Fibril Structure Research Articles

Biophysical Properties of the Fibril Structure of the Toxic Conformer of Amyloid-β42: Characterization by Atomic Force Microscopy in Liquid and Molecular Docking.

Alzheimer's disease is associated with the aggregation of the misfolded neuronal peptide, amyloid-β42 (Aβ42). Evidence has suggested that several reasons are responsible for the toxicity caused by the aggregation of Aβ42, including the conformational restriction of Aβ42. In this study, one of the toxic conformers of Aβ42, which contains a Glu-to-Pro substitution (E22P-Aβ42), was explored using atomic force microscopy and molecular docking to study the aggregation dynamics. We proposed a systematic model of fibril formation to better understand the molecular basis of conformational transitions in the Aβ42 species. Our results demonstrated the formation of amorphous aggregates in E22P-Aβ42 that are stem-based, network-like structures, while the formation of mature fibrils occurred in the less toxic conformer of Aβ42, E22-Aβ42, that are sphere-like flexible structures. A comparison was made between the biophysical properties of E22P-Aβ42 and E22-Aβ42 that revealed that E22P-Aβ42 had greater stiffness, dihedral angle, number of β sheets involved, and elasticity, compared with E22-Aβ42. These findings will have considerable implications toward our understanding of the structural basis of the toxicity caused by conformational diversity in Aβ42 species.

Capacitive Electrode-Based Electric Field Treatments on Redox-Toxic Iron Deposits in Transgenic AD Mouse Models: The Electroceutical Targeting of Alzheimer's Disease Feasibility Study.

Iron accumulation in the brain accelerates Alzheimer's disease progression. To cure iron toxicity, we assessed the therapeutic effects of noncontact transcranial electric field stimulation to the brain on toxic iron deposits in either the Aβ fibril structure or the Aβ plaque in a mouse model of Alzheimer's disease (AD) as a pilot study. A capacitive electrode-based alternating electric field (AEF) was applied to a suspension of magnetite (Fe3O4) to measure field-sensitized reactive oxygen species (ROS) generation. The increase in ROS generation compared to the untreated control was both exposure-time and AEF-frequency dependent. The frequency-specific exposure of AEF to 0.7-1.4 V/cm on a magnetite-bound Aβ-fibril or a transgenic Alzheimer's disease (AD) mouse model revealed the degradation of the Aβ fibril or the removal of the Aβ-plaque burden and ferrous magnetite compared to the untreated control. The results of the behavioral tests show an improvement in impaired cognitive function following AEF treatment on the AD mouse model. Tissue clearing and 3D-imaging analysis revealed no induced damage to the neuronal structures of normal brain tissue following AEF treatment. In conclusion, our results suggest that the effective degradation of magnetite-bound amyloid fibrils or plaques in the AD brain by the electro-Fenton effect from electric field-sensitized magnetite offers a potential electroceutical treatment option for AD.

Ultrasound‒treated soy protein fibrils: A potential vehicle for curcumin with improved water solubility, antioxidant activity and sustained‒release property

Plant protein fibrils have recently attracted considerable attention as delivery vehicles for hydrophobic bioactive compounds. In this work, ultrasound treatment was applied to improve the structure of protein fibrils. The aim of this work was to investigate the impacts of ultrasound treatment on the structure of soy protein fibrils (SPF), and further explore the interaction between ultrasound‒treated SPF (USPF) and curcumin (Cur) to clarify the impacts of ultrasound on SPF as delivery vehicles. Thioflavin T fluorescence, SDS‒PAGE, Fourier transform infrared (FTIR) spectroscopy, and far‒UV circular dichroism spectroscopy revealed no significant changes in the primary and secondary structure of SPF after ultrasound treatment. Compared with SPF, USPF presented a more uniform particle size and higher surface hydrophobicity. Fluorescence quenching, ultraviolet–visible spectroscopy, isothermal titration calorimetry, and FTIR spectroscopy indicated USPF10 (ultrasonication for 10 min) possessed a high affinity toward curcumin via hydrophobic interactions and hydrogen bonds. The curcumin solubility in USPF10-Cur (133.19 μg/mL) complex was much higher than SPI-Cur (78.26 μg/mL) and SPF-Cur (116.22 μg/mL) complexes. The USPF10‒Cur complex exhibited good colloidal stability and thermal stability. The DPPH and ABTS scavenging capacities of the USPF10‒Cur complex were approximately 1.4 and 2.7 folds higher than those of free curcumin, respectively. An In vitro digestion assay suggested that the USPF10‒Cur complex showed a much more stable and sustainable release of curcumin and provided chances for curcumin release in the intestine. This study indicated that USPF might be promising delivery vehicles for hydrophobic bioactives as stable and functional ingredients in acidic food systems.

