Serum-Induced Proliferation of Human Cardiac Stem Cells Is Modulated via TGFßRI/II and SMAD2/3.

The ageing phenotype is strongly driven by the exhaustion of adult stem cells (ASCs) and the accumulation of senescent cells. Cardiovascular diseases (CVDs) and heart failure (HF) are strongly linked to the ageing phenotype and are the leading cause of death. As the human heart is considered as an organ with low regenerative capacity, treatments targeting the rejuvenation of human cardiac stem cells (hCSCs) are of great interest. In this study, the beneficial effects of human blood serum on proliferation and senescence of hCSCs have been investigated at the molecular level. We show the induction of a proliferation-related gene expression response by human blood serum at the mRNA level. The concurrent differential expression of the TGFß target and inhibitor genes indicates the participation of TGFß signalling in this context. Surprisingly, the application of TGFß1 as well as the inhibition of TGFß type I and type II receptor (TGFßRI/II) signalling strongly increased the proliferation of hCSCs. Likewise, both human blood serum and TGFß1 reduced the senescence in hCSCs. The protective effect of serum on senescence in hCSCs was enhanced by simultaneous TGFßRI/II inhibition. These results strongly indicate a dual role of TGFß signalling in terms of the serum-mediated effects on hCSCs. Further analysis via RNA sequencing (RNA-Seq) revealed the participation of Ras-inactivating genes wherefore a prevention of hyperproliferation upon serum-treatment in hCSCs via TGFß signalling and Ras-induced senescence is suggested. These insights may improve treatments of heart failure in the future.

Serum Induces the Subunit-Specific Activation of NF-κB in Proliferating Human Cardiac Stem Cells.

Cardiovascular diseases (CVDs) are often linked to ageing and are the major cause of death worldwide. The declined proliferation of adult stem cells in the heart often impedes its regenerative potential. Thus, an investigation of the proliferative potential of adult human cardiac stem cells (hCSCs) might be of great interest for improving cell-based treatments of cardiovascular diseases. The application of human blood serum was already shown to enhance hCSC proliferation and reduce senescence. Here, the underlying signalling pathways of serum-mediated hCSC proliferation were studied. We are the first to demonstrate the involvement of the transcription factor NF-κB in the serum-mediated proliferative response of hCSCs by utilizing the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). RNA-Sequencing (RNA-Seq) revealed ATF6B, COX5B, and TNFRSF14 as potential targets of NF-κB that are involved in serum-induced hCSC proliferation.

EPO regulates neuronal differentiation of adult human neural-crest derived stem cells in a sex-specific manner.

BACKGROUND: Sexual differences in the biology of human stem cells are increasingly recognized to influence their proliferation, differentiation and maturation. Especially in neurodegenerative diseases such as Alzheimers disease (AD), Parkinson's disease (PD) or ischemic stroke, sex is a key player for disease progression and recovery of damaged tissue. Recently, the glycoprotein hormone erythropoietin (EPO) has been implicated as a regulator of neuronal differentiation and maturation in female rats. METHODS: In this study, we used adult human neural crest-derived stem cells (NCSCs) as a model system for exploring potential sex specific effects of EPO on human neuronal differentiation. We started with expression validation of the specific EPO receptor (EPOR) by performing PCR analysis in the NCSCs. Next, EPO mediated activation of nuclear factor-κB (NF-κB) via Immunocytochemistry (ICC) was performed, followed by investigating the sex-specific effects of EPO on neuronal differentiation by determining morphological changes in axonal growth and neurite formation accompanied by ICC. RESULTS: Undifferentiated male and female NCSCs showed a ubiquitous expression of the EPO receptor (EPOR). EPO treatment resulted in a statistically profound (male p = 0.0022, female p = 0.0012) nuclear translocation of NF-κB RELA in undifferentiated NCSCs of both sexes. But after one week of neuronal differentiation, we could show a highly significant (p = 0,0079) increase of nuclear NF-κB RELA in females only. In contrast, we observed a strong decrease (p = 0,0022) of RELA activation in male neuronal progenitors. Extending the view on the role of sex during human neuronal differentiation, here we demonstrate a significant increase of axon lengths in female NCSCs-derived neurons upon EPO-treatment (+ EPO: 167,73 (SD = 41,66) µm, w/o EPO: 77,68 (SD = 18,31) µm) compared to their male counterparts (+ EPO: 68,37 (SD = 11,97) µm, w/o EPO: 70,23 (SD = 12,89) µm). CONCLUSION: Our present findings therefore show for the first time an EPO-driven sexual dimorphism in neuronal differentiation of human neural-crest derived stem cells and emphasize sex-specific variability as a crucial parameter in stem cell biology and for treating neurodegenerative diseases.

