Synthetic Enzymology and the Fountain of Youth: Repurposing Biology for Longevity.

Caloric restriction (CR) is an intervention that can increase maximal lifespan in organisms, but its application to humans remains challenging. A more feasible approach to achieve lifespan extension is to develop CR mimetics that target biochemical pathways affected by CR. Recent studies in the engineering and structural characterization of polyketide synthases (PKSs) have facilitated their use as biocatalysts to produce novel polyketides. Here, we show that by establishing a combinatorial biosynthetic route in Escherichia coli and exploring the substrate promiscuity of a mutant PKS from alfalfa, 413 potential anti-ageing polyketides were biosynthesized. In this approach, novel acyl-coenzyme A (CoA) precursors generated by promiscuous acid-CoA ligases were utilized by PKS to generate polyketides which were then fed to Caenorhabditis elegans to study their potential efficacy in lifespan extension. It was found that CR mimetics like resveratrol can counter the age-associated decline in mitochondrial function and increase the lifespan of C. elegans. Using the mitochondrial respiration profile of C. elegans supplemented for 8 days with 50 µM resveratrol as a blueprint, we can screen our novel polyketides for potential CR mimetics with improved potency. This study highlights the utility of synthetic enzymology in the development of novel anti-ageing therapeutics.

Anhydrous polymer-based coating with sustainable controlled release functionality for facile, efficacious impregnation, and delivery of antimicrobial peptides.

Anhydrous polymers are actively explored as alternative materials to overcome limitations of conventional hydrogel-based antibacterial coating. However, the requirement for strong organic solvent in polymerization reactions often necessitates extra protection steps for encapsulation of target biomolecules, lowering encapsulation efficiency, and increasing process complexity. This study reports a novel coating strategy that allows direct solvation and encapsulation of antimicrobial peptides (HHC36) into anhydrous polycaprolactone (PCL) polymer-based dual layer coating. A thin 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) film is layered onto the peptide-impregnated PCL as a diffusion barrier, to modulate and enhance release kinetics. The impregnated peptides are eventually released in a controlled fashion. The use of 2,2,2-trifluoroethanol (TFE), as polymerization and solvation medium, induces the impregnated peptides to adopt highly stable turned conformation, conserving peptide integrity, and functionality during both encapsulation and subsequent release processes. The dual layer coating showed sustained antibacterial functionality, lasting for 14 days. In vivo assessment using an experimental mouse wounding model demonstrated good biocompatibility and significant antimicrobial efficacy of the coating under physiological conditions. The coating was translated onto silicone urinary catheters and showed promising antibacterial efficacy, even outperforming commercial silver-based Dover cather. This anhydrous polymer-based platform holds immense potential as an effective antibacterial coating to prevent clinical device-associated infections. The simplicity of the coating process enhances its industrial viability.

Microscale Bioreactors for in situ characterization of GI epithelial cell physiology.

The development of in vitro artificial small intestines that realistically mimic in vivo systems will enable vast improvement of our understanding of the human gut and its impact on human health. Synthetic in vitro models can control specific parameters, including (but not limited to) cell types, fluid flow, nutrient profiles and gaseous exchange. They are also "open" systems, enabling access to chemical and physiological information. In this work, we demonstrate the importance of gut surface topography and fluid flow dynamics which are shown to impact epithelial cell growth, proliferation and intestinal cell function. We have constructed a small intestinal bioreactor using 3-D printing and polymeric scaffolds that mimic the 3-D topography of the intestine and its fluid flow. Our results indicate that TEER measurements, which are typically high in static 2-D Transwell apparatuses, is lower in the presence of liquid sheer and 3-D topography compared to a flat scaffold and static conditions. There was also increased cell proliferation and discovered localized regions of elevated apoptosis, specifically at the tips of the villi, where there is highest sheer. Similarly, glucose was actively transported (as opposed to passive) and at higher rates under flow.

A photopolymerized antimicrobial hydrogel coating derived from epsilon-poly-L-lysine.

