Complexes of ß-lactoglobulin and high methyl-esterified pectin as a one-shot delivery system for reinforcing oil/water interfaces.

Electrostatic complexation of negatively charged polysaccharides with ß-lactoglobulin (ß-lg) has been shown to bolster the protein films at oil/water interfaces thereby improving emulsion stability. However, recent sub-phase exchange experiments demonstrated that highly charged polysaccharides such as low methyl-esterified pectin are complementary only if sequentially introduced to a pre-formed interfacial ß-lg film. In this study, results of transient interfacial shear rheology show that, by using high-methylesterified pectins instead, complexes can be formed in pre-mixed solutions with ß-lg at pH 4 that can lead to reinforced protein films at dodecane/water interfaces. Using this one-shot adsorption of such complexes, pectins as well as short chain polysaccharides like homogalacturonan nearly doubled the steady state shear elastic moduli as compared to that of a pure ß-lg film. The lag times of film formation were established to be primarily decided by the charge density and pattern on the polysaccharide. Based on the results from mixed solutions of ß-lg monomers, it is proposed that the polysaccharide at pH 4 strengthens the resulting interfacial layer by concatenating adsorbed ß-lg molecules thereby establishing cross-links in the aqueous phase.

Medicinal psychedelics for mental health and addiction: Advancing research of an emerging paradigm.

The medical use of psychedelic substances (e.g. psilocybin, ayahuasca, lysergic acid diethylamide and 3,4-methylenedioxymethamphetamine) is attracting renewed interest, driven by a pressing need for research and development of novel therapies for psychiatric disorders, as well as promising results of contemporary studies. In this Viewpoint, we reflect upon the 'Clinical Memorandum on Psychedelics' recently released by the Royal Australian and New Zealand College of Psychiatrists and note subsequent developments including the application for down-scheduling of psilocybin and 3,4-methylenedioxymethamphetamine presently being considered by the Therapeutic Goods Administration and approvals for access via the Special Access Scheme. We suggest that this field is worthy of rigorous research to assess potential benefits, address safety parameters and clarify therapeutic mechanisms. To this end, we outline recent research findings, provide an overview of current knowledge relating to mechanisms of action and discuss salient aspects of the psychedelic-assisted psychotherapy treatment model. The sum of this research points towards medicinal psychedelics as a potential new class of psychiatric treatments when used within a medically supervised framework with integrated psychotherapeutic support. However, before widespread translation into clinical use can occur, appropriately designed and sufficiently powered trials are required to detect both potential positive and negative outcomes. Unique safety and regulatory challenges also need to be addressed. As for any new medical therapy, psychedelic research needs to be conducted in a rigorous manner, through the dispassionate lens of scientific enquiry. Carte blanche availability to practitioners, without specific protocols and appropriate training, would be potentially harmful to individuals and detrimental to the field.

Rapid Elaboration of Fragments into Leads by X-ray Crystallographic Screening of Parallel Chemical Libraries (REFiLX).

A bottleneck in fragment-based lead development is the lack of systematic approaches to elaborate the initial fragment hits, which usually bind with low affinity to their target. Herein, we describe an analysis using X-ray crystallography of a diverse library of compounds prepared using microscale parallel synthesis. This approach yielded an 8-fold increase in affinity and detailed structural information for the resulting complex, providing an efficient and broadly applicable approach to early fragment development.

Experience of Music Used With Psychedelic Therapy: A Rapid Review and Implications.

