Open Access

Protein associated with Myc (PAM) is a giant E3 ubiquitin ligase of 510 kDa. Although the role of PAM during neuronal development is well established, very little is known about its function in the regulation of synaptic strength. Here we used multiepitope ligand cartography (MELC) to study protein network profiles associated with PAM during the modulation of synaptic strength. MELC is a novel imaging technology that utilizes biomathematical tools to describe protein networks after consecutive immunohistochemical visualization of up to 100 proteins on the same sample. As an in vivo model to modulate synaptic strength we used the formalin test, a common model for acute and inflammatory pain. MELC analysis was performed with 37 different antibodies or fluorescence tags on spinal cord slices and led to the identification of 1390 PAM-related motifs that distinguish untreated and formalin-treated spinal cords. The majority of these motifs related to ubiquitin-dependent processes and/or the actin cytoskeleton. We detected an intermittent colocalization of PAM and ubiquitin with TSC2, a known substrate of PAM, and the glutamate receptors mGluR5 and GLUR1. Importantly these complexes were detected exclusively in the presence of F-actin. A direct PAM/F-actin interaction was confirmed by colocalization and cosedimentation. The binding of PAM toward F-actin varied strongly between the PAM splice forms found in rat spinal cords. PAM did not ubiquitylate actin or alter actin polymerization and depolymerization. However, F-actin decreased the ubiquitin ligase activity of purified PAM. Because PAM activation is known to involve its translocation, the binding of PAM to F-actin may serve to control its subcellular localization as well as its activity. Taken together we show that defining protein network profiles by topological proteomics analysis is a useful tool to identify previously unknown protein/protein interactions that underlie synaptic processes.

The importance of biologically active lipid mediators, such as prostanoids, leukotrienes, and specialized pro-resolving mediators, in the regulation of inflammation is well established. While the relevance of cholesterol in the context of atherosclerosis is also widely accepted, the role of cholesterol and its biosynthetic precursors on inflammatory processes is less comprehensively described. In the present mini-review, we summarize the current understanding of the inflammation-regulatory properties of cholesterol and relevant biosynthetic intermediates taking into account the implications of different subcellular distributions. Finally, we discuss the inflammation-regulatory effect of cholesterol homeostasis in the context of SARS-CoV-2 infections.

NADPH oxidases produce reactive oxygen species that differ in localization, type and concentration. Within the Nox family only Nox4 produces H2O2 which can directly oxidize cysteine residues. With this post-translational modification, activity, stability, localization and protein-protein interactions of the affected protein is altered. Nox4 controls differentiation, cellular homeostasis and prevents inflammation. Therefore, is likely that epigenetic mechanisms contribute to the effects of Nox4. One group of epigenetic modifiers are class IIa histone deacetylases (HDACs). We hypothesize that Nox4-derived H2O2 oxidizes HDACs and analyzed whether HDACs can be differentially oxidized by Nox4. As an artificial system, we utilized HEK293 cells, overexpressing Nox4 in a tetracycline-inducible manner. HDAC4 was oxidized upon Nox4 overexpression. Additionally, Nox4 overexpression increased HDAC4 phosphorylation on Ser632. H2O2 disrupted HDAC4/Mef2A complex, which de-represses Mef2A. In endothelial cells such as HUVECs and HMECs, overexpression of HDAC4 significantly reduced tube formation. Overexpression of a redox insensitive HDAC4 had no effect on endothelial tube formation. Treatment with H2O2, induction of Nox4 expression by treatment of the cells with TGFβ and co-overexpression of Nox4 not only induced phosphorylation of HDAC4, but also restored the repressive effect of HDAC4 for tube formation, while overexpression of a redox dead mutant of Nox4 did not.

