Biology subjects

Wagner, K.

Publications and source records attributed to Wagner, K..

16 recordsLinked to original sources

bioRxiv2025

TRIM28 regulates pre-mRNA splicing via phosphorylation and SUMOylation networks

Pre-mRNA splicing is a highly regulated process orchestrated by splicing factors, cis-acting elements, and interconnected cellular processes such as transcription and chromatin remodeling. Here, we identify a novel regulatory axis involving phosphorylation and SUMOylation that governs the function of Tripartite motif-containing 28 protein (TRIM28) and its role in pre-mRNA splicing. We demonstrate that TRIM28 interacts with the spliceosomal protein USP39 in a phosphorylation-dependent manner, with non-phosphorylated TRIM28 promoting USP39 SUMOylation at defined lysine residues. This post-translational modification enhances USP39s role within the U4/U6.U5 tri-snRNP complex. Functionally, TRIM28 knockdown induces widespread alterations in alternative splicing patterns, underscoring its importance in splicing regulation. Together, our findings uncover a mechanistic link between TRIM28-mediated post-translational modifications and the modulation of spliceosomal activity, offering new insights into how splicing decisions are integrated with cellular signaling pathways.

cell biology↗

bioRxiv2024

AER-270 and TGN-020 are not aquaporin-4 water channel blockers

Aquaporin-4 (AQP4) is the most abundant water channel protein in the brain. It controls water homeostasis, facilitates glymphatic function and is a drug target for brain edema following injury or stroke. Dysregulation of brain water homeostasis affects millions of people every year leading to death, disability and cognitive decline, for which no medicines are available. Two compounds, AER-270 and TGN-020, are sold as AQP4 inhibitors and a prodrug of AER-270 is currently in a phase I human trial. However, the direct effect of these compounds on AQP4 function has not been unequivocally demonstrated. Our data across multiple cellular and molecular assay systems demonstrate, unexpectedly, that AER-270 and TGN-020 do not inhibit AQP4. Although we observed an apparent inhibitory effect of AER-270 and TGN-020 in the Xenopus laevis oocyte assay, there was no effect in assays using reconstituted recombinant AQP4 or mammalian cells expressing exogenous or endogenous AQP4. We identify alternative mechanisms of action for both molecules that may explain previously reported in vivo results that were interpreted in the context of AQP4 inhibition. Overall, we conclude that AER-270 and TGN-020 should not be used to investigate the AQP4-dependence of biological processes in the brain.

biophysics↗

bioRxiv2024

Identification of targetable vulnerabilities of PLK1-overexpressing cancers by synthetic dosage lethality

Tumor heterogeneity poses a significant challenge in combating treatment resistance. Despite Polo-like kinase 1 (PLK1) being universally overexpressed in cancers and contributing to chromosomal instability (CIN), direct PLK1 inhibition hasnt yielded clinical progress. To address this, we utilized the synthetic dosage lethality (SDL) approach, targeting PLK1s genetic interactions for selective killing of overexpressed tumor cells while mitigating heterogeneity-associated challenges. Employing computational methods, we conducted a genome-wide shRNA screen, identifying 105 SDL candidates. Further in vivo CRISPR screening in a breast cancer xenograft model and in vitro CRISPR analysis validated these candidates. Employing Perturb-seq revealed IGF2BP2/IMP2 as a key SDL hit eliminating PLK1-overexpressing cells. Suppression of IGF2BP2, genetically or pharmacologically, downregulated PLK1 and limited tumor growth. Our findings strongly propose targeting PLK1s genetic interactions as a promising therapeutic approach, holding broad implications across multiple cancers where PLK1 is overexpressed.

cancer biology↗

bioRxiv2024

Protective function of ex vivo expanded CD8 T cells in a mouse model of adoptive therapy for cytomegalovirus infection depends on integrin beta 1 but not CXCR3, CTLA4, or PD-1 expression