The Role of Blood-Derived Factors in Protection and Regeneration of Aged Tissues.

Tissue regeneration substantially relies on the functionality of tissue-resident endogenous adult stem cell populations. However, during aging, a progressive decline in organ function and regenerative capacities impedes endogenous repair processes. Especially the adult human heart is considered as an organ with generally low regenerative capacities. Interestingly, beneficial effects of systemic factors carried by young blood have been described in diverse organs including the heart, brain and skeletal muscle of the murine system. Thus, the interest in young blood or blood components as potential therapeutic agents to target age-associated malignancies led to a wide range of preclinical and clinical research. However, the translation of promising results from the murine to the human system remains difficult. Likewise, the establishment of adequate cellular models could help to study the effects of human blood plasma on the regeneration of human tissues and particularly the heart. Facing this challenge, this review describes the current knowledge of blood plasma-mediated protection and regeneration of aging tissues. The current status of preclinical and clinical research examining blood borne factors that act in stem cell-based tissue maintenance and regeneration is summarized. Further, examples of cellular model systems for a more detailed examination of selected regulatory pathways are presented.

The Diminishment of Novel Endometrial Carcinoma-Derived Stem-like Cells by Targeting Mitochondrial Bioenergetics and MYC.

Cancer stem cells (CSCs) are a small subpopulation of tumor cells harboring properties that include self-renewal, multi-lineage differentiation, tumor reconstitution, drug resistance and invasiveness, making them key players in tumor relapse. In the present paper, we develop new CSC models and analyze the molecular pathways involved in survival to identify targets for the establishment of novel therapies. Endometrial carcinoma-derived stem-like cells (ECSCs) were isolated from carcinogenic gynecological tissue and analyzed regarding their expression of prominent CSC markers. Further, they were treated with the MYC-signaling inhibitor KJ-Pyr-9, chemotherapeutic agent carboplatin and type II diabetes medication metformin. ECSC populations express common CSC markers, such as Prominin-1 and CD44 antigen as well as epithelial-to-mesenchymal transition markers, Twist, Snail and Slug, and exhibit the ability to form free-floating spheres. The inhibition of MYC signaling and treatment with carboplatin as well as metformin significantly reduced the cell survival of ECSC-like cells. Further, treatment with metformin significantly decreased the mitochondrial membrane potential of ECSC-like cells, while the extracellular lactate concentration was increased. The established ECSC-like populations represent promising in vitro models to further study the contribution of ECSCs to endometrial carcinogenesis. Targeting MYC signaling as well as mitochondrial bioenergetics has shown promising results in the diminishment of ECSCs, although molecular signaling pathways need further investigations.

Targeting Key Signaling Pathways in Glioblastoma Stem Cells for the Development of Efficient Chemo- and Immunotherapy.

