Biology subjects

Klaus, A.

Publications and source records attributed to Klaus, A..

8 recordsLinked to original sources

bioRxiv2026

Scalable generation of human stem cell-derived ureteric bud and collecting duct organoids in stirred bioreactors

Emerging efforts to engineer multicellular kidney tissues for drug discovery, disease modeling, and therapeutic repair require scalable generation of kidney organoids derived from human induced pluripotent stem cells (hiPSCs). Here, we report a method for generating kidney ureteric bud and collecting duct epithelial organoids in stirred tank bioreactors. We demonstrate that extracellular matrix microcarriers facilitate organoid expansion and are compatible with suspension bioreactor methods. The combination of cell-coated matrix microcarriers with stirred bioreactors promotes maturation marker expression compared to static controls comprising matrix-embedded organoids. Our method offers promise for generating epithelial organoids that serve as multicellular models and organ building blocks for scalable biofabrication.

bioengineering↗

bioRxiv2024

Abnormal hyperactivity of specific striatal ensembles encodes distinct dyskinetic behaviors revealed by high-resolution clustering

L-DOPA-induced dyskinesia (LID) is a debilitating complication of dopamine replacement therapy in Parkinso[n]s disease and the most common hyperkinetic disorder of basal ganglia origin. Abnormal activity of striatal D1 and D2 spiny projection neurons (SPNs) is critical for LID, yet the link between SPN activity patterns and specific dyskinetic movements remains unknown. To explore this, we developed a novel method for clustering movements based on high-resolution motion sensors and video recordings. In a mouse model of LID, this method identified two main dyskinesia types and pathological rotations, all absent during normal behavior. Using single-cell resolution imaging, we found that specific sets of both D1 and D2-SPNs were abnormally active during these pathological movements. Under baseline conditions, the same SPN sets were active during behaviors sharing physical features with LID movements. These findings indicate that ensembles of behavior-encoding D1- and D2-SPNs form new combinations of hyperactive neurons mediating specific dyskinetic movements.

neuroscience↗

bioRxiv2024

Expanding the BonnMu sequence-indexed repository of transposon induced maize (Zea mays L.) mutations in dent and flint germplasm

The BonnMu resource is a transposon-tagged mutant collection designed for functional genomics studies in maize. To expand this resource, we crossed an active Mutator (Mu) line with dent (B73, Co125) and flint (DK105, EP1 and F7) germplasm, resulting in the generation of 8,064 mutagenized BonnMu F2-families. Sequencing of these Mu-tagged families revealed 425,924 heritable Mu insertions affecting 36,612 (83%) of the 44,303 high-confidence gene models of maize (B73v5). On average, we observed 12 Mu insertions per gene (425,924 total insertions/ 36,612 affected genes) and 53 insertions per BonnMu F2-family (425,924 total insertions/ 8,064 families). Mu insertions and photos of seedling phenotypes from segregating BonnMu F2-families can be accessed through the Maize Genetics and Genomics Database (MaizeGDB). Downstream examination via the automated Mutant-seq Workflow Utility (MuWU) identified 94% of the germinal insertion sites in genic regions and only a small fraction of 6% inserting in non-coding intergenic sequences of the genome. Consistently, Mu insertions aligned with gene-dense chromosomal arms. In total, 42% of all BonnMu insertions were located in the 5 untranslated region (UTR) of genes, corresponding to accessible chromatin. Furthermore, for 38% of the insertions (163,843 of 425,924 total insertions) Mu1, Mu8 and MuDR were confirmed to be the causal Mu elements. Our publicly accessible European BonnMu resource has archived insertions covering two major germplasm groups, thus facilitating both forward and reverse genetics studies.

genomics↗

bioRxiv2024

Targeting MYCN upregulates L1CAM tumor antigen in MYCN-dysregulated neuroblastoma to increase CAR T cell efficacy

BackgroundCurrent treatment protocols have only limited success in pediatric patients with neuroblastomas harboring amplifications of the central oncogene, MYCN. Adoptive T cell therapy presents an innovative strategy to improve cure rates. However, L1CAM-targeting CAR T cells achieved only limited response against refractory/relapsed neuroblastoma in an ongoing phase I trial to date. Here, we investigate how oncogenic MYCN levels influence tumor cell response to CAR T cells, as one possible factor limiting success in trials. MethodsHigh MYCN levels were induced in SK-N-AS cells harboring the normal diploid MYCN complement using a tetracycline-inducible system. The inducible MYCN cell model or MYCN-amplified neuroblastoma cell lines were cocultured with L1CAM-CAR T cells. CAR T cell effector function was assessed via activation marker expression (flow cytometry), cytokine release and tumor cytotoxicity (biophotonic signal assessment). The cell model was characterized using RNA sequencing, and our data compared to publicly available RNA and proteomic data sets from neuroblastomas. ChIP-sequencing data was used to determine transcriptional L1CAM regulation by MYCN using public data sets. Synergism between CAR T cells and the MLN8237 AURKA inhibitor, which indirectly inhibits MYCN activity, was assessed in vitro using the Bliss model and in vivo in an immunocompromised mouse model. ResultsInducing high MYCN levels in the neuroblastoma cell model reduced L1CAM expression and, consequently, L1CAM-CAR T cell effector function (activation, cytokine release and cytotoxicity) in vitro. Primary neuroblastomas possessing high MYCN levels expressed lower levels of both the L1CAM transcript and L1CAM tumor antigen. Indirectly inhibiting MYCN via AURKA using MLN8237 treatment restored L1CAM expression on tumor cells in vitro and restored L1CAM-CAR T cell effector function. Combining MLN8237 and L1CAM-CAR T cell treatment synergistically increased neuroblastoma-directed killing in MYCN-overexpressing cells in vitro and in vivo concomitant with severe in vivo toxicity. ConclusionWe shed new light on a primary resistance mechanism in MYCN-driven neuroblastoma against L1CAM-CAR T cells via target antigen downregulation. These data suggest that combining L1CAM-CAR T cell therapy with pharmacological MYCN inhibition may benefit patients with high-risk neuroblastomas harboring MYCN amplifications.

