Designing Coiled Coils for Heterochiral Complexation to Enhance Binding and Enzymatic Stability.

Coiled coils, commonly found in native proteins, are helical motifs important for mediating intermolecular interactions. While coiled coils are attractive for use in new therapies and biomaterials, the lack of enzymatic stability of naturally occurring l-peptides may limit their implementation in biological environments. d-peptides are of interest for biomedical applications as they are resistant to enzymatic degradation and recent reports indicate that stereochemistry-driven interactions, achieved by blending d- and l-peptides, yield access to a greater range of binding affinities and a resistance to enzymatic degradation compared to l-peptides alone. To our knowledge, this effect has not been studied in coiled coils. Here, we investigate the effects of blending heterochiral E/K coiled coils, which are a set of coiled coils widely used in biomaterials. We found that we needed to redesign the coiled coils from a repeating pattern of seven amino acids (heptad) to a repeating pattern of 11 amino acids (hendecad) to make them more amenable to heterochiral complex formation. The redesigned hendecad coiled coils form both homochiral and heterochiral complexes, where the heterochiral complexes have stronger heats of binding between the constituent peptides and are more enzymatically stable than the analogous homochiral complexes. Our results highlight the ability to design peptides to make them amenable to heterochiral complexation, so as to achieve desirable properties like increased enzymatic stability and stronger binding. Looking forward, understanding how to engineer peptides to utilize stereochemistry as a materials design tool will be important to the development of next-generation therapeutics and biomaterials.

Peptide Stereocomplexation Orchestrates Supramolecular Assembly of Hydrogel Biomaterials.

Stereocomplexation, or specific interactions among complementary stereoregular macromolecules, is burgeoning as an increasingly impactful design tool, exerting exquisite control of material structure and properties. Since stereocomplexation of polymers produces remarkable transformations in mechanics, morphology, and degradation, we sought to leverage stereocomplexation to tune these properties in peptide-based biomaterials. We found that blending the pentapeptides l- and d-KYFIL triggers dual mechanical and morphological transformations from stiff fibrous hydrogels into less stiff networks of plates, starkly contrasting prior reports that blending l- and d-peptides produces stiffer fibrous hydrogels than the individual constituents. The morphological transformation of KYFIL in phosphate-buffered saline from fibers that entangle into hydrogels to plates that cannot entangle explains the accompanying mechanical transformation. Moreover, the blends shield l-KYFIL from proteolytic degradation, producing materials with comparable proteolytic stability to d-KYFIL but with distinct 2D plate morphologies that in biomaterials may promote unique therapeutic release profiles and cell behavior. To confirm that these morphological, mechanical, and stability changes arise from differences in molecular packing as in polymer stereocomplexation, we acquired X-ray diffraction patterns, which showed l- and d-KYFIL to be amorphous and their blends to be crystalline. Stereocomplexation is particularly apparent in pure water, where l- and d-KYFIL are soluble random coils, and their blends form ß-sheets and gel within minutes. Our results highlight the role of molecular details, such as peptide sequence, in determining the material properties resulting from stereocomplexation. Looking forward, the ability of stereocomplexation to orchestrate supramolecular assembly and tune application-critical properties champions stereochemistry as a compelling design consideration.

The chemistry of ynol and thioynol ethers.

Alkynyl ethers and alkynyl thioethers ('ynol ethers' and 'thioynol ethers') are appealing building-blocks in synthetic chemistry due to their ease of manipulation and predictable reactivity. Until recently however, their potential has remained underexploited due to difficulties in preparation and isolation. Although recent advances in synthetic chemistry have highlighted various applications for ynol ethers, the equivalent thioynol examples have been rather less exploited despite a unique and fascinating reactivity profile. Although superficially the chemistry of alkynyl ethers and their sulfide counterparts are similar, close examination of their chemistry reveals important differences which can be exploited by the synthetic chemist. This review will examine the preparation of both classes of compound and examine their reactivity to highlight their powerful synthetic applications. Particular focus will be made of thiynol ethers whose chemistry exhibits some fascinating differences compared to their oxygen counterparts and have immense untapped potential for synthetic chemistry.

