Proteotransciptomics of the Most Popular Host Sea Anemone Entacmaea quadricolor Reveals Not All Toxin Genes Expressed by Tentacles Are Recruited into Its Venom Arsenal.

While the unique symbiotic relationship between anemonefishes and sea anemones is iconic, it is still not fully understood how anemonefishes can withstand and thrive within the venomous environment of their host sea anemone. In this study, we used a proteotranscriptomics approach to elucidate the proteinaceous toxin repertoire from the most common host sea anemone, Entacmaea quadricolor. Although 1251 different toxin or toxin-like RNA transcripts were expressed in E. quadricolor tentacles (0.05% of gene clusters, 1.8% of expression) and 5375 proteins were detected in milked venom, only 4% of proteins detected in venom were putative toxins (230), and they only represent on average 14% of the normalised protein expression in the milked venom samples. Thus, most proteins in milked venom do not appear to have a toxin function. This work raises the perils of defining a dominant venom phenotype based on transcriptomics data alone in sea anemones, as we found that the dominant venom phenotype differs between the transcriptome and proteome abundance data. E. quadricolor venom contains a mixture of toxin-like proteins of unknown and known function. A newly identified toxin protein family, Z3, rich in conserved cysteines of unknown function, was the most abundant at the RNA transcript and protein levels. The venom was also rich in toxins from the Protease S1, Kunitz-type and PLA2 toxin protein families and contains toxins from eight venom categories. Exploring the intricate venom toxin components in other host sea anemones will be crucial for improving our understanding of how anemonefish adapt to the venomous environment.

Proteomic profiling of paired human liver homogenate and tissue derived extracellular vesicles.

Advances in technologies to isolate extracellular vesicles (EVs) and detect/quantify their cargo underpin the novel potential of these circulating particles as a liquid biopsy to understand physiology and disease. One organ of particular interest in terms of utilizing EVs as a liquid biopsy is the liver. The extent to which EVs originating from the liver reflect the functional status of this organ remains unknown. This is an important knowledge gap that underpins the utility of circulating liver derived EVs as a liquid biopsy. The primary objective of this study was to characterize the proteomic profile of EVs isolated from the extracellular space of liver tissue (LEV) and compare this profile to that of paired tissue (LH). LCMS analyses detected 2892 proteins in LEV and 2673 in LH. Of the 2673 proteins detected in LH, 1547 (58%) were also detected in LEV. Bioinformatic analyses demonstrated comparable representation of proteins in terms of biological functions and cellular compartments. Although, enriched representation of membrane proteins and associated functions was observed in LEV, while representation of nuclear proteins and associated functions was depleted in LEV. These data support the potential use of circulating liver derived EVs as a liquid biopsy for this organ.

Circular RNAs drive oncogenic chromosomal translocations within the MLL recombinome in leukemia.

The first step of oncogenesis is the acquisition of a repertoire of genetic mutations to initiate and sustain the malignancy. An important example of this initiation phase in acute leukemias is the formation of a potent oncogene by chromosomal translocations between the mixed lineage leukemia (MLL) gene and one of 100 translocation partners, known as the MLL recombinome. Here, we show that circular RNAs (circRNAs)-a family of covalently closed, alternatively spliced RNA molecules-are enriched within the MLL recombinome and can bind DNA, forming circRNA:DNA hybrids (circR loops) at their cognate loci. These circR loops promote transcriptional pausing, proteasome inhibition, chromatin re-organization, and DNA breakage. Importantly, overexpressing circRNAs in mouse leukemia xenograft models results in co-localization of genomic loci, de novo generation of clinically relevant chromosomal translocations mimicking the MLL recombinome, and hastening of disease onset. Our findings provide fundamental insight into the acquisition of chromosomal translocations by endogenous RNA carcinogens in leukemia.

The Nasal Innate Immune Proteome After Saline Irrigation: A Pilot Study in Healthy Individuals.

