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1.  Multiplex single-molecule interaction profiling of DNA barcoded proteins 
Nature  2014;515(7528):554-557.
In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.
doi:10.1038/nature13761
PMCID: PMC4246050  PMID: 25252978
2.  Mutations that disrupt PHOXB interaction with the neuronal calcium sensor HPCAL1 impede cellular differentiation in neuroblastoma 
Oncogene  2013;33(25):3316-3324.
Heterozygous germline mutations in PHOX2B, a transcriptional regulator of sympathetic neuronal differentiation, predispose to diseases of the sympathetic nervous system, including neuroblastoma and congenital central hypoventilation syndrome (CCHS). Whereas the PHOX2B variants in CCHS largely involve expansions of the second polyalanine repeat within the C-terminus of the protein, those associated with neuroblastic tumors are nearly always frameshift and truncation mutations. To test the hypothesis that the neuroblastoma-associated variants exert their effects through loss or gain of protein-protein interactions, we performed a large-scale yeast two-hybrid screen using both wild-type (WT) and six different mutant PHOX2B proteins against over 10,000 human genes. The neuronal calcium sensor protein HPCAL1 (VILIP-3) exhibited strong binding to WT PHOX2B and a CCHS-associated polyalanine expansion mutant, but only weakly or not at all to neuroblastoma-associated frameshift and truncation variants. We demonstrate that both WT PHOX2B, the neuroblastoma-associated R100L missense and the CCHS-associated alanine expansion variants induce nuclear translocation of HPCAL1 in a Ca2+-independent manner, while the neuroblastoma-associated 676delG frameshift and K155X truncation mutants impair subcellular localization of HPCAL1, causing it to remain in the cytoplasm. HPCAL1 did not appreciably influence the ability of WT PHOX2B to transactivate the DBH promoter, nor did it alter the decreased transactivation potential of PHOX2B variants in 293T cells. Abrogation of the PHOX2B-HPCAL1 interaction by shRNA knockdown of HPCAL1 in neuroblastoma cells expressing PHOX2B led to impaired neurite outgrowth with transcriptional profiles indicative of inhibited sympathetic neuronal differentiation. Our results suggest that certain PHOX2B variants associated with neuroblastoma pathogenesis, because of their inability to bind to key interacting proteins such as HPCAL1, may predispose to this malignancy by impeding the differentiation of immature sympathetic neurons.
doi:10.1038/onc.2013.290
PMCID: PMC4040330  PMID: 23873030
PHOX2B; HPCAL1; neuroblastoma; neuronal calcium sensor; differentiation; network perturbation
3.  Methodology for the inference of gene function from phenotype data 
BMC Bioinformatics  2014;15(1):405.
Background
Biomedical ontologies are increasingly instrumental in the advancement of biological research primarily through their use to efficiently consolidate large amounts of data into structured, accessible sets. However, ontology development and usage can be hampered by the segregation of knowledge by domain that occurs due to independent development and use of the ontologies. The ability to infer data associated with one ontology to data associated with another ontology would prove useful in expanding information content and scope. We here focus on relating two ontologies: the Gene Ontology (GO), which encodes canonical gene function, and the Mammalian Phenotype Ontology (MP), which describes non-canonical phenotypes, using statistical methods to suggest GO functional annotations from existing MP phenotype annotations. This work is in contrast to previous studies that have focused on inferring gene function from phenotype primarily through lexical or semantic similarity measures.
Results
We have designed and tested a set of algorithms that represents a novel methodology to define rules for predicting gene function by examining the emergent structure and relationships between the gene functions and phenotypes rather than inspecting the terms semantically. The algorithms inspect relationships among multiple phenotype terms to deduce if there are cases where they all arise from a single gene function.
We apply this methodology to data about genes in the laboratory mouse that are formally represented in the Mouse Genome Informatics (MGI) resource. From the data, 7444 rule instances were generated from five generalized rules, resulting in 4818 unique GO functional predictions for 1796 genes.
Conclusions
We show that our method is capable of inferring high-quality functional annotations from curated phenotype data. As well as creating inferred annotations, our method has the potential to allow for the elucidation of unforeseen, biologically significant associations between gene function and phenotypes that would be overlooked by a semantics-based approach. Future work will include the implementation of the described algorithms for a variety of other model organism databases, taking full advantage of the abundance of available high quality curated data.
Electronic supplementary material
The online version of this article (doi:10.1186/s12859-014-0405-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12859-014-0405-z
PMCID: PMC4302099  PMID: 25495798
Gene ontology; Mammalian phenotype ontology; Function prediction; Ontology development
4.  Temporal Signatures of Taste Quality Driven by Active Sensing 
The Journal of Neuroscience  2014;34(22):7398-7411.
