This is to certify that the work presented in the dissertation entitled: Amyloid Like Property of Large Protein Fibers & its application as a biomaterial submitted by Kaustuv Das, Roll Number 216BM2014, is a record of original research conducted by him under the supervision and our guidance in partially fulfilling the requirements of the M.Tech Biotechnology degree in the Department of Biotechnology and Medical Engineering. Mukesh Kumar Gupta, Head, Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela for the support.
31 Fig 4.18 (A) Scanning electron micrograph of clay nest fibers for. understanding the structural architecture of the nest. 48 Fig 4.38 X-ray diffraction curve (A) Large amyloid filaments (B) Wasp nest filaments. C) Muddy nest threads (D) Yellowjacket nest threads.
CONTENTS
Introduction
Materials & Methods
INTRODUCTION
- Background study and Significance of Work
- Pepsin - An Acid Protease
- Synthetically Prepared Amyloid
- Large Amyloid Fibers
- Application of Amyloid as a Biomaterial
- Non-Mammalian Amyloid like Structures
- Wasp Nest
- Emulsions
The shape of fibers can be rectangular or cylindrical with tapered ends depending on the distribution of the hydrophobic amino acids. Construction of amyloid hydrogels used for the differentiation of the mesenchymal stem cells to neurons[14].
Objectives
Literature Review
Amyloids are widely used as biomaterials. Some applications of amyloid as a biomaterial are listed below. Amyloid fibers were made from lysozyme and β-lactoglobulin. The aim of the experiment was to create a new form of hybrid nano-composite that exhibits bone mimetic properties. The amyloid fibers made the HAP suspension and grinds colloidally stable in water by adsorption of platelets on the surface.
By varying the fibril fraction, the moduli and density of the composite were observed to be comparable to those of normal cancellous bone. Two aspartate residues are present in the active site of the molecules and cleave peptide bonds between aromatic or hydrophobic amino acids. Oil droplets in water, when shaken, form globules in it, resulting in an emulsion, but after a while they flow together again to form separate phases, leading to destabilization of the emulsion.
This emulsion destabilization is mainly attributed to the 4 factors of Brownian flocculation, creaming, sedimentation and disproportionation. The component that prevents the coalescence of organic droplets is known as an emulsifier.
Materials & Methods
- Preparation and Characterization of Emulsion - The emulsion was prepared by mixing Hexane as the organic solvent with that of amyloid solution without any additional dilution
- Emulsion Stability - The stability of the formed emulsion was evaluated by using centrifugation and also by inverted tube assay. The emulsions were inverted to check both the
- Sample Preparation of Natural Fibers
- Scanning Electron Microscopy - Electron microscopy images of pepsin amyloid, YNF, WNF and MNF was done under Scanning Electron Microscope ( Nova Nanosem 450) Pepsin
- Environmental Scanning Electron Microscope - ESEM (FEI,USA) was done for visualizing the amyloids around the hexane droplets as it provides the provision for allowing
- Confocal Laser Scanning Microscopy (CLSM) – Binding of Thioflavin T dye to the amyloid Fibers prepared from pepsin was checked through CLSM. The sample along with the
- Biocompatibility
- Hemolysis Assay
- Alkaline Phosphatase Activity
- Release of Sulfated Glycosaminoglycan
The drying of the sample and also due to the focussing of the electron beam in the system resulted in puffed air bubbles due to the escape of the volatile hexane solvent. The dye working solution was prepared by diluting the stock solution in phosphate buffer at a ratio of 1:50. To determine the distribution of secondary structures, deconvolution of the amide I region (1600-1700cm-1) was performed.
Thioflavin T and Congo red both specific amyloid dyes have been used for visualization and confirmation of amyloids stabilizing emulsion droplets. The biocompatible nature of naturally occurring fiber samples was assessed by performing a cell viability assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The sample was washed and sterilized followed by incubation with diluted blood for 60 minutes at 37°c.
