Biology subjects

Offen, D.

Publications and source records attributed to Offen, D..

6 recordsLinked to original sources

bioRxiv2026

CytoLight: A Rapid and Versatile Fluorescent-Based Labeling Method for Extracellular Vesicle Characterization and Tracking

Efficient, aggregation-free extracellular vesicles (EVs) labeling is essential for studying their dynamics in-vitro and in-vivo. However, traditional dyes introduce limitations including aggregation, membrane intercalation, fluorescence transfer and inconsistent performance across EV sources thus distorting quantification, altering surface properties and confounding uptake and biodistribution analyses. Here, we systematically evaluated CytoLight, a luminal dye traditionally used for live-cell imaging, as an alternative for EV quantification, characterization, uptake analysis and in-vivo tracking, benchmarking it against PKH26, CFSE and ExoBrite across multiple platforms. CytoLight generated stable, intravesicular fluorescence without aggregation or membrane alteration, eliminating artifacts characteristic of conventional dyes. Using fluorescence-NTA and single-EV flow cytometry, CytoLight showed more consistent labeling across EV types than CFSE or ExoBrite, while avoiding PKH-related micelle-driven artifacts and exhibited compatibility with CD81 dual-detection. In uptake assays, CytoLight produced EV-specific endocytosis-dependent internalization signals exceeding labeled-BPS/protein controls. In-vivo, CytoLight-labeled EVs enabled fluorescent biodistribution mapping showing conventional EV tropism patterns distinguishable from labeled-PBS controls. These findings establish CytoLight as an effective, aggregation-free EV-labeling strategy. Its stability, specificity, compatibility with single-EV platforms and reliable performance in both cellular uptake and biodistribution studies position CytoLight as a practical, scalable alternative to current dyes, providing a stronger foundation for standardized and reproducible EV research.

cell biology↗

bioRxiv2024

Exosomes can modulate the early hyperexcitability in cortical neurons with ASD-associated Shank3 mutation.

Extracellular vesicles (EVs) are lipid membrane-bound structures that mediate intercellular communication by transferring diverse cargoes, including RNA and proteins. Shank3, a synaptic scaffolding protein critical for synapse structure and function, is implicated in autism spectrum disorder (ASD) and Phelan-McDermid Syndrome (PMS). Early hyperexcitability in cortical neurons is a recognized endophenotype in ASD. Here, we investigated EV-mediated effects in the context of Shank3 deficiency using human iPSC-derived cortical neurons and Shank3B-/- mice. Switching EVs between Shank3 mutant and control neurons revealed that Shank3 mutant-derived EVs transferred the hyperexcitability and accelerated maturation phenotypes to control neurons. This was driven by enriched synaptic proteins (e.g., ACTB, CFL1, AGRN, CLSTN1) in Shank3 mutant-derived EVs as confirmed by proteomic analysis. Conversely, control EVs failed to rescue mutant phenotypes consistent with their lower enrichment for synaptic proteins and related pathways. Further, EVs from mesenchymal stem cells (MSCs) and healthy donor iPSCs, containing synaptic modulators such as complement proteins (C1R, C1S), plasticity-related proteins (MDK, IGFBP3), and homeostatic regulators (FGF2, SFRP1), rescued the hyperexcitability and normalized the maturation in Shank3 mutant neurons. Moreover, intranasal administration of iPSC-derived EVs in Shank3B-/- mice significantly ameliorated ASD-like behavioral deficits, underscoring their therapeutic potential. Together, these findings reveal a novel EV-mediated mechanism for modulating dysregulated excitability and synaptic maturation, addressing a critical unmet need in ASD and related neurodevelopmental disorders treatment.

neuroscience↗

bioRxiv2024

The DJ-1-derived peptide, ND-13, confers cardioprotection by inhibiting mitochondrial fission and preserving mitochondrial bioenergetics via the RhoA-ROCK1-Drp1 pathway

BackgroundAcute myocardial infarction (AMI) and post-infarct heart failure (HF) are among the leading causes of death and disability worldwide. As such, new treatments are urgently needed to protect the heart against the detrimental effects of acute ischemia/reperfusion injury (IRI), in order to prevent the onset of HF and improve clinical outcomes following AMI. Given that mitochondrial dysfunction is a key determinant of IRI-induced cardiomyocyte death in AMI, we investigated ND-13, a 13-amino acid peptide derived from the pro-survival protein DJ-1, as a novel mitoprotective strategy for limiting myocardial infarct size (IS) following AMI. Methods and ResultsIn isolated adult Dendra-2 mice cardiomyocytes subjected to simulated IRI, treatment with ND-13 peptide reduced cell death by 42%, decreased phosphorylation of Drp1 at Ser616 and inhibited mitochondrial fission, improved mitochondrial respiratory function, decreased oxidative stress, and preserved ATP levels. Treatment of cardiomyocytes with ND-13 decreased levels of RhoA and reduced activity of downstream ROCK1, the latter of which is known to phosphorylate Drp1 at Ser616. In ex vivo Langendorff-perfused hearts subjected to IRI, treatment with ND-13 at reperfusion reduced IS by 43%, attenuated oxidative stress and preserved post-infarct cardiac contractile function. Finally, in vivo administration of ND-13 peptide at reperfusion in mice subjected to acute myocardial IRI reduced IS by 35% at 72-hours of reperfusion, and restored mitochondrial bioenergetics in cardiomyocytes isolated from the area-at-risk following 2-hours reperfusion as evidenced by preservation of key metabolites involved in glucose oxidation and fatty acid oxidation. The cardioprotective phenotype translated to improved cardiac function and less adverse left ventricular remodelling at 28 days post-infarction. ConclusionsWe show for the first time the mitoprotective effects of the DJ-1-derived peptide, ND-13, administered at the onset of reperfusion following AMI. We found that ND-13 protects mitochondria by inhibiting IRI-induced mitochondrial fission and preserved mitochondrial function following IRI via the RhoA-ROCK-Drp1 pathway. These findings highlight ND-13 as a novel mitoprotective agent which has the therapeutic potential for limiting IS and preventing HF in patients with AMI. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIAdministration of the DJ-1-derived peptide, ND-13, at the onset of reperfusion reduced myocardial infarct size and preserved cardiac contractile function following acute myocardial ischemia/reperfusion injury (IRI). C_LIO_LIThe cardioprotective effect of ND-13 treatment was mediated by inhibition of IRI-induced mitochondrial fission and improvement in mitochondrial bioenergetics via the RhoA-ROCK-Drp1 pathway. C_LI What are the Clinical Implications?O_LITreatment with ND-13 peptide at the onset of reperfusion has the therapeutic potential to reduce myocardial infarct size and prevent the onset of heart failure in ST-segment elevation myocardial infarction patients undergoing primary percutaneous coronary intervention. C_LI

