Visualization and Quantification of IgG Antibody Adsorbed at the Cellulose-Liquid Interface.

Quantification of adsorbed biomolecules (enzymes, proteins) at the cellulose interface is a major challenge in developing eco-friendly biodiagnostics. Here, a novel methodology is developed to visualize and quantify the adsorption of antibody from solution to the cellulose-liquid interface. The concept is to deuterate cellulose by replacing all nonexchangeable hydrogens from the glucose rings with deuterium in order to enhance the scattering contrast between the cellulose film surface and adsorbed antibody molecules. Deuterated cellulose (DC) was obtained from bacterial (Gluconacetobacter xylinus strain) cellulose, which was grown in heavy water (D2O) media with a deuterated glycerol as a carbon source. For comparison, hydrogenated cellulose (HC) was obtained from cellulose acetate. Both HC and DC thin films were prepared on silicon substrate by spin coating. X-ray reflectivity (XR) shows the formation of homogeneous and smooth film. Neutron reflectivity (NR) at the liquid/film interface reveals swelling of the cellulose film by a factor of 2-3× its initial thickness. An Immunoglobulin G (IgG), used as a model antibody, was adsorbed at the liquid-solid interface of cellulose (HC) and deuterated cellulose (DC) films under equilibrium and surface saturation conditions. NR measurements of the IgG antibody layer adsorbed onto the DC film can clearly be visualized, in sharp contrast in comparison to the HC film. The average thickness of the IgG adsorbed layer onto cellulose films is 127 ± 5 Å and a partial monolayer is formed. Visualization and quantification of adsorbed IgG is shown by large difference in scattering length density (SLD) between DC (7.1 × 10-6 Å-2) and IgG (4.1 × 10-6 Å-2) in D2O, which enhanced the scattering contrast in NR. Quartz crystal measurements (QCM-D) were used as a complementary method to NR to quantify the adsorbed IgG over the cellulose interface.

BioPEGylation of polyhydroxybutyrate promotes nerve cell health and migration.

This study reports on the superior suitability of Polyhydroxybutyrate-polyethylene glycol hybrid polymers biosynthesised by Cupriavidus necator over PHB as biomaterials for tissue engineering. Incorporation of PEG106 (DEG) during PHB biosynthesis reduced crystallinity, molecular weight, and hydrophobicity while improving mechanical properties. In vitro olfactory ensheathing cell (OEC) proliferation was enhanced by cultivation on PHB-b-DEG films. Cultivation on PHB and PHB-b-DEG films showed no cytotoxic responses and cell viability and membrane integrity was sustained. PHB-b-DEG films promoted OECs entering into the DNA replication (S) phase and mitotic (G2-M) phase during the cell growth cycle and apoptosis was low. This study also confirmed an association between the level of neurite-outgrowth inhibitory protein (Nogo) and receptor pair Ig-like receptor B (PirB) expression and cell proliferation, both being down-regulated in cells grown on hybrid films when compared with PHB and asynchronous growth. Thus, DEG-terminated PHB-based biomaterials have great potential as biological scaffolds supporting nerve repair.

Deuterated polymers for probing phase separation using infrared microspectroscopy.

Infrared (IR) microspectroscopy has the capacity to determine the extent of phase separation in polymer blends. However, a major limitation in the use of this technique has been its reliance on overlapping peaks in the IR spectra to differentiate between polymers of similar chemical compositions in blends. The objective of this study was to evaluate the suitability of deuteration of one mixture component to separate infrared (IR) absorption bands and provide image contrast in phase separated materials. Deuteration of poly(3-hydroxyoctanoate) (PHO) was achieved via microbial biosynthesis using deuterated substrates, and the characteristic C-D stretching vibrations provided distinct signals completely separated from the C-H signals of protonated poly(3-hydroxybutyrate) (PHB). Phase separation was observed in 50:50 (% w/w) blends as domains up to 100 µm through the film cross sections, consistent with earlier reports of phase separation observed by scanning electron microscopy (SEM) of freeze-fractured protonated polymer blends. The presence of deuterated phases throughout the film suggests there is some miscibility at smaller length scales, which increased with increasing PHB content. These investigations indicate that biodeuteration combined with IR microspectroscopy represents a useful tool for mapping the phase behavior of polymer blends.

