Smart Galactosidase-Responsive Antimicrobial Dendron: Towards More Biocompatible Membrane-Disruptive Agents.

Antimicrobial resistance is a global healthcare challenge that urgently needs the development of new therapeutic agents. Antimicrobial peptides and mimics thereof are promising candidates but mostly suffer from inherent toxicity issues due to the non-selective binding of cationic groups with mammalian cells. To overcome this toxicity issue, this work herein reports the synthesis of a smart antimicrobial dendron with masked cationic groups (Gal-Dendron) that could be uncaged in the presence of ß-galactosidase enzyme to form the activated Enz-Dendron and confer antimicrobial activity. Enz-Dendron show bacteriostatic activity toward Gram-negative (P. aeruginosa and E. coli) and Gram-positive (S. aureus) bacteria with minimum inhibitory concentration values of 96 µm and exerted its antimicrobial mechanism via a membrane disruption pathway, as indicated by inner and outer membrane permeabilization assays. Crucially, toxicity studies confirmed that the masked prodrug Gal-Dendron exhibited low hemolysis and is at least 2.4 times less toxic than the uncaged cationic Enz-Dendron, thus demonstrating the advantage of masking the cationic groups with responsive immolative linkers to overcome toxicity and selectivity issues. Overall, this study highlights the potential of designing new membrane-disruptive antimicrobial agents that are more biocompatible via the amine uncaging strategy.

Development of an Albumin-Polymer Bioconjugate via Covalent Conjugation and Supramolecular Interactions.

Preexisting serum albumin-polymer bioconjugates have been formed either through covalent conjugation or supramolecular interactions. However, the viability of producing a bioconjugate where both covalent conjugation and supramolecular interactions have been adopted is yet to be explored. In this work, the noncovalent interaction of two polymers bearing fatty acid-based end-functionalities were compared and the superior binder was carried forward for testing with serum albumin that possessed a polymer conjugated to its Cys34 residue. The studies demonstrated that an albumin-polymer bioconjugate equipped with polymers via both covalent and supramolecular interactions can be successfully achieved.

Cross-linking Mass Spectrometry Analysis of the Yeast Nucleus Reveals Extensive Protein-Protein Interactions Not Detected by Systematic Two-Hybrid or Affinity Purification-Mass Spectrometry.

Saccharomyces cerevisiae has the most comprehensively characterized protein-protein interaction network, or interactome, of any eukaryote. This has predominantly been generated through multiple, systematic studies of protein-protein interactions by two-hybrid techniques and of affinity-purified protein complexes. A pressing question is to understand how large-scale cross-linking mass spectrometry (XL-MS) can confirm and extend this interactome. Here, intact yeast nuclei were subject to cross-linking with disuccinimidyl sulfoxide (DSSO) and analyzed using hybrid MS2-MS3 methods. XlinkX identified a total of 2,052 unique residue pair cross-links at 1% FDR. Intraprotein cross-links were found to provide extensive structural constraint data, with almost all intralinks that mapped to known structures and slightly fewer of those mapping to homology models being within 30 Å. Intralinks provided structural information for a further 366 proteins. A method for optimizing interprotein cross-link score cut-offs was developed, through use of extensive known yeast interactions. Its application led to a high confidence, yeast nuclear interactome. Strikingly, almost half of the interactions were not previously detected by two-hybrid or AP-MS techniques. Multiple lines of evidence existed for many such interactions, whether through literature or ortholog interaction data, through multiple unique interlinks between proteins, and/or through replicates. We conclude that XL-MS is a powerful means to measure interactions, that complements two-hybrid and affinity-purification techniques.

Light-sheet microscopy as a tool to understanding the behaviour of Polyion complex micelles for drug delivery.

Polyelectrolyte-protein complexes are widely used to deliver therapeutic proteins. Here, we present a method for imaging the release of drugs from polyion complex (PIC) micelles in 3D tumour spheroids using light-sheet microscopy. A negatively charged block copolymer was condensed with a positively charged model drug, hen egg white lysozyme (HEWL) by electrostatic interaction. We were able to observe the distribution of polymer and protein within the entire tumour spheroid, showing that the protein was released from the polyelectrolyte complex upon cell internalization at the peripheral cell layer of the spheroid.