Learning in the Single-Cell Organism Physarum polycephalum: Effect of Propofol.

Propofol belongs to a class of molecules that are known to block learning and memory in mammals, including rodents and humans. Interestingly, learning and memory are not tied to the presence of a nervous system. There are several lines of evidence indicating that single-celled organisms also have the capacity for learning and memory which may be considered as basal intelligence. Here, we introduce a new experimental model for testing the learning ability of Physarum polycephalum, a model organism frequently used to study single-celled "intelligence". In this study, the impact of propofol on Physarum's "intelligence" was tested. The model consists of a labyrinth of subsequent bifurcations in which food (oat flakes soaked with coconut oil-derived medium chain triglycerides [MCT] and soybean oil-derived long chain triglycerides [LCT]) or propofol in MCT/LCT) is placed in one of each Y-branch. In this setting, it was tested whether Physarum memorized the rewarding branch. We saw that Physarum was a quick learner when capturing the first bifurcations of the maze; thereafter, the effect decreased, perhaps due to reaching a state of satiety. In contrast, when oat flakes were soaked with propofol, Physarum's preference for oat flakes declined significantly. Several possible actions, including the blocking of gamma-aminobutyric acid (GABA) receptor signaling, are suggested to account for this behavior, many of which can be tested in our new model.

Repurposing of the antibiotic nitroxoline for the treatment of mpox.

The antiviral drugs tecovirimat, brincidofovir, and cidofovir are considered for mpox (monkeypox) treatment despite a lack of clinical evidence. Moreover, their use is affected by toxic side-effects (brincidofovir, cidofovir), limited availability (tecovirimat), and potentially by resistance formation. Hence, additional, readily available drugs are needed. Here, therapeutic concentrations of nitroxoline, a hydroxyquinoline antibiotic with a favourable safety profile in humans, inhibited the replication of 12 mpox virus isolates from the current outbreak in primary cultures of human keratinocytes and fibroblasts and a skin explant model by interference with host cell signalling. Tecovirimat, but not nitroxoline, treatment resulted in rapid resistance development. Nitroxoline remained effective against the tecovirimat-resistant strain and increased the anti-mpox virus activity of tecovirimat and brincidofovir. Moreover, nitroxoline inhibited bacterial and viral pathogens that are often co-transmitted with mpox. In conclusion, nitroxoline is a repurposing candidate for the treatment of mpox due to both antiviral and antimicrobial activity.

Functional Downregulation of PD-L1 and PD-L2 by CpG and non-CpG Oligonucleotides in Melanoma Cells.

The clinical application of immune checkpoint inhibitors represents a breakthrough progress in the treatment of metastasized melanoma and other tumor entities. In the present study, it was hypothesized that oligonucleotides (ODNs), known as modulators of the immune response, have an impact on the endogenous expression of checkpoint molecules, namely PD-L1 and PD-L2 (PD-L1/2). IFNγ-stimulated melanoma cells (A375, SK-Mel-28) were treated with different synthetically manufactured oligonucleotides which differed in sequence, length and backbone composition. It was found that a variety of different ODN sequences significantly suppressed PD-L1/2 expression. This effect was dependent on length and phosphorothioate (PTO) backbone. In particular, a sequence containing solely guanines (nCpG-6-PTO) was highly effective in downregulating PD-L1/2 at the protein, mRNA and promoter levels. Mechanistically, we gave evidence that ODNs with G-quartet-forming motifs suppress the interferon signaling axis (JAK/STAT/IRF1). Our findings identify a subset of ODNs as interesting pharmacological compounds that could expand the arsenal of targeted therapies to combat the immunological escape of tumor cells.

Increased inducible nitric oxide synthase (iNOS) expression in human myocardial infarction.

Increased inducible nitric oxide synthase (iNOS) expression has been reported in heart failure, cardiomyopathies, and arteriosclerosis. iNOS is expressed in the heart upon inflammatory stimuli and produces excessive amounts of nitric oxide (NO). The overproduction of NO is cytotoxic and involved in cardiovascular diseases. Furthermore, iNOS produces superoxide anion which proceeds with NO to the harmful oxidant peroxynitrite, causing oxidative stress in the heart. The aim of the study was to gain new insights into the role of iNOS in human myocardial infarction (MI) and its contribution to oxidative stress in the heart. Furthermore, we investigated the unaffected myocardium of the infarction hearts, to study if iNOS expression is increased, probably as an indicator for oxidative stress. Our results show a significant increase (p = 0.013) of the iNOS expression in the affected regions of MI hearts (n = 9) in comparison with healthy control hearts (n = 4). In the unaffected regions of MI hearts, an increase in the iNOS expression in some samples was found as well. Our study demonstrated the direct detection of iNOS mRNA in human myocardial tissue. The balance between beneficial and deleterious effects of iNOS may be particularly influenced by the presence or absence of concurrent oxidative stress.