The adoptive transfer of virus-specific T cells (VSTs) represents a therapeutic option for viral infection treatment in immunocompromised patients. Before administration, ex vivo culture enables VST expansion. However, it is unclear how ex vivo expansion affects the circulation, homing, and intra-tissue migration of administered VSTs. We established a model of VST immunotherapy of acute cytomegalovirus infection using adoptive transfer of ex vivo expanded OT-I CD8 T cells (recognizing SIINFEKL peptide) into Rag2-/- mice infected with murine cytomegalovirus (MCMV) encoding for the SIINFEKL peptide. Ex vivo expansion induced an effector T cell phenotype and affected the expression of integrins and chemokine receptors. CRISPR/Cas9-mediated gene deletions enabled us to address the role of selected genes in the homing of VSTs following intravenous administration. We found that deletion of Itgb1, encoding for integrin beta 1, prevented OT-I cells from entering infected organs and drastically reduced their number in blood, suggesting that adoptively transferred VSTs primarily expand in the infected tissues. In contrast, Cxcr3-/- OT-I cells provided equal protection as their Cxcr3+/+ counterparts, indicating that this chemokine receptor does not contribute to VST entry into infected organs. Further, Pdcd1 and Ctla4 deletion did not impair the transferred OT-I cells ability to protect mice from MCMV, arguing against quick exhaustion of VSTs with an effector T cell phenotype. Together, these data indicate that ex vivo expansion affects migration and activation properties of VSTs and suggest that future clinical evaluation of adoptive T cell therapy efficacy should include homing molecule expression assessment.

immunology↗

bioRxiv2024

Efficient targeting of heart lesions with cardiac myofibroblasts: Combined gene and cell therapy enhanced by magnetic steering

1The cardiac scar is a collagen-rich area, which is populated by myofibroblasts and has proven little amenable for therapeutic interventions. Herein, we have established an efficient targeting strategy for cardiac lesions by genetically manipulating embryonic cardiac myofibroblasts (mFB) in vitro, load the cells with magnetic nanoparticles and inject these into infarcted mouse hearts using magnetic steering. This yields strongly increased numbers ([~]4 fold compared to other cell types) of engrafted mFB. The injected mFB and endogenous myofibroblast (endoFB) population remain separate in the scar, but grafted mFB enhance the proliferation rate of endoFB by [~]4 fold. We also tested the functional impact of this approach by grafting lentiviral (LV)-transduced Connexin43 (Cx43) overexpressing mFB into the cardiac lesion. Prominent engraftment of Cx43+ mFB provides strong protection against post-infarct ventricular tachycardia (VT) in vivo, as VT incidence is reduced by [~]50 % at two and eight weeks after cell injection. Thus, ex vivo gene and subsequent in vivo cell therapy combined with magnetic steering of cardiac mFB enable efficient functional targeting of the cardiac scar.

physiology↗

bioRxiv2023

COMPLETE GENOME SEQUENCE OF VIBRIO SYNGNATHI SP. NOV., A FISH PATHOGEN, ISOLATED FROM THE KIEL FJORD

A new Vibrio strain K08M4T was isolated from the broad-nosed pipefish Syngnathus typhle in the Kiel Fjord. Infection experiments revealed that K08M4T is highly virulent for juvenile pipefish. Cells of strain K08M4T are Gram-stain-negative, curved rod-shaped and motile by means of a single polar flagellum. The strain can grow aerobically at 9 to 40{degrees}C, at pH 4 to 10.5 and tolerates up to 12% (w/v) NaCl. The most prevalent (> 10%) cellular fatty acids of K08M4T were C16:1{omega} 7c and C16:0. Whole-genome comparisons revealed that K08M4T represents a separate evolutionary lineage which is distinct from other Vibrio species and falls within the Vibrio Splendidus clade. The genome is 4,886,292 bp in size, consists of two circular chromosomes (3,298,328 bp, 1,587,964 bp), and comprises 4,178 protein-coding genes and 175 RNA genes. In this study, we describe the phenotypic features of the new isolate and present the annotation and analysis of its complete genome sequence. Based on these data, the new isolate represents a new species for which we propose the name Vibrio syngnathi. The type strain is K08M4T (=DSM 109818T). Supplementary materialOne supplementary figure and six supplementary table are available with the online version of the Manuscript.

