Unnatural amino acid analogues of membrane-active helical peptides with anti-mycobacterial activity and improved stability

Objectives The emergence of MDR-TB, coupled with shrinking antibiotic pipelines, has increased demands for new antimicrobials with novel mechanisms of action. Antimicrobial peptides have increasingly been explored as promising alternatives to antibiotics, but their inherent poor in vivo stability remains an impediment to their clinical utility. We therefore systematically evaluated unnatural amino acid-modified peptides to design analogues with enhanced anti-mycobacterial activities. Methods Anti-mycobacterial activities were evaluated in vitro and intracellularly against drug-susceptible and MDR isolates of Mycobacterium tuberculosis using MIC, killing efficacy and intracellular growth inhibition studies. Toxicity profiles were assessed against mammalian cells to verify cell selectivity. Anti-mycobacterial mechanisms were investigated using microfluidic live-cell imaging with time-lapse fluorescence microscopy and confocal laser-scanning microscopy. Results Unnatural amino acid incorporation was well tolerated without an appreciable effect on toxicity profiles and secondary conformations of the synthetic peptides. The modified peptides also withstood proteolytic digestion by trypsin. The all D-amino acid peptide, i(llkk)2i (II-D), displayed superior activity against all six mycobacterial strains tested, with a 4-fold increase in selectivity index as compared with the unmodified L-amino acid peptide in broth. II-D effectively reduced the intracellular bacterial burden of both drug-susceptible and MDR clinical isolates of M. tuberculosis after 4 days of treatment. Live-cell imaging studies demonstrated that II-D permeabilizes the mycobacterial membrane, while confocal microscopy revealed that II-D not only permeates the cell membrane, but also accumulates within the cytoplasm. Conclusions Unnatural amino acid modifications not only decreased the susceptibility of peptides to proteases, but also enhanced mycobacterial selectivity

DNA bipedal motor walking dynamics: an experimental and theoretical study of the dependency on step size

We present a detailed coarse-grained computer simulation and single molecule fluorescence study of the walking dynamics and mechanism of a DNA bipedal motor striding on a DNA origami. In particular, we study the dependency of the walking efficiency and stepping kinetics on step size. The simulations accurately capture and explain three different experimental observations. These include a description of the maximum possible step size, a decrease in the walking efficiency over short distances and a dependency of the efficiency on the walking direction with respect to the origami track. The former two observations were not expected and are non-trivial. Based on this study, we suggest three design modifications to improve future DNA walkers. Our study demonstrates the ability of the oxDNA model to resolve the dynamics of complex DNA machines, and its usefulness as an engineering tool for the design of DNA machines that operate in the three spatial dimensions

Disruption of drug-resistant biofilms using de novo designed short α-helical antimicrobial peptides with idealized facial amphiphilicity

The escalating threat of antimicrobial resistance has increased pressure to develop novel therapeutic strategies to tackle drug-resistant infections. Antimicrobial peptides have emerged as a promising class of therapeutics for various systemic and topical clinical applications. In this study, the de novo design of α-helical peptides with idealized facial amphiphilicities, based on an understanding of the pertinent features of protein secondary structures, is presented. Synthetic amphiphiles composed of the backbone sequence (X1Y1Y2X2)n, where X1 and X2 are hydrophobic residues (Leu or Ile or Trp), Y1 and Y2 are cationic residues (Lys), and n is the number repeat units (2 or 2.5 or 3), demonstrated potent broad-spectrum antimicrobial activities against clinical isolates of drug-susceptible and multi-drug resistant bacteria. Live-cell imaging revealed that the most selective peptide, (LKKL)3, promoted rapid permeabilization of bacterial membranes. Importantly, (LKKL)3 not only suppressed biofilm growth, but effectively disrupted mature biofilms after only 2 h of treatment. The peptides (LKKL)3 and (WKKW)3 suppressed the production of LPS-induced pro-inflammatory mediators to levels of unstimulated controls at low micromolar concentrations. Thus, the rational design strategies proposed herein can be implemented to develop potent, selective and multifunctional α-helical peptides to eradicate drug-resistant biofilm-associated infections

Analysis of current research addressing complementary use of life-cycle assessment and risk assessment for engineered nanomaterials: have lessons been learned from previous experience with chemicals?

While it is generally agreed that successful strategies to address the health and environmental impacts of engineered nanomaterials (NM) should consider the well-established frameworks for conducting life cycle assessment (LCA) and risk assessment (RA), scientific research and specific guidance on how to practically apply these methods are still very much under development. This paper evaluates how research efforts have applied LCA and RA together for NM, particularly reflecting on previous experiences with applying these methods to chemicals. Through a literature review and a separate analysis of research focused on applying LCA and RA together for NM, it appears that current research efforts have taken into account some key “lessons learned” from previous experience with chemicals while many key challenges remain for practically applying these methods to NM. We identified two main approaches for using these methods together for NM: “LC-based RA” (traditional RA applied in a life cycle perspective) and “RA-complemented LCA” (conventional LCA supplemented by RA in specific life cycle steps). Hence, the latter is the only identified approach which genuinely combines LC- and RA-based methods for NM-risk research efforts to date as the former is rather a continuation of normal RA according to standard assessment procedures (e.g. REACH). Both these approaches along with recommendations for using LCA and RA together for NM are similar to those made previously for chemicals, and thus, there does not appear to be much progress made specific for NM. We have identified one issue in particular that may be specific for NM when applying LCA and RA at this time: the need to establish proper dose metrics within both methods.JRC.I.4-Nanobioscience