Sally L Gras

University of Melbourne, Melbourne, Victoria, Australia

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Publications (49)179.67 Total impact

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    ABSTRACT: Amyloid fibrils are ordered, insoluble protein aggregates that are associated with neurodegenerative conditions such as Alzheimer's disease. The fibrils have a common rod-like core structure, formed from an elongated stack of β-strands, and have a rigidity similar to that of silk (Young's modulus of 0.2-14 GPa). They also exhibit high thermal and chemical stability and can be assembled in vitro from short synthetic non-disease-related peptides. As a result, they are of significant interest in the development of self-assembled materials for bionanotechnology applications. Synthetic DNA molecules have previously been used to form intricate structures and organize other materials such as metal nanoparticles and could in principle be used to nucleate and organize amyloid fibrils. Here, we show that DNA origami nanotubes can sheathe amyloid fibrils formed within them. The fibrils are built by modifying the synthetic peptide fragment corresponding to residues 105-115 of the amyloidogenic protein transthyretin and a DNA origami construct is used to form 20-helix DNA nanotubes with sufficient space for the fibrils inside. Once formed, the fibril-filled nanotubes can be organized onto predefined two-dimensional platforms via DNA-DNA hybridization interactions.
    Nature Nanotechnology 06/2014; · 31.17 Impact Factor
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    ABSTRACT: The physicochemical and rheological properties of yoghurt made from unstandardised unhomogenised buffalo milk were investigated during fermentation and 28 days of storage and compared to the properties of yoghurt made from homogenised fortified bovine milk. A number of differences observed in the gel network can be linked to differences in milk composition. The microstructure of buffalo yoghurt, as assessed by confocal laser scanning microscopy (CLSM) and cryo scanning electron microscopy (cryo-SEM), was interrupted by large fat globules and featured more serum pores. These fat globules have a lower surface area and bind less protein than the homogenised fat globules in bovine milk. These microstructural differences likely lead to the higher syneresis observed for buffalo yoghurt with an increase from 17.4 % (w/w) to 19.7 % (w/w) in the weight of whey generated at days 1 and 28 of the storage. The higher concentration of total calcium in buffalo milk resulted in the release of more ionic calcium during fermentation. Gelation was also slower but the strength of the two gels was similar due to similar protein and total solids concentrations. Buffalo yoghurt was more viscous, less able to recover from deformation and less Newtonian than bovine yoghurt with a thixotropy of 3,035 Pa.s−1 measured for buffalo yoghurt at the end of the storage, at least four times higher than the thixotropy of bovine yoghurt. While the titratable acidity, lactose consumption and changes in organic acid concentrations were similar, differences were recorded in the viability of probiotic bacteria with a lower viability of Lactobacillus acidophilus of 5.17 log (CFU/g) recorded for buffalo yoghurt at day 28 of the storage. Our results show that factors other than the total solids content and protein concentration of milk affect the structural properties of yoghurt. They also illustrate the physicochemical reasons why buffalo and bovine yoghurt are reported to have different sensory properties and provide insight into how compositional changes can be used to alter the microstructure and properties of dairy products.
    Food and Bioprocess Technology 04/2014; · 4.12 Impact Factor
  • Food Hydrocolloids 01/2014; 40:16–21. · 3.49 Impact Factor
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    ABSTRACT: Porous microspheres capable of delivering high payloads of biomolecules with suitable biodegradability and biocompatibility would be valuable in delivery systems to aid tissue regeneration. This study describes a facile, scalable technique to produce biodegradable porous microspheres by combining continuous ink-jetting through a piezoelectric nozzle with thermally induced phase separation (TIPS). A selection of biomaterials is investigated to suit delivery in tissue engineering, the synthetic polyesters poly(lactic-co-glycolic acid) (PLGA), and poly caprolactone (PCL) and a natural polymer, gelatin. The parameters governing the microsphere production are determined experimentally and compared to theoretical predictions derived from the fluid mechanics and heat transfer during the ink-jetting process. The microspheres produced have open interconnected pores with mean particle diameters of 80–200 μm and no significant skin region. The physical properties, such as the mean particle diameter, pore size, and surface area could be controlled by varying production parameters including the ink-jetting pressure, nozzle height, and the size and oscillation frequency of the nozzle. The technique is demonstrated to successfully encapsulate a model hydrophobic molecule during microsphere production with uniform distribution. Porous PLGA microspheres are also used to achieve much higher adsorption capacities of a short peptide than non-porous microspheres of the same material.
