Ultra High-plex Spatial Proteogenomic Investigation of Giant Cell Glioblastoma Multiforme Immune Infiltrates Reveals Distinct Protein and RNA Expression Profiles.

A deeper understanding of complex biological processes, including tumor development and immune response, requires ultra high-plex, spatial interrogation of multiple "omes". Here we present the development and implementation of a novel spatial proteogenomic (SPG) assay on the GeoMx Digital Spatial Profiler platform with next-generation sequencing readout that enables ultra high-plex digital quantitation of proteins (>100-plex) and RNA (whole transcriptome, >18,000-plex) from a single formalin-fixed paraffin-embedded (FFPE) sample. This study highlighted the high concordance, R > 0.85 and <15% change in sensitivity between the SPG assay and the single-analyte assays on various cell lines and tissues from human and mouse. Furthermore, we demonstrate that the SPG assay was reproducible across multiple users. When used in conjunction with advanced cellular neighborhood segmentation, distinct immune or tumor RNA and protein targets were spatially resolved within individual cell subpopulations in human colorectal cancer and non-small cell lung cancer. We used the SPG assay to interrogate 23 different glioblastoma multiforme (GBM) samples across four pathologies. The study revealed distinct clustering of both RNA and protein based on pathology and anatomic location. The in-depth investigation of giant cell glioblastoma multiforme (gcGBM) revealed distinct protein and RNA expression profiles compared with that of the more common GBM. More importantly, the use of spatial proteogenomics allowed simultaneous interrogation of critical protein posttranslational modifications alongside whole transcriptomic profiles within the same distinct cellular neighborhoods. Significance: We describe ultra high-plex spatial proteogenomics; profiling whole transcriptome and high-plex proteomics on a single FFPE tissue section with spatial resolution. Investigation of gcGBM versus GBM revealed distinct protein and RNA expression profiles.

Glucocerebrosidase reduces the spread of protein aggregation in a Drosophila melanogaster model of neurodegeneration by regulating proteins trafficked by extracellular vesicles.

Abnormal protein aggregation within neurons is a key pathologic feature of Parkinson's disease (PD). The spread of brain protein aggregates is associated with clinical disease progression, but how this occurs remains unclear. Mutations in glucosidase, beta acid 1 (GBA), which encodes glucocerebrosidase (GCase), are the most penetrant common genetic risk factor for PD and dementia with Lewy bodies and associate with faster disease progression. To explore how GBA mutations influence pathogenesis, we previously created a Drosophila model of GBA deficiency (Gba1b) that manifests neurodegeneration and accelerated protein aggregation. Proteomic analysis of Gba1b mutants revealed dysregulation of proteins involved in extracellular vesicle (EV) biology, and we found altered protein composition of EVs from Gba1b mutants. Accordingly, we hypothesized that GBA may influence pathogenic protein aggregate spread via EVs. We found that accumulation of ubiquitinated proteins and Ref(2)P, Drosophila homologue of mammalian p62, were reduced in muscle and brain tissue of Gba1b flies by ectopic expression of wildtype GCase in muscle. Neuronal GCase expression also rescued protein aggregation both cell-autonomously in brain and non-cell-autonomously in muscle. Muscle-specific GBA expression reduced the elevated levels of EV-intrinsic proteins and Ref(2)P found in EVs from Gba1b flies. Perturbing EV biogenesis through neutral sphingomyelinase (nSMase), an enzyme important for EV release and ceramide metabolism, enhanced protein aggregation when knocked down in muscle, but did not modify Gba1b mutant protein aggregation when knocked down in neurons. Lipidomic analysis of nSMase knockdown on ceramide and glucosylceramide levels suggested that Gba1b mutant protein aggregation may depend on relative depletion of specific ceramide species often enriched in EVs. Finally, we identified ectopically expressed GCase within isolated EVs. Together, our findings suggest that GCase deficiency promotes accelerated protein aggregate spread between cells and tissues via dysregulated EVs, and EV-mediated trafficking of GCase may partially account for the reduction in aggregate spread.