BACKGROUND: Previous research has shown diminished nasal immune function following nasal saline irrigation (NSI), returning to baseline at 6 hours. The aim of this study was to examine the immune nasal proteome before and after 14 days of nasal irrigation. METHODS: Seventeen healthy volunteers received either isotonic (IsoSal) or low salt (LowNa) NSI. Nasal secretions were collected before and 30 min after NSI at baseline and again after 14 days. Specimens were analyzed using mass spectrometry to detect proteins of relevance to nasal immune function. RESULTS: One thousand eight hundred and sixty-five proteins were identified with significant changes in 71 proteins, of which 23 were identified as part of the innate immune system. Baseline analysis demonstrated an increase of 9 innate proteins after NSI, most after IsoSal. After 14 days, a greater increase in innate peptides was present, with most now in the LowNa group. When NSI solutions were compared, a significant increase in 4 innate proteins, including a 211% in lysozyme, was detected in the LowNa group. CONCLUSION: LowNa NSI demonstrates evidence of improving the innate immune secretions, especially lysozyme, in healthy volunteers.

Immunisation of ferrets and mice with recombinant SARS-CoV-2 spike protein formulated with Advax-SM adjuvant protects against COVID-19 infection.

The development of a safe and effective vaccine is a key requirement to overcoming the COVID-19 pandemic. Recombinant proteins represent the most reliable and safe vaccine approach but generally require a suitable adjuvant for robust and durable immunity. We used the SARS-CoV-2 genomic sequence and in silico structural modelling to design a recombinant spike protein vaccine (Covax-19™). A synthetic gene encoding the spike extracellular domain (ECD) was inserted into a baculovirus backbone to express the protein in insect cell cultures. The spike ECD was formulated with Advax-SM adjuvant and first tested for immunogenicity in C57BL/6 and BALB/c mice. Covax-19 vaccine induced high spike protein binding antibody levels that neutralised the original lineage B.1.319 virus from which the vaccine spike protein was derived, as well as the variant B.1.1.7 lineage virus. Covax-19 vaccine also induced a high frequency of spike-specific CD4 + and CD8 + memory T-cells with a dominant Th1 phenotype associated with the ability to kill spike-labelled target cells in vivo. Ferrets immunised with Covax-19 vaccine intramuscularly twice 2 weeks apart made spike receptor binding domain (RBD) IgG and were protected against an intranasal challenge with SARS-CoV-2 virus given two weeks after the last immunisation. Notably, ferrets that received the two higher doses of Covax-19 vaccine had no detectable virus in their lungs or in nasal washes at day 3 post-challenge, suggesting that in addition to lung protection, Covax-19 vaccine may have the potential to reduce virus transmission. This data supports advancement of Covax-19 vaccine into human clinical trials.

Proteomic analysis of nasal mucus samples of healthy patients and patients with chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) has a complex and multifactorial pathogenesis with a heterogeneous inflammatory profile. Proteomic analysis of nasal mucus may enable further understanding of protein abundances and biologic processes present in CRS and its endotypes compared with in healthy patients. OBJECTIVE: Our aim was to determine differences in the nasal mucus proteome of healthy patients and patients with CRS. METHODS: Nasal mucus was obtained from healthy patients, patients with CRS without nasal polyps (CRSsNP), and patients with CRS with nasal polyps (CRSwNP) before surgery. Gel electrophoresis was performed to fractionate the complex protein extracts before mass spectrometry analysis. Gene set enrichment analysis was performed on differentially expressed proteins. RESULTS: A total of 33 patients were included in this study (12 healthy, 10 with CRSsNP, and 11 with CRSwNP). In all, 1142 proteins were identified in mucus samples from healthy patients, 761 in mucus samples from patients with CRSsNP, and 998 in mucus samples from patients with CRSwNP. Dysfunction in immunologic pathways, reduced cellular signaling, and increased cellular metabolism with associated tissue remodeling pathways were present in patients with CRS compared with in healthy patients. CONCLUSION: Significant downregulation of mucosal immunity and antioxidant pathways with increased tissue modeling processes may account for the clinical manifestations of CRS. Ultimately, the differing proteome and biologic processes provide further insight into CRS pathogenesis and its endotypes.

