Long-Chain Hydrocarbons from Nonthermal Plasma-Driven Biogas Upcycling.

The burgeoning necessity to discover new methodologies for the synthesis of long-chain hydrocarbons and oxygenates, independent of traditional reliance on high-temperature, high-pressure, and fossil fuel-based carbon, is increasingly urgent. In this context, we introduce a nonthermal plasma-based strategy for the initiation and propagation of long-chain carbon growth from biogas constituents (CO2 and CH4). Utilizing a plasma reactor operating at atmospheric room temperature, our approach facilitates hydrocarbon chain growth up to C40 in the solid state (including oxygenated products), predominantly when CH4 exceeds CO2 in the feedstock. This synthesis is driven by the hydrogenation of CO2 and/or amalgamation of CHx radicals. Global plasma chemistry modeling underscores the pivotal role of electron temperature and CHx radical genesis, contingent upon varying CO2/CH4 ratios in the plasma system. Concomitant with long-chain hydrocarbon production, the system also yields gaseous products, primarily syngas (H2 and CO), as well as liquid-phase alcohols and acids. Our finding demonstrates the feasibility of atmospheric room-temperature synthesis of long-chain hydrocarbons, with the potential for tuning the chain length based on the feed gas composition.

Effect of mango butter on the physicochemical properties of beeswax-Moringa seed oil-based oleogels for topical application.

OBJECTIVE: The purpose of this research was to determine any connections between the characteristics of oleogels made of beeswax and the impact of mango butter. METHODS: Oleogel was prepared through inverted tube methods, and optimized through oil binding capacity. Other evaluations like bright field and polarized microscopy, Fourier-transform infrared (FTIR) spectroscopy, crystallization kinetics, mechanical study, and X-ray diffractometry (XRD). The drug release kinetic studies and in vitro antibacterial studies were performed. RESULTS: FTIR study reveals that the gelation process does not significantly alter the chemical composition of the individual components. Prepared gel exhibiting fluid-like behavior or composed of brittle networks is particularly vulnerable to disruptions in their network design. The incorporation of mango butter increases the drug permeation. In-vitro microbial efficacy study was found to be excellent. CONCLUSION: The studies revealed that mango butter can be used to modify the physico-chemical properties of the oleogels.

Binding of a pyrimidine RNA base-mimic to SARS-CoV-2 nonstructural protein 9.

The coronaviral nonstructural protein 9 (Nsp9) is essential for viral replication; it is the primary substrate of Nsp12's pseudokinase domain within the viral replication transcription complex, an association that also recruits other components during different stages of RNA reproduction. In the unmodified state, Nsp9 forms an obligate homodimer via an essential GxxxG protein-interaction motif, but its ssRNA-binding mechanism remains unknown. Using structural biological techniques, here we show that a base-mimicking compound identified from a small molecule fragment screen engages Nsp9 via a tetrameric Pi-Pi stacking interaction that induces the formation of a parallel trimer-of-dimers. This oligomerization mechanism allows an interchange of "latching" N-termini, the charges of which contribute to a series of electropositive channels that suggests a potential interface for viral RNA. The identified pyrrolo-pyrimidine compound may also serve as a potential starting point for the development of compounds seeking to probe Nsp9's role within SARS-CoV-2 replication.

Side-Chain Interactions in d/l Peptide Nanotubes: Studies by Crystallography, NMR Spectroscopy and Molecular Dynamics.

Our understanding of the factors affecting the stability of cyclic d/l peptide (CP) nanotubes remains underdeveloped. In this work, we investigate the impact of side chain alignment, hydrophobicity and charge on CP nanotube stability through X-ray crystallography, NMR spectroscopy and molecular dynamics (MD) simulations. We characterise the distinct CP-CP alignments that can form and identify stable and unstable dimers by MD simulation. We measure H-bond half-lives of synthesised CPs by 1 H-D exchange experiments and find good correlation with predicted CP-CP stabilities. We find that hydrophobic amino acids improve CP dimer stability but experimentally reduce solubility. Charged amino acids either increase or decrease CP dimer stability depending on the relative orientation and composition of charged groups. X-ray crystal structures are solved for two CPs, revealing non-tubular folded conformations. Ultimately, this work will assist the educated design of stable tubular structures for potential applications in biomedicine.

A ligand-induced structural change in fatty acid-binding protein 1 is associated with potentiation of peroxisome proliferator-activated receptor α agonists.

