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E-cigarette liquids are complex mixtures of chemicals consisting of humectants, such as propylene glycol (PG) and vegetable glycerin (VG), with nicotine or flavorings added. Published literature emphasizes the toxicity of e-cigarette aerosols with flavorings whereas much less attention has been given to the biologic effects of humectants. The purpose of the current study was to provide a comprehensive view of the acute biologic effects of e-cigarette aerosols on rat bronchoalveolar lavage (BAL) using mass spectrometry-based global proteomics. Sprague–Dawley rats were exposed to e-cigarette aerosol for 3 h/day for three consecutive days. Groups included: PG/VG alone, PG/VG + 2.5% nicotine (N), or PG/VG + N + 3.3% vanillin (V). Right lung lobes were lavaged for BAL and supernatants prepared for proteomics. Extracellular BAL S100A9 concentrations and BAL cell staining for citrullinated histone H3 (citH3) were also performed. From global proteomics, ∼2,100 proteins were identified from rat BAL. The greatest change in number of BAL proteins occurred with PG/VG exposures alone compared with controls with biological pathways enriched for acute phase responses, extracellular trap formation, and coagulation. Extracellular BAL S100A9 concentrations and the number of citH3 + BAL cells also increased significantly in PG/VG and PG/VG + 2.5% N. In contrast to PG/VG or PG/VG + N, the addition of vanillin to PG/VG + N increased BAL neutrophilia and downregulated lipid transport proteins. In summary, global proteomics support e-cigarette aerosol exposures to PG/VG alone as having a significant biologic effect on the lung independent of nicotine or flavoring with increased markers of extracellular trap formation.

Proteinaceous aggregates containing α-synuclein protein called Lewy bodies in the substantia nigra is a hallmark of Parkinson's disease. The molecular mechanisms of Lewy body formation and associated neuronal loss remain largely unknown. To gain insights into proteins and pathways associated with Lewy body pathology, we performed quantitative profiling of the proteome. We analyzed substantia nigra tissue from 51 subjects arranged into three groups: cases with Lewy body pathology, Lewy body-negative controls with matching neuronal loss, and controls with no neuronal loss. Using a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we characterized the proteome both in terms of protein abundances and peptide modifications. Statistical testing for differential abundance of the most abundant 2963 proteins, followed by pathway enrichment and Bayesian learning of the causal network structure, was performed to identify likely drivers of Lewy body formation and dopaminergic neuronal loss. The identified pathways include (1) Arp2/3 complex-mediated actin nucleation; (2) synaptic function; (3) poly(A) RNA binding; (4) basement membrane and endothelium; and (5) hydrogen peroxide metabolic process. According to the data, the endothelial/basement membrane pathway is tightly connected with both pathologies and likely to be one of the drivers of neuronal loss. The poly(A) RNA-binding proteins, including the ones relevant to other neurodegenerative disorders (e.g., TDP-43 and FUS), have a strong inverse correlation with Lewy bodies and may reflect an alternative mechanism of nigral neurodegeneration.

The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 hours in nylon bags within the rumen of cannulated dairy cows. Across a gene catalogue covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of polysaccharides such as hemicellulose, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo- and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8 and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryote-selected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks ofin siturecalcitrant fiber deconstruction, and importantly, suggests that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.

Protein posttranslational modifications (PTMs) by O-GlcNAc globally rise during pressure-overload hypertrophy (POH). However, only a few specific proteins with altered O-GlcNAc levels during POH are known primarily because this PTM is easily lost during standard mass spectrometry (MS) conditions used for protein identification. Methodologies have recently emerged to stabilize the O-GlcNAc moiety for MS analysis. Accordingly, our goal was to determine the proteins undergoing changes in O-GlcNAc levels during POH. We used C57/Bl6 mice subjected to Sham or transverse aortic constriction (TAC) to create POH. From the hearts, we stabilized and labelled the O-GlcNAc moiety with tetramethylrhodamine azide (TAMRA) before enriching by TAMRA immunoprecipitation (IP). We used LC-MS to identify the captured O-GlcNAcylated proteins. We identified a total of 707 O-GlcNAcylated proteins in Sham and POH. Two hundred thirty-three of these proteins were significantly increased in POH over Sham whereas no proteins were significantly decreased in POH. We examined two MS identified proteins, CPT1B and PDH, to validate the MS data by immunoprecipitation. We corroborated increased O-GlcNAc levels during POH for the metabolic enzymes CPT1B and PDH. Enzyme activity assays showed higher O-GlcNAcylation increased CPT1 activity and decreased PDH activity. In summary, we generated the first comprehensive list of proteins with changes in O-GlcNAc levels during POH and, to our knowledge, the largest list for any cardiac pathology. Our results demonstrate the large number of proteins and cellular processes affected by O-GlcNAc during POH and serve as a guide for testing specific O-GlcNAc-regulated mechanisms.

