Mansy, S. S. | bioRxiv Science

bioRxiv2025

Protometabolically generated NADH mediates material properties of aqueous dispersions to coacervate microdroplets

Macromolecular assembly between biomolecules dictates the material state of aqueous dispersions like the cytoplasm. The formation of protein precipitates, fibres, or liquid droplets have been associated with the regulation of biochemical function and disease. However, the effect of small molecules and metabolites on tuning phase behaviour remains underexplored. Here we use the protometabolic reduction of NAD+ to NADH by pyruvate to study the effect of NADH production on the phase properties of polyarginine in bicarbonate buffer. Our results show that reduction of NAD+ in the presence of polyarginine can tune the material properties of the dispersion between precipitates, homogeneous solution and liquid droplets depending on the buffer concentration. In-situ droplet formation results in 2-3 times higher reaction rate and NADH yield, compared to homogeneous solution. Our results demonstrate how small molecules can significantly impact material properties in aqueous dispersions and the positive effect of heterogeneous dispersions on primitive metabolic reactions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/642830v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@19261c8org.highwire.dtl.DTLVardef@151aae5org.highwire.dtl.DTLVardef@aaa110org.highwire.dtl.DTLVardef@342e16_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Integrating extracellular vesicle and circulating cell-free DNA analysis on a single plasma aliquot from breast cancer patients improves the detection of HER2 positivity

BackgroundMulti-analyte liquid biopsies represents an emerging opportunity for non-invasive cancer assessment. We developed ONCE (ONe Aliquot for Circulating Elements), a novel multi-analytes liquid biopsy approach for the isolation of extracellular vesicles (EVs) and cell-free DNA (cfDNA) from a single aliquot of blood. MethodsWe assessed ONCE performance to classify HER2-positive early-stage breast cancer (BrCa) patients by combining RNA and DNA signals on n=64 healthy donors (HD) and non-metastatic BrCa patients. Specifically, we investigated EVs-derived RNA (EV-RNA) and cfDNA by next-generation sequencing (NGS) and by digital droplet PCR (ddPCR). Additionally, we utilized imaging flow cytometry to evaluate EVs as potential carriers of the HER2 protein. ResultsWestern blot analysis and immunocapture assay revealed that EVs-enriched proteins were detected at similar levels among the HER2+ and HER2- subtypes. Sequencing of cfDNA and EV-RNA from HER2- and HER2+ patients demonstrated concordance with in situ molecular analyses of matched tissues. Combined analysis of the two circulating analytes by ddPCR showed increased sensitivity in ERBB2/HER2 detection compared to single nucleic acid components. Multi-analyte liquid biopsy prediction performance was comparable to tissue-based sequencing results from TCGA. Also, we observed HER2 protein on the surface of EVs isolated from the HER2+ BrCa plasma, thus corroborating the potential relevance of studying EVs as companion analyte to cfDNA. ConclusionsThis data confirms the relevance of combining cfDNA and EV-RNA analytes for cancer assessment and supports the ONCE approach as a valuable tool for multi-analytes liquid biopsies clinical implementation.

cancer biology↗