Biology subjects

Nazarenko, I.

Publications and source records attributed to Nazarenko, I..

6 recordsLinked to original sources

bioRxiv2026

Reference-free single-vesicle profiling of small extracellular vesicles from liquid biopsies with the PICO assay

Small extracellular vesicles (sEV) are membrane-enclosed nanoparticles found in body fluids that carry molecular cargo from their cells of origin. Their stability and disease-associated molecular signatures make them promising targets for the development of non-, or minimally invasive liquid biopsies, yet scalable approaches enabling single-vesicle quantification of sEV while resolving their heterogeneity remain limited. Here, we present PICO (Protein Interaction Coupling), a reference-free quantitative assay adapted for sensitive multiplex profiling of individual intact vesicles. PICO detects vesicle markers by requiring colocalization of two or more copies of the same protein or of distinct proteins (e.g., CD9 or CD9/CD63) on individual vesicles, using DNA-barcoded antibodies and digital PCR (dPCR) for quantitative readout. We demonstrate that this unique architecture of the assay provides high specificity by distinguishing EV-bound proteins from soluble counterparts, and can be adapted to target either surface-exposed or intravesicular biomarkers. PICO requires minimal sample input (1 {micro}l) and no specialized instrumentation beyond standard digital PCR. In a head-to-head comparison with nano-flow cytometry, PICO achieved a comparable limit of detection for sEV subpopulations. Profiling sEV isolates from blood for canonical markers (CD9, CD63 and CD81) and HER2 demonstrates precise, high-resolution quantification of sEV subpopulations in complex clinical samples and supports integration of scalable single-EV analysis into research and diagnostic workflows.

molecular biology↗

bioRxiv2026

PHoNUPS: Open-Source Software for Standardized Analysis and Visualization of Multi-Instrument Extracellular Vesicle Measurements

Accurate and transparent characterization of extracellular vesicle (EV) preparations is essential to ensure reproducibility, comparability, and adherence to MISEV reporting standards. However, data outputs from commonly used instruments for assessing EV size, concentration, and surface charge (zeta potential) vary widely in format and structure, complicating standardized analysis and integration across platforms. We present PHoNUPS (Plotting the Histogram of Non-Uniform Particles Sizes), free and open-source software (FOSS) developed in R, that enables unified processing, analysis, and visualization of EV characterization data. PHoNUPS computes statistics and generates standardized histograms and contour plots (for size against zeta potential) suitable for transparent reporting and cross-study comparison. The software produces high-quality, publication-ready figures. Third-party graphical editing tools allow users to refine and annotate visualizations for presentation or manuscript preparation. PHoNUPS supports multiple measurement file formats, thereby facilitating dataset integration from different instruments. PHoNUPS was developed with extensibility at its core, providing a basis for user-driven growth. We invite the EV community--researchers, analysts, and tool developers--to use PHoNUPS, share feedback on their experience and needs, and contribute to the platform by integrating additional input data formats, analytical routines, and visualization functionalities. Graphical abstractThe free software PHoNUPS processes the outputs of several different EV characterization instruments and it is extensible with further ones. It computes statistics of particle size and zeta potential distributions and it plots the corresponding histograms or contour plots. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/702479v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@b2b3a1org.highwire.dtl.DTLVardef@2f2907org.highwire.dtl.DTLVardef@2ec521org.highwire.dtl.DTLVardef@903624_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

VDisk: Microfluidic Cartridge for Multimodal High-Yield, High-Purity Isolation of Extracellular Vesicles from up to 1 mL of Plasma

Blood-derived extracellular vesicles (EVs) hold strong diagnostic potential, yet conventional isolation methods such as ultracentrifugation and size-exclusion chromatography (SEC) involve manual handling steps and show substantial run-to-run variability, hindering clinical translation. This study presents the Vesicle Disk (VDisk), a centrifugal microfluidics-based EV purification platform that combines cation-exchange chromatography, sequential filtration, and multimodal chromatography for automated, label-free EV isolation from up to 1 mL of plasma. VDisk configurations differing in filter membrane and plasma volume (0.1-1.0 mL) are benchmarked against SEC for yield, purity, reproducibility and robustness. VDisk matches SEC in EV yield and exceeds it in EV/contaminant ratios at reduced input volumes, while achieving greater reproducibility (intra-donor CV < 5% for CD9 and CD81, versus up to 16% for SEC) and maintaining consistent yield and contaminant removal under both fasting and postprandial sampling conditions. The platform is application-tunable: processing up to 1 mL of plasma maximizes EV yield and concentration, whereas processing 0.5 mL achieves approximately 2-3-fold higher EV/total protein ratios than SEC. These findings establish the VDisk as an automated, robust and adaptable alternative to existing EV isolation methods for both research and clinical translation.

bioengineering↗

bioRxiv2025

Chlamydial membrane vesicles deliver the chlamydial porin OmpA to mitochondria to inhibit apoptosis

