Modelling corneal transparency with reference to stromal architecture

The arrangement of corneal collagen fibrils within lamellae was investigated by comparing fibril positions obtained from electron microscopy with distorted hexagonal, quasi-random and aperiodic arrays. By calculating the wavelength dependence and Fourier transforms of these various arrays it was determined that an aperiodic array based on the sunflower seed head is the most compatible with corneal ultrastructure. An investigation of corneal light scattering away from the central axis was undertaken for the first time. Experimentally it was shown that corneal transmission decreases peripherally, particularly in the far periphery near the limbus. This was shown to be theoretically compatible with calculated positional changes in refractive index and fibril radius, by calculating transmission using the direct summation of scattered fields method. In swollen human corneas, it was determined that there was a notable change in wavelength dependence in the peripheral regions, possibly suggesting an increase in the size and relative number fibril free voids. . Corneal infrared transmission is poorly studied. In this part of the spectrum, the cornea acts as an absorber of incident radiation. It was hypothesised that there should be a systematic variation between corneal hydration and infrared light transmission. Experimentally, by Fourier transform infrared spectroscopy a convenient linear relationship between hydration and transmission was found. Riboflavin-UVA crosslinking is used to treat keratoconus, a degenerative corneal disorder. A swelling experiment was performed on porcine corneas in order to elucidate whether the crosslinking mechanism is intra- or inter-fibrillar. Swelling rates for the treated and untreated tissue were not statistically significant, excluding interfibrillar crosslinking. The penetration depth of the riboflavin molecule into corneal stroma was also examined by visible spectroscopy of thin segments of tissue. It was demonstrated that when riboflavin infiltrates a full thickness cornea, the highest concentration of riboflavin is present in the anterior tissue segment.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Modelling corneal transparency with reference to stromal architecture

The arrangement of corneal collagen fibrils within lamellae was investigated by comparing fibril positions obtained from electron microscopy with distorted hexagonal, quasi-random and aperiodic arrays. By calculating the wavelength dependence and Fourier transforms of these various arrays it was determined that an aperiodic array based on the sunflower seed head is the most compatible with corneal ultrastructure. An investigation of corneal light scattering away from the central axis was undertaken for the first time. Experimentally it was shown that corneal transmission decreases peripherally, particularly in the far periphery near the limbus. This was shown to be theoretically compatible with calculated positional changes in refractive index and fibril radius, by calculating transmission using the direct summation of scattered fields method. In swollen human corneas, it was determined that there was a notable change in wavelength dependence in the peripheral regions, possibly suggesting an increase in the size and relative number fibril free voids. . Corneal infrared transmission is poorly studied. In this part of the spectrum, the cornea acts as an absorber of incident radiation. It was hypothesised that there should be a systematic variation between corneal hydration and infrared light transmission. Experimentally, by Fourier transform infrared spectroscopy a convenient linear relationship between hydration and transmission was found. Riboflavin-UVA crosslinking is used to treat keratoconus, a degenerative corneal disorder. A swelling experiment was performed on porcine corneas in order to elucidate whether the crosslinking mechanism is intra- or inter-fibrillar. Swelling rates for the treated and untreated tissue were not statistically significant, excluding interfibrillar crosslinking. The penetration depth of the riboflavin molecule into corneal stroma was also examined by visible spectroscopy of thin segments of tissue. It was demonstrated that when riboflavin infiltrates a full thickness cornea, the highest concentration of riboflavin is present in the anterior tissue segment

The variation in transparency of amniotic membrane used in ocular surface regeneration

