Biology subjects

Metkar, S.

Publications and source records attributed to Metkar, S..

3 recordsLinked to original sources

bioRxiv2026

Structural Tuning of HEWL Amyloid Polymorphs Enhances Antibacterial Activity Against Gram-Positive and Gram-Negative Pathogens

Amyloid fibrils are traditionally associated with protein misfolding disorders; however, increasing evidence indicates that they can also perform beneficial biological functions, including antimicrobial defense. Here, we investigated whether structurally distinct amyloid polymorphs of hen egg white lysozyme (HEWL) exhibit enhanced antibacterial activity compared with the native protein. HEWL was converted into two amyloid polymorphs, flexible fibrils (FFs) and rigid fibrils (RFs), and their antibacterial activities were evaluated against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacteria Escherichia coli (Top10) and Salmonella Typhimurium. Fibril formation was confirmed by circular dichroism (CD) spectroscopy, thioflavin T (ThT) fluorescence, and transmission electron microscopy (TEM), demonstrating morphologically distinct amyloid assemblies with different secondary-structure organizations. Fluorescence-based bacterial growth assays showed that native HEWL exhibited only moderate antibacterial activity, whereas both amyloid polymorphs produced potent, concentration-dependent bacterial growth inhibition. FFs and RFs consistently displayed greater antibacterial efficacy than native HEWL across all tested strains, with FFs exhibiting slightly stronger activity against S. Typhimurium. At concentrations of 600-800 M, FFs achieved >90% growth inhibition for all bacterial species examined. Cytotoxicity studies using SH-SY5Y human neuroblastoma cells demonstrated minimal toxicity for native HEWL, modest effects for FFs, and substantially greater toxicity for RFs, indicating that amyloid polymorphism influences both antimicrobial activity and mammalian cell compatibility. Collectively, these findings establish a direct relationship between amyloid structure, antibacterial efficacy, and cytotoxicity. The combination of potent antibacterial activity and relatively low cytotoxicity identifies FFs as a promising functional amyloid biomaterial for the development of next-generation antimicrobial materials.

bioengineering↗

bioRxiv2025

Humanized TfR1 and transferrin gene-replacement rats for in vivo evaluation of BBB transport

The transferrin receptor 1 (TfR1)-transferrin (TF) axis plays a central role in iron homeostasis and has long been recognized as a promising route for delivering biologics across the blood-brain barrier (BBB). We have developed a class of human-specific anti-TfR1 nanobodies (NewroBus) that exploit this transport pathway. However, the lack of cross-reactivity with rodent TfR1 limits the utility of standard animal models for preclinical testing. To overcome this challenge, we generated knock-in (KI) rats in which the coding sequences of the endogenous Tfrc and Tf genes were replaced with human coding sequences, yielding animals that express human TfR1 and/or human TF under physiological control. Rats homozygous for both humanized alleles were viable and fertile, indicating that the human proteins can functionally replace their rodent equivalents. Nonetheless, these double homozygous rats exhibited erythropoietic abnormalities and tissue-specific alterations in iron distribution--characterized by decreased splenic and increased hepatic iron--suggesting incomplete functional compensation. In contrast, heterozygous rats showed only mild, subclinical hematologic changes (microcytosis and hypochromia). These findings demonstrate that the humanized TfR1-TF axis is compatible with life and iron regulation, albeit with varying degrees of compensation depending on gene dosage. Importantly, these KI rats provide a translationally relevant platform for evaluating pharmacokinetics, CNS penetration, and safety of human-specific BBB-targeting therapeutics, including NewroBus-based biologics and other TfR1-mediated delivery strategies.

neuroscience↗

bioRxiv2025

NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

Effective delivery of therapeutics to the brain is restricted by the blood-brain barrier (BBB). A strategy to overcome this limitation involves taking advantage of receptor-mediated transcytosis pathways, such as those mediated by transferrin receptor 1 (TfR1), which is highly expressed on brain endothelial cells and naturally transports iron-bound transferrin across the BBB. To exploit this mechanism, we immunized camelids with human TfR1 and cloned 470 VHH nanobody sequences from their B cells. From this repertoire, 24 nanobodies (TfR1b-Nbs) were identified that bind human TfR1 on the cell membrane. These nanobodies were screened for binding to human TfR1, lack of interference with transferrin binding and TfR1-mediated iron uptake, and the ability to cross the BBB via human TfR1-mediated transcytosis in newly generated humanized Tfr1h knock-in rats. To improve developability and reduce potential immunogenicity, selected TfR1b-Nbs were humanized and optimized with computational and artificial intelligence (AI) algorithms, enhancing humanness, solubility, and VHH-nativeness. Eight optimized TfR1b-Nbs retained BBB permeability and were fused to humanized anti-TNF nanobody inhibitors (TNFI- or TNFI-{beta}), generating 16 heterodimers. Fusion to these TNFIs served as a functional readout, confirming that TfR1b-Nbs can shuttle biologically active, BBB-impermeable payloads into the central nervous system (CNS). All heterodimers demonstrated CNS delivery after intravenous administration, and selected constructs also reached the brain via subcutaneous injection, maintaining high serum and cerebrospinal fluid (CSF) levels for up to 72 hours. A pilot study with one heterodimer showed that chronic administration in rats humanized for both transferrin and TfR1 caused no hematological toxicity or signs of anemia - a key safety concern when targeting TfR1. These results establish humanized TfR1b-Nbs - designated NewroBus - as promising BBB shuttles for the safe and effective therapeutic delivery of biologics to the brain.

bioengineering↗