2020

Cost-effectiveness of combinatorial pharmacogenomic testing for depression from the Canadian public payer perspective.

Pharmacogenomics 2020 06 17;21(8):521-531. Epub 2020 Apr 17.

Myriad Genetics Inc., Salt Lake City, UT 84108, USA.

Evaluate the cost-effectiveness of combinatorial pharmacogenomic (PGx) testing, versus treatment as usual (TAU), to guide treatment for patients with depression, from the Canadian public healthcare system perspective. Clinical and economic data associated with depression were extracted from published literature. Clinical (quality-adjusted life years; QALYs) and economic (incremental cost-effectiveness ratio) outcomes were modeled using combinatorial PGx and TAU treatment strategies across a 5-year time horizon. With the combinatorial PGx strategy to guide treatment, patients were projected to gain 0.14-0.19 QALYs versus TAU. Accounting for test price, combinatorial PGx saved CAD $1,687-$3,056 versus TAU. Incremental cost-effectiveness ratios ranged from -$11,861 to -$16,124/QALY gained. Combinatorial PGx testing was more efficacious and less costly compared with the TAU for depression.
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http://dx.doi.org/10.2217/pgs-2020-0012DOI Listing
June 2020

The active contribution of OPCs to neuroinflammation is mediated by LRP1.

Acta Neuropathol 2020 02 24;139(2):365-382. Epub 2019 Sep 24.

Department of Neuroscience, Center for Brain Immunology and Glia, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA.

Oligodendrocyte progenitor cells (OPCs) account for about 5% of total brain and spinal cord cells, giving rise to myelinating oligodendrocytes that provide electrical insulation to neurons of the CNS. OPCs have also recently been shown to regulate inflammatory responses and glial scar formation, suggesting functions that extend beyond myelination. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifaceted phagocytic receptor that is highly expressed in several CNS cell types, including OPCs. Here, we have generated an oligodendroglia-specific knockout of LRP1, which presents with normal myelin development, but is associated with better outcomes in two animal models of demyelination (EAE and cuprizone). At a mechanistic level, LRP1 did not directly affect OPC differentiation into mature oligodendrocytes. Instead, animals lacking LRP1 in OPCs in the demyelinating CNS were characterized by a robust dampening of inflammation. In particular, LRP1-deficient OPCs presented with impaired antigen cross-presentation machinery, suggesting a failure to propagate the inflammatory response and thus promoting faster myelin repair and neuroprotection. Our study places OPCs as major regulators of neuroinflammation in an LRP1-dependent fashion.
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http://dx.doi.org/10.1007/s00401-019-02073-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994364PMC
February 2020

Publisher Correction: Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease.

Nature 2018 12;564(7734):E7

Center for Brain Immunology and Glia (BIG), University of Virginia, Charlottesville, VA, USA.

Change history: In this Article, Extended Data Fig. 9 was appearing as Fig. 2 in the HTML, and in Fig. 2, the panel labels 'n' and 'o' overlapped the figure; these errors have been corrected online.
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http://dx.doi.org/10.1038/s41586-018-0689-7DOI Listing
December 2018

Improving network inference algorithms using resampling methods.

BMC Bioinformatics 2018 Oct 12;19(1):376. Epub 2018 Oct 12.

Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, Washington, USA.

Background: Relatively small changes to gene expression data dramatically affect co-expression networks inferred from that data which, in turn, can significantly alter the subsequent biological interpretation. This error propagation is an underappreciated problem that, while hinted at in the literature, has not yet been thoroughly explored. Resampling methods (e.g. bootstrap aggregation, random subspace method) are hypothesized to alleviate variability in network inference methods by minimizing outlier effects and distilling persistent associations in the data. But the efficacy of the approach assumes the generalization from statistical theory holds true in biological network inference applications.

Results: We evaluated the effect of bootstrap aggregation on inferred networks using commonly applied network inference methods in terms of stability, or resilience to perturbations in the underlying expression data, a metric for accuracy, and functional enrichment of edge interactions.

Conclusion: Bootstrap aggregation results in improved stability and, depending on the size of the input dataset, a marginal improvement to accuracy assessed by each method's ability to link genes in the same functional pathway.
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http://dx.doi.org/10.1186/s12859-018-2402-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6186128PMC
October 2018

CNS lymphatic drainage and neuroinflammation are regulated by meningeal lymphatic vasculature.

Nat Neurosci 2018 10 17;21(10):1380-1391. Epub 2018 Sep 17.

Center for Brain Immunology and Glia (BIG), University of Virginia, Charlottesville, VA, USA.