Peroxisome proliferator-activated receptor α (PPARα) is a transcriptional regulator of lipid metabolism. GW7647 is a potent PPARα agonist that must reach the nucleus to activate this receptor. In cells expressing human fatty acid-binding protein 1 (FABP1), GW7647 treatment increases FABP1's nuclear localization and potentiates GW7647-mediated PPARα activation; GW7647 is less effective in cells that do not express FABP1. To elucidate the underlying mechanism, here we substituted residues in FABP1 known to dictate lipid signaling by other intracellular lipid-binding proteins. Substitutions of Lys-20 and Lys-31 to Ala in the FABP1 helical cap affected neither its nuclear localization nor PPARα activation. In contrast, Ala substitution of Lys-57, Glu-77, and Lys-96, located in the loops adjacent to the ligand-binding portal region, abolished both FABP1 nuclear localization and GW7647-induced PPARα activation but had little effect on GW7647-FABP1 binding affinity. Using solution NMR spectroscopy, we determined the WT FABP1 structure and analyzed the dynamics in the apo and GW7647-bound structures of both the WT and the K57A/E77A/K96A triple mutant. We found that GW7647 binding causes little change in the FABP1 backbone, but solvent exposes several residues in the loops around the portal region, including Lys-57, Glu-77, and Lys-96. These residues also become more solvent-exposed upon binding of FABP1 with the endogenous PPARα agonist oleic acid. Together with previous observations, our findings suggest that GW7647 binding stabilizes a FABP1 conformation that promotes its interaction with PPARα. We conclude that full PPARα agonist activity of GW7647 requires FABP1-dependent transport and nuclear localization processes.

NMR fragment screening reveals a novel small molecule binding site near the catalytic surface of the disulfide-dithiol oxidoreductase enzyme DsbA from Burkholderia pseudomallei.

The presence of suitable cavities or pockets on protein structures is a general criterion for a therapeutic target protein to be classified as 'druggable'. Many disease-related proteins that function solely through protein-protein interactions lack such pockets, making development of inhibitors by traditional small-molecule structure-based design methods much more challenging. The 22 kDa bacterial thiol oxidoreductase enzyme, DsbA, from the gram-negative bacterium Burkholderia pseudomallei (BpsDsbA) is an example of one such target. The crystal structure of oxidized BpsDsbA lacks well-defined surface pockets. BpsDsbA is required for the correct folding of numerous virulence factors in B. pseudomallei, and genetic deletion of dsbA significantly attenuates B. pseudomallei virulence in murine infection models. Therefore, BpsDsbA is potentially an attractive drug target. Herein we report the identification of a small molecule binding site adjacent to the catalytic site of oxidized BpsDsbA. 1HN CPMG relaxation dispersion NMR measurements suggest that the binding site is formed transiently through protein dynamics. Using fragment-based screening, we identified a small molecule that binds at this site with an estimated affinity of KD ~ 500 µM. This fragment inhibits BpsDsbA enzymatic activity in vitro. The binding mode of this molecule has been characterized by NMR data-driven docking using HADDOCK. These data provide a starting point towards the design of more potent small molecule inhibitors of BpsDsbA.

Structural and biochemical insights into the disulfide reductase mechanism of DsbD, an essential enzyme for neisserial pathogens.

The worldwide incidence of neisserial infections, particularly gonococcal infections, is increasingly associated with antibiotic-resistant strains. In particular, extensively drug-resistant Neisseria gonorrhoeae strains that are resistant to third-generation cephalosporins are a major public health concern. There is a pressing clinical need to identify new targets for the development of antibiotics effective against Neisseria-specific processes. In this study, we report that the bacterial disulfide reductase DsbD is highly prevalent and conserved among Neisseria spp. and that this enzyme is essential for survival of N. gonorrhoeae DsbD is a membrane-bound protein that consists of two periplasmic domains, n-DsbD and c-DsbD, which flank the transmembrane domain t-DsbD. In this work, we show that the two functionally essential periplasmic domains of Neisseria DsbD catalyze electron transfer reactions through unidirectional interdomain interactions, from reduced c-DsbD to oxidized n-DsbD, and that this process is not dictated by their redox potentials. Structural characterization of the Neisseria n- and c-DsbD domains in both redox states provides evidence that steric hindrance reduces interactions between the two periplasmic domains when n-DsbD is reduced, thereby preventing a futile redox cycle. Finally, we propose a conserved mechanism of electron transfer for DsbD and define the residues involved in domain-domain recognition. Inhibitors of the interaction of the two DsbD domains have the potential to be developed as anti-neisserial agents.

