Echinobase: a resource to support the echinoderm research community.

Echinobase (www.echinobase.org) is a model organism knowledgebase serving as a resource for the community that studies echinoderms, a phylum of marine invertebrates that includes sea urchins and sea stars. Echinoderms have been important experimental models for over 100 years and continue to make important contributions to environmental, evolutionary, and developmental studies, including research on developmental gene regulatory networks. As a centralized resource, Echinobase hosts genomes and collects functional genomic data, reagents, literature, and other information for the community. This third-generation site is based on the Xenbase knowledgebase design and utilizes gene-centric pages to minimize the time and effort required to access genomic information. Summary gene pages display gene symbols and names, functional data, links to the JBrowse genome browser, and orthology to other organisms and reagents, and tabs from the Summary gene page contain more detailed information concerning mRNAs, proteins, diseases, and protein-protein interactions. The gene pages also display 1:1 orthologs between the fully supported species Strongylocentrotus purpuratus (purple sea urchin), Lytechinus variegatus (green sea urchin), Patiria miniata (bat star), and Acanthaster planci (crown-of-thorns sea star). JBrowse tracks are available for visualization of functional genomic data from both fully supported species and the partially supported species Anneissia japonica (feather star), Asterias rubens (sugar star), and L. pictus (painted sea urchin). Echinobase serves a vital role by providing researchers with annotated genomes including orthology, functional genomic data aligned to the genomes, and curated reagents and data. The Echinoderm Anatomical Ontology provides a framework for standardizing developmental data across the phylum, and knowledgebase content is formatted to be findable, accessible, interoperable, and reusable by the research community.

New hypotheses of cell type diversity and novelty from orthology-driven comparative single cell and nuclei transcriptomics in echinoderms.

Cell types are the building blocks of metazoan biodiversity and offer a powerful perspective for inferring evolutionary phenomena. With the development of single-cell transcriptomic techniques, new definitions of cell types are emerging. This allows a conceptual reassessment of traditional definitions of novel cell types and their evolution. Research in echinoderms, particularly sea star and sea urchin embryos has contributed significantly to understanding the evolution of novel cell types, through the examination of skeletogenic mesenchyme and pigment cells, which are found in sea urchin larvae, but not sea star larvae. This paper outlines the development of a gene expression atlas for the bat sea star, Patiria miniata, using single nuclear RNA sequencing (snRNA-seq) of embryonic stages. The atlas revealed 23 cell clusters covering all expected cell types from the endoderm, mesoderm, and ectoderm germ layers. In particular, four distinct neural clusters, an immune-like cluster, and distinct right and left coelom clusters were revealed as distinct cell states. A comparison with Strongylocentrotus purpuratus embryo single-cell transcriptomes was performed using 1:1 orthologs to anchor and then compare gene expression patterns. The equivalent of S. purpuratus piwil3+ Cells were not detected in P. miniata, while the Left Coelom of P. miniata has no equivalent cell cluster in S. purpuratus. These differences may reflect changes in developmental timing between these species. While considered novel morphologically, the Pigment Cells of S. purpuratus map to clusters containing Immune-like Mesenchyme and Neural cells of P. miniata, while the Skeletogenic Mesenchyme of S. purpuratus are revealed as orthologous to the Right Coelom cluster of P. miniata. These results suggest a new interpretation of the evolution of these well-studied cell types and a reflection on the definition of novel cell types.

Multicolor plate reader fluorescence calibration.

Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.

Echinobase: leveraging an extant model organism database to build a knowledgebase supporting research on the genomics and biology of echinoderms.

