Robust dengue virus infection in bat cells and limited innate immune responses coupled with positive serology from bats in IndoMalaya and Australasia.

Natural reservoir hosts can sustain infection of pathogens without succumbing to overt disease. Multiple bat species host a plethora of viruses, pathogenic to other mammals, without clinical symptoms. Here, we detail infection of bat primary cells, immune cells, and cell lines with Dengue virus. While antibodies and viral RNA were previously detected in wild bats, their ability to sustain infection is not conclusive. Old-world fruitbat cells can be infected, producing high titres of virus with limited cellular responses. In addition, there is minimal interferon (IFN) response in cells infected with MOIs leading to dengue production. The ability to support in vitro replication/production raises the possibility of bats as a transient host in the life cycle of dengue or similar flaviviruses. New antibody serology evidence from Asia/Pacific highlights the previous exposure and raises awareness that bats may be involved in flavivirus dynamics and infection of other hosts.

Breast Cancer-Derived Exosomes Reflect the Cell-of-Origin Phenotype.

A manner in which cells can communicate with each other is via secreted nanoparticles termed exosomes. These vesicles contain lipids, nucleic acids, and proteins, and are said to reflect the cell-of-origin. However, for the exosomal protein content, there is limited evidence in the literature to verify this statement. Here, proteomic assessment combined with pathway-enrichment analysis is used to demonstrate that the protein cargo of exosomes reflects the epithelial/mesenchymal phenotype of secreting breast cancer cells. Given that epithelial-mesenchymal plasticity is known to implicate various stages of cancer progression, the results suggest that breast cancer subtypes with distinct epithelial and mesenchymal phenotypes may be distinguished by directly assessing the protein content of exosomes. Additionally, the work is a substantial step toward verifying the statement that cell-derived exosomes reflect the phenotype of the cells-of-origin.

Proteomic Analysis of Rhizoctonia solani Identifies Infection-specific, Redox Associated Proteins and Insight into Adaptation to Different Plant Hosts.

Rhizoctonia solaniis an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about howR. solanicauses disease. This study capitalizes on recent genomic studies by applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Many of the proteins found in the culture filtrate had predicted functions relating to modification of the plant cell wall, a major activity required for pathogenesis on the plant host, including a number found only under infection conditions. Other infection related proteins included a high proportion of proteins with redox associated functions and many novel proteins without functional classification. The majority of infection only proteins tested were confirmed to show transcript up-regulation during infection including a thaumatin which increased susceptibility toR. solaniwhen expressed inNicotiana benthamiana In addition, analysis of expression during infection of different plant hosts highlighted how the infection strategy of this broad host range pathogen can be adapted to the particular host being encountered. Data are available via ProteomeXchange with identifier PXD002806.

Oncogenic transformation of lung cells results in distinct exosome protein profile similar to the cell of origin.

Lung cancer is responsible for the highest rate of cancer mortality worldwide. Lung cancer patients are often ineligible for tumor biopsies due to comorbidities. As a result, patients may not have the most effective treatment regimens administered. Patients with mutations in the epidermal growth factor receptor (EGFR) have improved survival in response to EGFR tyrosine kinase inhibitors. A noninvasive method of determining EGFR mutations in patients would have promising clinical applications. Exosomes have the potential to be noninvasive novel diagnostic markers in cancer. Using MS analysis, we identify differentially abundant cell and exosome proteins induced by mutations in p53 and EGFR in lung cells. Importantly, mutations in p53 and EGFR alter cell and exosome protein content compared to an isogenic normal lung epithelial cell. For some proteins, mutation had similar effects in the cell of origin and exosomes. Differences between the cells of origin and exosomes were also apparent, which may reflect specific packaging of proteins into exosomes. These findings that mutations alter protein abundance in exosomes suggest that analysis of exosomes may be beneficial in the diagnosis of oncogenic mutations.

Metagenomic and metaproteomic analyses of Accumulibacter phosphatis-enriched floccular and granular biofilm.