Transition-metal-free synthesis of ynol ethers and thioynol ethers via displacement at sp centers: a revised mechanistic pathway.

We present here valuable extensions to our previous work in preparing highly functionalized, heteroatom-substituted alkynes via displacement at an sp center. Our results show that a wide range of ynol ethers can be prepared by the same methodology and that the same protocol can be applied to the synthesis of synthetically useful thioynol ethers. We also present new observations that have led us to revise our original hypothesis in favor of a pathway involving radical intermediates.

Net anionic poly(ß-amino ester)s: synthesis, pH-dependent behavior, and complexation with cationic cargo.

As hydrolytically-labile, traditionally-cationic polymers, poly(ß-amino ester)s (PBAEs) adeptly complex anionic compounds such as nucleic acids, and release their cargo as the polymer degrades. To engineer fully-degradable polyelectrolyte complexes and delivery vehicles for cationic therapeutics, we sought to invert PBAE net charge to generate net anionic PBAEs. Since PBAEs can carry up to a net charge of +1 per tertiary amine, we synthesized a series of alkyne-functionalized PBAEs that allowed installation of 2 anionic thiol-containing molecules per tertiary amine via a radical thiol-yne reaction. Finding dialysis in aqueous solution to lead to PBAE degradation, we developed a preparative size exclusion chromatography method to remove unreacted thiol from the net anionic PBAEs without triggering hydrolysis. The net anionic PBAEs display non-monotonic solution behavior as a function of pH, being more soluble at pH 4 and 10 than in intermediate pH ranges. Like cationic PBAEs, these net anionic PBAEs degrade in aqueous environments with hydrophobic content-dependent hydrolysis, as determined by 1H NMR spectroscopy. Further, these net anionic PBAEs form complexes with the cationic peptide (GR)10, which disintegrate over time as the polymer hydrolyzes. Together, these studies outline a synthesis and purification route to make previously inaccessible net anionic PBAEs with tunable solution and degradation behavior, allowing for user-determined complexation and release rates and providing opportunities for degradable polyelectrolyte complexes and cationic therapeutic delivery.

Disparate Regions of the Human Chemokine CXCL10 Exhibit Broad-Spectrum Antimicrobial Activity against Biodefense and Antibiotic-Resistant Bacterial Pathogens.

CXCL10 is a pro-inflammatory chemokine produced by the host in response to microbial infection. In addition to canonical, receptor-dependent actions affecting immune-cell migration and activation, CXCL10 has also been found to directly kill a broad range of pathogenic bacteria. Prior investigations suggest that the bactericidal effects of CXCL10 occur through two distinct pathways that compromise the cell envelope. These observations raise the intriguing notion that CXCL10 features a separable pair of antimicrobial domains. Herein, we affirm this possibility through peptide-based mapping and structure/function analyses, which demonstrate that discrete peptides derived from the N- and C-terminal regions of CXCL10 mediate bacterial killing. The N-terminal derivative, peptide P1, exhibited marked antimicrobial activity against Bacillus anthracis vegetative bacilli and spores, as well as antibiotic-resistant clinical isolates of Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecium, and Staphylococcus aureus, among others. At bactericidal concentrations, peptide P1 had a minimal degree of chemotactic activity, but did not cause red blood cell hemolysis or cytotoxic effects against primary human cells. The C-terminal derivative, peptide P9, exhibited antimicrobial effects, but only against Gram-negative bacteria in low-salt medium─conditions under which the peptide can adopt an α-helical conformation. The introduction of a hydrocarbon staple induced and stabilized α-helicity; accordingly, stapled peptide P9 displayed significantly improved bactericidal effects against both Gram-positive and Gram-negative bacteria in media containing physiologic levels of salt. Together, our findings identify and characterize the antimicrobial regions of CXCL10 and functionalize these novel determinants as discrete peptides with potential therapeutic utility against difficult-to-treat pathogens.

Transition-metal-free synthesis of aryl-substituted tert-butyl ynol ethers through addition/elimination substitution at an sp centre.