Collagen I Promotes Adipocytogenesis in Adipose-Derived Stem Cells In Vitro.

A hallmark of ageing is the redistribution of body fat. Particularly, subcutaneous fat decreases paralleled by a decrease of skin collagen I are typical for age-related skin atrophy. In this paper, we hypothesize that collagen I may be a relevant molecule stimulating the differentiation of adipose-derived stem cells (ASCs) into adipocytes augmenting subcutaneous fat. In this context lipogenesis, adiponectin, and collagen I receptor expression were determined. Freshly isolated ASCs were characterized by stemness-associated surface markers by FACS analysis and then transdifferentiated into adipocytes by specific medium supplements. Lipogenesis was evaluated using Nile Red staining and documented by fluorescence microscopy or quantitatively measured by using a multiwell spectrofluorometer. Expression of adiponectin was measured by real-time RT-PCR and in cell-free supernatants by ELISA, and expression of collagen I receptors was observed by western blot analysis. It was found that supports coated with collagen I promote cell adhesion and lipogenesis of ASCs. Interestingly, a reverse correlation to adiponectin expression was observed. Moreover, we found upregulation of the collagen receptor, discoidin domain-containing receptor 2; receptors of the integrin family were absent or downregulated. These findings indicate that collagen I is able to modulate lipogenesis and adiponectin expression and therefore may contribute to metabolic dysfunctions associated with ageing.

Fatty acid receptor GPR120: a novel marker for human melanoma.

The correlation between ultraviolet radiation of the skin and melanoma incidence in humans is well established. Interestingly, epidemiologic data suggest also a correlation to an increased BMI pointing to metabolic trigger factors in melanoma pathogenesis. To substantiate this connection, we studied the expression of G-protein-coupled receptor 120 (GPR120), a receptor sensitive to unsaturated long-chain free fatty acids in melanoma tissues. One-hundred fourteen tissue sections histologically confirmed as nevi (n=32), primary melanoma (n=39), and melanoma metastasis (n=43) were immunohistochemically stained against GPR120. The staining was evaluated by three trained dermatopathologists and independently scored. Compared with nevi, primary melanoma and melanoma metastasis showed significantly higher levels of GPR120 staining. Only three out of 32 nevi showed strong GPR120 expression [median immunoreactivity-scoring system (IRS) score: 1, range: 0-10], whereas in primary melanomas 14 out of 39 were highly GPR120-positive (median IRS score: 7, range: 0-12) and in melanoma metastasis 27 out of 43 were highly GPR120-positive (median IRS score: 9, range: 0-12). GPR120 expression and tumor thickness (mm) show a statistically significant correlation in primary melanoma (P=0.011). Moreover, GPR120-positive staining was found throughout the epidermis and in sebaceous and sweat glands, which is yet not described. This study identified GPR120 as a novel marker for melanoma, indicating that melanoma cells are sensitive to free fatty acids. It is tempting to speculate that pharmacologically interfering with GPR120 signaling might improve melanoma therapy.

Activation of PKB/Akt and p44/42 by mechanical stretch utilizes desmosomal structures and the keratin filament.

BACKGROUND: Mechanical stress is an ubiquitous challenge of human cells with fundamental impact on cell physiology. Previous studies have shown that stretching promotes signalling cascades involved in proliferation and tissue enlargement. OBJECTIVE: The present study is dedicated to learn more about cellular structures contributing to perception and signal transmission of cell stretch. In particular, we hypothesized that desmosmal contacts and the adjacent keratin filament build an intercellular matrix providing information about the mechanical load. METHODS: Epidermal cells with different keratin equipment were seeded on flexible silicon dishes and stretched. As read out parameter the activation of PKB/Akt and p44/42 was monitored by Western blotting. Likewise desomosomal contacts were manipulated by depletion or addition of calcium. Moreover, desmoglein 3 and desmocollin 3 were blocked by either specific antibodies or siRNA. RESULTS: It was found that the omission of calcium from the medium, a necessary cofactor for desmosomal cadherins, inhibited stretch mediated activation of PKB/Akt and p44/42. The relevance of desmosomes in this context was further substantiated by experiments using a desmoglein 3 blocking antibody (AK23) and siRNA against desmocollin 3. Moreover, disruption of the keratin filament by sodium orthovanadate also abrogates PKB/Akt and p44/42 activation in response to stretch. Likewise, KEB-7 keratinocytes harbouring a mutation in the keratin 14 gene and genetically modified keratinocytes devoid of any keratin show an altered signalling after stretch indicating the relevance of the keratin filament in this context. CONCLUSION: Besides their important role in cell architecture our results identify desmosomes and keratins as mechanosensing structures.