microbiology↗

bioRxiv2023

Single cell spatial transcriptomic and translatomic profiling of dopaminergic neurons in health, ageing and disease

The brain is spatially organized and contains unique cell types, each performing diverse functions, and exhibiting differential susceptibility to neurodegeneration. This is exemplified in Parkinsons disease with the preferential loss of dopaminergic neurons of the substantia nigra pars compacta. Using a Parkinsons transgenic model, we conducted a single-cell spatial transcriptomic and dopaminergic neuron translatomic analysis of young and old mouse brains. Through the high resolving capacity of single-cell spatial transcriptomics, we provide a deep characterization of the expression features of dopaminergic neurons and 27 other cell types within their spatial context, identifying markers of healthy and aging cells, spanning Parkinsons-relevant pathways. We integrate gene enrichment and GWAS data to prioritize putative causative genes for disease investigation, identifying CASR as a novel regulator of dopaminergic calcium handling. These datasets (see: spatialbrain.org) represent the largest public resource for the investigation of spatial gene expression in brain cells in health, aging and disease.

molecular biology↗

bioRxiv2022

Advanced physiological maturation of iPSC-derived human cardiomyocytes using an algorithm-directed optimization of defined media components

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold tremendous promise for in vitro modeling to assess native myocardial function and disease mechanisms as well as testing drug safety and efficacy. However, current iPSC- CMs are functionally immature, resembling in vivo CMs of fetal or neonatal developmental states. The use of targeted culture media and organoid formats have been identified as potential high-yield contributors to improve CM maturation. This study presents a novel iPSC-CM maturation medium formulation, designed using a differential evolutionary approach targeting metabolic functionality for iterative optimization. Relative to gold-standard reference formulations, our medium significantly matured morphology, Ca2+ handling, electrophysiology, and metabolism, which was further validated by multiomic screening, for cells in either pure or co-cultured microtissue formats. Together, these findings not only provide a reliable workflow for highly functional iPSC-CMs for downstream use, but also demonstrate the power of high-dimensional optimization processes in evoking advanced biological function in vitro.

bioengineering↗

bioRxiv2022

MHC class Ia molecules facilitate MCK2-dependent MCMV infection of macrophages and virus dissemination to the salivary gland

Murine cytomegalovirus (MCMV) infection of macrophages relies on MCMV-encoded chemokine 2 (MCK2) through one or more unknown cellular receptors while infection of fibroblast occurs independent of MCK2 and is mediated by cell-expressed neuropilin 1. Applying a CRISPR screen, we now identified that the MHC-Ia/Beta-2-microglobulin (B2m) complex serves as an entry port for MCK2-mediated infection of macrophages. Further analyses revealed that MCK2-dependent infection requires expression of the MHC-Ia haplotypes H-2b and H-2d but not H-2k. The importance of the MCK2-MHC-I-pathway for primary infection and viral dissemination was highlighted by experiments with B2m-deficient mice, which lack surface expression of MHC-I molecules. In those mice, intranasally administered MCK2-proficient MCMV could not infect alveolar macrophages and subsequently failed to disseminate into the salivary glands. The identified molecular pathway used by MCMV to infect lung resident macrophages provides essential knowledge for understanding cytomegalovirus-induced pathogenesis, tissue targeting, and virus dissemination.

immunology↗

bioRxiv2021

Tissue-Specific Biochemical Differences Between Chronic Wasting Disease Prions Isolated From Free Ranging, White-Tailed Deer (Odocoileus virginianus)