    Particle and Particle Systems Characterization 01/2014; · 0.86 Impact Factor
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    ABSTRACT: Nanotechnology promises to affect many aspects of our lives with its development being greeted with both excitement and fear. The debate concerning nanotechnology has echoed that of genetically engineered organisms and their introduction into the environment and the food chain. Nanotechnology offers many potential advantages in the processing and manufacture of foods: enhanced bioavailability, colour and flavour; novel food textures; new delivery mechanisms; and access to biosensors to enhance food safety. In fact, many of the foods we have been consuming for centuries already contain nanostructures, leading many to assume that they are safe. The extent to which novel nanostructures may afford new risks has not been adequately resolved, however, leading to concern within some consumer groups. In this article, we use proteins as a case study to explore our current understanding of nanostructures in foods and the extent to which novel nanostructures may introduce new properties. It is well recognised that some protein nanostructures are toxic and are associated with disease, so there is legitimate concern as to whether such species should be deliberately introduced into our foods. This review highlights current literature on protein nanostructures in food and possible risks associated with their use. We aim to provide a balanced assessment to inform future decision-making regarding the utilisation of nanostructures in food.
    Trends in Food Science & Technology 01/2014; · 4.14 Impact Factor
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    ABSTRACT: The amyloid fibril-forming ability of two closely related antifungal and antimicrobial peptides derived from plant defensin proteins has been investigated. As assessed by sequence analysis, thioflavin T binding, transmission electron microscopy, atomic force microscopy and X-ray fibre diffraction, a 19 amino acid fragment from the C-terminal region of Raphanus sativus antifungal protein, known as RsAFP-19, is highly amyloidogenic. Further, its fibrillar morphology can be altered by externally controlled conditions. Freezing and thawing led to amyloid fibril formation which was accompanied by loss of RsAFP-19 antifungal activity. A second, closely related antifungal peptide displayed no fibril-forming capacity. It is concluded that while fibril formation is not associated with the antifungal properties of these peptides, the peptide RsAFP-19 is of potential use as a controllable, highly amyloidogenic small peptide for investigating the structure of amyloid fibrils and their mechanism of formation.
    Biochimica et Biophysica Acta 05/2013; · 4.66 Impact Factor
  • Elizabeth B Sawyer, Sally L Gras
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    ABSTRACT: Amyloid fibrils are attractive targets for applications in biotechnology. These thin, nanoscale protein fibers are highly ordered structures that self-assemble from their component proteins or peptides. This chapter describes the use of several biophysical techniques to monitor the formation of amyloid fibrils including a common dye-binding assay, turbidity assay, and small-angle X-ray scattering. These techniques provide information about the assembly mechanism, the rate and reproducibility of assembly, as well as the size of species along the assembly pathway.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 996:77-101. · 1.29 Impact Factor
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    ABSTRACT: Large bowel luminal short chain fatty acids (SCFA) are protective against gut diseases such as colorectal cancer, Crohn’s disease and ulcerative colitis. High amylose maize starches acylated with acetic, propionic or butyric were previously shown to deliver SCFA to the gut and increase gut SCFA. This study examines the ability of five Bifidobacterium spp. (B. longum, B. breve, B. infantis, B. adolescentis and B. bifidum), Ruminococcus bromii and Faecalibacterium prausnitzii to degrade starches acylated with SCFA. Release of SCFA from modified starches was not observed, suggesting no preferential hydrolysis of the SCFA ester bond over the glycosidic bond. Acetylated and propionated starches are more readily degraded than either the base or butyrated starches in tested cultures. These observed differences may depend on modification to the starch structure. These structural differences alter the kinetics of starch digestion, irrespective of bacterial type, which may impact current approaches of prebiotic selection.