Echinobase (www.echinobase.org) is a third generation web resource supporting genomic research on echinoderms. The new version was built by cloning the mature Xenopus model organism knowledgebase, Xenbase, refactoring data ingestion pipelines and modifying the user interface to adapt to multispecies echinoderm content. This approach leveraged over 15 years of previous database and web application development to generate a new fully featured informatics resource in a single year. In addition to the software stack, Echinobase uses the private cloud and physical hosts that support Xenbase. Echinobase currently supports six echinoderm species, focused on those used for genomics, developmental biology and gene regulatory network analyses. Over 38 000 gene pages, 18 000 publications, new improved genome assemblies, JBrowse genome browser and BLAST + services are available and supported by the development of a new echinoderm anatomical ontology, uniformly applied formal gene nomenclature, and consistent orthology predictions. A novel feature of Echinobase is integrating support for multiple, disparate species. New genomes from the diverse echinoderm phylum will be added and supported as data becomes available. The common code development design of the integrated knowledgebases ensures parallel improvements as each resource evolves. This approach is widely applicable for developing new model organism informatics resources.

Classifying domain-specific text documents containing ambiguous keywords.

A keyword-based search of comprehensive databases such as PubMed may return irrelevant papers, especially if the keywords are used in multiple fields of study. In such cases, domain experts (curators) need to verify the results and remove the irrelevant articles. Automating this filtering process will save time, but it has to be done well enough to ensure few relevant papers are rejected and few irrelevant papers are accepted. A good solution would be fast, work with the limited amount of data freely available (full paper body may be missing), handle ambiguous keywords and be as domain-neutral as possible. In this paper, we evaluate a number of classification algorithms for identifying a domain-specific set of papers about echinoderm species and show that the resulting tool satisfies most of the abovementioned requirements. Echinoderms consist of a number of very different organisms, including brittle stars, sea stars (starfish), sea urchins and sea cucumbers. While their taxonomic identifiers are specific, the common names are used in many other contexts, creating ambiguity and making a keyword search prone to error. We try classifiers using Linear, Naïve Bayes, Nearest Neighbor, Tree, SVM, Bagging, AdaBoost and Neural Network learning models and compare their performance. We show how effective the resulting classifiers are in filtering irrelevant articles returned from PubMed. The methodology used is more dependent on the good selection of training data and is a practical solution that can be applied to other fields of study facing similar challenges. Database URL: The code and date reported in this paper are freely available at http://xenbaseturbofrog.org/pub/Text-Topic-Classifier/.

CLARINET: efficient learning of dynamic network models from literature.

Motivation: Creating or extending computational models of complex systems, such as intra- and intercellular biological networks, is a time and labor-intensive task, often limited by the knowledge and experience of modelers. Automating this process would enable rapid, consistent, comprehensive and robust analysis and understanding of complex systems. Results: In this work, we present CLARINET (CLARIfying NETworks), a novel methodology and a tool for automatically expanding models using the information extracted from the literature by machine reading. CLARINET creates collaboration graphs from the extracted events and uses several novel metrics for evaluating these events individually, in pairs, and in groups. These metrics are based on the frequency of occurrence and co-occurrence of events in literature, and their connectivity to the baseline model. We tested how well CLARINET can reproduce manually built and curated models, when provided with varying amount of information in the baseline model and in the machine reading output. Our results show that CLARINET can recover all relevant interactions that are present in the reading output and it automatically reconstructs manually built models with average recall of 80% and average precision of 70%. CLARINET is highly scalable, its average runtime is at the order of ten seconds when processing several thousand interactions, outperforming other similar methods. Availability and implementation: The data underlying this article are available in Bitbucket at https://bitbucket.org/biodesignlab/clarinet/src/master/. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Protein Proximity Observed Using Fluorogen Activating Protein and Dye Activated by Proximal Anchoring (FAP-DAPA) System.

