Nanoscale FasL Organization on DNA Origami to Decipher Apoptosis Signal Activation in Cells

Cell signaling is initiated by characteristic protein patterns in the plasma membrane, but tools to decipher their molecular organization and activation are hitherto lacking. Among the well-known signaling pattern is the death inducing signaling complex with a predicted hexagonal receptor architecture. To probe this architecture, DNA origami-based nanoagents with nanometer precise arrangements of the death receptor ligand FasL are introduced and presented to cells. Mimicking different receptor geometries, these nanoagents act as signaling platforms inducing fastest time-to-death kinetics for hexagonal FasL arrangements with 10 nm inter-molecular spacing. Compared to naturally occurring soluble FasL, this trigger is faster and 100x more efficient. Nanoagents with different spacing, lower FasL number or higher coupling flexibility impede signaling. The results present DNA origami as versatile signaling scaffolds exhibiting unprecedented control over molecular number and geometry. They define molecular benchmarks in apoptosis signal initiation and constitute a new strategy to drive particular cell responses

OxPhos defects cause hypermetabolism and reduce lifespan in cells and in patients with mitochondrial diseases

Patients with primary mitochondrial oxidative phosphorylation (OxPhos) defects present with fatigue and multi-system disorders, are often lean, and die prematurely, but the mechanistic basis for this clinical picture remains unclear. By integrating data from 17 cohorts of patients with mitochondrial diseases (n = 690) we find evidence that these disorders increase resting energy expenditure, a state termed hypermetabolism. We examine this phenomenon longitudinally in patient-derived fibroblasts from multiple donors. Genetically or pharmacologically disrupting OxPhos approximately doubles cellular energy expenditure. This cell-autonomous state of hypermetabolism occurs despite near-normal OxPhos coupling efficiency, excluding uncoupling as a general mechanism. Instead, hypermetabolism is associated with mitochondrial DNA instability, activation of the integrated stress response (ISR), and increased extracellular secretion of age-related cytokines and metabokines including GDF15. In parallel, OxPhos defects accelerate telomere erosion and epigenetic aging per cell division, consistent with evidence that excess energy expenditure accelerates biological aging. To explore potential mechanisms for these effects, we generate a longitudinal RNASeq and DNA methylation resource dataset, which reveals conserved, energetically demanding, genome-wide recalibrations. Taken together, these findings highlight the need to understand how OxPhos defects influence the energetic cost of living, and the link between hypermetabolism and aging in cells and patients with mitochondrial diseases

Fluctuations in active membranes

Active contributions to fluctuations are a direct consequence of metabolic energy consumption in living cells. Such metabolic processes continuously create active forces, which deform the membrane to control motility, proliferation as well as homeostasis. Membrane fluctuations contain therefore valuable information on the nature of active forces, but classical analysis of membrane fluctuations has been primarily centered on purely thermal driving. This chapter provides an overview of relevant experimental and theoretical approaches to measure, analyze and model active membrane fluctuations. In the focus of the discussion remains the intrinsic problem that the sole fluctuation analysis may not be sufficient to separate active from thermal contributions, since the presence of activity may modify membrane mechanical properties themselves. By combining independent measurements of spontaneous fluctuations and mechanical response, it is possible to directly quantify time and energy-scales of the active contributions, allowing for a refinement of current theoretical descriptions of active membranes.Comment: 38 pages, 9 figures, book chapte

Frequent mechanical stress suppresses proliferation of mesenchymal stem cells from human bone marrow without loss of multipotency

Mounting evidence indicated that human mesenchymal stem cells (hMSCs) are responsive not only to biochemical but also to physical cues, such as substrate topography and stiffness. To simulate the dynamic structures of extracellular environments of the marrow in vivo, we designed a novel surrogate substrate for marrow derived hMSCs based on physically cross-linked hydrogels whose elasticity can be adopted dynamically by chemical stimuli. Under frequent mechanical stress, hMSCs grown on our hydrogel substrates maintain the expression of STRO-1 over 20 d, irrespective of the substrate elasticity. On exposure to the corresponding induction media, these cultured hMSCs can undergo adipogenesis and osteogenesis without requiring cell transfer onto other substrates. Moreover, we demonstrated that our surrogate substrate suppresses the proliferation of hMSCs by up to 90% without any loss of multiple lineage potential by changing the substrate elasticity every 2nd days. Such “dynamic in vitro niche” can be used not only for a better understanding of the role of dynamic mechanical stresses on the fate of hMSCs but also for the synchronized differentiation of adult stem cells to a specific lineage

OxPhos Defects Cause Hypermetabolism and Reduce Lifespan in Cells and in Patients With Mitochondrial Diseases

