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PubMed2026

Novel and High-Throughput Method of Isolating Single Fetal Cells Using FACS for NIPT.

OBJECTIVE: To evaluate fluorescence activated cell sorting (FACS) as a method of single-cell isolation of rare circulating fetal cells from maternal blood for use in cell-based non-invasive prenatal testing (cbNIPT). METHOD: Blood samples (30 mL) were collected from 75 'low-risk' pregnant women (gestational age 10-15 weeks). Fetal cells were enriched and stained using magnetic activated cell sorting. Following enrichment, single fetal cells were sorted in individual PCR tubes by FACS. After cell lysis, verification of fetal cell origin was performed using short tandem repeat (STR) analysis with the GlobalFiler PCR Amplification kit. RESULTS: An average of 13.7 cells were sorted using FACS. STR analysis identified 8.2 fetal cells on average, representing 60.2% of the sorted cells. The four-step single-cell isolation procedure facilitated an overall enrichment of approximately 16-million-fold. One sample did not render any fetal cell, corresponding to 1.3% of the samples. CONCLUSION: FACS, which is typically used for segregation of large populations of cells, can be used for single-cell isolation of rare fetal cells in an automated setup. This not only helps in making cell isolation faster and high throughput but also provides fetal cells for a more comprehensive genetic analysis of the fetus.

Humans↗

PubMed2025

FACS-Proteomics strategy toward extracellular vesicles single-phenotype characterization in biological fluids: exploring the role of leukocyte-derived EVs in multiple sclerosis.

BACKGROUND: The isolation and proteomics characterization of extracellular vesicles (EVs) from body fluids is challenging due to their vast heterogeneity. We have recently demonstrated that Fluorescence-activated Cell Sorting (FACS) efficiently isolates the whole EV circulating compartment directly from untouched body fluids enabling a comprehensive EV proteomics analysis. RESULTS: Here, we characterized, for the first time, a single-phenotype EV subset by sorting leukocyte-derived EVs (Leuko EVs) from peripheral blood and tears of healthy volunteers. Using an optimized and patented staining protocol of the whole EV compartment we identified and excluded non-EV particles, debris and damaged EVs. We further isolated, using an anti-CD45 antibody, Leuko EVs (CD45+ EVs), reaching a high level of purity (> 90%). Purified Leuko EVs were characterized using atomic force microscopy, nanoparticle tracking, and shotgun proteomics analysis revealing a similar coded protein cargo in both biological fluids. Subsequently, the same workflow was applied to tears from Relapsing-Remitting Multiple Sclerosis (RRMS) patients, revealing a Leuko EVs protein cargo enrichment that reflects the neuroinflammatory condition characteristics of RRMS. This enrichment was evidenced by the activation of upstream regulators TGFB1 and NFE2L2, which are associated with inflammatory responses. Additionally, the analysis identified markers indicative of endothelial cell proliferation and the development of enhanced vascular networks, with AGNPT2 and VEGF emerging as activated upstream regulators. These findings indicate the complex interplay between inflammation and angiogenesis in RRMS. CONCLUSIONS: In conclusion, our combined FACS-Proteomics strategy offers a promising approach for biomarker discovery, analysing cell-specific EV phenotypes directly from untouched body fluids, advancing the clinical value of tears EVs and improving the understanding of EV-mediated processes in vivo. Data are available via ProteomeXchange with the identifier PXD049036 and in EV-TRACK knowledgebase with ID: EV240150.

Humans↗

PubMed2024

Revealing differential expression patterns of piRNA in FACS blood cells of SARS-CoV-2 infected patients.