Nucleophilic attack of an alkoxide on an alkynyl sulfonamide leads to displacement of the sulfonamide at the sp centre and isolation of the ynol ether in good yield in a single operation. The mechanistic pathway has been probed by the use of coordinating additives, (13)C-labelling experiments and ab initio calculations, which indicated that an addition/elimination mechanism is in operation.

Microbial utilization of crude glycerol for the production of value-added products.

Energy fuels for transportation and electricity generation are mainly derived from finite and declining reserves of fossil hydrocarbons. Fossil hydrocarbons are also used to produce a wide range of organic carbon-based chemical products. The current global dependency on fossil hydrocarbons will not be environmentally or economically sustainable in the long term. Given the future pessimistic prospects regarding the complete dependency on fossil fuels, political and economic incentives to develop carbon neutral and sustainable alternatives to fossil fuels have been increasing throughout the world. For example, interest in biodiesel has undergone a revival in recent times. However, the disposal of crude glycerol contaminated with methanol, salts, and free fatty acids as a by-product of biodiesel production presents an environmental and economic challenge. Although pure glycerol can be utilized in the cosmetics, tobacco, pharmaceutical, and food industries (among others), the industrial purification of crude glycerol is not economically viable. However, crude glycerol could be used as an organic carbon substrate for the production of high-value chemicals such as 1,3-propanediol, organic acids, or polyols. Microorganisms have been employed to produce such high-value chemicals and the objective of this article is to provide an overview of studies on the utilization of crude glycerol by microorganisms for the production of economically valuable products. Glycerol as a by-product of biodiesel production could be used a feedstock for the manufacture of many products that are currently produced by the petroleum-based chemical industry.

Biomaterials via peptide assembly: Design, characterization, and application in tissue engineering.

A core challenge in biomaterials, with both fundamental significance and technological relevance, concerns the rational design of bioactive microenvironments. Designed properly, peptides can undergo supramolecular assembly into dynamic, physical hydrogels that mimic the mechanical, topological, and biochemical features of native tissue microenvironments. The relatively facile, inexpensive, and automatable preparation of peptides, coupled with low batch-to-batch variability, motivates the expanded use of assembling peptide hydrogels for biomedical applications. Integral to realizing dynamic peptide assemblies as functional biomaterials for tissue engineering is an understanding of the molecular and macroscopic features that govern assembly, morphology, and biological interactions. In this review, we first discuss the design of assembling peptides, including primary structure (sequence), secondary structure (e.g., α-helix and ß-sheets), and molecular interactions that facilitate assembly into multiscale materials with desired properties. Next, we describe characterization tools for elucidating molecular structure and interactions, morphology, bulk properties, and biological functionality. Understanding of these characterization methods enables researchers to access a variety of approaches in this ever-expanding field. Finally, we discuss the biological properties and applications of peptide-based biomaterials for engineering several important tissues. By connecting molecular features and mechanisms of assembling peptides to the material and biological properties, we aim to guide the design and characterization of peptide-based biomaterials for tissue engineering and regenerative medicine. STATEMENT OF SIGNIFICANCE: Engineering peptide-based biomaterials that mimic the topological and mechanical properties of natural extracellular matrices provide excellent opportunities to direct cell behavior for regenerative medicine and tissue engineering. Here we review the molecular-scale features of assembling peptides that result in biomaterials that exhibit a variety of relevant extracellular matrix-mimetic properties and promote beneficial cell-biomaterial interactions. Aiming to inspire and guide researchers approaching this challenge from both the peptide biomaterial design and tissue engineering perspectives, we also present characterization tools for understanding the connection between peptide structure and properties and highlight the use of peptide-based biomaterials in neural, orthopedic, cardiac, muscular, and immune engineering applications.

User-defined, temporal presentation of bioactive molecules on hydrogel substrates using supramolecular coiled coil complexes.