Hydrogels made from epsilon-poly-l-lysine-graft-methacrylamide (EPL-MA) have been found to have impressive wide spectrum antimicrobial activity against both bacteria (specifically Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens and Staphylococcus aureus) and fungi (specifically Candida albicans and Fusarium solani). The EPL-MA hydrogel also possesses in vitro biocompatibility and EPL-MA solution is relatively non-hemolytic: the concentration needed for onset of human red blood cell (hRBC) hemolysis is 12,500 µg/mL so that the selectivity for the pathogenic microorganisms over hRBCs is 230-1560. Further, EPL-MA hydrogel can be conveniently ultraviolet-immobilized onto plasma-treated plastic surfaces to form thin highly adherent antimicrobial hydrogel coatings for medical devices and implants.

A polycationic antimicrobial and biocompatible hydrogel with microbe membrane suctioning ability.

Despite advanced sterilization and aseptic techniques, infections associated with medical implants have not been eradicated. Most present coatings cannot simultaneously fulfil the requirements of antibacterial and antifungal activity as well as biocompatibility and reusability. Here, we report an antimicrobial hydrogel based on dimethyldecylammonium chitosan (with high quaternization)-graft-poly(ethylene glycol) methacrylate (DMDC-Q-g-EM) and poly(ethylene glycol) diacrylate, which has excellent antimicrobial efficacy against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Fusarium solani. The proposed mechanism of the antimicrobial activity of the polycationic hydrogel is by attraction of sections of anionic microbial membrane into the internal nanopores of the hydrogel, like an 'anion sponge', leading to microbial membrane disruption and then microbe death. We have also demonstrated a thin uniform adherent coating of the hydrogel by simple ultraviolet immobilization. An animal study shows that DMDC-Q-g-EM hydrogel coating is biocompatible with rabbit conjunctiva and has no toxicity to the epithelial cells or the underlying stroma.

High potency and broad-spectrum antimicrobial peptides synthesized via ring-opening polymerization of alpha-aminoacid-N-carboxyanhydrides.

Antimicrobial peptides (AMPs), particularly those effective against methicillin-resistant Staphylococcus aureus ( S. aureus ) and antibiotic-resistant Pseudomonas aeruginosa ( P. aeruginosa ), are important alternatives to antibiotics. Typical peptide synthesis methods involving solid-phase sequential synthesis are slow and costly, which are obstacles to their more widespread application. In this paper, we synthesize peptides via ring-opening polymerization of alpha-amino acid N-carboxyanhydrides (NCA) using a transition metal initiator. This method offers high potential for inexpensive synthesis of substantial quantities of AMPs. Lysine (K) was chosen as the hydrophilic amino acid and alanine (A), phenylalanine (F), and leucine (L) as the hydrophobic amino acids. We synthesized five series of AMPs (i.e., P(KA), P(KL), P(KF), P(KAL), and P(KFL)), varied the hydrophobic amino acid content from 0 to 100%, and determined minimal inhibitory concentrations (MICs) against clinically important Gram-negative and Gram-positive bacteria and fungi (i.e., Escherichia coli ( E. coli ), P. aeruginosa , Serratia marcescens ( S. marcescens ), and Candida albicans ( C. albicans ). We found that P(K(10)F(7.5)L(7.5)) and P(K(10)F(15)) show the broadest activity against all five pathogens and have the lowest MICs against these pathogens. For P(K(10)F(7.5)L(7.5)), the MICs against E. coli , P. aeruginosa , S. marcescens , S. aureus , and C. albicans are 31 microg/mL, 31 microg/mL, 250 microg/mL, 31 microg/mL, and 62.5 microg/mL, while for P(K(10)F(15)) the respective MICs are 31 microg/mL, 31 microg/mL, 250 microg/mL, 31 microg/mL, and 125 microg/mL. These are lower than the MICs of many naturally occurring AMPs. The membrane depolarization and SEM assays confirm that the mechanism of microbe killing by P(K(10)F(7.5)L(7.5)) copeptide includes membrane disruption, which is likely to inhibit rapid induction of AMP-resistance in pathogens.