Biology subjects

Kristensen, M.

Publications and source records attributed to Kristensen, M..

7 recordsLinked to original sources

bioRxiv2025

Therapeutic Potential of Neural Rosette Stem Cell-Derived Small Extracellular Vesicles in the Management of Brain Edema

Small extracellular vesicles (sEVs) derived from neural stem cells (NSCs) have gained significant interest for their potential roles in neuroprotection, brain edema management, and the preservation of blood-brain barrier (BBB) integrity. These nanoscale particles facilitate intercellular communication and carry a diverse array of bioactive molecules, including proteins and RNAs, which influence various neurobiological processes. The composition of their cargo depends on the parental cell type, affecting their interactions with recipient cells. In this study, we characterized the cargo of sEVs derived from neural rosette stem cells (NRSCs) at both transcriptomic and proteomic levels and compared it to sEVs derived from cardiomyocyte progenitor cells (CMPCs) and human pluripotent stem cells (hPSCs). Furthermore, we assessed the therapeutic effects of NRSC-derived sEVs in a rat model of transient middle cerebral artery occlusion (tMCAO) to investigate neuronal injury and the progression of brain edema. Our proteomic and miRNA analyses revealed that the sEVs from NRSCs, CMPCs, and hPSCs possess distinct cargo compositions. In an in vitro neuronal ischemia model, NRSC-derived sEVs significantly enhanced cell viability, while in an endothelial model, they expedited the restoration of cell barrier integrity. Furthermore, the in vivo studies utilizing the rat model of tMCAO showed that intravenous administration of NRSC-derived sEVs at 3, 24 and 48 hours after reperfusion resulted in a significantly faster resolution of brain edema. These findings underscore the influence of the parental cell type on sEV composition and highlight the therapeutic potential of NRSC-derived sEVs in treating brain edema, paving the way for novel therapeutic strategies.

neuroscience↗

bioRxiv2025

Efficient Generation of Expandable Dorsal Forebrain Neural Rosette Stem Cell Lines

Neural stem cells (NSCs) represent an interesting option for developing in vitro disease models and drug screening assays due to their differentiation capacity into neurons and glial cells. Additionally, NSCs are under investigation in on-going clinical trials for treatment of various human neurological disorders. NSCs can be isolated from the central nervous system or derived in vitro from human pluripotent stem cells (hPSCs). However, the current methods for generating NSCs typically include a phase of neural rosette formation and subsequent manual isolation of these tiny structures. As this is a laborious process characterized by operator-dependent variability and scalability challenges, there is a pressing need to develop optimized and scalable protocols to obtain pure NSC populations. In this study, we present a new method for generating highly pure and expandable dorsal forebrain FOXG1+OTX2+TLE4+SOX5+ neural rosette stem cell (NRSC) lines without the necessity for manual isolation of rosette structures. Our findings demonstrate the reproducibility of this protocol through the characterization of different NRSC lines over multiple passages, highlighting the robustness of the process. These NRSCs can be expanded for at least 12 passages without compromising their rosette-formation capacity or their initial dorsal forebrain identity. Furthermore, we show the differentiation capacity of these NRSCs to generate pure populations of TUBB3+ neurons, and under specific conditions, their ability to differentiate into early glial progenitor cells including GFAP+ astrocytes and O4+ oligodendrocytes. Collectively, these results show the capabilities of our protocol to generate an expandable NRSC population suitable for in vitro disease modeling and drug screening, while also suggesting a viable strategy for scalable NRSC production for clinical application.

neuroscience↗

bioRxiv2025

Reshaping the progranulin/sortilin interaction for targeted degradation of extracellular proteins