Neuroinflammatory diseases, such as multiple sclerosis, are characterized by invasion of the brain by autoreactive T cells. The mechanism for how T cells acquire their encephalitogenic phenotype and trigger disease remains, however, unclear. The existence of lymphatic vessels in the meninges indicates a relevant link between the CNS and peripheral immune system, perhaps affecting autoimmunity. Here we demonstrate that meningeal lymphatics fulfill two critical criteria: they assist in the drainage of cerebrospinal fluid components and enable immune cells to enter draining lymph nodes in a CCR7-dependent manner. Unlike other tissues, meningeal lymphatic endothelial cells do not undergo expansion during inflammation, and they express a unique transcriptional signature. Notably, the ablation of meningeal lymphatics diminishes pathology and reduces the inflammatory response of brain-reactive T cells during an animal model of multiple sclerosis. Our findings demonstrate that meningeal lymphatics govern inflammatory processes and immune surveillance of the CNS and pose a valuable target for therapeutic intervention.
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http://dx.doi.org/10.1038/s41593-018-0227-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214619PMC
October 2018

Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease.

Nature 2018 08 25;560(7717):185-191. Epub 2018 Jul 25.

Center for Brain Immunology and Glia (BIG), University of Virginia, Charlottesville, VA, USA.

Ageing is a major risk factor for many neurological pathologies, but its mechanisms remain unclear. Unlike other tissues, the parenchyma of the central nervous system (CNS) lacks lymphatic vasculature and waste products are removed partly through a paravascular route. (Re)discovery and characterization of meningeal lymphatic vessels has prompted an assessment of their role in waste clearance from the CNS. Here we show that meningeal lymphatic vessels drain macromolecules from the CNS (cerebrospinal and interstitial fluids) into the cervical lymph nodes in mice. Impairment of meningeal lymphatic function slows paravascular influx of macromolecules into the brain and efflux of macromolecules from the interstitial fluid, and induces cognitive impairment in mice. Treatment of aged mice with vascular endothelial growth factor C enhances meningeal lymphatic drainage of macromolecules from the cerebrospinal fluid, improving brain perfusion and learning and memory performance. Disruption of meningeal lymphatic vessels in transgenic mouse models of Alzheimer's disease promotes amyloid-β deposition in the meninges, which resembles human meningeal pathology, and aggravates parenchymal amyloid-β accumulation. Meningeal lymphatic dysfunction may be an aggravating factor in Alzheimer's disease pathology and in age-associated cognitive decline. Thus, augmentation of meningeal lymphatic function might be a promising therapeutic target for preventing or delaying age-associated neurological diseases.
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http://dx.doi.org/10.1038/s41586-018-0368-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085146PMC
August 2018

Neuronal integrity and complement control synaptic material clearance by microglia after CNS injury.

J Exp Med 2018 07 25;215(7):1789-1801. Epub 2018 Jun 25.

Center for Brain Immunology, and Glia (BIG), University of Virginia, Charlottesville, VA

Phagocytosis of synaptic material by microglia is critical for central nervous system development. Less well understood is this microglial function in the injured adult brain. Assay of microglial phagocytosis is challenging, because peripheral myeloid cells engraft the site of injury, which could obscure interpretation of microglial roles. The model used here, optic nerve crush injury, results in degeneration of synapses in the dorsal lateral geniculate nucleus (dLGN), which stimulates rapid activation and engulfment of synaptic material by resident microglia without myeloid cell engraftment. Pharmacological depletion of microglia causes postinjury accumulation of synaptic debris, suggesting that microglia are the dominant postinjury phagocytes. Genetic or pharmacological manipulations revealed that neuronal activity does not trigger microglia phagocytosis after injury. RNA sequencing reveals C1q and CD11b/CR3 involvement in clearance of debris by dLGN-resident microglia. Indeed, and mice exhibit impaired postinjury debris clearance. Our results show how neurodegenerative debris is cleared by microglia and offers a model for studying its mechanisms and physiological roles.
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http://dx.doi.org/10.1084/jem.20172244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028515PMC
July 2018

Species-specific transcriptomic network inference of interspecies interactions.

ISME J 2018 08 24;12(8):2011-2023. Epub 2018 May 24.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.