Patients with primary mitochondrial oxidative phosphorylation (OxPhos) defects present with fatigue and multi-system disorders, are often lean, and die prematurely, but the mechanistic basis for this clinical picture remains unclear. By integrating data from 17 cohorts of patients with mitochondrial diseases (n = 690) we find evidence that these disorders increase resting energy expenditure, a state termed hypermetabolism. We examine this phenomenon longitudinally in patient-derived fibroblasts from multiple donors. Genetically or pharmacologically disrupting OxPhos approximately doubles cellular energy expenditure. This cell-autonomous state of hypermetabolism occurs despite near-normal OxPhos coupling efficiency, excluding uncoupling as a general mechanism. Instead, hypermetabolism is associated with mitochondrial DNA instability, activation of the integrated stress response (ISR), and increased extracellular secretion of age-related cytokines and metabokines including GDF15. In parallel, OxPhos defects accelerate telomere erosion and epigenetic aging per cell division, consistent with evidence that excess energy expenditure accelerates biological aging. To explore potential mechanisms for these effects, we generate a longitudinal RNASeq and DNA methylation resource dataset, which reveals conserved, energetically demanding, genome-wide recalibrations. Taken together, these findings highlight the need to understand how OxPhos defects influence the energetic cost of living, and the link between hypermetabolism and aging in cells and patients with mitochondrial diseases

2D versus 3D human induced pluripotent stem cell-derived cultures for neurodegenerative disease modelling

Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS), affect millions of people every year and so far, there are no therapeutic cures available. Even though animal and histological models have been of great aid in understanding disease mechanisms and identifying possible therapeutic strategies, in order to find disease-modifying solutions there is still a critical need for systems that can provide more predictive and physiologically relevant results. One possible avenue is the development of patient-derived models, e.g. by reprogramming patient somatic cells into human induced pluripotent stem cells (hiPSCs), which can then be differentiated into any cell type for modelling. These systems contain key genetic information from the donors, and therefore have enormous potential as tools in the investigation of pathological mechanisms underlying disease phenotype, and progression, as well as in drug testing platforms. hiPSCs have been widely cultured in 2D systems, but in order to mimic human brain complexity, 3D models have been proposed as a more advanced alternative. This review will focus on the use of patient-derived hiPSCs to model AD, PD, HD and ALS. In brief, we will cover the available stem cells, types of 2D and 3D culture systems, existing models for neurodegenerative diseases, obstacles to model these diseases in vitro, and current perspectives in the field

Measuring fast stochastic displacements of bio-membranes with dynamic optical displacement spectroscopy

Stochastic displacements or fluctuations of biological membranes are increasingly recognized as an important aspect of many physiological processes, but hitherto their precise quantification in living cells was limited due to a lack of tools to accurately record them. Here we introduce a novel technique—dynamic optical displacement spectroscopy (DODS), to measure stochastic displacements of membranes with unprecedented combined spatiotemporal resolution of 20 nm and 10 μs. The technique was validated by measuring bending fluctuations of model membranes. DODS was then used to explore the fluctuations in human red blood cells, which showed an ATP-induced enhancement of non-Gaussian behaviour. Plasma membrane fluctuations of human macrophages were quantified to this accuracy for the first time. Stimulation with a cytokine enhanced non-Gaussian contributions to these fluctuations. Simplicity of implementation, and high accuracy make DODS a promising tool for comprehensive understanding of stochastic membrane processes

Do patients with diabetes type 2 or chronic heart failure understand a medication plan?

A standardized medication plan (MP) was recently enacted into German law (§ 31a SGB V). The purpose of our study was to assess if patients with chronic diseases requiring polymedication understand the standardized MP and can transfer the given information into practice. 100 patients who took at least five medicines regularly were prospectively included in a cross-sectional study: 50 patients with the primary diagnosis chronic heart failure (CHF), and 50 with diabetes mellitus type 2 (DMT2). We performed a structured test-scenario studying the handling of a provided MP then evaluated the execution of the information on the MP by filling pill boxes and requested patients' opinion. An established weighted scoring system, the "Evaluation Tool to test the handling of the Medication Plan" (ET-MP) was applied to quantitate the ability of the patients to handle the MP. In addition, signs of depression, cognitive function and self-care behavior in chronic heart failure were characterized using the PHQ-9, Mini-Cog, and G9-EHFScB-9 questionnaires, respectively. The understanding of the MP was poor and irrespective of the underlying primary diagnosis. Only 32% of all patients were able to handle the MP without difficulties (ET-MP score >90%), the median ET-MP score was 83 [(IQR) 50-98]. Comprehension of the MP was better in patients aged <70 years compared to ≥70 years (p<0.01). Patients ≥10 years of education achieved higher ET-MP results than patients with <10 years of education (p<0.01). Patients with signs of cognitive impairment exhibited significantly lower ET-MP scores than patients without cognitive impairment (p<0.001). There were no significant correlations of the ET-MP score with number of daily medications, living situation, sex, the Charlson Comorbidity Index, the PHQ-9 score, and use of a dosing aid or possession of a medication list