Mild conditions for deuteration of primary and secondary arylamines for the synthesis of deuterated optoelectronic organic molecules.

Deuterated arylamines demonstrate great potential for use in optoelectronic devices, but their widespread utility requires a method for large-scale synthesis. The incorporation of these deuterated materials into optoelectronic devices also provides the opportunity for studies of the functioning device using neutron reflectometry based on the difference in the scattering length density between protonated and deuterated compounds. Here we report mild deuteration conditions utilising standard laboratory glassware for the deuteration of: diphenylamine, N-phenylnaphthylamine, N-phenyl-o-phenylenediamine and 1-naphthylamine (via H/D exchange in D2O at 80 °C, catalysed by Pt/C and Pd/C). These conditions were not successful in the deuteration of triphenylamine or N,N-dimethylaniline, suggesting that these mild conditions are not suitable for the deuteration of tertiary arylamines, but are likely to be applicable for the deuteration of other primary and secondary arylamines. The deuterated arylamines can then be used for synthesis of larger organic molecules or polymers with optoelectronic applications.

Structural characterization of a model gram-negative bacterial surface using lipopolysaccharides from rough strains of Escherichia coli.

Lipopolysaccharides (LPS) make up approximately 75% of the Gram-negative bacterial outer membrane (OM) surface, but because of the complexity of the molecule, there are very few model OMs that include LPS. The LPS molecule consists of lipid A, which anchors the LPS within the OM, a core polysaccharide region, and a variable O-antigen polysaccharide chain. In this work we used RcLPS (consisting of lipid A plus the first seven sugars of the core polysaccharide) from a rough strain of Escherichia coli to form stable monolayers of LPS at the air-liquid interface. The vertical structure RcLPS monolayers were characterized using neutron and X-ray reflectometry, while the lateral structure was investigated using grazing incidence X-ray diffraction and Brewster angle microscopy. It was found that RcLPS monolayers at surface pressures of 20 mN m(-1) and above are resolved as hydrocarbon tails, an inner headgroup, and an outer headgroup of polysaccharide with increasing solvation from tails to outer headgroups. The lateral organization of the hydrocarbon lipid chains displays an oblique hexagonal unit cell at all surface pressures, with only the chain tilt angle changing with surface pressure. This is in contrast to lipid A, which displays hexagonal or, above 20 mN m(-1), distorted hexagonal packing. This work provides the first complete structural analysis of a realistic E. coli OM surface model.

Synthesis of deuterated [D32 ]oleic acid and its phospholipid derivative [D64 ]dioleoyl-sn-glycero-3-phosphocholine.

Oleic acid and its phospholipid derivatives are fundamental to the structure and function of cellular membranes. As a result, there has been increasing interest in the availability of their deuterated forms for many nuclear magnetic resonance, infrared, mass spectroscopy and neutron scattering studies. Here, we present for the first time a straightforward, large-scale (gram quantities) synthesis of highly deuterated [D32 ]oleic acid by using multiple, yet simple and high yielding reactions. The precursors for the synthesis of [D32 ]oleic acid are [D14 ]azelaic acid and [D17 ]nonanoic acid, which were obtained by complete deuteration (>98% D) of their (1) H forms by using metal catalysed hydrothermal H/D exchange reactions. The oleic acid was produced with ca. 94% D isotopic purity and with no contamination by the trans-isomer (elaidic acid). The subsequent synthesis of [D64 ]dioleoyl-sn-glycero-3-phosphocholine from [D32 ]oleic acid is also described.

