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Background In addition to known allergens, other proteins in pollen can aid the development of an immune response in allergic individuals. The contribution of the “unknown” protein allergens is apparent in phylogenetically related species where, despite of high homology of the lead allergens, the degree of allergenic potential can vary greatly. The aim of this study was to identify other potentially allergenic proteins in pollen of three common and highly related allergenic tree species: birch (Betula pendula), hazel (Corylus avellana) and alder (Alnus glutinosa). Methods For that purpose, we carried out a comprehensive, comparative proteomic screening of the pollen from the three species. In order to maximize protein recovery and coverage, different protein extraction and isolation strategies during sample preparation were employed. Results As a result, we report 2500–3000 identified proteins per each of the pollen species. Identified proteins were further used for a number of annotation steps, providing insight into differential distribution of peptidases, peptidase inhibitors and other potential allergenic proteins across the three species. Moreover, we carried out functional enrichment analyses that, interestingly, corroborated high species similarity in spite of their relatively distinct protein profiles. Conclusion We provide to our knowledge first insight into proteomes of two very important allergenic pollen types, hazel and alder, where not even transcriptomics data are available, and compared them to birch. Datasets from this study can be readily used as protein databases and as such serve as basis for further functional studies., This study aims to identify and annotate novel potentially allergenic proteins in the three highly related pollen species: birch, hazel, and alder. Immunoblotting of pollen protein extracts with serum from allergenic patients corroborates differential allergenic potential despite of the high similarity of the three species. Comprehensive proteomics analysis provides the first available protein database of pollens of alder and hazel and enables thorough annotation of proteases and allergens as well as comparative functional analysis of expressed proteins across the three pollens. Abbreviations: LC‐MS, liquid chromatography‐mass spectrometry; SDS‐PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Background: Atrial fibrillation (AF), the most common sustained arrhythmia, is associated with increased morbidity, mortality, and health care costs. AF develops over many years and is often related to substantial atrial structural and electrophysiological remodeling. AF may lack symptoms at onset, and atrial biopsy samples are generally obtained in subjects with advanced disease, so it is difficult to study earlier stage pathophysiology in humans. Methods: Here, we characterized comprehensively the transcriptomic (miRNA-seq and mRNA-seq) changes in the left atria of 2 robust canine AF models after 1 week of electrically maintained AF, without or with ventricular rate control via atrioventricular node-ablation/ventricular pacing. Results: Our RNA-sequencing experiments identified thousands of genes that are differentially expressed, including a majority that have never before been implicated in AF. Gene set enrichment analyses highlighted known (eg, extracellular matrix structure organization) but also many novel pathways (eg, muscle structure development, striated muscle cell differentiation) that may play a role in tissue remodeling and cellular trans-differentiation. Of interest, we found dysregulation of a cluster of noncoding RNAs, including many microRNAs but also the MEG3 long noncoding RNA orthologue, located in the syntenic region of the imprinted human DLK1-DIO3 locus. Interestingly (in the light of other recent observations), our analysis identified gene targets of differentially expressed microRNAs at the DLK1-DIO3 locus implicating glutamate signaling in AF pathophysiology. Conclusions: Our results capture molecular events that occur at an early stage of disease development using well-characterized animal models and may, therefore, inform future studies that aim to further dissect the causes of AF in humans.

BackgroundAtrial fibrillation (AF), the most common sustained arrhythmia, is associated with increased morbidity, mortality, and health-care costs. AF develops over many years and is often related to substantial atrial structural and electrophysiological remodeling. AF may lack symptoms at onset and atrial biopsy samples are generally obtained in subjects with advanced disease, so it is difficult to study earlier-stage pathophysiology in humans.MethodsHere, we characterized comprehensively the transcriptomic (miRNAseq and mRNAseq) changes in the left atria of two robust canine AF-models after one week of electrically-maintained AF, without or with ventricular rate-control via atrioventricular node-ablation/ventricular pacing.ResultsOur RNA-sequencing experiments identified thousands of genes that are differentially expressed, including a majority that have never before been implicated in AF. Gene-set enrichment analyses highlighted known (e.g. extracellular matrix structure organization) but also many novel pathways (e.g. muscle structure development, striated muscle cell differentiation) that may play a role in tissue remodeling and/or cellular transdifferentiation. Of interest, we found dysregulation of a cluster of non-coding RNAs, including many microRNAs but also theMEG3long non-coding RNA orthologue, located in the syntenic region of the imprinted humanDLK1-DIO3locus. Interestingly (in the light of other recent observations), our analysis identified gene-targets of differentially expressed microRNAs at theDLK1-DIO3locus implicating glutamate signaling in AF pathophysiology.ConclusionsOur results capture molecular events that occur at an early stage of disease development using well-characterized animal models, and may therefore inform future studies that aim to further dissect the causes of AF in humans.