Chlamydiae are obligate intracellular bacteria that inhibit mitochondrial apoptosis to maintain integrity of the host cell. We have previously reported that a chlamydial outer membrane {beta}-barrel protein, the porin OmpA, can during ectopic expression inhibit mitochondrial apoptosis through direct interaction with the BCL-2-family effectors BAX and BAK. We here show that OmpA from Chlamydia trachomatis (Ctr) uses membrane vesicles for its delivery to the outer mitochondrial membrane during Ctr infection. Using a number of imaging and fractionation techniques, we show that OmpA during infection reaches mitochondria and is inserted into mitochondrial membranes. Purified membrane vesicles (MV) from Ctr-infected cells contained OmpA. When added to uninfected cells, MV fused with mitochondrial membranes, causing the interaction of OmpA with BAK and the cytosolic retro-translocation of BAX. MV addition to uninfected cells protected the cells against apoptosis. We propose a structural model of this BAK inhibition by OmpA that reenacts the inhibition of BAK by the mitochondrial porin VDAC2. The results provide evidence that the porin from Chlamydia, as well as the structurally similar porin from the related Simkania, specifically exploits its relationship to mitochondrial porins to protect the infected cell against apoptosis and to enable intracellular growth of the bacteria in human cells.

cell biology↗

bioRxiv2024

Advanced median-based genetic similarity analysis in Kazakh Tazy dogs: A novel approach for breed conformity assessment

The breed conformity evaluation is crucial for the preservation of the traits that characterise each dog breed. The use of genetic markers for this purpose provides a precision and objectivity that can surpass the reliability of phenotypic evaluations. In this study, we present a new simple algorithm for assessing breed conformity. The algorithm creates a similarity matrix based on genotypic data and then uses the median to calculate the percentage of genetic similarity that an individual has in relation to the genetic diversity of the breed. To validate the proposed algorithm, we applied it to the genotypic data of 18 microsatellites and 43,691 single nucleotide polymorphisms (SNPs) of the Kazakh Tazy dog, a breed of great cultural and historical importance to Kazakhstan that is now threatened with extinction due to crossbreeding. The algorithm showed a moderate correlation between the microsatellite and SNP genotyping methods, reflecting the different aspects of genetic similarity. In particular, the SNP-based evaluations agreed better with the expert judgements, highlighting their potential for accurate analysis of breed conformity. The proposed algorithm provides easily interpretable results, is flexible, adapts to different genetic markers and may provide an evaluation mechanism for breed conformity in situations where there is no reference population, incomplete pedigrees, unidentified meta-founders and high genetic diversity in the population. Author summaryOur research was initiated by the urgent concern for the possible extinction of the Kazakh Tazy dog, a breed with deep historical roots and cultural significance in Kazakhstan. Information at the DNA level may lead to faster genetic improvement of the breed than relying only on phenotypic data and pedigrees. Genotypic data can be processed to provide valuable insights into genetic diversity, relatedness and ancestry. However, existing methods do not provide a measure of percentage similarity that can be used to assess how closely a particular individual matches the typical genetic composition of the breed. These challenges have led us to propose an approach that overcomes the limitations of existing methods and allows genetic similarity to be assessed based on genotypes. It is based on a median-based approach to analyze genetic data and has been applied to microsatellite and SNP markers but can also be adapted to other genetic markers. This method provides results even in the absence of a defined reference population, complete pedigrees or identified meta-pedigrees and during high genetic diversity within a breed. It can provide breeders and researchers with a tool to maintain the genetic purity of unique breeds.

genomics↗

bioRxiv2020

Airway Basal Cells show a dedifferentiated KRT17high Phenotype and promote Fibrosis in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with limited treatment options. In this study we focus on the profibrotic properties of airway basal cells (ABC) obtained from patients with IPF (IPF-ABC). Single cell RNA sequencing of bronchial brushes revealed extensive reprogramming of IPF-ABC towards a KRT17high PTENlow dedifferentiated cell type. In the 3D organoid model, compared to ABC obtained from healthy volunteers, IPF-ABC give rise to more bronchospheres, de novo bronchial structures resembling lung developmental processes, induce fibroblast proliferation and extracellular matrix deposition in co-culture. Intratracheal application of IPF-ABC into minimally injured lungs of Rag2-/- or NRG mice causes severe fibrosis, remodeling of the alveolar compartment, and formation of honeycomb cyst-like structures. Connectivity MAP analysis of scRNA seq of bronchial brushings suggested that gene expression changes in IPF-ABC can be reversed by SRC inhibition. After demonstrating enhanced SRC expression and activity in these cells, and in IPF lungs, we tested the effects of saracatinib, a potent SRC inhibitor previously studied in humans. We demonstrated that saracatinib modified in-vitro and in-vivo the profibrotic changes observed in our 3D culture system and novel mouse xenograft model.

cell biology↗