Penicillin binding protein 2a (PBP2a)-dependent resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is regulated by the activity of the tricarboxylic acid (TCA) cycle via a poorly understood mechanism. We report that mutations in sucC and sucD, but not other TCA cycle enzymes, negatively impact β-lactam resistance without changing PBP2a expression. Increased intracellular levels of succinyl coenzyme A (succinyl-CoA) in the sucC mutant significantly perturbed lysine succinylation in the MRSA proteome. Suppressor mutations in sucA or sucB, responsible for succinyl-CoA biosynthesis, reversed sucC mutant phenotypes. The major autolysin (Atl) was the most succinylated protein in the proteome, and increased Atl succinylation in the sucC mutant was associated with loss of autolytic activity. Although PBP2a and PBP2 were also among the most succinylated proteins in the MRSA proteome, peptidoglycan architecture and cross-linking were unchanged in the sucC mutant. These data reveal that perturbation of the MRSA succinylome impacts two interconnected cell wall phenotypes, leading to repression of autolytic activity and increased susceptibility to β-lactam antibiotics. IMPORTANCEmecA-dependent methicillin resistance in MRSA is subject to regulation by numerous accessory factors involved in cell wall biosynthesis, nucleotide signaling, and central metabolism. Here, we report that mutations in the TCA cycle gene, sucC, increased susceptibility to β-lactam antibiotics and was accompanied by significant accumulation of succinyl-CoA, which in turn perturbed lysine succinylation in the proteome. Although cell wall structure and cross-linking were unchanged, significantly increased succinylation of the major autolysin Atl, which was the most succinylated protein in the proteome, was accompanied by near complete repression of autolytic activity. These findings link central metabolism and levels of succinyl-CoA to the regulation of β-lactam antibiotic resistance in MRSA through succinylome-mediated control of two interlinked cell wall phenotypes. Drug-mediated interference of the SucCD-controlled succinylome may help overcome β-lactam resistance.

Significance Anaerobic gut fungi are important members of the gut microbiome of herbivores, yet they exist in small numbers relative to bacteria. Here, we show that these early-branching fungi produce a wealth of secondary metabolites (natural products) that may act to regulate the gut microbiome. We use an integrated 'omics'-based approach to classify the biosynthetic genes predicted from fungal genomes, determine transcriptionally active genes, and verify the presence of their enzymatic products. Our analysis reveals that anaerobic gut fungi are an untapped reservoir of bioactive compounds that could be harnessed for biotechnology., Anaerobic fungi (class Neocallimastigomycetes) thrive as low-abundance members of the herbivore digestive tract. The genomes of anaerobic gut fungi are poorly characterized and have not been extensively mined for the biosynthetic enzymes of natural products such as antibiotics. Here, we investigate the potential of anaerobic gut fungi to synthesize natural products that could regulate membership within the gut microbiome. Complementary 'omics' approaches were combined to catalog the natural products of anaerobic gut fungi from four different representative species: Anaeromyces robustus (A. robustus), Caecomyces churrovis (C. churrovis), Neocallimastix californiae (N. californiae), and Piromyces finnis (P. finnis). In total, 146 genes were identified that encode biosynthetic enzymes for diverse types of natural products, including nonribosomal peptide synthetases and polyketide synthases. In addition, N. californiae and C. churrovis genomes encoded seven putative bacteriocins, a class of antimicrobial peptides typically produced by bacteria. During standard laboratory growth on plant biomass or soluble substrates, 26% of total core biosynthetic genes in all four strains were transcribed. Across all four fungal strains, 30% of total biosynthetic gene products were detected via proteomics when grown on cellobiose. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of fungal supernatants detected 72 likely natural products from A. robustus alone. A compound produced by all four strains of anaerobic fungi was putatively identified as the polyketide-related styrylpyrone baumin. Molecular networking quantified similarities between tandem mass spectrometry (MS/MS) spectra among these fungi, enabling three groups of natural products to be identified that are unique to anaerobic fungi. Overall, these results support the finding that anaerobic gut fungi synthesize natural products, which could be harnessed as a source of antimicrobials, therapeutics, and other bioactive compounds.