A guided tour through α-helical peptide antibiotics and their targets.

Nowadays, not only biologists, but also researchers from other disciplines such as chemistry, pharmacy, material sciences, or physics are working with antimicrobial peptides. This review is written for researchers and students working in or interested in the field of antimicrobial peptides - and especially those who do not have a profound biological background. To lay the ground for a thorough discussion on how AMPs act on cells, the architectures of mammalian and bacterial cell envelopes are described in detail because they are important targets of AMPs and provide the basis for their selectivity. The modes of action of α-helical AMPs (αAMPs) are not limited to different models of membrane permeabilization, but also include the disruption of intracellular processes, as well as the formation of fibrillary structures and their potential implications for antimicrobial activity. As biofilm-related infections are very difficult to treat with conventional antibiotics, they pose a major problem in the clinic. Therefore, this review also discusses the biological background of biofilm infections and the mode of actions of αAMPs against biofilms. The last chapter focusses on the design of αAMPs by providing an overview of historic milestones in αAMP design. It describes how modern αAMP design is aiming to produce peptides suitable to be applied in the clinic. Hence, the article concludes with a section on translational research discussing the prospects of αAMPs and remaining challenges on their way into the clinic.

Design and synthesis of multi-functional small-molecule based inhibitors of amyloid-β aggregation: Molecular modeling and in vitro evaluation.

Amyloid-β1-42 (Aβ42) peptide aggregate formation in the brain plays a crucial role in the onset and progression of Alzheimer's disease. According to published research, the Aβ monomer's amino acid residues KLVFF (16-20) self-associate to create antiparallel β-sheet fibrils. Small compounds can prevent self-assembly and destroy Aβ fibrils by attaching to the Aβ16-20 regions of Aβ42. To enhance biological characteristics and binding affinity to the amyloid beta peptide, β-sheet breaker small molecules can be developed and modified with various scaffolds. In the current study, a novel series of 2,3-disubstitutedbenzofuran derivatives was designed and created by fusing the benzofuran core of a known iron chelator, neuroprotective, and neurorestorative agent, like VK-28, with a motif found in the structure of a known muscarinic inhibitor and amyloid binding agent, like SKF-64346. Measurements of the binding affinity and in vitro aggregation inhibition of the Aβ42 peptide were made using the thioflavin T (ThT) test. Using AutoDock 4.2 software, molecular docking studies of the synthesized compounds were performed on the monomer and fibril structures of amyloid beta peptide. The compounds 8a-8g exhibited strong binding energy and affinity to Aβ fibrils as well as a 50%-67% reduction of the growth of Aβ aggregation. Finally, the positive traits of our recently synthesized compounds make them excellent candidates for additional in vivo testing as a "β-sheet breaking agent."

Inhibitive and Destructive Mechanisms of Chronic Traumatic Encephalopathy-Related R3-R4 Tau Peptide Chains and Protofibril by Epigallocatechin Gallate: Evidence from Molecular Dynamics Simulation.