Background/aims: Scant consideration has been given to the variation in structure of the human amniotic membrane (AM) at source or to the significance such differences might have on its clinical transparency. Therefore, we applied our experience of quantifying corneal transparency to AM. Methods: Following elective caesarean, AM from areas of the fetal sac distal and proximal (ie, adjacent) to the placenta was compared with freeze-dried AM. The transmission of light through the AM samples was quantified spectrophotometrically; also, tissue thickness was measured by light microscopy and refractive index by refractometry. Results: Freeze-dried and freeze-thawed AM samples distal and proximal to the placenta differed significantly in thickness, percentage transmission of visible light and refractive index. The thinnest tissue (freeze-dried AM) had the highest transmission spectra. The thickest tissue (freeze-thawed AM proximal to the placenta) had the highest refractive index. Using the direct summation of fields method to predict transparency from an equivalent thickness of corneal tissue, AM was found to be up to 85% as transparent as human cornea. Conclusion: When preparing AM for ocular surface reconstruction within the visual field, consideration should be given to its original location from within the fetal sac and its method of preservation, as either can influence corneal transparency

An introduction to classical molecular dynamics simulation for experimental scattering users

Classical molecular dynamics simulations are a common component of multi-modal analyses from scattering measurements, such as small-angle scattering and diffraction. Users of these experimental techniques often have no formal training in the theory and practice of molecular dynamics simulation, leading to the possibility of these simulations being treated as a "black box" analysis technique. In this article, we describe an open educational resource (OER) designed to introduce classical molecular dynamics to users of scattering methods. This resource is available as a series of interactive web pages, which can be easily accessed by students, and as an open source software repository, which can be freely copied, modified, and redistributed by educators. The topic covered in this OER includes classical atomistic modelling, parameterising interatomic potentials, molecular dynamics simulations, typical sources of error, and some of the approaches to using simulations in the analysis of scattering data.Comment: Electronic Supplementary Information (ESI) available: All analysis/plotting scripts and figure files, allowing for a fully reproducible, and automated, analysis workflow for the work presented is available at \url{https://github.com/arm61/sim_and_scat_paper} (DOI: 10.5281/zenodo.2556826) under a CC BY-SA 4.0 licens

The effect of polymer end-group on the formation of styrene – maleic acid lipid particles (SMALPs)

A series of block copolymers comprising styrene and maleic acid (SMA) has been prepared using RAFT polymerisation. RAFT often results in a large hydrophobic alkylthiocarbonylthio end group and this work examines its effect on the solution behaviour of the copolymers. SMA variants with, and without, this end group were synthesised and their behaviour compared with a commercially-available random copolymer of similar molecular weight. Dynamic light scattering and surface tension measurements found the RAFT-copolymers preferentially self-assembled into higher-order aggregates in aqueous solution. Small angle neutron scattering using deuterated styrene varients add support to the accepted model that these agreggates comprise a solvent-protected styrenic core with an acid-rich shell. Replacing the hydrophobic RAFT end group with a more hydrophilic nitrile caused differences in the resulting surface activity, attributed to the ability of the adjoining styrene homoblock to drive aggregation. Each of the copolymers formed SMALP nanodiscs with DMPC lipids, which were found to encapsulate a model membrane protein, gramicidin. However, end group variation affected solubilisition of DPPC, a lipid with a higher phase transition temperature. When using RAFT-copolymers terminated with a hydrophobic group, swelling of the bilayer and greater penetration of the homoblock into the nanodisc core occurred with increasing homoblock length. Conversely, commercial and nitrile-terminated RAFT-copolymers produced nanodisc sizes that stayed constant, instead indicating interaction at the edge of the lipid patch. The results highlight how even minor changes to the copolymer can modify the amphiphilic balance between regions, knowledge useful towards optimising copolymer structure to enhance and control nanodisc formation

A multipronged approach to understanding the form and function of hStaufen protein