Bronchiolitis obliterans (BO) is a devastating lung disease seen commonly after lung transplant, following severe respiratory tract infection or chemical inhalation exposure. Diacetyl (DA; 2,3-butanedione) is a highly reactive alpha-diketone known to cause BO when inhaled, however, the mechanisms of how inhalation exposure leads to BO development remains poorly understood. In the current work, we combined two clinically relevant models for studying the pathogenesis of DA-induced BO: (1) an in vivo rat model of repetitive DA vapor exposures with recovery and (2) an in vitro model of primary human airway epithelial cells exposed to pure DA vapors. Rats exposed to 5 consecutive days 200 parts-per-million DA 6 h per day had worsening survival, persistent hypoxemia, poor weight gain, and histologic evidence of BO 14 days after DA exposure cessation. At the end of exposure, increased expression of the ubiquitin stress protein ubiquitin-C accumulated within DA-exposed rat lung homogenates and localized primarily to the airway epithelium, the primary site of BO development. Lung proteasome activity increased concurrently with ubiquitin-C expression after DA exposure, supportive of significant proteasome stress. In primary human airway cultures, global proteomics identified 519 significantly modified proteins in DA-exposed samples relative to controls with common pathways of the ubiquitin proteasome system, endosomal reticulum transport, and response to unfolded protein pathways being upregulated and cell–cell adhesion and oxidation–reduction pathways being downregulated. Collectively, these two models suggest that diacetyl inhalation exposure causes abundant protein damage and subsequent ubiquitin proteasome stress prior to the development of chemical-induced BO pathology.

Temperate coniferous forests sustain the highest levels of biomass of all terrestrial ecosystems and belong to the major carbon sinks on Earth. However, the community composition and its functional diversity depending on the habitat have yet to be unveiled. Here, we analyzed the proteomes from litter, plant roots, rhizosphere, and bulk soil in a temperate coniferous forest at two time points to improve the understanding of the interplay between bacterial and eukaryotic communities in different habitats. Our metaproteomic approach yielded a total of 139,127 proteins that allowed to differentiate the contribution of microbial taxa to protein expression as well as the general functionality based on KEGG Orthology in each habitat. The pool of expressed carbohydrate-active enzymes (CAZymes) was dominated by fungal proteins. While CAZymes in roots and litter targeted mostly the structural biopolymers of plant origin such as lignin and cellulose, the majority of CAZymes in bulk and rhizosphere soil targeted oligosaccharides, starch, and glycogen. Proteins involved in nitrogen cycling were mainly of bacterial origin. Most nitrogen cycling proteins in litter and roots participated in ammonium assimilation while those performing nitrification were the most abundant in bulk and rhizosphere soil. Together, our results indicated niche differentiation of the microbial involvement in carbon and nitrogen cycling in a temperate coniferous forest topsoil.

Navigating through menu structures to access functions and search for information on small displays in cellular phones and other handheld devices is difficult. Visual momentum is a display technique that may help users integrate information across successive displays, and thus improve navigation performance for devices with small screens. This study investigated the effects of visual momentum and menu structure on the learnability of menus on small displays. Learnability was assessed using a trials-to-mastery measure. Mastery criteria regarding search time and errors were set during a pilot study and the number of trials it took for participants to meet these criteria during the Main Experiment were recorded. It was found that providing preview information in the display did not help users learn to navigate through hierarchical menus, while providing motion in the display did help.