Simulation of synthetic gecko arrays shearing on rough surfaces

PubMed Central

Gillies, Andrew G.; Fearing, Ronald S.

2014-01-01

To better understand the role of surface roughness and tip geometry in the adhesion of gecko synthetic adhesives, a model is developed that attempts to uncover the relationship between surface feature size and the adhesive terminal feature shape. This model is the first to predict the adhesive behaviour of a plurality of hairs acting in shear on simulated rough surfaces using analytically derived contact models. The models showed that the nanoscale geometry of the tip shape alters the macroscale adhesion of the array of fibres by nearly an order of magnitude, and that on sinusoidal surfaces with amplitudes much larger than the nanoscale features, spatula-shaped features can increase adhesive forces by 2.5 times on smooth surfaces and 10 times on rough surfaces. Interestingly, the summation of the fibres acting in concert shows behaviour much more complex that what could be predicted with the pull-off model of a single fibre. Both the Johnson–Kendall–Roberts and Kendall peel models can explain the experimentally observed frictional adhesion effect previously described in the literature. Similar to experimental results recently reported on the macroscale features of the gecko adhesive system, adhesion drops dramatically when surface roughness exceeds the size and spacing of the adhesive fibrillar features. PMID:24694893

Immunodetection of nucleolar proteins and ultrastructure of nucleoli of soybean root meristematic cells treated with chilling stress and after recovery.

PubMed

Stepiński, Dariusz

2009-03-01

The nucleolar proteins, fibrillarin and nucleophosmin, have been identified immunofluorescently in the root meristematic cells of soybean seedlings under varying experimental conditions: at 25 degrees C (control), chilling at 10 degrees C for 3 h and 4 days and recovery from the chilling stress at 25 degrees C. In each experimental variant, the immunofluorescence signals were present solely at the nucleolar territories. Fluorescent staining for both proteins was mainly in the shape of circular domains that are assumed to correspond to the dense fibrillar component of the nucleoli. The fewest fluorescent domains were observed in the nucleoli of chilled plants, and the highest number was observed in the plants recovered after chilling. This difference in the number of circular domains in the nucleoli of each variant may indicate various levels of these proteins in each variant. Both the number of circular domains and the level of these nucleolar proteins changed with changes in the transcriptional activity of the nucleoli, with the more metabolically active cell having higher numbers of active areas in the nucleolus and higher levels of nucleolar proteins, and conversely. Electron microscopic studies revealed differences in the ultrastructure of the nucleoli in all experimental variants and confirmed that the number of fibrillar centres surrounded by dense fibrillar component was the lowest in the nucleoli of chilled plants, and the highest in the nucleoli of recovered seedlings.

Nanomechanical properties of distinct fibrillar polymorphs of the protein α-synuclein.

PubMed

Makky, Ali; Bousset, Luc; Polesel-Maris, Jérôme; Melki, Ronald

2016-11-30

Alpha-synuclein (α-Syn) is a small presynaptic protein of 140 amino acids. Its pathologic intracellular aggregation within the central nervous system yields protein fibrillar inclusions named Lewy bodies that are the hallmarks of Parkinson's disease (PD). In solution, pure α-Syn adopts an intrinsically disordered structure and assembles into fibrils that exhibit considerable morphological heterogeneity depending on their assembly conditions. We recently established tightly controlled experimental conditions allowing the assembly of α-Syn into highly homogeneous and pure polymorphs. The latter exhibited differences in their shape, their structure but also in their functional properties. We have conducted an AFM study at high resolution and performed a statistical analysis of fibrillar α-Syn shape and thermal fluctuations to calculate the persistence length to further assess the nanomechanical properties of α-Syn polymorphs. Herein, we demonstrated quantitatively that distinct polymorphs made of the same protein (wild-type α-Syn) show significant differences in their morphology (height, width and periodicity) and physical properties (persistence length, bending rigidity and axial Young's modulus).

Solid-state NMR studies of metal-free SOD1 fibrillar structures.

PubMed

Banci, Lucia; Blaževitš, Olga; Cantini, Francesca; Danielsson, Jens; Lang, Lisa; Luchinat, Claudio; Mao, Jiafei; Oliveberg, Mikael; Ravera, Enrico

2014-06-01

Copper-zinc superoxide dismutase 1 (SOD1) is present in the protein aggregates deposited in motor neurons of amyotrophic lateral sclerosis (ALS) patients. ALS is a neurodegenerative disease that can be either sporadic (ca. 90%) or familial (fALS). The most widely studied forms of fALS are caused by mutations in the sequence of SOD1. Ex mortuo SOD1 aggregates are usually found to be amorphous. In vitro SOD1, in its immature reduced and apo state, forms fibrillar aggregates. Previous literature data have suggested that a monomeric SOD1 construct, lacking loops IV and VII, (apoSODΔIV-VII), shares the same fibrillization properties of apoSOD1, both proteins having the common structural feature of the central β-barrel. In this work, we show that structural information can be obtained at a site-specific level from solid-state NMR. The residues that are sequentially assignable are found to be located at the putative nucleation site for fibrillar species formation in apoSOD, as detected by other experimental techniques.

Nanomechanical properties of distinct fibrillar polymorphs of the protein α-synuclein

NASA Astrophysics Data System (ADS)

Makky, Ali; Bousset, Luc; Polesel-Maris, Jérôme; Melki, Ronald

2016-11-01

Alpha-synuclein (α-Syn) is a small presynaptic protein of 140 amino acids. Its pathologic intracellular aggregation within the central nervous system yields protein fibrillar inclusions named Lewy bodies that are the hallmarks of Parkinson’s disease (PD). In solution, pure α-Syn adopts an intrinsically disordered structure and assembles into fibrils that exhibit considerable morphological heterogeneity depending on their assembly conditions. We recently established tightly controlled experimental conditions allowing the assembly of α-Syn into highly homogeneous and pure polymorphs. The latter exhibited differences in their shape, their structure but also in their functional properties. We have conducted an AFM study at high resolution and performed a statistical analysis of fibrillar α-Syn shape and thermal fluctuations to calculate the persistence length to further assess the nanomechanical properties of α-Syn polymorphs. Herein, we demonstrated quantitatively that distinct polymorphs made of the same protein (wild-type α-Syn) show significant differences in their morphology (height, width and periodicity) and physical properties (persistence length, bending rigidity and axial Young’s modulus).

Competition between surface adsorption and folding of fibril-forming polypeptides

NASA Astrophysics Data System (ADS)

Ni, Ran; Kleijn, J. Mieke; Abeln, Sanne; Cohen Stuart, Martien A.; Bolhuis, Peter G.

2015-02-01

Self-assembly of polypeptides into fibrillar structures can be initiated by planar surfaces that interact favorably with certain residues. Using a coarse-grained model, we systematically studied the folding and adsorption behavior of a β -roll forming polypeptide. We find that there are two different folding pathways depending on the temperature: (i) at low temperature, the polypeptide folds in solution into a β -roll before adsorbing onto the attractive surface; (ii) at higher temperature, the polypeptide first adsorbs in a disordered state and folds while on the surface. The folding temperature increases with increasing attraction as the folded β -roll is stabilized by the surface. Surprisingly, further increasing the attraction lowers the folding temperature again, as strong attraction also stabilizes the adsorbed disordered state, which competes with folding of the polypeptide. Our results suggest that to enhance the folding, one should use a weakly attractive surface. They also explain the recent experimental observation of the nonmonotonic effect of charge on the fibril formation on an oppositely charged surface [C. Charbonneau et al., ACS Nano 8, 2328 (2014), 10.1021/nn405799t].

Metal ions differentially influence the aggregation and deposition of Alzheimer's beta-amyloid on a solid template.

PubMed

Ha, Chanki; Ryu, Jungki; Park, Chan Beum

2007-05-22

The abnormal deposition and aggregation of beta-amyloid (Abeta) on brain tissues are considered to be one of the characteristic neuropathological features of Alzheimer's disease (AD). Environmental conditions such as metal ions, pH, and cell membranes are associated with Abeta deposition and plaque formation. According to the amyloid cascade hypothesis of AD, the deposition of Abeta42 oligomers as diffuse plaques in vivo is an important earliest event, leading to the formation of fibrillar amyloid plaques by the further accumulation of soluble Abeta under certain environmental conditions. In order to characterize the effect of metal ions on amyloid deposition and plaque growth on a solid surface, we prepared a synthetic template by immobilizing Abeta oligomers onto a N-hydroxysuccinimide ester-activated solid surface. According to our study using ex situ atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), and thioflavin T (ThT) fluorescence spectroscopy, Cu2+ and Zn2+ ions accelerated both Abeta40 and Abeta42 deposition but resulted only in the formation of "amorphous" aggregates. In contrast, Fe3+ induced the deposition of "fibrillar" amyloid plaques at neutral pH. Under mildly acidic environments, the formation of fibrillar amyloid plaques was not induced by any metal ion tested in this work. Using secondary ion mass spectroscopy (SIMS) analysis, we found that binding Cu ions to Abeta deposits on a solid template occurred by the possible reduction of Cu ions during the interaction of Abeta with Cu2+. Our results may provide insights into the role of metal ions on the formation of fibrillar or amorphous amyloid plaques in AD.

Dense fibrillar collagen is a potent inducer of invadopodia via a specific signaling network

PubMed Central

Swatkoski, Stephen; Matsumoto, Kazue; Campbell, Catherine B.; Petrie, Ryan J.; Dimitriadis, Emilios K.; Li, Xin; Mueller, Susette C.; Bugge, Thomas H.; Gucek, Marjan

2015-01-01

Cell interactions with the extracellular matrix (ECM) can regulate multiple cellular activities and the matrix itself in dynamic, bidirectional processes. One such process is local proteolytic modification of the ECM. Invadopodia of tumor cells are actin-rich proteolytic protrusions that locally degrade matrix molecules and mediate invasion. We report that a novel high-density fibrillar collagen (HDFC) matrix is a potent inducer of invadopodia, both in carcinoma cell lines and in primary human fibroblasts. In carcinoma cells, HDFC matrix induced formation of invadopodia via a specific integrin signaling pathway that did not require growth factors or even altered gene and protein expression. In contrast, phosphoproteomics identified major changes in a complex phosphosignaling network with kindlin2 serine phosphorylation as a key regulatory element. This kindlin2-dependent signal transduction network was required for efficient induction of invadopodia on dense fibrillar collagen and for local degradation of collagen. This novel phosphosignaling mechanism regulates cell surface invadopodia via kindlin2 for local proteolytic remodeling of the ECM. PMID:25646088

Non-fibrillar amyloid-{beta} peptide reduces NMDA-induced neurotoxicity, but not AMPA-induced neurotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niidome, Tetsuhiro, E-mail: tniidome@pharm.kyoto-u.ac.jp; Goto, Yasuaki; Kato, Masaru

2009-09-04

Amyloid-{beta} peptide (A{beta}) is thought to be linked to the pathogenesis of Alzheimer's disease. Recent studies suggest that A{beta} has important physiological roles in addition to its pathological roles. We recently demonstrated that A{beta}42 protects hippocampal neurons from glutamate-induced neurotoxicity, but the relationship between A{beta}42 assemblies and their neuroprotective effects remains largely unknown. In this study, we prepared non-fibrillar and fibrillar A{beta}42 based on the results of the thioflavin T assay, Western blot analysis, and atomic force microscopy, and examined the effects of non-fibrillar and fibrillar A{beta}42 on glutamate-induced neurotoxicity. Non-fibrillar A{beta}42, but not fibrillar A{beta}42, protected hippocampal neurons frommore » glutamate-induced neurotoxicity. Furthermore, non-fibrillar A{beta}42 decreased both neurotoxicity and increases in the intracellular Ca{sup 2+} concentration induced by N-methyl-D-aspartate (NMDA), but not by {alpha}-amino-3-hydrozy-5-methyl-4-isoxazole propionic acid (AMPA). Our results suggest that non-fibrillar A{beta}42 protects hippocampal neurons from glutamate-induced neurotoxicity through regulation of the NMDA receptor.« less

Nanomechanical properties of distinct fibrillar polymorphs of the protein α-synuclein

PubMed Central

Makky, Ali; Bousset, Luc; Polesel-Maris, Jérôme; Melki, Ronald

2016-01-01

Alpha-synuclein (α-Syn) is a small presynaptic protein of 140 amino acids. Its pathologic intracellular aggregation within the central nervous system yields protein fibrillar inclusions named Lewy bodies that are the hallmarks of Parkinson’s disease (PD). In solution, pure α-Syn adopts an intrinsically disordered structure and assembles into fibrils that exhibit considerable morphological heterogeneity depending on their assembly conditions. We recently established tightly controlled experimental conditions allowing the assembly of α-Syn into highly homogeneous and pure polymorphs. The latter exhibited differences in their shape, their structure but also in their functional properties. We have conducted an AFM study at high resolution and performed a statistical analysis of fibrillar α-Syn shape and thermal fluctuations to calculate the persistence length to further assess the nanomechanical properties of α-Syn polymorphs. Herein, we demonstrated quantitatively that distinct polymorphs made of the same protein (wild-type α-Syn) show significant differences in their morphology (height, width and periodicity) and physical properties (persistence length, bending rigidity and axial Young’s modulus). PMID:27901068

Binding, internalization and fate of Huntingtin Exon1 fibrillar assemblies in mitotic and nonmitotic neuroblastoma cells.

PubMed

Ruiz-Arlandis, G; Pieri, L; Bousset, L; Melki, R

2016-02-01

The aggregation of Huntingtin (HTT) protein and of its moiety encoded by its Exon1 (HTTExon1) into fibrillar structures inside neurons is the molecular hallmark of Huntington's disease. Prion-like transmission of these aggregates between cells has been demonstrated. The cell-to-cell transmission mechanisms of these protein aggregates and the susceptibility of different kinds of neuronal cells to these toxic assemblies still need assessment. Here, we documented the binding to and internalization by differentiated and undifferentiated neuroblastoma cells of exogenous fibrillar HTTExon1 and polyglutamine (polyQ) polypeptides containing the same number of glutamines. We assessed the contribution of endocytosis to fibrillar HTTExon1 uptake, their intracellular localization and fate. We observed that undifferentiated neuroblastoma cells were more susceptible to fibrillar HTTExon1 and polyQ than their differentiated counterparts. Furthermore, we demonstrated that exogenous HTTExon1 aggregates are mainly taken up by endocytosis and directed to lysosomal compartments in both mitotic and quiescent cells. These data suggest that the rates of endocytic processes that differ in mitotic and quiescent cells strongly impact the uptake of exogenous HTTExon1 and polyQ fibrils. This may be either the consequence of distinct metabolisms or distributions of specific protein partners for amyloid-like assemblies at the surface of highly dividing versus quiescent cells. Our results highlight the importance of endocytic processes in the internalization of exogenous HTTExon1 fibrils and suggest that a proportion of those assemblies reach the cytosol where they can amplify by recruiting the endogenous protein after escaping, by yet an unknown process, from the endo-lysosomal compartments. © 2015 British Neuropathological Society.

A study of the native cell wall structures of the marine alga Ventricaria ventricosa (Siphonocladales, Chlorophyceae) using atomic force microscopy.

PubMed

Eslick, Enid M; Beilby, Mary J; Moon, Anthony R

2014-04-01

A substantial proportion of the architecture of the plant cell wall remains unknown with a few cell wall models being proposed. Moreover, even less is known about the green algal cell wall. Techniques that allow direct visualization of the cell wall in as near to its native state are of importance in unravelling the spatial arrangement of cell wall structures and hence in the development of cell wall models. Atomic force microscopy (AFM) was used to image the native cell wall of living cells of Ventricaria ventricosa (V. ventricosa) at high resolution under physiological conditions. The cell wall polymers were identified mainly qualitatively via their structural appearance. The cellulose microfibrils (CMFs) were easily recognizable and the imaging results indicate that the V. ventricosa cell wall has a cross-fibrillar structure throughout. We found the native wall to be abundant in matrix polysaccharides existing in different curing states. The soft phase matrix polysaccharides susceptible by the AFM scanning tip existed as a glutinous fibrillar meshwork, possibly incorporating both the pectic- and hemicellulosic-type substances. The hard phase matrix producing clearer images, revealed coiled fibrillar structures associated with CMFs, sometimes being resolved as globular structures by the AFM tip. The coiling fibrillar structures were also seen in the images of isolated cell wall fragments. The mucilaginous component of the wall was discernible from the gelatinous cell wall matrix as it formed microstructural domains over the surface. AFM has been successful in imaging the native cell wall and revealing novel findings such as the 'coiling fibrillar structures' and cell wall components which have previously not been seen, that is, the gelatinous matrix phase.

Fibrillar Adhesive for Climbing Robots

NASA Technical Reports Server (NTRS)

Pamess, Aaron; White, Victor E.

2013-01-01

A climbing robot needs to use its adhesive patches over and over again as it scales a slope. Replacing the adhesive at each step is generally impractical. If the adhesive or attachment mechanism cannot be used repeatedly, then the robot must carry an extra load of this adhesive to apply a fresh layer with each move. Common failure modes include tearing, contamination by dirt, plastic deformation of fibers, and damage from loading/ unloading. A gecko-like fibrillar adhesive has been developed that has been shown useful for climbing robots, and may later prove useful for grasping, anchoring, and medical applications. The material consists of a hierarchical fibrillar structure that currently contains two levels, but may be extended to three or four levels in continuing work. The contacting level has tens of thousands of microscopic fibers made from a rubberlike material that bend over and create intimate contact with a surface to achieve maximum van der Waals forces. By maximizing the real area of contact that these fibers make and minimizing the bending energy necessary to achieve that contact, the net amount of adhesion has been improved dramatically.

Novel aminobenzanthrone dyes for amyloid fibril detection

NASA Astrophysics Data System (ADS)

Vus, Kateryna; Trusova, Valeriya; Gorbenko, Galyna; Kirilova, Elena; Kirilov, Georgiy; Kalnina, Inta; Kinnunen, Paavo

2012-04-01

A series of novel fluorescent aminobenzanthrone dyes have been tested for their ability to identify and characterize the oligomeric and fibrillar aggregates of lysozyme. The parameters of the dye binding to native, oligomeric and fibrillar protein have been calculated from the results of fluorimetric titration. Furthermore, several additional quantities reflecting the preference of the probe to either pre-fibrillar or fibrillar protein aggregates, have been evaluated. Based on the comparative analysis of the recovered parameters, AM4 was recommended for selective detection of protein pre-fibrillar assemblies, while the dyes AM1, AM2, AM3 were selected as the most prospective amyloid tracers.

Adsorption and decontamination of α-synuclein from medically and environmentally-relevant surfaces.