The development and function of tissues, blood, and the immune system is dependent upon proximity for cellular recognition and communication. However, the detection of cell-to-cell contacts is limited due to a lack of reversible, quantitative probes that can function at these dynamic sites of irregular geometry. Described here is a novel chemo-genetic tool developed for fluorescent detection of protein-protein proximity and cell apposition that utilizes the Fluorogen Activating Protein (FAP) in combination with a Dye Activated by Proximal Anchoring (DAPA). The FAP-DAPA system has two protein components, the HaloTag and FAP, expressed on separate protein targets or in separate cells. The proteins function to bind and activate a compound that has the hexyl chloride (HexCl) ligand connected to malachite green (MG), the FAP fluorogen, via a poly(ethylene glycol) spacer spanning up to 28 nm. The dehalogenase protein, HaloTag, covalently binds the HexCl ligand, locally concentrating the attached MG. If the FAP is within range of the anchored fluorogen, it will bind and activate MG specifically when the bath concentration is too low to saturate the FAP receptor. A new FAP variant was isolated with a 1000-fold reduced KD of ∼10-100 nM so that the fluorogen activation reports proximity without artificially enhancing it. The system was characterized using purified FRB and FKBP fusion proteins and showed a doubling of fluorescence upon rapamycin induced complex formation. In cocultured HEK293 cells (HaloTag and FAP-expressing) fluorescence increased at contact sites across a broad range of labeling conditions, more reliably providing contact-specific fluorescence activation with the lower-affinity FAP variant. When combined with suitable targeting and expression constructs, this labeling system may offer significant improvements in on-demand detection of intercellular contacts, potentially applicable in neurological and immunological synapse measurements and other transient, dynamic biological appositions that can be perturbed using other labeling methods that stabilize these interactions.

FLUTE: Fast and reliable knowledge retrieval from biomedical literature.

State-of-the-art machine reading methods extract, in hours, hundreds of thousands of events from the biomedical literature. However, many of the extracted biomolecular interactions are incorrect or not relevant for computational modeling of a system of interest. Therefore, rapid, automated methods are required to filter and select accurate and useful information. The FiLter for Understanding True Events (FLUTE) tool uses public protein interaction databases to filter interactions that have been extracted by machines from databases such as PubMed and score them for accuracy. Confidence in the interactions allows for rapid and accurate model assembly. As our results show, FLUTE can reliably determine the confidence in the biomolecular interactions extracted by fast machine readers and at the same time provide a speedup in interaction filtering by three orders of magnitude. Database URL: https://bitbucket.org/biodesignlab/flute.

Enhanced Hybridization Selectivity Using Structured GammaPNA Probes.

High affinity nucleic acid analogues such as gammaPNA (γPNA) are capable of invading stable secondary and tertiary structures in DNA and RNA targets but are susceptible to off-target binding to mismatch-containing sequences. We introduced a hairpin secondary structure into a γPNA oligomer to enhance hybridization selectivity compared with a hairpin-free analogue. The hairpin structure features a five base PNA mask that covers the proximal five bases of the γPNA probe, leaving an additional five γPNA bases available as a toehold for target hybridization. Surface plasmon resonance experiments demonstrated that the hairpin probe exhibited slower on-rates and faster off-rates (i.e., lower affinity) compared with the linear probe but improved single mismatch discrimination by up to a factor of five, due primarily to slower on-rates for mismatch vs. perfect match targets. The ability to discriminate against single mismatches was also determined in a cell-free mRNA translation assay using a luciferase reporter gene, where the hairpin probe was two-fold more selective than the linear probe. These results validate the hairpin design and present a generalizable approach to improving hybridization selectivity.

Analysis of In Vitro Cytotoxicity of Carbohydrate-Based Materials Used for Dissolvable Microneedle Arrays.