Bacterial biofilms are polymicrobial aggregates embedded in a self-produced matrix, which consists of extracellular polymeric substances (EPS). In a clinical context, bacterial biofilms are related to the development of chronic infections. Notably, their unique properties confer persistence in host niches despite antimicrobial treatment and the host immune response, where their distinct virulence exacerbates disease progression. In vitro models for bacterial biofilms have been developed. Simple models typically provide artificial growth environments, resulting in biofilm development diverging from in vivo biofilms. Consequently, sophisticated models incorporate growth conditions mimicking the environment of chronic infections. Still, they lack host cells, as the pathogenic bacteria cause cytotoxic effects during biofilm formation. In this context, electrospinning represents a promising approach, due to the ability to design nanofibrous scaffolds that closely mimic biological matrices, while maintaining mechanical strength for non-destructive handling in in vitro experiments. The objective of this thesis was to develop an in vitro biofilm model for chronic wound infections based on electrospun scaffolds, offering high clinical relevance through its close representation of in vivo conditions, the capacity to explore host-biofilm interfaces, and the incorporation of multiple bacterial species within a single biofilm. Three-dimensional electrospun scaffolds of cellulose acetate (CA) and gelatin were fabricated as scaffolds for biofilm development. The scaffolds were designed to possess composition and physicochemical properties similar to native biofilm microenvironments. Furthermore, high mechanical strength was required to enable the transfer of mature biofilms. The CA-based scaffolds, alone and in combination with gelatin, fulfilled the necessary criteria for physicochemical properties. Gelatin fiber scaffolds were limited due to rapid dissolution in aqueous environments. P. aeruginosa colonization of CA scaffolds did not result in three-dimensional model systems. In contrast, fiber scaffolds of CA and gelatin promoted bacterial distribution throughout their entire dimensions and enabled matrix production. Along with the formation of aggregates and a dense biofilm morphology, this confirmed their maturation. Their high antibiotic tolerance demonstrated the functional properties of mature biofilms. Subsequently, the high mechanical strength of the biofilm models on blend scaffolds enabled the transfer of mature biofilms to ex vivo human skin wounds, imitating a chronic state of wound infection. The novel biofilm model based on nanofibrous scaffolds addressed the major limitations of existing biofilm models by mimicking the native microenvironment of infectious biofilms and facilitating the examination of biofilm-host interfaces. In the next step, the developed biofilm model system was used to evaluate host biofilm interactions. For this study, in vitro and ex vivo models of human skin wounds were infected with pre-cultivated biofilms of P. aeruginosa. Regarding the structural characterization of biofilm infected wound models, all models enabled biofilm growth in close contact to the wound bed, creating ideal conditions for biofilm host interactions. In the case of viable wound models, an active immune response was demonstrated with cytokine gene expression patterns consistent with in vivo observations. Furthermore, the tissue viability was found to impact bacterial colonization behavior in the wound bed. These observations underscored the relevance of using viable models for studying direct biofilm-host-interactions. The new biofilm model, combined with human wound models of various complexities, demonstrated its applicability across a spectrum of research fields, from basic investigations to translational approaches. Biofilms in persistent wound infections typically exhibit a polymicrobial nature. Previous investigations have largely concentrated on single species due to the challenges of co-cultivating biofilms of multiple pathogens in vitro. P. aeruginosa and S. aureus were selected to create mono and dual-species biofilms. P. aeruginosa mono-species biofilms and dual-species biofilms exhibited rapid biofilm development. In contrast, the development of S. aureus biofilms was delayed. Initially, the presence of P. aeruginosa stimulated the adhesion of S. aureus in the dual-species biofilms. However, P. aeruginosa became increasingly dominant, revealing both cooperative and competitive interactions. In dual-species biofilms, both species existed in close proximity, essential for interbacterial interactions. All models exhibited high tolerance to antibiotic treatment. The biofilm matrix acting as a penetration barrier was demonstrated as a major factor. Constituents of P. aeruginosa mono-species and dual-species biofilms impaired wound healing by stimulating host immune responses and reducing cell viability. In contrast, S. aureus biofilms appeared to exhibit proliferative effects with no impairment of wound healing. The biofilm model facilitated the co-cultivation of multiple species in one biofilm, underscoring its similarity to the in vivo environment.

Editorial on the Research Topic: Clinical application and impact of blood-flow-restriction training