Staufen is a dsRNA binding protein that plays an essential role in many aspects of RNA regulation, such as mRNA transport, Staufen-mediated mRNA decay and the regulation of mRNA translation. Staufen is a modular protein characterized by the presence of conserved consensus amino acid sequences that fold into double-stranded RNA binding domains (RBDs) as well as degenerated RBDs that maintain the α-β-β-β-α fold but are unable to bind RNA and are instead involved in protein-protein interactions. The variety of biological processes in which Staufen participates in the cell suggests that this protein associates with many diverse RNA targets, some of which have been identified experimentally. Staufen binding mediates the recruitment of effectors via protein-protein and protein-RNA interactions. The structural determinants of a number of these interactions, as well as the structure of full-length Staufen, remain unknown. Here, we present the first solution structure models for full-length human Staufen155, showing that its domains are arranged as beads-on-a-string in the absence of RNA

Tuneable peptide cross-linked nanogels for enzyme-triggered protein delivery

Many diseases are associated with the dysregulated activity of enzymes, such as matrix metalloproteinases (MMPs). This dysregulation can be leveraged in drug delivery to achieve disease- or site-specific cargo release. Self-assembled polymeric nanoparticles are versatile drug carrier materials due to the accessible diversity of polymer chemistry. However, efficient loading of sensitive cargo, such as proteins, and introducing functional enzyme-responsive behaviour remain challenging. Herein, peptide-crosslinked, temperature-sensitive nanogels for protein delivery were designed to respond to MMP-7, which is overexpressed in many pathologies including cancer and inflammatory diseases. The incorporation of N-cyclopropylacrylamide (NCPAM) into N-isopropylacrylamide (NIPAM)-based copolymers enabled us to tune the polymer lower critical solution temperature from 33 to 44 °C, allowing the encapsulation of protein cargo and nanogel-crosslinking at slightly elevated temperatures. This approach resulted in nanogels that were held together by MMP-sensitive peptides for enzyme-specific protein delivery. We employed a combination of cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), small angle neutron scattering (SANS), and fluorescence correlation spectroscopy (FCS) to precisely decipher the morphology, self-assembly mechanism, enzyme-responsiveness, and model protein loading/release properties of our nanogel platform. Simple variation of the peptide linker sequence and combining multiple different crosslinkers will enable us to adjust our platform to target specific diseases in the future

Coupling Lipid Nanoparticle Structure and Automated Single Particle Composition Analysis to Design Phospholipase Responsive Nanocarriers

Lipid nanoparticles (LNPs) are versatile structures with tunable physicochemical properties that are ideally suited as a platform for vaccine delivery and RNA therapeutics. A key barrier to LNP rational design is the inability to relate composition and structure to intracellular processing and function. Here we combine Single Particle Automated Raman Trapping Analysis (SPARTA®) with small angle scattering (SAXS / SANS) techniques to link LNP composition with internal structure and morphology and to monitor dynamic LNP - phospholipase D (PLD) interactions. Our analysis demonstrates that phospholipase D, a key intracellular trafficking mediator, can access the entire LNP lipid membrane to generate stable, anionic LNPs. PLD activity on vesicles with matched amounts of enzyme substrate was an order of magnitude lower, indicating that the LNP lipid membrane structure can be used to control enzyme interactions. This represents an opportunity to design enzyme-responsive LNP solutions for stimuli-responsive delivery and diseases where PLD is dysregulated

Using solution history to control hydrogel properties of a perylene bisimide

pH dependence on water soluble aggregates is well-known in the field of low molecular weight gelators (LMWGs), with different aggregates sometimes having very different properties depending on their final pH. This aggregation determines their applications and performance. Here, we investigate the pH dependence of perylene bisimide gels; initially solutions are formed at a high pH and gels form as the pH is decreased. We find it is not only the final pH but also the starting pH that can impact the resulting gel. We use small angle neutron scattering (SANS), rheology, 1H NMR spectroscopy and absorption spectroscopy to examine the effect of starting pH on gelation kinetics and final gel properties. Adjusting the solution from pH 9 (where there are few or no aggregates) to pH 6 results in the formation of different worm-like micelles than the ones directly formed at pH 6, leading to again gels with different mechanical properties. This work highlights the importance of controlling the pH of solutions before gelation, but also opens up more possible morphologies and therefore more properties from the same molecule