Using Humidity to Control the Morphology and Properties of Electrospun BioPEGylated Polyhydroxybutyrate Scaffolds.

Electrospinning produces nanofibrous scaffolds with potential for tissue engineering and wound repair. Spinning parameters control scaffold morphology and properties. BioPEGylation of polyhydroxybutyrate (PHB) introduces terminal hydrophilic groups into the hydrophobic chain, making this natural-synthetic hybrid copolymer more susceptible to humidity. Varying the humidity from 10 to 50% RH during electrospinning had a relatively little effect on polyhydroxybutyrate (PHB) average fiber and pore diameters, which remained around 3.0 and 8.7 µm, respectively. In contrast, fiber and pore diameters for electrospun bioPEGylated PHB scaffolds varied significantly with humidity, peaking at 30% RH (5.5 and 14.1 µm, respectively). While scaffolds showed little change, hydrophobicity decreased linearly with humidity during electrospinning. Compared to solvent-cast films, electrospun scaffolds showed significantly greater average cell spread. A 108% increase for olfactory ensheathing cells (OECs) cultivated on bioPEGylated PHB scaffolds was proportionally greater than their counterparts on electrospun PHB scaffolds, (70%). OECS grown on BioPEGylated PHB scaffolds were over twice the size, 260 ± 20 µm diameter, than those on PHB electrospun scaffolds, 110 ± 18 µm diameter. Electrospun scaffolds also promoted cell health compared to their solvent-cast counterparts, with increases in the mitochondrial activity of 165 ± 13 and 196 ± 13% for PHB and bioPEGylated PHB, respectively. OECS cultivated on electrospun scaffolds of bioPEGylated PHB had significantly better membrane integrities compared to their counterparts on solvent-cast films, 47 ± 5% reducing to 17 ± 6%. The combination of bioPEGylation and humidity during electrospinning permitted significant controllable changes to scaffold morphology and properties. These changes resulted in the significantly greater promotion of cell growth on electrospun bioPEGylated PHB scaffolds compared to their solvent-cast counterparts and electrospun PHB.

A rapid MS/MS method to assess the deuterium kinetic isotope effect and associated improvement in the metabolic stability of deuterated biological and pharmacological molecules as applied to an imaging agent.

The deuterium kinetic isotope effect has been known for a period of 40 years, but it is only relatively recently that new drug entities (NDEs) incorporating deuterium demonstrating beneficial pharmacokinetics, pharmacodynamics, and toxicology have arrived to market. Determination of the precise location to deuterate and subsequently any evaluation for a kinetic isotope effect (KIE) is challenging. Typically, such an evaluation would be performed in an in vitro metabolic assay (e.g. liver microsomes) in separate reaction media for both the deuterated and non-deuterated analogues. Here, we have devised an approach whereby we incubate a 1:1 ratio of both the deuterated and protio-form of an imaging agent together in the same liver microsomal assay and determine the relative rate of consumption of both moieties, based upon specific MS-MS transitions unique to both molecules without the need for liquid chromatography-mass spectrometry (LC-MS) separation and quantification. Any deviation of the ratio of the MS transitions from the initial starting point indicated an observable KIE. A site specific deuteration of PBR111, a neuroinflammation imaging agent, was chosen for a proof-of-concept study. Based upon prior mechanistic knowledge of PBR111, two locations were selected for deuteration; an active and inactive site, to corroborate that there was no significant KIE for the inactive site and confirm the efficacy of the developed methodology.

Small-angle neutron scattering characterization of polyhydroxyalkanoates and their BioPEGylated hybrids in solution.