PURPOSE: Dissolvable microneedle arrays (MNAs) can be used to realize enhanced transdermal and intradermal drug delivery. Dissolvable MNAs are fabricated from biocompatible and water-soluble base polymers, and the biocargo to be delivered is integrated with the base polymer when forming the MNAs. The base polymer is selected to provide mechanical strength, desired dissolution characteristics, and compatibility with the biocargo. However, to satisfy regulatory requirements and be utilized in clinical applications, cytotoxicity of the base polymers should also be thoroughly characterized. This study systematically investigated the cytotoxicity of several important carbohydrate-based base polymers used for production of MNAs, including carboxymethyl cellulose (CMC), maltodextrin (MD), trehalose (Treh), glucose (Gluc), and hyaluronic acid (HA). METHODS: Each material was evaluated using in vitro cell-culture methods on relevant mouse and human cells, including MPEK-BL6 mouse keratinocytes, NIH-3T3 mouse fibroblasts, HaCaT human keratinocytes, and NHDF human fibroblasts. A common laboratory cell line, human embryonic kidney cells HEK-293, was also used to allow comparisons to various cytotoxicity studies in the literature. Dissolvable MNA materials were evaluated at concentrations ranging from 3 mg/mL to 80 mg/mL. RESULTS: Qualitative and quantitative analyses of cytotoxicity were performed using optical microscopy, confocal fluorescence microscopy, and flow cytometry-based assays for cell morphology, viability, necrosis and apoptosis. Results from different methods consistently demonstrated negligible in vitro cytotoxicity of carboxymethyl cellulose, maltodextrin, trehalose and hyaluronic acid. Glucose was observed to be toxic to cells at concentrations higher than 50 mg/mL. CONCLUSIONS: It is concluded that CMC, MD, Treh, HA, and glucose (at low concentrations) do not pose challenges in terms of cytotoxicity, and thus, are good candidates as MNA materials for creating clinically-relevant and well-tolerated biodissolvable MNAs.

Cross-talk between Colon Cells and Macrophages Increases ST6GALNAC1 and MUC1-sTn Expression in Ulcerative Colitis and Colitis-Associated Colon Cancer.

Patients with ulcerative colitis have an increased risk of developing colitis-associated colon cancer (CACC). Changes in glycosylation of the oncoprotein MUC1 commonly occur in chronic inflammation, including ulcerative colitis, and this abnormally glycosylated MUC1 promotes cancer development and progression. It is not known what causes changes in glycosylation of MUC1. Gene expression profiling of myeloid cells in inflamed and malignant colon tissues showed increased expression levels of inflammatory macrophage-associated cytokines compared with normal tissues. We analyzed the involvement of macrophage-associated cytokines in the induction of aberrant MUC1 glycoforms. A coculture system was used to examine the effects of M1 and M2 macrophages on glycosylation-related enzymes in colon cancer cells. M2-like macrophages induced the expression of the glycosyltransferase ST6GALNAC1, an enzyme that adds sialic acid to O-linked GalNAc residues, promoting the formation of tumor-associated sialyl-Tn (sTn) O-glycans. Immunostaining of ulcerative colitis and CACC tissue samples confirmed the elevated number of M2-like macrophages as well as high expression of ST6GALNAC1 and the altered MUC1-sTn glycoform on colon cells. Cytokine arrays and blocking antibody experiments indicated that the macrophage-dependent ST6GALNAC1 activation was mediated by IL13 and CCL17. We demonstrated that IL13 promoted phosphorylation of STAT6 to activate transcription of ST6GALNAC1. A computational model of signaling pathways was assembled and used to test IL13 inhibition as a possible therapy. Our findings revealed a novel cellular cross-talk between colon cells and macrophages within the inflamed and malignant colon that contributes to the pathogenesis of ulcerative colitis and CACC.See related Spotlight on p. 160.

Fluorescence-Based Quantitative Synapse Analysis for Cell Type-Specific Connectomics.