Non-coding RNA expression has shown to have cell type-specificity. The regulatory characteristics of these molecules are impacted by changes in their expression levels. We performed next-generation sequencing and examined small RNA-seq data obtained from 6 different types of blood cells separated by fluorescence-activated cell sorting of severe COVID-19 patients and healthy control donors. In addition to examining the behavior of piRNA in the blood cells of severe SARS-CoV-2 infected patients, our aim was to present a distinct piRNA differential expression portrait for each separate cell type. We observed that depending on the type of cell, different sorted control cells (erythrocytes, monocytes, lymphocytes, eosinophils, basophils, and neutrophils) have altering piRNA expression patterns. After analyzing the expression of piRNAs in each set of sorted cells from patients with severe COVID-19, we observed 3 significantly elevated piRNAs - piR-33,123, piR-34,765, piR-43,768 and 9 downregulated piRNAs in erythrocytes. In lymphocytes, all 19 piRNAs were upregulated. Monocytes were presented with a larger amount of statistically significant piRNA, 5 upregulated (piR-49039 piR-31623, piR-37213, piR-44721, piR-44720) and 35 downregulated. It has been previously shown that piR-31,623 has been associated with respiratory syncytial virus infection, and taking in account the major role of piRNA in transposon silencing, we presume that the differential expression patterns which we observed could be a signal of indirect antiviral activity or a specific antiviral cell state. Additionally, in lymphocytes, all 19 piRNAs were upregulated.

Humans↗

PubMed2026

Genome-Wide Characterization of PEBP, FD, and GRF Families in Amomum villosum Lour. and Their Potential Roles in Flowering.

A detailed understanding of the molecular mechanisms governing the flowering time of Amomum villosum Lour., a medicinal plant within the Zingiberaceae family, is currently lacking. In modern plants, the florigen activation complex (FAC), which includes PEBP, FD/bZIP, and GRF proteins, is known to regulate flowering. In this study, we identified 13 PEBP, 5 FD, and 19 GRF genes within the A. villosum genome and conducted phylogenetic, structural and promoter analysis. Notably, cross-species protein-protein interaction predictions and yeast two-hybrid assays uncovered an unexpected interaction pattern: an AREB3-like FD protein (AvFD5) and a GRF protein (AvGRF13) directly interact with specific PEBP members, whereas canonical FD-like proteins (AvFD1 and AvFD4) did not, which contrasts with the classical rice FAC model (Hd3a-14-3-3-OsFD1). These results imply that FAC assembly in A. villosum may involve alternative components or regulatory mechanisms, potentially indicating lineage-specific divergence within monocots. This research represents the first systematic characterization of FAC core gene families in A. villosum and Zingiberaceae, laying the groundwork for understanding flowering time regulation and facilitating future molecular breeding efforts in this economically significant plant.

Amomum villosum↗

PubMed2026

Clinical efficacy and brain mechanism characteristics of guide chi and regulate spirit tuina therapy in the treatment of post-stroke walking dysfunction: A randomized controlled trial based on fNIRS.

BACKGROUND: This study aims to preliminarily evaluate the role of Guide Chi and Regulate Spirit(GCRS) Tuina in enhancing walking function in post-stroke patients with walking dysfunction; secondly, by using functional near-infrared spectroscopy (fNIRS), it investigates the effect of GCRS Tuina on the restoration of brain function in this patient population. METHODS: Participants in the control group received 4-week rehabilitation treatment, while those in the Combined Tuina Group (CTG) additionally received GCRS Tuina therapy for another 4 weeks on this basis. Functional Ambulation Category (FAC), Fugl - Meyer Assessment Scale for Lower Extremity Motor Function (FMA - LE), and Modified Barthel Index (MBI) were evaluated at the baseline and after 4 treatment weeks. A gait and motion analysis system was used to measure step length, stride, walking speed, and step frequency. FNIRS was used to measure the resting-state functional connectivity(FC) strength, as well as the β - value and HbO2 concentration mean during the walking task. RESULTS: A total of 60 participants completed the randomized, and 53 completed the trial and entered the statistical analysis. Compared with the Single Rehabilitation Group(SRG), the CTG group had higher FAC, FMA-LE, and MBI scores after 4 weeks. After the treatment course, the standardized step length, stride, walking speed, and step frequency of the CTG were higher than SRG. At the Region of Interest(ROI) level, the CTG exhibited 13 inter-ROI FC strengths that were higher than SRG, and the differences could survive the FDR correction (PFDR<0.05). Under the walking task, the CTG group had higher β values in 18 channels and higher Oxyhemoglobin(HbO2) concentrations in 19 channels than the SRG (PFDR <0.05). CONCLUSION: GCRS Tuina therapy can significantly improve patients' walking function, enhance lower limb motor ability and daily living ability. It can improve walking efficiency. Tuina can significantly increase the FC and enhance the activation levels and HbO2 concentrations. The stimulation of Tuina may help reconstruct the brain's motor control network, restore impaired motor function, promote the occurrence of neural plasticity, strengthen the neural circuits in the cognitive-motor-sensory cortex to improve walking function. TRIAL REGISTRATION: This study has been registered with the International Traditional Medicine Clinical Trial Registry (ITMCTR2024000654).

