Biology subjects

Ghiran, I.

Publications and source records attributed to Ghiran, I..

7 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

Real-time, automated, standardized, and transparent analysis of microfluidic nanoparticle data with RPSPASS

Extracellular vesicles (EVs) are lipid spheres released from cells. Research utilizing EVs has met several hurdles owing to the small size of the majority of EVs and other nanoparticles (<150 nm) and the lack of detection technologies capable of providing high-throughput single particle measurements at this scale. The use of high-throughput single particle measurements is critical for the assessment of EV heterogeneity and abundance which are features often used to assess the development of isolation protocols or particle characterization. The Coulter principle, known in the field as resistive pulse sensing (RPS), has been used for several decades to size and count cells. More recently, this technology has evolved to accommodate nanoparticle analysis. In the last decade a platform utilizing microfluidic resistive pulse sensing (MRPS) has been demonstrated for nanoparticles, offering ergonomic characterization of nanoparticles along with utilizing open format data. To date, assessment of MRPS accuracy and reporting standards have not been assessed. With the aim of increasing data accuracy, ergonomics, and reporting transparency, we developed a microfluidic resistive pulse sensing post-acquisition analysis software (RPSPASS) application for automated cohort calibration, population gating, statistical output, QC plot generation, alternative data file outputs, and standardized reporting templates.

bioengineering↗

bioRxiv2024

Cannabinoids shift the basal ganglia miRNA m6A methylation profile towards an anti-inflammatory phenotype in SIV-infected Rhesus macaques

BackgroundEpitranscriptomic modifications modulate diverse biological processes like regulation of gene expression, abundance, location and function. N6-methyladenosine (m6A) methylation has been shown to regulate various diseases, including cancer and inflammation. While there is evidence that m6A modification is functionally relevant in neural development and differentiation, the role of m6A modification in HIV neuropathogenesis is unknown. MethodsHere, we used anti-N6-methyladenosine (m6A) antibody immunoprecipitation and microarray profiling to identify m6A modifications in miRNAs in basal ganglia (BG) of Rhesus macaques (RMs) that were uninfected (VEH) and SIV-infected on combination anti-retroviral therapy (cART) and either VEH-treated (VEH/SIV/cART), or THC:CBD-treated (THC:CBD/SIV/cART). Ingenuity pathway analysis was conducted to understand the biological implications of miRNA m6A methylation in HIV neuropathogenesis. Finally, to understand the functional significance of m6A modifications in miRNAs, we overexpressed FAM-labeled wild-type or m6A-modified miR-194-5p in SCC-25 cells and determined its impact on the expression of its target, STAT1, an interferon-stimulated transcription factor known to drive persistent neuroinflammation in several neurodegenerative diseases. ResultsHIV/SIV infection promoted an overall hypomethylated miRNA m6A profile. While the overall hypomethylated m6A profile was not significantly impacted by THC:CBD, specific miRNAs predicted to target proinflammatory genes showed markedly reduced m6A methylation levels compared to the VEH-treated RMs. Additionally, specific BG tissue miRNAs bearing m6A epi-transcriptomic marks were transferred and detected in BG-derived extracellular vesicles. Mechanistically, the DRACH motif in the seed region of miR-194-5p was significantly m6A hypomethylated in THC:CBD/SIV/cART RMs. In SCC-25 cells, unlike wild-type miR-194-5p, transfected m6A-modified miR-194-5p mimics failed to downregulate STAT1 protein expression. Further, compared to VEH/SIV/cART RMs, THC:CBD administration significantly reduced m6A methylation of 44 miRNAs directly involved in regulating CNS network genes. ConclusionsThese results underscore the need for investigating the qualitative, and posttranscriptional modifications in RNA along with the more traditional, quantitative alterations in pathological conditions or in response to disease modifying treatments. Our findings indicate that m6A epitranscriptomic marks in the seed nucleotide region can impair miRNA function and that cannabinoids may preserve it by reducing m6A methylation levels. Finally, these findings provide a novel mechanistic (miRNA m6A hypomethylation) explanation underlying the anti-neuroinflammatory effects of phytocannabinoids in HIV/SIV infection.

neuroscience↗

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↗

bioRxiv2022

Possible Mechanism of Porosome-Mediated Exosome Release

Supramolecular cup-shaped lipoprotein structures called porosomes embedded in the cell plasma membrane mediate fractional release of intra-vesicular contents from cells during secretion. The presence of porosomes, have been documented in many cell types including neurons, acinar cells of the exocrine pancreas, growth hormone secreting cells of the pituitary, and mouse insulin-secreting beta cell insulinomas called Min6. Electron micrographs of the nerve terminal in rat brain and of Min6 cells, suggest that besides the docking, fusion and content release of neurotransmitter containing synaptic vesicles and insulin containing granules, multi-vesicular bodies and exosome-like vesicles are released at the porosome complex. To test this hypothesis, the porosome-associated calcium transporting ATPase 1 [SPCA1] encoded by the ATP2C1 gene, was knocked-out in Min6 cells using CRISPR, to study glucose-stimulated insulin and exosome release. In agreement with electron micrographs, results from the study demonstrate a loss of both glucose-stimulated insulin and exosome release in the ATP2C1 knockout Min6 cells. These results further confirm the role of the porosome complex in insulin secretion and establishes a new paradigm in porosome-mediated exosome release in beta cells of the endocrine pancreas.

cell 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↗

bioRxiv2017

Accuracy, Reproducibility And Bias Of Next Generation Sequencing For Quantitative Small RNA Profiling: A Multiple Protocol Study Across Multiple Laboratories

Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols.

genomics↗