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  1. Free, publicly-accessible full text available June 1, 2025
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  3. Free, publicly-accessible full text available April 1, 2025
  4. Abstract

    Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to how acid/base chemistry drives drug efflux. Here, we uncover the proton-coupling mechanism of theStaphylococcus aureusefflux pump NorA by elucidating structures in various protonation states of two essential acidic residues using cryo-EM. Protonation of Glu222 and Asp307 within the C-terminal domain stabilized the inward-occluded conformation by forming hydrogen bonds between the acidic residues and a single helix within the N-terminal domain responsible for occluding the substrate binding pocket. Remarkably, deprotonation of both Glu222 and Asp307 is needed to release interdomain tethering interactions, leading to opening of the pocket for antibiotic entry. Hence, the two acidic residues serve as a “belt and suspenders” protection mechanism to prevent simultaneous binding of protons and drug that enforce NorA coupling stoichiometry and confer antibiotic resistance.

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  5. Free, publicly-accessible full text available January 1, 2025
  6. Abstract

    Extracellular vesicles (EVs) secreted by human‐induced pluripotent stem cells (hiPSCs) have great potential as cell‐free therapies in various diseases, including prevention of blood–brain barrier senescence and stroke. However, there are still challenges in pre‐clinical and clinical use of hiPSC‐EVs due to the need for large‐scale production of a large quantity. Vertical‐Wheel bioreactors (VWBRs) have design features that allow the biomanufacturing of hiPSC‐EVs using a scalable aggregate or microcarrier‐based culture system under low shear stress. EV secretion by undifferentiated hiPSCs expanded as 3‐D aggregates and on Synthemax II microcarriers in VWBRs were investigated. Additionally, two types of EV collection media, mTeSR and HBM, were compared. The hiPSCs were characterized by metabolite and transcriptome analysis as well as EV biogenesis markers. Protein and microRNA cargo were analysed by proteomics and microRNA‐seq, respectively. Thein vitrofunctional assays of microglia stimulation and proliferation were conducted. HiPSCs expanded as 3‐D aggregates and on microcarriers had comparable cell number, while microcarrier culture had higher glucose consumption, higher glycolysis and lower autophagy gene expression based on mRNA‐seq. The microcarrier cultures had at least 17–23 fold higher EV secretion, and EV collection in mTeSR had 2.7–3.7 fold higher yield than HBM medium. Microcarrier culture with mTeSR EV collection had a smaller EV size than other groups, and the cargo was enriched with proteins (proteomics) and miRNAs (microRNA‐seq) reducing apoptosis and promoting cell proliferation (e.g. Wnt‐related pathways). hiPSC‐EVs demonstrated the ability of stimulating proliferation and M2 polarization of microgliain vitro. HiPSC expansion on microcarriers produces much higher yields of EVs than hiPSC aggregates in VWBRs. EV collection in mTeSR increases yield compared to HBM. The biomanufactured EVs from microcarrier culture in mTeSR have exosomal characteristics and are functional in microglia stimulation, which paves the ways for future in vivo anti‐aging study.

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    Free, publicly-accessible full text available January 1, 2025
  7. Free, publicly-accessible full text available December 10, 2024
  8. Abstract

    Metamaterials are architected cellular networks with solid struts, plates, or shells that constitute the edges and faces of building cells. Certain metamaterial designs can balance light weight and high stiffness requirements, which are otherwise mutually exclusive in their bulk form. Existing studies on these materials typically focus on their mechanical response under uniaxial compression, but it is unclear whether a strut-based metastructure design with high compressive stiffness can exhibit high torsional stiffness simultaneously. Designing lightweight metastructures with both high compressive and torsional stiffnesses could save time and cost in future material development.

    To explore the effect of unit cell design, unit cell number, and density distribution on both compressive and torsional stiffnesses, a computational design space was presented. Seven different unit cells, including three basic building blocks: body-centered cubic (BCC), face-centered cubic (FCC), and simple cubic (SC) were analyzed. All samples had a relative density of approximately 7%. It was found that a high compressive stiffness required a high concentration of struts along the loading direction, while a high torsional stiffness needed diagonal struts distributed on the outer face. Increasing unit cell numbers from 1 to 64 affected stiffness by changing the stress distribution globally. Non-uniform metastructure designs with strengthened vertical and diagonal struts towards the outer surface exhibited higher stiffness under either compressive or torsional loading. This study provides valuable guidelines for designing and manufacturing metamaterials for complex mechanical environments.

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  9. Free, publicly-accessible full text available April 1, 2025
  10. Free, publicly-accessible full text available July 3, 2024