The uncharacterized bacterial protein YejG has the same architecture as domain III of elongation factor G.

InterPro family IPR020489 comprises ~1000 uncharacterized bacterial proteins. Previously we showed that overexpressing the Escherichia coli representative of this family, EcYejG, conferred low-level resistance to aminoglycoside antibiotics. In an attempt to shed light on the biochemical function of EcYejG, we have solved its structure using multinuclear solution NMR spectroscopy. The structure most closely resembles that of domain III from elongation factor G (EF-G). EF-G catalyzes ribosomal translocation and mutations in EF-G have also been associated with aminoglycoside resistance. While we were unable to demonstrate a direct interaction between EcYejG and the ribosome, the protein might play a role in translation.

Controlled Construction of Cyclic d†/†l Peptide Nanorods.

Cyclic d / l peptides (CPs) assemble spontaneously via backbone H-bonding to form extended nanostructures. These modular materials have great potential as versatile bionanomaterials. However, the useful development of CP nanomaterials requires practical methods to direct and control their assembly. In this work, we present novel, heterogeneous, covalently linked CP tetramers that achieve local control over the CP subunit order and composition through coupling of amino acid side-chains using copper-activated azide-alkyne cycloaddition and disulfide bond formation. Cryo-transmission electron microscopy revealed the formation of highly ordered, fibrous nanostructures, while NMR studies showed that these systems have strong intramolecular H-bonding in solution. The introduction of inter-CP tethers is expected to enable the development of complex nanomaterials with controllable chemical properties, facilitating the development of precisely functionalized or "decorated" peptide nanostructures.

The first total synthesis and solution structure of a polypeptin, PE2, a cyclic lipopeptide with broad spectrum antibiotic activity.

The first total synthesis of a polypeptin, PE2, as well as its solution structure is reported. Synthesis in optically pure form confirms the proposed stereochemistry of the polypeptins at the 3-position on the 3-hydroxy depsipeptide moiety. We have also determined the NMR structure of PE2 in aqueous solution, showing it to form a stable ring conformation. The synthetic peptide shows anti-bacterial activity consistent with reports for naturally derived counterparts.

Determination of ligand binding modes in weak protein-ligand complexes using sparse NMR data.

We describe a general approach to determine the binding pose of small molecules in weakly bound protein-ligand complexes by deriving distance constraints between the ligand and methyl groups from all methyl-containing residues of the protein. We demonstrate that using a single sample, which can be prepared without the use of expensive precursors, it is possible to generate high-resolution data rapidly and obtain the resonance assignments of Ile, Leu, Val, Ala and Thr methyl groups using triple resonance scalar correlation data. The same sample may be used to obtain Met εCH3 assignments using NOESY-based methods, although the superior sensitivity of NOESY using [U-13C,15N]-labeled protein makes the use of this second sample more efficient. We describe a structural model for a weakly binding ligand bound to its target protein, DsbA, derived from intermolecular methyl-to-ligand nuclear Overhauser enhancements, and demonstrate that the ability to assign all methyl resonances in the spectrum is essential to derive an accurate model of the structure. Once the methyl assignments have been obtained, this approach provides a rapid means to generate structural models for weakly bound protein-ligand complexes. Such weak complexes are often found at the beginning of programs of fragment based drug design and can be challenging to characterize using X-ray crystallography.

Structure and Functional Characterization of the Conserved JAK Interaction Region in the Intrinsically Disordered N-Terminus of SOCS5.

SOCS5 can negatively regulate both JAK/STAT and EGF-receptor pathways and has therefore been implicated in regulating both the immune response and tumorigenesis. Understanding the molecular basis for SOCS5 activity may reveal novel ways to target key components of these signaling pathways. The N-terminal region of SOCS5 coordinates critical protein interactions involved in inhibition of JAK/STAT signaling, and a conserved region within the N-terminus of SOCS5 mediates direct binding to the JAK kinase domain. Here we have characterized the solution conformation of this conserved JAK interaction region (JIR) within the largely disordered N-terminus of SOCS5. Using nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements, and NOE analysis, we demonstrate the presence of preformed structural elements in the JIR of mouse SOCS5 (mSOCS5175-244), consisting of an α-helix encompassing residues 224-233, preceded by a turn and an extended structure. We have identified a phosphorylation site (Ser211) within the JIR of mSOCS5 and have investigated the role of phosphorylation in modulating JAK binding using site-directed mutagenesis.