Background Nanoparticles, which are exposed to biological fluids are rapidly interacting with proteins and other biomolecules forming a corona. In addition to dimension, charge and material the distinct protein corona influences the interplay of nanoparticles with tissue barriers. In this study we were focused on the impact of in situ formed human plasma protein corona on the transfer of 80 nm polystyrene nanoparticles (PS-particles) across the human placenta. To study materno-to fetal PS transfer we used the human ex vivo placental perfusion approach, which represents an intact and physiological tissue barrier. To analyze the protein corona of PS particles we performed shotgun proteomics of isolated nanoparticles before and after tissue exposure. Results Human plasma incubated with PS-particles of 80 nm and subsequent formed protein corona enhanced the transfer across the human placenta compared to PS-corona formed by bovine serum albumin and dextran which served as a control. Quantitative and qualitative changes of plasma proteins determined the changes in PS transfer across the barrier. Based on the analysis of the PS-proteome two candidate proteins, namely human albumin and immunoglobulin G were tested if these proteins may account for the enhanced PS-transfer across the placenta. Interestingly, the protein corona formed by human albumin significantly induced the transfer of PS-particles across the tissue compared to the formed IgG-corona. Conclusion In total we demonstrate the PS corona dynamically and significantly evolves upon crossing the human placenta. Thus, the initial composition of PS particles in the maternal circulation is not predictive for their transfer characteristics and performance once beyond the barrier of the placenta. The precise mechanism of these effects remains to be elucidated but highlights the importance of using well designed biological models when testing nanoparticles for biomedical applications.

Heart failure with preserved ejection fraction (HFpEF) is a major health problem without effective therapies. This study assessed the effects of histone deacetylase (HDAC) inhibition on cardiopulmonary structure, function, and metabolism in a large mammalian (feline) model of pressure overload that recapitulates features of diastolic dysfunction common to human HFpEF. Male domestic short hair felines (n=31, aged 2 mo), underwent a sham procedure (n=10) or loose aortic banding (n=21), resulting in slow-progressive pressure overload during growth. Two months post-banding, animals were treated daily with suberoylanilide hydroxamic acid (b+SAHA, 10 mg/kg, n=8), a Food and Drug Administration (FDA)-approved pan-HDAC inhibitor, or vehicle (b+veh, n=8) for 2 months. Echocardiography at 4-months post-banding revealed that b+SAHA animals had significantly reduced left ventricular hypertrophy (LVH) (P < 0.0001) and left atrium size (P < 0.0001) vs b+veh animals. Left ventricular end-diastolic pressure and mean pulmonary arterial pressure were significantly reduced in b+SAHA (P < 0.01) vs b+veh. SAHA increased myofibril relaxation ex vivo, which correlated with in vivo improvements of LV relaxation. Furthermore, SAHA treatment preserved lung structure, compliance, blood oxygenation, and reduced perivascular fluid cuffs around extra-alveolar vessels, suggesting attenuated alveolar-capillary stress failure. Acetylation proteomics revealed that SAHA altered lysine acetylation of mitochondrial metabolic enzymes. HDAC inhibition reduces LVH, LV diastolic dysfunction, and atrial and pulmonary remodeling in a feline model of diastolic dysfunction. These results suggest that acetylation defects in hypertrophic stress can be reversed by HDAC inhibitors, with implications for improving cardiac structure and function in patients.

Background In the settings of primary and secondary prevention for coronary artery disease (CAD), a crucial role is played by some key molecules involved in triglyceride (TG) metabolism, such as ApoCIII. Fatty acid (FA) intake is well recognized as a main determinant of plasma lipids, including plasma TG concentration. Objectives The aim was to investigate the possible relations between the intakes of different FAs, estimated by their plasma concentrations, and circulating amounts of ApoCIII. Methods Plasma samples were obtained from 1370 subjects with or without angiographically demonstrated CAD (mean ± SD age: 60.6 ± 11.0 y; males: 75.8%; BMI: 25.9 ± 4.6 kg/m2; CAD: 73.3%). Plasma lipid, ApoCIII, and FA concentrations were measured. Data were analyzed by regression models adjusted for FAs and other potential confounders, such as sex, age, BMI, diabetes, smoking, and lipid-lowering therapies. The in vitro effects of FAs were tested by incubating HepG2 hepatoma cells with increasing concentrations of selected FAs, and the mRNA and protein contents in the cells were quantified by real-time RT-PCR and LC-MS/MS analyses. Results Among all the analyzed FAs, myristic acid (14:0) showed the most robust correlations with both TGs (R = 0.441, P = 2.6 × 10-66) and ApoCIII (R = 0.327, P = 1.1 × 10-31). By multiple regression analysis, myristic acid was the best predictor of both plasma TG and ApoCIII variability. Plasma TG and ApoCIII concentrations increased progressively at increasing concentrations of myristic acid, independently of CAD diagnosis and gender. Consistent with these data, in the in vitro experiments, an ∼2-fold increase in the expression levels of the ApoCIII mRNA and protein was observed after incubation with 250 μM myristic acid. A weaker effect (∼30% increase) was observed for palmitic acid, whereas incubation with oleic acid did not affect ApoCIII protein or gene expression. Conclusions Plasma myristic acid is associated with increased ApoCIII concentrations in cardiovascular patients. In vitro experiments indicated that myristic acid stimulates ApoCIII expression in HepG2 cells.