Small-angle neutron scattering was used to probe the molecular conformation of various polyhydroxyalkanoates (PHAs) and their bioPEGylated counterparts (PHA- b-PEG). Analysis of neutron scattering profiles of these polymers dissolved in deuterated chloroform at various concentrations from dilute (approximately 0.1% w/v) to semidilute (approximately 7% w/v) showed the two distinct regimes and established overlap concentrations around 4-9 mg mL(-1). Scattering profiles were similar for all polymers investigated; power laws of approximately Q(-1.66) at high Q demonstrated that chloroform behaves as a good solvent for PHAs and suggests that under conditions synonymous with processing the solvated chains were swollen rather than in Gaussian conformation as previously reported. A gradual change to Guinier knees was followed by slopes of Q(-3) suggesting the presence of supramolecular structures at larger length scales. These observations in both the dilute and semidilute concentrations have not been previously reported. Zimm analysis of the data provided gyration radii and absolute molecular weights consistent with trends established using light scattering but showed some variation in their second virial coefficients. While natural-synthetic hybrids of PHA- b-PEG can self-assemble into microporous films, they showed no noticeable differences in chain conformation when in solution, the fabricating medium. This suggests that some form of entropic inducement is required.

Production and use of deuterated polyhydroxyoctanoate in structural studies of PHO inclusions.

This work reports on the biosynthesis of polyhydroxyalkanoates with medium chain length alkyl substituents in the side chain by Pseudomonas oleovorans using hydrogenated and deuterated substrates. These investigations aimed to obtain polyhydroxyalkanoates with varying degrees of deuterium substitution, and establish whether they are suitable analogues for structural investigation. In order to understand the formation and structure of inclusions in their native state, whole inclusions were isolated from microbial cells and were analysed using Small Angle Neutron Scattering. A contrast variation study was conducted on hydrogenated and deuterated inclusions of polyhydroxyoctanoate, as well as inclusions resulting from co-feeding or sequentially feeding different precursors. The data indicated a core/shell structure resulting from feeding hydrogenated followed by perdeuterated PHO precursor, and demonstrated the utility of this analysis for characterising chemically similar systems.

Quantitative analysis of deuterium using the isotopic effect on quaternary (13)C NMR chemical shifts.

Quantitative analysis of specifically deuterated compounds can be achieved by a number of conventional methods, such as mass spectroscopy, or by quantifying the residual (1)H NMR signals compared to signals from internal standards. However, site specific quantification using these methods becomes challenging when dealing with non-specifically or randomly deuterated compounds that are produced by metal catalyzed hydrothermal reactions in D2O, one of the most convenient deuteration methods. In this study, deuterium-induced NMR isotope shifts of quaternary (13)C resonances neighboring deuterated sites have been utilized to quantify the degree of isotope labeling of molecular sites in non-specifically deuterated molecules. By probing (13)C NMR signals while decoupling both proton and deuterium nuclei, it is possible to resolve (13)C resonances of the different isotopologues based on the isotopic shifts and the degree of deuteration of the carbon atoms. We demonstrate that in different isotopologues, the same quaternary carbon, neighboring partially deuterated carbon atoms, are affected to an equal extent by relaxation. Decoupling both nuclei ((1)H, (2)H) resolves closely separated quaternary (13)C signals of the different isotopologues, and allows their accurate integration and quantification under short relaxation delays (D1 = 1 s) and hence fast accumulative spectral acquisition. We have performed a number of approaches to quantify the deuterium content at different specific sites to demonstrate a convenient and generic analysis method for use in randomly deuterated molecules, or in cases of specifically deuterated molecules where back-exchange processes may take place during work up.

Deuterium Labeling Strategies for Creating Contrast in Structure-Function Studies of Model Bacterial Outer Membranes Using Neutron Reflectometry.