Kv1.3 channel-blocking immunomodulatory peptides from parasitic worms: implications for autoimmune diseases.

The voltage-gated potassium (Kv) 1.3 channel is widely regarded as a therapeutic target for immunomodulation in autoimmune diseases. ShK-186, a selective inhibitor of Kv1.3 channels, ameliorates autoimmune diseases in rodent models, and human phase 1 trials of this agent in healthy volunteers have been completed. In this study, we identified and characterized a large family of Stichodactyla helianthus toxin (ShK)-related peptides in parasitic worms. Based on phylogenetic analysis, 2 worm peptides were selected for study: AcK1, a 51-residue peptide expressed in the anterior secretory glands of the dog-infecting hookworm Ancylostoma caninum and the human-infecting hookworm Ancylostoma ceylanicum, and BmK1, the C-terminal domain of a metalloprotease from the filarial worm Brugia malayi. These peptides in solution adopt helical structures closely resembling that of ShK. At doses in the nanomolar-micromolar range, they block native Kv1.3 in human T cells and cloned Kv1.3 stably expressed in L929 mouse fibroblasts. They preferentially suppress the proliferation of rat CCR7(-) effector memory T cells without affecting naive and central memory subsets and inhibit the delayed-type hypersensitivity (DTH) response caused by skin-homing effector memory T cells in rats. Further, they suppress IFNγ production by human T lymphocytes. ShK-related peptides in parasitic worms may contribute to the potential beneficial effects of probiotic parasitic worm therapy in human autoimmune diseases.

Development and characterization of itraconazole-loaded solid lipid nanoparticles for ocular delivery.

The purpose of this study was to investigate the feasibility of entrapping water-insoluble drug itraconazole into solid lipid nanoparticles (SLNs) for topical ocular delivery. The drug-loaded SLNs were prepared from stearic acid and palmitic acid using different concentrations of polyvinyl alcohol employed as emulsifier. SLNs were prepared by the melt-emulsion sonication and low temperature-solidification method and characterized for particle size, zeta potential, drug loading and drug entrapment efficiency. The mean particle size of SLNs prepared with stearic acid ranged from 139 to 199 nm, while the SLNs prepared with palmitic acid had particle size in the range of 126-160 nm. The SLNs were spherical in shape. Stearic acid-SLNs showed higher entrapment of drug compared with palmitic acid-SLNs. Differential scanning calorimetry (DSC) and X-ray diffraction measurements showed decrease in crystallinity of drug in the SLN formulations. The modified Franz-diffusion cell and freshly excised goat corneas were used to test drug corneal permeability. Permeation of itraconazole from stearic acid-SLNs was higher than that obtained with palmitic acid-SLNs. The SLNs showed clear zone of inhibition against Aspergillus flavus indicating antimicrobial efficacy of formulations.

Application of fragment-based screening to the design of inhibitors of Escherichia coli DsbA.

The thiol-disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram-negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell-based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.

The relaxin receptor (RXFP1) utilizes hydrophobic moieties on a signaling surface of its N-terminal low density lipoprotein class A module to mediate receptor activation.

The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true "ligand" of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation.

The structure of integrin α1I domain in complex with a collagen-mimetic peptide.

We have determined the structure of the human integrin α1I domain bound to a triple-helical collagen peptide. The structure of the α1I-peptide complex was investigated using data from NMR, small angle x-ray scattering, and size exclusion chromatography that were used to generate and validate a model of the complex using the data-driven docking program, HADDOCK (High Ambiguity Driven Biomolecular Docking). The structure revealed that the α1I domain undergoes a major conformational change upon binding of the collagen peptide. This involves a large movement in the C-terminal helix of the αI domain that has been suggested to be the mechanism by which signals are propagated in the intact integrin receptor. The structure suggests a basis for the different binding selectivity observed for the α1I and α2I domains. Mutational data identify residues that contribute to the conformational change observed. Furthermore, small angle x-ray scattering data suggest that at low collagen peptide concentrations the complex exists in equilibrium between a 1:1 and 2:1 α1I-peptide complex.