Animals actively acquire sensory information from the outside world, with rodents sniffing to smell and whisking to feel. Licking, a rapid motor sequence used for gustation, serves as the primary means of controlling stimulus access to taste receptors in the mouth. Using a novel taste-quality discrimination task in head-restrained mice, we measured and compared reaction times to four basic taste qualities (salt, sour, sweet, and bitter) and found that certain taste qualities are perceived inherently faster than others, driven by the precise biomechanics of licking and functional organization of the peripheral gustatory system. The minimum time required for accurate perception was strongly dependent on taste quality, ranging from the sensory-motor limits of a single lick (salt, ∼100 ms) to several sampling cycles (bitter, >500 ms). Further, disruption of sensory input from the anterior tongue significantly impaired the speed of perception of some taste qualities, with little effect on others. Overall, our results show that active sensing may play an important role in shaping the timing of taste-quality representations and perception in the gustatory system.
doi:10.1523/JNEUROSCI.0213-14.2014
PMCID: PMC4035510  PMID: 24872546
5.  Antitumor Effects of Synthetic 6,7-Annulated-4-substituted Indole Compounds in L1210 Leukemic Cells In Vitro 
Anticancer research  2012;32(11):4671-4684.
Background
Because annulated indoles have almost no representation in the PubChem or MLSMR databases, an unprecedented class of an indole-based library was constructed, using the indole aryne methodology, and screened for antitumor activity. Sixty-six novel 6,7-annulated-4-substituted indole compounds were synthesized, using a strategic combination of 6,7-indolyne cycloaddition and cross-coupling reactions under both Suzuki-Miyaura and Buchwald-Hartwig conditions, and tested for their effectiveness against murine L1210 tumor cell proliferation in vitro.
Materials and Methods
Various markers of tumor cell metabolism, DNA degradation, mitotic disruption, cytokinesis and apoptosis were assayed in vitro to evaluate drug cytotoxicity.
Results
Most compounds inhibited the metabolic activity of leukemic cells in a time- and concentration-dependent manner but only 9 of them were sufficiently potent to inhibit L1210 tumor cell proliferation by 50% in the low-μM range after 2 (IC50: 4.5–20.4 μM) and 4 days (0.5–4.0 μM) in culture. However, the antiproliferative compounds that were the most effective at day 4 were not necessarily the most potent at day 2, suggesting different speeds of action. A 3-h treatment with antiproliferative annulated indole was sufficient to inhibit, in a concentration-dependent manner, the rate of DNA synthesis measured in L1210 cells over a 0.5-h period of pulse-labeling with 3H-thymidine. Four of the antiproliferative compounds had weak DNA-binding activities but one compound reduced the fluorescence of the ethidium bromide-DNA complex by up to 53%, suggesting that some annulated indoles might directly interact with double-stranded DNA to disrupt its integrity and prevent the dye from intercalating into DNA base pairs. However, all 9 antiproliferative compounds induced DNA cleavage at 24 h in L1210 cells, containing 3H-thymidine-prelabeled DNA, suggesting that these antitumor annulated indoles might trigger an apoptotic pathway of DNA fragmentation. Indeed the antiproliferative annulated indoles caused a time-dependent increase of caspase-3 activity with a peak at 6 h. Interestingly, the compounds with the most potent antiproliferative IC50 values at day 2 were consistently the most effective at inhibiting DNA synthesis at 3 h and inducing DNA fragmentation at 24 h. After 24–48 h, antiproliferative concentrations of annulated indoles increased the mitotic index of L1210 cells and stimulated the formation of many bi-nucleated cells, multi-nucleated cells, apoptotic cells and micronuclei, suggesting that these antitumor compounds might enhance mitotic abnormality, induce chromosomal damage or missegregation, and block cytokinesis to induce apoptosis.
Conclusion
Although annulated indoles may have interesting bioactivity, novel derivatives with different substitutions must be synthesized to elucidate structure-activity relationships, identify more potent antitumor lead compounds, and investigate their molecular targets and mechanisms of action.
PMCID: PMC4175989  PMID: 23155229
Annulated indoles; tumor cell proliferation; DNA synthesis; interaction and fragmentation; cells with mitotic figures; several nuclei and micronuclei; cytokinesis; apoptosis
6.  Lipid Profile Remodeling in Response to Nitrogen Deprivation in the Microalgae Chlorella sp. (Trebouxiophyceae) and Nannochloropsis sp. (Eustigmatophyceae) 
PLoS ONE  2014;9(8):e103389.
Many species of microalgae produce greatly enhanced amounts of triacylglycerides (TAGs), the key product for biodiesel production, in response to specific environmental stresses. Improvement of TAG production by microalgae through optimization of growth regimes is of great interest. This relies on understanding microalgal lipid metabolism in relation to stress response in particular the deprivation of nutrients that can induce enhanced TAG synthesis. In this study, a detailed investigation of changes in lipid composition in Chlorella sp. and Nannochloropsis sp. in response to nitrogen deprivation (N-deprivation) was performed to provide novel mechanistic insights into the lipidome during stress. As expected, an increase in TAGs and an overall decrease in polar lipids were observed. However, while most membrane lipid classes (phosphoglycerolipids and glycolipids) were found to decrease, the non-nitrogen containing phosphatidylglycerol levels increased considerably in both algae from initially low levels. Of particular significance, it was observed that the acyl composition of TAGs in Nannochloropsis sp. remain relatively constant, whereas Chlorella sp. showed greater variability following N-deprivation. In both algae the overall fatty acid profiles of the polar lipid classes were largely unaffected by N-deprivation, suggesting a specific FA profile for each compartment is maintained to enable continued function despite considerable reductions in the amount of these lipids. The changes observed in the overall fatty acid profile were due primarily to the decrease in proportion of polar lipids to TAGs. This study provides the most detailed lipidomic information on two different microalgae with utility in biodiesel production and nutraceutical industries and proposes the mechanisms for this rearrangement. This research also highlights the usefulness of the latest MS-based approaches for microalgae lipid research.