Proteomic profiling of precipitated Clostridioides difficile toxin A and B antibodies.

Clostridioides difficile infection is the leading cause of nosocomial diarrhoea globally. Immune responses to toxins produced by C. difficile are important in disease progression and outcome. Here, we analysed the anti-toxin A and anti-toxin B serum antibody proteomes following natural infection or vaccination with a C. difficile toxoid A/toxoid B vaccine using a modified miniaturised proteomic approach based on de novo mass spectrometric sequencing. Analysis of immunoglobulin variable region (IgV) subfamily expression in immunoprecipitated toxin A and toxin B antibodies from four and seven participants of a vaccine trial, respectively, revealed a polyclonal proteome with restricted IGHV, IGKV and IGLV subfamily usage. No dominant IGHV subfamily was observed in the toxin A response, however the dominant anti-toxin B heavy (H)-chain was encoded by IGHV3-23. Light (L)-chain usage was convergent for both anti-toxin A and anti-toxin B proteomes with IGKV3-11, 3-15, 3-20 and 4-1 shared among all subjects in both cohorts. Peptide mapping of common IgV families showed extensive public and private amino acid substitutions. The cohort responses to toxin A and toxin B showed limited similarity in shared IGHV subfamilies. L-chain subfamily usage was more similar in the anti-toxin A and anti-toxin B responses, however the mutational signatures for each subfamily were toxin-dependent. Samples taken both post vaccination (n = 5) or at baseline, indicating previous exposure (n = 2), showed similar anti-toxin B IgV subfamily usage and mutational profiles. In summary, this study provides the first sequence-based proteomic analysis of the antibody response to the major disease-mediating toxins of C. difficile, toxin A and toxin B, and demonstrates that despite the potential for extreme diversity, the immunoglobulin repertoire can raise convergent responses to specific pathogens whether through natural infection or following vaccination.

Abundance of Synaptic Vesicle-Related Proteins in Alpha-Synuclein-Containing Protein Inclusions Suggests a Targeted Formation Mechanism.

Proteinaceous α-synuclein-containing inclusions are found in affected brain regions in patients with Parkinson's disease (PD), Dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). These appear in neurons as Lewy bodies in both PD and DLB and as glial cytoplasmic inclusions (GCIs) in oligodendrocytes in MSA. The role they play in the pathology of the diseases is unknown, and relatively little is still known about their composition. By purifying the inclusions from the surrounding tissue and comprehensively analysing their protein composition, vital clues to the formation mechanism and role in the disease process may be found. In this study, Lewy bodies were purified from postmortem brain tissue from DLB cases (n = 2) and GCIs were purified from MSA cases (n = 5) using a recently improved purification method, and the purified inclusions were analysed by mass spectrometry. Twenty-one percent of the proteins found consistently in the GCIs and LBs were synaptic-vesicle related. Identified proteins included those associated with exosomes (CD9), clathrin-mediated endocytosis (clathrin, AP-2 complex, dynamin), retrograde transport (dynein, dynactin, spectrin) and synaptic vesicle fusion (synaptosomal-associated protein 25, vesicle-associated membrane protein 2, syntaxin-1). This suggests that the misfolded or excess α-synuclein may be targeted to inclusions via vesicle-mediated transport, which also explains the presence of the neuronal protein α-synuclein within GCIs.

Molecular Profiling and Clonal Tracking of Secreted Rheumatoid Factors in Primary Sjögren's Syndrome.

