Broad-Spectrum Antiviral Peptides and Polymers.

As the human cost of the pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still being witnessed worldwide, the development of broad-spectrum antiviral agents against emerging and re-emerging viruses is seen as a necessity to hamper the spread of infections. Various targets during the viral life-cycle can be considered to inhibit viral infection, from viral attachment to viral fusion or replication. Macromolecules represent a particularly attractive class of therapeutics due to their multivalency and versatility. Although several antiviral macromolecules hold great promise in clinical applications, the emergence of resistance after prolonged exposure urges the need for improved solutions. In the present article, the recent advancement in the discovery of antiviral peptides and polymers with diverse structural features and antiviral mechanisms is reviewed. Future perspectives, such as, the development of virucidal peptides/polymers and their coatings against SARS-CoV-2 infection, standardization of antiviral testing protocols, and use of artificial intelligence or machine learning as a tool to accelerate the discovery of antiviral macromolecules, are discussed.

Microbial biodiesel production from industrial organic wastes by oleaginous microorganisms: Current status and prospects.

This review aims to encourage the technical development of microbial biodiesel production from industrial-organic-wastes-derived volatile fatty acids (VFAs). To this end, this article summarizes the current status of several key technical steps during microbial biodiesel production, including (1) acidogenic fermentation of bio-wastes for VFA collection, (2) lipid accumulation in oleaginous microorganisms, (3) microbial lipid extraction, (4) transesterification of microbial lipids into crude biodiesel, and (5) crude biodiesel purification. The emerging membrane-based bioprocesses such as electrodialysis, forward osmosis and membrane distillation, are promising approaches as they could help tackle technical challenges related to the separation and recovery of VFAs from the fermentation broth. The genetic engineering and metabolic engineering approaches could be applied to design microbial species with higher lipid productivity and rapid growth rate for enhanced fatty acids synthesis. The enhanced in situ transesterification technologies aided by microwave, ultrasound and supercritical solvents are also recommended for future research. Technical limitations and cost-effectiveness of microbial biodiesel production from bio-wastes are also discussed, in regard to its potential industrial development. Based on the overview on microbial biodiesel technologies, an integrated biodiesel production line incorporating all the critical technical steps is proposed for unified management and continuous optimization for highly efficient biodiesel production.

The type VII secretion system protects Staphylococcus aureus against antimicrobial host fatty acids.

The Staphylococcus aureus type VII secretion system (T7SS) exports several proteins that are pivotal for bacterial virulence. The mechanisms underlying T7SS-mediated staphylococcal survival during infection nevertheless remain unclear. Here we report that S. aureus lacking T7SS components are more susceptible to host-derived antimicrobial fatty acids. Unsaturated fatty acids such as linoleic acid (LA) elicited an increased inhibition of S. aureus mutants lacking T7SS effectors EsxC, EsxA and EsxB, or the membrane-bound ATPase EssC, compared to the wild-type (WT). T7SS mutants generated in different S. aureus strain backgrounds also displayed an increased sensitivity to LA. Analysis of bacterial membrane lipid profiles revealed that the esxC mutant was less able to incorporate LA into its membrane phospholipids. Although the ability to bind labelled LA did not differ between the WT and mutant strains, LA induced more cell membrane damage in the T7SS mutants compared to the WT. Furthermore, proteomic analyses of WT and mutant cell fractions revealed that, in addition to compromising membranes, T7SS defects induce oxidative stress and hamper their response to LA challenge. Thus, our findings indicate that T7SS contribute to maintaining S. aureus membrane integrity and homeostasis when bacteria encounter antimicrobial fatty acids.

Methane yield enhancement of mesophilic and thermophilic anaerobic co-digestion of algal biomass and food waste using algal biochar: Semi-continuous operation and microbial community analysis.

The impact of algal biochar addition on mesophilic and thermophilic anaerobic co-digestion of algal biomass and food waste was investigated with a focus on semi-continuous operations and functional microbial communities. Under batch co-digestion, the highest co-digestion synergy was observed for a mixture of 25% food waste and 75% algal biomass. During semi-continuous co-digestion of 25% food waste-75% algal biomass mixture, biochar amended digesters exhibited a 12-54% increase in average methane yield (275.8-394.6 mL/gVS) compared to the controls. Elevated temperature induced narrow distributions of volatile fatty acids (VFAs) by inhibiting the production of branched VFAs. Genus Proteiniphilum was selectively enriched by 3.2 folds in mesophilic digesters with biochar amendment while genus Defluviitoga was selectively enriched in thermophilic digesters due to elevated temperature. Methanogenic communities were significantly different in mesophilic and thermophilic digesters. Biochar amendment contributed to shifts in the predominant methanogens leading to a more balanced state of two methanogenic pathways.

Stimuli-responsive membrane activity of cyclic-peptide-polymer conjugates.

Cyclic peptide nanotubes (CPNT) consisting of an even number of amino acids with an alternating chirality are highly interesting materials in a biomedical context due to their ability to insert themselves into cellular membranes. However, unwanted unspecific interactions between CPNT and non-targeted cell membranes are a major drawback. To solve this issue we have synthetized a series of CPNT-polymer conjugates with a cleavable covalent connection between macromolecule and peptide. As a result, the polymers form a stabilizing and shielding shell around the nanotube that can be cleaved on demand to generate membrane active CPNT from non-active conjugates. This approach enables us to control the stacking and lateral aggregation of these materials, thus leading to stimuli responsive membrane activity. Moreover, upon activation, the systems can be adjusted to form nanotubes with an increased length instead of aggregates. We were able to study the dynamics of these systems in detail and prove the concept of stimuli responsive membrane interaction using CPNT-polymer conjugates to permeabilize liposomes as well as mammalian cell membranes.

