Secondary Structure Stabilization of Macrocyclic Antimicrobial Peptides via Cross-Link Swapping.

Lactam cross-links have been employed to stabilize the helical secondary structure and enhance the activity and physiological stability of antimicrobial peptides; however, stabilization of ß-sheets via lactamization has not been observed. In the present study, lactams between the side chains of C- and N-terminal residues have been used to stabilize the ß-sheet conformation in a short ten-residue analogue of chicken angiogenin-4. Designed using a combination of molecular dynamics simulations and Markov state models, the lactam cross-linked peptides are shown to adopt stabilized ß-sheet conformations consistent with simulated structures. Replacement of the peptide side-chain Cys-Cys disulfide by a lactam cross-link enhanced the broad-spectrum antibacterial activity compared to the parent peptide and exhibited greater propensity to induce proinflammatory activity in macrophages. The combination of molecular simulations and conformational and biological analyses of the synthetic peptides provides a useful paradigm for the rational design of therapeutically active peptides with constrained ß-sheet structures.

Antibacterial activities of physiologically stable, self-assembled peptide nanoparticles.

In this study, we report that host defense protein-derived ten amino acid long disulfide-linked peptides self-assemble in the form of ß-sheets and ß-turns, and exhibit concentration-dependent self-assembly in the form of nanospheres, termed as disulfide linked nanospheres (DSNs). As expected, bare DSNs are prone to aggregation in ionic solutions and in the presence of serum proteins. To yield physiologically stable self-assembled peptide-based materials, DSNs are stabilized in the form of supramolecular assemblies using ß-cyclodextrins (ß-CD) and fucoidan, as delivery carriers. The inclusion complexes of DSNs with ß-CD (ß-CD-DSN) and electrostatic complexation of fucoidan with DSNs (FC-DSN) stabilizes the secondary structure of DSNs. Comparison of ß-CD-DSNs with FC-DSNs reveals that inclusion complexes of DSNs formed in the presence of ß-CD are highly stable under physiological conditions, show high cellular uptake, exhibit bacterial flocculation, and enhance antibacterial efficacies of DSNs in a range of Gram-positive and Gram-negative bacteria.

Bacterial-Specific Aggregation and Killing of Immunomodulatory Host Defense Peptides.

This study involves the design and development of disulfide bridge-linked antimicrobial peptides using the host defense protein Angiogenin 4 (chAng4) as a template. The mini peptides derived from chAng4 (mCA4s) were evaluated for their antibacterial efficacies in various pathogenic bacterial strains, and the role of the oxidation state of thiols in the peptide sequence and its implication on antibacterial properties were explored. A remarkable property of these synthetic mCA4 peptides is their capability to flocculate bacteria and mediate bacterial-specific killing, in the absence of any other external stimulus. mCA4s were further evaluated for their cellular uptake, hemolytic activities, toxicities, and immunomodulatory activities in different eukaryotic cell lines. The results indicate that disulfide bridge-containing cationic amphipathic peptides show superior antibacterial efficacies, are nontoxic and nonhemolytic, and mediate bacterial flocculation and killing, in the absence of external stimuli.

Quantification of landfill gas generation and renewable energy potential in arid countries: Case study of Bahrain.

Sustainable solid waste management can provide pathways for renewable energy generation. The Kingdom of Bahrain has witnessed burgeoning municipal solid waste (MSW) generation rate due to socio-economic development. The authorities of this Small Island Developing State, which is located in arid environment, plan to produce 5% of the total electricity demand from renewable energy sources by 2025 and then double it to 10% by 2035. The US Environmental Protection Agency's Landfill Gas Emission Model software was used to estimate the generation of biogas from MSW at the Askar Landfill site. Results envisaged that maximum landfill gas (LFG) emission rates will be in 2020 following landfill closure by the end of 2019, as an intentional scenario, with a maximum electricity generation potential of 57.4 GWh that could provide power to 488 households. Revenues from carbon credits and electricity sales were US$97.8 million and US$64.8 million, respectively, for the period 2020-2035. The internal combustion engine exhibited the most viable option based on economic analysis of the cost of alternative LFG energy recovery technologies. Our work highlights the potential to use LFG-to-energy technologies to reduce the carbon footprint in arid climates for developing countries with substantial electricity subsidization.

Identification, tissue characterization, and innate immune role of Angiogenin-4 expression in young broiler chickens.

Intestinal epithelial cells are major producers of antimicrobial proteins, which play an important role in innate immunity. In addition to defensins, the Ribonuclease A superfamily includes important antimicrobial proteins involved in host-defense mechanisms in vertebrates. Angiogenin-4 (Ang4), a member of this RNase superfamily, has been demonstrated to be secreted by Paneth cells in mice. We have successfully cloned and characterized a new chicken gene (chAng4), found for the first time in a nonmammalian species, from intestinal epithelial and lymphoid cells. Characterization of chAng4 revealed 99% nucleotide and 97% amino acid sequence homology to mouse Ang4. Similar functional regions were identified, suggesting a role in innate immunity and regulation of gut microbiota. Furthermore, the mRNA expression pattern of chAng4 was studied in broilers in the presence or absence of beneficial bacteria (probiotics) and organic acids. The results showed that one-day-old chickens expressed low levels of Ang4 in almost all the evaluated tissues (crop, proventriculus, duodenum, jejunum, ileum, and cecal tonsils), except in the bursa of Fabricius that presented the highest expression level. The addition of probiotics and organic acids for either 7 or 14 consecutive days demonstrated a direct effect of probiotics and organic acids on chAng4 expression; moreover, broilers receiving probiotics and organic acids for only 7 D showed higher levels of chAng4 expression compared with those treated for 14 D. Broilers without treatment had a constant high level of expression in cecal tonsils and bursa. In conclusion, we were able to identify and characterize a new antimicrobial gene in chickens (chAng4) throughout the gastrointestinal tract. chAng4 mRNA gene expression was associated with the presence of naturally occurring and supplemented (probiotic) bacteria. The encoded protein might have a potential bactericidal effect against intestinal nonpathogenic and pathogenic microbes, modulating the intestinal microbiota and the innate immunity, and thereby may help minimize the use of antibiotics in poultry feed.

Diatoms embedded, self-assembled carriers for dual delivery of chemotherapeutics in cancer cell lines.

The concept of dual drug delivery to treat relapsing tumors is a well-studied approach to improve the antitumor efficacies and to reduce the side effects of single drug chemotherapeutic treatments. One of the major issues with dual drug delivery to treat drug resistant tumors is the concentration and ratio dependent antagonistic behavior of two drugs, which may reverse the anticancer efficacies of individual chemotherapeutics and stimulate the growth of tumor cells. In this paper, we address this issue by developing diatomaceous earth embedded core shell materials, which are capable of encapsulating two chemotherapeutic drugs at constant molar ratios, in different compartments of a single drug delivery carrier. The encapsulation of each drug in different compartments of delivery carrier (core of diatoms versus shell of cyclodextrin) then controls the release rate of both drugs in situ, and maintains the optimal molar ratios required for their synergistic outcomes in vitro.