The advent of high-throughput 'omics approaches coupled with computational analyses to reconstruct individual genomes from metagenomes provides a basis for species-resolved functional studies. Here, a mutual information approach was applied to build a gene association network of a commensal consortium, in which a unicellular cyanobacterium Thermosynechococcus elongatus BP1 supported the heterotrophic growth of Meiothermus ruber strain A. Specifically, we used the context likelihood of relatedness (CLR) algorithm to generate a gene association network from 25 transcriptomic datasets representing distinct growth conditions. The resulting interspecies network revealed a number of linkages between genes in each species. While many of the linkages were supported by the existing knowledge of phototroph-heterotroph interactions and the metabolism of these two species several new interactions were inferred as well. These include linkages between amino acid synthesis and uptake genes, as well as carbohydrate and vitamin metabolism, terpenoid metabolism and cell adhesion genes. Further topological examination and functional analysis of specific gene associations suggested that the interactions are likely to center around the exchange of energetically costly metabolites between T. elongatus and M. ruber. Both the approach and conclusions derived from this work are widely applicable to microbial communities for identification of the interactions between species and characterization of community functioning as a whole.
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http://dx.doi.org/10.1038/s41396-018-0145-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052077PMC
August 2018

Peripherally derived macrophages can engraft the brain independent of irradiation and maintain an identity distinct from microglia.

J Exp Med 2018 06 11;215(6):1627-1647. Epub 2018 Apr 11.

Center for Brain Immunology and Glia (BIG), University of Virginia, Charlottesville, VA

Peripherally derived macrophages infiltrate the brain after bone marrow transplantation and during central nervous system (CNS) inflammation. It was initially suggested that these engrafting cells were newly derived microglia and that irradiation was essential for engraftment to occur. However, it remains unclear whether brain-engrafting macrophages (beMφs) acquire a unique phenotype in the brain, whether long-term engraftment may occur without irradiation, and whether brain function is affected by the engrafted cells. In this study, we demonstrate that chronic, partial microglia depletion is sufficient for beMφs to populate the niche and that the presence of beMφs does not alter behavior. Furthermore, beMφs maintain a unique functional and transcriptional identity as compared with microglia. Overall, this study establishes beMφs as a unique CNS cell type and demonstrates that therapeutic engraftment of beMφs may be possible with irradiation-free conditioning regimens.
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http://dx.doi.org/10.1084/jem.20180247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987928PMC
June 2018

Microbiota alteration is associated with the development of stress-induced despair behavior.

Sci Rep 2017 03 7;7:43859. Epub 2017 Mar 7.

Center for Brain Immunology and Glia, University of Virginia, Charlottesville, VA, 22904, USA.

Depressive disorders often run in families, which, in addition to the genetic component, may point to the microbiome as a causative agent. Here, we employed a combination of behavioral, molecular and computational techniques to test the role of the microbiota in mediating despair behavior. In chronically stressed mice displaying despair behavior, we found that the microbiota composition and the metabolic signature dramatically change. Specifically, we observed reduced Lactobacillus and increased circulating kynurenine levels as the most prominent changes in stressed mice. Restoring intestinal Lactobacillus levels was sufficient to improve the metabolic alterations and behavioral abnormalities. Mechanistically, we identified that Lactobacillus-derived reactive oxygen species may suppress host kynurenine metabolism, by inhibiting the expression of the metabolizing enzyme, IDO1, in the intestine. Moreover, maintaining elevated kynurenine levels during Lactobacillus supplementation diminished the treatment benefits. Collectively, our data provide a mechanistic scenario for how a microbiota player (Lactobacillus) may contribute to regulating metabolism and resilience during stress.
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http://dx.doi.org/10.1038/srep43859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339726PMC
March 2017

Characterization of meningeal type 2 innate lymphocytes and their response to CNS injury.

J Exp Med 2017 02 19;214(2):285-296. Epub 2016 Dec 19.

Center for Brain Immunology and Glia (BIG), University of Virginia, Charlottesville, VA 22908

The meningeal space is occupied by a diverse repertoire of immune cells. Central nervous system (CNS) injury elicits a rapid immune response that affects neuronal survival and recovery, but the role of meningeal inflammation remains poorly understood. Here, we describe type 2 innate lymphocytes (ILC2s) as a novel cell type resident in the healthy meninges that are activated after CNS injury. ILC2s are present throughout the naive mouse meninges, though are concentrated around the dural sinuses, and have a unique transcriptional profile. After spinal cord injury (SCI), meningeal ILC2s are activated in an IL-33-dependent manner, producing type 2 cytokines. Using RNAseq, we characterized the gene programs that underlie the ILC2 activation state. Finally, addition of wild-type lung-derived ILC2s into the meningeal space of IL-33R animals partially improves recovery after SCI. These data characterize ILC2s as a novel meningeal cell type that responds to SCI and could lead to new therapeutic insights for neuroinflammatory conditions.
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http://dx.doi.org/10.1084/jem.20161982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5294864PMC
February 2017

Bayesian Model Averaging for Ensemble-Based Estimates of Solvation-Free Energies.