Bonny Method of Guided Imagery and Music emerged following discontinuation of psychedelic therapy research in the early 1970s, but psychedelic therapy research has since revived. Music remains a vital component. This study examined participants' experiences of music in psychedelic therapy research. A rapid review of qualitative and quantitative journal articles in four major databases was conducted in February to April, 2019, using the terms hallucinogens, psychedelic, "lysergic acid diethylamide," psilocybin, ayahuasca, music, and/or "music therapy." Of 406 articles retrieved, 10 were included (n = 180; 18-69 years old). Participants had varied backgrounds. Music was widely considered integral for meaningful emotional and imagery experiences and self-exploration during psychedelic therapy. Music transformed through its elicitation of anthropomorphic, transportive, synesthetic, and material sensations. Music could convey love, carry listeners to other realms, be something to "hold," inspire, and elicit a deep sense of embodied transformation. Therapeutic influence was especially evident in music's dichotomous elicitations: Music could simultaneously anchor and propel. Participant openness to music and provision of participant-centered music were associated with optimal immediate and longer-term outcomes. Many studies reported scarce details about the music used and incidental findings of music experienced. Further understanding of participants' idiosyncratic and shared responses to music during drug therapy phases will inform optimal development of flexible music protocols which enhance psychedelic therapy. Music therapists could be involved in the psychedelic therapy research renaissance through assisting with research to optimize music-based protocols used. If psychedelics become approved medicines, music therapists may be involved in offering psychedelic therapy as part of therapeutic teams.

Molecular Dynamics Simulations Illuminate the Role of Counterion Condensation in the Electrophoretic Transport of Homogalacturonans.

Homogalacturonans (HGs) are polysaccharide copolymers of galacturonic acid and its methylesterified counterpart. The inter- and intramolecular distributions of the methylesterifed residues are vital behavior-determining characteristics of a sample's structure, and much experimental effort has been directed to their measurement. While many techniques are able to measure the sample-averaged degree of methylesterification (DM), the measurement of inter- and intramolecular charge distributions are challenging. Here, molecular dynamics (MD) simulations are used to calculate the electrophoretic mobilities of HGs that have different amounts and distributions of charges placed along the backbone. The simulations are shown to capture experimental results well, even for low-DM samples that possess high charge densities. In addition, they illuminate the role that local counterion condensation can play in the determination of the electrophoretic mobility of heterogeneous blocky polyelectrolytes that cannot be adequately described by a single chain-averaged charge spacing.

Morphology of complexes formed between ß-lactoglobulin nanofibrils and pectins is influenced by the pH and structural characteristics of the pectins.

For the optimal use of ß-lactoglobulin nanofibrils as a raw material in biological composites an in-depth knowledge of their interactions with other constituents is necessary. To understand the effect of electrostatic interactions on the morphology of resulting complexes, ß-lactoglobulin nanofibrils were allowed to interact with pectins in which the amount of available negative charge was controlled by selecting their degree of methylesterification. In this study, citrus pectins having different degrees of methylesterification (∼48, 67, 86, and 97%) were selected and interacted with nanofibrils at pH 2 and pH 3, where they possess a net positive charge. Electrostatic complexes formed between ß-lactoglobulin nanofibrils and all pectin types, except for the sample having a degree of methylesterification of 97%. The morphology of these complexes, however, differed significantly with the degree of methylesterification of the pectin, its concentration, and the pH of the medium, revealing that distinct desired biological architectures can be attained relatively easily through manipulating the electrostatic interactions. Interestingly, the pectin with a degree of methylesterification of 86% was found to crosslink the ß-lactoglobulin nanofibrils into ordered 'nanotapes'.

ß-Lactoglobulin nanofibrils can be assembled into nanotapes via site-specific interactions with pectin.

