The avian seminal fluid (SF) is a protein-rich fluid, derived from the testis, the rudimentary epididymis and, finally, from the cloacal gland. The SF interacts with spermatozoa and the inner cell lining of the female genital tract, to modulate sperm functions and female immune responsiveness. Its complex proteome might either be free or linked to extracellular vesicles (EVs) as it is the case in mammals, where EVs depict the tetraspanin CD9; and where those EVs derived from the epididymis (epididymosomes) also present the receptor CD44. In the present study, sperm-free SF from Red Jungle Fowl, White Leghorn and an advanced intercross (AIL, 12th generation) were studied using flow cytometry of the membrane marker tetraspanin CD9, Western blotting of the membrane receptor CD44 and electron microscopy in non-enriched (whole SF) or enriched fractions obtained by precipitation using a commercial kit (Total Exosome Precipitation Solution). Neither CD9- nor CD44 could be detected, and the ultrastructure confirmed the relative absence of EVs, raising the possibility that avian SF interacts differently with the female genitalia as compared to the seminal plasma of mammals.

Quorum sensing (QS) is a mode of cell-cell communication that bacteria use to sense population density and orchestrate collective behaviors. The common opportunistic human pathogen Pseudomonas aeruginosa employs QS to regulate a large set of genes involved in virulence and host-pathogen interactions. The Las circuit positioned on the top of the QS hierarchy in P. aeruginosa makes use of N-acyl-L-homoserine lactones (AHLs) as signal molecules, like N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C12-HSL). Disabling QS circuits by certain small-molecule compounds, known as quorum-sensing inhibitors (QSIs), has been proposed as a strategy to attenuate bacterial pathogenicity. In this study, four new AHL analogs were designed by incorporating a tert-butoxycarbonyl Boc group in amide and beta-keto (3-oxo) moiety. Compounds were evaluated on a molecular and phenotypic basis as a QSI using the screening strategy linked to the assignment of the Las QS system in P. aeruginosa. Using a LasR-based bioreporter, we found that the compounds decreased LasR-controlled light activity and competed efficiently with natural 3O-C12-HSL. The compounds reduced the production of the cognate 3O-C12-HSL and certain virulence traits, like total protease activity, elastase activity, pyocyanin production, and extracellular DNA release. Furthermore, a quantitative proteomic approach was used to study the effect of the compounds on QS-regulated extracellular proteins. Among the four compounds tested, one of them showed the most significant difference in the appearance of the 3O-C12-HSL-responsive reference proteins related to QS communication and virulence, i.e., a distinct activity as a QSI. Moreover, by combining experimental data with computational chemistry, we addressed the effect of LasR protein flexibility on docking precision and assessed the advantage of using a multi-conformational docking procedure for binding mode prediction of LasR modulators. Thus, the four new AHL compounds were tested for their interaction with the AHL-binding site in LasR to identify the key interferences with the activity of LasR. Our study provides further insight into molecular features that are required for small-molecule modulation of LasR-dependent QS communication in P. aeruginosa. This should facilitate rational design of the next generation of antivirulence tools to study and manipulate QS-controlled fitness in bacteria and, thereby, handle bacterial infections in a new way.

Epigenetic reprogramming of innate immune cells by beta-glucan in a process called trained immunity, leads to an enhanced host response to a secondary infection. beta-glucans are structural components of plants, algae, fungi and bacteria and thus recognized as non-self by human macrophages. We selected the beta-glucans curdlan from Alcaligenes faecalis, WGP dispersible from Saccharomyces cerevisiae, and beta-glucan-rich culture supernatant of Alternaria and investigated whether they could produce trained immunity effects leading to an increased control of virulent Mycobacterium tuberculosis. We observed a significant M. tuberculosis growth-reduction in macrophages trained with curdlan and Alternaria, which also correlated with increased IL-6 and IL-1 beta release. WGP dispersible-trained macrophages were stratified into non responders and responders, according to their ability to control M. tuberculosis, with responders producing higher IL-6 levels. The addition of neutrophils to infected macrophage cultures further enhanced macrophage control of virulent M. tuberculosis, but not in a stimuli-dependent manner. Pathway enrichment analysis of DNA methylome data also highlighted hypomethylation of genes in pathways associated with signaling and cellular reorganization and motility, and responders to WGP-training were enriched in the interferon-gamma signaling pathway. This study adds evidence that certain beta-glucans show promise as immune training agents.