Glioblastoma multiforme (GBM) is the most aggressive and most common malignant brain tumor with poor patient survival despite therapeutic intervention. On the cellular level, GBM comprises a rare population of glioblastoma stem cells (GSCs), driving therapeutic resistance, invasion, and recurrence. GSCs have thus come into the focus of therapeutic strategies, although their targeting remains challenging. In the present study, we took advantage of three GSCs-populations recently established in our lab to investigate key signaling pathways and subsequent therapeutic strategies targeting GSCs. We observed that NF-κB, a crucial transcription factor in GBM progression, was expressed in all CD44+/CD133+/Nestin+-GSC-populations. Exposure to TNFα led to activation of NF-κB-RELA and/or NF-κB-c-REL, depending on the GBM type. GSCs further expressed the proto-oncogene MYC family, with MYChigh GSCs being predominantly located in the tumor spheres ("GROW"-state) while NF-κB-RELAhigh GSCs were migrating out of the sphere ("GO"-state). We efficiently targeted GSCs by the pharmacologic inhibition of NF-κB using PTDC/Bortezomib or inhibition of MYC by KJ-Pyr-9, which significantly reduced GSC-viability, even in comparison to the standard chemotherapeutic drug temozolomide. As an additional cell-therapeutic strategy, we showed that NK cells could kill GSCs. Our findings offer new perspectives for developing efficient patient-specific chemo- and immunotherapy against GBM.

Hepatic Vasculopathy and Regenerative Responses of the Liver in Fatal Cases of COVID-19.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects the nasopharynx and lungs and causes coronavirus disease-2019 (COVID-19). It may impact the heart, brain, kidney, and liver.1 Although functional impairment of the liver has been correlated with worse clinical outcomes, little is known about the pathophysiology of hepatic injury and repair in COVID-19.2,3 Histologic evaluation has been limited to small numbers of COVID-19 cases with no control subjects2,4 and demonstrated largely heterogeneous patterns of pathology.2,3.

Hyperosmolality in CHO culture: Effects on cellular behavior and morphology.

Exposure of Chinese hamster ovary cells (CHO) to highly concentrated feed solution during fed-batch cultivation is known to result in an unphysiological osmolality increase (>300 mOsm/kg), affecting cell physiology and morphology. Extending previous observation on osmotic adaptation, the present study investigates for the first time potential effects of hyperosmolality on CHO cells on both population and single-cell level. We intentionally exposed CHO cells to hyperosmolality of up to 545 mOsm/kg during fed-batch cultivation. In concordance with existing research data, hyperosmolality-exposed CHO cells showed a nearly triplicated volume accompanied by ablation of proliferation. On the molecular level, we observed a strong hyperosmolality-dependent increase in mitochondrial activity in CHO cells compared to control. In contrast to mitochondrial activity, hyperosmolality-dependent proliferation arrest of CHO cells was not accompanied by DNA accumulation or caspase-3/7-mediated apoptosis. Notably, we demonstrate for the first time a formation of up to eight multiple, small nuclei in single hyperosmolality-stressed CHO cells. The here presented observations reveal previously unknown hyperosmolality-dependent morphological changes in CHO cells and support existing data on the osmotic response in mammalian cells.

Chronic inflammation of middle ear cholesteatoma promotes its recurrence via a paracrine mechanism.

BACKGROUND: Cholesteatoma disease is an expanding lesion in the middle ear. Hearing loss and facial paralysis alongside with other intracranial complications are found. No pharmaceutical treatment is available today and recurrence after surgical extraction occurs. We investigated possible TLR4-based mechanisms promoting recurrence and explore possible treatments strategies. METHODS: We isolated fibroblasts and epidermal stem cells from cholesteatoma tissue and healthy auditory canal skin. Subsequently, their expression under standard culture conditions and after stimulation with LPS was investigated by RT-qPCR. Cell metabolism and proliferation were analysed upon LPS treatment, with and without TLR4 antagonist. An indirect co-culture of fibroblasts and epidermal stem cells isolated from cholesteatoma tissue was utilized to monitor epidermal differentiation upon LPS treatment by RT-qPCR and immunocytochemistry. RESULTS: Under standard culture conditions, we detected a tissue-independent higher expression of IL-1ß and IL-8 in stem cells, an upregulation of KGF and IGF-2 in both cell types derived from cholesteatoma and higher expression of TLR4 in stem cells derived from cholesteatoma tissue. Upon LPS challenge, we could detect a significantly higher expression of IL-1α, IL-1ß, IL-6 and IL-8 in stem cells and of TNF-a, GM-CSF and CXCL-5 in stem cells and fibroblasts derived from cholesteatoma. The expression of the growth factors KGF, EGF, EREG, IGF-2 and HGF was significantly higher in fibroblasts, particularly when derived from cholesteatoma. Upon treatment with LPS the metabolism was elevated in stem cells and fibroblasts, proliferation was only enhanced in fibroblasts derived from cholesteatoma. This could be reversed by the treatment with a TLR4 antagonist. The cholesteatoma fibroblasts could be triggered by LPS to promote the epidermal differentiation of the stem cells, while no LPS treatment or LPS treatment without the presence of fibroblasts did not result in such a differentiation. CONCLUSION: We propose that cholesteatoma recurrence is based on TLR4 signalling imprinted in the cholesteatoma cells. It induces excessive inflammation of stem cells and fibroblasts, proliferation of perimatrix fibroblasts and the generation of epidermal cells from stem cells thru paracrine signalling by fibroblasts. Treatment of the operation site with a TLR4 antagonist might reduce the chance of cholesteatoma recurrence. Video Abstract.