Controlling the self-assembly of individual supramolecular entities, such as amyloid fibrils, into hierarchical architectures enables the 'bottom-up' fabrication of useful bionanomaterials. Here, we present the hierarchical assembly of ß-lactoglobulin nanofibrils into the form of 'nanotapes' in the presence of a specific pectin with a high degree of methylesterification. The nanotapes produced were highly ordered, and had an average width of 180 nm at pH 3. Increasing the ionic strength or the pH of the medium led to the disassembly of nanotapes, indicating that electrostatic interactions stabilised the nanotape architecture. Small-angle X-ray scattering experiments conducted on the nanotapes showed that adequate space is available between adjacent nanofibrils to accommodate pectin molecules. To locate the interaction sites on the pectin molecule, it was subjected to endopolygalacturonase digestion, and the resulting products were analysed using capillary electrophoresis and size-exclusion chromatography for their charge and molecular weight, respectively. Results suggested that the functional pectin molecules carry short (<10 residues) enzyme-susceptible blocks of negatively charged, non-methylesterified galacturonic acid residues in the middle of their homogalacturonan backbones (and possibly near their ends), that specifically bind to sites on the nanofibrils. Blocking the interaction sites on the nanofibril surface using small oligomers of non-methylesterified galacturonic acid residues similar in size to the interaction sites of the pectin molecule decreased the nanotape formation, indicating that site-specific electrostatic interactions are vital for the cross-linking of nanofibrils. We propose a structural model for the pectin-cross-linked ß-lactoglobulin nanotapes, the elements of which will inform the future design of bionanomaterials.

Zooming in: Structural Investigations of Rheologically Characterized Hydrogen-Bonded Low-Methoxyl Pectin Networks.

Self-assembled hydrogen-bonded networks of the polysaccharide pectin, a mechanically functional component of plant cell walls, have been of recent interest as biomimetic exemplars of physical gels, and the microrheological and strain-stiffening behaviors have been previously investigated. Despite this detailed rheological characterization of preformed gels, little is known about the fundamental arrangement of the polymers into cross-linking junction zones, the size of these bonded regions, and the resultant network architecture in these hydrogen-bonded materials, especially in contrast to the plethora of such information available for their well-known calcium-assembled counterparts. In this work, in concert with pertinent rheological measurements, an in-depth structural study of the hydrogen-bond-mediated gelation of pectins is provided. Gels were realized by using glucona-delta-lactone to decrease the pH of solutions of pectic polymers that had a (blockwise) low degree of methylesterification. Small-angle X-ray scattering and transmission electron microscopy were utilized to access structural information on length scales on the order of nanometers to hundreds of nanometers, while complementary mechanical properties were measured predominantly using small amplitude oscillatory shear rheology.

Structural mechanism of complex assemblies: characterisation of beta-lactoglobulin and pectin interactions.

Knowledge of how proteins and polysaccharides interact is the key to understanding encapsulation and emulsification in these composite systems and ultimately to understanding the structures of many biological network systems. As a model system we have studied ß-lactoglobulin A (ßLgA) interacting with pectins of various amounts and distribution patterns of charge. The studies were conducted at pH 4 at minimal ionic strength, where the ßLgA and the pectins are oppositely charged, resulting in an electrostatic attraction to each other. Isothermal titration calorimetry (ITC) experiments were performed to determine the thermodynamics associated with ßLgA-pectin titration. It was found that ßLgA only interacted with pectins with an adequate amount of charge, and that the complexation between ßLgA and pectin was a two-step process initially involving binding of the protein to available sites on the pectin, and subsequently binding of the protein onto the bound protein that has previously adsorbed. Circular dichroism (CD) and intrinsic tryptophan fluorescence were also measured of ßLgA during its interaction with the pectin samples, and show that the binding leads to significant conformational changes in ßLgA. An increase in the turbidity of the solution of the resultant complexes indicates the formation of large-scale interpolymer associations of the primary complexes mediated by protein-rich domains.

Characterization of gold kiwifruit pectin from fruit of different maturities and extraction methods.

Studies on gold kiwifruit pectins are limited. In this work, the characterization of pectin isolated from two different stages of maturity of gold kiwifruit, namely early harvested fruit (EHF) and main harvested fruit (MHF) isolated by three methods (acid, water, enzymatic) was carried out. Pectins isolated from MHF were higher in galacturonic acid content (52-59% w/w) and weight-average molecular weights (Mw, 1.7-3.8 × 10(6)g/mol) compared with EHF pectins (29-49% w/w and 0.2-1.7 × 10(6)g/mol respectively). Enzymatic treatment gave the highest yield but lowest in Mw, viscosity and mechanical spectra for both maturities. The pectin of both maturities was classified as high-methoxyl pectin with the degree of esterification ranged from 82% to 90%. Water-extracted MHF pectin molecules had the highest RMS radius (182.7 nm) and Mw (3.75 × 10(6)g/mol). The water extraction method appeared to retain the native state of pectin molecules compared with acid and enzymatic extraction methods based on the Mw and viscosity data.

