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.

To investigate the molecular mechanism underlying the onset of choroidal neovascularization (CNV).
Integrated transcriptomic and proteomic analyses of retinas in mice with laser-induced CNV were performed using RNA sequencing and tandem mass tag. In addition, the laser-treated mice received systemic interferon-β (IFN-β) therapy. Measurements of CNV lesions were acquired by the confocal analysis of stained choroidal flat mounts. The proportions of T helper 17 (Th17) cells were determined by flow cytometric analysis.
A total of differentially expressed 186 genes (120 up-regulated and 66 down-regulated) and 104 proteins (73 up-regulated and 31 down-regulated) were identified. The gene ontology and KEGG pathway analyses indicated that CNV was mainly associated with immune and inflammatory responses, such as cellular response to IFN-β and Th17 cell differentiation. Moreover, the key nodes of the protein-protein interaction network mainly involved up-regulated proteins, including alpha A crystallin and fibroblast growth factor 2, and were verified by Western blotting. To confirm the changes in gene expression, real-time quantitative PCR was performed. Furthermore, levels of IFN-β in both the retina and plasma, as measured by enzyme-linked immunosorbent assay (ELISA), were significantly lower in the CNV group than in the control group. IFN-β treatment significantly reduced CNV lesion size and promoted the proliferation of Th17 cells in laser-treated mice.
This study demonstrates that the occurrence of CNV might be associated with the dysfunction of immune and inflammatory processes and that IFN-β could serve as a potential therapeutic target.

True water bugs (Nepomorpha) are mostly predacious insects that live in aquatic habitats. They use their piercing-sucking mouthparts to inject venomous saliva that facilitates the capture and extra-oral digestion of prey animals, but their venom can also be deployed for defence. In Central Europe, nepomorph species representing different families coexist in the same habitat. However, their feeding ecology, including venom composition and deployment, has not been investigated in detail. We used an integrated proteotranscriptomic and bioactivity-based approach to test whether venom composition and activity differ between four water bug species sharing the same habitat but occupying different ecological niches. We found considerable species-dependent differences in the composition of digestive enzymes and venom components that probably evolved as adaptations to particular food sources, foraging strategies and/or microhabitats. The venom of Corixa punctata differed substantially from that of the three strictly predatory species (Ilyocoris cimicoides, Notonecta glauca and Nepa cinerea), and the abundance of herbivory-associated proteins confirms a mostly plant-based diet. Our findings reveal independent adaptations of the digestive and defensive enzyme repertoires accompanied by the evolution of distinct feeding strategies in aquatic bugs.

Since the introduction of a highly pathogenic genotype II isolate of the African swine fever virus (ASFV) into Georgia in 2007, African swine fever (ASF) has gone panzootic. Outbreaks have been reported in Europe, Asia and, more recently, Latin America. Thus, ASFV has become a major threat to the pig industry worldwide, as broadly applicable vaccines are not available. While the majority of ASFV strains show high virulence in domestic pigs and wild boar, variations within the ASFV genome have resulted in the emergence of attenuated strains with low or moderate virulence. However, the molecular basis of the differences in virulence has not yet been discovered. To reveal virulence-associated protein expression patterns, we analysed the proteomes of the natural target cells of ASFV, primary porcine macrophages, after infection with two genotype II ASFV strains displaying high (Armenia 2008) and moderate (Estonia 2014) virulence using quantitative mass spectrometry. Very similar expression patterns were observed for the viral genes, and any differences were limited to the deletions within the Estonia 2014 genome. In addition to the canonical ASFV proteins, twelve novel protein products from recently described transcripts were confirmed in both isolates. Pathway analyses showed that both isolates evoked a similar host proteome response, despite their difference in virulence. However, subtle differences in the manipulation of the proteins involved in the proinflammatory response mediated by the MAPK14/p38 signalling cascade were observed.

Applications in omics research, such as comparative transcriptomics and proteomics, require the knowledge of the species-specific gene sequence and benefit from a comprehensive high-quality annotation of the coding genes to achieve high coverage. While protein-coding genes can in simple cases be detected by scanning the genome for open reading frames, in more complex genomes exonic sequences are separated by introns. Despite advances in sequencing technologies that allow for ever-growing numbers of genomes, the quality of many of the provided genome assemblies do not reach reference quality. These non-contiguous assemblies with gaps and the necessity to predict splice sites limit accurate gene annotation from solely genomic data. In contrast, the transcriptome only contains transcribed gene regions, is devoid of introns and thus provides the optimal basis for the identification of open reading frames. The additional integration of proteomics data to validate predicted protein-coding genes further enriches for accurate gene models. This review outlines the principles of the proteotranscriptomics approach, discusses common challenges and suggests methods for improvement.