Immersive augmented reality system for the training of pattern classification control with a myoelectric prosthesis.

BACKGROUND: Hand amputation can have a truly debilitating impact on the life of the affected person. A multifunctional myoelectric prosthesis controlled using pattern classification can be used to restore some of the lost motor abilities. However, learning to control an advanced prosthesis can be a challenging task, but virtual and augmented reality (AR) provide means to create an engaging and motivating training. METHODS: In this study, we present a novel training framework that integrates virtual elements within a real scene (AR) while allowing the view from the first-person perspective. The framework was evaluated in 13 able-bodied subjects and a limb-deficient person divided into intervention (IG) and control (CG) groups. The IG received training by performing simulated clothespin task and both groups conducted a pre- and posttest with a real prosthesis. When training with the AR, the subjects received visual feedback on the generated grasping force. The main outcome measure was the number of pins that were successfully transferred within 20 min (task duration), while the number of dropped and broken pins were also registered. The participants were asked to score the difficulty of the real task (posttest), fun-factor and motivation, as well as the utility of the feedback. RESULTS: The performance (median/interquartile range) consistently increased during the training sessions (4/3 to 22/4). While the results were similar for the two groups in the pretest, the performance improved in the posttest only in IG. In addition, the subjects in IG transferred significantly more pins (28/10.5 versus 14.5/11), and dropped (1/2.5 versus 3.5/2) and broke (5/3.8 versus 14.5/9) significantly fewer pins in the posttest compared to CG. The participants in IG assigned (mean ± std) significantly lower scores to the difficulty compared to CG (5.2 ± 1.9 versus 7.1 ± 0.9), and they highly rated the fun factor (8.7 ± 1.3) and usefulness of feedback (8.5 ± 1.7). CONCLUSION: The results demonstrated that the proposed AR system allows for the transfer of skills from the simulated to the real task while providing a positive user experience. The present study demonstrates the effectiveness and flexibility of the proposed AR framework. Importantly, the developed system is open source and available for download and further development.

Neuroprotection Mediated by Human Blood Plasma in Mouse Hippocampal Slice Cultures and in Oxidatively Stressed Human Neurons.

Neuroprotection from oxidative stress is critical during neuronal development and maintenance but also plays a major role in the pathogenesis and potential treatment of various neurological disorders and neurodegenerative diseases. Emerging evidence in the murine system suggests neuroprotective effects of blood plasma on the aged or diseased brain. However, little is known about plasma-mediated effects on human neurons. In the present study, we demonstrate the neuroprotective effect mediated by human plasma and the most abundant plasma-protein human serum albumin against oxidative stress in glutamatergic neurons differentiated from human neural crest-derived inferior turbinate stem cells. We observed a strong neuroprotective effect of human plasma and human serum albumin against oxidative stress-induced neuronal death on the single cell level, similar to the one mediated by tumor necrosis factor alpha. Moreover, we detected neuroprotection of plasma and human serum albumin against kainic acid-induced excitatory stress in ex vivo cultured mouse hippocampal tissue slices. The present study provides deeper insights into plasma-mediated neuroprotection ultimately resulting in the development of novel therapies for a variety of neurological and, in particular, neurodegenerative diseases.