Biofilms are ubiquitous in nature, forming diverse adherent microbial communities that perform a plethora of functions. Here we operated two laboratory-scale sequencing batch reactors enriched with Candidatus Accumulibacter phosphatis (Accumulibacter) performing enhanced biological phosphorus removal. Reactors formed two distinct biofilms, one floccular biofilm, consisting of small, loose, microbial aggregates, and one granular biofilm, forming larger, dense, spherical aggregates. Using metagenomic and metaproteomic methods, we investigated the proteomic differences between these two biofilm communities, identifying a total of 2022 unique proteins. To understand biofilm differences, we compared protein abundances that were statistically enriched in both biofilm states. Floccular biofilms were enriched with pathogenic secretion systems suggesting a highly competitive microbial community. Comparatively, granular biofilms revealed a high-stress environment with evidence of nutrient starvation, phage predation pressure, and increased extracellular polymeric substance and cell lysis. Granular biofilms were enriched in outer membrane transport proteins to scavenge the extracellular milieu for amino acids and other metabolites, likely released through cell lysis, to supplement metabolic pathways. This study provides the first detailed proteomic comparison between Accumulibacter-enriched floccular and granular biofilm communities, proposes a conceptual model for the granule biofilm, and offers novel insights into granule biofilm formation and stability.

The human respiratory syncytial virus nonstructural protein 1 regulates type I and type II interferon pathways.

Respiratory syncytial viruses encode a nonstructural protein (NS1) that interferes with type I and III interferon and other antiviral responses. Proteomic studies were conducted on human A549 type II alveolar epithelial cells and type I interferon-deficient Vero cells (African green monkey kidney cells) infected with wild-type and NS1-deficient clones of human respiratory syncytial virus to identify other potential pathway and molecular targets of NS1 interference. These analyses included two-dimensional differential gel electrophoresis and quantitative Western blotting. Surprisingly, NS1 was found to suppress the induction of manganese superoxide dismutase (SOD2) expression in A549 cells and to a much lesser degree Vero cells in response to infection. Because SOD2 is not directly inducible by type I interferons, it served as a marker to probe the impact of NS1 on signaling of other cytokines known to induce SOD2 expression and/or indirect effects of type I interferon signaling. Deductive analysis of results obtained from cell infection and cytokine stimulation studies indicated that interferon-γ signaling was a potential target of NS1, possibly as a result of modulation of STAT1 levels. However, this was not sufficient to explain the magnitude of the impact of NS1 on SOD2 induction in A549 cells. Vero cell infection experiments indicated that NS1 targeted a component of the type I interferon response that does not directly induce SOD2 expression but is required to induce another initiator of SOD2 expression. STAT2 was ruled out as a target of NS1 interference using quantitative Western blot analysis of infected A549 cells, but data were obtained to indicate that STAT1 was one of a number of potential targets of NS1. A label-free mass spectrometry-based quantitative approach is proposed as a means of more definitive identification of NS1 targets.

An epithelial-to-mesenchymal transition induced extracellular vesicle prognostic signature in non-small cell lung cancer.

Despite significant therapeutic advances, lung cancer remains the leading cause of cancer-related death worldwide1. Non-small cell lung cancer (NSCLC) patients have a very poor overall five-year survival rate of only 10-20%. Currently, TNM staging is the gold standard for predicting overall survival and selecting optimal initial treatment options for NSCLC patients, including those with curable stages of disease. However, many patients with locoregionally-confined NSCLC relapse and die despite curative-intent interventions, indicating a need for intensified, individualised therapies. Epithelial-to-mesenchymal transition (EMT), the phenotypic depolarisation of epithelial cells to elongated, mesenchymal cells, is associated with metastatic and treatment-refractive cancer. We demonstrate here that EMT-induced protein changes in small extracellular vesicles are detectable in NSCLC patients and have prognostic significance. Overall, this work describes a novel prognostic biomarker signature that identifies potentially-curable NSCLC patients at risk of developing metastatic NSCLC, thereby enabling implementation of personalised treatment decisions.

Mass-spectrometry data for Rhizoctonia solani proteins produced during infection of wheat and vegetative growth.

Rhizoctonia solani is an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato, legumes and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about how R. solani causes disease. The data described in this article is derived from applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Comparisons of the data for sample types in this set will be useful to identify metabolic pathway changes as the fungus switches from saprophytic to a pathogenic lifestyle or pathogenicity related proteins contributing to the ability to cause disease on wheat. The data set is deposited in the PRIDE archive under identifier PRIDE: PXD002806.

Discovery and characterisation of a new insect-specific bunyavirus from Culex mosquitoes captured in northern Australia.