doi:10.1371/journal.pone.0103389
PMCID: PMC4149361  PMID: 25171084
7.  Low utilization of extra embryos in donor oocyte in vitro fertilization cycles: an ethical dilemma to donor management 
Purpose
To explore outcomes of donor In Vitro Fertilization (IVF) cycles with regards to cryopreservation and utilization of extra embryos after fresh transfer.
Methods
A database search was performed to identify all consecutive fresh donor oocyte cycles from January 1, 2000 to December 31, 2010 at a private fertility laboratory. Parameters analyzed included: number of oocytes retrieved, number of patients choosing embryo cryopreservation, number of patients returning for frozen embryo transfer (FET), and pregnancy outcomes.
Results
A total of 1070 fresh oocyte donor cycles were identified. Average number of oocytes retrieved was 16.9 ± 7.9, and average number of embryos transferred was 2.3 ± 0.96. Sixty-six percent of patients cryopreserved excess embryos following fresh transfer, and only 40 % of these patients ultimately returned for FET. Patients who conceived in their fresh cycle were much less likely to return for FET than those who did not (25 % v 65 %, p < 0.001), however chance of conceiving with FET was no different between these two groups (38 % v 38 %, NS).
Conclusions
An unexpectedly low number of patients undergoing a donor oocyte IVF cycle will ultimately return to utilize extra embryos from their fresh cycle. This is concerning considering the high numbers of oocytes retrieved and the known complications from hyperstimulation, especially in light of the relatively high pregnancy rates associated with donor cycles. This raises concerns not only for donor management, but also raises ethical dilemmas when considering the large numbers of remaining embryos that will never be utilized.
doi:10.1007/s10815-013-0038-6
PMCID: PMC3790114  PMID: 23812846
Embryo donation; Embryo utilization; Cryopreservation; Embryo surplus
8.  Systematic identification of pathological lamin A interactors 
Molecular Biology of the Cell  2014;25(9):1493-1510.
As essential components of the cell nucleus, lamins play key roles in organizing genomes and as protein–protein interaction platforms. Mutations in lamin A cause a diverse set of human diseases. This work describes the identification of lamin A partners and assesses how interactions are affected by a comprehensive set of lamin A disease mutations.
Laminopathies are a collection of phenotypically diverse diseases that include muscular dystrophies, cardiomyopathies, lipodystrophies, and premature aging syndromes. Laminopathies are caused by >300 distinct mutations in the LMNA gene, which encodes the nuclear intermediate filament proteins lamin A and C, two major architectural elements of the mammalian cell nucleus. The genotype–phenotype relationship and the basis for the pronounced tissue specificity of laminopathies are poorly understood. Here we seek to identify on a global scale lamin A–binding partners whose interaction is affected by disease-relevant LMNA mutations. In a screen of a human genome–wide ORFeome library, we identified and validated 337 lamin A–binding proteins. Testing them against 89 known lamin A disease mutations identified 50 disease-associated interactors. Association of progerin, the lamin A isoform responsible for the premature aging disorder Hutchinson–Gilford progeria syndrome, with its partners was largely mediated by farnesylation. Mapping of the interaction sites on lamin A identified the immunoglobulin G (IgG)–like domain as an interaction hotspot and demonstrated that lamin A variants, which destabilize the Ig-like domain, affect protein–protein interactions more globally than mutations of surface residues. Analysis of a set of LMNA mutations in a single residue, which result in three phenotypically distinct diseases, identified disease-specific interactors. The results represent a systematic map of disease-relevant lamin A interactors and suggest loss of tissue-specific lamin A interactions as a mechanism for the tissue-specific appearance of laminopathic phenotypes.
doi:10.1091/mbc.E14-02-0733
PMCID: PMC4004598  PMID: 24623722
9.  Representing Kidney Development Using the Gene Ontology 
PLoS ONE  2014;9(6):e99864.
Gene Ontology (GO) provides dynamic controlled vocabularies to aid in the description of the functional biological attributes and subcellular locations of gene products from all taxonomic groups (www.geneontology.org). Here we describe collaboration between the renal biomedical research community and the GO Consortium to improve the quality and quantity of GO terms describing renal development. In the associated annotation activity, the new and revised terms were associated with gene products involved in renal development and function. This project resulted in a total of 522 GO terms being added to the ontology and the creation of approximately 9,600 kidney-related GO term associations to 940 UniProt Knowledgebase (UniProtKB) entries, covering 66 taxonomic groups. We demonstrate the impact of these improvements on the interpretation of GO term analyses performed on genes differentially expressed in kidney glomeruli affected by diabetic nephropathy. In summary, we have produced a resource that can be utilized in the interpretation of data from small- and large-scale experiments investigating molecular mechanisms of kidney function and development and thereby help towards alleviating renal disease.
doi:10.1371/journal.pone.0099864
PMCID: PMC4062467  PMID: 24941002
10.  A method for increasing expressivity of Gene Ontology annotations using a compositional approach 
BMC Bioinformatics  2014;15:155.