The nosocomial opportunistic Gram-negative bacterial pathogen Acinetobacter baumannii is resistant to multiple antimicrobial agents and an emerging global health problem. The polymyxin antibiotic colistin, targeting the negatively charged lipid A component of the lipopolysaccharide on the bacterial cell surface, is often considered as the last-resort treatment, but resistance to colistin is unfortunately increasing worldwide. Notably, colistin-susceptible A. baumannii can also develop a colistin dependence after exposure to this drug in vitro. Colistin dependence might represent a stepping stone to resistance also in vivo. However, the mechanisms are far from clear. To address this issue, we combined proteogenomics, high-resolution microscopy, and lipid profiling to characterize and compare A. baumannii colistin-susceptible clinical isolate (Ab-S) of to its colistin-dependent subpopulation (Ab-D) obtained after subsequent passages in moderate colistin concentrations. Incidentally, in the colistin-dependent subpopulation the lpxA gene was disrupted by insertion of ISAjo2, the lipid A biosynthesis terminated, and Ab-D cells displayed a lipooligosaccharide (LOS)-deficient phenotype. Moreover, both mlaD and pldA genes were perturbed by insertions of ISAjo2 and ISAba13, and LOS-deficient bacteria displayed a capsule with decreased thickness as well as other surface imperfections. The major changes in relative protein abundance levels were detected in type 6 secretion system (T6SS) components, the resistance-nodulation-division (RND)-type efflux pumps, and in proteins involved in maintenance of outer membrane asymmetry. These findings suggest that colistin dependence in A. baumannii involves an ensemble of mechanisms seen in resistance development and accompanied by complex cellular events related to insertional sequences (ISs)-triggered LOS-deficiency. To our knowledge, this is the first study demonstrating the involvement of ISAjo2 and ISAba13 IS elements in the modulation of the lipid A biosynthesis and associated development of dependence on colistin.

Mitochondria play crucial roles in cellular metabolism, signaling, longevity, and immune defense. Recent evidences have revealed that the host microbiota, including bacterial pathogens, impact mitochondrial behaviors and activities in the host. The pathogenicity of Pseudomonas aeruginosa requires quorum sensing (QS) cell-cell communication allowing the bacteria to sense population density and collectively control biofilm development, virulence traits, adaptation and interactions with the host. QS molecules, like N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C-12-HSL), can also modulate the behavior of host cells, e.g., epithelial barrier properties and innate immune responses. Here, in two types of cells, fibroblasts and intestinal epithelial cells, we investigated whether and how P. aeruginosa 3O-C-12-HSL impacts the morphology of mitochondrial networks and their energetic characteristics, using high-resolution transmission electron microscopy, fluorescence live-cell imaging, assay for mitochondrial bioenergetics, and quantitative mass spectrometry for mitoproteomics and bioinformatics. We found that 3O-C-12-HSL induced fragmentation of mitochondria, disruption of cristae and inner membrane ultrastructure, altered major characteristics of respiration and energetics, and decreased mitochondrial membrane potential, and that there are distinct cell-type specific details of these effects. Moreover, this was mechanistically accompanied by differential expression of both common and cell-type specific arrays of components in the mitochondrial proteome involved in their structural organization, electron transport chain complexes and response to stress. We suggest that this effect of 3O-C-12-HSL on mitochondria may represent one of the events in the interaction between P. aeruginosa and host mitochondria and may have an impact on the pathogens strategy to hijack host cell activities to support their own survival and spreading.