Targeting intracellular, multi-drug resistant Staphylococcus aureus with guanidinium polymers by elucidating the structure-activity relationship.

Intracellular persistence of bacteria represents a clinical challenge as bacteria can thrive in an environment protected from antibiotics and immune responses. Novel targeting strategies are critical in tackling antibiotic resistant infections. Synthetic antimicrobial peptides (SAMPs) are interesting candidates as they exhibit a very high antimicrobial activity. We first compared the activity of a library of ammonium and guanidinium polymers with different sequences (statistical, tetrablock and diblock) synthesized by RAFT polymerization against methicillin-resistant S. aureus (MRSA) and methicillin-sensitive strains (MSSA). As the guanidinium SAMPs were the most potent, they were used to treat intracellular S. aureus in keratinocytes. The diblock structure was the most active, reducing the amount of intracellular MSSA and MRSA by two-fold. We present here a potential treatment for intracellular, multi-drug resistant bacteria, using a simple and scalable strategy.

Investigating Cell Uptake of Guanidinium-Rich RAFT Polymers: Impact of Comonomer and Monomer Distribution.

A range of well-defined guanidinium-rich linear polymers with demonstrable efficiency for cellular internalization were developed. A protected guanidinium-functional acrylamide monomer (di-Boc-guanidinium ethyl acrylamide, GEAdiBoc) was synthesized and then polymerized via RAFT polymerization to yield well-defined homopolymers, which were then deprotected and functionalized with a fluorescein dye to observe and quantify their cellular uptake. The cellular uptake of these homopolymers was first compared to analogous polyarginines, which are commonly used in modern drug delivery. Following this, a range of well-defined guanidinium-rich copolymers were prepared in which the monomer distribution was varied using a convenient one-pot sequential RAFT polymerization approach. Systematic quantification of the cell uptake of these compounds, supported by fluorescent confocal microscopy data, revealed that while the overall hydrophobicity of the resulting copolymers has a direct impact on the amount of copolymer taken up by cells, the distribution of monomers has an influence on both the extent of uptake and the relative extent to which each route of internalization (endocytosis vs direct translocation) is exploited.

Sequence Control as a Powerful Tool for Improving the Selectivity of Antimicrobial Polymers.

Antimicrobial polymers appear as a promising alternative to tackle the current development of bacterial resistance against conventional antibiotics as they rely on bacterial membrane disruption. This study investigates the effect of segmentation of hydrophobic and cationic functionalities on antimicrobial polymers over their selectivity between bacteria and mammalian cells. Using RAFT technology, statistical, diblock, and highly segmented multiblock copolymers were synthesized in a controlled manner. Polymers were analyzed by HPLC, and the segmentation was found to have a significant influence on their overall hydrophobicity. In addition, the amount of incorporated cationic comonomer was varied to yield a small library of bioactive macromolecules. The antimicrobial properties of these compounds were probed against pathogenic bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis), and their biocompatibility was tested using hemolysis and erythrocyte aggregation assays, as well as mammalian cell viability assays. In all cases, diblock and multiblock copolymers were found to outperform statistical copolymers, and for polymers with a low content of cationic comonomer, the multiblock showed a tremendously increased selectivity for P. aeruginosa and S. epidermidis compared to its statistical and diblock analogue. This work highlights the remarkable effect of segmentation on both the physical properties of the materials as well as their interaction with biological systems. Due to the outstanding selectivity of multiblock copolymers toward certain bacteria strains, the presented materials are a promising platform for the treatment of infections and a valuable tool to combat antimicrobial resistance.

Antimicrobial Polymers: Mimicking Amino Acid Functionali ty, Sequence Control and Three-dimensional Structure of Host-defen se Peptides.

Peptides and proteins control and direct all aspects of cellular function and communication. Having been honed by nature for millions of years, they also typically display an unsurpassed specificity for their biological targets. This underlies the continued focus on peptides as promising drug candidates. However, the development of peptides into viable drugs is hampered by their lack of chemical and pharmacokinetic stability and the cost of large scale production. One method to overcome such hindrances is to develop polymer systems that are able to retain the important structural features of these biologically active peptides, while being cheaper and easier to produce and manipulate chemically. This review illustrates these principles using examples of polymers designed to mimic antimicrobial host-defence peptides. The host-defence peptides have been identified as some of the most important leads for the next generation of antibiotics as they typically exhibit broad spectrum antimicrobial ability, low toxicity toward human cells and little susceptibility to currently known mechanisms of bacterial resistance. Their movement from the bench to clinic is yet to be realised, however, due to the limitations of these peptides as drugs. The literature provides a number of examples of polymers that have been able to mimic these peptides through all levels of structure, starting from specific amino acid sidechains, through to more global features such as overall charge, molecular weight and threedimensional structure (e.g. α-helical). The resulting optimised polymers are able retain the activity profile of the peptides, but within a synthetic macromolecular construct that may be better suited to the development of a new generation of antimicrobial therapeutics. Such work has not only produced important new leads to combat the growing threat of antibiotic resistance, but may also open up new ways for polymers to mimic other important classes of biologically active peptides.