The OD of the supernatant with the lysed RBCs was calculated using spectrophotometer at 545nm. Samples were initially seeded with cells and incubated for 7 days at 37°c and 5% CO2. After 7 days, 20μl of the media with GAG was taken and added with 200μl of prepared DMMB dye. The absorptivity reading was taken at 525nm.
Results & Discussion
Formation of pepsin amyloid
Morphology of Synthetically Prepared amyloid
4.1 - (A) Scanning electron microscopy images of porcine pepsin flipped amyloid fibrils. B) Large amyloid fibers observed under a scanning electron microscope after incubation for 7 days at 40°C.
X-Ray Diffraction
The cross-linked β conformation accounts for the rigidity and robustness of amyloid along with its property to resist degradation by any enzymes. The orientation of the β strands perpendicular to the axis of the plates is the reason for the cross β architecture, which is also the reason for the binding of thioflavin T.
Dye Binding Assay
In our study, we presented the T study along with CD spectra analysis, SEM and X-ray diffraction to validate the formation of amyloid fibers from pepsin.
Confocal Laser Scanning Microcopy (CLSM)
Pepsin Assay
Role of Pepsin Amyloid as an Emulsion Stabilizer
- Emulsion Preparation
- Validation of the Role of Amyloids as an Emulsifier
- Emulsion Stability
- Droplet Characterization
Figure (4.7) shows that pepsin lost its ability to retain organic solvent over time and that the phases separated. When they slowly begin to return to their original states, they lose the ability to retain organic solvent in the aqueous environment, which leads to phase separation. The presence of amyloid fibers surrounding the inflated hexane droplets can be seen in Figure (4.10).
Centrifugation leads to coalescence of the organic solvent droplets, which ultimately leads to destabilization of the oil-in-water emulsion. Fig (4.8) shows that with time, native pepsin's ability to stabilize organic solvent decreased, ultimately leading to destabilization of the emulsion, leading to phase separation. The thioflavin T fluorescence in fig. (4.9) provides support for the presence of amyloids in a surrounding organic solvent. ESEM imaging was performed for visual confirmation of the amyloids in their role in stabilizing the organic solvent.
The presence of the amyloid fibers around a single inflated droplet on which the electron beam was focused proves its occurrence. However, the presence of denatured protein aggregates may also have been responsible for the emulsifying property of the amyloid solution.
Nest Samples and its corresponding Insects
Surface Characterization of the Natural fibers
- Wasp Nest
- Wasp Nest after Boiling in in Sodium Carbonate
- Mud dauber Nest
- Yellow Jacket Nest
- Comparison of the diameters of YNF, MNF and WNF
Mud nest as observed under a scanning electron microscope with fibrous structure is depicted in Fig (4.18A) which gives a probable idea of how it is able to bind the soil particles around it. The membranous sheet as shown in Fig (4.18B) may be finer fibers in the nanometer range that hold the framework. It should be noted that the mud floater nests possess these fibrous structures within the nest, unlike the other hives where the fibers are exposed.
The nest fibers are present in the clay soil which actually binds and holds the soli particles in it. Similar to WNF and MNF scanning electron micrograph imaging showed the structural morphology of the yellowjacket nest. The presence of membranous skin similar to MNF is also visible in Fig (4.19B) which may be the connecting structure of between the fibers that confers the nest's overall structural integrity.
A comparison was made for YNF, WNF and MNF based on the diameters of the fibers that make up the nest, to understand their significance according to their place of occurrence. The standard deviation of YNF indicates the presence of different fibers of different diameters in nested structures.
Structural Study of the Natural Fibers
- X-Ray Diffraction
The major peak at 1632 cm-1 as shown in Fig (4.22) indicates the presence of β-sheet conformations. Both 1629 and 1639, which fall within the amide I region, indicate the presence of native β-sheet structures along with the presence of antiparallel β-sheets or turns. As reported previously for silk fibroin 35 spider silk5, similar observations for the presence of β-sheet distribution were observed with transmission peaks in the amide I region at 1630 cm-1 and 1624 cm-1, respectively.