Background
The Gene Ontology project integrates data about the function of gene products across a diverse range of organisms, allowing the transfer of knowledge from model organisms to humans, and enabling computational analyses for interpretation of high-throughput experimental and clinical data. The core data structure is the annotation, an association between a gene product and a term from one of the three ontologies comprising the GO. Historically, it has not been possible to provide additional information about the context of a GO term, such as the target gene or the location of a molecular function. This has limited the specificity of knowledge that can be expressed by GO annotations.
Results
The GO Consortium has introduced annotation extensions that enable manually curated GO annotations to capture additional contextual details. Extensions represent effector–target relationships such as localization dependencies, substrates of protein modifiers and regulation targets of signaling pathways and transcription factors as well as spatial and temporal aspects of processes such as cell or tissue type or developmental stage. We describe the content and structure of annotation extensions, provide examples, and summarize the current usage of annotation extensions.
Conclusions
The additional contextual information captured by annotation extensions improves the utility of functional annotation by representing dependencies between annotations to terms in the different ontologies of GO, external ontologies, or an organism’s gene products. These enhanced annotations can also support sophisticated queries and reasoning, and will provide curated, directional links between many gene products to support pathway and network reconstruction.
doi:10.1186/1471-2105-15-155
PMCID: PMC4039540  PMID: 24885854
Gene Ontology; Functional annotation; Annotation extension; Manual curation
11.  A novel approach to quantifying ovarian cell lipid content and lipid accumulation in vitro by confocal microscopy in lean women undergoing ovarian stimulation for in vitro fertilization (IVF) 
Purpose
To quantify intracellular lipid levels in cumulus cells (CCs) and mural granulosa cells (MGCs) of lean women undergoing gonadotropin therapy for in vitro fertilization (IVF), based upon different cell preparation methods.
Methods
CCs and MGCs from 16 lean women undergoing ovarian stimulation for IVF were studied. Cells were pooled by cell type, with each type of cell separated into two groups for determination of initial lipid content (Method 1) and subsequent lipid accumulation in vitro (Method 2). Cells for initial lipid content were immediately fixed at the time of the oocyte retrieval with 4 % paraformaldehyde in suspension, while those for subsequent lipid accumulation in vitro were cultured for 4 h with 5 % fetal calf serum and then fixed. Cells were treated with lipid fluorescent dye BODIPY® FL C16 and nuclear marker DAPI. Intracellular lipid was quantified by confocal microscopy, using ImageJ software analysis.
Results
There was no significant effect of cell type (P = 0.2) or cell type-cell preparation method interaction (P = 0.8) on cell area (Method 1: CC 99.7 ± 5.1, MGC 132.8 ± 5.8; Method 2: CC 221.9 ± 30.4, MGC 265.1 ± 48.5 μm2). The mean area of all cells combined was significantly less for cells prepared by Method 1 (116.2 ± 4.9 μm2) vs. Method 2 (243.5 ± 22.5 μm2, P < 0.00005). Intracellular lipid level, however, was significantly altered by cell preparation method (P < 0.05; cell preparation method-cell type interaction, P < 0.00001). Initial lipid content was significantly lower in CC (74.5 ± 9.3) than MGC (136.3 ± 16.7 fluorescence/cell area, P < 0.00005), while subsequent lipid accumulation in vitro was significantly higher in CC (154.0 ± 9.1) than MGC (104.6 ± 9.9 fluorescence/cell area, P < 0.00001). The relatively diminished initial CC lipid content compared to subsequent CC lipid accumulation in vitro (P < 0.00001), and the opposite pattern for MGC (P < 0.05), significantly lowered the CC/MGC lipid ratio in Method 1 (0.55 ± 0.04) vs. Method 2 (1.58 ± 0.10, P < 0.00001).
Conclusions
Differential uptake or utilization of lipid by CC and MGC occurs during oocyte maturation and steroidogenesis, respectively, with the amount of lipid present in ovarian cells a function of both the follicular microenvironment at the time of the oocyte retrieval and the capacity of these cells to accumulate lipid in vitro over time.
doi:10.1007/s10815-013-9976-2
PMCID: PMC3663971  PMID: 23512091
Lipid; Confocal microscopy; Cumulus cell; Mural granulosa cell; In vitro fertilization
12.  PhylOPDb: a 16S rRNA oligonucleotide probe database for prokaryotic identification 
In recent years, high-throughput molecular tools have led to an exponential growth of available 16S rRNA gene sequences. Incorporating such data, molecular tools based on target-probe hybridization were developed to monitor microbial communities within complex environments. Unfortunately, only a few 16S rRNA gene-targeted probe collections were described. Here, we present PhylOPDb, an online resource for a comprehensive phylogenetic oligonucleotide probe database. PhylOPDb provides a convivial and easy-to-use web interface to browse both regular and explorative 16S rRNA-targeted probes. Such probes set or subset could be used to globally monitor known and unknown prokaryotic communities through various techniques including DNA microarrays, polymerase chain reaction (PCR), fluorescent in situ hybridization (FISH), targeted gene capture or in silico rapid sequence identification. PhylOPDb contains 74 003 25-mer probes targeting 2178 genera including Bacteria and Archaea.
