As high‐throughput techniques including proteomics become more accessible to individual laboratories, there is an urgent need for a user‐friendly bioinformatics analysis system. Here, we describe ...FunRich, an open access, standalone functional enrichment and network analysis tool. FunRich is designed to be used by biologists with minimal or no support from computational and database experts. Using FunRich, users can perform functional enrichment analysis on background databases that are integrated from heterogeneous genomic and proteomic resources (>1.5 million annotations). Besides default human specific FunRich database, users can download data from the UniProt database, which currently supports 20 different taxonomies against which enrichment analysis can be performed. Moreover, the users can build their own custom databases and perform the enrichment analysis irrespective of organism. In addition to proteomics datasets, the custom database allows for the tool to be used for genomics, lipidomics and metabolomics datasets. Thus, FunRich allows for complete database customization and thereby permits for the tool to be exploited as a skeleton for enrichment analysis irrespective of the data type or organism used. FunRich (http://www.funrich.org) is user‐friendly and provides graphical representation (Venn, pie charts, bar graphs, column, heatmap and doughnuts) of the data with customizable font, scale and color (publication quality).
Background
Diagnostic tests for fish allergy are hampered by the large number of under‐investigated fish species. Four salmon allergens are well‐characterized and registered with the WHO/IUIS while ...no catfish allergens have been described so far. In 2008, freshwater‐cultured catfish production surpassed that of salmon, the globally most‐cultured marine species. We aimed to identify, quantify, and compare all IgE‐binding proteins in salmon and catfish.
Methods
Seventy‐seven pediatric patients with clinically confirmed fish allergy underwent skin prick tests to salmon and catfish. The allergen repertoire of raw and heated protein extracts was evaluated by immunoblotting using five allergen‐specific antibodies and patients' serum followed by mass spectrometric analyses.
Results
Raw and heated extracts from catfish displayed a higher frequency of IgE‐binding compared to those from salmon (77% vs 70% and 64% vs 53%, respectively). The major fish allergen parvalbumin demonstrated the highest IgE‐binding capacity (10%‐49%), followed by triosephosphate isomerase (TPI; 19%‐34%) in raw and tropomyosin (6%‐32%) in heated extracts. Six previously unidentified fish allergens, including TPI, were registered with the WHO/IUIS. Creatine kinase from salmon and catfish was detected by IgE from 14% and 10% of patients, respectively. Catfish L‐lactate dehydrogenase, glyceraldehyde‐3‐phosphate dehydrogenase, pyruvate kinase, and glucose‐6‐phosphate isomerase showed IgE‐binding for 6%‐13% of patients. In salmon, these proteins could not be separated successfully.
Conclusions
We detail the allergen repertoire of two highly farmed fish species. IgE‐binding to fish tropomyosins and TPIs was demonstrated for the first time in a large patient cohort. Tropomyosins, in addition to parvalbumins, should be considered for urgently needed improved fish allergy diagnostics.
Catfish raw and heated extracts demonstrate higher prevalence and diversity of IgE‐binding as compared to salmon. PV, TM and TPI are the most prominent IgE‐binding fish proteins, followed by Eno, Ald and GAPDH. TM is the second most abundant protein, after PV, in heated extracts and up to 49% of 77 fish‐allergic children demonstrate IgE‐binding.
Abbreviations: Ald, aldolase; Eno, enolase; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; MS, mass spectrometry, PV, parvalbumin, SDS‐PAGE, sodium dodecyl sulphate‐polyacrylamide gel electrophoresis; TM, tropomyosin, TPI, triosephosphate isomerase
Here, we discovered an endogenous dafachronic acid (DA) in the socioeconomically important parasitic nematode Haemonchus contortus. We demonstrate that DA promotes larval exsheathment and development ...in this nematode via a relatively conserved nuclear hormone receptor (DAF-12). This stimulatory effect is dose- and time-dependent, and relates to a modulation of dauer-like signalling, and glycerolipid and glycerophospholipid metabolism, likely via a negative feedback loop. Specific chemical inhibition of DAF-9 (cytochrome P450) was shown to significantly reduce the amount of endogenous DA in H. contortus; compromise both larval exsheathment and development in vitro; and modulate lipid metabolism. Taken together, this evidence shows that DA plays a key functional role in the developmental transition from the free-living to the parasitic stage of H. contortus by modulating the dauer-like signalling pathway and lipid metabolism. Understanding the intricacies of the DA-DAF-12 system and associated networks in H. contortus and related parasitic nematodes could pave the way to new, nematode-specific treatments.