Animal venoms are a rich source of novel biomolecules with potential applications in medicine and agriculture. Ants are one of the most species-rich lineages of venomous animals. However, only a fraction of their biodiversity has been studied so far. Here, we investigated the venom components of two myrmicine (subfamily Myrmicinae) ants: Myrmica rubra and Myrmica ruginodis. We applied a venomics workflow based on proteotranscriptomics and found that the venoms of both species are composed of several protein classes, including venom serine proteases, cysteine-rich secretory protein, antigen 5 and pathogenesis-related 1 (CAP) superfamily proteins, Kunitz-type serine protease inhibitors and venom acid phosphatases. Several of these protein classes are known venom allergens, and for the first time we detected phospholipase A1 in the venom of M. ruginodis. We also identified two novel epidermal growth factor (EGF) family toxins in the M. ruginodis venom proteome and an array of additional EGF-like toxins in the venom gland transcriptomes of both species. These are similar to known toxins from the related myrmicine ant, Manica rubida, and the myrmecine (subfamily Myrmeciinae) Australian red bulldog ant Myrmecia gullosa, and are possibly deployed as weapons in defensive scenarios or to subdue prey. Our work suggests that M.rubra and M. ruginodis venoms contain many enzymes and other high-molecular-weight proteins that cause cell damage. Nevertheless, the presence of EGF-like toxins suggests that myrmicine ants have also recruited smaller peptide components into their venom arsenal. Although little is known about the bioactivity and function of EGF-like toxins, their presence in myrmicine and myrmecine ants suggests they play a key role in the venom systems of the superfamily Formicoidea. Our work adds to the emerging picture of ant venoms as a source of novel bioactive molecules and highlights the need to incorporate such taxa in future venom bioprospecting programs.

BACKGROUND: The process of identifying all coding regions in a genome is crucial for any study at the level of molecular biology, ranging from single-gene cloning to genome-wide measurements using RNA-seq or mass spectrometry. While satisfactory annotation has been made feasible for well-studied model organisms through great efforts of big consortia, for most systems this kind of data is either absent or not adequately precise.
RESULTS: Combining in-depth transcriptome sequencing and high resolution mass spectrometry, we here use proteotranscriptomics to improve gene annotation of protein-coding genes in the Bombyx mori cell line BmN4 which is an increasingly used tool for the analysis of piRNA biogenesis and function. Using this approach we provide the exact coding sequence and evidence for more than 6200 genes on the protein level. Furthermore using spatial proteomics, we establish the subcellular localization of thousands of these proteins. We show that our approach outperforms current Bombyx mori annotation attempts in terms of accuracy and coverage.
CONCLUSIONS: We show that proteotranscriptomics is an efficient, cost-effective and accurate approach to improve previous annotations or generate new gene models. As this technique is based on de-novo transcriptome assembly, it provides the possibility to study any species also in the absence of genome sequence information for which proteogenomics would be impossible.

Spiders use venom to subdue their prey, but little is known about the diversity of venoms in different spider families. Given the limited data available for orb-weaver spiders (Araneidae), we selected the wasp spider Argiope bruennichi for detailed analysis. Our strategy combined a transcriptomics pipeline based on multiple assemblies with a dual proteomics workflow involving parallel mass spectrometry techniques and electrophoretic profiling. We found that the remarkably simple venom of A. bruennichi has an atypical composition compared to other spider venoms, prominently featuring members of the cysteine-rich secretory protein, antigen 5 and pathogenesis-related protein 1 (CAP) superfamily and other, mostly high-molecular-weight proteins. We also detected a subset of potentially novel toxins similar to neuropeptides. We discuss the potential function of these proteins in the context of the unique hunting behavior of wasp spiders, which rely mostly on silk to trap their prey. We propose that the simplicity of the venom evolved to solve an economic dilemma between two competing yet metabolically expensive weapon systems. This study emphasizes the importance of cutting-edge methods to encompass the lineages of smaller venomous species that have yet to be characterized in detail, allowing us to understand the biology of their venom systems and to mine this prolific resource for translational research.

Numerous clinical trials of drug candidates for Alzheimer\'s disease (AD) have failed, and computational drug repositioning approaches using omics data have been proposed as effective alternative approaches to the discovery of drug candidates. However, little multi-omics data is available for AD, due to limited availability of brain tissues. Even if omics data exist, systematic drug repurposing study for AD has suffered from lack of big data, insufficient clinical information, and difficulty in data integration on account of sample heterogeneity derived from poor diagnosis or shortage of qualified post-mortem tissue. In this study, we developed a proteotranscriptomic-based computational drug repositioning method named Drug Repositioning Perturbation Score/Class (DRPS/C) based on inverse associations between disease- and drug-induced gene and protein perturbation patterns, incorporating pharmacogenomic knowledge. We constructed a Drug-induced Gene Perturbation Signature Database (DGPSD) comprised of 61,019 gene signatures perturbed by 1,520 drugs from the Connectivity Map (CMap) and the L1000 CMap. Drugs were classified into three DRPCs (High, Intermediate, and Low) according to DRPSs that were calculated using drug- and disease-induced gene perturbation signatures from DGPSD and The Cancer Genome Atlas (TCGA), respectively. The DRPS/C method was evaluated using the area under the ROC curve, with a prescribed drug list from TCGA as the gold standard. Glioblastoma had the highest AUC. To predict anti-AD drugs, DRPS were calculated using DGPSD and AD-induced gene/protein perturbation signatures generated from RNA-seq, microarray and proteomic datasets in the Synapse database, and the drugs were classified into DRPCs. We predicted 31 potential anti-AD drug candidates commonly belonged to high DRPCs of transcriptomic and proteomic signatures. Of these, four drugs classified into the nervous system group of Anatomical Therapeutic Chemical (ATC) system are voltage-gated sodium channel blockers (bupivacaine, topiramate) and monamine oxidase inhibitors (selegiline, iproniazid), and their mechanism of action was inferred from a potential anti-AD drug perspective. Our approach suggests a shortcut to discover new efficacy of drugs for AD.