Database URL: http://g2im.u-clermont1.fr/phylopdb/
doi:10.1093/database/bau036
PMCID: PMC4000467  PMID: 24771669
13.  A randomized controlled trial of the effectiveness of a pre-recruitment primer letter to increase participation in a study of colorectal screening and surveillance 
Background
Recruiting cancer patients is a barrier often encountered in research trials. However, very few randomized trials explore strategies to improve participation rates. The purpose of this study was to evaluate the effectiveness of a pre-recruitment primer letter to recruit persons diagnosed with colorectal cancer for a research trial.
Methods
Potentially eligible participants were identified by the Victorian Cancer Registry. A total of 1,062 participants were randomized to receive either a mailed explanatory primer letter designed to encourage research participation, or no primer letter. Two weeks after the intervention, the Victorian Cancer Registry sought permission from patients to release their contact details to researchers. Those who agreed were contacted and invited to the study.
Results
Pre-recruitment encouragement was not effective at increasing recruitment, with no significant differences demonstrated between experimental groups. Overall, 40% (n = 425) consented to participate, 25% (n = 243) refused and 35% (n = 394) did not respond.
Conclusions
While this study demonstrated disappointing outcomes, pre-recruitment letters should not be ruled out as an approach altogether. Rather, future research should explore whether other factors to increase motivation, such as intensity and timing, are feasible and acceptable for contacting cancer patients.
Trial registration
Australian and New Zealand Clinical Trials Registry, ACTRN12609000628246
doi:10.1186/1471-2288-14-44
PMCID: PMC3975453  PMID: 24690533
Colorectal cancer; Patient recruitment; Population registers; Randomized controlled trials
14.  Application of 6,7-Indole Aryne Cycloaddition and Pd(0)-Catalyzed Suzuki-Miyaura and Buchwald-Hartwig Cross-Coupling Reactions for the Preparation of Annulated Indole Libraries 
ACS combinatorial science  2011;13(5):443-448.
The construction of an unprecedented class of an indole-based library, namely, a 6,7-annulated-4-substituted 93-member indole library, using a strategic combination of 6,7-indolyne cycloaddition and cross-coupling reactions under both Suzuki-Miyaura and Buchwald-Hartwig conditions is described. This work represents the first example of library development that employs the indole aryne methodology. Annulated indoles, with the exception of only a few biologically active natural products (i.e., the trikentrins, herbindoles, teleocidins, and nodulisporic acids), have no representation in the PubChem or MLSMR databases. These structural entities are therefore predicted to have unique chemical property space characteristics and a high probability of exhibiting interesting biological activity.
doi:10.1021/co2000289
PMCID: PMC3959986  PMID: 21668016
Indole; aryne; indolyne; cycloaddition; Suzuki-Miyaura; Buchwald-Hartwig; libraries
15.  TSLP-elicited basophil responses can mediate the pathogenesis of eosinophilic esophagitis 
Nature medicine  2013;19(8):1005-1013.
Eosinophilic esophagitis (EoE) is a food allergy-associated inflammatory disease characterized by esophageal eosinophilia. EoE has become increasingly common, but current management strategies are nonspecific. Thus, there is an urgent need to identify specific immunological pathways that could be targeted to treat this disease. EoE is associated with polymorphisms in the gene that encodes thymic stromal lymphopoietin (TSLP), a cytokine that promotes allergic inflammation, but how TSLP might contribute to EoE disease pathogenesis remains unknown. Here, we describe a new mouse model of EoE-like disease that developed independently of IgE but was dependent on TSLP-elicited basophils. Therapeutic TSLP neutralization or basophil depletion also ameliorated established EoE-like disease. Critically, in human subjects with EoE, we observed elevated TSLP levels and exaggerated basophil responses in esophageal biopsies, and a gain-of-function TSLP polymorphism was associated with increased basophil responses. Together, these data suggest that the TSLP-basophil axis could be therapeutically targeted to treat EoE.
doi:10.1038/nm.3281
PMCID: PMC3951204  PMID: 23872715
16.  A Biophysical Basis for Mucus Solids Concentration as a Candidate Biomarker for Airways Disease 
PLoS ONE  2014;9(2):e87681.