J Phys Chem B 2017 04 4;121(15):3458-3472. Epub 2017 Jan 4.

Division of Applied Mathematics, Brown University , Providence, Rhode Island 02912, United States.

This paper applies the Bayesian Model Averaging statistical ensemble technique to estimate small molecule solvation free energies. There is a wide range of methods available for predicting solvation free energies, ranging from empirical statistical models to ab initio quantum mechanical approaches. Each of these methods is based on a set of conceptual assumptions that can affect predictive accuracy and transferability. Using an iterative statistical process, we have selected and combined solvation energy estimates using an ensemble of 17 diverse methods from the fourth Statistical Assessment of Modeling of Proteins and Ligands (SAMPL) blind prediction study to form a single, aggregated solvation energy estimate. Methods that possess minimal or redundant information are pruned from the ensemble and the evaluation process repeats until aggregate predictive performance can no longer be improved. We show that this process results in a final aggregate estimate that outperforms all individual methods by reducing estimate errors by as much as 91% to 1.2 kcal mol accuracy. This work provides a new approach for accurate solvation free energy prediction and lays the foundation for future work on aggregate models that can balance computational cost with prediction accuracy.
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http://dx.doi.org/10.1021/acs.jpcb.6b09198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5398953PMC
April 2017

Network analysis of transcriptomics expands regulatory landscapes in Synechococcus sp. PCC 7002.

Nucleic Acids Res 2016 Oct 27;44(18):8810-8825. Epub 2016 Aug 27.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA

Cyanobacterial regulation of gene expression must contend with a genome organization that lacks apparent functional context, as the majority of cellular processes and metabolic pathways are encoded by genes found at disparate locations across the genome and relatively few transcription factors exist. In this study, global transcript abundance data from the model cyanobacterium Synechococcus sp. PCC 7002 grown under 42 different conditions was analyzed using Context-Likelihood of Relatedness (CLR). The resulting network, organized into 11 modules, provided insight into transcriptional network topology as well as grouping genes by function and linking their response to specific environmental variables. When used in conjunction with genome sequences, the network allowed identification and expansion of novel potential targets of both DNA binding proteins and sRNA regulators. These results offer a new perspective into the multi-level regulation that governs cellular adaptations of the fast-growing physiologically robust cyanobacterium Synechococcus sp. PCC 7002 to changing environmental variables. It also provides a methodological high-throughput approach to studying multi-scale regulatory mechanisms that operate in cyanobacteria. Finally, it provides valuable context for integrating systems-level data to enhance gene grouping based on annotated function, especially in organisms where traditional context analyses cannot be implemented due to lack of operon-based functional organization.
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http://dx.doi.org/10.1093/nar/gkw737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5062996PMC
October 2016

Unexpected role of interferon-γ in regulating neuronal connectivity and social behaviour.

Nature 2016 07 13;535(7612):425-9. Epub 2016 Jul 13.

Immune dysfunction is commonly associated with several neurological and mental disorders. Although the mechanisms by which peripheral immunity may influence neuronal function are largely unknown, recent findings implicate meningeal immunity influencing behaviour, such as spatial learning and memory. Here we show that meningeal immunity is also critical for social behaviour; mice deficient in adaptive immunity exhibit social deficits and hyper-connectivity of fronto-cortical brain regions. Associations between rodent transcriptomes from brain and cellular transcriptomes in response to T-cell-derived cytokines suggest a strong interaction between social behaviour and interferon-γ (IFN-γ)-driven responses. Concordantly, we demonstrate that inhibitory neurons respond to IFN-γ and increase GABAergic (γ-aminobutyric-acid) currents in projection neurons, suggesting that IFN-γ is a molecular link between meningeal immunity and neural circuits recruited for social behaviour. Meta-analysis of the transcriptomes of a range of organisms reveals that rodents, fish, and flies elevate IFN-γ/JAK-STAT-dependent gene signatures in a social context, suggesting that the IFN-γ signalling pathway could mediate a co-evolutionary link between social/aggregation behaviour and an efficient anti-pathogen response. This study implicates adaptive immune dysfunction, in particular IFN-γ, in disorders characterized by social dysfunction and suggests a co-evolutionary link between social behaviour and an anti-pathogen immune response driven by IFN-γ signalling.
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http://dx.doi.org/10.1038/nature18626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961620PMC
July 2016

Prediction of multi-drug resistance transporters using a novel sequence analysis method.