Processive pectin methylesterases: the role of electrostatic potential, breathing motions and bond cleavage in the rectification of Brownian motions.

Pectin methylesterases (PMEs) hydrolyze the methylester groups that are found on the homogalacturonan (HG) chains of pectic polysaccharides in the plant cell wall. Plant and bacterial PMEs are especially interesting as the resulting de-methylesterified (carboxylated) sugar residues are found to be arranged contiguously, indicating a so-called processive nature of these enzymes. Here we report the results of continuum electrostatics calculations performed along the molecular dynamics trajectory of a PME-HG-decasaccharide complex. In particular it was observed that, when the methylester groups of the decasaccharide were arranged in order to mimic the just-formed carboxylate product of de-methylesterification, a net unidirectional sliding of the model decasaccharide was subsequently observed along the enzyme's binding groove. The changes that occurred in the electrostatic binding energy and protein dynamics during this translocation provide insights into the mechanism by which the enzyme rectifies Brownian motions to achieve processivity. The free energy that drives these molecular motors is thus demonstrated to be incorporated endogenously in the methylesterified groups of the HG chains and is not supplied exogenously.

Substrate dynamics in enzyme action: rotations of monosaccharide subunits in the binding groove are essential for pectin methylesterase processivity.

The dynamical behavior of biomacromolecules is a fundamental property regulating a large number of biological processes. Protein dynamics have been widely shown to play a role in enzyme catalysis; however, the interplay between substrate dynamics and enzymatic activity is less understood. We report insights into the role of dynamics of substrates in the enzymatic activity of PME from Erwinia chrysanthemi, a processive enzyme that catalyzes the hydrolysis of methylester groups from the galacturonic acid residues of homogalacturonan chains, the major component of pectin. Extensive molecular dynamics simulations of this PME in complex with decameric homogalacturonan chains possessing different degrees and patterns of methylesterification show how the carbohydrate substitution pattern governs the dynamics of the substrate in the enzyme's binding cleft, such that substrate dynamics represent a key prerequisite for the PME biological activity. The analyses reveal that correlated rotations around glycosidic bonds of monosaccharide subunits at and immediately adjacent to the active site are a necessary step to ensure substrate processing. Moreover, only substrates with the optimal methylesterification pattern attain the correct dynamical behavior to facilitate processive catalysis. This investigation is one of the few reported examples of a process where the dynamics of a substrate are vitally important.

Innovative enzymatic approach to resolve homogalacturonans based on their methylesterification pattern.

Three series of model homogalacturonans (HGs) covering a large range of degree of methylesterification (DM) were prepared by chemical and/or enzymatic means. Randomly demethylesterified HGs, HGs containing a few long demethylesterified galacturonic acid stretches, and HGs with numerous but short demethylesterified blocks were recovered. The analysis of the degradation products generated by the action of a purified pectin lyase allowed the definition of two new parameters, the degree of blockiness, and the absolute degree of blockiness of the highly methylesterified stretches (DBMe and DB(abs)Me, respectively). By combining this information with that obtained by the more traditional endopolygalacturonase digestion, the total proportion of degradable zones for a given DM was measured and was shown to permit a clear differentiation of the three types of HG series over a large range of DM. This double enzymatic approach will be of interest to discriminate industrial pectin samples exhibiting different functionalities and to evaluate pectin fine structure dynamics in vivo in the plant cell wall, where pectin plays a key mechanical role.

Using SAXS to reveal the degree of bundling in the polysaccharide junction zones of microrheologically distinct pectin gels.