    Journal of Functional Foods. 01/2013;
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    Zhenyu Shi, Anthony G Wedd, Sally L Gras
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    ABSTRACT: The development of synthetic biology requires rapid batch construction of large gene networks from combinations of smaller units. Despite the availability of computational predictions for well-characterized enzymes, the optimization of most synthetic biology projects requires combinational constructions and tests. A new building-brick-style parallel DNA assembly framework for simple and flexible batch construction is presented here. It is based on robust recombination steps and allows a variety of DNA assembly techniques to be organized for complex constructions (with or without scars). The assembly of five DNA fragments into a host genome was performed as an experimental demonstration.
    PLoS ONE 01/2013; 8(2):e56854. · 3.73 Impact Factor
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    ABSTRACT: A novel combination of site-specific isotope labelling, polarised infrared spectroscopy and molecular combing reveals local orientational ordering in the fibril-forming peptide YTIAALLSPYSGGRADS. Use of (13)C-(18)O labelled alanine residues demonstrates that the N-terminal end of the peptide is incorporated into the cross-beta structure, while the C-terminal end shows orientational disorder.
    Chemical Communications 09/2012; · 6.38 Impact Factor
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    ABSTRACT: The effect of milk pH at renneting on the microstructure, composition and texture of Cheddar cheese was investigated. Four different Cheddar cheeses were made by the addition of rennet to milk pre-acidified to pH 6.7, pH 6.5, pH 6.3 or pH 6.1. The gel renneted at pH 6.1 showed a dense protein network when observed using confocal laser scanning microscopy and cryo scanning electron microscopy. This structure became more compact after cooking, forming an irregular and coarse matrix with lower porosity than in other treatments. There was less fat lost to the whey during the draining of curd after cooking and higher fat lost to the whey during pressing in samples made from milk renneted at a lower pH. The texture of the Cheddar cheese made using milk renneted at pH 6.1 was altered, with lower chewiness, gumminess, cohesiveness and springiness when compared to cheese made using milk renneted at pH 6.7 or pH 6.5. The yield in dry matter of cheese renneted at pH 6.1 or pH 6.3 was 11–13% higher than for cheese renneted at pH 6.7. These results indicate that the pH of milk at renneting is a process variable that can be used to increased yield and alter the texture of Cheddar cheese.
    Food Research International. 08/2012; 48(1):119–130.
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    ABSTRACT: Many bacteria produce protein fibrils that are structurally analogous to those associated with protein misfolding diseases such as Alzheimer's disease. However, unlike fibrils associated with disease, bacterial amyloids have beneficial functions including conferring stability to biofilms, regulating development or imparting virulence. In the present review, we consider what makes amyloid fibrils so suitable for these roles and discuss recent developments in the study of bacterial amyloids, in particular the chaplins from Streptomyces coelicolor. We also consider the broader impact of the study of bacterial amyloids on our understanding of infection and disease and on developments in nanotechnology.
    Biochemical Society Transactions 08/2012; 40(4):728-34. · 2.59 Impact Factor
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    ABSTRACT: Protein nanofibers are emerging as useful biological nanomaterials for a number of applications, but to realize these applications requires a cheap and readily available source of fibril-forming protein material. We have identified fish lens crystallins as a feedstock for the production of protein nanofibers and report optimized methods for their production. Altering the conditions of formation leads to individual protein nanofibers assembling into much larger structures. The ability to control the morphology and form higher order structures is a crucial step in bottom up assembly of bionanomaterials. Cell toxicity assays suggest no adverse impact of these structures on mammalian cell proliferation. There are many possible applications for protein nanofibers; here we illustrate their potential as templates for nanowire formation, with a simple gold plating process.