Anatomical methods for determining cell type-specific connectivity are essential to inspire and constrain our understanding of neural circuit function. We developed genetically-encoded reagents for fluorescence-synapse labeling and connectivity analysis in brain tissue, using a fluorogen-activating protein (FAP)-coupled or YFP-coupled, postsynaptically-localized neuroligin-1 (NL-1) targeting sequence (FAP/YFPpost). FAPpost expression did not alter mEPSC or mIPSC properties. Sparse AAV-mediated expression of FAP/YFPpost with the cell-filling, red fluorophore dTomato (dTom) enabled high-throughput, compartment-specific detection of putative synapses across diverse neuron types in mouse somatosensory cortex. We took advantage of the bright, far-red emission of FAPpost puncta for multichannel fluorescence alignment of dendrites, FAPpost puncta, and presynaptic neurites in transgenic mice with saturated labeling of parvalbumin (PV), somatostatin (SST), or vasoactive intestinal peptide (VIP)-expressing neurons using Cre-reporter driven expression of YFP. Subtype-specific inhibitory connectivity onto layer 2/3 (L2/3) neocortical pyramidal (Pyr) neurons was assessed using automated puncta detection and neurite apposition. Quantitative and compartment-specific comparisons show that PV inputs are the predominant source of inhibition at both the soma and the dendrites and were particularly concentrated at the primary apical dendrite. SST inputs were interleaved with PV inputs at all secondary-order and higher-order dendritic branches. These fluorescence-based synapse labeling reagents can facilitate large-scale and cell-type specific quantitation of changes in synaptic connectivity across development, learning, and disease states.

PI3K class II α regulates δ-opioid receptor export from the trans-Golgi network.

The interplay between signaling and trafficking by G protein-coupled receptors (GPCRs) has focused mainly on endocytic trafficking. Whether and how surface delivery of newly synthesized GPCRs is regulated by extracellular signals is less understood. Here we define a signaling-regulated checkpoint at the trans-Golgi network (TGN) that controls the surface delivery of the delta opioid receptor (δR). In PC12 cells, inhibition of phosphoinositide-3 kinase (PI3K) activity blocked export of newly synthesized δR from the Golgi and delivery to the cell surface, similar to treatment with nerve growth factor (NGF). Depletion of class II phosphoinositide-3 kinase α (PI3K C2A), but not inhibition of class I PI3K, blocked δR export to comparable levels and attenuated δR-mediated cAMP inhibition. NGF treatment displaced PI3K C2A from the Golgi and optogenetic recruitment of the PI3K C2A kinase domain to the TGN-induced δR export downstream of NGF. Of importance, PI3K C2A expression promotes export of endogenous δR in primary trigeminal ganglion neurons. Taken together, our results identify PI3K C2A as being required and sufficient for δR export and surface delivery in neuronal cells and suggest that it could be a key modulator of a novel Golgi export checkpoint that coordinates GPCR delivery to the surface.

RNA G-Quadruplex Invasion and Translation Inhibition by Antisense γ-Peptide Nucleic Acid Oligomers.

We have examined the abilities of three complementary γ-peptide nucleic acid (γPNA) oligomers to invade an RNA G-quadruplex and potently inhibit translation of a luciferase reporter transcript containing the quadruplex-forming sequence (QFS) within its 5'-untranslated region. All three γPNA oligomers bind with low nanomolar affinities to an RNA oligonucleotide containing the QFS. However, while all probes inhibit translation with low to midnanomolar IC50 values, the γPNA designed to hybridize to the first two G-tracts of the QFS and adjacent 5'-overhanging nucleotides was 5-6 times more potent than probes directed to either the 3'-end or internal regions of the target at 37 °C. This position-dependent effect was eliminated after the probes and target were preincubated at an elevated temperature prior to translation, demonstrating that kinetic effects exert significant control over quadruplex invasion and translation inhibition. We also found that antisense γPNAs exhibited similarly potent effects against luciferase reporter transcripts bearing QFS motifs having G2, G3, or G4 tracts. Finally, our results indicate that γPNA oligomers exhibit selectivity and/or potency higher than those of other antisense molecules such as standard PNA and 2'-OMe RNA previously reported to target G-quadruplexes in RNA.