Chronic traumatic encephalopathy (CTE), a unique tauopathy, is pathologically associated with the aggregation of hyperphosphorylated tau protein into fibrillar aggregates. Inhibiting tau aggregation and disaggregating tau protofibril might be promising strategies to prevent or delay the development of CTE. Newly resolved tau fibril structures from deceased CTE patients' brains show that the R3-R4 fragment of tau forms the core of the fibrils and the structures are distinct from other tauopathies. An in vitro experiment finds that epigallocatechin gallate (EGCG) can effectively inhibit human full-length tau aggregation and disaggregate preformed fibrils. However, its inhibitive and destructive effects on the CTE-related R3-R4 tau and the underlying molecular mechanisms remain elusive. In this study, we performed extensive all-atom molecular dynamics simulations on the CTE-related R3-R4 tau dimer/protofibril with and without EGCG. The results reveal that EGCG could reduce the β-sheet structure content of the dimer, induce the dimer to form loosely packed conformations, and impede the interchain interactions, thus inhibiting the further aggregation of the two peptide chains. Besides, EGCG could reduce the structural stability, decrease the β-sheet structure content, reduce the structural compactness, and weaken local residue-residue contacts of the protofibril, hence making the protofibril disaggregated. We also identified the dominant binding sites and pivotal interactions. EGCG preferentially binds with hydrophobic, aromatic, and positively/negatively charged residues of the dimer, while it tends to bind with polar, hydrophobic, aromatic, and positively charged residues of the protofibril. Hydrophobic, hydrogen-bonding, π-π stacking, and cation-π interactions synergistically drive the binding of EGCG on both the dimer and the protofibril, but anion-π interaction only exists in the interaction of EGCG with the dimer. Our work unravels EGCG's inhibitive and destructive effects on the CTE-related R3-R4 tau dimer/protofibril and the underlying molecular mechanisms, which provides useful implications for the design of drugs to prevent or delay the progression of CTE.

The in-tissue molecular architecture of β-amyloid pathology in the mammalian brain

Amyloid plaques composed of Aβ fibrils are a hallmark of Alzheimer’s disease (AD). However, the molecular architecture of amyloid plaques in the context of fresh mammalian brain tissue is unknown. Here, using cryogenic correlated light and electron tomography we report the in situ molecular architecture of Aβ fibrils in the AppNL-G-F familial AD mouse model containing the Arctic mutation and an atomic model of ex vivo purified Arctic Aβ fibrils. We show that in-tissue Aβ fibrils are arranged in a lattice or parallel bundles, and are interdigitated by subcellular compartments, extracellular vesicles, extracellular droplets and extracellular multilamellar bodies. The Arctic Aβ fibril differs significantly from an earlier AppNL-F fibril structure, indicating a striking effect of the Arctic mutation. These structural data also revealed an ensemble of additional fibrillar species, including thin protofilament-like rods and branched fibrils. Together, these results provide a structural model for the dense network architecture that characterises β-amyloid plaque pathology.

α-Synuclein fibril and synaptic vesicle interactions lead to vesicle destruction and increased lipid-associated fibril uptake into iPSC-derived neurons

Monomeric alpha-synuclein (aSyn) is a well characterised protein that importantly binds to lipids. aSyn monomers assemble into amyloid fibrils which are localised to lipids and organelles in insoluble structures found in Parkinson’s disease patient’s brains. Previous work to address pathological aSyn-lipid interactions has focused on using synthetic lipid membranes, which lack the complexity of physiological lipid membranes. Here, we use physiological membranes in the form of synaptic vesicles (SV) isolated from rodent brain to demonstrate that lipid-associated aSyn fibrils are more easily taken up into iPSC-derived cortical i3Neurons. Lipid-associated aSyn fibril characterisation reveals that SV lipids are an integrated part of the fibrils and while their fibril morphology differs from aSyn fibrils alone, the core fibril structure remains the same, suggesting the lipids lead to the increase in fibril uptake. Furthermore, SV enhance the aggregation rate of aSyn, yet increasing the SV:aSyn ratio causes a reduction in aggregation propensity. We finally show that aSyn fibrils disintegrate SV, whereas aSyn monomers cause clustering of SV using small angle neutron scattering and high-resolution imaging. Disease burden on neurons may be impacted by an increased uptake of lipid-associated aSyn which could enhance stress and pathology, which in turn may have fatal consequences for neurons.