immunology↗

bioRxiv2022

High robustness of cytosolic glutathione redox potential under combined salt and osmotic stress in barley as revealed by the biosensor Grx1-roGFP2

O_LIBarley is a staple crop of major global importance and relatively resilient to a wide range of stress factors in the field. Transgenic reporter lines to investigate physiological parameters during stress treatments remain scarce. C_LIO_LIWe generated and characterized stable homozygous barley lines (cv. Golden Promise Fast) expressing the genetically-encoded biosensor Grx1-roGFP2, which indicates the redox potential of the major antioxidant glutathione in the cytosol. C_LIO_LIOur results demonstrate functionality of the sensor in living barley plants. We determined the glutathione redox potential (EGSH) of the cytosol to be in the range of -308 to -320 mV. EGSH was robust against a combined NaCl (150 mM) and water deficit treatment (-0.8 MPa) that caused growth retardation and showed only a minor oxidation after 96 h of treatment. C_LIO_LIWe conclude that the generated reporter lines are a novel resource to study stress resilience in barley. C_LI One sentence summaryGeneration and characterization of barley plants stably expressing Grx1-roGFP2 reveal high robustness of cytosolic glutathione redox potential (EGSH) under combined salt and osmotic stress.

plant biology↗

bioRxiv2022

ENHANCED GRAVITROPISM 2 coordinates molecular adaptations to gravistimulation in the elongation zone of barley roots

O_LIRoot gravitropism includes gravity perception in the root cap, signal transduction between root cap and elongation zone, and curvature response in the elongation zone. The barley (Hordeum vulgare) mutant enhanced gravitropism 2 (egt2) displays a hypergravitropic root phenotype. C_LIO_LIWe compared the transcriptomic reprogramming of the root cap, the meristem and the elongation zone of wild type and egt2 seminal roots upon gravistimulation in a time-course experiment and identified direct interaction partners of EGT2 by yeast-two-hybrid screening and bimolecular fluorescence complementation (BiFC) validation. C_LIO_LIWe demonstrated that the elongation zone is subjected to most transcriptomic changes after gravistimulation. Here, 35% of graviregulated genes are also transcriptionally controlled by EGT2, suggesting a central role of this gene in controlling the molecular networks associated with gravitropic bending. Gene co-expression analyses suggested a role of EGT2 in cell wall and reactive oxygen species (ROS)-related processes, in which direct interaction partners of EGT2 regulated by EGT2 and gravity might be involved. C_LIO_LITaken together, this study demonstrated the central role of EGT2 and its interaction partners in the networks controlling root zone-specific transcriptomic reprogramming of barley roots upon gravistimulation. These findings can contribute to the development of novel root idiotypes leading to improved crop performance. C_LI

plant biology↗

bioRxiv2022

Dynamic refinement of behavioral structure mediates dopamine-dependent credit assignment

Animals exhibit a diverse behavioral repertoire when exploring new environments and can learn which actions or action sequences produce positive outcomes. Dopamine release upon encountering reward is critical for reinforcing reward-producing actions1-3. However, it has been challenging to understand how credit is assigned to the exact action that produced dopamine release during continuous behavior. We investigated this problem with a novel self-stimulation paradigm in which specific spontaneous movements triggered optogenetic stimulation of dopaminergic neurons. Dopamine self-stimulation rapidly and dynamically changes the structure of the entire behavioral repertoire. Initial stimulations reinforced not only the stimulation-producing target action, but also actions similar to target and actions that occurred a few seconds before stimulation. Repeated pairings led to gradual refinement of the behavioral repertoire to home in on the target. Reinforcement of action sequences revealed further temporal dependencies of refinement. Action pairs spontaneously separated by long time intervals promoted a stepwise credit assignment, with early refinement of actions most proximal to stimulation and subsequent refinement of more distal actions. Thus, a retrospective reinforcement mechanism promotes not only reinforcement, but gradual refinement of the entire behavioral repertoire to assign credit to specific actions and action sequences that lead to dopamine release.

neuroscience↗

bioRxiv2020

Generation of a new Tbx6-inducible reporter mouse line to trace presomitic mesoderm derivatives throughout development and in adults

The presomitic mesoderm (PSM) is initially an unsegmented structure localized on each side of the neural tube of the developing embryo, which progressively segments to form the somites. The somites will segregate and partition to generate the dorsal dermomyotome and the ventral sclerotome. Endothelial and myogenic cells of both the trunk and limbs are derived from the somites. There is a lack of efficient reporter mouse models to label and trace the PSM derivatives, despite their crucial contribution to many developmental processes. In this study, we generated a tamoxifen inducible transgenic Tbx6 mouse line, Tg(Tbx6_Cre/ERT2)/ROSA-eYFP, to tag and follow PSM-derivatives from early embryonic stages until adulthood. After induction, endothelial and myogenic cells can be easily identified within the trunk and limbs with proper expression patterns. Since our Tg(Tbx6_Cre/ERT2)/ROSA-eYFP model allows to permanently label the PSM-derived cells, their progeny can be studied at long-term, opening the possibility to perform lineage tracing of stem cells upon aging.

developmental biology↗