Nikfarjam, S. | Search bioRxiv

bioRxiv2025

BRCA2-deficiency Causes Global Transcriptomic Alterations in Endothelial Cells

This study aims to explore the alterations in gene expression following BRCA2 loss in endothelial cells and to investigate the potential contribution of BRCA2 in regulating the endothelial protein-coding transcriptome. Cultured human umbilical vein endothelial cells (HUVECs) were transfected with siBRCA2 or non-targeting scrambled RNA as control. Total RNA was extracted and used for RNA sequencing following purification and quality check. Transcription profiles and differentially expressed genes (DEGs) were identified. Enrichment of functions and signaling pathways analysis were performed based on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In total, 3196 DEGs with (cut-off 1.5-fold) were identified, of which 1852 genes were upregulated, and 1344 genes were downregulated in BRCA2-deficient HUVECs. Cell cycle exit and neuronal differentiation 1 (CEND1) and mannose receptor C-type 1 (MRC1) were the most significantly up- and downregulated genes, respectively, in BRCA2-deficient endothelial cells. The expression of top DEGs were further validated by RT-qPCR in HUVECs and in human coronary artery endothelial cells. The GO and KEGG analysis suggest that cell adhesion molecules and cytokine-cytokine receptor interaction may play an important role in BRCA2-deficient endothelial cells. Overall, these findings contribute to a greater understanding of the mechanisms involved in BRCA2-mediated endothelial function.

genomics↗

bioRxiv2025

Rapid Antibody Fragment Production and Binding Analysis Using Cell-Free Protein Synthesis Combined with Fluorescence Correlation Spectroscopy

This study investigates the efficient development and production of single-chain variable fragments (scFvs) and antibody fragments (Fabs) using an E. coli-based cell-free protein synthesis system. Validation of the methodology was performed using a fluorescence correlation spectroscopy (FCS)-based assay to determine binding equilibrium constants (KD) between antibodies and the receptor binding domain (RBD) of SARS-CoV-2 Spike protein. An initial assessment employed two conventionally cell-produced anti-RBD antibodies. To optimize cell-free production, folding strategies were developed to enhance the solubility and yields of scFvs, including a two-stage refolding protocol that successfully recovered active proteins from misfolded precipitates. Fab fragments were also produced and characterized, with their binding properties analyzed to assess functionality. This study highlights the potential of cell-free systems for the rapid and efficient production of functional antibody fragments. The integration of advanced techniques, such as FCS-based kinetic measurements, underscores the versatility and applicability of cell-free platforms for antibody development and high-throughput screening. These findings offer a promising avenue for accelerating therapeutic antibody research and production. SIGNIFICANCE of WORKThis study highlights the transformative potential of integrating cell-free protein synthesis (CFPS) with fluorescence correlation spectroscopy (FCS) for the rapid and scalable production and characterization of functional antibody fragments. By leveraging an E. coli-based CFPS platform, we successfully designed and optimized the production of single-chain variable fragments (scFvs) compared to Fab fragments, addressing some common challenges of low solubility and yield. The development of a cost-efficient two-stage refolding strategy further enhanced the scalability and functionality of scFvs, enabling higher recovery of active proteins from the unfolded state. Additionally, FCS provided a sensitive, rapid method for accurately quantifying antigen-antibody binding kinetics across a range of affinities. This work establishes CFPS and FCS as versatile and complementary tools, offering a robust framework for accelerating antibody fragment development, particularly in time-sensitive scenarios like infectious disease outbreaks.

biophysics↗