In Vitro Reversible Translation Control Using γPNA Probes.

On-demand regulation of gene expression in living cells is a central goal of chemical biology and antisense therapeutic development. While significant advances have allowed regulatory modulation through inserted genetic elements, on-demand control of the expression/translation state of a given native gene by complementary sequence interactions remains a technical challenge. Toward this objective, we demonstrate the reversible suppression of a luciferase gene in cell-free translation using Watson-Crick base pairing between the mRNA and a complementary γ-modified peptide nucleic acid (γPNA) sequence with a noncomplementary toehold. Exploiting the favorable thermodynamics of γPNA-γPNA interactions, the antisense sequence can be removed by hybridization of a second, fully complementary γPNA, through a strand displacement reaction, allowing translation to proceed. Complementary RNA is also shown to displace the bound antisense γPNA, opening up possibilities of in vivo regulation by native gene expression.

Genetically targeted fluorogenic macromolecules for subcellular imaging and cellular perturbation.

The alteration of cellular functions by anchoring macromolecules to specified organelles may reveal a new area of therapeutic potential and clinical treatment. In this work, a unique phenotype was evoked by influencing cellular behavior through the modification of subcellular structures with genetically targetable macromolecules. These fluorogen-functionalized polymers, prepared via controlled radical polymerization, were capable of exclusively decorating actin, cytoplasmic, or nuclear compartments of living cells expressing localized fluorgen-activating proteins. The macromolecular fluorogens were optimized by establishing critical polymer architecture-biophysical property relationships which impacted binding rates, binding affinities, and the level of internalization. Specific labeling of subcellular structures was realized at nanomolar concentrations of polymer, in the absence of membrane permeabilization or transduction domains, and fluorogen-modified polymers were found to bind to protein intact after delivery to the cytosol. Cellular motility was found to be dependent on binding of macromolecular fluorogens to actin structures causing rapid cellular ruffling without migration.

Rapid, specific, no-wash, far-red fluorogen activation in subcellular compartments by targeted fluorogen activating proteins.

Live cell imaging requires bright photostable dyes that can target intracellular organelles and proteins with high specificity in a no-wash protocol. Organic dyes possess the desired photochemical properties and can be covalently linked to various protein tags. The currently available fluorogenic dyes are in the green/yellow range where there is high cellular autofluorescence and the near-infrared (NIR) dyes need to be washed out. Protein-mediated activation of far-red fluorogenic dyes has the potential to address these challenges because the cell-permeant dye is small and nonfluorescent until bound to its activating protein, and this binding is rapid. In this study, three single chain variable fragment (scFv)-derived fluorogen activating proteins (FAPs), which activate far-red emitting fluorogens, were evaluated for targeting, brightness, and photostability in the cytosol, nucleus, mitochondria, peroxisomes, and endoplasmic reticulum with a cell-permeant malachite green analog in cultured mammalian cells. Efficient labeling was achieved within 20-30 min for each protein upon the addition of nM concentrations of dye, producing a signal that colocalized significantly with a linked mCerulean3 (mCer3) fluorescent protein and organelle specific dyes but showed divergent photostability and brightness properties dependent on the FAP. These FAPs and the ester of malachite green dye (MGe) can be used as specific, rapid, and wash-free labels for intracellular sites in live cells with far-red excitation and emission properties, useful in a variety of multicolor experiments.

Fluorogen activating protein-affibody probes: modular, no-wash measurement of epidermal growth factor receptors.