Insect-specific viruses belonging to significant arboviral families have recently been discovered. These viruses appear to be maintained within the insect population without the requirement for replication in a vertebrate host. Mosquitoes collected from Badu Island in the Torres Strait in 2003 were analysed for insect-specific viruses. A novel bunyavirus was isolated in high prevalence from Culex spp. The new virus, provisionally called Badu virus (BADUV), replicated in mosquito cells of both Culex and Aedes origin, but failed to replicate in vertebrate cells. Genomic sequencing revealed that the virus was distinct from sequenced bunyavirus isolates reported to date, but phylogenetically clustered most closely with recently discovered mosquito-borne, insect-specific bunyaviruses in the newly proposed Goukovirus genus. The detection of a functional furin cleavage motif upstream of the two glycoproteins in the M segment-encoded polyprotein suggests that BADUV may employ a unique strategy to process the virion glycoproteins.

TRAF2 recruitment via T61 in CD30 drives NFκB activation and enhances hESC survival and proliferation.

CD30 (TNFRSF8), a tumor necrosis factor receptor family protein, and CD30 variant (CD30v), a ligand-independent form encoding only the cytoplasmic signaling domain, are concurrently overexpressed in transformed human embryonic stem cells (hESCs) or hESCs cultured in the presence of ascorbate. CD30 and CD30v are believed to increase hESC survival and proliferation through NFκB activation, but how this occurs is largely unknown. Here we demonstrate that hESCs that endogenously express CD30v and hESCs that artificially overexpress CD30v exhibit increased ERK phosphorylation levels, activation of the canonical NFκB pathway, down-regulation of the noncanonical NFκB pathway, and reduced expression of the full-length CD30 protein. We further find that CD30v, surprisingly, resides predominantly in the nucleus of hESC. We demonstrate that alanine substitution of a single threonine residue at position 61 (T61) in CD30v abrogates CD30v-mediated NFκB activation, CD30v-mediated resistance to apoptosis, and CD30v-enhanced proliferation, as well as restores normal G2/M-checkpoint arrest upon H2O2 treatment while maintaining its unexpected subcellular distribution. Using an affinity purification strategy and LC-MS, we identified TRAF2 as the predominant protein that interacts with WT CD30v but not the T61A-mutant form in hESCs. The identification of Thr-61 as a critical residue for TRAF2 recruitment and canonical NFκB signaling by CD30v reveals the substantial contribution that this molecule makes to overall NFκB activity, cell cycle changes, and survival in hESCs.

Exploration of peptides bound to MHC class I molecules in melanoma.

Advancements in high-resolution HPLC and mass spectrometry have reinvigorated the application of this technology to identify peptides eluted from immunopurified MHC class I molecules. Three melanoma cell lines were assessed using w6/32 isolation, peptide elution and HPLC purification; peptides were identified by mass spectrometry. A total of 13,829 peptides were identified; 83-87% of these were 8-11 mers. Only approximately 15% have been described before. Subcellular locations of the source proteins showed even sampling; mRNA expression and total protein length were predictive of the number of peptides detected from a single protein. HLA-type binding prediction for 10,078 9/10 mer peptides assigned 88-95% to a patient-specific HLA subtype, revealing a disparity in strength of predicted binding. HLA-B*27-specific isolation successfully identified some peptides not found using w6/32. Sixty peptides were selected for immune screening, based on source protein and predicted HLA binding; no new peptides recognized by antimelanoma T cells were discovered. Additionally, mass spectrometry was unable to identify several epitopes targeted ex vivo by one patient's T cells.

Mass spectrometry identification of age-associated proteins from the malaria mosquitoes Anopheles gambiae s.s. and Anopheles stephensi.

This study investigated proteomic changes occurring in Anopheles gambiae and Anopheles stephensi during adult mosquito aging. These changes were evaluated using two-dimensional difference gel electrophoresis (2D-DIGE) and the identities of aging related proteins were determined using capillary high-pressure liquid chromatography (capHPLC) coupled with a linear ion-trap (LTQ)-Orbitrap XL hybrid mass spectrometry (MS). Here, we have described the techniques used to determine age associated proteomic changes occurring in heads and thoraces across three age groups; 1, 9 and 17 d old A. gambiae and 4 age groups; 1, 9, 17 and 34 d old A. stephensi. We have provided normalised spot volume raw data for all protein spots that were visible on 2D-DIGE images for both species and processed Orbitrap mass spectrometry data. For public access, mass spectrometry raw data are available via ProteomeXchange with identifier PXD002153. A detailed description of this study has been described elsewhere [1].

Proteomic changes occurring in the malaria mosquitoes Anopheles gambiae and Anopheles stephensi during aging.