Analysis of Several Pathways for Efficient Killing of Prostate Cancer Stem Cells: A Central Role of NF-κB RELA.

Prostate cancer is a common cause of death worldwide. Here, we isolated cancer stem cells (CSCs) from four adenocarcinomas of the prostate (Gleason scores from 3 + 3 up to 4 + 5). CSCs were characterized by the expression of the stem cell markers TWIST, the epithelial cell adhesion molecule (EPCAM), the transcription factors SNAI1 (SNAIL) and SNAI2 (SLUG) and cancer markers such as CD44 and prominin-1 (CD133). All investigated CSC populations contained a fraction highly positive for aldehyde dehydrogenase (ALDH) function and displayed robust expressions of programmed cell death 1 (PD-1) ligands. Furthermore, we investigated immunotherapeutic approaches but had no success even with the clinically used PD-1 inhibitor pembrolizumab. In addition, we studied another death-inducing pathway via interferon gamma signaling and detected high-level upregulations of human leukocyte antigen A (HLA-A) and beta 2-microglobulin (B2M) with only moderate killing efficacy. To examine further killing mechanisms in prostate cancer stem cells (PCSCs), we analyzed NF-κB signaling. Surprisingly, two patient-specific populations of PCSCs were found: one with canonical NF-κB signaling and another one with blunted NF-κB activation, which can be efficiently killed by tumor necrosis factor (TNF). Thus, culturing of PCSCs and analysis of respective NF-κB induction potency after surgery might be a powerful tool for optimizing patient-specific treatment options, such as the use of TNF-inducing chemotherapeutics and/or NF-κB inhibitors.

IKK1/2 protect human cells from TNF-mediated RIPK1-dependent apoptosis in an NF-κB-independent manner.

TNF signaling is directly linked to cancer development and progression. A broad range of tumor cells is able to evade cell death induced by TNF impairing the potential anti-cancer value of TNF in therapy. Although sensitizing cells to TNF-induced death therefore has great clinical implications, detailed mechanistic insights into TNF-mediated human cell death still remain unknown. Here, we analyzed human cells by applying CRISPR/Cas9n to generate cells deficient of IKK1, IKK2, IKK1/2 and RELA. Despite stimulation with TNF resulted in impaired NF-κB activation in all genotypes compared to wildtype cells, increased cell death was observable only in IKK1/2-double-deficient cells. Cell death could be detected by Caspase-3 activation and binding of Annexin V. TNF-induced programmed cell death in IKK1/2-/- cells was further shown to be mediated via RIPK1 in a predominantly apoptotic manner. Our findings demonstrate the IKK complex to protect from TNF-induced cell death in human cells independently to NF-κB RelA suggesting IKK1/2 to be highly promising targets for cancer therapy.

Natural and synthetic nanopores directing osteogenic differentiation of human stem cells.

Bone regeneration is a highly orchestrated process crucial for endogenous healing procedures after accidents, infections or tumor therapy. Changes in surface nanotopography are known to directly affect the formation of osteogenic cell types, although no direct linkage to the endogenous nanotopography of bone was described so far. Here we show the presence of pores of 31.93 ±â€¯0.97 nm diameter on the surface of collagen type I fibers, the organic component of bone, and demonstrate these pores to be sufficient to induce osteogenic differentiation of adult human stem cells. We further applied SiO2 nanoparticles thermally cross-linked to a nanocomposite to artificially biomimic 31.93 ±â€¯0.97 nm pores, which likewise led to in vitro production of bone mineral by adult human stem cells. Our findings show an endogenous mechanism of directing osteogenic differentiation of adult stem cells by nanotopological cues and provide a direct application using SiO2 nanocomposites with surface nanotopography biomimicking native bone architecture.

1,8-Cineole potentiates IRF3-mediated antiviral response in human stem cells and in an ex vivo model of rhinosinusitis.