The tumor suppressor p16INK4A induces cell cycle arrest and senescence in response to oncogenic transformation and is therefore frequently lost in cancer. p16INK4A is also known to accumulate under conditions of oxidative stress. Thus, we hypothesized it could potentially be regulated by reversible oxidation of cysteines (redox signaling). Here we report that oxidation of the single cysteine in p16INK4A in human cells occurs under relatively mild oxidizing conditions and leads to disulfide-dependent dimerization. p16INK4A is an all α-helical protein, but we find that upon cysteine-dependent dimerization, p16INK4A undergoes a dramatic structural rearrangement and forms aggregates that have the typical features of amyloid fibrils, including binding of diagnostic dyes, presence of cross-β sheet structure, and typical dimensions found in electron microscopy. p16INK4A amyloid formation abolishes its function as a Cyclin Dependent Kinase 4/6 inhibitor. Collectively, these observations mechanistically link the cellular redox state to the inactivation of p16INK4A through the formation of amyloid fibrils., Graphical abstract Image 1, Highlights • The only cysteine in human p16INK4A is prone to oxidation in vitro and in vivo. • p16INK4A forms disulfide-dependent homodimers upon oxidation. • The all α-helical p16INK4A protein aggregates as β-amyloids upon oxidation. • Oxidation and subsequent β-amyloid formation impair p16INK4A function.

Background Cardiac troponins are often elevated in patients with skeletal muscle disease who have no evidence of cardiac disease. Objectives The goal of this study was to characterize cardiac troponin concentrations in patients with myopathies and derive insights regarding the source of elevated troponin T measurements. Methods Cardiac troponin T (cTnT) and cardiac troponin I (cTnI) concentrations were determined by using high sensitivity assays in 74 patients with hereditary and acquired skeletal myopathies. Patients underwent comprehensive cardiac evaluation, including 12-lead electrocardiogram, 24-h electrocardiogram, cardiac magnetic resonance imaging, and coronary artery computed tomography. cTnT and cTnI protein expression was determined in skeletal muscle samples of 9 patients and in control tissues derived from autopsy using antibodies that are used in commercial assays. Relevant Western blot bands were subjected to liquid chromatography tandem mass spectrometry for protein identification. Results Levels of cTnT (median: 24 ng/l; interquartile range: 11 to 54 ng/l) were elevated (>14 ng/l) in 68.9% of patients; cTnI was elevated (>26 ng/l) in 4.1% of patients. Serum cTnT levels significantly correlated with creatine kinase and myoglobin (r = 0.679 and 0.786, respectively; both p Conclusions Measured cTnT concentrations were chronically elevated in the majority of patients with skeletal myopathies, whereas cTnI elevation was rare. Our data indicate that cross-reaction of the cTnT immunoassay with skeletal muscle troponin isoforms was the likely cause.

Technical advances including liquid chromatography–tandem mass spectrometry and its data analysis enable detailed proteomic analysis of the nasal mucus. Alterations of the nasal mucus proteome may provoke substantial changes of the nasal physiology and have already been associated with rhinologic diseases such as allergic rhinitis. This study was conducted as a pilot study to map the olfactory cleft proteome using current techniques for proteomic analysis. Furthermore, we aimed to investigate proteomic changes as potential biomarkers in patients suffering from idiopathic and postinfectious olfactory disorders compared to healthy controls. Seven patients with idiopathic hyposmia and anosmia, seven patients with postinfectious hyposmia and anosmia and seven healthy controls were included in this study. In total, 1117 different proteins were detected in at least five patients in at least one group. Results of this study did not reveal significant differences regarding the proteomic composition of the olfactory cleft mucus between patients versus healthy controls. Among proteins involved in olfactory perception the G protein family was detected but also found unchanged between groups. Investigation of protein composition by liquid chromatography–tandem mass spectrometry enabled us to perform an in–depth analysis of the olfactory cleft mucus proteome regarding the diversity of different proteins in individual patients. However untargeted proteomics of the olfactory cleft mucus may not be an applicable approach to develop biomarkers for olfactory disorders. Targeted analyses of distinct proteins known to be involved in olfactory perception but not detected by our approach, e.g. odorant binding proteins, may provide more information regarding pathophysiology of olfactory diseases.