PubMed

Phan, Hanh T M; Bartz, Jason C; Ayers, Jacob; Giasson, Benoit I; Schubert, Mathias; Rodenhausen, Keith B; Kananizadeh, Negin; Li, Yusong; Bartelt-Hunt, Shannon L

2018-06-01

The assembly and accumulation of α-synuclein fibrils are implicated in the development of several neurodegenerative disorders including multiple system atrophy and Parkinson's disease. Pre-existing α-synuclein fibrils can recruit and convert soluble non-fibrillar α-synuclein to the fibrillar form similar to what is observed in prion diseases. This raises concerns regarding attachment of fibrillary α-synuclein to medical instruments and subsequent exposure of patients to α-synuclein similar to what has been observed in iatrogenic transmission of prions. Here, we evaluated adsorption and desorption of α-synuclein to two surfaces: stainless steel and a gold surface coated with a 11-Amino-1-undecanethiol hydrochloride self-assembled-monolayer (SAM) using in-situ combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry. α-Synuclein was found to attach to both surfaces, however, increased α-synuclein adsorption was observed onto the positively charged SAM surface compared to the stainless steel surface. Dynamic light scattering data showed that larger α-synuclein fibrils were preferentially attached to the stainless steel surface when compared with the distributions in the original α-synuclein solution and on the SAM surface. We determined that after attachment, introduction of a 1N NaOH solution could completely remove α-synuclein adsorbed on the stainless steel surface while α-synuclein was retained on the SAM surface. Our results indicate α-synuclein can bind to multiple surface types and that decontamination is surface-dependent. Copyright © 2018 Elsevier B.V. All rights reserved.

Prion Protein-mediated Toxicity of Amyloid-β Oligomers Requires Lipid Rafts and the Transmembrane LRP1*

PubMed Central

Rushworth, Jo V.; Griffiths, Heledd H.; Watt, Nicole T.; Hooper, Nigel M.

2013-01-01

Soluble oligomers of the amyloid-β (Aβ) peptide cause neurotoxicity, synaptic dysfunction, and memory impairments that underlie Alzheimer disease (AD). The cellular prion protein (PrPC) was recently identified as a high affinity neuronal receptor for Aβ oligomers. We report that fibrillar Aβ oligomers recognized by the OC antibody, which have been shown to correlate with the onset and severity of AD, bind preferentially to cells and neurons expressing PrPC. The binding of Aβ oligomers to cell surface PrPC, as well as their downstream activation of Fyn kinase, was dependent on the integrity of cholesterol-rich lipid rafts. In SH-SY5Y cells, fluorescence microscopy and co-localization with subcellular markers revealed that the Aβ oligomers co-internalized with PrPC, accumulated in endosomes, and subsequently trafficked to lysosomes. The cell surface binding, internalization, and downstream toxicity of Aβ oligomers was dependent on the transmembrane low density lipoprotein receptor-related protein-1 (LRP1). The binding of Aβ oligomers to cell surface PrPC impaired its ability to inhibit the activity of the β-secretase BACE1, which cleaves the amyloid precursor protein to produce Aβ. The green tea polyphenol (−)-epigallocatechin gallate and the red wine extract resveratrol both remodeled the fibrillar conformation of Aβ oligomers. The resulting nonfibrillar oligomers displayed significantly reduced binding to PrPC-expressing cells and were no longer cytotoxic. These data indicate that soluble, fibrillar Aβ oligomers bind to PrPC in a conformation-dependent manner and require the integrity of lipid rafts and the transmembrane LRP1 for their cytotoxicity, thus revealing potential targets to alleviate the neurotoxic properties of Aβ oligomers in AD. PMID:23386614

Decreased expression of microRNA-29 family in leiomyoma contributes to increased major fibrillar collagen production.

PubMed

Marsh, Erica E; Steinberg, Marissa L; Parker, J Brandon; Wu, Ju; Chakravarti, Debabrata; Bulun, Serdar E

2016-09-01

To determine the expression and function of the microRNA-29 family (miRNA-29a, miRNA-29b, miRNA-29c) in human leiomyoma and myometrium. Basic science experimental design. Academic medical center. Women undergoing surgery for symptomatic uterine fibroids. Overexpression and knockdown of miRNA-29a, miRNA-29b, and miRNA-29c in primary leiomyoma and myometrial cells. [1] Expression of the miRNA-29 family members in vivo in leiomyoma versus myometrium; [2] Major fibrillar collagen (I, II, III) expression in leiomyoma and myometrial cells with manipulation of miRNA-29 species. Members of the miRNA-29 family (29a, 29b, 29c) are all down-regulated in leiomyoma versus myometrium in vivo. The expression of the miRNA-29 family can be successfully modulated in primary leiomyoma and myometrial cells. Overexpression of the miRNA-29 family in leiomyoma cells results in down-regulation of the major fibrillar collagens. Down-regulation of the miRNA-29 species in myometrium results in an increase in collagen type III deposition. The miRNA-29 family is consistently down-regulated in leiomyoma compared to matched myometrial tissue. This down-regulation contributes to the increased collagen seen in leiomyomas versus myometrium. When miRNA-29 members are overexpressed in leiomyoma cells, protein levels of all of the major fibrillar collagens decrease. The miRNA-29 members are potential therapeutic targets in this highly prevalent condition. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of [alpha]- and PPII-helices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Patel, Manisha H.

2010-08-18

Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 {angstrom}) crystal structure of the A{sub 3}VP{sub 1} fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 {angstrom}) through the interaction of two noncontiguous regions in the primary sequence. The A{sub 3} repeat of the alanine-rich domain adopts an extended {alpha}-helix that intertwines with the P{submore » 1} repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A{sub 3} and P{sub 1} helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.« less

Improving controllable adhesion on both rough and smooth surfaces with a hybrid electrostatic/gecko-like adhesive

PubMed Central

Ruffatto, Donald; Parness, Aaron; Spenko, Matthew

2014-01-01

This paper describes a novel, controllable adhesive that combines the benefits of electrostatic adhesives with gecko-like directional dry adhesives. When working in combination, the two technologies create a positive feedback cycle whose adhesion, depending on the surface type, is often greater than the sum of its parts. The directional dry adhesive brings the electrostatic adhesive closer to the surface, increasing its effect. Similarly, the electrostatic adhesion helps engage more of the directional dry adhesive fibrillar structures, particularly on rough surfaces. This paper presents the new hybrid adhesive's manufacturing process and compares its performance to three other adhesive technologies manufactured using a similar process: reinforced PDMS, electrostatic and directional dry adhesion. Tests were performed on a set of ceramic tiles with varying roughness to quantify its effect on shear adhesive force. The relative effectiveness of the hybrid adhesive increases as the surface roughness is increased. Experimental data are also presented for different substrate materials to demonstrate the enhanced performance achieved with the hybrid adhesive. Results show that the hybrid adhesive provides up to 5.1× greater adhesion than the electrostatic adhesive or directional dry adhesive technologies alone. PMID:24451392

Elasto-capillarity in insect fibrillar adhesion.

PubMed

Gernay, Sophie; Federle, Walter; Lambert, Pierre; Gilet, Tristan

2016-08-01

The manipulation of microscopic objects is challenging because of high adhesion forces, which render macroscopic gripping strategies unsuitable. Adhesive footpads of climbing insects could reveal principles relevant for micro-grippers, as they are able to attach and detach rapidly during locomotion. However, the underlying mechanisms are still not fully understood. In this work, we characterize the geometry and contact formation of the adhesive setae of dock beetles (Gastrophysa viridula) by interference reflection microscopy. We compare our experimental results to the model of an elastic beam loaded with capillary forces. Fitting the model to experimental data yielded not only estimates for seta adhesion and compliance in agreement with previous direct measurements, but also previously unknown parameters such as the volume of the fluid meniscus and the bending stiffness of the tip. In addition to confirming the primary role of surface tension for insect adhesion, our investigation reveals marked differences in geometry and compliance between the three main kinds of seta tips in leaf beetles. © 2016 The Author(s).

Fibrillar amyloid-β burden in cognitively normal people at 3 levels of genetic risk for Alzheimer's disease

PubMed Central

Reiman, Eric M.; Chen, Kewei; Liu, Xiaofen; Bandy, Daniel; Yu, Meixiang; Lee, Wendy; Ayutyanont, Napatkamon; Keppler, Jennifer; Reeder, Stephanie A.; Langbaum, Jessica B. S.; Alexander, Gene E.; Klunk, William E.; Mathis, Chester A.; Price, Julie C.; Aizenstein, Howard J.; DeKosky, Steven T.; Caselli, Richard J.

2009-01-01

Fibrillar amyloid-beta (Aβ) is found in the brains of many cognitively normal older people. Whether or not this reflects a predisposition to Alzheimer's disease (AD) is unknown. We used Pittsburgh Compound B (PiB) PET to characterize the relationship between fibrillar Aβ burden and this predisposition in cognitively normal older people at 3 mean levels of genetic risk for AD. Dynamic PiB PET scans, the Logan method, statistical parametric mapping, and automatically labeled regions of interest (ROIs) were used to characterize and compare cerebral-to-cerebellar PIB distribution volume ratios, reflecting fibrillar Aβ burden, in 28 cognitively normal persons (mean age, 64 years) with a reported family history of AD and 2 copies, 1 copy, and no copies of the apolipoprotein E (APOE) ε4 allele. The 8 ε4 homozygotes, 8 heterozygotes, and 12 noncarriers did not differ significantly in terms of age, sex, or cognitive scores. Fibrillar Aβ was significantly associated with APOE ε4 carrier status and ε4 gene dose in AD-affected mean cortical, frontal, temporal, posterior cingulate-precuneus, parietal, and basal ganglia ROIs, and was highest in an additional homozygote who had recently developed mild cognitive impairment. These findings suggest that fibrillar Aβ burden in cognitively normal older people is associated with APOE ε4 gene dose, the major genetic risk factor for AD. Additional studies are needed to track fibrillar Aβ accumulation in persons with different kinds and levels of AD risk; to determine the extent to which fibrillar Aβ, alone or in combination with other biomarkers and risk factors, predicts rates of cognitive decline and conversion to clinical AD; and to establish the role of fibrillar Aβ imaging in primary prevention trials. PMID:19346482

Walking the Line: A Fibronectin Fiber-Guided Assay to Probe Early Steps of (Lymph)angiogenesis

PubMed Central

Mitsi, Maria; Schulz, Martin Michael Peter; Gousopoulos, Epameinondas; Ochsenbein, Alexandra Michaela; Detmar, Michael; Vogel, Viola

2015-01-01

Angiogenesis and lymphangiogenesis are highly complex morphogenetic processes, central to many physiological and pathological conditions, including development, cancer metastasis, inflammation and wound healing. While it is described that extracellular matrix (ECM) fibers are involved in the spatiotemporal regulation of angiogenesis, current angiogenesis assays are not specifically designed to dissect and quantify the underlying molecular mechanisms of how the fibrillar nature of ECM regulates vessel sprouting. Even less is known about the role of the fibrillar ECM during the early stages of lymphangiogenesis. To address such questions, we introduced here an in vitro (lymph)angiogenesis assay, where we used microbeads coated with endothelial cells as simple sprouting sources and deposited them on single Fn fibers used as substrates to mimic fibrillar ECM. The fibers were deposited on a transparent substrate, suitable for live microscopic observation of the ensuing cell outgrowth events at the single cell level. Our proof-of-concept studies revealed that fibrillar Fn, compared to Fn-coated surfaces, provides far stronger sprouting and guidance cues to endothelial cells, independent of the tested mechanical strains of the Fn fibers. Additionally, we found that VEGF-A, but not VEGF-C, stimulates the collective outgrowth of lymphatic endothelial cells (LEC), while the collective outgrowth of blood vascular endothelial cells (HUVEC) was prominent even in the absence of these angiogenic factors. In addition to the findings presented here, the modularity of our assay allows for the use of different ECM or synthetic fibers as substrates, as well as of other cell types, thus expanding the range of applications in vascular biology and beyond. PMID:26689200

Structure–Function Relationships of Pre-Fibrillar Protein Assemblies in Alzheimer's Disease and Related Disorders

PubMed Central

Rahimi, F.; Shanmugam, A.; Bitan, G.

2010-01-01

Several neurodegenerative diseases, including Alzheimer's, Parkinson's, Huntington's and prion diseases, are characterized pathognomonically by the presence of intra- and/or extracellular lesions containing proteinaceous aggregates, and by extensive neuronal loss in selective brain regions. Related non-neuropathic systemic diseases, e.g., light-chain and senile systemic amyloidoses, and other organ-specific diseases, such as dialysis-related amyloidosis and type-2 diabetes mellitus, also are characterized by deposition of aberrantly folded, insoluble proteins. It is debated whether the hallmark pathologic lesions are causative. Substantial evidence suggests that these aggregates are the end state of aberrant protein folding whereas the actual culprits likely are transient, pre-fibrillar assemblies preceding the aggregates. In the context of neurodegenerative amyloidoses, the proteinaceous aggregates may eventuate as potentially neuroprotective sinks for the neurotoxic, oligomeric protein assemblies. The pre-fibrillar, oligomeric assemblies are believed to initiate the pathogenic mechanisms that lead to synaptic dysfunction, neuronal loss, and disease-specific regional brain atrophy. The amyloid β-protein (Aβ), which is believed to cause Alzheimer's disease (AD), is considered an archetypal amyloidogenic protein. Intense studies have led to nominal, functional, and structural descriptions of oligomeric Aβ assemblies. However, the dynamic and metastable nature of Aβ oligomers renders their study difficult. Different results generated using different methodologies under different experimental settings further complicate this complex area of research and identification of the exact pathogenic assemblies in vivo seems daunting. Here we review structural, functional, and biological experiments used to produce and study pre-fibrillar Aβ assemblies, and highlight similar studies of proteins involved in related diseases. We discuss challenges that contemporary researchers are facing and future research prospects in this demanding yet highly important field. PMID:18537546

Fibrillar collagen scoring by second harmonic microscopy: a new tool in the assessment of liver fibrosis.

PubMed

Gailhouste, Luc; Le Grand, Yann; Odin, Christophe; Guyader, Dominique; Turlin, Bruno; Ezan, Frédéric; Désille, Yoann; Guilbert, Thomas; Bessard, Anne; Frémin, Christophe; Theret, Nathalie; Baffet, Georges

2010-03-01

Imaging of supramolecular structures by multiphoton microscopy offers significant advantages for studying specific fibrillar compounds in biological tissues. In this study, we aimed to demonstrate the relevance of Second Harmonic Generation (SHG) for assessing and quantifying, without staining, fibrillar collagen in liver fibrosis. We first showed the relationship between SHG signal and collagen forms over-produced and accumulated during fibrosis progression. Taking this property into consideration, we developed an innovative method to precisely quantify the fibrosis area in histological slices by scoring of fibrillar collagen deposits (Fibrosis-SHG index). The scoring method was routinely applied to 119 biopsies from patients with chronic liver disease allowing a fast and accurate measurement of fibrosis correlated with the Fibrosis-Metavir score (rho=0.75, p<0.0001). The technique allowed discriminating patients with advanced (moderate to severe) fibrosis (AUROC=0.88, p<0.0001) and cirrhosis (AUROC=0.89, p<0.0001). Taking advantage of its continuous gradation, the Fibrosis-SHG index also allowed the discrimination of several levels of fibrosis within the same F-Metavir stage. The SHG process presented several advantages such as a high reliability and sensitivity that lead to a standardized evaluation of hepatic fibrosis in liver biopsies without staining and pathological examination. Second harmonic microscopy emerges as an original and powerful tool in the assessment of liver fibrosis and offers new possibilities for the evaluation of experimental protocols. We expect that this technology could easily be applicable in the study of other fibro-proliferative pathologies. Copyright (c) 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Decorin Core Protein (Decoron) Shape Complements Collagen Fibril Surface Structure and Mediates Its Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Orgel, Joseph P.R.O.; Eid, Aya; Antipova, Olga

Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e1 bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule,more » which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e{sub 1} bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.« less

Decorin core protein (decoron) shape complements collagen fibril surface structure and mediates its binding.

PubMed

Orgel, Joseph P R O; Eid, Aya; Antipova, Olga; Bella, Jordi; Scott, John E

2009-09-15

Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e(1) bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e(1) bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.

A fibril-specific, conformation-dependent antibody recognizes a subset of Abeta plaques in Alzheimer disease, Down syndrome and Tg2576 transgenic mouse brain.

PubMed

Sarsoza, Floyd; Saing, Tommy; Kayed, Rakez; Dahlin, Robert; Dick, Malcolm; Broadwater-Hollifield, Camille; Mobley, Scott; Lott, Ira; Doran, Eric; Gillen, Daniel; Anderson-Bergman, Clifford; Cribbs, David H; Glabe, Charles; Head, Elizabeth

2009-10-01

Beta-amyloid (Abeta) is thought to be a key contributor to the pathogenesis of Alzheimer disease (AD) in the general population and in adults with Down syndrome (DS). Different assembly states of Abeta have been identified that may be neurotoxic. Abeta oligomers can assemble into soluble prefibrillar oligomers, soluble fibrillar oligomers and insoluble fibrils. Using a novel antibody, OC, recognizing fibrils and soluble fibrillar oligomers, we characterized fibrillar Abeta deposits in AD and DS cases. We further compared human specimens to those obtained from the Tg2576 mouse model of AD. Our results show that accumulation of fibrillar immunoreactivity is significantly increased in AD relative to nondemented aged subjects and those with select cognitive impairments (p < 0.0001). Further, there was a significant correlation between the extent of frontal cortex fibrillar deposit accumulation and dementia severity (MMSE r = -0.72). In DS, we observe an early age of onset and age-dependent accumulation of fibrillar OC immunoreactivity with little pathology in similarly aged non-DS individuals. Tg2576 mice show fibrillar accumulation that can be detected as young as 6 months. Interestingly, fibril-specific immunoreactivity was observed in diffuse, thioflavine S-negative Abeta deposits in addition to more mature neuritic plaques. These results suggest that fibrillar deposits are associated with disease in both AD and in adults with DS and their distribution within early Abeta pathology associated with diffuse plaques and correlation with MMSE suggest that these deposits may not be as benign as previously thought.

Load sharing in bioinspired fibrillar adhesives with backing layer interactions and interfacial misalignment

NASA Astrophysics Data System (ADS)

Bacca, Mattia; Booth, Jamie A.; Turner, Kimberly L.; McMeeking, Robert M.