This paper discusses the successful adoption of a subscription-based, open-access model of service delivery for a mass spectrometry and proteomics facility. In 2009, the Mass Spectrometry and ...Proteomics Facility at the University of Melbourne (Australia) moved away from the standard fee for service model of service provision. Instead, the facility adopted a subscription- or membership-based, open-access model of service delivery. For a low fixed yearly cost, users could directly operate the instrumentation but, more importantly, there were no limits on usage other than the necessity to share available instrument time with all other users. All necessary training from platform staff and many of the base reagents were also provided as part of the membership cost. These changes proved to be very successful in terms of financial outcomes for the facility, instrument access and usage, and overall research output. This article describes the systems put in place as well as the overall successes and challenges associated with the operation of a mass spectrometry/proteomics core in this manner.
Graphical abstract
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The maintenance of the Golgi ribbon relies on a dynamic balance between the actin and microtubule networks, however the pathways controlling actin networks remain poorly defined. Previously, we ...showed that the TGN membrane tether/golgin, GCC88, modulates the Golgi ribbon architecture (Gosavi et al., 2018). Here we show that dispersal of the Golgi ribbon by GCC88 is dependent on actin and the involvement of non-muscle myosin IIA. We have identified the long isoform of intersectin-1 (ITSN-1), a guanine nucleotide exchange factor for Cdc42, as a novel Golgi component and an interaction partner of GCC88 responsible for mediating the actin-dependent dispersal of the Golgi ribbon. We show that perturbation of Golgi morphology by changes in membrane flux, mediated by silencing the retromer subunit Vps26, or in a model of neurodegeneration, induced by Tau overexpression, are also dependent on the ITSN-1-GCC88 interaction. Overall, our study reveals a role for a TGN golgin and ITSN-1 in linking to the actin cytoskeleton and regulating the balance between a compact Golgi ribbon and a dispersed Golgi, a pathway with relevance to pathophysiological conditions.
Antigen-presenting molecules, encoded by the major histocompatibility complex (MHC) and CD1 family, bind peptide- and lipid-based antigens, respectively, for recognition by T cells. ...Mucosal-associated invariant T (MAIT) cells are an abundant population of innate-like T cells in humans that are activated by an antigen(s) bound to the MHC class I-like molecule MR1. Although the identity of MR1-restricted antigen(s) is unknown, it is present in numerous bacteria and yeast. Here we show that the structure and chemistry within the antigen-binding cleft of MR1 is distinct from the MHC and CD1 families. MR1 is ideally suited to bind ligands originating from vitamin metabolites. The structure of MR1 in complex with 6-formyl pterin, a folic acid (vitamin B9) metabolite, shows the pterin ring sequestered within MR1. Furthermore, we characterize related MR1-restricted vitamin derivatives, originating from the bacterial riboflavin (vitamin B2) biosynthetic pathway, which specifically and potently activate MAIT cells. Accordingly, we show that metabolites of vitamin B represent a class of antigen that are presented by MR1 for MAIT-cell immunosurveillance. As many vitamin biosynthetic pathways are unique to bacteria and yeast, our data suggest that MAIT cells use these metabolites to detect microbial infection.
Hydrophilic interaction liquid chromatography (HILIC) glycopeptide enrichment is an indispensable tool for the high-throughput characterization of glycoproteomes. Despite its utility, HILIC ...enrichment is associated with a number of shortcomings, including requiring large amounts of starting materials, potentially introducing chemical artifacts such as formylation when high concentrations of formic acid are used, and biasing/undersampling specific classes of glycopeptides. Here, we investigate HILIC enrichment-independent approaches for the study of bacterial glycoproteomes. Using three Burkholderia species (Burkholderia cenocepacia, Burkholderia Dolosa, and Burkholderia ubonensis), we demonstrate that short aliphatic O-linked glycopeptides are typically absent from HILIC enrichments, yet are readily identified in whole proteome samples. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS) fractionation, we show that at high compensation voltages (CVs), short aliphatic glycopeptides can be enriched from complex samples, providing an alternative means to identify glycopeptide recalcitrant to hydrophilic-based enrichment. Combining whole proteome and FAIMS analyses, we show that the observable glycoproteome of these Burkholderia species is at least 25% larger than what was initially thought. Excitingly, the ability to enrich glycopeptides using FAIMS appears generally applicable, with the N-linked glycopeptides of Campylobacter fetus subsp. fetus also being enrichable at high FAIMS CVs. Taken together, these results demonstrate that FAIMS provides an alternative means to access glycopeptides and is a valuable tool for glycoproteomic analysis.