In human airways diseases, including cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), host defense is compromised and airways inflammation and infection often result. Mucus clearance and trapping of inhaled pathogens constitute key elements of host defense. Clearance rates are governed by mucus viscous and elastic moduli at physiological driving frequencies, whereas transport of trapped pathogens in mucus layers is governed by diffusivity. There is a clear need for simple and effective clinical biomarkers of airways disease that correlate with these properties. We tested the hypothesis that mucus solids concentration, indexed as weight percent solids (wt%), is such a biomarker. Passive microbead rheology was employed to determine both diffusive and viscoelastic properties of mucus harvested from human bronchial epithelial (HBE) cultures. Guided by sputum from healthy (1.5–2.5 wt%) and diseased (COPD, CF; 5 wt%) subjects, mucus samples were generated in vitro to mimic in vivo physiology, including intermediate range wt% to represent disease progression. Analyses of microbead datasets showed mucus diffusive properties and viscoelastic moduli scale robustly with wt%. Importantly, prominent changes in both biophysical properties arose at ∼4 wt%, consistent with a gel transition (from a more viscous-dominated solution to a more elastic-dominated gel). These findings have significant implications for: (1) penetration of cilia into the mucus layer and effectiveness of mucus transport; and (2) diffusion vs. immobilization of micro-scale particles relevant to mucus barrier properties. These data provide compelling evidence for mucus solids concentration as a baseline clinical biomarker of mucus barrier and clearance functions.
doi:10.1371/journal.pone.0087681
PMCID: PMC3928107  PMID: 24558372
17.  Impact of Chorda Tympani Nerve Injury on Cell Survival, Axon Maintenance, and Morphology of the Chorda Tympani Nerve Terminal Field in the Nucleus of the Solitary Tract 
The Journal of comparative neurology  2012;520(11):2395-2413.
Chorda tympani nerve transection (CTX) has been useful to study the relationship between nerve and taste buds in fungiform papillae. This work demonstrated that the morphological integrity of taste buds depends on their innervation. Considerable research focused on the effects of CTX on peripheral gustatory structures, but much less research has focused on the central effects. Here, we explored how CTX affects ganglion cell survival, maintenance of injured peripheral axons, and the chorda tympani nerve terminal field organization in the nucleus of the solitary tract (NTS). After CTX in adult rats, the chorda tympani nerve was labeled with biotinylated dextran amine at 3, 7, 14, 30, and 60 days post-CTX to allow visualization of the terminal field associated with peripheral processes. There was a significant and persistent reduction of the labeled chorda tympani nerve terminal field volume and density in the NTS following CTX. Compared with controls, the volume of the labeled terminal field was not altered at 3 or 7 days post-CTX; however, it was significantly reduced by 44% and by 63% at 30 and 60 days post-CTX, respectively. Changes in the density of labeled terminal field in the NTS paralleled the terminal field volume results. The dramatic decrease in labeled terminal field size post-CTX cannot be explained by a loss of geniculate ganglion neurons or degeneration of central axons. Instead, the function and/or maintenance of the peripheral axonal process appear to be affected. These new results have implications for long-term functional and behavioral alterations.
doi:10.1002/cne.23044
PMCID: PMC3905605  PMID: 22237830
taste; gustatory; anterograde; axotomy; biotinylated dextran amine
18.  Large Scale Explorative Oligonucleotide Probe Selection for Thousands of Genetic Groups on a Computing Grid: Application to Phylogenetic Probe Design Using a Curated Small Subunit Ribosomal RNA Gene Database 
The Scientific World Journal  2014;2014:350487.
Phylogenetic Oligonucleotide Arrays (POAs) were recently adapted for studying the huge microbial communities in a flexible and easy-to-use way. POA coupled with the use of explorative probes to detect the unknown part is now one of the most powerful approaches for a better understanding of microbial community functioning. However, the selection of probes remains a very difficult task. The rapid growth of environmental databases has led to an exponential increase of data to be managed for an efficient design. Consequently, the use of high performance computing facilities is mandatory. In this paper, we present an efficient parallelization method to select known and explorative oligonucleotide probes at large scale using computing grids. We implemented a software that generates and monitors thousands of jobs over the European Computing Grid Infrastructure (EGI). We also developed a new algorithm for the construction of a high-quality curated phylogenetic database to avoid erroneous design due to bad sequence affiliation. We present here the performance and statistics of our method on real biological datasets based on a phylogenetic prokaryotic database at the genus level and a complete design of about 20,000 probes for 2,069 genera of prokaryotes.
doi:10.1155/2014/350487
PMCID: PMC3913353  PMID: 24516366
19.  The influence of commensal bacteria-derived signals on basophil-associated allergic inflammation 
Gut Microbes  2013;4(1):76-83.
Commensal bacteria that colonize mammalian mucosal surfaces are reported to influence T helper type 2 (TH2) cytokine-dependent inflammation and susceptibility to allergic disease. However, the mechanisms that underlie these observations are only beginning to be understood. We recently utilized studies of murine model systems and atopic patient populations to elucidate a mechanism by which commensal bacteria-derived signals limit serum immunoglobulin E levels, influence basophil development and steady-state circulating basophil populations and regulate basophil-associated TH2 cell responses and allergic inflammation. In this addendum, we summarize the findings of our recent work and other developments in the field, discuss the broader implications of these findings and generate new hypotheses regarding our understanding of host-commensal relationships. These areas of investigation may be applicable to the development of new preventative or therapeutic approaches to reduce the burden of allergic disease.
doi:10.4161/gmic.22759
PMCID: PMC3555891  PMID: 23137965
commensal bacteria; allergy; atopy; basophil; hematopoiesis
20.  Comparative analysis of virus-host interactomes with a mammalian high- throughput protein complementation assay based on Gaussia princeps luciferase 
Methods (San Diego, Calif.)  2012;58(4):349-359.