physiology↗

bioRxiv2023

Computationally Engineered CRISPR-SpyCas9 High-Fidelity Variants with Improved Specificity and Reduced Non-specific DNA Damage

The CRISPR-Cas system holds great promise in the treatment of diseases caused by genetic variations. As wildtype SpyCas9 is known to generate many off-target effects, its use in the clinic remains controversial due to safety concerns. Several high-fidelity Cas9 variants with greater specificity have been developed using rational design and directed evolution. However, the enhancement of specificity by these methods is limited by factors like selection pressure and library diversity. Thus, in-silico protein engineering may provide a more efficient route for enhancing specificity, although computationally testing these proteins remains challenging. We recently demonstrated the advantage of normal mode analysis to simulate and predict the enzymatic function of SpyCas9 in the presence of mismatches. Here, we report several mathematical models describing the entropy and functionality relationships in the CRISPR-Cas9 system. We demonstrate the invariant characteristics of these models across different conformational structures. Based on these invariant models, we developed ComPE, a novel computational protein engineering method to modify the protein and measure the vibrational entropy of wildtype or variant SpyCas9 in complex with its sgRNA and target DNA. Using this platform, we discovered novel high-fidelity Cas9 variants with improved specificity. We functionally validated the improved specificity of four variants, and the intact on-target activity in one of them. Lastly, we demonstrate their reduced off-target editing and non-specific gRNA-independent DNA damage, highlighting their advantages for clinical applications. The described method could be applied to a wide range of proteins, from CRISPR-Cas orthologs to distinct proteins in any field where engineered proteins can improve biological processes.

bioengineering↗

bioRxiv2021

Induced neural differentiation of human mesenchymal stem cells affects lipid metabolism pathways

Neuronal membranes contain exceptionally high concentrations of long-chain polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA) and arachidonic acid (ARA), which are essential for neuronal development and function. Adult bone-marrow-derived mesenchymal stem cells (MSC) can be induced to possess some neuronal characteristics. Here we examined the effects of neuronal induction on the PUFA metabolism specific pathways. Differentiated cells contained ~30% less ARA than MSC. The expression of specific ARA metabolizing enzymes was upregulated, notably that of prostaglandin E2 synthase which increased more than 15-fold, concomitantly with a 3-fold increase in the concentration of PGE2 in the medium. Moreover, induced differentiation was associated with enhanced incorporation of exogenous DHA, upregulation of acyl-CoA synthases, fatty acid binding proteins, choline kinase (CK) and phosphatidylserine synthases as well as increased total cellular phospholipids (PL). These findings suggest that active ARA metabolites may be important in the differentiation process and that neuronal induction prepares the resulting cells for increased DHA incorporation through the action of specific enzymes.

neuroscience↗

bioRxiv2020

Prediction of synonymous corrections by the BE-FF computational tool expands the targeting scope of base editing

Base editing is a genome-editing approach that employs the CRISPR/Cas system to precisely install point mutations within the genome. A cytidine or adenosine deaminase enzyme is fused to a deactivated Cas and converts C to T or A to G, respectively. The diversified repertoire of base editors, varied in their Cas and deaminase proteins, provides a wide range of functionality. However, existing base-editors can only induce transition substitutions in a specified region determined by the base editor, thus, they are incompatible for many point mutations. Here, we present BE-FF (Base Editors Functional Finder), a novel computational tool that identifies suitable base editors to correct the translated sequence erred by a given single nucleotide variation. Even if a perfect correction of the single nucleotide variation is not possible, BE-FF detects synonymous corrections to produce the reference protein. To assess the potential of BE-FF, we analysed a database of human pathogenic point mutations and found suitable base editors for 60.9% of the transition mutations. Importantly, 19.4% of them were made possible only by synonymous corrections. Moreover, we detected 298 cases in which pathogenic mutations caused by transversions were potentially repairable by base editing via synonymous corrections, although it had been thought impractical. The BE-FF tool and the database are available at https://www.danioffenlab.com/be-ff. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/890244v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@36330borg.highwire.dtl.DTLVardef@514ca0org.highwire.dtl.DTLVardef@17dbc56org.highwire.dtl.DTLVardef@1d7d8d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