Human Adenovirus Type 26 Induced IL-6 Gene Expression in an αvß3 Integrin- and NF-κB-Dependent Manner.

The low seroprevalent human adenovirus type 26 (HAdV26)-based vaccine vector was the first adenovirus-based vector to receive marketing authorization from European Commission. HAdV26-based vaccine vectors induce durable humoral and cellular immune responses and, as such, represent a highly valuable tool for fighting infectious diseases. Despite well-described immunogenicity in vivo, the basic biology of HAdV26 still needs some refinement. The aim of this study was to determine the pro-inflammatory cytokine profile of epithelial cells infected with HAdV26 and then investigate the underlying molecular mechanism. The expression of studied genes and proteins was assessed by quantitative polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. Confocal microscopy was used to visualize HAdV26 cell uptake. We found that HAdV26 infection in human epithelial cells triggers the expression of pro-inflammatory cytokines and chemokines, namely IL-6, IL-8, IL-1ß, and TNF-α, with the most pronounced difference shown for IL-6. We investigated the underlying molecular mechanism and observed that HAdV26-induced IL-6 gene expression is αvß3 integrin dependent and NF-κB mediated. Our findings provide new data regarding pro-inflammatory cytokine and chemokine expression in HAdV26-infected epithelial cells, as well as details concerning HAdV26-induced host signaling pathways. Information obtained within this research increases our current knowledge of HAdV26 basic biology and, as such, can contribute to further development of HAdV26-based vaccine vectors.

Parallel reaction monitoring on a Q Exactive mass spectrometer increases reproducibility of phosphopeptide detection in bacterial phosphoproteomics measurements.

Increasing number of studies report the relevance of protein Ser/Thr/Tyr phosphorylation in bacterial physiology, yet the analysis of this type of modification in bacteria still presents a considerable challenge. Unlike in eukaryotes, where tens of thousands of phosphorylation events likely occupy more than two thirds of the proteome, the abundance of protein phosphorylation is much lower in bacteria. Even the state-of-the-art phosphopeptide enrichment protocols fail to remove the high background of abundant unmodified peptides, leading to low signal intensity and undersampling of phosphopeptide precursor ions in consecutive data-dependent MS runs. Consequently, large-scale bacterial phosphoproteomic datasets often suffer from poor reproducibility and a high number of missing values. Here we explore the application of parallel reaction monitoring (PRM) on a Q Exactive mass spectrometer in bacterial phosphoproteome analysis, focusing especially on run-to-run sampling reproducibility. In multiple measurements of identical phosphopeptide-enriched samples, we show that PRM outperforms data-dependent acquisition (DDA) in terms of detection frequency, reaching almost complete sampling efficiency, compared to 20% in DDA. We observe a similar trend over multiple heterogeneous phosphopeptide-enriched samples and conclude that PRM shows a great promise in bacterial phosphoproteomics analyses where reproducible detection and quantification of a relatively small set of phosphopeptides is desired. SIGNIFICANCE: Bacterial phosphorylated peptides occur in low abundance compared to their unmodified counterparts, and are therefore rarely reproducibly detected in shotgun (DDA) proteomics measurements. Here we show that parallel reaction monitoring complements DDA analyses and makes detection of known, targeted phosphopeptides more reproducible. This will be of significance in replicated MS measurements that have a goal to reproducibly detect and quantify phosphopeptides of interest.