Amyloid-like aggregation of recombinant β-lactoglobulin at pH 3.5 and 7.0: Is disulfide bond removal the key to fibrillation?

Functional nanofibrils from globular proteins are usually formed by heating for several hours at pH 2.0, which induces acidic hydrolysis and consecutive self-association. The functional properties of these micro-metre-long anisotropic structures are promising for biodegradable biomaterials and food applications, but their stability at pH > 2.0 is low. The results presented here show that modified β-lactoglobulin can also form nanofibrils by heating at neutral pH without prior acidic hydrolysis; the key is removing covalent disulfide bonds via precision fermentation. The aggregation behaviour of various recombinant β-lactoglobulin variants was systemically studied at pH 3.5 and 7.0. The suppression of intra- and intermolecular disulfide bonds by eliminating one to three out of the five cysteines makes the non-covalent interactions more prevalent and allow for structural rearrangement. This stimulated the linear growth of worm-like aggregates. Full elimination of all five cysteines led to the transformation of worm-like aggregates into actual fibril structures (several hundreds of nanometres long) at pH 7.0. This understanding of the role of cysteine in protein-protein interactions will help to identify proteins and protein modifications to form functional aggregates at neutral pH.

Abundant Aβ fibrils in ultracentrifugal supernatants of aqueous extracts from Alzheimer’s disease brains

Soluble oligomers of amyloid β-protein (Aβ) have been defined as aggregates in supernatants following ultracentrifugation of aqueous extracts from Alzheimer's disease (AD) brains and are believed to be upstream initiators of synaptic dysfunction, but little is known about their structures. We now report the unexpected presence of Aβ fibrils in synaptotoxic high-speed supernatants from AD brains extracted by soaking in an aqueous buffer. The fibrils did not appear to form during preparation, and their counts by EM correlated with AβELISA quantification. Cryo-EM structures of aqueous Aβ fibrils were identical to those from sarkosyl-insoluble homogenates. The fibrils in aqueous extracts were labeled by lecanemab, an Aβ aggregate-directed antibody reported to improve AD cognitive outcomes. Lecanemab provided protection against aqueous fibril synaptotoxicity. We conclude that fibrils are abundant in aqueous extracts from AD brains and have the same structures as those from plaques. These findings have implications for AD pathogenesis and drug design.

An electrochemically active textile current collector with a high areal capacity and a strong energy recovery effect using an interfacial interaction assembly

Conventional current collectors in lithium-ion batteries (LIBs) are generally nonactive components. However, enhancing their electroactive properties and increasing the electroactive surface area can significantly improve the areal energy performance of next-generation battery electrodes. Herein, we introduce an electrochemically active textile current collector that delivers high energy storage performance, achieved through interfacial interaction assembly-induced electroplating. We first prepared metal nanoparticle/multiwalled carbon nanotube multilayer-incorporated cotton textiles using complementary interaction-mediated layer-by-layer assembly, and subsequently electroplated them with Cu. The resulting textile exhibited a high areal capacity of ∼3.27 mA h cm−2 at 0.875 mA cm−2, excellent cycling stability, and a strong energy recovery effect, thanks to the synergistic contributions of the large active surface area of the fibril structure, the robust interfacial assembly, and the formation of a metal oxide NP/pseudocapacitive polymeric gel-like phase at the electrode/electrolyte interface. Moreover, when incorporating Li4Ti5O12 with a theoretical capacity of 175 mA h g − 1 into our textile current collector, the specific capacity and areal capacity of the LIB anode can be increased up to ∼573 mA h g − 1 and 8.60 mA h cm−2 (at 15 mg cm−2 LTO), respectively, outperforming those of previously reported LTO-based anodes.