Chronic wasting disease (CWD) is an invariably fatal prion disease affecting cervid species world-wide. Prions can manifest as distinct strains that can influence disease pathology and transmission. CWD is profoundly lymphotropic and most infected cervids likely shed peripheral prions replicated in lymphoid organs. However, CWD is a neurodegenerative disease and most research on prion strains has focused on neurogenic prions. Thus, a knowledge gap exists comparing prions in the brain to prions in the lymph node. In this study we compared prions from the obex and lymph node of naturally exposed white-tailed deer to identify potential biochemical strain differences. Here, we report biochemical evidence of strain differences between the brain and lymph node from these animals. Future work should examine the biological and zoonotic impact of these biochemical differences and examine more cervids from multiple locations to see if these differences are conserved across species and locations.

pathology↗

bioRxiv2019

3D microenvironment stiffness regulates tumor spheroid growth and mechanics via p21 and ROCK

Mechanical properties of cancer cells and their microenvironment contribute to breast cancer progression. While mechanosensing has been extensively studied using two-dimensional (2D) substrates, much less is known about it in a physiologically more relevant 3D context. Here we demonstrate that breast cancer tumor spheroids, growing in 3D polyethylene glycol-heparin hydrogels, are sensitive to their environment stiffness. During tumor spheroid growth, compressive stresses of up to 2 kPa built up, as quantitated using elastic polymer beads as stress sensors. Atomic force microscopy (AFM) revealed that tumor spheroid stiffness increased with hydrogel stiffness. Also, constituent cell stiffness increased in a ROCK- and F-actin-dependent manner. Increased hydrogel stiffness correlated with attenuated tumor spheroid growth, a higher proportion of cells in G0/G1 phase and elevated levels of the cyclin-dependent kinase inhibitor p21. Drug-mediated ROCK inhibition reversed not only cell stiffening upon culture in stiff hydrogels but also increased tumor spheroid growth. Taken together, we reveal here a mechanism by which the growth of a tumor spheroid can be regulated via cytoskeleton rearrangements in response to its mechanoenvironment. Thus, our findings contribute to a better understanding of how cancer cells react to compressive stress when growing under confinement in stiff environments and provide the basis for a more in-depth exploration of the underlying mechanosensory response.

bioengineering↗

bioRxiv2018

Acute but not inherited demyelination in mouse models leads to brain tissue stiffness changes

The alteration or decrease of axonal myelination is an important hallmark of aging and disease. Demyelinated axons are impaired in their function and degenerate over time. Oligodendrocytes, the cells responsible for myelination of axons, are sensitive to mechanical properties of their environment. Growing evidence indicates that mechanical properties of demyelinating lesions are different from the healthy state and thus have the potential to affect myelinating potential of oligodendrocytes. We performed a high-resolution spatial mapping of the mechanical heterogeneity of demyelinating lesions using Atomic Force Microscope enabled indentation. Our results indicate that the stiffness of specific regions of mouse brain tissue is influenced by age and degree of myelination. Here we specifically demonstrate that acute but not inherited demyelination leads to decreased tissue stiffness, which could lower remyelination potential of oligodendrocytes. We also demonstrate that specific brain regions have unique ranges of stiffness in white and grey matter. Our ex vivo findings may help the design of future in vitro models to mimic mechanical environment of the brain in healthy and disease state. Reported here, mechanical properties of demyelinating lesions may facilitate novel approaches in treating demyelinating diseases such as multiple sclerosis.

biophysics↗

bioRxiv2018

Polyacrylamide Bead Sensors for in vivo Quantification of Cell-Scale Stress in Zebrafish Development