The extensive presence of such a β-sheet structure can be attributed to the properties of nested fibers to have stiffness and tensile strength. In both cases of spider silk and silk fibroin, the distribution of alanine and glycine residues is cited as the reason for the presence of such a β-sheet structure. In Figure (4.23B), the first peak at 1625 cm-1 indicates the presence of amyloid β-sheet conformation[35], at 1642 cm-1 such as the structure of Blatne dlak and Wasp's nest, indicates the presence of natural β-sheet structures.
However, at 1658 cm-1 the peak indicating the presence of alpha-helical structure is a unique understanding. The corresponding d-spacing between the two peaks is at 6.55 Å and 3.81 Å, indicating the presence of β-sheet [36]-like structure along with distribution of α-helix patterns.
Binding of Amyloid Specific Dye
Yellow Jacket is also a form of wasp whose saliva secretion is said to be necessary for the assembly of the nest fiber structure. All 3 structures show similarity in the d-spacing indicating the bias towards β-sheet conformations that provide rigidity to the structures. FT-IR spectra and X-ray diffraction data obtained from the naturally occurring fibers discussed above support the distribution of β-sheet structures and the fluorescence of Thioflavin T when the fibers bind, by which we can say that the naturally occurring nest fibers are one or other form has similar amyloid-like structure.
The membranous layer between the fibers in Mud dauber nest and Yellow Jacket also took up fluorescence, indicating the amyloid-like nature of the nanometer-sized fibers present. The laser intensity of both dyes with respect to all the samples was kept constant.
Cell Viability Assay
Hemolysis Assay
Cell Attachment Study
Release of Sulfated Glycosaminoglycan (GAG)
Alkaline Phosphatase Activity
Structural Comparison of Large Amyloid Fibers and Natural Wasp Nest Fibers
- Scanning Electron Microscopy
- X-Ray Diffraction
- Fourier Transform Infrared Spectroscopy
- Confocal Laser Scanning Microscopy
The initial peak of 10.5° of amyloid fibers is close to 13° of the natural fibers, indicating the presence of β-sheets as this corresponds to interstrand spacing. From the initial findings, it can be concluded that the presence of β-sheets more similar to silk fibers is present, but their structural arrangement cannot be deduced requiring further investigation. Fourier transform Infrared spectroscopy of amyloid fibers shows a peak between 1605 cm-1 to 1625 cm-1, indicating the presence of β-sheets in amyloid-like conformation.
The results show the presence of native β-sheet conformation that is different from the amyloid cross-β arrangement. The findings indicate the presence of native β-sheets that mimic silk fibers rather than amyloid structural arrangement. Thioflavin T-linked amyloids when excited at 454nm, the amyloid fibers were visible which proved that apart from electron microscopy CLSM can also be a procedure of amyloid identification.
Now, XRD and FT-IR proved the presence of native β-strands, different from cross-β conformation of amyloids. The CLSM study of the natural fibers using Thioflavin T may be due to the presence of some amyloid-like structures within the fibers that remains to be investigated, otherwise it may be an ionic interaction.
Conclusion
YNF will be used in tissue engineering except WNF which had significantly less ALP activity. The cell adhesion study of all samples using the osteosarcoma cell line was validated by the combination of CLSM and phase contrast microscopy. In addition, the preliminary investigation of wasp fibers on their ability to allow osteosarcoma cells to secrete Alkaline Phosphatase and Glycosaminoglycans shows their perspective in tissue engineering as effective biomaterials.
An in-depth study of the structural properties of the fibers and further evaluation of the properties of the fibers to act as effective biomaterials is what remains to be done.
Characterization of large amyloid fibrils and ribbons by Fourier transform infrared (FT-IR) and Raman spectroscopy. Chemical composition of nest walls and nesting behavior of Ropalidia (Icarielia) opifex van der Vecht, 1962 (Hymenoptera: . Vespidae), a social wasp of Southeast Asia with transparent nests.