Studying the outer membrane of Gram-negative bacteria is challenging due to the complex nature of its structure. Therefore, simplified models are required to undertake structure-function studies of processes that occur at the outer membrane/fluid interface. Model membranes can be created by immobilizing bilayers to solid supports such as gold or silicon surfaces, or as monolayers on a liquid support where the surface pressure and fluidity of the lipids can be controlled. Both model systems are amenable to having their structure probed by neutron reflectometry, a technique that provides a one-dimensional depth profile through a membrane detailing its thickness and composition. One of the strengths of neutron scattering is the ability to use contrast matching, allowing molecules containing hydrogen and those enriched with deuterium to be highlighted or matched out against the bulk isotopic composition of the solvent. Lipopolysaccharides, a major component of the outer membrane, can be isolated for incorporation into model membranes. Here, we describe the deuteration of lipopolysaccharides from rough strains of Escherichia coli for incorporation into model outer membranes, and how the use of deuterated materials enhances structural analysis of model membranes by neutron reflectometry.

Smooth deuterated cellulose films for the visualisation of adsorbed bio-macromolecules.

Novel thin and smooth deuterated cellulose films were synthesised to visualize adsorbed bio-macromolecules using contrast variation neutron reflectivity (NR) measurements. Incorporation of varying degrees of deuteration into cellulose was achieved by growing Gluconacetobacter xylinus in deuterated glycerol as carbon source dissolved in growth media containing D2O. The derivative of deuterated cellulose was prepared by trimethylsilylation(TMS) in ionic liquid(1-butyl-3-methylimidazolium chloride). The TMS derivative was dissolved in toluene for thin film preparation by spin-coating. The resulting film was regenerated into deuterated cellulose by exposure to acidic vapour. A common enzyme, horseradish peroxidase (HRP), was adsorbed from solution onto the deuterated cellulose films and visualized by NR. The scattering length density contrast of the deuterated cellulose enabled accurate visualization and quantification of the adsorbed HRP, which would have been impossible to achieve with non-deuterated cellulose. The procedure described enables preparing deuterated cellulose films that allows differentiation of cellulose and non-deuterated bio-macromolecules using NR.

Synthesis of Perdeuterated 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ([D82 ]POPC) and Characterisation of Its Lipid Bilayer Membrane Structure by Neutron Reflectometry.

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), an unsaturated acyl chain containing lipid, is often the predominant lipid in eukaryotic cell membranes in which it is crucial for the fluidity of membranes under physiological conditions. Commercially available, partially deuterated [D31 ]1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ([D31 ]POPC) does not provide sufficient isotopic contrast for detailed structural studies of multicomponent membranes through neutron techniques. Herein, a relatively straightforward and generic chemical deuteration method is discussed for the asymmetric synthesis of perdeuterated [D31 ]1-palmitoyl-[D33 ]2-oleoyl-sn-[D5 ]glycero-[D13 ]3-phosphocholine ([D82 ]POPC) that also allows selective deuteration of any of its constituent groups. Neutron reflectivity of a [D82 ]POPC-supported bilayer was used to experimentally determine the neutron scattering length density profile of the lipid. The acyl chains of [D82 ]POPC are closely contrast-matched to heavy water, whereas the very high scattering length density of the deuterated glycerophosphocholine head groups provides good contrast to membrane-binding agents in both deuterated and non-deuterated solvent environments.

Robust high-yield methodologies for (2)H and (2)H/(15)N/(13)C labeling of proteins for structural investigations using neutron scattering and NMR.

We have developed a method that has proven highly reliable for the deuteration and triple labeling ((2)H/(15)N/(13)C) of a broad range of proteins by recombinant expression in Escherichia coli BL21. Typical biomass yields are 40-80g/L wet weight, yielding 50-500mg/L purified protein. This method uses a simple, relatively inexpensive defined medium, and routinely results in a high-yield expression without need for optimization. The key elements are very tight control of expression, careful starter culture adaptation steps, media composition, and strict maintenance of aerobic conditions ensuring exponential growth. Temperature is reduced as required to prevent biological oxygen demand exceeding maximum aeration capacity. Glycerol is the sole carbon source. We have not encountered an upper limit for the size of proteins that can be expressed, achieving excellent expression for proteins from 11 to 154kDa and the quantity produced at 1L scale ensures that no small-angle neutron scattering, nuclear magnetic resonance, or neutron crystallography experiment is limited by the amount of deuterated material. Where difficulties remain, these tend to be cases of altered protein solubility due to high protein concentration and a D2O-based environment.

