Tamhankar, S. | bioRxiv Science

bioRxiv2026

Forward Programming Identifies Inducers of Blood-Brain Barrier Properties in Human Pluripotent Stem Cell-Derived Endothelial Cells

Brain microvascular endothelial cells (BMECs) forming the blood-brain barrier (BBB) maintain brain homeostasis through specialized properties such as tight junctions, efflux transporters, and low levels of transcytosis. However, mechanisms governing induction of BBB properties during development remain poorly understood. We mined single-cell RNA sequencing datasets to identify transcription factors (TFs) critical for BBB development. Forty-four TFs were overexpressed in human pluripotent stem cell-derived endothelial cells cultured in the presence of the Wnt pathway agonist CHIR99021 to identify TFs capable of directing acquisition of BBB properties via forward programming. Individual TFs, including KLF2, KLF4, FOXF1, FOXF2, ZIC2, ZIC3, NR4A1, NR4A2, FOXC1, and FOXQ1, induced distinct BBB-like gene expression patterns. Combinations of these TFs induced many canonical BBB genes, yielding ECs with reduced endocytosis, increased efflux activity, and improved barrier function. The resultant forward programmed CNS-like ECs (fpCECs) offer promising tools for modeling human BBB development and neurovascular disease and for drug screening.

cell biology↗

bioRxiv2025

Cost of altered translation accuracy shapes adaptation to antibiotics in E. coli

Protein synthesis, while central to cellular function, is error-prone. The resulting mistranslation is generally costly, but we do not know how these costs compare or interact with the costs imposed by external selection pressures such as antibiotics. We also do not know whether and how these costs are compensated during evolution. It is important to answer these questions, since mistranslation is ubiquitous and antibiotic exposure, widespread. We quantified the growth cost of genetically increasing and decreasing mistranslation rates, and exposure to low antibiotic concentrations, in Escherichia coli. Mistranslation costs were generally lower than the cost imposed by antibiotics, and exacerbated in a strain-specific manner under antibiotic exposure. All strains quickly compensated the antibiotic cost during experimental evolution, via antibiotic- and genotype-specific mutations. In contrast, mistranslation costs were significantly reduced only in some cases, without clear causal mutations. Control populations that evolved without antibiotics consistently compensated the cost of accuracy, and evolved increased antibiotic resistance as a by-product. Our work demonstrates that even when the cost of mistranslation is weak, altered translation accuracy can shape adaptive outcomes and underlying genetic mechanisms, with strong collateral fitness effects for apparently unrelated phenotypes such as antibiotic resistance.

evolutionary biology↗