The results of microrheological studies carried out on ionotropic pectin gels, particularly the manifest power law behavior observed at high frequencies, indicate that by using different assembly conditions gels can be formed in which the elementary network strands have different stiffnesses. It has been hypothesized that these differences reflect different network architectures, the extreme cases of which might be described as (i) dimeric calcium-chelating junction-zones of limited extent, linked by considerably longer, flexible, single-chain sections, or (ii) semiflexible bundles consisting of extensively aggregated dimeric junction zones that latterly become entangled and cross-linked. To test this hypothesis directly, microrheologically distinct pectin gels have been generated using different assembly modalities, in particular by using different concentrations of polymer and cross-linking ions and by contrasting the controlled-release of ions or ion-binding groups, and the resulting systems have been studied by small-angle X-ray scattering. The results straightforwardly reveal that gels that are clearly more semiflexible from a microrheological point-of-view contain larger scattering entities than those with a more flexible character. Furthermore, a more detailed interpretation of the scattering data with the aid of molecular modeling suggests that for the gels formed here those with a semiflexible microrheological signature consist predominantly of network filaments consisting of four or more chains, whereas those with a more flexible signature are predominantly single-chain sections linked by dimeric associations with no more that a few percent of the chains bundled to any higher extent. The ability to generate differing network architectures from the same polymer that fulfill different functional requirements, either in vivo in the plant cell wall, where pectin plays a crucial structural and mechanical role, or in vitro in a myriad of applications, makes these biomimetic biopolymer networks of considerable interest.

Enzymatic modification of a model homogalacturonan with the thermally tolerant pectin methylesterase from Citrus: 1. Nanostructural characterization, enzyme mode of action, and effect of pH.

Methyl ester distribution in pectin homogalacturonan has a major influence on functionality. Enzymatic engineering of the pectin nanostructure for tailoring functionality can expand the role of pectin as a food-formulating agent and the use of in situ modification in prepared foods. We report on the mode of action of a unique citrus thermally tolerant pectin methylesterase (TT-PME) and the nanostructural modifications that it produces. The enzyme was used to produce a controlled demethylesterification series from a model homogalacturonan. Oligogalacturonides released from the resulting demethylesterified blocks introduced by TT-PME using a limited endopolygalacturonase digestion were separated and quantified by high-pressure anion-exchange chromatography (HPAEC) coupled to an evaporative light-scattering detector (ELSD). The results were consistent with the predictions of a numerical simulation, which assumed a multiple-attack mechanism and a degree of processivity ∼10, at both pH 4.5 and 7.5. The average demethylesterified block size (0.6-2.8 nm) and number of average-sized blocks per molecule (0.8-1.9) differed, depending upon pH of the enzyme treatment. The mode of action of this enzyme and consequent nanostructural modifications of pectin differ from a previously characterized citrus salt-independent pectin methylesterase (SI-PME).

Mechanically interlocked gold and silver nanoparticles using metallosupramolecular catenane chemistry.

We have employed the toolbox of metallosupramolecular chemistry to mechanically interlock gold and silver nanoparticles. A specifically designed PEGthiol-functionalized bis(phenanthroline)copper(I) complex acts to 'catenate' the nanoparticles. The interlocked assemblies were characterised by three complementary techniques: DLS, SERS and TEM.

Rheo-NMR studies of an enzymatic reaction: evidence of a shear-stable macromolecular system.

Understanding the effects of shear forces on biopolymers is key to understanding how biological systems function. Although currently there is good agreement between theoretical predictions and experimental measurements of the behavior of DNA and large multimeric proteins under shear flow, applying the same arguments to globular proteins leads to the prediction that they should only exhibit shear-induced conformational changes at extremely large shear rates. Nevertheless, contradictory experimental evidence continues to appear, and the effect of shear on these biopolymers remains contentious. Here, a custom-built rheo-NMR cell was used to investigate whether shear flow modifies enzyme action compared with that observed quiescently. Specifically, (1)H NMR was used to follow the kinetics of the liberation of methanol from the methylesterified polysaccharide pectin by pectinmethylesterase enzymes. Two different demethylesterifying enzymes, known to have different action patterns, were used. In all experiments performed, Couette flows with shear rates of up to 1570 s(-1) did not generate detectable differences in the rate of methanol liberation compared to unsheared samples. This study provides evidence for a shear-stable macromolecular system consisting of a largely beta-sheet protein and a polysaccharide, in line with current theoretical predictions, but in contrast to some other experimental work on other proteins.

