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  1. van Veen, Hendrik W. (Ed.)
    Antibiotic resistance is a major public health concern. The shrinking selection of effective antibiotics and lack of new development is making the situation worse. Gram-negative bacteria more specifically pose serious threat because of their double layered cell envelope and effective efflux systems, which is a challenge for drugs to penetrate. One promising approach to breach this barrier is the “Trojan horse strategy”. In this technique, an antibiotic molecule is conjugated with a nutrient molecule that helps the antibiotic to enter the cell through dedicated transporters for the nutrient. Here, we explored the approach using biotin conjugation with a florescent molecule Atto565 to determine if biotinylation enhances accumulation. Biotin is an essential vitamin for bacteria and is obtained through either synthesis or uptake from the environment. We found that biotinylation enhanced accumulation of Atto565 in E . coli . However, the enhancement did not seem to be due to uptake through biotin transporters since the presence of free biotin had no observable impact on accumulation. Accumulated compound was mostly in the periplasm, as determined by cell fractionation studies. This was further confirmed through the observation that expression of streptavidin in the periplasm specifically enhanced the accumulation of biotinylated Atto565. This enhancement was not observed when streptavidin was expressed in the cytoplasm indicating no significant distribution of the compound inside the cytoplasm. Using gene knockout strains, plasmid complementation and mutagenesis studies we demonstrated that biotinylation made the compound a better passenger through OmpC, an outer membrane porin. Density functional theory (DFT)-based evaluation of the three-dimensional geometries showed that biotinylation did not directly stabilize the conformation of the compound to make it favorable for the entry through a pore. Further studies including molecular dynamics simulations are necessary to determine the possible mechanisms of enhanced accumulation of the biotinylated Atto565. 
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  2. Abstract

    The airborne nature of coronavirus transmission makes it critical to develop new barrier technologies that can simultaneously reduce aerosol and viral spread. Here, we report nanostructured membranes with tunable thickness and porosity for filtering coronavirus-sized aerosols, combined with antiviral enzyme functionalization that can denature spike glycoproteins of the SARS-CoV-2 virus in low-hydration environments. Thin, asymmetric membranes with subtilisin enzyme and methacrylic functionalization show more than 98.90% filtration efficiency for 100-nm unfunctionalized and protein-functionalized polystyrene latex aerosol particles. Unfunctionalized membranes provided a protection factor of 540 ± 380 for coronavirus-sized particle, above the Occupational Safety and Health Administration’s standard of 10 for N95 masks. SARS-CoV-2 spike glycoprotein on the surface of coronavirus-sized particles was denatured in 30 s by subtilisin enzyme-functionalized membranes with 0.02-0.2% water content on the membrane surface.

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  3. null (Ed.)
    The cell envelope structure of Gram-negative bacteria is unique, composed of two lipid bilayer membranes and an aqueous periplasmic space sandwiched in between. The outer membrane constitutes an extra barrier to limit the exchange of molecules between the cells and the exterior environment. Donnan potential is a membrane potential across the outer membrane, resulted from the selective permeability of the membrane, which plays a pivotal role in the permeability of many antibiotics. In this review, we discussed factors that affect the intensity of the Donnan potential, including the osmotic strength and pH of the external media, the osmoregulated periplasmic glucans trapped in the periplasmic space, and the displacement of cell surface charges. The focus of our discussion is the impact of Donnan potential on the cellular permeability of selected antibiotics including fluoroquinolones, tetracyclines, β-lactams, and trimethoprim. 
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  4. null (Ed.)
    The RND family efflux pump AcrAB-TolC in E. coli and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM efforts, the dynamic assembly process remains poorly understood. Here, we tested the effect of overexpressing functionally defective pump components in wild type E. coli cells to probe the pump assembly process. Incorporation of a defective component is expected to reduce the efflux efficiency of the complex, leading to the so called “dominant negative” effect. Being one of the most intensively studied bacterial multidrug efflux pumps, many AcrA and AcrB mutations have been reported that disrupt efflux through different mechanisms. We examined five groups of AcrB and AcrA mutants, defective in different aspects of assembly and substrate efflux. We found that none of them demonstrated the expected dominant negative effect, even when expressed at concentrations many folds higher than their genomic counterpart. The assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of futile pump complex. 
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  8. Aquaporins are water channel proteins in cell membrane, highly specific for water molecules while restricting the passage of contaminants and small molecules, such as urea and boric acid. Cysteine functional groups were installed on aquaporin Z for covalent attachment to the polymer membrane matrix so that the proteins could be immobilized to the membranes and aligned in the direction of the flow. Depth profiling using x-ray photoelectron spectrometer (XPS) analysis showed the presence of functional groups corresponding to aquaporin Z modified with cysteine (Aqp-SH). Aqp-SH modified membranes showed a higher salt rejection as compared to unmodified membranes. For 2 M NaCl and CaCl2 solutions, the rejection obtained from Aqp-SH membranes was 49.3 ± 7.5% and 59.1 ± 5.1%. On the other hand, the rejections obtained for 2 M NaCl and CaCl2 solutions from unmodified membranes were 0.8 ± 0.4% and 1.3 ± 0.2% respectively. Furthermore, Aqp-SH membranes did not show a significant decrease in salt rejection with increasing feed concentrations, as was observed with other membranes. Through simulation studies, it was determined that there was approximately 24% capping of membrane pores by dispersed aquaporins. 
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