Sensitization of Melanoma Cells for Death Ligand TRAIL Is Based on Cell Cycle Arrest, ROS Production, and Activation of Proapoptotic Bcl-2 Proteins.

The death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) represents a promising strategy for melanoma due to significant expression of TRAIL receptor 1 in melanoma metastases and high TRAIL sensitivity through this receptor. However, prevalent and inducible resistance are limiting its clinical use. In previous work, we and others have described multiple strategies leading to TRAIL sensitization; however, the common principles of these strategies remained elusive. Here, we demonstrate in melanoma cell lines (TRAIL-sensitive, TRAIL-resistant, and TRAIL-selected cells with acquired resistance) that cell cycle arrest clearly correlates with enhanced TRAIL sensitivity. Cell cycle arrest was induced by high cell confluence, serum starvation, or cyclin-dependent kinase (CDK) 4/6 inhibition. Addressing the signaling pathways revealed disruption of mitochondrial membrane potential and production of reactive oxygen species (ROS) in response to antiproliferative conditions alone. Activation of the proapoptotic Bcl-2 protein Bax and inhibition of apoptosis by Bcl-2 overexpression or by the antioxidant N-acetyl cysteine underlined the critical involvement of mitochondrial apoptosis pathways and of ROS, respectively. Most pronounced was the upregulation of small proapoptotic Bcl-2 proteins (Puma and Bcl-xS). These data provide a general understanding on TRAIL sensitization as well as an alternative view on CDK inhibitors and may suggest selective targeting of melanoma cells by cell cycle inhibition and TRAIL.

Methylprednisolone blocks autoantibody-induced tissue damage in experimental models of bullous pemphigoid and epidermolysis bullosa acquisita through inhibition of neutrophil activation.

Corticosteroids are regularly used to treat autoimmune diseases, such as bullous pemphigoid (BP). In BP, autoantibodies bind to type XVII collagen (COL17), located at the dermal-epidermal junction. A crucial role of neutrophils in experimental BP has been established. Specifically, reactive oxygen species and proteolytic granule enzymes mediate tissue injury. Therefore, we investigated the effects of methylprednisolone (MP) on neutrophils, which are likely to be affected by topical treatment. First, MP inhibited dermal-epidermal separation ex vivo in cryosections of the human skin induced by co-incubation of BP autoantibodies with neutrophils from healthy volunteers. Next, MP inhibited neutrophil activation in vitro induced by immune complexes (ICs) of COL17 and autoantibodies. This neutrophil activation was associated with phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and Akt. In turn, inhibition of ERK1/2, p38 MAPK, or Akt phosphorylation inhibited neutrophil activation by IC in vitro and dermal-epidermal separation ex vivo. In addition, we observed an increase of p38 MAPK phosphorylation in dermal infiltrates of BP patients. Treatment of mice with either MP or inhibitors of p38-MAPK or ERK1/2 phosphorylation impaired induction of autoantibody- or irritant-induced neutrophil-dependent inflammation. We here identify the inhibition of Akt, ERK1/2, and p38 MAPK phosphorylation as molecular mechanisms to promote MP's therapeutic effects.

Endothelial cells are highly heterogeneous at the level of cytokine-induced insulin resistance.

The endothelial wall plays a crucial role in various diseases as it serves as the barrier between circulatory system and organ tissue. Inflammation-driven insulin resistance and subsequent endothelial dysfunction represent a pathomechanism in cardiovascular diseases such as atherosclerosis and myocardial infarction. It was recently suggested that insulin resistance also contributes to the pathogenesis of psoriasis, a chronic inflammatory disease of the skin. However, it is not clear whether similar mechanisms at the endothelium contribute to the disease. In this study, we ask which endothelial cells are most suitable to address this question. We investigated the insulin response of four cell types (primary cells and cell lines) representing different vascular beds (micro- and macrovascular cells) in the presence of different pro-inflammatory cytokines. All four cell types used responded well to insulin; however, the ability to become resistant to insulin due to an inflammatory stimulus by cytokines involved in psoriasis (e.g. IL-1ß, IL-12, IL-17, IL-23 and TNF-α) was very heterogeneous and could not be attributed to the differential expression of the cognate cytokine receptors. We conclude that this disparity is due to the different origins and properties of the endothelial cells used. Thus, endothelial cells should be carefully selected for the purpose of the respective study, particularly when it comes to analysing the pathogenesis of a disease and the search of new molecular targets for innovative therapies.