F1000Res 2015 9;4:60. Epub 2015 Mar 9.

Biological Sciences, Pacific Northwest National Laboratory, Washington, WA, 99352, USA.

There are many examples of groups of proteins that have similar function, but the determinants of functional specificity may be hidden by lack of sequence similarity, or by large groups of similar sequences with different functions. Transporters are one such protein group in that the general function, transport, can be easily inferred from the sequence, but the substrate specificity can be impossible to predict from sequence with current methods. In this paper we describe a linguistic-based approach to identify functional patterns from groups of unaligned protein sequences and its application to predict multi-drug resistance transporters (MDRs) from bacteria. We first show that our method can recreate known patterns from PROSITE for several motifs from unaligned sequences. We then show that the method, MDRpred, can predict MDRs with greater accuracy and positive predictive value than a collection of currently available family-based models from the Pfam database. Finally, we apply MDRpred to a large collection of protein sequences from an environmental microbiome study to make novel predictions about drug resistance in a potential environmental reservoir.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4743146PMC
http://dx.doi.org/10.12688/f1000research.6200.2DOI Listing
February 2016

ChIP-Seq Analysis of the σE Regulon of Salmonella enterica Serovar Typhimurium Reveals New Genes Implicated in Heat Shock and Oxidative Stress Response.

PLoS One 2015 21;10(9):e0138466. Epub 2015 Sep 21.

Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, Oregon, United States of America.

The alternative sigma factor σE functions to maintain bacterial homeostasis and membrane integrity in response to extracytoplasmic stress by regulating thousands of genes both directly and indirectly. The transcriptional regulatory network governed by σE in Salmonella and E. coli has been examined using microarray, however a genome-wide analysis of σE-binding sites in Salmonella has not yet been reported. We infected macrophages with Salmonella Typhimurium over a select time course. Using chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq), 31 σE-binding sites were identified. Seventeen sites were new, which included outer membrane proteins, a quorum-sensing protein, a cell division factor, and a signal transduction modulator. The consensus sequence identified for σE in vivo binding was similar to the one previously reported, except for a conserved G and A between the -35 and -10 regions. One third of the σE-binding sites did not contain the consensus sequence, suggesting there may be alternative mechanisms by which σE modulates transcription. By dissecting direct and indirect modes of σE-mediated regulation, we found that σE activates gene expression through recognition of both canonical and reversed consensus sequence. New σE regulated genes (greA, luxS, ompA and ompX) are shown to be involved in heat shock and oxidative stress responses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0138466PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4577112PMC
May 2016

The Pacific Northwest National Laboratory library of bacterial and archaeal proteomic biodiversity.

Sci Data 2015 18;2:150041. Epub 2015 Aug 18.

Biological Sciences Division, Pacific Northwest National Laboratory , Richland, Washington 99354, USA.

This Data Descriptor announces the submission to public repositories of the PNNL Biodiversity Library, a large collection of global proteomics data for 112 bacterial and archaeal organisms. The data comprises 35,162 tandem mass spectrometry (MS/MS) datasets from ~10 years of research. All data has been searched, annotated and organized in a consistent manner to promote reuse by the community. Protein identifications were cross-referenced with KEGG functional annotations which allows for pathway oriented investigation. We present the data as a freely available community resource. A variety of data re-use options are described for computational modelling, proteomics assay design and bioengineering. Instrument data and analysis files are available at ProteomeXchange via the MassIVE partner repository under the identifiers PXD001860 and MSV000079053.
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http://dx.doi.org/10.1038/sdata.2015.41DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540001PMC
December 2015

Integrated in silico Analyses of Regulatory and Metabolic Networks of Synechococcus sp. PCC 7002 Reveal Relationships between Gene Centrality and Essentiality.

Life (Basel) 2015 Mar 27;5(2):1127-40. Epub 2015 Mar 27.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.