Humans↗

PubMed2025

Live-Cell Monitoring and Omics Analysis of Liquid-Solid Transitions of Biomolecular Condensates.

Biomolecular condensates, or so-called membraneless organelles, transition from liquid into more solid-like states over time, contributing to the development of pathological conditions. The present study proposes a simple method using photoactive yellow protein (PYP) and its specific fluorescent covalent ligands to distinguish between the liquid and solid states of protein condensates in live cells. The method, compatible with fluorescence-activated cell sorting (FACS), correlates the stiffness of specific protein condensates with their accessibility to PYP ligands, enabling quantitative multicolor monitoring of condensate solidification. We applied this technique to 12 phase-separating proteins and their mutants, finding that TDP-43, particularly its A315T mutant linked to familial amyotrophic lateral sclerosis, most readily forms solid aggregates. Furthermore, this FACS-compatible strategy enabled the isolation of distinct cell populations based on condensate states, allowing for subsequent proteomic and transcriptomic analyses. Our findings demonstrate that condensate solidification is accompanied by the upregulated expression of extracellular matrix proteins, suggesting a previously unrecognized link between solid aggregate formation and extracellular matrix hardening.

Humans↗

PubMed2026

Defining active and repressive chromatin states in neural crest cells using low-input CUT&RUN.

The transition of neural crest cells (NCCs) from a multipotent state to lineage-restricted derivatives, including melanocytes, is governed by tightly regulated epigenetic mechanisms that orchestrate cell type specific gene expression programs. Histone post-translational modifications (PTMs), in particular, play an important role in modulating chromatin accessibility, enhancer activation, and transcription factor occupancy, thereby facilitating dynamic chromatin and transcriptional reprogramming required during development. However, profiling such chromatin states in rare and transient Neural Crest Cell (NCC) populations in vivo remains technically challenging. To address this, we present an optimized low-input Cleavage Under Targets and Release Using Nuclease (CUT&RUN) workflow tailored for fluorescence-activated cell sorting (FACS) isolated NCCs from zebrafish embryos. This approach enables high-resolution and low-background mapping of key histone modifications, including H3K27ac, H3K4me3, and H3K27me3, from limited cell numbers. Collectively, these methodologies provide a robust framework for dissecting chromatin state dynamics in developmental systems and can also offer insights into epigenetic dysregulation associated with disease.

Animals↗

PubMed2026

Genome-wide CRISPRi screen in human iNeurons identifies novel negative mTOR regulator genes associated with focal cortical dysplasia.

Focal cortical dysplasia (FCD) is a common cause of focal epilepsy that typically results from brain mosaic mutations in the mTOR cell signaling pathway. To identify new potential FCD genes, we developed an in vitro CRISPRi screen in human neurons and used FACS enrichment based on the FCD biomarker, phosphorylated S6 ribosomal protein (pS6). Using whole-genome (110,000 gRNAs) and candidate (129 gRNAs) libraries, we discovered 6 new genes in which loss of function significantly increases pS6 levels: LRRC4, EIF3A, TSN, HIP1, PIK3R3, and URI1. Further analysis of the mTOR pathway showed that only two of the genes, PIK3R3 and HIP1, caused hyperphosphorylation throughout the AKT/mTOR/S6 signaling pathway. Importantly, potential pathogenic variants in these two genes have been reported in resected brain tissue from a single FCD patient each, supporting the predictive validity of our screen. Knocking down each of the 6 genes in iNeurons made mTOR signaling resistant to the loss of neurotrophic factor signaling, specifically GDNF; even without GDNF, pS6 levels remained comparable to GDNF-stimulated controls. Thus, we have identified negative regulators of neuronal mTOR signaling in the context of lost neurotrophic factor support. Our data expand the set of genes that are likely to regulate mTOR pathway signaling in neurons, provide biological confirmation for candidate genes identified in human tissue, and suggest additional targets for investigating somatic gene variants in resected FCD tissues. The identification of novel mTOR regulators using iNeurons also highlights the importance of genetic screening in disease-related cell types.