Fluorescence is essential for dynamic live cell imaging, and affinity reagents are required for quantification of endogenous proteins. Various fluorescent dyes can report on different aspects of biological trafficking, but must be independently conjugated to affinity reagents and characterized for specific biological readouts. Here we present the characterization of a new modular platform for small anti-EGFR affinity probes for studying rapid changes in receptor pools. A protein domain (FAP dL5**) that binds to malachite-green (MG) derivatives for fluorescence activation was expressed as a recombinant fusion to one or two copies of the compact EGFR binding affibody ZEGFR:1907. This is a recombinant and fluorogenic labeling reagent for native EGFR molecules. In vitro fluorescence assays demonstrated that the binding of these dyes to the FAP-affibody fusions produced thousand-fold fluorescence enhancements, with high binding affinity and fast association rates. Flow cytometry assays and fluorescence microscopy demonstrated that these probes label endogenous EGFR on A431 cells without disruption of EGFR function, and low nanomolar surface Kd values were observed with the double-ZEGFR:1907 constructs. The application of light-harvesting fluorogens (dyedrons) significantly improved the detected fluorescence signal. Altering the order of addition of the ligand, probe, and dyes allowed differentiation between surface and endocytotic pools of receptors to reveal the rapid dynamics of endocytic trafficking. Therefore, FAP/affibody coupling provides a new approach to construct compact and modular affinity probes that label endogenous proteins on living cells and can be used for studying rapid changes in receptor pools involved in trafficking.

Fluorogen-activating proteins provide tunable labeling densities for tracking FcεRI independent of IgE.

Crosslinking of IgE bound FcεRI on mast cells and basophils by multivalent antigen leads to degranulation and the release of key inflammatory mediators that stimulate the allergic response. Here, we present and characterize the use of fluorogen-activating proteins (FAPs) for single particle tracking of FcεRI to investigate how receptor mobility is influenced after IgE-induced changes in mast cell behavior. FAPs are genetically encoded tags that bind a fluorogen dye and increase its brightness upon binding up to 20,000-fold. We demonstrate that, by titrating fluorogen concentration, labeling densities from ensemble to single particle can be achieved, independent of expression level and without the need for wash steps or photobleaching. The FcεRI γ-subunit fused to a FAP (FAP-γ) provides, for the first time, an IgE-independent probe for tracking this signaling subunit of FcεRI at the single molecule level. We show that the FcεRI γ-subunit dynamics are controlled by the IgE-binding α-subunit and that the cytokinergic IgE, SPE-7, induces mast cell activation without altering FcεRI mobility or promoting internalization. We take advantage of the far-red emission of the malachite green (MG) fluorogen to track FcεRI relative to dynamin-GFP and find that immobilized receptors readily correlate with locations of dynamin recruitment only under conditions that promote rapid endocytosis. These studies demonstrate the usefulness of the FAP system for single molecule studies and have provided new insights into the relationship among FcεRI structure, activity, and mobility.

Detection and assignment of CYP21 mutations using peptide mass signature genotyping.

Congenital adrenal hyperplasia (CAH) is a common inborn error of steroidogenesis. The clinical spectrum of CAH ranges from the severe classical form, which can be fatal in the newborn, to simple virilizing forms or a milder non-classical form which is often not diagnosed until puberty. Recessive mutations in the autosomal gene encoding 21-hydroxylase (CYP21) are responsible for approximately 95% of CAH cases. Since CYP21 genotype is generally predictive of the presence and severity of the disorder, accurate CYP21 genotyping is of clear medical significance. Determining the CYP21 genotype of an individual, using standard methods, is difficult due to the presence of a nearly identical pseudogene (CYP21P) in close proximity to the functional gene. To address the need for a comprehensive test for mutations in the CYP21 gene, we developed a multiplexed peptide mass signature genotyping (PMSG) assay and applied the assay to 151 DNA samples. CAH patients had been previously characterized for the 10 most common mutations. The PMSG assay detected all common mutations; in addition it identified six known rare mutations and also discovered four new mutations (two frameshifts in the first half of the gene, P42fs and S171fs, and two point mutations, H365Y and R479L). This assay has the potential to provide high-throughput, cost-effective analysis of the CYP21 gene to detect known mutations and identify novel variants in samples obtained from patients with CAH, individuals suspected to have CAH, and heterozygous carriers.