Comparative interactomics is a strategy for inferring potential interactions among orthologous proteins or “iginterologs”. Herein we focus, in contrast to standard homology-based inference, on the divergence of protein interaction profiles among closely related organisms, showing that the approach can correlate specific traits to phenotypic differences. As a model, this new comparative interactomic approach was applied at a large scale to human papillomaviruses (HPVs) proteins. The oncogenic potential of HPVs is mainly determined by the E6 and E7 early proteins. We have mapped and overlapped the virus-host protein interaction networks of E6 and E7 proteins from 11 distinct HPV genotypes, selected for their different tropisms and pathologies. We generated robust and comprehensive datasets by combining two orthogonal protein interation assays: yeast two-hybrid (Y2H), and our recently described “High-Throughput Gaussia princeps Protein Complementation Assay” (HT-GPCA). HT-GPCA detects protein interaction by measuring the interaction-mediated reconstitution of activity of a split Gaussia princeps luciferase. Hierarchical clustering of interaction profiles recapitulated HPV phylogeny and was used to correlate specific virus-host interaction profiles with pathological traits, reflecting the distinct carcinogenic potentials of different HPVs. This comparative interactomics constitutes a reliable and powerful strategy to decipher molecular relationships in virtually any combination of microorganism-host interactions.
doi:10.1016/j.ymeth.2012.07.029
PMCID: PMC3546263  PMID: 22898364
comparative interactomics; HPV; E6; E7; interactome; complementation assay
21.  Mycobacterium tuberculosis Type VII Secreted Effector EsxH Targets Host ESCRT to Impair Trafficking 
PLoS Pathogens  2013;9(10):e1003734.
Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D'Arcy Hart showed that Mtb avoids delivery to lysosomes, but the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown. Here, we describe a systematic strategy to identify bacterial virulence factors by looking for interactions between the Mtb secretome and host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform. Using this approach we identified an interaction between EsxH, which is secreted by the Esx-3 TSSS, and human hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). ESCRT has a well-described role in directing proteins destined for lysosomal degradation into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), ensuring degradation of the sorted cargo upon MVB-lysosome fusion. Here, we show that ESCRT is required to deliver Mtb to the lysosome and to restrict intracellular bacterial growth. Further, EsxH, in complex with EsxG, disrupts ESCRT function and impairs phagosome maturation. Thus, we demonstrate a role for a TSSS and the host ESCRT machinery in one of the central features of tuberculosis pathogenesis.
Author Summary
Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis, one of the world's most deadly infections. The host immune system can't eradicate Mtb because it grows within macrophages, cells that normally kill bacteria. One of the intracellular survival strategies of Mtb is to avoid delivery to lysosomes, a phenomenon described over 40 years ago, but for which the mechanism and molecular details remain incomplete. Mtb possess specialized secretion systems (Type VII secretion systems; TSSS) that transfer particular proteins out of the bacteria, but how these proteins promote infection is not well understood. In this study, we used a high stringency yeast two-hybrid system to identify interactions between secreted effectors from Mtb and human host factors. We identified ninety-nine such interactions and focused our attention on the interaction between EsxH, secreted by Esx-3, a TSSS of Mtb, and Hrs, a component of the host ESCRT machinery. We provide evidence that Mtb EsxH directly targets host Hrs to disrupt delivery of bacteria to lysosomes. Thus, this study demonstrates the role of a TSSS effector and the ESCRT machinery in what is one of the central features of tuberculosis pathogenesis, thereby providing molecular insight into why humans can't clear Mtb infection.
doi:10.1371/journal.ppat.1003734
PMCID: PMC3814348  PMID: 24204276
22.  Extensive Rewiring and Complex Evolutionary Dynamics in a C. elegans Multiparameter Transcription Factor Network 
Molecular cell  2013;51(1):116-127.
SUMMARY
Gene duplication results in two identical paralogs that diverge through mutation, leading to loss or gain of interactions with other biomolecules. Here, we comprehensively characterize such network rewiring for C. elegans transcription factors (TFs) within and across four newly delineated molecular networks. Remarkably, we find that even highly similar TFs often have different interaction degree and partners. In addition, we find that most TF families have a member that is highly connected in multiple networks. Further, different TF families have opposing correlations between network connectivity and phylogenetic age, suggesting that they are subject to different evolutionary pressures. Finally, TFs that have similar partners in one network generally do not in another, indicating a lack of pressure to retain cross-network similarity. Our multiparameter analyses provide an unprecedented glimpse into the evolutionary dynamics that shaped TF networks.
doi:10.1016/j.molcel.2013.05.018
PMCID: PMC3794439  PMID: 23791784
23.  Disentangling Coordination among Functional Traits Using an Individual-Centred Model: Impact on Plant Performance at Intra- and Inter-Specific Levels 
PLoS ONE  2013;8(10):e77372.