Interfacial Dynamics and Growth Modes of β2-Microglobulin Dimers.

Protein aggregation is a complex process, strongly dependent on environmental conditions and highly structurally heterogeneous, both at the final level of fibril structure and intermediate level of oligomerization. Since the first step in aggregation is the formation of a dimer, it is important to clarify how certain properties of the latter (e.g., stability or interface geometry) may play a role in self-association. Here, we report a simple model that represents the dimer's interfacial region by two angles and combine it with a simple computational method to investigate how modulations of the interfacial region occurring on the ns-μs time scale change the dimer's growth mode. To illustrate the proposed methodology, we consider 15 different dimer configurations of the β2m D76N mutant protein equilibrated with long Molecular Dynamics simulations and identify which interfaces lead to limited and unlimited growth modes, having, therefore, different aggregation profiles. We found that despite the highly dynamic nature of the starting configurations, most polymeric growth modes tend to be conserved within the studied time scale. The proposed methodology performs remarkably well taking into consideration the nonspherical morphology of the β2m dimers, which exhibit unstructured termini detached from the protein's core, and the relatively weak binding affinities of their interfaces, which are stabilized by nonspecific apolar interactions. The proposed methodology is general and can be applied to any protein for which a dimer structure has been experimentally determined or computationally predicted.

How Single Site Mutations Can Help Understanding Structure Formation of Amyloid β1−40

Back Cover: The image is a schematic representation of amyloid β1.40 fibrils, in which the important intrastrand contact Phe19–Leu34 is modified to alter the physical forces governing fibril formation. In this way, information on their influence on fibrillation kinetics, morphology, structure, and dynamics as well as toxicity can be obtained. In article 2200489 by Benedikt Schwarze and Daniel Huster reviews work done in this context.

Tendon collagen fibril morphology and nano-mechanics via atomic force microscopy

Due to the hierarchical structure of tendon tissue (fascicles→fibers→fibrils), it is essential to investigate morphology and mechanics at the nano-scopic level to better understand whole body function. Previous studies investigating changes in tendon mechanics after exercise/disease/unloading have focused on the whole tendon and fascicle levels, but limited data exists on adaptations at the collagen fibril level. Objective: Provide a new method to study previously frozen tendon tissue by investigating collagen fibril 1) morphology, 2) nano-mechanical properties, and 3) elasticity maps using atomic force microscopy (AFM). Hypothesis: This method will provide a reliable way to assess tendon structure and function at the nano-scale. Methods: C57BL/6J mouse tendons from NASA Rodent Research-1 (-80°C) were chemically separated and mounted onto glass slides then mechanically separated into tendon subunit sheets and left to air-dry. Collagen fibril morphology was tested with a JPK Nanowizard 4a in tapping mode with a Tap300Al-G cantilever (tip radius=10nm, k=40N/m). Samples were then rehydrated for mechanical testing in Quantitative Imaging (QI) mode with an SNL-10D cantilever (tip radius=2nm, k=0.06N/m). Force data acquisition took place in liquid with a set point of 2nN and Z-speed of 40μm/s. Force-microscopy (FM) and structural data were processed using JPK Data Processing Software and Gwyddion, respectively. Data: Tendon collagen fibrils exhibited uniform D-periods across 38 fibril sections (D-period: 66nm±2.1, diameter: 63nm±23.5). Quantitative analysis of the force curves (n=30) along the highest point of the fibril revealed an average Young’s Modulus (YM) of 2.53MPa±0.31. Elasticity maps (n=3) highlighting the distribution of mechanical properties were generated using FM data. Results Summary: Our tissue preparation protocol combined with AFM reliably determines collagen fibril morphology and nano-mechanical properties of mouse tendons that were previously frozen. As evidence, we present systematic YM within and across different data sets. In addition, the YM values were found to be consistent with previous measurements in tendon tissue under similar conditions. Elasticity maps produced by analysis of FM data indicate the similarity of YM values across the tendon surface. Tendon fibrils were found to have similar D-periodicity despite the variation in fibril size, which is consistent with previous literature. Conclusions: This research provides a new method to test tendon collagen fibril structure and mechanical properties at the nano-scale, presenting a novel analysis (elasticity maps) showing contrast in YM across the fibril surface. Funding: CSUPERB New Investigator Grant to JRB This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Amyloid fibril formation, structure and domain swapping of acyl-coenzyme A thioesterase-7.

