Antibiotic resistance is projected as one of the greatest threats to human health in the future and hence alternatives are being explored to combat resistance. Antimicrobial peptides (AMPs) have shown great promise, because use of AMPs leads bacteria to develop no or low resistance. In this review, we discuss the diversity, history and the various mechanisms of action of AMPs. Although many AMPs have reached clinical trials, to date not many have been approved by the US Food and Drug Administration (FDA) due to issues with toxicity, protease cleavage and short half-life. Some of the recent strategies developed to improve the activity and biocompatibility of AMPs, such as chemical modifications and the use of delivery systems, are also reviewed in this article.

Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine isotopic labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.

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Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria.

Functionalized anionic polystyrene latex particles with ATRP initiators were synthesized by surfactant-free shell-growth emulsion polymerization of styrene and 2-(2′-chloropropionato)ethyl acrylate (HEA-Cl). N-Isopropylacrylamide (NIPAM) was polymerized from these particles by surfaceinitiated aqueous ATRP using PMDETA/CuCl and HMTETA/CuCl catalysts to synthesize poly(Nisopropylacrylamide) (PNIPAM) brushes. The grafted latexes were characterized for molecular weight of the PNIPAM chains, grafting density, and hydrodynamic thickness of the grafted polymer layer. Molecular weights of the grafted PNIPAM chains depended on the monomer concentration, concentration of copper(II) complex, and the presence of external initiator in the reaction medium. M n of the grafted chains increases with increase in the monomer concentration and decreases with addition of copper(II) complex and external initiator. The HMTETA/CuCl catalyst produces higher molecular weight chains than PMDETA/CuCl. Molecular weights from ∼50 000 to 800 000 with low polydispersities, between 1.25 and 1.4, were achieved. The grafting density of PNIPAM on the surface increases with increasing monomer concentration and decreases with addition of copper(II) catalyst and external initiator. Block copolymerization of N,Ndimethylacrylamide from PNIPAM-grafted latex demonstrated that the chains are terminated with a chlorine atom, and the grafting reactions are taking place by the ATRP mechanism. The hydrodynamic thickness (HT) of the grafted PNIPAM layer scales as DP 0.66 (where DP ) degree of polymerization) at constant grafting density (chains/nm 2 ). The HT values for PNIPAM brushes are sensitive to temperature and salt concentration. Since the transition from extended coil to collapsed structure occurs over a range of temperature and salt concentration, it follows a second-order transition, as predicted by theory. The thickness of the collapsed brush is sensitive to the type of stimulus used to induce the phase transition.

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Phospholipids in the cell membranes of all eukaryotic cells contain phosphatidyl choline (PC) as the headgroup. Here we show that hyperbranched polyglycerols (HPGs) decorated with the 'PC-inverse' choline phosphate (CP) in a polyvalent fashion can electrostatically bind to a variety of cell membranes and to PC-containing liposomes, the binding strength depending on the number density of CP groups per macromolecule. We also show that HPG-CPs can cause cells to adhere with varying affinity to other cells, and that binding can be reversed by subsequent exposure to low molecular weight HPGs carrying small numbers of PCs. Moreover, PC-rich membranes adsorb and rapidly internalize fluorescent HPG-CP but not HPG-PC molecules, which suggests that HPG-CPs could be used as drug-delivery agents. CP-decorated polymers should find broad use, for instance as tissue sealants and in the self-assembly of lipid nanostructures.

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