OBJECTIVE: Rheumatoid factors (RFs) are associated with systemic disease in primary Sjögren's syndrome (SS) and may be pathogenic as mixed cryoglobulins. Current detection methods cannot resolve RFs at a molecular level. This study was undertaken to perform the first proteomic and transcriptomic analysis of secreted and membrane-bound IgM-RF in primary SS and identify unique heavy-chain peptide signatures for RF clonotype tracking. METHODS: Purified heavy chains of serum RFs from 15 patients with primary SS were subjected to de novo mass spectrometric sequencing. The circulating B cell Ig repertoire was determined by massively parallel sequencing of IGH RNA from matched peripheral blood mononuclear cells (n = 7). RF-specific heavy-chain third complementarity-determining region (CDR3) peptides were identified by searching RF heavy-chain peptide sequences against the corresponding IGH RNA sequence libraries. Heavy-chain CDR3 peptides were used as biomarkers to track serum RF clonotypes using quantitative multiple reaction monitoring. RESULTS: Serum RFs were clonally restricted and composed of shared sets of IgM heavy-chain variable region (Ig VH ) 1-69, 3-15, 3-7, and 3-74 subfamilies. Cryoprecipitable RFs from patients with mixed cryoglobulinemia (MC) were distinguishable from nonprecipitating RFs by a higher frequency of amino acid substitutions and identification of stereotypic heavy-chain CDR3 transcripts. Potentially pathogenic RF clonotypes were detected in serum by multiple reaction monitoring years before patients presented with MC. Levels of Ig VH 4-34 IgM-RF decreased following immunosuppression and remission of MC. CONCLUSION: Cryoprecipitable RF clonotypes linked to vasculitis in primary SS have different molecular profiles than nonprecipitating RFs, suggesting different underlying mechanisms of production. The combined omics workflow presented herein provides molecular biomarkers for tracking and removal of pathogenic RF clones.

Proteomic analysis of influenza haemagglutinin-specific antibodies following vaccination reveals convergent immunoglobulin variable region signatures.

Analysis of the anti-haemagglutinin serum antibody proteome from six H1N1pdm09 influenza A vaccinated subjects demonstrated restricted IgG1 heavy chain species encoded by IGHV5-51 and IGHV3-7 gene families in 2 subjects and either IGHV5-51 or IGHV3-7 in 4 individuals. All subjects exhibited a dominant IGKV3-20 light chain, however 5 subjects also exhibited IGKV3-11 and IGKV4-1 families. Sequences were closely aligned with the matched germline sequence, with few shared mutations. This study illustrates the feasibility of using a proteomic approach to determine the expressed V region signatures of serum antibodies induced by vaccination.

IgV peptide mapping of native Ro60 autoantibody proteomes in primary Sjögren's syndrome reveals molecular markers of Ro/La diversification.

We have used high-resolution mass spectrometry to sequence precipitating anti-Ro60 proteomes from sera of patients with primary Sjögren's syndrome and compare immunoglobulin variable-region (IgV) peptide signatures in Ro/La autoantibody subsets. Anti-Ro60 were purified by elution from native Ro60-coated ELISA plates and subjected to combined de novo amino acid sequencing and database matching. Monospecific anti-Ro60 Igs comprised dominant public and minor private sets of IgG1 kappa and lambda restricted heavy and light chains. Specific IgV amino acid substitutions stratified anti-Ro60 from anti-Ro60/La responses, providing a molecular fingerprint of Ro60/La determinant spreading and suggesting that different forms of Ro60 antigen drive these responses. Sequencing of linked anti-Ro52 proteomes from individual patients and comparison with their anti-Ro60 partners revealed sharing of a dominant IGHV3-23/IGKV3-20 paired clonotype but with divergent IgV mutational signatures. In summary, anti-Ro60 IgV peptide mapping provides insights into Ro/La autoantibody diversification and reveals serum-based molecular markers of humoral Ro60 autoimmunity.

Secreted autoantibody repertoires in Sjögren's syndrome and systemic lupus erythematosus: A proteomic approach.

The structures of epitopes bound by autoantibodies against RNA-protein complexes have been well-defined over several decades, but little is known of the clonality, immunoglobulin (Ig) variable (V) gene usage and mutational status of the autoantibodies themselves at the level of the secreted (serum) proteome. A novel proteomic workflow is presented based on affinity purification of specific Igs from serum, high-resolution two-dimensional gel electrophoresis, and de novo and database-driven sequencing of V-region proteins by mass spectrometry. Analysis of anti-Ro52/Ro60/La proteomes in primary Sjögren's syndrome (SS) and anti-Sm and anti-ribosomal P proteomes in systemic lupus erythematosus (SLE) has revealed that these antibody responses are dominated by restricted sets of public (shared) clonotypes, consistent with common pathways of production across unrelated individuals. The discovery of shared sets of specific V-region peptides can be exploited for diagnostic biomarkers in targeted mass spectrometry platforms and for tracking and removal of pathogenic clones.

