Biology subjects

Tigges, J.

Publications and source records attributed to Tigges, J..

5 recordsLinked to original sources

bioRxiv2026

Advancing Nano-Flow Cytometry: High-Precision Sorting and Analysis of Extracellular Vesicles

Extracellular Vesicles (EVs) are small membrane-bound particles secreted by cells that play key roles in intercellular communication, gene regulation and modulation of cell function. They are involved in both physiological and pathological processes and, due to their ability to transport biomolecules across biological barriers, have emerged as promising tools for use as drug delivery vehicles and biomarkers with diagnostic and prognostic applications. Various methodologies are currently employed for the isolation, characterization, and analysis of EVs, including Ultracentrifugation (UC), Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and Flow Cytometry. Flow Cytometry has emerged as a powerful technique capable of providing a multiparametric analysis of individual EVs. Recent advancements have led to the development of cytometers with higher sensitivity and increased limit of detection, enabling the detection and sorting of nanoscale particles--a technique known as Nano-Flow Cytometry. In this study, we show the optimization of small particle sorting, termed nanoFACS, via the CytoFLEX SRT. This method enables sorting based on size or fluorescence, enhancing reproducibility and broadening the potential for application in biological and clinical assays. Furthermore, we demonstrate the utility of nanoFACS in isolating nanoparticles from complex biofluids and in detecting miRNA using molecular beacons (MBs) highlighting its potential in both basic research and translational applications.

bioengineering↗

bioRxiv2026

Evaluating the Utility of a Nanoscale Flow Cytometer for Detection of Surface Proteins on HIV and Extracellular Vesicles

BackgroundFlow virometry (FV) - the application of flow cytometry to viruses - has historically been hindered by the inability of cytometers to detect particles below [~]300 nm in size. However, advances in optics and fluidics have enabled cytometers primarily designed for cells to detect viruses and extracellular vesicles (EVs) through light scatter alone. In 2024, the CytoFLEX nano was released, marketed for the detection of particles as small as 40 nm; however, its performance has yet to be compared to a conventional instrument for FV. MethodsFV was utilized to evaluate performance of the CytoFLEX nano and a conventional flow cytometer (CytoFLEX S). Instrument scatter sensitivity was assessed using NIST beads (40-400 nm), and virus stocks [human immunodeficiency virus (HIV), human coronaviruses (HCoV)-229E and HCoV-OC43]. For fluorescence analysis, HIV virions were stained with PE- and BV421-conjugated antibodies targeting virion incorporated proteins (CD38, CD44), individually and in combination. Finally, HIV stocks were labeled with antibodies against the envelope (Env) glycoprotein and tetraspanins (CD9, CD81) to assess EVs within virus preparations. ResultsCompared to the CytoFLEX S, the CytoFLEX nano exhibited substantially greater scatter sensitivity, reflected by up to 50-fold higher signal-to-noise ratio across NIST-traceable beads and virus samples. This enabled clearer resolution of smaller populations, including bead populations < 70 nm that were undetectable on the CytoFLEX S, as well as improved resolution across all viruses. While both instruments reliably detected stained proteins on HIV virions, the CytoFLEX nano revealed a distinct population of tetraspanin-positive EVs within HIV stocks that was undetected on the CytoFLEX S. Using GFP-tagged HIV, we identified Env+ particles lacking GFP, indicating the presence of Env on EVs. ConclusionsThe CytoFLEX nano exhibited markedly improved scatter sensitivity compared to the CytoFLEX S, improving detection of viruses and enabling detection of EV populations that were undetectable on the conventional instrument. While both platforms performed similarly for surface protein labeling, additional consideration of spectral overlap was needed with the CytoFLEX nano in multicolor experiments. These findings highlight that the complementary strengths of each platform can be utilized to more comprehensively characterize virus and EV populations, providing new opportunities to investigate nanoparticle heterogeneity.

microbiology↗

bioRxiv2025

Preconditioning of human iPSCs with doxorubicin causes genome-wide transcriptional reprogramming in iPSC-derived cardiomyocytes linked to mitochondrial dysfunction and impaired cardiac regeneration

