Jangwook P. Jung

Cell fusion generates hybrid cells with unique traits. To understand the transcriptional and signaling alterations after fusion, we analyzed a published single-cell RNA-sequencing dataset of fused murine cardiomyocytes (mHL1) and mesenchymal stromal/stem cells (mMSC). Our analysis showed that fused cells exhibit a transcriptional trajectory suggesting a rapid change that stabilizes over time. We observed asymmetric plasticity. Initially, at Day 1, fusion hybrids resembled mMSCs (mesenchymal reprogramming), but by Day 3, their gene expression shifted to resemble mHL1 cells (myogenic reprogramming). Our analysis also identified distinct transcriptional subpopulations, including a subset enriched for tenascin (extracellular matrix remodeling), accompanied by dynamic changes in cell adhesion and intercellular communication. We also saw a significant shift in signaling pathways over time. At Day 1, Wnt and Melanogenesis (regenerative/antioxidant) signaling were downregulated. By Day 3, stress resistance and cellular adaptation pathways became enriched. Gene regulatory network analysis revealed key changes in master regulators; genes associated with chromatin remodeling (Hmga2), circadian rhythm (Arntl), and mesenchymal identity (Prrx1) became more active by Day 3. Collectively, our findings demonstrate that cell fusion is a dynamic reprogramming process, where evolving gene regulatory and signaling networks generate novel hybrid cell states, creating cellular diversity.

Cell fusion, a natural process occurring between similar or dissimilar cell types, often confers new or enhanced functionality, yet its impact on cellular communication and gene regulation remains poorly understood. Here, we used recent analytical frameworks to investigate accidental cell fusion between murine cardiomyocytes (mHL1) and murine mesenchymal stromal/stem cells (mMSC) leveraging previously published single-cell RNA sequencing data. After fusion, we observed a biased distribution of gene expression and acquired phenotypes in fused hybrids. Trajectory inference showed that hybrids with a more mMSC-like transcriptome diverged more significantly from parental cells relative to hybrids with a more mHL1-like transcriptome. We also observed dynamic changes in cell-cell communication, with early (Day 1) downregulation of Wnt signaling and Melanogenesis evolving into the upregulation of pathways like Endocrine resistance and Focal adhesion by Day 3. Notably, ECM (extracellular matrix)-receptor interactions were largely consistent whether annotation or unsupervised Clustree methods were used. Furthermore, our analysis indicated the emergence of various cancer-associated signaling mechanisms. Our findings highlight the remarkable plasticity of cellular identity following fusion and lay the groundwork for future research into the precise molecular mechanisms driving these transformations and the potential of cell fusion for generating novel cell types.

Antioxidant therapies are of interest in the prevention and management of ocular disorders such as cataracts. Although an active area of interest, topical therapy with antioxidants for the treatment of cataracts is complicated by multiple ocular anatomical barriers, product stability, and solubility. Entrapment and delivery of antioxidants with poly(lactic-co-glycolic acid) nanoparticles is a possible solution to these challenges, however, little is known regarding their effects in vitro or in vivo. Our first aim was to investigate the impact of blank and lutein loaded PLGA nanoparticles on viability and development of reactive oxygen species in lens epithelial cells in vitro. Photo-oxidative stress was induced by ultraviolet light exposure with cell viability and reactive oxygen species monitored. Next, an in vivo, selenite model was utilized to induce cataract formation in rodents. Eyes were treated topically with both free lutein and lutein loaded nanoparticles (LNP) at varying concentrations. Eyes were monitored for the development of anterior segment changes and cataract formation. The ability of nanodelivered lutein to reach the anterior segment of the eye was evaluated by liquid chromatography coupled to mass spectrometry of aqueous humor samples and liquid chromatography coupled to tandem mass spectrometry (targeted LC-MS/MS) of lenses. LNP had a minimal impact on the viability of lens epithelial cells during the short exposure timeframe (24 h) and at concentrations < 0.2 μg LNP/μl. A significant reduction in the development of reactive oxygen species was also noted. Animals treated with LNPs at an equivalent lutein concentration of 1,278 μg /mL showed the greatest reduction in cataract scores. Lutein delivery to the anterior segment was confirmed through evaluation of aqueous humor and lens sample evaluation. Topical treatment was not associated with the development of secondary keratitis or anterior uveitis when applied once daily for one week. LNPs may be an effective in the treatment of cataracts.