Mechanical stress exerted and experienced by cells during tissue morphogenesis and organ formation plays an important role in embryonic development. While techniques to quantify mechanical stresses in vitro are available, few methods exist for studying stresses in living organisms. Here, we describe and characterize cell-like polyacrylamide (PAAm) bead sensors with well-defined elastic properties and size for in vivo quantification of cell-scale stresses. The beads were injected into developing zebrafish embryos and their deformations were computationally analyzed to delineate spatio-temporal local acting stresses. With this computational analysis-based cell-scale stress sensing (COMPAX) we are able to detect pulsatile pressure propagation in the developing neural rod potentially originating from polarized midline cell divisions and continuous tissue flow. COMPAX is expected to provide novel spatiotemporal insight into developmental processes at the local tissue level and to facilitate quantitative investigation and a better understanding of morphogenetic processes.

biophysics↗

bioRxiv2018

Connectivity and network state-dependent recruitment of long-range VIP-GABAergic neurons in the mouse hippocampus

GABAergic interneurons in the hippocampus provide for local and long-distance coordination of neurons in functionally connected areas. Vasoactive intestinal peptide-expressing (VIP+) interneurons occupy a distinct niche in circuitry as many of them specialize in innervating GABAergic cells, thus providing network disinhibition. In the CA1 hippocampus, VIP+ interneuron-selective cells target local interneurons. Here, we discovered a novel type of VIP+ neuron whose axon innervates CA1 and also projects to the subiculum (VIP-LRPs). VIP-LRPs showed specific molecular properties and targeted interneurons within the CA1 area but both interneurons and pyramidal cells within subiculum. They were interconnected through gap junctions but demonstrated sparse spike coupling in vitro. In awake mice, VIP-LRPs decreased their activity during theta-run epochs and were more active during quiet wakefulness but not coupled to sharp-wave ripples. Together, the data provide new evidence for VIP interneuron molecular diversity and functional specialization in controlling cell ensembles along the hippocampo-subicular axis.

neuroscience↗

bioRxiv2018

Standardized microgel beads as elastic cell mechanical probes

Cell mechanical measurements are gaining increasing interest in biological and biomedical studies. However, there are no standardized calibration particles available that permit the cross-comparison of different measurement techniques operating at different stresses and time-scales. Here we present the rational design, production, and comprehensive characterization of poly-acylamide (PAAm) microgel beads mimicking biological cells. We produced mono-disperse beads at rates of 20 - 60 kHz by means of a microfluidic droplet generator, where the pre-gel composition was adjusted to tune the beads elasticity in the range of cell and tissue relevant mechanical properties. We verified bead homogeneity by optical diffraction tomography and Brillouin microscopy. Consistent elastic behavior of microgel beads at different shear rates was confirmed by AFM-enabled nanoindentation and real-time deformability cytometry (RT-DC). The remaining inherent variability in elastic modulus was rationalized using polymer theory and effectively reduced by sorting based on forward-scattering using conventional flow cytometry. Our results show that PAAm microgel beads can be standardized as mechanical probes, to serve not only for validation and calibration of cell mechanical measurements, but also as cell-scale stress sensors.\n\nSignificance StatementOften vastly different cell mechanical properties are reported even for the same cell type when employing different measurement techniques. This discrepancy shows the urgent need for standardized calibration particles to cross-compare and validate techniques. Microgel beads can serve this purpose, but they have to fulfil specific requirements such as homogeneity, sizes and elasticities in the range of the cells, and they have to provide comparable results independent of the method applied. Here we demonstrate the standardized production of polyacrylamide microgel beads with all the features an elastic cell-mimic should have. These can not only be used as method calibration particles, but can also serve as cell-scale sensors to quantify normal and shear stresses exerted by other cells and inside tissues, enabling many new applications.

biophysics↗

bioRxiv2017

Real-time fluorescence and deformability cytometry - flow cytometry goes mechanics

Cell mechanical characterization has recently approached the throughput of conventional flow cytometers. However, this very sensitive, label-free approach still lacks the specificity of molecular markers. Here we combine real-time 1D-imaging fluorescence and deformability cytometry (RT-FDC) to merge the two worlds in one instrument -- opening many new research avenues. We demonstrate its utility using sub-cellular fluorescence localization to identify mitotic cells and test for their mechanical changes in an RNAi screen.

biophysics↗