A putative enoyl-CoA hydratase contributes to biofilm formation and the antibiotic tolerance of Achromobacter xylosoxidans.

Achromobacter xylosoxidans has attracted increasing attention as an emerging pathogen in patients with cystic fibrosis. Intrinsic resistance to several classes of antimicrobials and the ability to form robust biofilms in vivo contribute to the clinical manifestations of persistent A. xylosoxidans infection. Still, much of A. xylosoxidans biofilm formation remains uncharacterized due to the scarcity of existing genetic tools. Here we demonstrate a promising genetic system for use in A. xylosoxidans; generating a transposon mutant library which was then used to identify genes involved in biofilm development in vitro. We further described the effects of one of the genes found in the mutagenesis screen, encoding a putative enoyl-CoA hydratase, on biofilm structure and tolerance to antimicrobials. Through additional analysis, we find that a fatty acid signaling compound is essential to A. xylosoxidans biofilm ultrastructure and maintenance. This work describes methods for the genetic manipulation of A. xylosoxidans and demonstrated their use to improve our understanding of A. xylosoxidans pathophysiology.

Long-lived and short-lived reactive species produced by a cold atmospheric pressure plasma jet for the inactivation of Pseudomonas aeruginosa and Staphylococcus aureus.

Different chemical pathways leading to the inactivation of Pseudomonas aeruginosa and Staphylococcus aureus by a cold atmospheric pressure plasma jet (APPJ) in buffered and non-buffered solutions are reported. As APPJs produce a complex mixture of reactive species in solution, a comprehensive set of diagnostics were used to assess the liquid phase chemistry. This includes absorption and electron paramagnetic resonance spectroscopy in addition to a scavenger study to assess the relative importance of the various plasma produced species involved in the inactivation of bacteria. Different modes of inactivation of bacteria were found for the same plasma source depending on the solution and the plasma feed gas. The inactivation of bacteria in saline is due to the production of short-lived species in the case of argon plasma when the plasma touches the liquid. Long-lived species (ClO-) formed by the abundant amount of O. radicals produced by the plasmas played a dominant role in the case of Ar + 1% O2 and Ar + 1% air plasmas when the plasma is not in direct contact with the liquid. Inactivation of bacteria in distilled water was found to be due to the generation of short-lived species: O. &O2.- for Ar + 1% O2 plasma and O2.- (and .OH in absence of saline) for Ar plasma.

Multiple roles for Enterococcus faecalis glycosyltransferases in biofilm-associated antibiotic resistance, cell envelope integrity, and conjugative transfer.

The emergence of multidrug-resistant bacteria and the limited availability of new antibiotics are of increasing clinical concern. A compounding factor is the ability of microorganisms to form biofilms (communities of cells encased in a protective extracellular matrix) that are intrinsically resistant to antibiotics. Enterococcus faecalis is an opportunistic pathogen that readily forms biofilms and also has the propensity to acquire resistance determinants via horizontal gene transfer. There is intense interest in the genetic basis for intrinsic and acquired antibiotic resistance in E. faecalis, since clinical isolates exhibiting resistance to multiple antibiotics are not uncommon. We performed a genetic screen using a library of transposon (Tn) mutants to identify E. faecalis biofilm-associated antibiotic resistance determinants. Five Tn mutants formed wild-type biofilms in the absence of antibiotics but produced decreased biofilm biomass in the presence of antibiotic concentrations that were subinhibitory to the parent strain. Genetic determinants responsible for biofilm-associated antibiotic resistance include components of the quorum-sensing system (fsrA, fsrC, and gelE) and two glycosyltransferase (GTF) genes (epaI and epaOX). We also found that the GTFs play additional roles in E. faecalis resistance to detergent and bile salts, maintenance of cell envelope integrity, determination of cell shape, polysaccharide composition, and conjugative transfer of the pheromone-inducible plasmid pCF10. The epaOX gene is located in a variable extended region of the enterococcal polysaccharide antigen (epa) locus. These data illustrate the importance of GTFs in E. faecalis adaptation to diverse growth conditions and suggest new targets for antimicrobial design.