The common cold is one of the most frequent human inflammatory diseases caused by viruses and can facilitate bacterial superinfections, resulting in sinusitis or pneumonia. The active ingredient of the drug Soledum, 1,8-cineole, is commonly applied for treating inflammatory diseases of the respiratory tract. However, the potential for 1,8-cineole to treat primary viral infections of the respiratory tract remains unclear. In the present study, we demonstrate for the first time that 1,8-cineole potentiates poly(I:C)-induced activity of the antiviral transcription factor interferon regulatory factor 3 (IRF3), while simultaneously reducing proinflammatory nuclear factor (NF)-κB activity in human cell lines, inferior turbinate stem cells (ITSCs) and in ex vivo cultivated human nasal mucosa. Co-treatment of cell lines with poly(I:C) and 1,8-cineole resulted in significantly increased IRF3 reporter gene activity compared with poly(I:C) alone, whereas NF-κB activity was reduced. Accordingly, 1,8-cineole- and poly(I:C) treatment led to increased nuclear translocation of IRF3 in ITSCs and a human ex vivo model of rhinosinusitis compared with the poly(I:C) treatment approach. Nuclear translocation of IRF3 was significantly increased in ITSCs and slice cultures treated with lipopolysaccharide (LPS) and 1,8-cineole compared with the LPS-treated cells mimicking bacterial infection. Our findings strongly suggest that 1,8-cineole potentiates the antiviral activity of IRF3 in addition to its inhibitory effect on proinflammatory NF-κB signalling, and may thus broaden its field of application.

Helium Ion Microscopy Visualizes Lipid Nanodomains in Mammalian Cells.

Cell membranes are composed of 2D bilayers of amphipathic lipids, which allow a lateral movement of the respective membrane components. These components are arranged in an inhomogeneous manner as transient micro- and nanodomains, which are believed to be crucially involved in the regulation of signal transduction pathways in mammalian cells. Because of their small size (diameter 10-200 nm), membrane nanodomains cannot be directly imaged using conventional light microscopy. Here, direct visualization of cell membrane nanodomains by helium ion microscopy (HIM) is presented. It is shown that HIM is capable to image biological specimens without any conductive coating and that HIM images clearly allow the identification of nanodomains in the ultrastructure of membranes with 1.5 nm resolution. The shape of these nanodomains is preserved by fixation of the surrounding unsaturated fatty acids while saturated fatty acids inside the nanodomains are selectively removed. Atomic force microscopy, fluorescence microscopy, 3D structured illumination microscopy, and direct stochastic optical reconstruction microscopy provide additional evidence that the structures in the HIM images of cell membranes originate from membrane nanodomains. The nanodomains observed by HIM have an average diameter of 20 nm and are densely arranged with a minimal nearest neighbor distance of ≈ 15 nm.

MicroRNAs in pluripotency, reprogramming and cell fate induction.

Pluripotent stem cells display a unique expression pattern of microRNAs (miRNAs). These ~22 nucleotide non-coding RNAs have established a crucial role in controlling gene expression of pluripotent stem cells at the post-transcriptional level. Recent studies made important advances in identifying miRNA regulated processes like de novo DNA methylation, progression of the cell cycle and regulation of cell fate decision. miRNAs have also the ability to reprogram somatic cells to pluripotent stem cells and on the other hand, to induce differentiation of pluripotent stem cells into distinct somatic lineages. Previously it was published that miRNAs can direct reprogramming on its own. Here we provide evidence and critically discuss that the effect of miRNA depends on co-expression of the classical reprogramming factors. During transition between these different cell fates distinct miRNAs adjust the levels of specific transcriptional programs and confer robustness to differentiation processes. This results in a complex network between miRNAs and their targets. The fact that miRNAs itself can also be regulated by its targets establishes complex regulatory loops. Based on bioinformatical predictions, each miRNA theoretically has hundreds of target genes making it even more challenging to understand the complete network between miRNAs and their targets.

1,8-Cineol inhibits nuclear translocation of NF-κB p65 and NF-κB-dependent transcriptional activity.