Extracting intramolecular sequence information from intermolecular distributions: highly nonrandom methylester substitution patterns in homogalacturonans generated by pectinmethylesterase.

The ratio of the two component sugar residues comprising the anionic polysaccharide homogalacturonan (HG; namely, methylesterified or unmethylesterifed galacturonic acid (GalA)) has been controlled chemically or enzymatically in order to produce samples comprised of varying degrees and distributions of methylesterification (DM). Capillary electrophoresis (CE) has been used to characterize the samples produced and, by mapping the measured electrophoretic mobilities to biopolymer charge density, intermolecular distributions of the DM have been extracted. For chemically modified samples with random intramolecular patterns of methylesterification, the experimentally extracted intermolecular DM distributions agree well with the predictions of calculations based on the binomial theorem, demonstrating that the random nature of the demethylesterification process and, hence, the intramolecular DM patterns themselves, are directly reflected in the intermolecular distribution. Furthermore, this principle has been demonstrated by extending the work to the study of substrates with highly nonrandom DM distributions generated using a processive plant-pectinmethylesterase (pPME). An ensemble of polymer chains, generated in silico by a simulation optimized to match the experimentally measured intermolecular DM distribution, contains all possible information regarding the substrate and can further be interrogated to obtain, for example, the full Gal-A blocklength distribution.

Electrophoretic behavior of copolymeric galacturonans including comments on the information content of the intermolecular charge distribution.

Capillary electrophoresis has been used to characterize samples of the copolymeric anionic polysaccharides homo- and rhamno-galacturonan (HG and RGI, respectively). In the case of HG, the ratios of the two component sugar residues, galacturonic acid and its methylesterified analogue, have been controlled chemically to produce samples comprised of varying degrees of methylesterification (DM). The mapping of the measured electrophoretic mobilities to the biopolymer charge density has been considered in some detail and for HG substrates with random intramolecular patterns of methylesterification it is shown that the experimentally extracted intermolecular DM distribution agrees well with the predictions of calculations based on the binomial theorem. This demonstrates that the intermolecular distribution of the methylesterification of pectin samples contains information on the intramolecular pattern by virtue of the fact that they are both manifestations of the same statistical process.

Microrheological investigations give insights into the microstructure and functionality of pectin gels.

Many of the functional attributes of pectin, whether in the plant cell wall or in engineered food materials, are linked to its gelling properties and in particular to its ability to assemble in the presence of calcium. Pectin's fine structure and local concentration relative to that of its cross-linking ion play a major role in determining resultant gel micro-structures, and consequently the mechanical and transport properties of pectin matrices. Recent studies have sought to probe the basic properties of such calcium-induced matrices, using a light scattering technique called diffusing wave spectroscopy (DWS). In addition to the low frequency mechanical behaviour, which provides information about the nature and density of cross-links, microrheological measurements carried out with DWS are able to determine the high frequency behaviour, which is closely linked to the response of the basic strands of the network. By using these microrheological measurements, two distinct regimes have been identified into which pectin gels appear to fall: one corresponding to the presence of semi-flexible networks, a generally accepted paradigm in biological gels, and another where flexible networks dominate. In order to explain the origin of these dramatically different networks, distinct assembly pathways have been proposed in which the relative importance of the free energy gained by association and the frictional barrier to polymeric re-arrangement during network formation can differ significantly. By manipulating the local environment in the plant cell wall it is possible that Nature makes full use of both of these network types for fulfilling different tasks; such as providing strain-hardening, maximizing local elastic properties or controlling macromolecular transport.