2016-11-01

Bio-inspired fibrillar adhesives rely on the utilization of short-range intermolecular forces harnessed by intimate contact at fibril tips. The combined adhesive strength of multiple fibrils can only be utilized if equal load sharing (ELS) is obtained at detachment. Previous investigations have highlighted that mechanical coupling of fibrils through a compliant backing layer gives rise to load concentration and the nucleation and propagation of interfacial flaws. However, misalignment of the adhesive and contacting surface has not been considered in theoretical treatments of load sharing with backing layer interactions. Alignment imperfections are difficult to avoid for a flat-on-flat interfacial configuration. In this work we demonstrate that interfacial misalignment can significantly alter load sharing and the kinematics of detachment in a model adhesive system. Load sharing regimes dominated by backing layer interactions and misalignment are revealed, the transition between which is controlled by the misalignment angle, fibril separation, and fibril compliance. In the regime dominated by misalignment, backing layer deformation can counteract misalignment giving rise to improved load sharing when compared to an identical fibrillar array with a rigid backing layer. This result challenges the conventional belief that stiffer (and thinner) backing layers consistently reduce load concentration among fibrils. Finally, we obtain analytically the fibril compliance distribution required to harness backing layer interactions to obtain ELS. Through fibril compliance optimization, ELS can be obtained even with misalignment. However, since misalignment is typically not deterministic, it is of greater practical significance that the array optimized for perfect alignment exhibits load sharing superior to that of a homogeneous array subject to misalignment. These results inform the design of fibrillar arrays with graded compliance capable of exhibiting improved load sharing over large areas.

Fibrillar α-Synuclein and Huntingtin Exon 1 Assemblies Are Toxic to the Cells

PubMed Central

Pieri, Laura; Madiona, Karine; Bousset, Luc; Melki, Ronald

2012-01-01

The aggregation of alpha-synuclein (α-syn) and huntingtin (htt) into fibrillar assemblies in nerve and glial cells is a molecular hallmark of Parkinson's and Huntington's diseases. Within the aggregation process, prefibrillar and fibrillar oligomeric species form. Prefibrillar assemblies rather than fibrils are nowadays considered cytotoxic. However, recent reports describing spreading of fibrillar assemblies from one cell to another, in cell cultures, animal models, and brains of grafted patients suggest a critical role for fibrillar assemblies in pathogenesis. Here we compare the cytotoxic effect of defined and comparable particle concentrations of on-assembly pathway oligomeric and fibrillar α-syn and Htt fragment corresponding to the first exon of the protein (HttEx1). We show that homogeneous populations of α-syn and HttEx1 fibrils, rather than their precursor on-assembly pathway oligomers, are highly toxic to cultured cells and induce apoptotic cell death. We document the reasons that make fibrils toxic. We show that α-syn and HttEx1 fibrils bind and permeabilize lipid vesicles. We also show that fibrils binding to the plasma membrane in cultured cells alter Ca2+ homeostasis. Overall, our data indicate that fibrillar α-syn and HttEx1, rather than their precursor oligomers, are highly cytotoxic, the toxicity being associated to their ability to bind and permeabilize the cell membranes. PMID:22735540

Analysis of Toxic Amyloid Fibril Interactions at Natively Derived Membranes by Ellipsometry

PubMed Central

Smith, Rachel A. S.; Nabok, Aleksey; Blakeman, Ben J. F.; Xue, Wei-Feng; Abell, Benjamin; Smith, David P.

2015-01-01

There is an ongoing debate regarding the culprits of cytotoxicity associated with amyloid disorders. Although small pre-fibrillar amyloid oligomers have been implicated as the primary toxic species, the fibrillar amyloid material itself can also induce cytotoxicity. To investigate membrane disruption and cytotoxic effects associated with intact and fragmented fibrils, the novel in situ spectroscopic technique of Total Internal Reflection Ellipsometry (TIRE) was used. Fibril lipid interactions were monitored using natively derived whole cell membranes as a model of the in vivo environment. We show that fragmented fibrils have an increased ability to disrupt these natively derived membranes by causing a loss of material from the deposited surface when compared with unfragmented fibrils. This effect was corroborated by observations of membrane disruption in live cells, and by dye release assay using synthetic liposomes. Through these studies we demonstrate the use of TIRE for the analysis of protein-lipid interactions on natively derived lipid surfaces, and provide an explanation on how amyloid fibrils can cause a toxic gain of function, while entangled amyloid plaques exert minimal biological activity. PMID:26172440

Fibrillar dimer formation of islet amyloid polypeptides

NASA Astrophysics Data System (ADS)

Chiu, Chi-cheng; de Pablo, Juan J.

2015-09-01

Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 - 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 - 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

The role of microglial cells and astrocytes in fibrillar plaque evolution in transgenic APP(SW) mice.

PubMed

Wegiel, J; Wang, K C; Imaki, H; Rubenstein, R; Wronska, A; Osuchowski, M; Lipinski, W J; Walker, L C; LeVine, H

2001-01-01

Ultrastructural reconstruction of 27 fibrillar plaques in different stages of formation and maturation was undertaken to characterize the development of fibrillar plaques in the brains of human APP(SW) transgenic mice (Tg2576). The study suggests that microglial cells are not engaged in Abeta removal and plaque degradation, but in contrast, are a driving force in plaque formation and development. Fibrillar Abeta deposition at the amyloid pole of microglial cells appears to initiate three types of neuropil response: degeneration of neurons, protective activation of astrocytes, and attraction and activation of microglial cells sustaining plaque growth. Enlargement of neuronal processes and synapses with accumulation of degenerated mitochondria, dense bodies, and Hirano-type bodies is the marker of toxic injury of neurons by fibrillar Abeta. Separation of amyloid cores from neurons and degradation of amyloid cores by cytoplasmic processes of hypertrophic astrocytes suggest the protective and defensive character of astrocytic response to fibrillar Abeta. The growth of cored plaque from a small plaque with one microglial cell with an amyloid star and a few dystrophic neurites to a large plaque formed by several dozen microglial cells seen in old mice is the effect of attraction and activation of microglial cells residing outside of the plaque perimeter. This mechanism of growth of plaques appears to be characteristic of cored plaques in transgenic mice. Other features in mouse microglial cells that are absent in human brain are clusters of vacuoles, probably of lysosomal origin. They evolve into circular cisternae and finally into large vacuoles filled with osmiophilic, amorphous material and bundles of fibrils that are poorly labeled with antibody to Abeta. Microglial cells appear to release large amounts of fibrillar Abeta and accumulate traces of fibrillar Abeta in a lysosomal pathway.

Increased functional load on mouse kidney proximal tubule epithelial cells causes changes in nucleolar 3-D architecture.

PubMed

Chelidze, P V; Dzidziguri, D V; Tumanishvili, G D

1998-05-01

Ultrastructural 3-D analysis of nucleolar architecture and Ag-NOR protein distribution in mouse kidney-cortex proximal-tubule epithelium has been performed. A principal scheme of structural changes of the nucleolus and organization of its components during the intensification of pre-rRNA synthesis (dynamic model of a nucleolus) based on computer spatial modelling has been advanced. According to the nucleolar composition, three groups of cells, which differ from each other by rRNA synthesis, are defined in normal kidney. Most nephron proximal-section cells (about 52%) are characterized by lower activity of RNA synthesis. Such kind of cells are defined as group I (nucleolar diameter 0.7-1.5 microm) and always contain resting, ring-shaped or close to ring-shaped dense nucleoli, which have 2 or 3 fibrillar centers. Nucleoli of group II cells (about 37%, nucleolar diameter 1.5-2.5 microm) have a higher level of activity, contain 4-7 fibrillar centers, and their structural organization is close to reticulated forms due to the first indications of vacuolar network (identified as prereticulated nucleoli). The most active cells of group III (about 11%, nucleolar diameter 2.5-3.5 microm) include cells with typical reticulated nucleoli with a well expressed vacuolar network and numerous fibrillar centers (18-22). Increased functional load of the epithelium caused by unilateral nephrectomy and diuretic (4-chlor-H [2-furylmethyl] 5-sulphamyl-antranic acid) injection changed the proportion of the different cell groups: group I decreased (about 25%), whereas groups II and III increased (about 8% and 17%, respectively). The increase of nucleolar activity first causes a deformation of the individual fibrillar centers as well as complication and growth of their surface. Further, a progressive fragmentation of the fibrillar centers and the growth of their total volume is observed. The complication and growth of the total volume of Ag-positive zones is another indication of the nucleolar activation. The vacuolar system develops by a gradual fusion of small isolated cavities into a united vacuolar network. Nucleoli with 2-7 fibrillar centers are considered to be intermediate forms reflecting successive stages of its activation or inactivation: from the resting ring-shaped nucleolus via transient stages of increasing functional activity to the active reticulated nucleoli and vice versa. The observed differences in the nucleolar ultrastructure are regarded as evidence of the functional heterogeneity of cell populations within one functional segment of nephron.

Surface Structure of Yeast Protoplasts

PubMed Central

Streiblová, Eva

1968-01-01

The fine structure of the yeast cell wall during protoplast formation was studied by means of phase-contrast microscopy and the freeze-etching technique. The freeze-etching results indicated that at least in some cases the entire wall substance was not removed from the surface of the protoplasts. After a treatment of 30 min to 3 hr with 2% snail enzymes, an innermost thin wall layer as well as remnants of the fibrillar middle layer sometimes could be demonstrated. Images PMID:4867751

Silicifying Biofilm Exopolymers on a Hot-Spring Microstromatolite: Templating Nanometer-Thick Laminae

NASA Astrophysics Data System (ADS)

Handley, Kim M.; Turner, Sue J.; Campbell, Kathleen A.; Mountain, Bruce W.

2008-08-01

Exopolymeric substances (EPS) are an integral component of microbial biofilms; however, few studies have addressed their silicification and preservation in hot-spring deposits. Through comparative analyses with the use of a range of microscopy techniques, we identified abundant EPS significant to the textural development of spicular, microstromatolitic, siliceous sinter at Champagne Pool, Waiotapu, New Zealand. Examination of biofilms coating sinter surfaces by confocal laser scanning microscopy (CLSM), environmental scanning electron microscopy (ESEM), cryo-scanning electron microscopy (cryo-SEM), and transmission electron microscopy (TEM) revealed contraction of the gelatinous EPS matrix into films (approximately 10 nm thick) or fibrillar structures, which is common in conventional SEM analyses and analogous to products of naturally occurring desiccation. Silicification of fibrillar EPS contributed to the formation of filamentous sinter. Matrix surfaces or dehydrated films templated sinter laminae (nanometers to microns thick) that, in places, preserved fenestral voids beneath. Laminae of similar thickness are, in general, common to spicular geyserites. This is the first report to demonstrate EPS templation of siliceous stromatolite laminae. Considering the ubiquity of biofilms on surfaces in hot-spring environments, EPS silicification studies are likely to be important to a better understanding of the origins of laminae in other modern and ancient stromatolitic sinters, and EPS potentially may serve as biosignatures in extraterrestrial rocks.

Clinorotation influences rDNA and NopA100 localization in nucleoli

NASA Astrophysics Data System (ADS)

Sobol, M. A.; González-Camacho, F.; Rodríguez-Vilariño, V.; Kordyum, E. L.; Medina, F. J.

The nucleolus is the transcription site of rRNA genes as well as the site of processing and initial packaging of their transcripts. The plant nucleolin homologue NopA100 is involved in the regulation of r-chromatin condensation/expansion and rDNA transcription as well as in rRNA processing. We have investigated with immunogold electron microscopy the location of nucleolar DNA and NopA100 in cress root meristematic cells grown under slow horizontal clinorotation, reproducing an important feature of microgravity, namely the absence of an orienting action of a gravity vector, compared to control conditions. We demonstrate redistribution of both rDNA and NopA100 in nucleolar subcomponents induced by clinorotation. Ribosomal DNA concentrated predominantly in fibrillar centers in the form of condensed r-chromatin inclusions and internal non condensed fibrils, redistributing from the dense fibrillar component and the transition zone between fibrillar centers and the dense fibrillar component, recognized as the loci of rDNA transcription. The content of NopA100 was much higher in the inner space of fibrillar centers and reduced in the dense fibrillar component as compared to the control. Based on these data, an effect of slow horizontal clinorotation in lowering the level of rDNA transcription as well as rRNA processing is suggested.

Biochemical and biophysical characterization of collagens of marine sponge, Ircinia fusca (Porifera: Demospongiae: Irciniidae).

PubMed

Pallela, Ramjee; Bojja, Sreedhar; Janapala, Venkateswara Rao

2011-07-01

Collagens were isolated and partially characterized from the marine demosponge, Ircinia fusca from Gulf of Mannar (GoM), India, with an aim to develop potentially applicable collagens from unused and under-used resources. The yield of insoluble, salt soluble and acid soluble forms of collagens was 31.71 ± 1.59, 20.69 ± 1.03, and 17.38 ± 0.87 mg/g dry weight, respectively. Trichrome staining, Scanning & Transmission Electron microscopic (SEM & TEM) studies confirmed the presence of collagen in the isolated, terminally globular irciniid filaments. The partially purified (gel filtration chromatography), non-fibrillar collagens appeared as basement type collagenous sheets under light microscopy whereas the purified fibrillar collagens appeared as fibrils with a repeated band periodicity of 67 nm under Atomic Force Microscope (AFM). The non-fibrillar and fibrillar collagens were seen to have affinity for anti-collagen type IV and type I antibodies raised against human collagens, respectively. The macromolecules, i.e., total protein, carbohydrate and lipid contents within the tissues were also quantified. The present information on the three characteristic irciniid collagens (filamentous, fibrillar and non-fibrillar) could assist the future attempts to unravel the therapeutically important, safer collagens from marine sponges for their use in pharmaceutical and cosmeceutical industries. Copyright © 2011 Elsevier B.V. All rights reserved.

Quantification and Comparison of Anti-Fibrotic Therapies by Polarized SRM and SHG-Based Morphometry in Rat UUO Model

PubMed Central

Weldon, Steve M.; Matera, Damian; Lee, ChungWein; Yang, Haichun; Fryer, Ryan M.; Fogo, Agnes B.; Reinhart, Glenn A.

2016-01-01

Renal interstitial fibrosis (IF) is an important pathologic manifestation of disease progression in a variety of chronic kidney diseases (CKD). However, the quantitative and reproducible analysis of IF remains a challenge, especially in experimental animal models of progressive IF. In this study, we compare traditional polarized Sirius Red morphometry (SRM) to novel Second Harmonic Generation (SHG)-based morphometry of unstained tissues for quantitative analysis of IF in the rat 5 day unilateral ureteral obstruction (UUO) model. To validate the specificity of SHG for detecting fibrillar collagen components in IF, co-localization studies for collagens type I, III, and IV were performed using IHC. In addition, we examined the correlation, dynamic range, sensitivity, and ability of polarized SRM and SHG-based morphometry to detect an anti-fibrotic effect of three different treatment regimens. Comparisons were made across three separate studies in which animals were treated with three mechanistically distinct pharmacologic agents: enalapril (ENA, 15, 30, 60 mg/kg), mycophenolate mofetil (MMF, 2, 20 mg/kg) or the connective tissue growth factor (CTGF) neutralizing antibody, EX75606 (1, 3, 10 mg/kg). Our results demonstrate a strong co-localization of the SHG signal with fibrillar collagens I and III but not non-fibrillar collagen IV. Quantitative IF, calculated as percent cortical area of fibrosis, demonstrated similar response profile for both polarized SRM and SHG-based morphometry. The two methodologies exhibited a strong correlation across all three pharmacology studies (r2 = 0.89–0.96). However, compared with polarized SRM, SHG-based morphometry delivered a greater dynamic range and absolute magnitude of reduction of IF after treatment. In summary, we demonstrate that SHG-based morphometry in unstained kidney tissues is comparable to polarized SRM for quantitation of fibrillar collagens, but with an enhanced sensitivity to detect treatment-induced reductions in IF. Thus, performing SHG-based morphometry on unstained kidney tissue is a reliable alternative to traditional polarized SRM for quantitative analysis of IF. PMID:27257917

Contribution of Electrostatics in the Fibril Stability of a Model Ionic-Complementary Peptide.

PubMed

Owczarz, Marta; Casalini, Tommaso; Motta, Anna C; Morbidelli, Massimo; Arosio, Paolo

2015-12-14

In this work we quantified the role of electrostatic interactions in the self-assembly of a model amphiphilic peptide (RADA 16-I) into fibrillar structures by a combination of size exclusion chromatography and molecular simulations. For the peptide under investigation, it is found that a net charge of +0.75 represents the ideal condition to promote the formation of regular amyloid fibrils. Lower net charges favor the formation of amorphous precipitates, while larger net charges destabilize the fibrillar aggregates and promote a reversible dissociation of monomers from the ends of the fibrils. By quantifying the dependence of the equilibrium constant of this reversible reaction on the pH value and the peptide net charge, we show that electrostatic interactions contribute largely to the free energy of fibril formation. The addition of both salt and a charged destabilizer (guanidinium hydrochloride) at moderate concentration (0.3-1 M) shifts the monomer-fibril equilibrium toward the fibrillar state. Whereas the first effect can be explained by charge screening of electrostatic repulsion only, the promotion of fibril formation in the presence of guanidinium hydrochloride is also attributed to modifications of the peptide conformation. The results of this work indicate that the global peptide net charge is a key property that correlates well with the fibril stability, although the peptide conformation and the surface charge distribution also contribute to the aggregation propensity.

Gelsolin Amyloidogenesis Is Effectively Modulated by Curcumin and Emetine Conjugated PLGA Nanoparticles

PubMed Central

Goel, Surbhi; Kundu, Bishwajit; Mishra, Prashant; Fnu, Ashish

2015-01-01

Small molecule based therapeutic intervention of amyloids has been limited by their low solubility and poor pharmacokinetic characteristics. We report here, the use of water soluble poly lactic-co-glycolic acid (PLGA)-encapsulated curcumin and emetine nanoparticles (Cm-NPs and Em-NPs, respectively), as potential modulators of gelsolin amyloidogenesis. Using the amyloid-specific dye Thioflavin T (ThT) as an indicator along with electron microscopic imaging we show that the presence of Cm-NPs augmented amyloid formation in gelsolin by skipping the pre-fibrillar assemblies, while Em-NPs induced non-fibrillar aggregates. These two types of aggregates differed in their morphologies, surface hydrophobicity and secondary structural signatures, confirming that they followed distinct pathways. In spite of differences, both these aggregates displayed reduced toxicity against SH-SY5Y human neuroblastoma cells as compared to control gelsolin amyloids. We conclude that the cytotoxicity of gelsolin amyloids can be reduced by either stalling or accelerating its fibrillation process. In addition, Cm-NPs increased the fibrillar bulk while Em-NPs defibrillated the pre-formed gelsolin amyloids. Moreover, amyloid modulation happened at a much lower concentration and at a faster rate by the PLGA encapsulated compounds as compared to their free forms. Thus, besides improving pharmacokinetic and biocompatible properties of curcumin and emetine, PLGA conjugation elevates the therapeutic potential of both small molecules against amyloid fibrillation and toxicity. PMID:25996685

Collagen fibril surface displays a constellation of sites capable of promoting fibril assembly, stability, and hemostasis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Orgel, J.P.; Antipova, O.; Sagi, I.