Natural plant-derived products are commonly applied to treat a broad range of human diseases, including cancer as well as chronic and acute airway inflammation. In this regard, the monoterpene oxide 1,8-cineol, the active ingredient of the clinically approved drug Soledum®, is well-established for the therapy of airway diseases, such as chronic sinusitis and bronchitis, chronic obstructive pulmonary disease and bronchial asthma. Although clinical trials underline the beneficial effects of 1,8-cineol in treating inflammatory diseases, the molecular mode of action still remains unclear. Here, we demonstrate for the first time a 1,8-cineol-depending reduction of NF-κB-activity in human cell lines U373 and HeLa upon stimulation using lipopolysaccharides (LPS). Immunocytochemistry further revealed a reduced nuclear translocation of NF-κB p65, while qPCR and western blot analyses showed strongly attenuated expression of NF-κB target genes. Treatment with 1,8-cineol further led to increased protein levels of IκBα in an IKK-independent matter, while FRET-analyses showed restoring of LPS-associated loss of interaction between NF-κB p65 and IκBα. We likewise observed reduced amounts of phosphorylated c-Jun N-terminal kinase 1/2 protein in U373 cells after exposure to 1,8-cineol. In addition, 1,8-cineol led to decreased amount of nuclear NF-κB p65 and reduction of its target gene IκBα at protein level in human peripheral blood mononuclear cells. Our findings suggest a novel mode of action of 1,8-cineol through inhibition of nuclear NF-κB p65 translocation via IκBα resulting in decreased levels of proinflammatory NF-κB target genes and may therefore broaden the field of clinical application of this natural drug for treating inflammatory diseases.

Quantitative single-molecule localization microscopy combined with rule-based modeling reveals ligand-induced TNF-R1 reorganization toward higher-order oligomers.

We report on the assembly of tumor necrosis factor receptor 1 (TNF-R1) prior to ligand activation and its ligand-induced reorganization at the cell membrane. We apply single-molecule localization microscopy to obtain quantitative information on receptor cluster sizes and copy numbers. Our data suggest a dimeric pre-assembly of TNF-R1, as well as receptor reorganization toward higher oligomeric states with stable populations comprising three to six TNF-R1. Our experimental results directly serve as input parameters for computational modeling of the ligand-receptor interaction. Simulations corroborate the experimental finding of higher-order oligomeric states. This work is a first demonstration how quantitative, super-resolution and advanced microscopy can be used for systems biology approaches at the single-molecule and single-cell level.

Inherent Antibacterial Properties of Biodegradable FeMnC(Cu) Alloys for Implant Application.

Implant-related infections or inflammation are one of the main reasons for implant failure. Therefore, different concepts for prevention are needed, which strongly promote the development and validation of improved material designs. Besides modifying the implant surface by, for example, antibacterial coatings (also implying drugs) for deterring or eliminating harmful bacteria, it is a highly promising strategy to prevent such implant infections by antibacterial substrate materials. In this work, the inherent antibacterial behavior of the as-cast biodegradable Fe69Mn30C1 (FeMnC) alloy against Gram-negative Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive Staphylococcus aureus is presented for the first time in comparison to the clinically applied, corrosion-resistant AISI 316L stainless steel. In the second step, 3.5 wt % Cu was added to the FeMnC reference alloy, and the microbial corrosion as well as the proliferation of the investigated bacterial strains is further strongly influenced. This leads for instance to enhanced antibacterial activity of the Cu-modified FeMnC-based alloy against the very aggressive, wild-type bacteria P. aeruginosa. For clarification of the bacterial test results, additional analyses were applied regarding the microstructure and elemental distribution as well as the initial corrosion behavior of the alloys. This was electrochemically investigated by a potentiodynamic polarization test. The initial degraded surface after immersion were analyzed by glow discharge optical emission spectrometry and transmission electron microscopy combined with energy-dispersive X-ray analysis, revealing an increase of degradation due to Cu alloying. Due to their antibacterial behavior, both investigated FeMnC-based alloys in this study are attractive as a temporary implant material.