    Biopolymers 08/2012; 97(8):595-606. · 2.88 Impact Factor
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    ABSTRACT: The milk fat globule membrane (MFGM) is a complex soft matter system that encapsulates the milk fat globule (MFG). It is important for the successful delivery of nutrients for newborns and it is also increasingly studied for its potential pharmaceutical benefits. Yet, the persistence and integrity of the MFGM through many steps common in the processing of milk for consumption and cheese manufacture is not completely understood and is an area of ongoing interest. In this study, atomic force microscopy (AFM) was used to characterize the MFGM membrane interface to demonstrate the effects of mechanical force and shear encountered during milk processing. The results showed that significant differences can be observed and such a soft biological system with complex constituents can be quantitatively characterized using AFM. Major differences were observed in the surface morphology and the roughness of the MFGM in raw milk, raw ultra filtrate (UF) retentate, homogenized milk, cheese milk and cream samples. The MFGM of the homogenized fat globules showed the greatest difference in appearance when compared to the native raw MFGM from damage largely due to the homogenization process. In addition, AFM elasticity and thickness measurements of the MFGM showed quantitative differences between the five different samples with an increase in the stiffness of the MFGM of milk samples that have undergone more processing steps. The quantitative results of the physical nature of the MFGM from the AFM study, combined with the qualitative results on the chemical nature of the MFGM, demonstrate that AFM can aid in the understanding of the interfacial behaviour of complex soft matter systems where access to detailed chemical composition is limited.
    RSC Advances 02/2012; 2(6):2384-2394. · 3.71 Impact Factor
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    ABSTRACT: The incorporation of low molecular weight drugs and therapeutic peptides into multilayer films assembled via the layer-by-layer technique can potentially provide means to deliver small molecules to target sites and to tune their release. This study describes the use of both hydrophobic and electrostatic interactions to incorporate a tridecapeptide antiinflammatory hormone, α-melanocyte stimulating hormone (α-MSH), as a building block at the base of a multilayer assembly of hyaluronic acid (HA) and chitosan (CS) on poly(lactic-co-glycolic acid) (PLGA) surfaces. A range of switching layers, including a neutral lipid, dioleylphosphatidylcholine (DOPC), a negatively charged lipid mixture DOPC/dioleylphosphatidylserine (DOPS) and a negatively charged polysaccharide, HA, were investigated for their ability to support subsequent HA and CS layers. Molecular dynamics simulations were performed to examine the structure and surface chemistry of α-MSH in solution and on surfaces to provide insights into the conditions most likely to support multilayer assembly. The multilayer assembly was stable at physiological pH and was successfully applied to particulate systems.
    The Journal of Physical Chemistry B 12/2011; 116(3):1120-33. · 3.61 Impact Factor
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    ABSTRACT: Mutations in the polypeptide sequence that forms the core structure of amyloid fibrils are known to impact on fibril assembly and stability but the effect of changes on noncore residues, particularly relating to functionalized fibrils where the fibril core is preserved, has not been systematically examined. In this study, the short peptide sequence TTR(105-115) (also known as TTR1) and the functionalized variants TTR1-RGD and TTR1-RAD are used as a model system to investigate the effect of noncore residues on the kinetics of fibril assembly. The noncore residues in TTR1-RGD and TTR1-RAD influence the rate of fibril assembly in non-seeded samples with the glycine residue at position 15 increasing the rate of aggregation compared to alanine. Mature TTR1-RGD fibrils were also found to fragment more readily, indicating possible differences in mechanical properties. Fragments of each type of fibril are capable of self- and cross-seeding, generating fibrils with a highly similar cross-β core structure. The similar rates of assembly observed for self-seeded samples reflect the similar free energy of elongation calculated for these peptides, while the morphology of cross-seeded fibrils is determined by the properties of the monomeric peptide and its macromolecular arrangement within the protofilaments and fibrils. These findings illustrate that noncore residues impact on fibril formation and fibril properties and demonstrate that the influence of noncore residues should be considered when designing sequences for the production of self-assembling functional fibrillar materials.