The ability to spatiotemporally control the presentation of relevant biomolecules in synthetic culture systems has gained significant attention as researchers strive to recapitulate the endogenous extracellular matrix (ECM) in vitro. With the biochemical composition of the ECM constantly in flux, the development of platforms that allow for user-defined control of bioactivity is desired. Here, we reversibly conjugate bioactive molecules to hydrogel-based substrates through supramolecular coiled coil complexes that form between complementary peptides. Our system employs a thiolated peptide for tethering to hydrogel surfaces (T-peptide) through a spatially-controlled photomediated click reaction. The complementary association peptide (A-peptide), containing the bioactive domain, forms a heterodimeric coiled coil complex with the T-peptide. Addition of a disruptor peptide (D-peptide) engineered specifically to target the A-peptide outcompetes the T-peptide for binding, and removes the A-peptide and the attached bioactive motif from the scaffold. We use this platform to demonstrate spatiotemporal control of biomolecule presentation within hydrogel systems in a repeatable process that can be extended to adhesive motifs for cell culture. NIH 3T3 fibroblasts seeded on hyaluronic acid hydrogels and polyethylene glycol-based fibrous substrates supramolecularly functionalized with an RGD motif demonstrated significant cell spreading over their nonfunctionalized counterparts. Upon displacement of the RGD motif, fibroblasts occupied less area and clustured on the substrates. Taken together, this platform enables facile user-defined incorporation and removal of biomolecules in a repeatable process for controlled presentation of bioactivity in engineered culture systems.

Molecular engineering of antimicrobial peptide (AMP)-polymer conjugates.

As antimicrobial resistance becomes an increasing threat, bringing significant economic and health burdens, innovative antimicrobial treatments are urgently needed. While antimicrobial peptides (AMPs) are promising therapeutics, exhibiting high activity against resistant bacterial strains, limited stability and toxicity to mammalian cells has hindered clinical development. Attaching AMPs to polymers provides opportunities to present AMPs in a way that maximizes bacterial killing while enhancing compatibility with mammalian cells, stability, and solubility. Conjugation of an AMP to a linear hydrophilic polymer yields the desired improvements in stability, mammalian cell compatibility, and solubility, yet often markedly reduces bactericidal effects. Non-linear polymer architectures and supramolecular assemblies that accommodate multiple AMPs per polymer chain afford AMP-polymer conjugates that strike a superior balance of antimicrobial activity, mammalian cell compatibility, stability, and solubility. Therefore, we review the design criteria, building blocks, and synthetic strategies for engineering AMP-polymer conjugates, emphasizing the connection between molecular architecture and antimicrobial performance to inspire and enable further innovation to advance this emerging class of biomaterials.

Stimuli-Responsive Pure Protein Organogel Sensors and Biocatalytic Materials.

Utilizing protein chemistry in organic solvents has important biotechnology applications. Typically, organic solvents negatively impact protein structure and function. Immobilizing proteins via cross-links to a support matrix or to other proteins is a common strategy to preserve the native protein function. Recently, we developed methods to fabricate macroscopic responsive pure protein hydrogels by lightly cross-linking the proteins with glutaraldehyde for chemical sensing and enzymatic catalysis applications. The water in the resulting protein hydrogel can be exchanged for organic solvents. The resulting organogel contains pure organic solvents as their mobile phases. The organogel proteins retain much of their native protein function, i.e., protein-ligand binding and enzymatic activity. A stepwise ethylene glycol (EG) solvent exchange was performed to transform these hydrogels into organogels with a very low vapor pressure mobile phase. These responsive organogels are not limited by solvent/mobile phase evaporation. The solvent exchange to pure EG is accompanied by a volume phase transition (VPT) that decreases the organogel volume compared to that of the hydrogel. Our organogel sensor systems utilize shifts in the particle spacing of an attached two-dimensional photonic crystal (2DPC) to report on the volume changes induced by protein-ligand binding. Our 2DPC bovine serum albumin (BSA) organogels exhibit VPT that swell the organogels in response to the BSA binding of charged ligands like ibuprofen and fatty acids. To our knowledge, this is the first report of a pure protein organogel VPT induced by protein-ligand binding. Catalytic protein organogels were also fabricated that utilize the enzyme organophosphorus hydrolase (OPH) to hydrolyze toxic organophosphate (OP) nerve agents. Our OPH organogels retain significant enzymatic activity. The OPH organogel rate of OP hydrolysis is ∼160 times higher than that of un-cross-linked OPH monomers in a 1:1 ethylene glycol/water mixture.

Whole genome sequence of Oscheius sp. TEL-2014 entomopathogenic nematodes isolated from South Africa.