Biopolymer deuteration for neutron scattering and other isotope-sensitive techniques.

The use of microbial biosynthesis to produced deuterated recombinant proteins is a well-established practice in investigations of the relationship between molecular structure and function using neutron scattering and nuclear magnetic resonance spectroscopy. However, there have been few reports of using microbial synthetic capacity to produce labeled native biopolymers. Here, we describe methods for the production of deuterated polyhydroxyalkanoate biopolyesters in bacteria, the polysaccharide chitosan in the yeast Pichia pastoris, and cellulose in the bacterium Gluconacetobacter xylinus. The resulting molecules offer not only multiple options in creating structural contrast in polymer blends and composites in structural studies but also insight into the biosynthetic pathways themselves.

The effect of sulfate-reducing bacteria on adsorption of 137Cs by soils from arid and tropical regions.

Soils from different climatic regions of Australia were studied to determine their adsorption of (137)Cs, and the effect of microbial sulfate reduction on this adsorption. The soils consisted of a surface and regolith samples from the site of a proposed low and intermediate level radioactive waste repository in arid South Australia, and two red earth loam soils from an experimental plot in the tropical Northern Territory. The process of bacterial sulfate reduction substantially decreased the adsorption of (137)Cs to the arid and tropical soils, although extended incubation resulted in greater adsorption to the regolith sample. This could have implications for the mobility of radionuclides entering these soil ecosystems.

Stereoselective synthesis of perdeuterated phytanic acid, its phospholipid derivatives and their formation into lipid model membranes for neutron reflectivity studies.

We describe a straightforward method, for synthesis of large scale (gram quantities) of highly deuterated phytanic acid from commercially available phytol while preserving the stereochemistry around the chiral centres. The subsequent synthesis of tail-deuterated analogues of the archeabacterial membrane lipids 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine (DPEPC) and 1,2-di(3RS,7R,11R-phytanoyl)-sn-glycero-3-phosphocholine (DPhyPC) from perdeuterated phytanic acid is also described. Both lipids were employed in construction of two different model membranes, namely Langmuir monolayers and a tethered bilayer membrane (TBM) on a solid substrate, characterised by pressure area isotherm and neutron reflectometry techniques. At 10 mN/m pressure the head-group thickness of both monolayers was similar while the thickness of the tail region was significantly larger for tail-deuterated DPhyPC, which was evident from a smaller area per molecule. At 20 mN/m the thickness of the head and tail regions in both lipids was comparable, yet the area per molecule of tail-deuterated DPhyPC was 10% smaller than tail-deuterated DPEPC. In the TBM bilayer model membrane, the thickness of the lipid tails in both inner and outer leaflets was 8.2 Å, giving a total of 16.4 Å. Deuteration enabled unambiguous determination of the relative proportion of the hydrogenous tether, phospholipid and subphase.

In vivo deuteration strategies for neutron scattering analysis of bacterial polyhydroxyoctanoate.

The cultivation of microorganisms on deuterated substrates has allowed us to control deuterium incorporation into biopolymer systems which is important for characterisation using neutron scattering techniques. Bacterial polyhydroxyoctanoate (PHO) is a polyester formed within inclusions inside bacterial cells and was deuterated in vivo under various conditions to characterise the formation of these inclusions by neutron scattering. Manipulation of deuterated media during microbial growth and PHO production phases resulted in polymer with partial or complete substitution of hydrogen by deuterium, as shown by gas chromatography. Sequential feeding of hydrogenated and deuterated forms of the same precursor was used to demonstrate that neutron scattering analysis could be used to differentiate between chemically similar phases in these polymer inclusions.