Fibrillar collagens form the structural basis of organs and tissues including the vasculature, bone, and tendon. They are also dynamic, organizational scaffolds that present binding and recognition sites for ligands, cells, and platelets. We interpret recently published X-ray diffraction findings and use atomic force microscopy data to illustrate the significance of new insights into the functional organization of the collagen fibril. These data indicate that collagen's most crucial functional domains localize primarily to the overlap region, comprising a constellation of sites we call the 'master control region.' Moreover, the collagen's most exposed aspect contains its most stable part - themore » C-terminal region that controls collagen assembly, cross-linking, and blood clotting. Hidden beneath the fibril surface exists a constellation of 'cryptic' sequences poised to promote hemostasis and cell - collagen interactions in tissue injury and regeneration. These findings begin to address several important, and previously unresolved, questions: How functional domains are organized in the fibril, which domains are accessible, and which require proteolysis or structural trauma to become exposed? Here we speculate as to how collagen fibrillar organization impacts molecular processes relating to tissue growth, development, and repair.« less

Assembly of collagen into microribbons: effects of pH and electrolytes.

PubMed

Jiang, Fengzhi; Hörber, Heinrich; Howard, Jonathon; Müller, Daniel J

2004-12-01

Collagen represents the major structural protein of the extracellular matrix. Elucidating the mechanism of its assembly is important for understanding many cell biological and medical processes as well as for tissue engineering and biotechnological approaches. In this work, conditions for the self-assembly of collagen type I molecules on a supporting surface were characterized. By applying hydrodynamic flow, collagen assembled into ultrathin ( approximately 3 nm) highly anisotropic ribbon-like structures coating the entire support. We call these novel collagen structures microribbons. High-resolution atomic force microscopy topographs show that subunits of these microribbons are built by fibrillar structures. The smallest units of these fibrillar structures have cross-sections of approximately 3 x 5nm, consistent with current models of collagen microfibril formation. By varying the pH and electrolyte of the buffer solution during the self-assembly process, the microfibril density and contacts formed within this network could be controlled. Under certain electrolyte compositions the microribbons and microfibers display the characteristic D-periodicity of approximately 65 nm observed for much thicker collagen fibrils. In addition to providing insight into the mechanism of collagen assembly, the ultraflat collagen matrices may also offer novel ways to bio-functionalize surfaces.

Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure

PubMed Central

Chen, Xiyi; Nadiarynkh, Oleg; Plotnikov, Sergey; Campagnola, Paul J

2013-01-01

Second-harmonic generation (SHG) microscopy has emerged as a powerful modality for imaging fibrillar collagen in a diverse range of tissues. Because of its underlying physical origin, it is highly sensitive to the collagen fibril/fiber structure, and, importantly, to changes that occur in diseases such as cancer, fibrosis and connective tissue disorders. We discuss how SHG can be used to obtain more structural information on the assembly of collagen in tissues than is possible by other microscopy techniques. We first provide an overview of the state of the art and the physical background of SHG microscopy, and then describe the optical modifications that need to be made to a laser-scanning microscope to enable the measurements. Crucial aspects for biomedical applications are the capabilities and limitations of the different experimental configurations. We estimate that the setup and calibration of the SHG instrument from its component parts will require 2–4 weeks, depending on the level of the user’s experience. PMID:22402635

The fibrillar collagen family.

PubMed

Exposito, Jean-Yves; Valcourt, Ulrich; Cluzel, Caroline; Lethias, Claire

2010-01-28

Collagens, or more precisely collagen-based extracellular matrices, are often considered as a metazoan hallmark. Among the collagens, fibrillar collagens are present from sponges to humans, and are involved in the formation of the well-known striated fibrils. In this review we discuss the different steps in the evolution of this protein family, from the formation of an ancestral fibrillar collagen gene to the formation of different clades. Genomic data from the choanoflagellate (sister group of Metazoa) Monosiga brevicollis, and from diploblast animals, have suggested that the formation of an ancestral alpha chain occurred before the metazoan radiation. Phylogenetic studies have suggested an early emergence of the three clades that were first described in mammals. Hence the duplication events leading to the formation of the A, B and C clades occurred before the eumetazoan radiation. Another important event has been the two rounds of "whole genome duplication" leading to the amplification of fibrillar collagen gene numbers, and the importance of this diversification in developmental processes. We will also discuss some other aspects of fibrillar collagen evolution such as the development of the molecular mechanisms involved in the formation of procollagen molecules and of striated fibrils.

Indentation versus Rolling: Dependence of Adhesion on Contact Geometry for Biomimetic Structures.

PubMed

Moyle, Nichole; He, Zhenping; Wu, Haibin; Hui, Chung-Yuen; Jagota, Anand

2018-04-03

Numerous biomimetic structures made from elastomeric materials have been developed to produce enhancement in properties such as adhesion, static friction, and sliding friction. As a property, one expects adhesion to be represented by an energy per unit area that is usually sensitive to the combination of shear and normal stresses at the crack front but is otherwise dependent only on the two elastic materials that meet at the interface. More specifically, one would expect that adhesion measured by indentation (a popular and convenient technique) could be used to predict adhesion hysteresis in the more practically important rolling geometry. Previously, a structure with a film-terminated fibrillar geometry exhibited dramatic enhancement of adhesion by a crack-trapping mechanism during indentation with a rigid sphere. Roughly isotropic structures such as the fibrillar geometry show a strong correlation between adhesion enhancement in indentation versus adhesion hysteresis in rolling. However, anisotropic structures, such as a film-terminated ridge-channel geometry, surprisingly show a dramatic divergence between adhesion measured by indentation versus rolling. We study this experimentally and theoretically, first comparing the adhesion of the anisotropic ridge-channel structure to the roughly isotropic fibrillar structure during indentation with a rigid sphere, where only the isotropic structure shows adhesion enhancement. Second, we examine in more detail the anomalous anisotropic film-terminated ridge-channel structure during indentation with a rigid sphere versus rolling to show why these structures show a dramatic adhesion enhancement for the rolling case and no adhesion enhancement for indentation.

Preparing Synthetic Aβ in Different Aggregation States

PubMed Central

Stine, W. Blaine; Jungbauer, Lisa; Yu, Chunjiang; LaDu, Mary Jo

2013-01-01

This chapter outlines protocols that produce homogenous preparations of oligomeric and fibrillar amyloid -β peptide (Aβ). While there are several isoforms of this peptide, the 42 amino acid form is the focus because of its genetic and pathological link to Alzheimer’s disease (AD). Past decades of AD research highlight the dependence of Aβ42 function on its structural assembly state. Biochemical, cellular and in vivo studies of Aβ42 usually begin with purified peptide obtained by chemical synthesis or recombinant expression. The initial steps to solubilize and prepare these purified dry peptide stocks are critical to controlling the structural assembly of Aβ. To develop homogenous Aβ42 assemblies, we initially monomerize the peptide, erasing any “structural history” that could seed aggregation, by using a strong solvent. It is this starting material that has allowed us to define and optimize conditions that consistently produce homogenous solutions of soluble oligomeric and fibrillar Aβ42 assemblies. These preparations have been developed and characterized by using atomic force microscopy (AFM) to identify the structurally discrete species formed by Aβ42 under specific solution conditions. These preparations have been used extensively to demonstrate a variety of functional differences between oligomeric and fibrillar Aβ42. We also present a protocol for fluorescently labeling oligomeric Aβ42 that does not affect structure, as measured by AFM, or function, as measured by a cellular uptake assay. These reagents are critical experimental tools that allow for defining specific structure/function connections. PMID:20967580

Hydrogels constructed via self-assembly of beta-hairpin molecules

NASA Astrophysics Data System (ADS)

Ozbas, Bulent

There is a recent and growing interest in hydrogel materials that are formed via peptide self-assembly for tissue engineering applications. Peptide based materials are excellent candidates for diverse applications in biomedical field due to their responsive behavior and complex self-assembled structures. However, there is very limited information on the self-assembly and resultant network and mechanical properties of these types of hydrogels. The main goal of this dissertation is to investigate the self-assembly mechanism and viscoelastic properties of hydrogels that can be altered by changing solution conditions as well as the primary structure of the peptide. These hydrogels are formed via intramolecular folding and consequent self-assembly of 20 amino acid long beta-hairpin peptide molecules (Max1). The peptide molecules are locally amphiphilic with two linear strands of alternating hydrophobic valine and hydrophilic lysine amino acids connected with a Dproline-LProline turn sequence. Circular dichroism and FTIR spectroscopy show that at physiological conditions peptides are unfolded in the absence of salt. By raising the ionic strength of the solution electrostatic interactions between charged lysines are screened and the peptide arms are forced into a beta-sheet secondary structure stabilized by the turn sequence. These folded molecules intermolecularly assemble via hydrophobic collapse and hydrogen bonding into a three dimensional network. Folding and self-assembly of these molecules can also be triggered by increasing temperature and/or pH of the peptide solution. In addition, the random-coil to beta-sheet transition of the beta-hairpin peptides is pH and, with proper changes in the peptide sequence, thermally reversible. Rheological measurements demonstrate that the resultant supramolecular structure forms an elastic material, whose structure, and thus modulus, can be tuned by magnitude of the stimulus. Hydrogels recover their initial viscoelastic properties after cessation of high magnitude of strain due to the physically crosslinked network structure and strong inter-fibrillar interactions. These interactions can be turned off by either condensing anions or covalently attaching PEG chains on lysine-decorated fibrillar surfaces. TEM, SANS, and rheological data reveal that the elasticity arises from a network consisting of semiflexible fibrillar assemblies that are monodisperse in width. The experimental results are compared with scaling relationships developed for permanently crosslinked semiflexible biopolymer networks. (Abstract shortened by UMI.)

Effect of Polymer Electrode Morphology on Performance of a Lithium/Polypyrrole Battery. M.S. Thesis

NASA Technical Reports Server (NTRS)

Nicholson, Marjorie Anne

1991-01-01

A variety of conducting polymer batteries were described in the recent literature. In this work, a Li/Polypyrrole secondary battery is described. The effect of controlling the morphology of the polymer on enhancement of counterion diffusion in the polymer phase is explored. A method of preparing conducting polymers was developed which yields high surface area per unit volume of electrode material. A porous membrane is used as a template in which to electrochemically polymerize pyrrole, then the membrane is dissolved, leaving the polymer in a fibrillar form. Conventionally, the polymer is electrochemically polymerized as a dense polymer film on a smooth Pt disk electrode. Previous work has shown that when the polymer is electrochemically polymerized in fribrillar form, charge transport rates are faster and charge capacities are greater than for dense, conventionally grown films containing the same amount of polymer. The purpose is to expand previous work by further investigating the possibilities of the optimization of transport rates in polypyrrole films by controlling the morphology of the films. The utility of fibrillar polypyrrole as a cathode material in a lithium/polymer secondary battery is then assessed. The performance of the fibrillar battery is compared to the performance of an analogous battery which employed a conventionally grown polypyrrole film. The study includes a comparison of cyclic voltammetry, shape of charge/discharge curves, discharge time and voltage, cycle life, coulombic efficiencies, charge capacities, energy densities, and energy efficiencies.

Transthyretin Interferes with Aβ Amyloid Formation by Redirecting Oligomeric Nuclei into Non-Amyloid Aggregates.

PubMed

Nilsson, Lina; Pamrén, Annelie; Islam, Tohidul; Brännström, Kristoffer; Golchin, Solmaz A; Pettersson, Nina; Iakovleva, Irina; Sandblad, Linda; Gharibyan, Anna L; Olofsson, Anders

2018-06-08

The pathological Aβ aggregates associated with Alzheimer's disease follow a nucleation-dependent path of formation. A nucleus represents an oligomeric assembly of Aβ peptides that acts as a template for subsequent incorporation of monomers to form a fibrillar structure. Nuclei can form de novo or via surface-catalyzed secondary nucleation, and the combined rates of elongation and nucleation control the overall rate of fibril formation. Transthyretin (TTR) obstructs Aβ fibril formation in favor of alternative non-fibrillar assemblies, but the mechanism behind this activity is not fully understood. This study shows that TTR does not significantly disturb fibril elongation; rather, it effectively interferes with the formation of oligomeric nuclei. We demonstrate that this interference can be modulated by altering the relative contribution of elongation and nucleation, and we show how TTR's effects can range from being essentially ineffective to almost complete inhibition of fibril formation without changing the concentration of TTR or monomeric Aβ. Copyright © 2018. Published by Elsevier Ltd.

Functional Hydrogel Materials Inspired by Amyloid

NASA Astrophysics Data System (ADS)

Schneider, Joel

2012-02-01

Protein assembly resulting in the formation of amyloid fibrils, assemblies rich in cross beta-sheet structure, is normally thought of as a deleterious event associated with disease. However, amyloid formation is also involved in a diverse array of normal biological functions such as cell adhesion, melanin synthesis, insect defense mechanism and modulation of water surface tension by fungi and bacteria. These findings indicate that Nature has evolved to take advantage of large, proteinaceous fibrillar assemblies to elicit function. We are designing functional materials, namely hydrogels, from peptides that self-assembled into fibrillar networks, rich in cross beta-sheet structure. These gels can be used for the direct encapsulation and delivery of small molecule-, protein- and cell-based therapeutics. Loaded gels exhibit shear-thinning/self-healing mechanical properties enabling their delivery via syringe. In addition to their use for delivery, we have found that some of these gels display antibacterial activity. Although cytocompatible towards mammalian cells, the hydrogels can kill a broad spectrum of bacteria on contact.

Molecular crowding of collagen: a pathway to produce highly-organized collagenous structures.

PubMed

Saeidi, Nima; Karmelek, Kathryn P; Paten, Jeffrey A; Zareian, Ramin; DiMasi, Elaine; Ruberti, Jeffrey W

2012-10-01

Collagen in vertebrate animals is often arranged in alternating lamellae or in bundles of aligned fibrils which are designed to withstand in vivo mechanical loads. The formation of these organized structures is thought to result from a complex, large-area integration of individual cell motion and locally-controlled synthesis of fibrillar arrays via cell-surface fibripositors (direct matrix printing). The difficulty of reproducing such a process in vitro has prevented tissue engineers from constructing clinically useful load-bearing connective tissue directly from collagen. However, we and others have taken the view that long-range organizational information is potentially encoded into the structure of the collagen molecule itself, allowing the control of fibril organization to extend far from cell (or bounding) surfaces. We here demonstrate a simple, fast, cell-free method capable of producing highly-organized, anistropic collagen fibrillar lamellae de novo which persist over relatively long-distances (tens to hundreds of microns). Our approach to nanoscale organizational control takes advantage of the intrinsic physiochemical properties of collagen molecules by inducing collagen association through molecular crowding and geometric confinement. To mimic biological tissues which comprise planar, aligned collagen lamellae (e.g. cornea, lamellar bone or annulus fibrosus), type I collagen was confined to a thin, planar geometry, concentrated through molecular crowding and polymerized. The resulting fibrillar lamellae show a striking resemblance to native load-bearing lamellae in that the fibrils are small, generally aligned in the plane of the confining space and change direction en masse throughout the thickness of the construct. The process of organizational control is consistent with embryonic development where the bounded planar cell sheets produced by fibroblasts suggest a similar confinement/concentration strategy. Such a simple approach to nanoscale organizational control of structure not only makes de novo tissue engineering a possibility, but also suggests a clearer pathway to organization for fibroblasts than direct matrix printing. Copyright © 2012 Elsevier Ltd. All rights reserved.

Glycosaminoglycans and fibrillar collagen in Priapulida: a histo- and cytochemical study.

PubMed

Welsch, U; Erlinger, R; Storch, V

1992-12-01

The distribution of glycosaminoglycans and fibrillar collagen was studied in various tissues of priapulids, which represent an ancient group of marine metazoa. Sulphated glycosaminoglycans, as demonstrated at the electron microscopical level by Cupromeronic blue, were predominantly found in the cuticle, in basement membranes and also in the narrow connective tissue space below epidermis and anterior intestine. On the basis of their morphology the Cupromeronic blue precipitates could be divided into several groups. Fibrillar collagen occurred in the connective tissue under the epidermis and the epithelium of the anterior intestine. The spatial interrelationship between fibrillar collagen and glycosaminoglycans lacked with some exceptions, the high regularity found in connective tissues of other invertebrates and of vertebrates. This might be related to the special skeletal system of priapulids, consisting mainly of a strong extracellular cuticle and the turgor of the fluid-filled body cavity. In such a system the usual supportive structures seem to be of less functional significance.

Location of rRNA transcription to the nucleolar components: disappearance of the fibrillar centers in nucleoli of regenerating rat hepatocytes.

PubMed

Montanaro, Lorenzo; Govoni, Marzia; Orrico, Catia; Treré, Davide; Derenzini, Massimo

2011-01-01

The precise location of rDNA transcription to the components of mammalian cell nucleolus is still debated. This was due to the fact that all the molecules necessary for rRNA synthesis are located in two of the three components, the fibrillar centers (FCs) and the dense fibrillar component (DFC), which together with the granular component (GC) are considered to be constantly present in mammalian cell nucleoli. In the present study we demonstrated that in nucleoli of many regenerating rat hepatocytes at 15 h after partial hepatectomy the FCs were no longer present, only the DFC and the GC being detected. At this time of regeneration the rRNA transcriptional activity was three fold that of resting hepatocytes, while the synthesis of DNA was not yet significantly increased, indicating that these nucleolar changes were due to the rRNA synthesis up-regulation. The DFC appeared to be organized in numerous, small, roundish tufts of fibrils. The silver staining procedure for AgNOR proteins, which are associated with the ribosomal genes, selectively and homogeneously stained these fibrillar tufts. Immuno-gold visualization of the Upstream Binding Factor (UBF), which is associated with the promoter region and the transcribed portion of the rRNA 45S gene, demonstrated that UBF was selectively located in the fibrillar tufts. We concluded that in proliferating rat hepatocytes the increased synthesis of rRNA induced an activation of the rRNA transcription machinery located in the fibrillar centers which, by becoming associated with the ribonucleoprotein transcripts, assumed the morphological pattern of the DFC.