We present the annotation of the draft genome sequence of Oscheius sp. TEL-2014 (Genbank accession number KM492926). This entomopathogenic nematode was isolated from grassland in Suikerbosrand Nature Reserve near Johannesburg in South Africa. Oscheius sp. Strain TEL has a genome size of 110,599,558 bp and a GC content of 42.24%. The genome sequence can be accessed at DDBJ/EMBL/GenBank under the accession number LNBV00000000.

Genome sequencing and annotation of Serratia sp. strain TEL.

We present the annotation of the draft genome sequence of Serratia sp. strain TEL (GenBank accession number KP711410). This organism was isolated from entomopathogenic nematode Oscheius sp. strain TEL (GenBank accession number KM492926) collected from grassland soil and has a genome size of 5,000,541 bp and 542 subsystems. The genome sequence can be accessed at DDBJ/EMBL/GenBank under the accession number LDEG00000000.

Draft Whole-Genome Sequence and Annotation of Xenorhabdus griffiniae Strain BMMCB Associated with the South African Entomopathogenic Nematode Steinernema khoisanae Strain BMMCB.

Xenorhabdus griffiniae strain BMMCB (LDNM00000000) belongs to the family Enterobacteriaceae and was isolated from the South African entomopathogenic nematode Steinernema khoisanae strain BMMCB (GenBank accession no. KT027382). Here, we report the draft whole-genome sequence of X. griffinae strain BMMCB with a genome size of 4,183,779 bp and 44.7% G+C content. The NCBI Prokaryotic Automatic Annotation Pipeline (PGAAP) revealed 3,970 genes.

Draft Whole-Genome Sequence and Annotation of the Entomopathogenic Bacterium Xenorhabdus khoisanae Strain MCB.

We report here the draft genome sequence of Xenorhabdus khoisanae strain MCB, a Gram-negative bacterium and symbiont of a Steinernema entomopathogenic nematode. The genome assembly consists of 266 contigs covering 4.68 Mb. Genome annotation revealed 3,869 protein-coding sequences, with a G+C content of 43.5%.

Draft Whole-Genome Sequence of Serratia sp. Strain TEL, Associated with Oscheius sp. TEL-2014 (Nematoda: Rhabditidae) Isolated from a Grassland in South Africa.

Here, we report on the draft genome sequence of Serratia sp. strain TEL, associated with Oscheius sp. TEL-2014 (Nematoda: Rhabditidae, KM492926) isolated from a grassland in Suikerbosrand Nature Reserve near Johannesburg in South Africa. Serratia sp. strain TEL has a genome size of 5,000,541 bp with 4,647 genes and a G+C content of 59.1%.

Draft Whole-Genome Sequence of Xenorhabdus sp. Strain GDc328, Isolated from the Indigenous South African Nematode Host Steinernema khoisanae.

Here, we describe the draft genome sequence of Xenorhabdus sp. GDc328, an endosymbiont of the native South African entomopathogenic nematode host, Steinernema khoisanae. The total genome size of the bacteria is 4.09 Mb. The genome comprises a total of 3,608 genes with a molecular G+C content of 44.64%.

Draft Genome Sequence and Assembly of Photorhabdus heterorhabditis Strain VMG, a Bacterial Symbiont Associated with the Entomopathogenic Nematode Heterorhabditis zealandica.

Here, we report the draft genome sequence of Photorhabdus heterorhabditis strain VMG, a symbiont of the entomopathogenic nematode Heterorhabditis zealandica in South Africa. The draft genome sequence is 4,878,919 bp long and contains 4,023 protein-coding genes. The genome assembly contains 262 contigs with a G+C content of 42.22%.

Draft Whole-Genome Sequence of Serratia marcescens Strain MCB, Associated with Oscheius sp. MCB (Nematoda: Rhabditidae) Isolated from South Africa.

Here we report on the draft genome sequence of Serratia marcescens strain MCB associated with Oscheius sp. MCB (Nematoda: Rhabditidae) isolated from South African soil. S. marcescens strain MCB has 5,304,212-bp genome size with 4,877 genes and a G+C content of 59.1%.