Acyl-coenzyme A thioesterase (Acot) enzymes are involved in a broad range of essential intracellular roles including cell signalling, lipid metabolism, inflammation and the opening of ion channels. Dysregulation in lipid metabolism has been linked to neuroinflammatory and neurological disorders such as Alzheimer's and Parkinson's diseases. Structurally, Acot enzymes adopt a circularised trimeric arrangement with each monomer containing an N- and a C-terminal hotdog domain. Acot7 spontaneously forms amyloid fibrils in vitro under physiological conditions. The resultant amyloid fibrillar structures were characterised by dye-binding fluorescence assays, far-UV circular dichroism spectroscopy, transmission electron microscopy and X-ray fibre diffraction. Acot7 has an unusual mechanism of aggregation with no lag phase. The initial phase (~ 18 h) of aggregation involves conformational rearrangement within the oligomers to form species of enhanced β-sheet character. The subsequent loss of α-helical structure is accompanied by large-scale amyloid fibril formation. The crystal structure of Acot7 revealed an unexpected arrangement of the two domains within the circularised trimeric structure, which is the basis for a proposed mechanism of amyloid fibril formation involving domain swapping during the initial phase of aggregation. Acot7 formed fibrils in the presence of its substrate arachidonoyl-CoA and its inhibitors and maintained its enzyme activity during fibril assembly. It is proposed that the Acot7 fibrillar form acts as functional amyloid.

Structural polymorphism and cytotoxicity of brain-derived β-amyloid extracts.

To date, more than 37 amyloidogenic proteins have been found to form toxic aggregates that are implicated in the progression of numerous debilitating protein misfolding diseases including Alzheimer's disease (AD). Extensive literature highlights the role of β-amyloid (Aβ) aggregates in causing excessive neuronal cell loss in the brains of AD patients. In fact, major advances in our understanding of Aβ aggregation process, including kinetics, toxicity, and structures of fibrillar aggregates have been revealed by examining in vitro preparations of synthetic Aβ peptides. However, ongoing research shows that brain-derived Aβ aggregates have specific characteristics that distinguish them from in vitro prepared species. Notably, the molecular structures of amyloid fibrils grown in the human brain were found to be markedly different than synthetic Aβ fibrils. In addition, recent findings report the existence of heterogeneous Aβ proteoforms in AD brain tissue in contrast to synthetically produced full-length aggregates. Despite their high relevance to AD progression, brain-derived Aβ species are less well-characterized compared with synthetic aggregates. The aim of this review is to provide an overview of the literature on brain-derived Aβ aggregates with particular focus on recent studies that report their structures as well as pathological roles in AD progression. The main motivation of this review is to highlight the importance of utilizing brain-derived amyloids for characterizing the structural and toxic effects of amyloid species. With this knowledge, brain-derived aggregates can be adopted to identify more relevant drug targets and validate potent aggregation inhibitors toward designing highly effective therapeutic strategies against AD.

Proliferative and Osteogenic Supportive Effect of VEGF-Loaded Collagen-Chitosan Hydrogel System in Bone Marrow Derived Mesenchymal Stem Cells.