BackgroundThe anthracycline doxorubicin (Dox) is a widely used genotoxic chemotherapeutic drug with known dose-limiting cardiotoxic effects. How Dox-induced damage either to cardiomyocytes or to cardiac stem cells, which may compromise cardiac regeneration, contributes to cardiotoxicity remains poorly understood. MethodsHere we used a human induced pluripotent stem cell (iPSC)-based model system to determine the sensitivity of stem cells (iPSCs) and iPSC-derived derived cardiomyocytes (iCMs) applying different treatment regimens of Dox. Next to a broad range of methods to determine cellular and mitochondrial functions we performed an in-depth whole genome transcriptome profiling in iPSCs as well as iCMs. ResultsAs compared to their differentiated counterparts, iPSCs are highly sensitive against even short pulse-treatments with low Dox concentrations. Using such rather mild treatment conditions, we observed major mitochondrial impairments as demonstrated by increased mitochondrial fragmentation, persistent loss of mitochondrial membrane potential, and reduced ATP levels, while neither a markedly increased nuclear DNA damage response nor apoptosis were detected. Albeit mitochondrial dysfunction was not accompanied by changes in mitochondrial ultrastructure or altered OXPHOS complex assembly, mitochondrial genome (mtDNA) organization was altered. This points to a possible role of mtDNA remodelling for contributing to the high susceptibility of iPSCs to Dox. Whole genome transcriptome profiling revealed major differences in the transcriptional response to Dox treatment between iPSCs and iCMs. We could show that a moderate and transient exposure of iPSCs to Dox is sufficient to cause major transcriptional changes as for example reflected by the downregulation of numerous pivotal genes regulating cellular homeostasis and energy metabolism in iPSCs. Furthermore, pulse-treatment with Dox at the iPSC stage, termed preconditioning here, shifts the global transcriptional landscape of iCMs towards the expression of genes associated with impaired cardiac muscle regeneration, disrupted energy metabolism, altered muscle contraction, and increased fibrosis. ConclusionsOur findings support the hypothesis that Dox-induced mitochondrial dysfunction and transcriptional preconditioning in stem cells results in an impaired regenerative capacity after differentiation. This highlights a potential critical role of stem cells in mediating Dox-induced cardiotoxicity.

cell biology↗

bioRxiv2023

Identification of post-transcriptional modifications in nucleic acid sequences using propose-designed molecular beacons

Post-transcriptional RNA modifications (PTxMs) present in small RNA species, specifically circulating extracellular RNAs, were recently identified as clinically relevant readouts, often more indicative of disease severity than the classical "up and down" changes in their copy number alone. While identification of PTxMs requires multiple and complex sample preparation steps, microgram-range amounts of RNA, followed by expensive and protracted bioinformatics analyses, the clinically relevant information is usually a yes/no for a particular genetic variant(s), and an up/down answer for relevant biomarkers. We have previously shown that molecular beacons (MBs) can identify specific nucleic acid sequences with picomolar sensitivity and single nucleotide specificity by exploiting the target-dependent change in their electrophoretic mobility profile. We now present a method for direct identification of miRNAs and isomiRs in cells and extracellular vesicles using gel electrophoresis, without the need for RNA isolation and purification. The detection is based on discreet changes in the hydrodynamic surface profile, the overall size, charge and charge distribution of the MB-target hybrid. Furthermore, using an RNA tertiary structure prediction algorithm (iFoldRNA) and a custom molecular dynamics simulation (DMD), we designed modified MBs specific for m6A-modified nucleotides in target RNA sequences. The sample preparation method coupled to the software package affords the design of specific MBs and sensitive, multiplex-type detection of targets in a wide variety of biofluids and cells, in a simple mix and read approach.

molecular biology↗

bioRxiv2019

Red blood cell-derived extracellular vesicles mediate intercellular communication in ischemic heart failure

Extracellular vesicles (EV) mediate intercellular signaling by transferring their cargo to recipient cells. Red blood cell (RBC)-derived EVs constitute a significant proportion of circulating EVs and have been implicated in regulating immune responses. Here, we describe a transgenic mouse model for fluorescent-based mapping of RBC-EV target cells based on the functional transfer of EV-contained Cre-recombinase to target cells. In a murine model of ischemic heart failure, we detect an increase in RBC-EV-targeted cardiomyocytes in the hearts and microglial cells in the brains. Cells targeted by RBC-EVs present an enrichment of genes implicated in cell proliferation and metabolism pathways compared to non-recombined (non-targeted) cells. Cardiomyocytes targeted by RBC-EVs are more likely to demonstrate cellular markers of DNA synthesis and proliferation, suggesting functional significance of EV-mediated signaling. In conclusion, we leverage our mouse model for mapping of RBC-EV targets in murine ischemic heart failure to demonstrate quantitative and qualitative changes in RBC-EV recipients.

cell biology↗