The specific granule glycoprotein olfactomedin-4 (Olfm4) marks a subset (1-70%) of human neutrophils and the Olfm4-high (Olfm4-H) proportion has been found to correlate with septic shock severity. The aim of this study was to decipher proteomic differences between the subsets in healthy individuals, hypothesizing that Olfm4-H neutrophils have a proteomic profile distinct from that of Olfm4 low (Olfm4-L) neutrophils. We then extended the investigation to septic shock. A novel protocol for the preparation of fixed, antibody-stained, and sorted neutrophils for LC-MS/MS was developed. In healthy individuals, 39 proteins showed increased abundance in Olfm4-H, including the small GTPases Rab3d and Rab11a. In Olfm4-L, 52 proteins including neutrophil defensin alpha 4, CXCR1, Rab3a, and S100-A7 were more abundant. The data suggest differences in important neutrophil proteins that might impact immunological processes. However, in vitro experiments revealed no apparent difference in the ability to control bacteria nor produce oxygen radicals. In subsets isolated from patients with septic shock, 24 proteins including cytochrome b-245 chaperone 1 had significantly higher abundance in Olfm4-H and 30 in Olfm4-L, including Fc receptor proteins. There was no correlation between Olfm4-H proportion and septic shock severity, but plasma Olfm4 concentration was elevated in septic shock. Thus, the Olfm4-H and Olfm4-L neutrophils have different proteomic profiles, but there was no evident functional significance of the differences in septic shock.

Patients with glioblastoma (GBM) have a poor outcome, but even among patients receiving the same therapies and with good prognostic factors, one can find those with exceptionally short and long survival. From the Nordic trial, which randomized GBM patients of 60 years or older between two radiotherapy arms (60 Gy or 34 Gy) or temozolomide (TMZ), we selected 59 with good prognostic factors. These selected GBM patients were equally distributed according to treatment and MGMT promoter methylation status but had long or short survival. Methylation profiling with the Illumina Infinium Methylation EPIC BeadChip arrays was performed and utilized for methylation-based CNS tumor classification, and pathway enrichment analysis of differentially methylated CpG sites (DMCs), as well as calculation of epigenetic age acceleration with three different algorithms, to compare the long and short survival groups. Samples identified by the classifier as non-GBM IDH wildtype were excluded. DMCs between long- and short-term survivors were found in patients with methylated MGMT promoter treated with TMZ (123,510), those with unmethylated MGMT treated with 60Gy radiotherapy (4,086), and with methylated MGMT promoter treated with 34Gy radiotherapy (39,649). Long-term survivors with methylated MGMT promoter treated with TMZ exhibited hypermethylation of the Wnt signaling and the platelet activation, signaling, and aggregation pathways. The joint analysis of radiotherapy arms revealed 319 DMCs between long- and short-term survivors with unmethylated MGMT and none for samples with methylated MGMT promoter. An analysis comparing epigenetic age acceleration between patients with long- and short-term survival across all treatment arms showed a decreased epigenetic age acceleration for the latter. We identified DMCs for both TMZ and RT-treated patients and epigenetic age acceleration as a potential prognostic marker, but further systematic analysis of larger patient cohorts is necessary for confirmation of their prognostic and/or predictive properties.