Single-molecule localization-based super-resolution microscopy techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) produce ...pointillist data sets of molecular coordinates. Although many algorithms exist for the identification and localization of molecules from raw image data, methods for analyzing the resulting point patterns for properties such as clustering have remained relatively under-studied. Here we present a model-based Bayesian approach to evaluate molecular cluster assignment proposals, generated in this study by analysis based on Ripley's K function. The method takes full account of the individual localization precisions calculated for each emitter. We validate the approach using simulated data, as well as experimental data on the clustering behavior of CD3ζ, a subunit of the CD3 T cell receptor complex, in resting and activated primary human T cells.
The objective of this study was to characterize colistin-resistant bacteria isolated from pigs on a farm in Great Britain following identification of a plasmid-borne colistin resistance mechanism in ...Escherichia coli from China.
Phenotypic antimicrobial susceptibility testing was undertaken by broth dilution and WGS was performed to detect the presence of genes encoding resistance and virulence. Transferable colistin resistance was investigated by conjugation.
Two E. coli and one Salmonella Typhimurium variant Copenhagen were shown to be MDR, including resistance to colistin, with one E. coli and the Salmonella carrying the mcr-1 gene; all three harboured chromosomal mutations in genes conferring colistin resistance and both E. coli harboured β-lactamase resistance. The Salmonella mcr-1 plasmid was highly similar to pHNSHP45, from China, while the E. coli mcr-1 plasmid only had the ISApII and mcr-1 genes in common. The frequency of mcr-1 plasmid transfer by conjugation to recipient Enterobacteriaceae from Salmonella was low, lying between 10(-7) and 10(-9) cfu/recipient cfu. We were unable to demonstrate mcr-1 plasmid transfer from the E. coli. Plasmid profiling indicated transfer of multiple plasmids from the Salmonella resulting in some MDR transconjugants.
Identification of the mcr-1 gene in Enterobacteriaceae from pigs confirms its presence in livestock in Great Britain. The results suggest dissemination of resistance through different horizontally transferable elements. The in vitro transfer of multiple plasmids carrying colistin and other resistances from the Salmonella isolate underlines the potential for wider dissemination and recombination.
Background
Commercial allergen extracts for allergy skin prick testing (SPT) are widely used for diagnosing fish allergy. However, there is currently no regulatory requirement for standardization of ...protein and allergen content, potentially impacting the diagnostic reliability of SPTs. We therefore sought to analyse commercial fish extracts for the presence and concentration of fish proteins and in vitro IgE reactivity using serum from fish‐allergic patients.
Methods
Twenty‐six commercial fish extracts from five different manufacturers were examined. The protein concentrations were determined, protein compositions analysed by mass spectrometry, followed by SDS‐PAGE and subsequent immunoblotting with antibodies detecting 4 fish allergens (parvalbumin, tropomyosin, aldolase and collagen). IgE‐reactive proteins were identified using serum from 16 children with confirmed IgE‐mediated fish allergy, with focus on cod, tuna and salmon extracts.
Results
The total protein, allergen concentration and IgE reactivity of the commercial extracts varied over 10‐fold between different manufacturers and fish species. The major fish allergen parvalbumin was not detected by immunoblotting in 6/26 extracts. In 7/12 extracts, five known fish allergens were detected by mass spectrometry. For cod and tuna, almost 70% of patients demonstrated the strongest IgE reactivity to collagen, tropomyosin, aldolase A or β‐enolase but not parvalbumin.
Conclusions
Commercial fish extracts often contain insufficient amounts of important allergens including parvalbumin and collagen, resulting in low IgE reactivity. A comprehensive proteomic approach for the evaluation of SPT extracts for their utility in allergy diagnostics is presented. There is an urgent need for standardized allergen extracts, which will improve the diagnosis and management of fish allergy.
Commercial skin prick test (SPT) extracts often contain insufficient amounts of important fish allergens, resulting in low IgE reactivity. Collagen, tropomyosin and aldolase A are of under‐recognized importance for the diagnosis of fish allergy. Understanding the molecular allergology of SPT allergen extracts is essential for best diagnostics.
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