COMPUTER SIMULATION STUDY OF AMYLOID FIBRIL FORMATION BY PALINDROMIC SEQUENCES IN PRION PEPTIDES

PubMed Central

Wagoner, Victoria; Cheon, Mookyung; Chang, Iksoo; Hall, Carol

2011-01-01

We simulate the aggregation of large systems containing palindromic peptides from the Syrian hamster prion protein SHaPrP 113–120 (AGAAAAGA) and the mouse prion protein MoPrP 111–120 (VAGAAAAGAV) and eight sequence variations: GAAAAAAG, (AG)4, A8, GAAAGAAA, A10, V10, GAVAAAAVAG, and VAVAAAAVAV The first two peptides are thought to act as the Velcro that holds the parent prion proteins together in amyloid structures and can form fibrils themselves. Kinetic events along the fibrillization pathway influence the types of structures that occur and variations in the sequence affect aggregation kinetics and fibrillar structure. Discontinuous molecular dynamics simulations using the PRIME20 force field are performed on systems containing 48 peptides starting from a random coil configuration. Depending on the sequence, fibrillar structures form spontaneously over a range of temperatures, below which amorphous aggregates form and above which no aggregation occurs. AGAAAAGA forms well organized fibrillar structures whereas VAGAAAAGAV forms less well organized structures that are partially fibrillar and partially amorphous. The degree of order in the fibrillar structure stems in part from the types of kinetic events leading up to its formation, with AGAAAAGA forming less amorphous structures early in the simulation than VAGAAAAGAV. The ability to form fibrils increases as the chain length and the length of the stretch of hydrophobic residues increase. However as the hydrophobicity of the sequence increases, the ability to form well-ordered structures decreases. Thus, longer hydrophobic sequences form slightly disordered aggregates that are partially fibrillar and partially amorphous. Subtle changes in sequence result in slightly different fibril structures. PMID:21557317

Positron Emission Tomography and Neuropathologic Estimates of Fibrillar Amyloid-β in a Patient With Down Syndrome and Alzheimer Disease

PubMed Central

Sabbagh, Marwan N.; Fleisher, Adam; Chen, Kewei; Rogers, Joseph; Berk, Camryn; Reiman, Eric; Pontecorvo, Michael; Mintun, Mark; Skovronsky, Daniel; Jacobson, Sandra A.; Sue, Lucia I.; Liebsack, Carolyn; Charney, Albert S.; Cole, Lauren; Belden, Christine; Beach, Thomas G.

2012-01-01

Background Down syndrome appears to be associated with a virtually certain risk of fibrillar amyloid-β (Aβ) pathology by the age of 40 and a very high risk of dementia at older ages. The positron emission tomography (PET) ligand florbetapir F18 has been shown to characterize fibrillar Aβ in the living human brain and to provide a close correlation with subsequent Aβ neuropathology in individuals proximate to and after the end of life. The extent to which the most frequently used PET ligands can be used to detect fibrillar Aβ in patients with Down syndrome remains to be determined. Objectives To characterize PET estimates of fibrillar Aβ burden in a Down syndrome patient very close to the end of life and to compare them with neuropathologic assessment made after his death. Design/Methods With the family’s informed consent, florbetapir PET was used to study a 55-year-old Down syndrome patient with Alzheimer disease near the end of life; his brain was donated for neuropathologic assessment when he died 14 days later. Visual ratings of cerebral florbetapir uptake were performed by trained readers who were masked to the patient’s diagnosis as part of a larger study, and an automated algorithm was used to characterize regional-to-cerebellar standard uptake value ratios in 6 cerebral regions of interest. Neuropathologic assessments were performed masked to the patient’s diagnosis or PET measurements. Results Visual ratings and automated analyses of the PET image revealed a heavy fibrillar Aβ burden in cortical, striatal, and thalamic regions, similar to that reported for patients with late-onset Alzheimer disease. This matched neuropathologic findings of frequent neuritic and diffuse plaques, as well as frequent amyloid angiopathy, except for neuropathologically demonstrated frequent cerebellar diffuse plaques and amyloid angiopathy that were not detected by the PET scan. Conclusions Florbetapir PET can be used to detect increased cerebral-to-cerebellar fibrillar Aβ burden in a Down syndrome patient with Alzheimer disease, even in the presence of frequent amyloid angiopathy and diffuse plaques in the cerebellum. Additional studies are needed to determine the extent to which PET could be used to detect and to track fibrillar Aβ and to evaluate investigational Aβ-modifying treatments in the presymptomatic and symptomatic stages of Alzheimer disease. PMID:22084131

Effect of curcumin and Cu 2+/Zn 2+ ions on the fibrillar aggregates formed by the amyloid peptide and other peptides at the organic-aqueous interface

NASA Astrophysics Data System (ADS)

Sanghamitra, Nusrat J. M.; Varghese, Neenu; Rao, C. N. R.

2010-08-01

Characteristic features of a perilous neuro-degenerative disease such as the Alzhiemer's disease is fibrillar plaque formation by the amyloid (Aβ) peptide. We have modelled the formation and disintegration of fibrils by studying the aggregate structures formed by Aβ structural motif diphenylalanine as well as insulin and bovine serum albumin at the organic-aqueous interface. Even small concentrations of curcumin in the organic medium or Cu 2+ and Zn 2+ ions in the aqueous medium are found to break down the fibrillar structures.

Reevaluation of the role of the polar groups of collagen in the platelet-collagen interaction.

PubMed Central

Chesney, C. M.; Pifer, D. D.; Crofford, L. J.; Huch, K. M.

1983-01-01

Chemical modification of collagen is a tool for exploring the platelet-collagen interaction. Since collagen must polymerize prior to the initiation of platelet aggregation and secretion, modification must be shown to affect platelet-collagen interaction and not collagen-collagen interaction. To address this point, the authors carried out the following chemical modifications on soluble monomeric collagen and preformed fibrillar collagen in parallel: 1) N-and O-acetylation, 2) esterification of the carboxyl groups, 3) succinylation of the free amino groups, 4) esterification of succinylated collagen. Intrinsic viscosity studies of the modified soluble collagens were consistent with normal triple helix conformation. Electron microscopy revealed all modified fibrillar collagen to maintain a fibrillar structure. Platelet aggregation and secretion of 14C-serotonin and platelet factor 4 by soluble and fibrillar collagen, respectively, were studied in human platelet-rich plasma. Neutralization of polar groups by 1) totally abolished aggregation and secretion by both collagens, while blocking acidic groups 2) resulted in enhanced aggregation and secretion by both soluble and fibrillar collagen. Blockage of amino groups by 3) abolished aggregation and secretion by both collagens. Esterified succinylated collagen 4) caused aggregation and secretion at relatively high collagen concentrations. These data support the theory that positive groups of collagen are important in platelet-collagen interaction. Images Figure 1 PMID:6881287

Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging: A Proof-of-Concept Study.

PubMed

Pirhonen, Juho; Arola, Johanna; Sädevirta, Sanja; Luukkonen, Panu; Karppinen, Sanna-Maria; Pihlajaniemi, Taina; Isomäki, Antti; Hukkanen, Mika; Yki-Järvinen, Hannele; Ikonen, Elina

2016-01-01

Early detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We tested whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD. We analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0-4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals. We found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III. This study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading.

Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders.

PubMed

Caughey, Byron; Lansbury, Peter T

2003-01-01

Many neurodegenerative diseases, including Alzheimer's and Parkinson's and the transmissible spongiform encephalopathies (prion diseases), are characterized at autopsy by neuronal loss and protein aggregates that are typically fibrillar. A convergence of evidence strongly suggests that protein aggregation is neurotoxic and not a product of cell death. However, the identity of the neurotoxic aggregate and the mechanism by which it disables and eventually kills a neuron are unknown. Both biophysical studies aimed at elucidating the precise mechanism of in vitro aggregation and animal modeling studies support the emerging notion that an ordered prefibrillar oligomer, or protofibril, may be responsible for cell death and that the fibrillar form that is typically observed at autopsy may actually be neuroprotective. A subpopulation of protofibrils may function as pathogenic amyloid pores. An analogous mechanism may explain the neurotoxicity of the prion protein; recent data demonstrates that the disease-associated, infectious form of the prion protein differs from the neurotoxic species. This review focuses on recent experimental studies aimed at identification and characterization of the neurotoxic protein aggregates.

A cytochemical and radioautographic study of the ultrastructural organization of puff-like fibrillar structures in plant interphase nuclei (Allium porrum).

PubMed

Lafontaine, J G; Luck, B T; Dontigny, D

1979-10-01

Loose, fibrillar, spherical structures have been observed during recent years in interphase nuclei of both animal and plant cells. These nuclear formations have been referred to as karyosomes, fibrillar bodies, micropuffs and centromeres. In order to gain further information on the nature of these structures, a cytochemical and radioautographic investigation was undertaken using plant meristematic cells (Allium porrum). For that purpose roots were fixed with either formaldehyde or glutaraldehyde in order to carry out cytochemical tests for DNA, RNA and proteins. Certain of the preparations were also first digested with DNase, RNase or proteinase K and then stained according to different procedures. Other specimens were labelled with thymidine for high-resolution radioautographic observations. Staining with diaminobenzidine (DAB) revealed that these nuclear puff-like formations consisted partly of a loose fibrillar meshwork containing nucleic acids. Part of this fine fibrillar reticulum persisted whether the preparations were digested with DNase or RNase before staining with DAB, thus indicating that these nuclear structures contained both DNA and RNA. The fact that these formations incorporate thymidine furnished additional support for the view that they correspond to specific chromosome segments. Staining with ethanolic phosphotungstic acid or digestion of specimens with proteinase K showed that these loose fibrillar structures also consisted of proteins. Judging from their ultrastructure, their association with the chromatin reticulum as well as from their cytochemical characteristics, these nuclear formations most likely correspond to centromeres. In view of the presence of DNA within these structures, it is possible to distinguish them from other equally spherical nuclear formations, observed in certain plant species, that have generally been referred to as karyosomes or micronucleoli and that appear to consist of ribonucleoproteins.

Stress relaxing hyaluronic acid-collagen hydrogels promote cell spreading, fiber remodeling, and focal adhesion formation in 3D cell culture.

PubMed

Lou, Junzhe; Stowers, Ryan; Nam, Sungmin; Xia, Yan; Chaudhuri, Ovijit

2018-02-01

The physical and architectural cues of the extracellular matrix (ECM) play a critical role in regulating important cellular functions such as spreading, migration, proliferation, and differentiation. Natural ECM is a complex viscoelastic scaffold composed of various distinct components that are often organized into a fibrillar microstructure. Hydrogels are frequently used as synthetic ECMs for 3D cell culture, but are typically elastic, due to covalent crosslinking, and non-fibrillar. Recent work has revealed the importance of stress relaxation in viscoelastic hydrogels in regulating biological processes such as spreading and differentiation, but these studies all utilize synthetic ECM hydrogels that are non-fibrillar. Key mechanotransduction events, such as focal adhesion formation, have only been observed in fibrillar networks in 3D culture to date. Here we present an interpenetrating network (IPN) hydrogel system based on HA crosslinked with dynamic covalent bonds and collagen I that captures the viscoelasticity and fibrillarity of ECM in tissues. The IPN hydrogels exhibit two distinct processes in stress relaxation, one from collagen and the other from HA crosslinking dynamics. Stress relaxation in the IPN hydrogels can be tuned by modulating HA crosslinker affinity, molecular weight of the HA, or HA concentration. Faster relaxation in the IPN hydrogels promotes cell spreading, fiber remodeling, and focal adhesion (FA) formation - behaviors often inhibited in other hydrogel-based materials in 3D culture. This study presents a new, broadly adaptable materials platform for mimicking key ECM features of viscoelasticity and fibrillarity in hydrogels for 3D cell culture and sheds light on how these mechanical and structural cues regulate cell behavior. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quantitative fibrosis parameters highly predict esophageal-gastro varices in primary biliary cirrhosis.

PubMed

Wu, Q-M; Zhao, X-Y; You, H

2016-01-01

Esophageal-gastro Varices (EGV) may develop in any histological stages of primary biliary cirrhosis (PBC). We aim to establish and validate quantitative fibrosis (qFibrosis) parameters in portal, septal and fibrillar areas as ideal predictors of EGV in PBC patients. PBC patients with liver biopsy, esophagogastroscopy and Second Harmonic Generation (SHG)/Two-photon Excited Fluorescence (TPEF) microscopy images were retrospectively enrolled in this study. qFibrosis parameters in portal, septal and fibrillar areas were acquired by computer-assisted SHG/TPEF imaging system. Independent predictor was identified using multivariate logistic regression analysis. PBC patients with liver biopsy, esophagogastroscopy and Second Harmonic Generation (SHG)/Two-photon Excited Fluorescence (TPEF) microscopy images were retrospectively enrolled in this study. qFibrosis parameters in portal, septal and fibrillar areas were acquired by computer-assisted SHG/TPEF imaging system. Independent predictor was identified using multivariate logistic regression analysis. Among the forty-nine PBC patients with qFibrosis images, twenty-nine PBC patients with both esophagogastroscopy data and qFibrosis data were selected out for EGV prognosis analysis and 44.8% (13/29) of them had EGV. The qFibrosis parameters of collagen percentage and number of crosslink in fibrillar area, short/long/thin strings number and length/width of the strings in septa area were associated with EGV (p < 0.05). Multivariate logistic analysis showed that the collagen percentage in fibrillar area ≥ 3.6% was an independent factor to predict EGV (odds ratio 6.9; 95% confidence interval 1.6-27.4). The area under receiver operating characteristic (ROC), diagnostic sensitivity and specificity was 0.9, 100% and 75% respectively. Collagen percentage in Collagen percentage in the fibrillar area as an independent predictor can highly predict EGV in PBC patients.

Fibrillar amyloid correlates of preclinical cognitive decline.

PubMed

Stonnington, Cynthia M; Chen, Kewei; Lee, Wendy; Locke, Dona E C; Dueck, Amylou C; Liu, Xiaofen; Roontiva, Auttawut; Fleisher, Adam S; Caselli, Richard J; Reiman, Eric M

2014-01-01

It is not known whether preclinical cognitive decline is associated with fibrillar β-amyloid (Aβ) deposition irrespective of apolipoprotein E (APOE) ε4 status. From a prospective observational study of 623 cognitively normal individuals, we identified all subjects who showed preclinical decline of at least 2 standard deviations beyond the decline of the entire group in memory or executive function. Fourteen decliners were matched by APOE ε4 gene dose, age, sex, and education with 14 nondecliners. Dynamic Pittsburgh compound B (PiB) positron emission tomography (PET) scans, the Logan method, statistical parametric mapping, and automatically labeled regions of interest were used to characterize and compare cerebral-to-cerebellar PiB distribution volume ratios (DVRs), reflecting fibrillar Aβ burden. At P < .005 (uncorrected), decliners had significantly greater DVRs in comparison to nondecliners. Asymptomatic longitudinal neuropsychological decline is associated with subsequent increased fibrillar amyloid deposition, even when controlling for APOE ε4 genotype. Copyright © 2014 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.

Fibril formation from pea protein and subsequent gel formation.

PubMed

Munialo, Claire Darizu; Martin, Anneke H; van der Linden, Erik; de Jongh, Harmen H J

2014-03-19

The objective of this study was to characterize fibrillar aggregates made using pea proteins, to assemble formed fibrils into protein-based gels, and to study the rheological behavior of these gels. Micrometer-long fibrillar aggregates were observed after pea protein solutions had been heated for 20 h at pH 2.0. Following heating of pea proteins, it was observed that all of the proteins were hydrolyzed into peptides and that 50% of these peptides were assembled into fibrils. Changes on a structural level in pea proteins were studied using circular dichroism, transmission electron microscopy, and particle size analysis. During the fibril assembly process, an increase in aggregate size was observed, which coincided with an increase in thioflavin T binding, indicating the presence of β-sheet aggregates. Fibrils made using pea proteins were more branched and curly. Gel formation of preformed fibrils was induced by slow acidification from pH 7.0 to a final pH of around pH 5.0. The ability of pea protein-based fibrillar gels to fracture during an amplitude sweep was comparable to those of soy protein and whey protein-based fibrillar gels, although gels prepared from fibrils made using pea protein and soy protein were weaker than those of whey protein. The findings show that fibrils can be prepared from pea protein, which can be incorporated into protein-based fibrillar gels.

Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging: A Proof-of-Concept Study

PubMed Central

Pirhonen, Juho; Arola, Johanna; Sädevirta, Sanja; Luukkonen, Panu; Karppinen, Sanna-Maria; Pihlajaniemi, Taina; Isomäki, Antti; Hukkanen, Mika

2016-01-01

Background and Aims Early detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We tested whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD. Methods We analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0–4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals. Results We found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III. Conclusions This study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading. PMID:26808140

Nonlinear Surface Dilatational Rheology and Foaming Behavior of Protein and Protein Fibrillar Aggregates in the Presence of Natural Surfactant.

PubMed

Wan, Zhili; Yang, Xiaoquan; Sagis, Leonard M C

2016-04-19

The surface and foaming properties of native soy glycinin (11S) and its heat-induced fibrillar aggregates, in the presence of natural surfactant steviol glycoside (STE), were investigated and compared at pH 7.0 to determine the impact of protein structure modification on protein-surfactant interfacial interactions. The adsorption at, and nonlinear dilatational rheological behavior of, the air-water interface were studied by combining drop shape analysis tensiometry, ellipsometry, and large-amplitude oscillatory dilatational rheology. Lissajous plots of surface pressure versus deformation were used to analyze the surface rheological response in terms of interfacial microstructure. The heat treatment generates a mixture of long fibrils and unconverted peptides. The presence of small peptides in 11S fibril samples resulted in a faster adsorption kinetics than that of native 11S. The addition of STE affected the adsorption of 11S significantly, whereas no apparent effect on the adsorption of the 11S fibril-peptide system was observed. The rheological response of interfaces stabilized by 11S-STE mixtures also differed significantly from the response for 11S fibril-peptide-STE mixtures. For 11S, the STE reduces the degree of strain hardening in extension and increases strain hardening in compression, suggesting the interfacial structure may change from a surface gel to a mixed phase of protein patches and STE domains. The foams generated from the mixtures displayed comparable foam stability to that of pure 11S. For 11S fibril-peptide mixtures STE only significantly affects the response in extension, where the degree of strain softening is decreased compared to the pure fibril-peptide system. The foam stability of the fibril-peptide system was significantly reduced by STE. These findings indicate that fibrillization of globular proteins could be a potential strategy to modify the complex surface and foaming behaviors of protein-surfactant mixtures.

