Biology subjects

Rudolf, R.

Publications and source records attributed to Rudolf, R..

8 recordsLinked to original sources

bioRxiv2026

Stage-resolved iPSC-to-motoneuron differentiation: Metabolic switch & mitochondrial remodeling

Development of motoneurons from stem cells is characterized by a change from glycolytic to oxidative metabolism. Since this transition remains poorly understood, we examined it at five distinct differentiation stages from hiPSC to motoneuron. While a direct comparison of hiPSCs and mature motoneurons confirmed the expected glycolytic-to-oxidative shift, the intermediate stages showed that the conversion was not monotonic. After an initial drop of glycolysis at the hiPSC-to-neuroepithelial transition, late neuroepithelial cells showed intermittent peaks of the glycolytic marker lactate dehydrogenase A and the metabolic regulator TIGAR. Furthermore, the lactate-produced-to-glucose-consumed ratio remained elevated. A fully oxidative phenotype was only assumed upon progress from neural progenitors to motoneurons, portrayed by a definitive drop of the lactate-produced-to-glucose-consumed ratio, an increase of mitochondrial membrane charging, and shifts from lactate dehydrogenase A to B, from pyruvate dehydrogenase to anaplerotic pyruvate carboxylase, and from Mitofusin 1 to 2. Together, our data show that metabolic maturation in human motoneurons does not occur as a simple switch. Instead, it unfolds through distinct stages in a directional yet nonlinear manner.

developmental biology↗

bioRxiv2026

Advanced 3D spheroid-based skin models and deep-learning based image analysis enable in-depth investigation of keratinocyte differentiation and barrier function

To date, a panel of different biological models have been used in skin research, ranging from in vivo testing to 2D and 3D cultures. Among these, organotypic skin models represent the current gold standard for preclinical dermatology and toxicology studies. However, they are variable in quality and require long maturation times and a lot of work and cells, the latter often of primary origin. Here, we propose dermal-epidermal spheroids as an alternative model that balances physiological relevance and throughput. Next to corresponding full thickness skin models, different fibroblast/keratinocyte coculture spheroids were generated. These used the commonly employed HaCaT cells as well as two recently immortalized keratinocyte cell lines, NHK-SV/TERT and NHK-E6/E7. To investigate their differentiation with detailed spatio-temporal resolution, a deep-learning segmentation-based pipeline, capable of revealing nuclear morphology and positioning as well as marker expression with single-cell precision, was developed and applied. Moreover, the formation of a functional barrier was assessed by live-imaging of Lucifer yellow diffusion. These experiments identified the NHK-E6/E7 cell line as the most and HaCaT cells as the least suitable alternative to primary keratinocytes in both spheroids and full thickness models. Furthermore, NHK-based coculture spheroids displayed functional maturation, including stratification, cornification, and barrier formation, closely recapitulating these features of corresponding full thickness models. Given the scalability and compatibility with automation, these micro-skin fibroblast/NHK-based 3D coculture spheroids might represent a promising new platform for pharmaceutical, cosmetic, and toxicological testing.

bioengineering↗

bioRxiv2024

Poly-alanine-tailing is a modifier of neurodegeneration caused by Listerin mutation

The surveillance of translation is critical for the fitness of organisms from bacteria to humans. Ribosome-associated Quality Control (RQC) is a surveillance mechanism that promotes the elimination of truncated polypeptides, byproducts of ribosome stalling during translation. In canonical mammalian RQC, NEMF binds to the large ribosomal subunit and recruits the E3 ubiquitin ligase Listerin, which marks the nascent-chains for proteasomal degradation. NEMF additionally extends the nascent-chains C-terminus with poly-alanine ( Ala-tail), exposing lysines in the ribosomal exit tunnel for ubiquitination. In an alternative, Listerin-independent RQC pathway, released nascent-chains are targeted by Ala-tail-binding E3 ligases. While mutations in Listerin or in NEMF selectively elicit neurodegeneration in mice and humans, the physiological significance of Ala-tailing and its role in disease have remained unknown. Here, we report the analysis of mice in which NEMFs Ala-tailing activity was selectively impaired. Whereas the Nemf homozygous mutation did not affect lifespan and only led to mild motor defects, genetic interaction analyses uncovered its synthetic lethal phenotype when combined with the lister neurodegeneration-causing mutation. Conversely, the lister phenotype was markedly improved when Ala-tailing capacity was partially reduced by a heterozygous Nemf mutation. Providing a plausible mechanism for this striking switch from early neuroprotection to subsequent neurotoxicity, we found that RQC substrates that evade degradation form amyloid-like aggregates in an Ala-tail dependent fashion. These findings uncover a critical role for Ala-tailing in mammalian proteostasis, and deepen our molecular understanding of pathophysiological roles of RQC in neurodegeneration.

biochemistry↗

bioRxiv2024

From in vitro to in silico: a pipeline for generating virtual tissue simulations from real image data