Targeted protein degradation (TPD) using PROteolysis TArgeting Chimeras (PROTACs) is a rapidly emerging therapeutic strategy for difficult-to-drug cytosolic proteins. PROTACs are heterobifunctional small molecules that bridge the target with an E3 ubiquitin ligase, destining it for degradation by the proteasome. They have the potential to be orally available and to act catalytically, switching the pharmacology from occupancy-driven to event-driven (1-3). Here we present a strategy for targeted degradation of extracellular proteins by reshaping the interaction between the broadly expressed lysosome sorting receptor sortilin and its ligand progranulin for engineering SORtilin-based lysosome TArgeting Chimeras (SORTACs). SORTACs induce ternary complex formation with the target and sortilin, followed by endocytosis and lysosomal degradation. SORTAC activity can be genetically encoded as demonstrated by converting an IgG binding nanobody to an IgG degrading nanobody or by chemical conjugation, enabling single step conversion of therapeutic antibodies from binding their target to driving its degradation. Importantly, using structure-based design, we generated small molecule SORTACs against the inflammatory cytokine TNFa with nanomolar range potency and with physicochemical properties like PROTACs. Our results demonstrate that SORTACs constitute a versatile and highly modular platform for rapid generation of degraders of in theory any extracellular target and with the potential to have wide impact in drug discovery.

biochemistry↗

bioRxiv2021

Green self-immolative polymer: molecular antenna to collect and propagate the signal for zymogen activation

Chemical zymogens of three different types were established herein around protein cysteinome, in each case converting the protein thiol into a disulfide linkage: zero length Z0, polyethylene glycol based ZPEG, and ZLA that features a fast-depolymerizing fuse polymer. The latter was a polydisulfide based on a naturally occurring water-soluble lipoic acid. Three zymogen designs were applied to cysteinyl proteases and a kinase and in each case, enzymatic activity was successfully masked in full and reactivated by small molecule reducing agents. However, only ZLA could be reactivated by protein activators, demonstrating that the macromolecular fuse escapes the steric bulk created by the protein globule, collects activation signal in solution, and relays it to the enzyme active site. This afforded first-in-class chemical zymogens that are activated via protein-protein interactions. For ZLA, we also document a "chain transfer" bioconjugation mechanism and a unique zymogen exchange reaction between two proteins.

biochemistry↗

bioRxiv2021

Human blood lipoprotein predictions from 1H NMR spectra: protocol, model performances and cage of covariance

Lipoprotein subfractions are biomarkers for early diagnosis of cardiovascular diseases. The reference method, ultracentrifugation, for measuring lipoproteins is time consuming and there is a need to develop a rapid method for cohort screenings. Here we present partial least squares regression models developed using 1H-NMR spectra and concentrations of lipoproteins as measured by ultracentrifugation on 316 healthy Danes. Different regions of the 1H-NMR spectra representing signals of the lipoproteins and different lipid species were investigated to develop parsimonious, reliable and best performing prediction models. 65 LP main and subfractions were predictable with an accuracy Q2 of > 0.6 on test set samples. The models were tested on an independent cohort of 290 healthy Swedes with predicted and reference values matching by up to 85-95%. The software was developed to predict lipoproteins in human blood using 1H-NMR spectra and made freely available to be applied for future cohort screenings.

systems biology↗

bioRxiv2020

SmartPeak automates targeted and quantitative metabolomics data processing

SmartPeak is an application that encapsulates advanced algorithms to enable fast, accurate, and automated processing of CE-, GC- and LC-MS(/MS) data, and HPLC data for targeted and semi-targeted metabolomics, lipidomics, and fluxomics experiments. HighlightsO_LINovel algorithms for retention time alignment, calibration curve fitting, and peak integration C_LIO_LIEnables reproducibility by reducing operator bias and ensuring high QC/QA C_LIO_LIAutomated pipeline for high throughput targeted and/or quantitative metabolomics, lipidomics, and fluxomics data processing from multiple analytical instruments C_LIO_LIManually curated data set of LC-MS/MS, GC-MS, and HPLC integrated peaks for further algorithm development and benchmarking C_LI

bioinformatics↗

bioRxiv2019

The metabolic response of Pseudomonas taiwanensis to NADH dehydrogenase deficiency

Obligate aerobic organisms rely on a functional electron transport chain for energy generation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions.\n\nWe here investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the generated single or double knockout strains displayed none or only a marginal phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants including quantitative physiological experiments, transcript analysis, proteomics and enzyme activity assays revealed distinct responses to type II and type I dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes by rerouting of metabolic fluxes.\n\nThis metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, versatile redirecting of carbon fluxes upon genetic interventions can frustrate metabolic engineering efforts.\n\nImportanceWhile Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and consequently competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here, provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. The observed great metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse towards a host with controlled and efficient supply of redox cofactors for product synthesis.

physiology↗