Background: Host innate immune cells have been identified as key players in the early eradication of Mycobacterium tuberculosis and in the maintenance of an anti-mycobacterial immune memory, which we and others have shown are induced through epigenetic reprogramming. Studies on human tuberculosis immunity are dominated by those using peripheral blood as surrogate markers for immunity. We aimed to investigate DNA methylation patterns in immune cells of the lung compartment by obtaining induced sputum from M. tuberculosis- exposed subjects including symptom-free subjects testing positively and negatively for latent tuberculosis as well as patients diagnosed with active tuberculosis. Alveolar macrophages and alveolar T cells were isolated from the collected sputum and DNA methylome analyses performed (Illumina Infinium Human Methylation 450 k).Results: Multidimensional scaling analysis revealed that DNA methylomes of cells from the tuberculosis-exposed subjects and controls appeared as separate clusters. The numerous genes that were differentially methylated between the groups were functionally connected and overlapped with previous findings of trained immunity and tuberculosis. In addition, analysis of the interferon-gamma release assay (IGRA) status of the subjects demonstrated that the IGRA status was reflected in the DNA methylome by a unique signature.Conclusions: This pilot study suggests that M. tuberculosis induces epigenetic reprogramming in immune cells of the lung compartment, reflected as a specific DNA methylation pattern. The DNA methylation signature emerging from the comparison of IGRA-negative and IGRA-positive subjects revealed a spectrum of signature strength with the TB patients grouping together at one end of the spectrum, both in alveolar macrophages and T cells. DNA methylation-based biosignatures could be considered for further development towards a clinically useful tool for determining tuberculosis infection status and the level of tuberculosis exposure.

Wound healing is regulated by complex crosstalk between keratinocytes and other cell types, including stem cells. In this study, a 7-day direct co-culture model of human keratinocytes and adipose-derived stem cells (ADSCs) was proposed to study the interaction between the two cell types, in order to identify regulators of ADSCs differentiation toward the epidermal lineage. As major mediators of cell communication, miRNome and proteome profiles in cell lysates of cultured human keratinocytes and ADSCs were explored through experimental and computational analyses. GeneChip(R) miRNA microarray, identified 378 differentially expressed miRNAs; of these, 114 miRNAs were upregulated and 264 miRNAs were downregulated in keratinocytes. According to miRNA target prediction databases and the Expression Atlas database, 109 skin-related genes were obtained. Pathway enrichment analysis revealed 14 pathways including vesicle-mediated transport, signaling by interleukin, and others. Proteome profiling showed a significant upregulation of the epidermal growth factor (EGF) and Interleukin 1-alpha (IL-1 alpha) compared to ADSCs. Integrated analysis through cross-matching the differentially expressed miRNA and proteins suggested two potential pathways for regulations of epidermal differentiation; the first is EGF-based through the downregulation of miR-485-5p and miR-6765-5p and/or the upregulation of miR-4459. The second is mediated by IL-1 alpha overexpression through four isomers of miR-30-5p and miR-181a-5p.

Introduction: The role of Adipose-derived mesenchymal stem cells (AD-MSCs) in skin wound healing remains to be fully characterized. This study aims to evaluate the regenerative potential of autologous AD-MSCs in a non-healing porcine wound model, in addition to elucidate key miRNA-mediated epigenetic regulations that underlie the regenerative potential of AD-MSCs in wounds.Methods: The regenerative potential of autologous AD-MSCs was evaluated in porcine model using histopathology and spatial frequency domain imaging. Then, the correlations between miRNAs and proteins of AD-MSCs were evaluated using an integration analysis in primary human AD-MSCs in comparison to primary human keratinocytes. Transfection study of AD-MSCs was conducted to validate the bioinformatics data.Results: Autologous porcine AD-MSCs improved wound epithelialization and skin properties in comparison to control wounds. We identified 26 proteins upregulated in human AD-MSCs, including growth and angiogenic factors, chemokines and inflammatory cytokines. Pathway enrichment analysis highlighted cell signalling-associated pathways and immunomodulatory pathways. miRNA-target modelling revealed regulations related to genes encoding for 16 upregulated proteins. miR-155-5p was predicted to regulate Fibroblast growth factor 2 and 7, C-C motif chemokine ligand 2 and Vascular cell adhesion molecule 1. Transfecting human AD-MSCs cell line with anti-miR-155 showed transient gene silencing of the four proteins at 24 h post-transfection.Discussion: This study proposes a positive miR-155-mediated gene regulation of key factors involved in wound healing. The study represents a promising approach for miRNA-based and cell-free regenerative treatment for difficult-to-heal wounds. The therapeutic potential of miR-155 and its identified targets should be further explored in-vivo.