The age of mosquitoes is a crucial determinant of their ability to transmit pathogens and their resistance to insecticides. We investigated changes to the abundance of proteins found in heads and thoraces of the malaria mosquitoes Anopheles gambiae and Anopheles stephensi as they aged. Protein expression changes were assessed using two-dimensional difference gel electrophoresis and the identity of differentially expressed proteins was determined by using either matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry or capillary high-pressure liquid chromatography coupled with a linear ion-trap (LTQ)-Orbitrap XL hybrid mass spectrometer. Protein biomarkers were validated by semi quantitative Western blot analysis. Nineteen and nine age dependent protein spots were identified for A. stephensi and A. gambiae, respectively. Among the proteins down-regulated with age were homologs of ADF/Cofilin, cytochome c1, heat shock protein-70 and eukaryotic translation initiation factor 5A (eIF5a). Proteins up-regulated with age included probable methylmalonate-semialdehyde dehydrogenase, voltage-dependent anion-selective channel and fructose bisphosphate aldolase. Semi quantitative Western blot analysis confirmed expression patterns observed by 2-D DIGE for eIF5a and ADF/Cofilin. Further work is recommended to determine whether these biomarkers are robust to infection, blood feeding and insecticide resistance. Robust biomarkers could then be incorporated into rapid diagnostic assays for ecological and epidemiological studies. BIOLOGICAL SIGNIFICANCE: In this study, we have identified several proteins with characteristic changes in abundance in both A. gambiae and A. stephensi during their aging process. These changes may highlight underlying mechanisms beneath the relationship between mosquito age and factors affecting Plasmodium transmission and mosquito control. The similarity of changes in protein abundance between these species and the primary dengue vector Aedes aegypti, has revealed conserved patterns of aging-specific protein regulation.

The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition.

Chikungunya virus (CHIKV) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs) previously generated towards the capsid protein (CP) of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1-35 and 140-210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

Rab GTPase prenylation hierarchy and its potential role in choroideremia disease.

Protein prenylation is a widespread post-translational modification in eukaryotes that plays a crucial role in membrane targeting and signal transduction. RabGTPases is the largest group of post-translationally C-terminally geranylgeranylated. All Rabs are processed by Rab geranylgeranyl-transferase and Rab escort protein (REP). Human genetic defects resulting in the loss one of two REP isoforms REP-1, lead to underprenylation of RabGTPases that manifests in retinal degradation and blindness known as choroideremia. In this study we used a combination of microinjections and chemo-enzymatic tagging to establish whether Rab GTPases are prenylated and delivered to their target cellular membranes with the same rate. We demonstrate that although all tested Rab GTPases display the same rate of membrane delivery, the extent of Rab prenylation in 5 hour time window vary by more than an order of magnitude. We found that Rab27a, Rab27b, Rab38 and Rab42 display the slowest prenylation in vivo and in the cell. Our work points to possible contribution of Rab38 to the emergence of choroideremia in addition to Rab27a and Rab27b.

Proteomic comparison of mcf-7 tumoursphere and monolayer cultures.

Breast cancer is a heterogeneous disease, composed of tumour cells with differing gene expressions and phenotypes. Very few antigens have been identified and a better understanding of tumour initiating-cells as targets for therapy is critically needed. Recently, a rare subpopulation of cells within tumours has been described with the ability to: (i) initiate and sustain tumour growth; (ii) resist traditional therapies and allow for secondary tumour dissemination; and (iii) display some of the characteristics of stem cells such as self-renewal. These cells are termed tumour-initiating cells or cancer stem cells, or alternatively, in the case of breast cancer, breast cancer stem cells. Previous studies have demonstrated that breast cancer stem cells can be enriched for in "tumoursphere" culture. Proteomics represents a novel way to investigate protein expression between cells. We hypothesise that characterisation of the proteome of the breast cancer line MCF-7 tumourspheres compared to adherent/differentiated cells identifies proteins of novel interest for further isolating or targeting breast cancer stem cells. We present evidence that: (i) the proteome of adherent cells is different to the proteome of cells grown in sphere medium from either early passage (passage 2) or late passage (passage 5) spheres; (ii) that spheres are enriched in expression of a variety of tumour-relevant proteins (including MUC1 and Galectin-3); and (iii) that targeting of one of these identified proteins (galectin-3) using an inhibitor (N-acetyllactosamine) decreases sphere formation/self-renewal of MCF-7 cancer stem cells in vitro and tumourigenicity in vivo. Hence, proteomic analysis of tumourspheres may find use in identifying novel targets for future therapy. The therapeutic targeting of breast cancer stem cells, a highly clinically relevant sub-population of tumour cells, has the potential to eliminate residual disease and may become an important component of a multi-modality treatment of cancer.