Cyanobacteria dynamically relay environmental inputs to intracellular adaptations through a coordinated adjustment of photosynthetic efficiency and carbon processing rates. The output of such adaptations is reflected through changes in transcriptional patterns and metabolic flux distributions that ultimately define growth strategy. To address interrelationships between metabolism and regulation, we performed integrative analyses of metabolic and gene co-expression networks in a model cyanobacterium, Synechococcus sp. PCC 7002. Centrality analyses using the gene co-expression network identified a set of key genes, which were defined here as "topologically important." Parallel in silico gene knock-out simulations, using the genome-scale metabolic network, classified what we termed as "functionally important" genes, deletion of which affected growth or metabolism. A strong positive correlation was observed between topologically and functionally important genes. Functionally important genes exhibited variable levels of topological centrality; however, the majority of topologically central genes were found to be functionally essential for growth. Subsequent functional enrichment analysis revealed that both functionally and topologically important genes in Synechococcus sp. PCC 7002 are predominantly associated with translation and energy metabolism, two cellular processes critical for growth. This research demonstrates how synergistic network-level analyses can be used for reconciliation of metabolic and gene expression data to uncover fundamental biological principles.
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http://dx.doi.org/10.3390/life5021127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4500133PMC
March 2015

Analysis of the Salmonella regulatory network suggests involvement of SsrB and H-NS in σ(E)-regulated SPI-2 gene expression.

Front Microbiol 2015 10;6:27. Epub 2015 Feb 10.

Biological Sciences Division, Pacific Northwest National Laboratory Richland, WA, USA.

The extracytoplasmic functioning sigma factor σ(E) is known to play an essential role for Salmonella enterica serovar Typhimurium to survive and proliferate in macrophages and mice. However, its regulatory network is not well-characterized, especially during infection. Here we used microarray to identify genes regulated by σ(E) in Salmonella grown in three conditions: a nutrient-rich condition and two others that mimic early and late intracellular infection. We found that in each condition σ(E) regulated different sets of genes, and notably, several global regulators. When comparing nutrient-rich and infection-like conditions, large changes were observed in the expression of genes involved in Salmonella pathogenesis island (SPI)-1 type-three secretion system (TTSS), SPI-2 TTSS, protein synthesis, and stress responses. In total, the expression of 58% of Salmonella genes was affected by σ(E) in at least one of the three conditions. An important finding is that σ(E) up-regulates SPI-2 genes, which are essential for Salmonella intracellular survival, by up-regulating SPI-2 activator ssrB expression at the early stage of infection and down-regulating SPI-2 repressor hns expression at a later stage. Moreover, σ(E) is capable of countering the silencing of H-NS, releasing the expression of SPI-2 genes. This connection between σ(E) and SPI-2 genes, combined with the global regulatory effect of σ(E), may account for the lethality of rpoE-deficient Salmonella in murine infection.
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http://dx.doi.org/10.3389/fmicb.2015.00027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4322710PMC
February 2015

Global analysis of Salmonella alternative sigma factor E on protein translation.

J Proteome Res 2015 Apr 27;14(4):1716-26. Epub 2015 Feb 27.

The alternative sigma factor E (σ(E)) is critical for response to extracytoplasmic stress in Salmonella. Extensive studies have been conducted on σ(E)-regulated gene expression, particularly at the transcriptional level. Increasing evidence suggests however that σ(E) may indirectly participate in post-transcriptional regulation. In this study, we conducted sample-matched global proteomic and transcriptomic analyses to determine the level of regulation mediated by σ(E) in Salmonella. Samples were analyzed from wild-type and isogenic rpoE mutant Salmonella cultivated in three different conditions: nutrient-rich and conditions that mimic early and late intracellular infection. We found that 30% of the observed proteome was regulated by σ(E) combining all three conditions. In different growth conditions, σ(E) affected the expression of a broad spectrum of Salmonella proteins required for miscellaneous functions. Those involved in transport and binding, protein synthesis, and stress response were particularly highlighted. By comparing transcriptomic and proteomic data, we identified genes post-transcriptionally regulated by σ(E) and found that post-transcriptional regulation was responsible for a majority of changes observed in the σ(E)-regulated proteome. Further, comparison of transcriptomic and proteomic data from hfq mutant of Salmonella demonstrated that σ(E)-mediated post-transcriptional regulation was partially dependent on the RNA-binding protein Hfq.
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http://dx.doi.org/10.1021/pr5010423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476319PMC
April 2015

ArrayInitiative - a tool that simplifies creating custom Affymetrix CDFs.

BMC Bioinformatics 2011 May 6;12:136. Epub 2011 May 6.

Department of Bioinformatics and Genomics, University of North Carolina - Charlotte, Charlotte, NC 28223-0001, USA.

Background: Probes on a microarray represent a frozen view of a genome and are quickly outdated when new sequencing studies extend our knowledge, resulting in significant measurement error when analyzing any microarray experiment. There are several bioinformatics approaches to improve probe assignments, but without in-house programming expertise, standardizing these custom array specifications as a usable file (e.g. as Affymetrix CDFs) is difficult, owing mostly to the complexity of the specification file format. However, without correctly standardized files there is a significant barrier for testing competing analysis approaches since this file is one of the required inputs for many commonly used algorithms. The need to test combinations of probe assignments and analysis algorithms led us to develop ArrayInitiative, a tool for creating and managing custom array specifications.