Directed evolution (DE) mimics natural selection to improve the functions of a target protein. Machine learning (ML) has significantly streamlined DE by aiding in several steps, which includes identifying starting variants, generating diverse libraries and modeling sequence-fitness relationships. To date, the majority of ML-assisted DE (MLDE) approaches has relied predominantly on sequence information due to the challenges and cost of obtaining protein structure information. Here, we introduce a structure-augmented MLDE (saMLDE) approach for selecting high fitness variants from a library of Protein G B1 domain. We adopted and applied a zero-shot sequence-based prediction method (offering the potential to discover new insights without extensive training data) to select an initial training library of 96 variants for the saMLDE campaign. To leverage protein structure information, we used protein structure prediction with AlphaFold2 and molecular docking simulations performed with Rosetta FlexPepDock, resulting in structure-based features derived with an induced fit model. After three rounds of the saMLDE campaign, we demonstrated that saMLDE incorporating structural information gradually improves the average fitness scores and the precision of predicted binders. In addition, we found that the initial library selection with zero-shot subset selection methods significantly impacted the average fitness scores and precision, consequently influencing the overall directed evolutionary trajectories.

We demonstrate a single-component hydrophilic photocrosslinkable copolymer system that incorporates all critical functionalities into one chain. This design allows for the creation of uniform functional organic coatings on a variety of substrates. The copolymers were composed of a poly(ethylene oxide)-containing monomer, a monomer that can release a primary amine upon UV light, and a monomer with reactive epoxide or cyclic dithiocarbonate with a primary amine. These copolymers are easily incorporated into the solution-casting process using polar solvents. Furthermore, the resulting coating can be readily stabilized through UV light-induced crosslinking, providing an advantage for controlling the surface properties of various substrates. The photocrosslinking capability further enables us to photolithographically define stable polymer domains in a desirable region. The resulting copolymer coatings were chemically versatile in immobilizing complex molecules by (i) post-crosslinking functionalization with the reactive groups on the surface and (ii) the formation of a composite coating by mixing varying amounts of a protein of interest, i.e., fish skin gelatin, which can form a uniform dual crosslinked network. The number of functionalization sites in a thin film could be controlled by tuning the composition of the copolymers. In photocrosslinking and subsequent functionalizations, we assessed the reactivity of the epoxide and cyclic dithiocarbonate with the generated primary amine. Moreover, the orthogonality of the possible reactions of the presented reactive functionalities in the crosslinked thin films with complex molecules is assessed. The resulting copolymer coatings were further utilized to define a hydrophobic surface or an active surface for the adhesion of biological objects.

The imaging and quantification of stained red blood cells (RBCs) are important for identifying RBCs in hematology and for diagnosing diseased RBCs or parasites in cytopathology.

Imaging and quantification of stained blood cells are important for identifying the cells in hematology and for diagnosing diseased cells or parasites in cytopathology. Romanowsky staining have been used traditionally to produce hues in blood cells using anionic eosin Y and cationic methylene blue. While Romanowsky stains have been widely used in cytopathology, end-users have experienced problems with varying results in staining due to premature precipitation or evaporation of methanol, leading to the inherent inconsistency of solution-based Romanowsky staining. Here, we demonstrate that staining and destaining of blood smear are controllable by the contact time of agarose gel stamps. While the extent of staining and destaining are discernable by hue values of stamped red blood cells in micrographs, quantification of adsorbed and desorbed Romanowsky dye molecules (in particular, eosin Y, methylene blue, and azure B) from and to the agarose gel stamps needs a model that can explain the sorption process. We find predictable sorption of the Romanowsky dye molecules from the pseudo-second-order kinetics models for adsorption and the one phase decay model for desorption. Thus, the method of agarose gel stamping demonstrated here could be an alternative to solution-based Romanowsky staining with predictable quantity of sorption and timing of contact.