Brain mosaicism↗

PubMed2026

Single-cell vector copy number analysis of phenotypically defined long-term hematopoietic stem cells for gene therapy safety assessment.

Hematopoietic stem cell (HSC)-based gene therapy has emerged as a transformative approach for the treatment of genetic diseases; however, accurate evaluation of vector copy number (VCN) remains critical for ensuring safety. Conventional bulk VCN assays, including quantitative PCR (qPCR) and droplet digital PCR (ddPCR), do not resolve clonal heterogeneity and cannot identify rare high-VCN cells that may contribute disproportionately to insertional mutagenesis risk. Here, we developed an accessible single-cell VCN profiling method by combining fluorescence-activated cell sorting (FACS) of phenotypically defined long-term HSCs (Lineage- CD34+ CD38- CD90+ CD45RA- cells) with whole-genome amplification followed by conventional qPCR. This approach enabled resolution of VCN distributions at single-cell level using standard laboratory techniques. Notably, single-cell analysis revealed a high VCN tail that bulk VCN analysis could not resolve. Furthermore, in a humanized mouse transplantation model, single-cell VCN profiling demonstrated that overall VCN distributions could be analyzed after engraftment, although inter-donor and inter-mouse variability was observed. Collectively, this method provides a rapid, cost-effective, and phenotypically resolved strategy for assessing VCN heterogeneity in gene-modified HSCs. Single-cell VCN profiling offers complementary insights beyond conventional bulk assays and may enhance preclinical safety evaluation of gene and cell therapy products.

lentiviral vector↗

PubMed2025

Unraveling cellular dynamic changes in tumor evolution induced by long-term low dose-rate radiation.

BACKGROUND: In recent years, there has been a steady increase in professionals engaged in radioactive work. The biological impacts of long-term exposure to low dose-rate radiation remain elusive, as there is a dearth of systematic research in this field. METHODS: BEAS-2B cells were used to establish a cell model with continuous passaging after radiation exposure, which was subsequently subjected to in vivo tumorigenesis assays and in vitro malignant phenotype experiments. By scRNA-seq, we conducted copy number variation analysis, cell trajectory analysis, and cell communication analysis. Furthermore, we used FACS, molecular docking, multiplex immunohistochemistry, qRT-PCR, and co-immunoprecipitation to validate and further explore the molecular mechanisms driving tumor evolution. RESULTS: Long-term low dose-rate exposure is associated with a higher degree of malignancy, as evidenced by the induction of more CNV and EMT events, as well as the delayed activation of DNA repair pathways, which trigger increased genomic instability. The long-term low dose-rate specific ligand-receptor pair, ANGPTL4-SDC4, enhances cell malignancy by promoting angiogenesis in newly formed lung tumor cells. CONCLUSIONS: This study not only provides the first evidence and mechanistic explanation that long-term low dose-rate radiation leads to increased cellular malignancy but also offers valuable theoretical insights into the dynamic processes of early tumor evolution in lung cancer within the realm of tumor biology.

Humans↗

PubMed2026

VINE-seq and MultiVINE-seq for single-nucleus and multiome profiling of the brain vasculature.

The human cerebrovasculature is a critical yet historically understudied component of neurological health. Dysfunction of the diverse endothelial, mural, and perivascular cells that comprise cerebral vessels is central to diseases ranging from stroke to Alzheimer's disease. However, characterizing these cell populations at a molecular level has proven exceptionally challenging. Encased within a robust basement membrane, vascular cells resist standard dissociation methods, leading to their systematic depletion and underrepresentation in existing single-nucleus genomic atlases. This has created a major blind spot in neuroscience. To overcome this barrier, we developed vessel isolation and nucleus extraction for sequencing (VINE-seq) and its advanced iteration, MultiVINE-seq. The protocol provides a robust, reproducible workflow for the enrichment and high-resolution profiling of vascular, perivascular, and immune cells from fresh or frozen human and mouse brain tissue. First, intact vessels (predominantly capillaries and small arterioles/venules, 100 µm in diameter) are isolated from homogenized brain tissue via dextran-based density-gradient centrifugation, separating the vascular pellet from myelin and the parenchymal fraction. Second, the collected vessels are rigorously washed over a cell strainer to remove trapped contaminants. A critical innovation lies in the third stage: the optimized extraction of nuclei from purified vessels using enzymatic digestion. After extraction, the protocol uses fluorescence-activated cell sorting (FACS) to ensure collection of high-purity nuclei suitable for widely used droplet-based sequencing platforms (e.g., 10x Genomics single cell 3' or multiome). This protocol requires 4-5 h to complete and can be carried out by researchers with single-cell and flow cytometry training.