Results: ArrayInitiative is a standalone, cross-platform, rich client desktop application for creating correctly formatted, custom versions of manufacturer-provided (default) array specifications, requiring only minimal knowledge of the array specification rules and file formats. Users can import default array specifications, import probe sequences for a default array specification, design and import a custom array specification, export any array specification to multiple output formats, export the probe sequences for any array specification and browse high-level information about the microarray, such as version and number of probes. The initial release of ArrayInitiative supports the Affymetrix 3' IVT expression arrays we currently analyze, but as an open source application, we hope that others will contribute modules for other platforms.

Conclusions: ArrayInitiative allows researchers to create new array specifications, in a standard format, based upon their own requirements. This makes it easier to test competing design and analysis strategies that depend on probe definitions. Since the custom array specifications are easily exported to the manufacturer's standard format, researchers can analyze these customized microarray experiments using established software tools, such as those available in Bioconductor.
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http://dx.doi.org/10.1186/1471-2105-12-136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3113937PMC
May 2011

Microarray Detection Call Methodology as a Means to Identify and Compare Transcripts Expressed within Syncytial Cells from Soybean (Glycine max) Roots Undergoing Resistant and Susceptible Reactions to the Soybean Cyst Nematode (Heterodera glycines).

J Biomed Biotechnol 2010 19;2010:491217. Epub 2010 May 19.

Department of Biological Sciences, Harned Hall, Mississippi State University, Mississippi State, MS 39762, USA.

Background. A comparative microarray investigation was done using detection call methodology (DCM) and differential expression analyses. The goal was to identify genes found in specific cell populations that were eliminated by differential expression analysis due to the nature of differential expression methods. Laser capture microdissection (LCM) was used to isolate nearly homogeneous populations of plant root cells. Results. The analyses identified the presence of 13,291 transcripts between the 4 different sample types. The transcripts filtered down into a total of 6,267 that were detected as being present in one or more sample types. A comparative analysis of DCM and differential expression methods showed a group of genes that were not differentially expressed, but were expressed at detectable amounts within specific cell types. Conclusion. The DCM has identified patterns of gene expression not shown by differential expression analyses. DCM has identified genes that are possibly cell-type specific and/or involved in important aspects of plant nematode interactions during the resistance response, revealing the uniqueness of a particular cell population at a particular point during its differentiation process.
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http://dx.doi.org/10.1155/2010/491217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875038PMC
September 2012

Laser capture microdissection (LCM) and comparative microarray expression analysis of syncytial cells isolated from incompatible and compatible soybean (Glycine max) roots infected by the soybean cyst nematode (Heterodera glycines).

Planta 2007 Nov 1;226(6):1389-409. Epub 2007 Aug 1.

United States Department of Agriculture, Soybean Genomics and Improvement Laboratory, Bldg. 006, Rm. 118, 10300 Baltimore Ave., Beltsville, MD 20705-2350, USA.

Syncytial cells in soybean (Glycine max cultivar [cv.] Peking) roots infected by incompatible and compatible populations of soybean cyst nematode (SCN [Heterodera glycines]) were collected using laser capture microdissection (LCM). Gene transcript abundance was assayed using Affymetrix soybean GeneChips, each containing 37,744 probe sets. Our analyses identified differentially expressed genes in syncytial cells that are not differentially expressed in the whole root analyses. Therefore, our results show that the mass of transcriptional activity occurring in the whole root is obscuring identification of transcriptional events occurring within syncytial cells. In syncytial cells from incompatible roots at three dpi, genes encoding lipoxygenase (LOX), heat shock protein (HSP) 70, superoxidase dismutase (SOD) were elevated almost tenfold or more, while genes encoding several transcription factors and DNA binding proteins were also elevated, albeit at lower levels. In syncytial cells formed during the compatible interaction at three dpi, genes encoding prohibitin, the epsilon chain of ATP synthase, allene oxide cyclase and annexin were more abundant. By 8 days, several genes of unknown function and genes encoding a germin-like protein, peroxidase, LOX, GAPDH, 3-deoxy-D-arabino-heptolosonate 7-phosphate synthase, ATP synthase and a thioesterase were abundantly expressed. These observations suggest that gene expression is different in syncytial cells as compared to whole roots infected with nematodes. Our observations also show that gene expression is different between syncytial cells that were isolated from incompatible and compatible roots and that gene expression is changing over the course of syncytial cell development as it matures into a functional feeding site.
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http://dx.doi.org/10.1007/s00425-007-0578-zDOI Listing
November 2007

A time-course comparative microarray analysis of an incompatible and compatible response by Glycine max (soybean) to Heterodera glycines (soybean cyst nematode) infection.