The 26S Proteasome Degrades the Soluble but Not the Fibrillar Form of the Yeast Prion Ure2p In Vitro

PubMed Central

Wang, Kai; Redeker, Virginie; Madiona, Karine; Melki, Ronald; Kabani, Mehdi

2015-01-01

Yeast prions are self-perpetuating protein aggregates that cause heritable and transmissible phenotypic traits. Among these, [PSI +] and [URE3] stand out as the most studied yeast prions, and result from the self-assembly of the translation terminator Sup35p and the nitrogen catabolism regulator Ure2p, respectively, into insoluble fibrillar aggregates. Protein quality control systems are well known to govern the formation, propagation and transmission of these prions. However, little is known about the implication of the cellular proteolytic machineries in their turnover. We previously showed that the 26S proteasome degrades both the soluble and fibrillar forms of Sup35p and affects [PSI +] propagation. Here, we show that soluble native Ure2p is degraded by the proteasome in an ubiquitin-independent manner. Proteasomal degradation of Ure2p yields amyloidogenic N-terminal peptides and a C-terminal resistant fragment. In contrast to Sup35p, fibrillar Ure2p resists proteasomal degradation. Thus, structural variability within prions may dictate their ability to be degraded by the cellular proteolytic systems. PMID:26115123

Dual role for the latent transforming growth factor-beta binding protein in storage of latent TGF-beta in the extracellular matrix and as a structural matrix protein

PubMed Central

1995-01-01

The role of the latent TGF-beta binding protein (LTBP) is unclear. In cultures of fetal rat calvarial cells, which form mineralized bonelike nodules, both LTBP and the TGF-beta 1 precursor localized to large fibrillar structures in the extracellular matrix. The appearance of these fibrillar structures preceded the appearance of type I collagen fibers. Plasmin treatment abolished the fibrillar staining pattern for LTBP and released a complex containing both LTBP and TGF-beta. Antibodies and antisense oligonucleotides against LTBP inhibited the formation of mineralized bonelike nodules in long-term fetal rat calvarial cultures. Immunohistochemistry of fetal and adult rat bone confirmed a fibrillar staining pattern for LTBP in vivo. These findings, together with the known homology of LTBP to the fibrillin family of proteins, suggest a novel function for LTBP, in addition to its role in matrix storage of latent TGF-beta, as a structural matrix protein that may play a role in bone formation. PMID:7593177

Synergy of combined Doxycycline/TUDCA treatment in lowering Transthyretin deposition and associated biomarkers: studies in FAP mouse models

PubMed Central

2010-01-01

Familial Amyloidotic Polyneuropathy (FAP) is a disorder characterized by the extracellular deposition of fibrillar Transthyretin (TTR) amyloid, with a special involvement of the peripheral nerve. We had previously shown that doxycycline administered for 3 months at 40 mg/Kg/ml in the drinking water, was capable of removing TTR amyloid deposits present in stomachs of old TTR-V30M transgenic mice; the removal was accompanied by a decrease in extracellular matrix remodeling proteins that accompany fibrillar deposition, but not of non-fibrillar TTR deposition and/or markers associated with pre-fibrillar deposits. On the other hand, Tauroursodeoxycholic acid (TUDCA), a biliary acid, administrated to the same mouse model was shown to be effective at lowering deposited non-fibrillar TTR, as well as the levels of markers associated with pre-fibrillar TTR, but only at young ages. In the present work we evaluated different doxycycline administration schemes, including different periods of treatment, different dosages and different FAP TTR V30M animal models. Evaluation included CR staining, immunohistochemistry for TTR, metalloproteinase 9 (MMP-9) and serum amyloid P component (SAP). We determined that a minimum period of 15 days of treatment with a 8 mg/Kg/day dosage resulted in fibril removal. The possibility of intermittent treatments was also assessed and a maximum period of 15 days of suspension was determined to maintain tissues amyloid-free. Combined cycled doxycycline and TUDCA administration to mice with amyloid deposition, using two different concentrations of both drugs, was more effective than either individual doxycycline or TUDCA, in significantly lowering TTR deposition and associated tissue markers. The observed synergistic effect of doxycycline/TUDCA in the range of human tolerable quantities, in the transgenic TTR mice models prompts their application in FAP, particularly in the early stages of disease. PMID:20673327

Deciphering Structural Intermediates and Genotoxic Fibrillar Aggregates of Albumins: A Molecular Mechanism Underlying for Degenerative Diseases

PubMed Central

Naeem, Aabgeena; Amani, Samreen

2013-01-01

The misfolding and aggregation of proteins is involved in some of the most prevalent neurodegenerative disorders. The importance of human serum albumin (HSA) stems from the fact that it is involved in bio-regulatory and transport phenomena. Here the effect of acetonitrile (ACN) on the conformational stability of HSA and by comparison, ovalbumin (OVA) has been evaluated in the presence and absence of NaCl. The results show the presence of significant amount of secondary structure in HSA at 70% ACN and in OVA at 50% ACN, as evident from far-UV Circular Dichroism (CD) and Attenuated Total Reflection Fourier transformed infra red spectroscopy (ATR-FTIR). Tryptophan and 8-Anilino-1-Naphthalene-Sulphonic acid (ANS) fluorescence indicate altered tryptophan environment and high ANS binding suggesting a compact “molten globule”-like conformation with enhanced exposure of hydrophobic surface area. However, in presence of NaCl no intermediate state was observed. Detection of aggregates in HSA and OVA was possible at 90% ACN. Aggregates possess extensive β-sheet structure as revealed by far-UV CD and ATR-FTIR. These aggregates exhibit increase Thioflavin T (Th T) fluorescence with a red shift of Congo red (CR) absorption spectrum. X-ray diffraction (XRD) and Scanning Electron Microscopy (SEM) analysis confirmed the presence of fibrillar aggregates. Single cell gel electrophoresis (SCGE) assay of these fibrillar aggregates showed the DNA damage resulting in cell necrosis confirming their genotoxic nature. Some proteins not related to any human disease form fibrils in vitro. In the present study ACN gives access to a model system to study the process of aggregation. PMID:23342075

Nucleolar changes after microinjection of antibodies to RNA polymerase I into the nucleus of mammalian cells.

PubMed

Benavente, R; Reimer, G; Rose, K M; Hügle-Dörr, B; Scheer, U

1988-01-01

After microinjection of antibodies against RNA polymerase I into the nuclei of cultured rat kangaroo (PtK2) and rat (RVF-SMC) cells alterations in nucleolar structure and composition were observed. These were detected by electron microscopy and double-label immunofluorescence microscopy using antibodies to proteins representative of the three major components of the nucleolus. The microinjected antibodies produced a progressive loss of the material of the dense fibrillar component (DFC) from the nucleoli which, at 4 h after injection, were transformed into bodies with purely granular component (GC) structure with attached fibrillar centers (FCs). Concomitantly, numerous extranucleolar aggregates appeared in the nucleoplasm which morphologically resembled fragments of the DFC and contained a protein (fibrillarin) diagnostic for this nucleolar structure. These observations indicate that the topological distribution of the material constituting the DFC can be experimentally influenced in interphase cells, apparently by modulating the transcriptional activity of the rRNA genes. These effects are different from nucleolar lesions induced by inhibitory drugs such as actinomycin D-dependent "nucleolar segregation". The structural alterations induced by antibodies to RNA polymerase I resemble, however, the initial events of nucleolar disintegration during mitotic prophase.

Multifunctional hybrid networks based on self assembling peptide sequences

NASA Astrophysics Data System (ADS)

Sathaye, Sameer

The overall aim of this dissertation is to achieve a comprehensive correlation between the molecular level changes in primary amino acid sequences of amphiphilic beta-hairpin peptides and their consequent solution-assembly properties and bulk network hydrogel behavior. This has been accomplished using two broad approaches. In the first approach, amino acid substitutions were made to peptide sequence MAX1 such that the hydrophobic surfaces of the folded beta-hairpins from the peptides demonstrate shape specificity in hydrophobic interactions with other beta-hairpins during the assembly process, thereby causing changes to the peptide nanostructure and bulk rheological properties of hydrogels formed from the peptides. Steric lock and key complementary hydrophobic interactions were designed to occur between two beta-hairpin molecules of a single molecule, LNK1 during beta-sheet fibrillar assembly of LNK1. Experimental results from circular dichroism, transmission electron microscopy and oscillatory rheology collectively indicate that the molecular design of the LNK1 peptide can be assigned the cause of the drastically different behavior of the networks relative to MAX1. The results indicate elimination or significant reduction of fibrillar branching due to steric complementarity in LNK1 that does not exist in MAX1, thus supporting the original hypothesis. As an extension of the designed steric lock and key complementarity between two beta-hairpin molecules of the same peptide molecule. LNK1, three new pairs of peptide molecules LP1-KP1, LP2-KP2 and LP3-KP3 that resemble complementary 'wedge' and 'trough' shapes when folded into beta-hairpins were designed and studied. All six peptides individually and when blended with their corresponding shape complement formed fibrillar nanostructures with non-uniform thickness values. Loose packing in the assembled structures was observed in all the new peptides as compared to the uniform tight packing in MAX1 by SANS analysis. This loose packing can be attributed to the designed wedge and trough shapes of the peptides disturbing formation of a uniform bilayer type structure proposed in the case of MAX1 with each hairpin having a flat hydrophobic surface. Although designed changes in hydrophobic shape of the peptide nanofibril core in the new peptides were found to significantly influence the self-assembled nanostructure and network rheological behavior, a lack of direct morphological and rheological evidence to prove shape specific hydrophobic interactions between wedge and trough shaped beta-hairpins was encountered. In the second approach, peptides with established differences in assembly kinetics and bulk mechanical properties of assembled peptide hydrogels were used to develop composite materials with diverse morphological and mechanical properties by blending with the biopolymer hyaluronic acid. The diverse properties of the composites have been correlated to the specific peptide hydrogels used to develop the composite and the different stages of peptide assembly at which blending with hyaluronic acid was carried out. Finally along with overall conclusions, the new area of co-assembly of peptides in solution has been explored and discussed as potential future work following the research discussed in this dissertation. Strategies such as construction of composite hydrogels from blends of MAX1/MAX8 peptide hydrogels and biologically important anionic species such as heparin biopolymer and DNA have been discussed. Another area of future work discussed is the design and study of peptides that can incorporate chemically crosslinkable functional groups in their hydrophobic amino acid side chains that can be covalently crosslinked after peptide assembly into fibrils. Such covalent crosslinking can potentially lead to stiffer individual peptide fibrils due to additional bond formation at the fibrillar core and therefore much stiffer hydrogels due to a synergistic effect. These enhanced stiffness values can render these new hydrogels excellent candidates for applications like development of extracellular mimetic materials and substrates with easily tunable stiffness values for stem cell differentiation studies.

Platelet aggregating material from equine arterial tissue

DOEpatents

Schneider, Morris D.

1983-02-22

Novel hemostatic agent comprises equine arterial fibrillar collagen in a carrier. The agent is useful for the aggregation of platelets for clinical diagnostic tests and for the clotting of blood, such as for controlling bleeding in warm blooded species. The fibrillar collagen is obtained by extracting homogenized equine arterial tissue with aqueous solutions followed by extensive dialysis.

The process of EDC-NHS cross-linking of reconstituted collagen fibres increases collagen fibrillar order and alignment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shepherd, D. V., E-mail: dvs23@cam.ac.uk; Shepherd, J. H.; Cameron, R. E.

We describe the production of collagen fibre bundles through a multi-strand, semi-continuous extrusion process. Cross-linking using an EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), NHS (N-hydroxysuccinimide) combination was considered. Atomic Force Microscopy and Raman spectroscopy focused on how cross-linking affected the collagen fibrillar structure. In the cross-linked fibres, a clear fibrillar structure comparable to native collagen was observed which was not observed in the non-cross-linked fibre. The amide III doublet in the Raman spectra provided additional evidence of alignment in the cross-linked fibres. Raman spectroscopy also indicated no residual polyethylene glycol (from the fibre forming buffer) or water in any of the fibres.

Detection of Mutant Huntingtin Aggregation Conformers and Modulation of SDS-Soluble Fibrillar Oligomers by Small Molecules

PubMed Central

Sontag, Emily Mitchell; Lotz, Gregor P.; Yang, Guocheng; Sontag, Christopher J.; Cummings, Brian J.; Glabe, Charles G.; Muchowski, Paul J.; Thompson, Leslie Michels

2012-01-01

The Huntington’s disease (HD) mutation leads to a complex process of Huntingtin (Htt) aggregation into multimeric species that eventually form visible inclusions in cytoplasm, nuclei and neuronal processes. One hypothesis is that smaller, soluble forms of amyloid proteins confer toxic effects and contribute to early cell dysfunction. However, analysis of mutant Htt aggregation intermediates to identify conformers that may represent toxic forms of the protein and represent potential drug targets remains difficult. We performed a detailed analysis of aggregation conformers in multiple in vitro, cell and ex vivo models of HD. Conformation-specific antibodies were used to identify and characterize aggregation species, allowing assessment of multiple conformers present during the aggregation process. Using a series of assays together with these antibodies, several forms could be identified. Fibrillar oligomers, defined as having a β-sheet rich conformation, are observed in vitro using recombinant protein and in protein extracts from cells in culture or mouse brain and shown to be globular, soluble and non-sedimentable structures. Compounds previously described to modulate visible inclusion body formation and reduce toxicity in HD models were also tested and consistently found to alter the formation of fibrillar oligomers. Interestingly, these compounds did not alter the rate of visible inclusion formation, indicating that fibrillar oligomers are not necessarily the rate limiting step of inclusion body formation. Taken together, we provide insights into the structure and formation of mutant Htt fibrillar oligomers that are modulated by small molecules with protective potential in HD models. PMID:24086178

Soft grippers using micro-fibrillar adhesives for transfer printing.

PubMed

Song, Sukho; Sitti, Metin

2014-07-23

The adhesive characteristics of fibrillar adhesives on a soft deformable membrane are reported. A soft gripper with an inflatable membrane covered by elastomer mushroom-shaped microfibers have a superior conformation to non-planar 3D part geometries, enabling the transfer printing of various parts serially or in parallel. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

N-terminal region of myelin basic protein reduces fibrillar amyloid-β deposition in Tg-5xFAD mice.

PubMed

Ou-Yang, Ming-Hsuan; Xu, Feng; Liao, Mei-Chen; Davis, Judianne; Robinson, John K; Van Nostrand, William E

2015-02-01

Alzheimer's disease is a progressive neurodegenerative disorder that is characterized by extensive deposition of fibrillar amyloid-β (Aβ) in the brain. Previously, myelin basic protein (MBP) was identified to be a potent inhibitor to Aβ fibril formation, and this inhibitory activity was localized to the N-terminal residues 1-64, a fragment designated MBP1. Here, we show that the modest neuronal expression of a fusion protein of the biologically active MBP1 fragment and the enhanced green fluorescent protein (MBP1-EGFP) significantly improved the performance of spatial learning memory in Tg-5xFAD mice, a model of pathologic Aβ accumulation in brain. The levels of insoluble Aβ and fibrillar amyloid were significantly reduced in bigenic Tg-5xFAD/Tg-MBP1-EGFP mice. Quantitative stereological analysis revealed that the reduction in amyloid was because of a reduction in the size of fibrillar plaques rather than a decrease in plaque numbers. The current findings support previous studies showing that MBP1 inhibits Aβ fibril formation in vitro and demonstrate the ability of MBP1 to reduce Aβ pathology and improve behavioral performance. Copyright © 2015 Elsevier Inc. All rights reserved.

Imaging Collagen in Scar Tissue: Developments in Second Harmonic Generation Microscopy for Biomedical Applications

PubMed Central

Mostaço-Guidolin, Leila; Rosin, Nicole L.; Hackett, Tillie-Louise

2017-01-01

The ability to respond to injury with tissue repair is a fundamental property of all multicellular organisms. The extracellular matrix (ECM), composed of fibrillar collagens as well as a number of other components is dis-regulated during repair in many organs. In many tissues, scaring results when the balance is lost between ECM synthesis and degradation. Investigating what disrupts this balance and what effect this can have on tissue function remains an active area of research. Recent advances in the imaging of fibrillar collagen using second harmonic generation (SHG) imaging have proven useful in enhancing our understanding of the supramolecular changes that occur during scar formation and disease progression. Here, we review the physical properties of SHG, and the current nonlinear optical microscopy imaging (NLOM) systems that are used for SHG imaging. We provide an extensive review of studies that have used SHG in skin, lung, cardiovascular, tendon and ligaments, and eye tissue to understand alterations in fibrillar collagens in scar tissue. Lastly, we review the current methods of image analysis that are used to extract important information about the role of fibrillar collagens in scar formation. PMID:28809791

Conducting polymers with immobilised fibrillar collagen for enhanced neural interfacing.

PubMed

Liu, Xiao; Yue, Zhilian; Higgins, Michael J; Wallace, Gordon G

2011-10-01

Conducting polymers with pendant functionality are advantageous in various bionic and organic bioelectronic applications, as they allow facile incorporation of bio-regulative cues to provide bio-mimicry and conductive environments for cell growth, differentiation and function. In this work, polypyrrole substrates doped with chondroitin sulfate (CS), an extracellular matrix molecule bearing carboxylic acid moieties, were electrochemically synthesized and conjugated with type I collagen. During the coupling process, the conjugated collagen formed a 3-dimensional fibrillar matrix in situ at the conducting polymer interface, as evidenced by atomic force microscopy (AFM) and fluorescence microscopy under aqueous physiological conditions. Cyclic voltammetry (CV) and impedance measurement confirmed no significant reduction in the electroactivity of the fibrillar collagen-modified conducting polymer substrates. Rat pheochromocytoma (nerve) cells showed increased differentiation and neurite outgrowth on the fibrillar collagen, which was further enhanced through electrical stimulation of the underlying conducting polymer substrate. Our study demonstrates that the direct coupling of ECM components such as collagen, followed by their further self-assembly into 3-dimensional matrices, has the potential to improve the neural-electrode interface of implant electrodes by encouraging nerve cell attachment and differentiation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Molecular and ultrastructural studies of a fibrillar collagen from octocoral (Cnidaria).

PubMed

Orgel, Joseph P R O; Sella, Ido; Madhurapantula, Rama S; Antipova, Olga; Mandelberg, Yael; Kashman, Yoel; Benayahu, Dafna; Benayahu, Yehuda

2017-09-15

We report here the biochemical, molecular and ultrastructural features of a unique organization of fibrillar collagen extracted from the octocoral Sarcophyton ehrenbergi Collagen, the most abundant protein in the animal kingdom, is often defined as a structural component of extracellular matrices in metazoans. In the present study, collagen fibers were extracted from the mesenteries of S. ehrenbergi polyps. These fibers are organized as filaments and further compacted as coiled fibers. The fibers are uniquely long, reaching an unprecedented length of tens of centimeters. The diameter of these fibers is 9±0.37 μm. The amino acid content of these fibers was identified using chromatography and revealed close similarity in content to mammalian type I and II collagens. The ultrastructural organization of the fibers was characterized by means of high-resolution microscopy and X-ray diffraction. The fibers are composed of fibrils and fibril bundles in the range of 15 to 35 nm. These data indicate a fibrillar collagen possessing structural aspects of both types I and II collagen, a highly interesting and newly described form of fibrillar collagen organization. © 2017. Published by The Company of Biologists Ltd.