Journal Article↗

PubMed2003

A role for VASP in RhoA-Diaphanous signalling to actin dynamics and SRF activity.

Vasodilator-stimulated phosphoprotein (VASP) is involved in multiple actin-mediated processes, including regulation of serum response factor (SRF) activity. We used the SRF transcriptional assay to define functional domains in VASP and to show that they coincide with those required for F-actin accumulation, as determined by a quantitative FACS assay. We identified inactive VASP mutants that can interfere both with F-actin assembly and with SRF activation by wild-type VASP. These VASP mutants also inhibit actin-based motility of Vaccinia virus and Shigella flexneri. VASP-induced F-actin accumulation and SRF activation require both functional Rho and its effector mDia, and conversely, mDia-mediated SRF activation is critically dependent on functional VASP. VASP and mDia also associate physically in vivo. These findings show that VASP and mDia function cooperatively downstream of Rho to control F-actin assembly and SRF activity.

3T3 Cells↗

PubMed2025

A genome-wide, CRISPR-based screen reveals new requirements for translation initiation and ubiquitination in driving adipogenic fate change.

In response to excess nutrients, white adipose tissue expands by both generating new adipocytes and upregulating lipogenesis in existing adipocytes. Here, we performed a genome-wide functional CRISPR screen to identify regulators of adipogenesis in the mouse 3T3-L1 preadipocyte model. In this pooled screening strategy, we used FACS to isolate populations based on lipid content, gating for fluorescence intensity of lipophilic fluorescent BODIPY dye. Additionally, we categorized whether the gene functions primarily during mitotic clonal expansion, lipogenesis, or both. We found that translation initiation and ubiquitin-dependent protein stability regulators drive both adipogenic fate change and lipogenesis. We further supported these findings with proteomics, demonstrating that essential changes in protein reprogramming can drive or inhibit 3T3-L1 adipogenesis independent of transcription. Furthermore, we demonstrated that specific branches of the hypusination pathway, a conserved regulator of translation initiation, are critical for translating adipogenic inducers of mitotic clonal expansion and that the neddylation/ubiquitin pathway modulates insulin sensitivity during lipogenesis.

Animals↗

PubMed2025

Simultaneous Visualization of Protein and Genomic Regions in Plant Nuclei.

Immunohistostaining (IHS) is a widely used technique in diagnostic and research laboratories in which specific antibodies are used to detect and visualize a protein of interest in cells or tissues. Similarly, with specific oligonucleotide probes, the fluorescence in situ hybridization (FISH) method allows one to visualize genomic regions and to analyze its localization in the nuclear space. Here, we describe a combined FISH-IHS technique that enables researchers to determine the localization of protein and genomic loci in plant nuclei simultaneously. This method can be applied to extracted nuclei and sections of paraffin-embedded tissues. It provides a valuable tool to improve our understanding of nuclear dynamics by revealing the spatial relationship between specific genomic loci and target proteins.

In Situ Hybridization, Fluorescence↗

PubMed2025

Genome editing with programmable base editors in human cells.

Genome editing has garnered significant attention over the last decade, resulting in a massive expansion of the genome engineering toolbox. Base editors encompass a class of tools that enable installing single-nucleotide changes in genomic DNA without the use of double-strand breaks. With the ever-increasing development of new and/or improved base editor systems, it is easy to be overwhelmed by the abundance of options. Here, we provide clear guidance to facilitate the selection of a base editor and to design guide RNAs (gRNAs) to suit various needs. Additionally, we describe in detail how to generate gRNA plasmids, transfect various mammalian cell types, and evaluate editing efficiencies. Finally, we give alternative methods and troubleshooting tips for some common pitfalls encountered during base editing.

Humans↗