Nanocrystalline cellulose as a reinforcing agent for poly (vinyl alcohol)/ gellan-gum-based composite film for moxifloxacin ocular delivery.

Nanocrystalline cellulose (NCC) is a star material in drug delivery applications due to its good biocompatibility, large specific surface area, high tensile strength (TS), and high hydrophilicity. Poly(Vinyl Alcohol)/Gellan-gum-based innovative composite film has been prepared using nanocrystalline cellulose (PVA/GG/NCC) as a strengthening agent for ocular delivery of moxifloxacin (MOX) via solvent casting method. Impedance analysis was studied using the capacitive sensing technique for examining new capacitance nature of the nanocomposite MOX film. Antimicrobial properties of films were evaluated using Pseudomonas aeruginosa and Staphylococcus aureus as gram-negative and gram-positive bacteria respectively by disc diffusion technique. XRD revealed the characteristic peak of NCC and the amorphous form of the drug. Sustained in vitro release and enhanced corneal permeation of drug were noticed in the presence of NCC. Polymer matrix enhanced the mechanical properties (tensile strength 22.05 to 28.41 MPa) and impedance behavior (resistance 59.23 to 213.23 Ω) in the film due to the presence of NCC rather than its absence (16.78 MPa and 39.03 Ω respectively). Occurrence of NCC brought about good antimicrobial behavior (both gram-positive and gram-negative) of the film. NCC incorporated poly(vinyl alcohol)/gellan-gum-based composite film exhibited increased mechanical properties and impedance behavior for improved ocular delivery of moxifloxacin.

Development of microemulgel formulations with varied permeation enhancers for transungual delivery of luliconazole in onychomycosis management.

Luliconazole-loaded microemulgels containing different permeation enhancers were formulated for transungual drug delivery for the management of onychomycosis, onychomycosis, which affects nails. The physicochemical properties like droplet size, zeta potential, pH, viscosity, spreadability, extrudability, oil binding capacity, drug content, and microscopic study were evaluated. The Pseudo-ternary phase diagram was constructed for the formulation of microemulsions (MEs) by keeping the Km ratio constant at 3:1 and characterized for clarity, mean droplet size, zeta potential, viscosity, pH, transmittance, refractive index, and stability. The ME mean droplet size and zeta potential were found in the range of 38.78 to 171.4 nm, and 0.00 to - 6.6 mV, respectively. Prepared MEs were converted into microemulgel by adding a 2.5% gelling agent (Carbapol 934) in the external phase, and a drug release study was conducted. Formulation E3 showed better drug release and was chosen as the control. Four different penetration enhancers were added separately within E3 and further evaluated for pH, viscosity, spreadability, extrudability, oil binding capacity, drug content, microscopic study, Compatibility study, XRD, and DSC. A favorable docking score was observed between luliconazole and Lanosterol 14-alpha-demethylase. In-vitro cumulative drug release at the end of 24 h from E3-SS, containing sodium sulfide as a penetration enhancer, was found to be 94.70% and was 2 times more than the control formulation. Ex-vivo transungual permeation studies through cutting nail clippings were found to be in the range of 28.18 - 36.52 µg/mm2. The microemulgels tagged as E3, E3-SS, and E3-SL showed a significant zone of inhibition against Candida albicans and Aspergillus fumigatus as compared to the marketed formulation.

Fluoromethylketone-Fragment Conjugates Designed as Covalent Modifiers of EcDsbA are Atypical Substrates.

Disulfide bond protein A (DsbA) is an oxidoreductase enzyme that catalyzes the formation of disulfide bonds in Gram-negative bacteria. In Escherichia coli, DsbA (EcDsbA) is essential for bacterial virulence, thus inhibitors have the potential to act as antivirulence agents. A fragment-based screen was conducted against EcDsbA and herein we describe the development of a series of compounds based on a phenylthiophene hit identified from the screen. A novel thiol reactive and "clickable" ethynylfluoromethylketone was designed for reaction with azide-functionalized fragments to enable rapid and versatile attachment to a range of fragments. The resulting fluoromethylketone conjugates showed selectivity for reaction with the active site thiol of EcDsbA, however unexpectedly, turnover of the covalent adduct was observed. A mechanism for this turnover was investigated and proposed which may have wider ramifications for covalent reactions with dithiol-disulfide oxidoreducatases.