Serum SmD autoantibody proteomes are clonally restricted and share variable-region peptides.

Recent advances in mass spectrometry-based proteomic methods have allowed variable (V)-region peptide signatures to be derived from human autoantibodies present in complex serum mixtures. Here, we analysed the clonality and V-region composition of immunoglobulin (Ig) proteomes specific for the immunodominant SmD protein subunit of the lupus-specific Sm autoantigen. Precipitating SmD-specific IgGs were eluted from native SmD-coated ELISA plates preincubated with sera from six patients with systemic lupus erythematosus (SLE) positive for anti-Sm/RNP. Heavy (H)- and light (L)-chain clonality and V-region sequences were analysed by 2-dimensional gel electrophoresis and combined de novo database mass spectrometric sequencing. SmD autoantibody proteomes from all six patients with SLE expressed IgG1 kappa restricted clonotypes specified by IGHV3-7 and IGHV1-69 H-chains and IGKV3-20 and IGKV2-28 L-chains, with shared and individual V-region amino acid replacement mutations. Clonotypic sharing and restricted V-region diversity of systemic autoimmunity can now be extended from the Ro/La cluster to Sm autoantigen and implies a common pathway of anti-Sm autoantibody production in unrelated patients with SLE.

Comparison of Stain-Free gels with traditional immunoblot loading control methodology.

Loading controls are necessary for semiquantitative Western blotting to compensate for loading errors. Loading control methods include the reprobing of membranes with an antibody against a constitutively expressed protein or staining the membrane with a total protein stain. We compared the loading control performance of recently released Stain-Free (SF) gels with Sypro Ruby (SR) and reprobing using ß-actin. SF gels demonstrated superior performance in that they were faster, required fewer steps and consumables, and allowed the quality of electrophoresis and Western transfer to be assessed before committing to costly and time-consuming Western blots.

Drosophila orthologue of WWOX, the chromosomal fragile site FRA16D tumour suppressor gene, functions in aerobic metabolism and regulates reactive oxygen species.

Common chromosomal fragile sites FRA3B and FRA16D are frequent sites of DNA instability in cancer, but their contribution to cancer cell biology is not yet understood. Genes that span these sites (FHIT and WWOX, respectively) are often perturbed (either increased or decreased) in cancer cells and both are able to suppress tumour growth. While WWOX has some tumour suppressor characteristics, its normal role and functional contribution to cancer has not been fully determined. We find that a significant proportion of Drosophila Wwox interactors identified by proteomics and microarray analyses have roles in aerobic metabolism. Functional relationships between Wwox and either CG6439/isocitrate dehydrogenase (Idh) or Cu-Zn superoxide dismutase (Sod) were confirmed by genetic interactions. In addition, altered levels of Wwox resulted in altered levels of endogenous reactive oxygen species. Wwox (like FHIT) contributes to pathways involving aerobic metabolism and oxidative stress, providing an explanation for the 'non-classical tumour suppressor' behaviour of WWOX. Fragile sites, and the genes that span them, are therefore part of a protective response mechanism to oxidative stress and likely contributors to the differences seen in aerobic glycolysis (Warburg effect) in cancer cells.

Know thy fly.

The generation and analysis of mutants is central to studies of gene function in model organisms. Methods for random mutagenesis in Drosophila melanogaster have been available for many years, but an alternative approach--targeted mutagenesis using homologous recombination--has only recently been developed. This approach has the advantage of specificity, because genes of interest can be altered. One might expect with a gene-targeting approach that the frequency of background mutations would be minimal. Unfortunately, we have found that this is not the case. Although the possibility of background mutations arising during homologous-recombination-based gene targeting has been raised in the literature, it is not routinely taken into account when using this technique. Our experience suggests that it can be a considerable problem but that it has a relatively simple solution.