    Journal of Molecular Biology 12/2011; 421(2-3):256-69. · 3.91 Impact Factor
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    ABSTRACT: Many biomaterials used in tissue engineering cause a foreign body response in vivo, which left untreated can severely reduce the effectiveness of tissue regeneration. In this study, an anti-inflammatory hormone α-melanocyte stimulating hormone (α-MSH) was physically adsorbed to the surface of biodegradable poly (lactic-co-glycolic) acid (PLGA) microspheres to reduce inflammatory responses to this material. The stability and adsorption isotherm of peptide binding were characterized. The peptide secondary structure was not perturbed by the adsorption and subsequent desorption process. The α-MSH payload was released over 72 h and reduced the expression of the inflammatory cytokine, Tumor necrosis factor-α (TNF-α) in lipopolysaccharide activated RAW 264.7 macrophage cells, indicating that the biological activity of α-MSH was preserved. α-MSH coated PLGA microspheres also appeared to reduce the influx of inflammatory cells in a subcutaneous implantation model in rats. This study demonstrates the potential of α-MSH coatings for anti-inflammatory delivery and this approach may be applied to other tissue engineering applications. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.
    Journal of Biomedical Materials Research Part A 11/2011; · 2.83 Impact Factor
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    ABSTRACT: Amino acid-based core cross-linked star (CCS) polymers (poly(L-lysine)(arm)poly(L-cystine)(core)) with peripheral allyl functionalities were synthesized by sequential ring-opening polymerization (ROP) of amino acid N-carboxyanhydrides (NCAs) via the arm-first approach, using N-(trimethylsilyl)allylamine as the initiator. Subsequent functionalization with a poly(ethylene glycol) (PEG)-folic acid conjugate via thiol-ene click chemistry afforded poly(PEG-b-L-lysine)(arm)poly(L-cystine)(core) stars with outer PEG coronas decorated with folic acid targeting moieties. Similarly, a control was prepared without folic acid, using just PEG. A fluorophore was used to track both star polymers incubated with breast cancer cells (MDA-MB-231) in vitro. Confocal microscopy and flow cytometry revealed that the stars could be internalized into the cells, and higher cell internalization was observed when folic acid moieties were present. Cytotoxicity studies indicate that both stars are nontoxic to MDA-MB-231 cells at concentrations of up to 50 μg/mL. These results make this amino acid-based star polymer an attractive candidate in targeted drug delivery applications including chemotherapy.
    Biomacromolecules 08/2011; 12(10):3469-77. · 5.37 Impact Factor
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    ABSTRACT: An experimental determination of the thermodynamic stabilities of a series of amyloid fibrils reveals that this structural form is likely to be the most stable one that protein molecules can adopt even under physiological conditions. This result challenges the conventional assumption that functional forms of proteins correspond to the global minima in their free energy surfaces and suggests that living systems are conformationally as well as chemically metastable.
    Journal of the American Chemical Society. 08/2011; 133(36).
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    ABSTRACT: An experimental determination of the thermodynamic stabilities of a series of amyloid fibrils reveals that this structural form is likely to be the most stable one that protein molecules can adopt even under physiological conditions. This result challenges the conventional assumption that functional forms of proteins correspond to the global minima in their free energy surfaces and suggests that living systems are conformationally as well as chemically metastable.
    Journal of the American Chemical Society 06/2011; 133(36):14160-3. · 10.68 Impact Factor

Publication Stats

307 Citations
179.67 Total Impact Points

Institutions

  • 2006–2014
    • University of Melbourne
      • • Department of Chemical and Biomolecular Engineering
      • • School of Chemistry
      • • Bio21 Molecular Science and Biotechnology Institute
      Melbourne, Victoria, Australia
  • 2009–2013
    • Victoria University Melbourne
      Melbourne, Victoria, Australia
  • 2011
    • University of Reading
      • Department of Chemistry
      Reading, ENG, United Kingdom
  • 2006–2011
    • University of Cambridge
      • • Department of Chemistry
      • • Department of Physics: Cavendish Laboratory
      Cambridge, ENG, United Kingdom