Planta 2007 Nov 25;226(6):1423-47. Epub 2007 Jul 25.

United States Department of Agriculture, Soybean Genomics and Improvement Laboratory, 10300 Baltimore Ave. Bldg 006, Beltsville, MD 20705, USA.

The development of an infection in soybean [Glycine max L. cultivar (cv.) Peking] roots by incompatible (I) and compatible (C) populations of soybean cyst nematode (SCN) (Heterodera glycines) was assayed using an AffymetriX soybean GeneChip. This time-course microarray analysis, using 37,744 probe sets, measured transcript abundance during I and C. These analyses reveal that infection by individual I and C H. glycines populations influence the transcription of G. max genes differently. A substantial difference in gene expression is present between I and C at 12 h post infection. Thus, G. max can differentiate between I and C nematode populations even before they have begun to select their feeding sites. The microarray analysis identified genes induced earlier in infection during I than C. MA also identified amplitude differences in transcript abundance between I and C reactions. Some of the probe sets measuring increased transcript levels during I represented no apical meristem (NAM) and WRKY transcription factors as well as NBS-LRR kinases. Later during I, heat shock protein (HSPs) probe sets (i.e. HSP90, HSP70, ClpB/HSP101) measured increased transcript abundance. These results demonstrate that G. max roots respond very differently to the different H. glycines races even before their feeding site selection has occurred. The ability of G. max to engage an I reaction, thus, appears to be dependent on the ability of root cells to recognize the different races of H. glycines because these experiments were conducted in the identical G. max genetic background.
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http://dx.doi.org/10.1007/s00425-007-0581-4DOI Listing
November 2007

A decline in transcript abundance for Heterodera glycines homologs of Caenorhabditis elegans uncoordinated genes accompanies its sedentary parasitic phase.

BMC Dev Biol 2007 Apr 19;7:35. Epub 2007 Apr 19.

United States Department of Agriculture, Soybean Genomics and Improvement Laboratory, Beltsville, MD 20705, USA.

Background: Heterodera glycines (soybean cyst nematode [SCN]), the major pathogen of Glycine max (soybean), undergoes muscle degradation (sarcopenia) as it becomes sedentary inside the root. Many genes encoding muscular and neuromuscular components belong to the uncoordinated (unc) family of genes originally identified in Caenorhabditis elegans. Previously, we reported a substantial decrease in transcript abundance for Hg-unc-87, the H. glycines homolog of unc-87 (calponin) during the adult sedentary phase of SCN. These observations implied that changes in the expression of specific muscle genes occurred during sarcopenia.

Results: We developed a bioinformatics database that compares expressed sequence tag (est) and genomic data of C. elegans and H. glycines (CeHg database). We identify H. glycines homologs of C. elegans unc genes whose protein products are involved in muscle composition and regulation. RT-PCR reveals the transcript abundance of H. glycines unc homologs at mobile and sedentary stages of its lifecycle. A prominent reduction in transcript abundance occurs in samples from sedentary nematodes for homologs of actin, unc-60B (cofilin), unc-89, unc-15 (paromyosin), unc-27 (troponin I), unc-54 (myosin), and the potassium channel unc-110 (twk-18). Less reduction is observed for the focal adhesion complex gene Hg-unc-97.

Conclusion: The CeHg bioinformatics database is shown to be useful in identifying homologs of genes whose protein products perform roles in specific aspects of H. glycines muscle biology. Our bioinformatics comparison of C. elegans and H. glycines genomic data and our Hg-unc-87 expression experiments demonstrate that the transcript abundance of specific H. glycines homologs of muscle gene decreases as the nematode becomes sedentary inside the root during its parasitic feeding stages.
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http://dx.doi.org/10.1186/1471-213X-7-35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1867819PMC
April 2007

Developing a systems biology approach to study disease progression caused by Heterodera glycines in Glycine max.

Gene Regul Syst Bio 2007 Jun 5;1:17-33. Epub 2007 Jun 5.

United States Department of Agriculture, Soybean Genomics and Improvement Laboratory, Bldg 006, Beltsville, MD 20705, USA.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759149PMC
June 2007