The use of hydrogel (HG) in regenerative medicine is an emerging field and thus several approaches have been proposed recently to find an appropriate hydrogel system. In this sense, this study developed a novel HG system using collagen, chitosan, and VEGF composites for culturing mesenchymal stem cells (MSCs), and investigated their ability for osteogenic differentiation and mineral deposition. Our results showed that the HG loaded with 100 ng/mL VEGF (HG-100) significantly supported the proliferation of undifferentiated MSCs, the fibrillary filament structure (HE stain), mineralization (alizarin red S and von Kossa stain), alkaline phosphatase, and the osteogenesis of differentiated MSCs compared to other hydrogels (loaded with 25 and 50 ng/mL VEGF) and control (without hydrogel). HG-100 showed a higher VEGF releasing rate from day 3 to day 7 than other HGs, which substantially supports the proliferative and osteogenic properties of HG-100. However, the HGs did not increase the cell growth in differentiated MSCs on days 14 and 21 due to the confluence state (reach stationary phase) and cell loading ability, regardless of the VEGF content. Similarly, the HGs alone did not stimulate the osteogenesis of MSCs; however, they increased the osteogenic ability of MSCs in presence of osteogenic supplements. Accordingly, a fabricated HG with VEGF could be used as an appropriate system to culture stem cells for bone and dental regeneration.

Chiral and Structural Polymorphism of Fibril Architectures of Homologous Lysozymes.

Amyloid fibrils are fascinating and complex structures with the multilayered chiral organization. Using the multimodal methodology, including VCD, ECD, cryo-EM, and TEM, we characterized in detail different levels of organization (secondary structure/protofilament/mesoscopic structure) of amyloid fibrils prepared from proteins highly homologous in the structure (hen egg white and human lysozymes). Our results demonstrate that small changes in the native protein structure or preparation conditions translate into significant differences in the handedness and architecture of the formed fibrils at various levels of their complexity. In particular, fibrils of hen egg white and human lysozymes obtained in vitro at the same preparation conditions, possess different secondary structure, protofilament twist and ultrastructure. Yet, formed fibrils adopted a relatively similar mesoscopic structure, as observed in high-resolution 3D cryo-EM, scarcely used up to now for fibrils obtained in vitro in denaturing condition. Our results add to other puzzling experiments implicating the indeterministic nature of fibril formation.

Stability of collagen gel after uv irradiation

Type I collagen is the most abundant extracellular matrix protein in the human body, as well as is the main structural element in tissues and regulates cellular functions. In lifetime, the organism and extracellular matrix components such as collagen are exposed to UV irradiation. The purpose of this work was to study the effect of UV irradiation on the stability and structure of collagen fibrils. It has been shown that UV irradiation has a stabilization effect on collagen gel at a concentration of 0.5 mg/ml. Scanning electron microscope images have shown that the diameter of the collagen fibrils is not changed after UV irradiation. The degree of spreading of cells cultured on collagen fibrils after UV irradiation is greater compared to those cultured on collagen fibrils unirradiated with UV light. UV irradiation promotes a shift of the collagen amide A band to lower frequency, indicating that it induces structural changes in collagen.

High numerical aperture imaging allows chirality measurement in individual collagen fibrils using polarization second harmonic generation microscopy.

Second harmonic generation (SHG) microscopy is a commonly used technique to study the organization of collagen within tissues. However, individual collagen fibrils, which have diameters much smaller than the resolution of most optical systems, have not been extensively investigated. Here we probe the structure of individual collagen fibrils using polarization-resolved SHG (PSHG) microscopy and atomic force microscopy. We find that longitudinally polarized light occurring at the edge of a focal volume of a high numerical aperture microscope objective illuminated with linearly polarized light creates a measurable variation in PSHG signal along the axis orthogonal to an individual collagen fibril. By comparing numerical simulations to experimental data, we are able to estimate parameters related to the structure and chirality of the collagen fibril without tilting the sample out of the image plane, or cutting tissue at different angles, enabling chirality measurements on individual nanostructures to be performed in standard PSHG microscopes. The results presented here are expected to lead to a better understanding of PSHG results from both collagen fibrils and collagenous tissues. Further, the technique presented can be applied to other chiral nanoscale structures such as microtubules, nanowires, and nanoribbons.