Background
Plant functional traits co-vary along strategy spectra, thereby defining trade-offs for resource acquisition and utilization amongst other processes. A main objective of plant ecology is to quantify the correlations among traits and ask why some of them are sufficiently closely coordinated to form a single axis of functional specialization. However, due to trait co-variations in nature, it is difficult to propose a mechanistic and causal explanation for the origin of trade-offs among traits observed at both intra- and inter-specific level.
Methodology/Principal Findings
Using the Gemini individual-centered model which coordinates physiological and morphological processes, we investigated with 12 grass species the consequences of deliberately decoupling variation of leaf traits (specific leaf area, leaf lifespan) and plant stature (height and tiller number) on plant growth and phenotypic variability. For all species under both high and low N supplies, simulated trait values maximizing plant growth in monocultures matched observed trait values. Moreover, at the intraspecific level, plastic trait responses to N addition predicted by the model were in close agreement with observed trait responses. In a 4D trait space, our modeling approach highlighted that the unique trait combination maximizing plant growth under a given environmental condition was determined by a coordination of leaf, root and whole plant processes that tended to co-limit the acquisition and use of carbon and of nitrogen.
Conclusion/Significance
Our study provides a mechanistic explanation for the origin of trade-offs between plant functional traits and further predicts plasticity in plant traits in response to environmental changes. In a multidimensional trait space, regions occupied by current plant species can therefore be viewed as adaptive corridors where trait combinations minimize allometric and physiological constraints from the organ to the whole plant levels. The regions outside this corridor are empty because of inferior plant performance.
doi:10.1371/journal.pone.0077372
PMCID: PMC3793938  PMID: 24130879
24.  The Gene Ontology (GO) Cellular Component Ontology: integration with SAO (Subcellular Anatomy Ontology) and other recent developments 
Background
The Gene Ontology (GO) (http://www.geneontology.org/) contains a set of terms for describing the activity and actions of gene products across all kingdoms of life. Each of these activities is executed in a location within a cell or in the vicinity of a cell. In order to capture this context, the GO includes a sub-ontology called the Cellular Component (CC) ontology (GO-CCO). The primary use of this ontology is for GO annotation, but it has also been used for phenotype annotation, and for the annotation of images. Another ontology with similar scope to the GO-CCO is the Subcellular Anatomy Ontology (SAO), part of the Neuroscience Information Framework Standard (NIFSTD) suite of ontologies. The SAO also covers cell components, but in the domain of neuroscience.
Description
Recently, the GO-CCO was enriched in content and links to the Biological Process and Molecular Function branches of GO as well as to other ontologies. This was achieved in several ways. We carried out an amalgamation of SAO terms with GO-CCO ones; as a result, nearly 100 new neuroscience-related terms were added to the GO. The GO-CCO also contains relationships to GO Biological Process and Molecular Function terms, as well as connecting to external ontologies such as the Cell Ontology (CL). Terms representing protein complexes in the Protein Ontology (PRO) reference GO-CCO terms for their species-generic counterparts. GO-CCO terms can also be used to search a variety of databases.
Conclusions
In this publication we provide an overview of the GO-CCO, its overall design, and some recent extensions that make use of additional spatial information. One of the most recent developments of the GO-CCO was the merging in of the SAO, resulting in a single unified ontology designed to serve the needs of GO annotators as well as the specific needs of the neuroscience community.
doi:10.1186/2041-1480-4-20
PMCID: PMC3852282  PMID: 24093723
Gene ontology; Cellular component ontology; Subcellular anatomy ontology; Neuroscience; Annotation; Ontology language; Ontology integration; Neuroscience information framework
25.  Postnatal Development of Chorda Tympani Axons in the Rat Nucleus of the Solitary Tract 
The Journal of comparative neurology  2012;520(14):3217-3235.
The chorda tympani nerve (CT), one of three nerves that convey gustatory information to nucleus of the solitary tract (NTS), displays terminal field reorganization after postnatal day 15 in the rat. Aiming to gain insight into mechanisms of this phenomenon, CT axon projection field and terminal morphology in NTS subdivisions were examined using tract tracing, light microscopy, and immuno-electron microscopy at four postnatal ages: P15, P25, P35, and adult. The CT axons that innervated NTS rostrolateral subdivision both in the adult and in P15 rats were morphologically distinct from those that innervated the rostrocentral, gustatory subdivision. In both subdivisions, CT terminals reached morphological maturity before P15. Rostrolateral, but not rostrocentral axons, went through substantial axonal branch elimination after P15. Rostrocentral CT synapses, however, redistribute onto postsynaptic targets in the following weeks. CT terminal preference for GABAergic postsynaptic targets was drastically reduced after P15. Furthermore, CT synapses became a smaller component of the total synaptic input to the rostrocentral NTS after P35. The results underlined that CT axons in rostrocentral and rostrolateral subdivisions represent two distinct populations of CT input, displaying different morphological properties and structural reorganization mechanisms during postnatal development.
doi:10.1002/cne.23093
PMCID: PMC3418376  PMID: 22430892
Development; Chorda Tympani; Gustatory; Taste; Electron Microscopy

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