Electrostatic interactions lead to the formation of asymmetric collagen-phosphophoryn aggregates.

PubMed

Dahl, Thomas; Veis, Arthur

2003-01-01

In bone and dentin the formation and mineralization of the extra cellular matrix structure is a complex process highly dependent on intermolecular interactions. In dentin, the phosphophoryns (PP) and type I collagen (COL1) are the major constituents implicated in mineralization. Thus, as a first step in understanding the tissue organization, we have initiated a study of their interaction as a function of pH, ionic strength, and relative concentrations or mixing ratios. Complex formation has been analyzed by dynamic light scattering to detect aggregate formation and by rotary shadowing electron microscopy (EM) to determine aggregate shape. The EM data showed that at the pH values studied, the PP-COL1 interaction leads to the formation of large fibrillar aggregates in which the PP are present along the fibril surfaces. The quantitative phase distribution data showed a 1/1 molar equivalence at the maximum aggregation point, not at electrostatic PP-COL1 equivalence. As the ionic strength was raised, the PP-COL1 aggregates became smaller but the binding and asymmetric fibrillar aggregation persisted. In EM, the PP appear as dense spheres. Along the surfaces of the collagen aggregates, the PP are larger and more open or extended, suggesting that COL1-bound PP may undergo a conformational change, opening up so that a single PP molecule might interact with and electrostatically link several COL1 molecules. This might have important implications for dentin structure, stability, and mineralization.

Toward understanding insulin fibrillation.

PubMed

Brange, J; Andersen, L; Laursen, E D; Meyn, G; Rasmussen, E

1997-05-01

Formation of insulin fibrils is a physical process by which partially unfolded insulin molecules interact with each other to form linear aggregates. Shielding of hydrophobic domains is the main driving force for this process, but formation of intermolecular beta-sheet may further stabilize the fibrillar structure. Conformational displacement of the B-chain C-terminal with exposure of nonpolar, aliphatic core residues, including A2, A3, B11, and B15, plays a crucial role in the fibrillation process. Recent crystal analyses and molecular modeling studies have suggested that when insulin fibrillates this exposed domain interacts with a hydrophobic surface domain formed by the aliphatic residues A13, B6, B14, B17, and B18, normally buried when three insulin dimers form a hexamer. In rabbit immunization experiments, insulin fibrils did not elicit an increased immune response with respect to formation of IgG insulin antibodies when compared with native insulin. In contrast, the IgE response increased with increasing content of insulin in fibrillar form. Strategies and practical approaches to prevent insulin from forming fibrils are reviewed. Stabilization of the insulin hexameric structure and blockage of hydrophobic interfaces by addition of surfactants are the most effective means of counteracting insulin fibrillation.

The hierarchical response of human corneal collagen to load.

PubMed

Bell, J S; Hayes, S; Whitford, C; Sanchez-Weatherby, J; Shebanova, O; Vergari, C; Winlove, C P; Terrill, N; Sorensen, T; Elsheikh, A; Meek, K M

2018-01-01

Fibrillar collagen in the human cornea is integral to its function as a transparent lens of precise curvature, and its arrangement is now well-characterised in the literature. While there has been considerable effort to incorporate fibrillar architecture into mechanical models of the cornea, the mechanical response of corneal collagen to small applied loads is not well understood. In this study the fibrillar and molecular response to tensile load was quantified using small and wide angle X-ray scattering (SAXS/WAXS), and digital image correlation (DIC) photography was used to calculate the local strain field that gave rise to the hierarchical changes. A molecular scattering model was used to calculate the tropocollagen tilt relative to the fibril axis and changes associated with applied strain. Changes were measured in the D-period, molecular tilt and the orientation and spacing of the fibrillar and molecular networks. These measurements were summarised into hierarchical deformation mechanisms, which were found to contribute at varying strains. The change in molecular tilt is indicative of a sub-fibrillar "spring-like" deformation mechanism, which was found to account for most of the applied strain under physiological and near-physiological loads. This deformation mechanism may play an important functional role in tissues rich in fibrils of high helical tilt, such as skin and cartilage. Collagen is the primary mediator of soft tissue biomechanics, and variations in its hierarchical structure convey the varying amounts of structural support necessary for organs to function normally. Here we have examined the structural response of corneal collagen to tensile load using X-rays to probe hierarchies ranging from molecular to fibrillar. We found a previously unreported deformation mechanism whereby molecules, which are helically arranged relative to the axis of their fibril, change in tilt akin to the manner in which a spring stretches. This "spring-like" mechanism accounts for a significant portion of the applied deformation at low strains (<3%). These findings will inform the future design of collagen-based artificial corneas being developed to address world-wide shortages of corneal donor tissue. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Cytochemical and immunocytochemical characterization of a fibrillar network (GP2) in pancreatic juice: possible role as a sieve in the pancreatic ductal system.

PubMed

Grondin, G; St-Jean, P; Beaudoin, A R

1992-04-01

The secretory product of the exocrine pancreas contains sedimentable and non-sedimentable materials. Electron microscopy of the pellet obtained after ultracentrifugation reveals two major components: microvesicles (pancreasomes) and a fibrillar network of small mesh size. Negative staining of an unfixed pellet demonstrated that these structures are not fixation artifacts. Cytochemical analysis showed that pancreasomes are reactive to osmication and uranyl acetate staining, whereas the fibrillar network was unreactive thereby indicating that the latter does not contain lipids; however, lead citrate staining reveals the network. Alcian blue, known to bind sulfate groups of mucosubstances, reacted strongly with the fibrillar network. The pellet was also characterized by immunocytochemistry with specific antibodies to amylase and glycoprotein 2 (GP2). Both antibodies were located only on the fibrillar network. Washing of the pellet with 100 mM KCl-250 mM NaBr had little effect on GP2 content, but reduced considerably alpha-amylase associated with the reticular matrix. It appeared that GP2 was the major component of the scaffolding that gives rise to the fibrillar network and that other proteins such as alpha-amylase could reversibly bind to it. When double-labeling immunocytochemistry was carried out on the unwashed pellet, labeling of the first antigen reduced the labeling of the second. Removal of amylase by washing the pellet increased the GP2 signal. These results indicate that amylase is bound on the GP2 network. Although the function of the GP2 network is still not clearly defined several possibilities could be envisaged at the level of the pancreatic duct system: 1) The network could drain off any aggregates or precipitates forming in small ducts. 2) The small mesh of the network would present a physical barrier to infecting bacteria that could enter into the duct system from the intestine, especially in conditions of low flow rates. 3) The network may exert a mechanical pressure on the membranes bordering the acinar lumen and small ducts thereby preventing their collapse in basal conditions.

β-sheet propensity controls the kinetic pathways and morphologies of seeded peptide aggregation

NASA Astrophysics Data System (ADS)

Morriss-Andrews, Alex; Bellesia, Giovanni; Shea, Joan-Emma

2012-10-01

The effect of seeds in templating the morphology of peptide aggregates is examined using molecular dynamics simulations and a coarse-grained peptide representation. Varying the nature of the aggregate seed between β-sheet, amorphous, and β-barrel seeds leads to different aggregation pathways and to morphologically different aggregates. Similar effects are seen by varying the β-sheet propensity of the free peptides. For a fibrillar seed and free peptides of high β-sheet propensity, fibrillar growth occurred by means of direct attachment (without structural rearrangement) of free individual peptides and small ordered oligomers onto the seed. For a fibrillar seed and free peptides of low β-sheet propensity, fibrillar growth occurred through a dock-lock mechanism, in which the free peptides first docked onto the seed, and then locked on, extending and aligning to join the fibril. Amorphous seeds absorbed free peptides into themselves indiscriminately, with any fibrillar rearrangement subsequent to this absorption by means of a condensation-ordering transition. Although the mechanisms observed by varying peptide β-sheet propensity are diverse, the initial pathways can always be broken down into the following steps: (i) the free peptides diffuse in the bulk and attach individually to the seed; (ii) the free peptides diffuse and aggregate among themselves; (iii) the free peptide oligomers collide with the seed; and (iv) the free oligomers merge with the seed and rearrange in a manner dependent on the backbone flexibility of both the free and seed peptides. Our simulations indicate that it is possible to sequester peptides from amorphous aggregates into fibrils, and also that aggregate morphology (and thus cytoxicity) can be controlled by introducing seeds of aggregate-compatible peptides with differing β-sheet propensities into the system.

Isolation of nucleoli from Ehrlich ascites tumor cells and dynamics of nascent RNA within isolated nucleoli.

PubMed

Thiry, Marc; Ploton, Dominique

2008-01-01

Here we describe a new, rapid method for isolating nucleoli from Ehrlich tumor cells that preserves their morphological integrity and high transcriptional activity. Until now, methods for isolation of nucleoli were generally assumed to empty one of their three main compartments, the fibrillar center, of its contents. This new method consists of sonicating cells in an isotonic medium containing MgSO(4), spermidine, and spermine, followed by separation of nucleoli through a Percoll density gradient. Using the nonisotopic approach of labelling with BrUTP, we have further investigated the dynamics of nascent ribosomal RNAs (rRNAs) within morphologically intact isolated nucleoli at the electron microscope level. We show that ribosomal transcripts are elongated in the cortex of the fibrillar center and then enter the surrounding dense fibrillar component.

A novel strategy towards designing a CdSe quantum dot-metallohydrogel composite material.

PubMed

Chatterjee, Sayantan; Maitra, Uday

2016-08-11

We have described here an efficient method to disperse hydrophobic CdSe quantum dots (QDs) in an aqueous phase using cetyltrimethylammonium bromide (CTAB) micelles without any surface ligand exchange. The water soluble QDs were then embedded in 3D self assembled fibrillar networks (SAFINs) of a hydrogel showing homogeneous dispersibility as evidenced from optical and electron microscopic techniques. The photophysical studies of the hydrogel-QD composite are reported for the first time. These composite materials may have potential applications in biology, optoelectronics, sensors, non-linear optics and materials science.

Mechanics of a fiber network within a non-fibrillar matrix: model and comparison with collagen-agarose co-gels

PubMed Central

Lake, Spencer P.; Hadi, Mohammad F.; Lai, Victor K.; Barocas, Victor H.

2013-01-01

While collagen is recognized as the predominant mechanical component of soft connective tissues, the role of the non-fibrillar matrix (NFM) is less well understood. Even model systems, such as the collagen-agarose co-gel, can exhibit complex behavior, making it difficult to identify relative contributions of specific tissue constituents. In the present study, we developed a two-component microscale model of collagen-agarose tissue analogs and used it to elucidate the interaction between collagen and NFM in uniaxial tension. Collagen fibers were represented with Voronoi networks, and the NFM was modeled as a neo-Hookean solid. Model predictions of total normal stress and Poisson’s ratio matched experimental observations well (including high Poisson’s values of ~3), and the addition of NFM led to composition-dependent decreases in volume change and increases in fiber stretch. Because the NFM was more resistant to volume change than the fiber network, extension of the composite led to pressurization of the NFM. Within a specific range of parameter values (low shear modulus and moderate Poisson’s ratio), the magnitude of the reaction force decreased relative to this pressurization component resulting in a negative (compressive) NFM stress in the loading direction, even though the composite tissue was in tension. PMID:22565816

Monomeric and fibrillar α-synuclein exert opposite effects on the catalytic cycle that promotes the proliferation of Aβ42 aggregates

PubMed Central

Chia, Sean; Habchi, Johnny; Lattanzi, Veronica; Dobson, Christopher M.; Knowles, Tuomas P. J.; Vendruscolo, Michele

2017-01-01

The coaggregation of the amyloid-β peptide (Aβ) and α-synuclein is commonly observed in a range of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. The complex interplay between Aβ and α-synuclein has led to seemingly contradictory results on whether α-synuclein promotes or inhibits Aβ aggregation. Here, we show how these conflicts can be rationalized and resolved by demonstrating that different structural forms of α-synuclein exert different effects on Aβ aggregation. Our results demonstrate that whereas monomeric α-synuclein blocks the autocatalytic proliferation of Aβ42 (the 42-residue form of Aβ) fibrils, fibrillar α-synuclein catalyses the heterogeneous nucleation of Aβ42 aggregates. It is thus the specific balance between the concentrations of monomeric and fibrillar α-synuclein that determines the outcome of the Aβ42 aggregation reaction. PMID:28698377

Primary structure and subcellular localization of two fimbrial subunit-like proteins involved in the biosynthesis of K99 fibrillae.

PubMed

Roosendaal, E; Jacobs, A A; Rathman, P; Sondermeyer, C; Stegehuis, F; Oudega, B; de Graaf, F K

1987-09-01

Analysis of the nucleotide sequence of the distal part of the fan gene cluster encoding the proteins involved in the biosynthesis of the fibrillar adhesin, K99, revealed the presence of two structural genes, fanG and fanH. The amino acid sequence of the gene products (FanG and FanH) showed significant homology to the amino acid sequence of the fibrillar subunit protein (FanC). Introduction of a site-specific frameshift mutation in fanG or fanH resulted in a simultaneous decrease in fibrillae production and adhesive capacity. Analysis of subcellular fractions showed that, in contrast to the K99 fibrillar subunit (FanC), both the FanH and the FanG protein were loosely associated with the outer membrane, possibly on the periplasmic side, but were not components of the fimbriae themselves.

Fibrillar films obtained from sodium soap fibers and polyelectrolyte multilayers.

PubMed

Zawko, Scott A; Schmidt, Christine E

2011-08-01

An objective of tissue engineering is to create synthetic polymer scaffolds with a fibrillar microstructure similar to the extracellular matrix. Here, we present a novel method for creating polymer fibers using the layer-by-layer method and sacrificial templates composed of sodium soap fibers. Soap fibers were prepared from neutralized fatty acids using a sodium chloride crystal dissolution method. Polyelectrolyte multilayers (PEMs) of polystyrene sulfonate and polyallylamine hydrochloride were deposited onto the soap fibers, crosslinked with glutaraldehyde, and then the soap fibers were leached with warm water and ethanol. The morphology of the resulting PEM structures was a dense network of fibers surrounded by a nonfibrillar matrix. Microscopy revealed that the PEM fibers were solid structures, presumably composed of polyelectrolytes complexed with residual fatty acids. These fibrillar PEM films were found to support the attachment of human dermal fibroblasts. Copyright © 2011 Wiley Periodicals, Inc.

Protein folding pathology in domestic animals*

PubMed Central

Gruys, Erik

2004-01-01

Fibrillar proteins form structural elements of cells and the extracellular matrix. Pathological lesions of fibrillar microanatomical structures, or secondary fibrillar changes in globular proteins are well known. A special group concerns histologically amorphous deposits, amyloid. The major characteristics of amyloid are: apple green birefringence after Congo red staining of histological sections, and non-branching 7–10 nm thick fibrils on electron microscopy revealing a high content of cross beta pleated sheets. About 25 different types of amyloid have been characterised. In animals, AA-amyloid is the most frequent type. Other types of amyloid in animals represent: AIAPP (in cats), AApoAI, AApoAII, localised AL-amyloid, amyloid in odontogenic or mammary tumors and amyloid in the brain. In old dogs Aβ and in sheep APrPsc-amyloid can be encountered. AA-amyloidosis is a systemic disorder with a precursor in blood, acute phase serum amyloid A (SAA). In chronic inflammatory processes AA-amyloid can be deposited. A rapid crystallization of SAA to amyloid fibrils on small beta-sheeted fragments, the ‘amyloid enhancing factor’ (AEF), is known and the AEF has been shown to penetrate the enteric barrier. Amyloid fibrils can aggregate from various precursor proteins in vitro in particular at acidic pH and when proteolytic fragments are formed. Molecular chaperones influence this process. Tissue data point to amyloid fibrillogenesis in lysosomes and near cell surfaces. A comparison can be made of the fibrillogenesis in prion diseases and in enhanced AA-amyloidosis. In the reactive form, acute phase SAA is the supply of the precursor protein, whereas in the prion diseases, cell membrane proteins form a structural source. Aβ-amyloid in brain tissue of aged dogs showing signs of dementia forms a canine counterpart of senile dementia of the Alzheimer type (ccSDAT) in man. Misfolded proteins remain potential food hazards. Developments concerning prevention of amyloidogenesis and therapy of amyloid deposits are shortly commented. PMID:15362194

Nonlinear laser scanning microscopy of human vocal folds.

PubMed

Miri, Amir K; Tripathy, Umakanta; Mongeau, Luc; Wiseman, Paul W

2012-02-01

The purpose of this work was to apply nonlinear laser scanning microscopy (NLSM) for visualizing the morphology of extracellular matrix proteins within human vocal folds. This technique may potentially assist clinicians in making rapid diagnoses of vocal fold tissue disease or damage. Microstructural characterization based on NLSM provides valuable information for better understanding molecular mechanisms and tissue structure. Experimental, ex vivo human vocal fold. A custom-built multimodal nonlinear laser scanning microscope was used to scan fibrillar proteins in three 4% formaldehyde-fixed cadaveric samples. Collagen and elastin, key extracellular matrix proteins in the vocal fold lamina propria, were imaged by two nonlinear microscopy modalities: second harmonic generation (SHG) and two-photon fluorescence (TPF), respectively. An experimental protocol was introduced to characterize the geometrical properties of the imaged fibrous proteins. NLSM revealed the biomorphology of the human vocal fold fibrous proteins. No photobleaching was observed for the incident laser power of ∼60 mW before the excitation objective. Types I and III fibrillar collagen were imaged without label in the tissue by intrinsic SHG. Imaging while rotating the incident laser light-polarization direction confirmed a helical shape for the collagen fibers. The amplitude, periodicity, and overall orientation were then computed for the helically distributed collagen network. The elastin network was simultaneously imaged via TPF and found to have a basket-like structure. In some regions, particularly close to the epithelium, colocalization of both extracellular matrix components were observed. A benchmark study is presented for quantitative real-time, ex vivo, NLSM imaging of the extracellular macromolecules in human vocal fold lamina propria. The results are promising for clinical applications. Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.

Hierarchical Formation of Fibrillar and Lamellar Self-Assemblies from Guanosine-Based Motifs

PubMed Central

Neviani, Paolo; Sarazin, Dominique; Schmutz, Marc; Blanck, Christian; Giuseppone, Nicolas; Spada, Gian Piero

2010-01-01

Here we investigate the supramolecular polymerizations of two lipophilic guanosine derivatives in chloroform by light scattering technique and TEM experiments. The obtained data reveal the presence of several levels of organization due to the hierarchical self-assembly of the guanosine units in ribbons that in turn aggregate in fibrillar or lamellar soft structures. The elucidation of these structures furnishes an explanation to the physical behaviour of guanosine units which display organogelator properties. PMID:20798860

Inverted formin 2 in focal adhesions promotes dorsal stress fiber and fibrillar adhesion formation to drive extracellular matrix assembly

PubMed Central

Skau, Colleen T.; Plotnikov, Sergey V.; Doyle, Andrew D.; Waterman, Clare M.