3D cell culture models replicate tissue complexity, aiming to study cellular interactions and responses in a more physiologically relevant environment compared to traditional 2D cultures. However, the spherical structure of these models makes it difficult to extract meaningful data, necessitating advanced techniques for proper analysis. In silico simulations enhance research by predicting cellular behaviors and therapeutic responses, providing a powerful tool to complement experimental approaches. Despite their potential, these simulations often require advanced computational skills and significant resources, creating a barrier for many researchers. To address these challenges, we developed an accessible pipeline using open-source software to facilitate virtual tissue simulations. Our approach employs the Cellular Potts Model, a versatile framework for simulating cellular behaviors in tissues. The simulations are constructed from real world 3D image stacks of cancer spheroids, ensuring the virtual models are rooted in experimental data. By introducing a new metric for parameter optimization, we enable the creation of realistic simulations without requiring extensive computational expertise. This pipeline benefits researchers wanting to incorporate computational biology into their methods, even if they do not possess extensive expertise in this area. By reducing the technical barriers associated with advanced computational modeling, our pipeline allows more researchers to utilize these powerful tools. Our approach aims to foster broader use of in silico methods in disease research, contributing to a deeper understanding of disease biology and the refinement of therapeutic interventions.

biophysics↗

bioRxiv2024

A Multiparametric Analysis Reveals Differential Behavior of Spheroid Cultures on Distinct Ultra-Low Attachment Plates Types

Spheroids have become principal three-dimensional biological models to study cancer, developmental processes, and drug efficacy. For spheroid generation, ultra-low attachment plates are noteworthy due to their simplicity, compatibility with automation, and experimental and commercial accessibility. Nonetheless, it is unknown whether and to what degree the plate type impacts spheroid formation and biology. This study employed automated brightfield microscopy to systematically compare the size and eccentricity of spheroids formed in six different plate types using four distinct human cell lines, i.e., CCD-1137Sk fibroblasts, HaCaT keratinocytes, and MDA-MB-231 and HT-29 cancer cells. Results showed that all plate types exhibited similar sphe-roid-forming capabilities, and the gross patterns of growth or shrinkage during four days after seeding were comparable. Yet, size and eccentricity varied systematically among specific cell lines and plate types. A confocal wholemount analysis by a novel pipeline of AI-based 3D-image analysis procedures revealed changes in cell proliferation, cell number, nuclear volume, and keratino-cyte differentiation, which were accompanied by altered YAP1-signals. The findings show that the plate type may influence the outcome of experimental campaigns. It is advisable to scan different plate types for the optimal configuration for a specific investigation instead of using one standard plate for all kinds of applications.

cancer biology↗

bioRxiv2023

Deep MALDI-MS Spatial Omics guided by Quantum Cascade Laser Mid-infrared Imaging Microscopy

In spatial omics, highly confident molecular identifications are indispensable for the investigation of complex biology and for spatial biomarker discovery. However, current mass spectrometry (MS)-based spatial omics must compromise between data acquisition speed and biochemical profiling depth, thus often leading to only "putative" molecular identifications. Here, we introduce fast quantum cascade laser mid-infrared imaging microscopy to guide MS imaging to confined tissue areas of high interest, e.g., multicellular spheroid cores or kidney glomeruli, for spatial lipidomics profiling at maximized analytical depth utilizing magnetic resonance-MS imaging at >106 resolution or prm-PASEF-MS2 fragmentation imaging. Instigating selective sulfatide accumulation in arylsulfatase A-deficient mice as ground truth concept, we demonstrate that deep QCL-infrared-guided on-tissue spatial omics unequivocally identifies 120 sulfatides. This approach enables identifications of odd-chain sulfatides and studies of structure-ion mobility-relationships that provide chemical rationales for improvements to current ion mobility prediction algorithms. Workflows and data processing tools are provided as community resources.

biochemistry↗

bioRxiv2023

Harnessing PROTAC technology to combat stress hormone receptor activation

Counteracting the overactivation of glucocorticoid receptors (GR) is an important therapeutic goal in stress-related psychiatry and beyond. The only clinically approved GR antagonist lacks selectivity and induces unwanted side effects. To complement existing tools of small-molecule-based inhibitors, we present a highly potent, novel catalytically-driven GR degrader, KH-103, based on proteolysis-targeting chimera technology. This selective degrader enables immediate and reversible GR depletion that is independent of genetic manipulation and circumvents transcriptional adaptations to inhibition. KH-103 achieves passive inhibition, preventing agonistic induction of gene expression, and significantly averts the GRs genomic effects compared to two currently available inhibitors. Application in primary-neuron cultures revealed the dependency of a glucocorticoid-induced increase in spontaneous calcium activity on GR. Finally, we present a proof of concept for application in-vivo. KH-103 opens opportunities for a more lucid interpretation of GR functions with translational potential.

molecular biology↗

bioRxiv2022

3D-Mass Spectrometry Imaging of Micro-scale 3D Cell Culture Models in Cancer Research

Three-dimensional (3D) human cell culture models have emerged as a key technology for personalized medicine and for phenotypic compound screening in more disease-like in-vitro systems. Mass spectrometry imaging (MSI) is one of the most versatile label-free techniques that enables simultaneous generation of spatial maps for multiple relevant molecules in these 3D-models. Here, we present an integrated platform for 3D-MSI of 3D-cell cultures comprising 3D-printed metal casting molds for freezing and embedding, MS imaging of 100 serial cryosections and their computational elastic 3D-reconstruction. With this platform, we monitored multiple lipids that were selectively associated with different cell-types or cell-cell interactions within 300 m-scale fibroblast and colon cancer biculture spheroids. Our findings suggest that 3D-printing-aided precise preparation of serial sections from small spheroids and visualization of marker molecules in 3D can provide a detailed overview of the cellular metabolic interplay in 3D cell culture models in cancer research and drug discovery.

cancer biology↗