Synthetic Peptide Libraries Designed From a Minimal Alpha-Helical Domain of AS-48-Bacteriocin Homologs Exhibit Potent Antibacterial Activity.

The circularized bacteriocin enterocin AS-48 produced by Enterococcus sp. exhibits antibacterial activity through membrane disruption. The membrane-penetrating activity of enterocin AS-48 has been attributed to a specific alpha-helical region on the circular peptide. Truncated, linearized forms containing these domains have been shown to preserve limited bactericidal activity. We utilized the amino acid sequence of the active helical domain of enterocin AS-48 to perform a homology-based search of similar sequences in other bacterial genomes. We identified similar domains in three previously uncharacterized AS-48-like bacteriocin genes in Clostridium sordellii, Paenibacillus larvae, and Bacillus xiamenensis. Enterocin AS-48 and homologs from these bacterial species were used as scaffolds for the design of a minimal peptide library based on the active helical domain of each bacteriocin sequence. 95 synthetic peptide variants of each scaffold peptide, designated Syn-enterocin, Syn-sordellicin, Syn-larvacin, and Syn-xiamensin, were designed and synthesized from each scaffold sequence based on defined biophysical parameters. A total of 384 total peptides were assessed for antibacterial activity against Gram-negative and Gram-positive bacteria. Minimal Inhibitory Concentrations (MICs) as low as 15.6 nM could be observed for the most potent peptide candidate tested, with no significant cytotoxicity to eukaryotic cells. Our work demonstrates for the first time a general workflow of using minimal domains of natural bacteriocin sequences as scaffolds to design and rapidly synthesize a library of bacteriocin-based antimicrobial peptide variants for evaluation.

Synthetic Antimicrobial Peptide Tuning Permits Membrane Disruption and Interpeptide Synergy.

The ribosomally produced antimicrobial peptides of bacteria (bacteriocins) represent an unexplored source of membrane-active antibiotics. We designed a library of linear peptides from a circular bacteriocin and show that pore-formation dynamics in bacterial membranes are tunable via selective amino acid substitution. We observed antibacterial interpeptide synergy indicating that fundamentally altering interactions with the membrane enables synergy. Our findings suggest an approach for engineering pore-formation through rational peptide design and increasing the utility of novel antimicrobial peptides by exploiting synergy.

Novel antimicrobial peptide discovery using machine learning and biophysical selection of minimal bacteriocin domains.

Bacteriocins, the ribosomally produced antimicrobial peptides of bacteria, represent an untapped source of promising antibiotic alternatives. However, bacteriocins display diverse mechanisms of action, a narrow spectrum of activity, and inherent challenges in natural product isolation making in vitro verification of putative bacteriocins difficult. A subset of bacteriocins exert their antimicrobial effects through favorable biophysical interactions with the bacterial membrane mediated by the charge, hydrophobicity, and conformation of the peptide. We have developed a pipeline for bacteriocin-derived compound design and testing that combines sequence-free prediction of bacteriocins using machine learning and a simple biophysical trait filter to generate 20 amino acid peptides that can be synthesized and evaluated for activity. We generated 28,895 total 20-mer candidate peptides and scored them for charge, α-helicity, and hydrophobic moment. Of those, we selected 16 sequences for synthesis and evaluated their antimicrobial, cytotoxicity, and hemolytic activities. Peptides with the overall highest scores for our biophysical parameters exhibited significant antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa. Our combined method incorporates machine learning and biophysical-based minimal region determination to create an original approach to swiftly discover bacteriocin candidates amenable to rapid synthesis and evaluation for therapeutic use.

Relating n-pentane isomerization activity to the tungsten surface density of WO(x)/ZrO2.

Zirconia-supported tungsten oxide (WO(x)/ZrO(2)) is considered an important supported metal oxide model acid catalyst, for which structure-property relationships have been studied for numerous acid-catalyzed reactions. The catalytic activity for xylene isomerization, alcohol dehydration, and aromatic acylation follows a volcano-shape dependence on tungsten surface density. However, WO(x)/ZrO(2) has not been studied for more acid-demanding reactions, like n-pentane isomerization, with regard to surface density dependence. In this work, WO(x)/ZrO(2) was synthesized using commercially available amorphous ZrO(x)(OH)(4-2x) and model crystalline ZrO(2) as support precursors. They were analyzed for n-pentane isomerization activity and selectivity as a function of tungsten surface density, catalyst support type, and calcination temperature. Amorphous ZrO(x)(OH)(4-2x) led to WO(x)/ZrO(2) (WZrOH) that exhibited maximum isomerization activity at ∼5.2 W·nm(-2), and the crystalline ZrO(2) led to a material (WZrO(2)) nearly inactive at all surface densities. Increasing the calcination temperature from 773 to 973 K increased the formation of 0.8-1 nm Zr-WO(x) clusters detected through direct imaging on an aberration-corrected high-resolution scanning transmission electron microscope (STEM). Calcination temperature further increased catalytic activity by at least two times. Brønsted acidity was not affected but Lewis acidity decreased in number, as quantified via pyridine adsorption infrared spectroscopy. WO(x)/ZrO(2) exhibited isomerization activity that peaked within the first 2 h time-on-stream, which may be due to Zr-WO(x) clusters undergoing an activation process.

Calcium intake and 10-year weight change in middle-aged adults.

OBJECTIVE: The goal of this study was to examine the purported effects of calcium on modulating body weight. DESIGN: Retrospective data was used to assess the relationship between calcium intake and weight change over an 8- to 12-year period among middle-aged adults. SUBJECTS: Participants were 10,591 men and women aged 53 to 57 years recruited during 2000-2002. STATISTICAL ANALYSIS: Linear regression was used to model associations of 10-year weight change and calcium intake, adjusted for weight at age 45 years, energy intake, physical activity, and other factors. RESULTS: Calcium intake was associated with 10-year weight change only in women. Women with current calcium supplement dose of >500 mg/day had a 10-year weight gain of 5.1 kg (95% confidence interval 4.7 to 5.5) compared to 6.9 kg (95% confidence interval 6.5 to 7.4) among nonusers (P for trend=0.001). Trends were similar for total calcium intake from diet plus supplements (P for trend=0.001). Dietary calcium alone had no significant effect on 10-year weight change. CONCLUSIONS: Increasing total calcium intake, in the form of calcium supplementation, may be beneficial to weight maintenance, especially in women during midlife.