2015-01-01

Actin filaments and integrin-based focal adhesions (FAs) form integrated systems that mediate dynamic cell interactions with their environment or other cells during migration, the immune response, and tissue morphogenesis. How adhesion-associated actin structures obtain their functional specificity is unclear. Here we show that the formin-family actin nucleator, inverted formin 2 (INF2), localizes specifically to FAs and dorsal stress fibers (SFs) in fibroblasts. High-resolution fluorescence microscopy and manipulation of INF2 levels in cells indicate that INF2 plays a critical role at the SF–FA junction by promoting actin polymerization via free barbed end generation and centripetal elongation of an FA-associated actin bundle to form dorsal SF. INF2 assembles into FAs during maturation rather than during their initial generation, and once there, acts to promote rapid FA elongation and maturation into tensin-containing fibrillar FAs in the cell center. We show that INF2 is required for fibroblasts to organize fibronectin into matrix fibers and ultimately 3D matrices. Collectively our results indicate an important role for the formin INF2 in specifying the function of fibrillar FAs through its ability to generate dorsal SFs. Thus, dorsal SFs and fibrillar FAs form a specific class of integrated adhesion-associated actin structure in fibroblasts that mediates generation and remodeling of ECM. PMID:25918420

Numerical simulation of electrically stimulated osteogenesis in dental implants.

PubMed

Vanegas-Acosta, J C; Garzón-Alvarado, D A; Lancellotti, V

2014-04-01

Cell behavior and tissue formation are influenced by a static electric field (EF). Several protocols for EF exposure are aimed at increasing the rate of tissue recovery and reducing the healing times in wounds. However, the underlying mechanisms of the EF action on cells and tissues are still a matter of research. In this work we introduce a mathematical model for electrically stimulated osteogenesis at the bone-dental implant interface. The model describes the influence of the EF in the most critical biological processes leading to bone formation at the bone-dental implant interface. The numerical solution is able to reproduce the distribution of spatial-temporal patterns describing the influence of EF during blood clotting, osteogenic cell migration, granulation tissue formation, displacements of the fibrillar matrix, and formation of new bone. In addition, the model describes the EF-mediated cell behavior and tissue formation which lead to an increased osteogenesis in both smooth and rough implant surfaces. Since numerical results compare favorably with experimental evidence, the model can be used to predict the outcome of using electrostimulation in other types of wounds and tissues. Copyright © 2013 Elsevier B.V. All rights reserved.

Morphological assessment of bone mineralization in tibial metaphyses of ascorbic acid-deficient ODS rats.

PubMed

Hasegawa, Tomoka; Li, Minqi; Hara, Kuniko; Sasaki, Muneteru; Tabata, Chihiro; de Freitas, Paulo Henrique Luiz; Hongo, Hiromi; Suzuki, Reiko; Kobayashi, Masatoshi; Inoue, Kiichiro; Yamamoto, Tsuneyuki; Oohata, Noboru; Oda, Kimimitsu; Akiyama, Yasuhiro; Amizuka, Norio

2011-08-01

Osteogenic disorder shionogi (ODS) rats carry a hereditary defect in ascorbic acid synthesis, mimicking human scurvy when fed with an ascorbic acid-deficient (aa-def) diet. As aa-def ODS rats were shown to feature disordered bone formation, we have examined the bone mineralization in this rat model. A fibrous tissue layer surrounding the trabeculae of tibial metaphyses was found in aa-def ODS rats, and this layer showed intense alkaline phosphatase activity and proliferating cell nuclear antigen-immunopositivity. Many osteoblasts detached from the bone surfaces and were characterized by round-shaped rough endoplasmic reticulum (rER), suggesting accumulation of malformed collagen inside the rER. Accordingly, fine, fragile fibrillar collagenous structures without evident striation were found in aa-def bones, which may result from misassembling of the triple helices of collagenous α-chains. Despite a marked reduction in bone formation, ascorbic acid deprivation seemed to have no effect on mineralization: while reduced in number, normal matrix vesicles and mineralized nodules could be seen in aa-def bones. Fine needle-like mineral crystals extended from these mineralized nodules, and were apparently bound to collagenous fibrillar structures. In summary, collagen mineralization seems unaffected by ascorbic acid deficiency in spite of the fine, fragile collagenous fibrils identified in the bones of our animal model.

Development of 3D printed fibrillar collagen scaffold for tissue engineering.

PubMed

Nocera, Aden Díaz; Comín, Romina; Salvatierra, Nancy Alicia; Cid, Mariana Paula

2018-02-27

Collagen is widely used in tissue engineering because it can be extracted in large quantities, and has excellent biocompatibility, good biodegradability, and weak antigenicity. In the present study, we isolated printable collagen from bovine Achilles tendon and examined the purity of the isolated collagen using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The bands obtained corresponded to α 1 , α 2 and β chains with little contamination from other small proteins. Furthermore, rheological measurements of collagen dispersions (60 mg per ml of PBS) at pH 7 revealed values of viscosity of 35.62 ± 1.42 Pa s at shear rate of 10 s - 1 and a shear thinning behavior. Collagen gels and solutions can be used for building scaffolds by three-dimensional (3D) printing. After designing and fabricating a low-cost 3D printer we assayed the collagen printing and obtaining 3D printed scaffolds of collagen at pH 7. The porosity of the scaffold was 90.22% ± 0.88% and the swelling ratio was 1437% ± 146%. The microstructure of the scaffolds was studied using scanning electron microscopy, and a porous mesh of fibrillar collagen was observed. In addition, the 3D printed collagen scaffold was not cytotoxic with cell viability higher than 70% using Vero and NIH 3 T3 cells. In vitro evaluation using both cells lines demonstrated that the collagen scaffolds had the ability to support cell attachment and proliferation. Also a fibrillar collagen mesh was observed after two weeks of culture at 37 °C. Overall, these results are promising since they show the capability of the presented protocol to obtain printable fibrillar collagen at pH 7 and the potential of the printing technique for building low-cost biocompatible 3D plotted structures which maintained the fibrillar collagen structure after incubation in culture media without using additional strategies as crosslinking.

Physical stress and bacterial colonization

PubMed Central

Otto, Michael

2014-01-01

Bacterial surface colonizers are subject to a variety of physical stresses. During the colonization of human epithelia such as on the skin or the intestinal mucosa, bacteria mainly have to withstand the mechanical stress of being removed by fluid flow, scraping, or epithelial turnover. To that end, they express a series of molecules to establish firm attachment to the epithelial surface, such as fibrillar protrusions (pili) and surface-anchored proteins that bind to human matrix proteins. In addition, some bacteria – in particular gut and urinary tract pathogens – use internalization by epithelial cells and other methods such as directed inhibition of epithelial turnover to ascertain continued association with the epithelial layer. Furthermore, many bacteria produce multi-layered agglomerations called biofilms with a sticky extracellular matrix, providing additional protection from removal. This review will give an overview over the mechanisms human bacterial colonizers have to withstand physical stresses with a focus on bacterial adhesion. PMID:25212723

Composite Pillars with a Tunable Interface for Adhesion to Rough Substrates

PubMed Central

2016-01-01

The benefits of synthetic fibrillar dry adhesives for temporary and reversible attachment to hard objects with smooth surfaces have been successfully demonstrated in previous studies. However, surface roughness induces a dramatic reduction in pull-off stresses and necessarily requires revised design concepts. Toward this aim, we introduce cylindrical two-phase single pillars, which are composed of a mechanically stiff stalk and a soft tip layer. Adhesion to smooth and rough substrates is shown to exceed that of conventional pillar structures. The adhesion characteristics can be tuned by varying the thickness of the soft tip layer, the ratio of the Young’s moduli and the curvature of the interface between the two phases. For rough substrates, adhesion values similar to those obtained on smooth substrates were achieved. Our concept of composite pillars overcomes current practical limitations caused by surface roughness and opens up fields of application where roughness is omnipresent. PMID:27997118

Collagen cross-linking: insights on the evolution of metazoan extracellular matrix.

PubMed

Rodriguez-Pascual, Fernando; Slatter, David Anthony

2016-11-23

Collagens constitute a large family of extracellular matrix (ECM) proteins that play a fundamental role in supporting the structure of various tissues in multicellular animals. The mechanical strength of fibrillar collagens is highly dependent on the formation of covalent cross-links between individual fibrils, a process initiated by the enzymatic action of members of the lysyl oxidase (LOX) family. Fibrillar collagens are present in a wide variety of animals, therefore often being associated with metazoan evolution, where the emergence of an ancestral collagen chain has been proposed to lead to the formation of different clades. While LOX-generated collagen cross-linking metabolites have been detected in different metazoan families, there is limited information about when and how collagen acquired this particular modification. By analyzing telopeptide and helical sequences, we identified highly conserved, potential cross-linking sites throughout the metazoan tree of life. Based on this analysis, we propose that they have importantly contributed to the formation and further expansion of fibrillar collagens.

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level.

PubMed

Thiry, M; Scheer, U; Goessens, G

1991-01-01

Nucleoli are the morphological expression of the activity of a defined set of chromosomal segments bearing rRNA genes. The topological distribution and composition of the intranucleolar chromatin as well as the definition of nucleolar structures in which enzymes of the rDNA transcription machinery reside have been investigated in mammalian cells by various immunogold labelling approaches at the ultrastructural level. The precise intranucleolar location of rRNA genes has been further specified by electron microscopic in situ hybridization with a non-autoradiographic procedure. Our results indicate that the fibrillar centers are the sole nucleolar structures where rDNA, core histones, RNA polymerase I and DNA topoisomerase I are located together. Taking into account the potential value and limitations of immunoelectron microscopic techniques, we propose that transcription of the rRNA genes takes place within the confines of the fibrillar centers, probably close to the boundary regions to the surrounding dense fibrillar component.

Osteoblast mineralization requires β1 integrin/ICAP-1–dependent fibronectin deposition

PubMed Central

Brunner, Molly; Millon-Frémillon, Angélique; Chevalier, Genevieve; Nakchbandi, Inaam A.; Mosher, Deane; Block, Marc R.

2011-01-01

The morphogenetic and differentiation events required for bone formation are orchestrated by diffusible and insoluble factors that are localized within the extracellular matrix. In mice, the deletion of ICAP-1, a modulator of β1 integrin activation, leads to severe defects in osteoblast proliferation, differentiation, and mineralization and to a delay in bone formation. Deposition of fibronectin and maturation of fibrillar adhesions, adhesive structures that accompany fibronectin deposition, are impaired upon ICAP-1 loss, as are type I collagen deposition and mineralization. Expression of β1 integrin with a mutated binding site for ICAP-1 recapitulates the ICAP-1–null phenotype. Follow-up experiments demonstrated that ICAP-1 negatively regulates kindlin-2 recruitment onto the β1 integrin cytoplasmic domain, whereas an excess of kindlin-2 binding has a deleterious effect on fibrillar adhesion formation. These results suggest that ICAP-1 works in concert with kindlin-2 to control the dynamics of β1 integrin–containing fibrillar adhesions and, thereby, regulates fibronectin deposition and osteoblast mineralization. PMID:21768292

Fibrillar disruption by AC electric field induced oscillation: A case study with human serum albumin.

PubMed

Sen, Shubhatam; Chakraborty, Monojit; Goley, Snigdha; Dasgupta, Swagata; DasGupta, Sunando

2017-07-01

The effect of oscillation induced by a frequency-dependent alternating current (AC) electric field to dissociate preformed amyloid fibrils has been investigated. An electrowetting-on-dielectric type setup has been used to apply the AC field of varying frequencies on preformed fibrils of human serum albumin (HSA). The disintegration potency has been monitored by a combination of spectroscopic and microscopic techniques. The experimental results suggest that the frequency of the applied AC field plays a crucial role in the disruption of preformed HSA fibrils. The extent of stress generated inside the droplet due to the application of the AC field at different frequencies has been monitored as a function of the input frequency of the applied AC voltage. This has been accomplished by assessing the morphology deformation of the oscillating HSA fibril droplets. The shape deformation of the oscillating droplets is characterized using image analysis by measuring the dynamic changes in the shape dependent parameters such as contact angle and droplet footprint radius and the amplitude. It is suggested that the cumulative effects of the stress generated inside the HSA fibril droplets due to the shape deformation induced hydrodynamic flows and the torque induced by the intrinsic electric dipoles of protein due to their continuous periodic realignment in presence of the AC electric field results in the destruction of the fibrillar species. Copyright © 2017. Published by Elsevier B.V.

Proteoglycan: collagen interactions in connective tissues. Ultrastructural, biochemical, functional and evolutionary aspects.

PubMed

Scott, J E

1991-06-01

Electron histochemical investigations of mammalian and echinoderm tissues, using cupromeronic blue to stain proteoglycans (PGs) specifically in critical electrolyte concentration methods, showed that collagen fibrils are associated with keratan sulphate and chondroitin (dermatan) sulphate ('tadpole') PGs at the a, c, d and e bands on the fibril surface, giving rise to the 'one proteoglycan: one binding site' hypothesis. Intra-fibrillar PGs have been observed, distributed in a regular way which suggests that collagen fibrils are aggregates of 'protofibrils', some of which carry PGs at their surfaces. A scheme for remodelling of collagen fibrils, based on recycling of these protofibrils, is outlined. The choice of which tadpole PG to use to carry out a given function is decided to a considerable extent by the availability of oxygen to the relevant tissue element.

Modeling of chemical inhibition from amyloid protein aggregation kinetics.

PubMed

Vázquez, José Antonio

2014-02-27

The process of amyloid proteins aggregation causes several human neuropathologies. In some cases, e.g. fibrillar deposits of insulin, the problems are generated in the processes of production and purification of protein and in the pump devices or injectable preparations for diabetics. Experimental kinetics and adequate modelling of chemical inhibition from amyloid aggregation are of practical importance in order to study the viable processing, formulation and storage as well as to predict and optimize the best conditions to reduce the effect of protein nucleation. In this manuscript, experimental data of insulin, Aβ42 amyloid protein and apomyoglobin fibrillation from recent bibliography were selected to evaluate the capability of a bivariate sigmoid equation to model them. The mathematical functions (logistic combined with Weibull equation) were used in reparameterized form and the effect of inhibitor concentrations on kinetic parameters from logistic equation were perfectly defined and explained. The surfaces of data were accurately described by proposed model and the presented analysis characterized the inhibitory influence on the protein aggregation by several chemicals. Discrimination between true and apparent inhibitors was also confirmed by the bivariate equation. EGCG for insulin (working at pH = 7.4/T = 37°C) and taiwaniaflavone for Aβ42 were the compounds studied that shown the greatest inhibition capacity. An accurate, simple and effective model to investigate the inhibition of chemicals on amyloid protein aggregation has been developed. The equation could be useful for the clear quantification of inhibitor potential of chemicals and rigorous comparison among them.

[Ultrastructure of nephridial systems in cyclophyllidean cestoda: Catenotaenia pusilla (Goeze, 1782), Hymenolepis diminuta (Rudolphi, 1819) and Inermicapsifer madagascariensis (Davaine, 1870) Baer, 1956].

PubMed

Swiderski, Z; Euzet, L; Schönenberger, N

1975-01-01

Electron microscopic study of nephridial systems in three cyclophyllidean cestodes indicates a resemblance in their ultrastructure. The walls of longitudinal, transverse and collecting ducts show a very similar pattern of organization. The surface of the anucleate epithelium lining the ducts is developed into microvilli. A relatively thick layer of fibrillar tissue underlies the basal membrane of the microvillar epithelium. The nucleated portions or "pericaryons", situated between the parenchymal cells, are directly connected with epithelium by cytoplasmic prolongations. The canalicular lumen extends through a single series of cells curved into a ring. The epithelial surface of the canalicular wall is developed into short, densly staining microvilli and the immediately underlying fibrillar tissue appears very compact. The cilia were never observed in any of the above ducts. The ultrastructure of protonephridia proper is comparable with those already described in other cestodes. There is a close association between the flame-cell and the cancalicular ending, enlarged into a nephridial funnel. A single row of nephridial rods of the flame-cell is surrounded by a row of digitiform prolongations of the nephridial funnel border. The prolongations alternate with the rods and their interlocking pattern appears clearly in cross-sections. A series of minute pores or "nephrostomes" providing a direct contact between the nephridial chamber and intercellular space of the paranchyma was shown. The problem of classification and definition between the "closed" protonephridia and open metanephridia is discussed. The structural unity of protonephridia in different groupes of Platyhelminthes is reviewed. The different number of flagella within the "flames" of different cestodes is compared and analyzed. The ultrastructural characteristics of duct-wall epithelium provides some confirmation of its high metabolic activity.

Surface contact and design of fibrillar ‘friction pads’ in stick insects (Carausius morosus): mechanisms for large friction coefficients and negligible adhesion

PubMed Central

Labonte, David; Williams, John A.; Federle, Walter

2014-01-01

Many stick insects and mantophasmids possess tarsal ‘heel pads’ (euplantulae) covered by arrays of conical, micrometre-sized hairs (acanthae). These pads are used mainly under compression; they respond to load with increasing shear resistance, and show negligible adhesion. Reflected-light microscopy in stick insects (Carausius morosus) revealed that the contact area of ‘heel pads’ changes with normal load on three hierarchical levels. First, loading brought larger areas of the convex pads into contact. Second, loading increased the density of acanthae in contact. Third, higher loads changed the shape of individual hair contacts gradually from circular (tip contact) to elongated (side contact). The resulting increase in real contact area can explain the load dependence of friction, indicating a constant shear stress between acanthae and substrate. As the euplantula contact area is negligible for small loads (similar to hard materials), but increases sharply with load (resembling soft materials), these pads show high friction coefficients despite little adhesion. This property appears essential for the pads’ use in locomotion. Several morphological characteristics of hairy friction pads are in apparent contrast to hairy pads used for adhesion, highlighting key adaptations for both pad types. Our results are relevant for the design of fibrillar structures with high friction coefficients but small adhesion. PMID:24554580

Inhibition of Alzheimer’s Amyloid Toxicity with a Tricyclic Pyrone Molecule In Vitro and In Vivo

PubMed Central

Hong, Hyun-Seok; Rana, Sandeep; Barrigan, Lydia; Shi, Aibin; Zhang, Yi; Zhou, Feimeng; Jin, Lee-Way; Hua, Duy H.

2009-01-01

Small amyloid β 1–42 aggregates are toxic to neurons and may be the primary toxic species in Alzheimer’s disease (AD). Methods to reduce the level of Aβ, prevent Aβ aggregation, and eliminate existing Aβ aggregates have been proposed for treatment of AD. A tricyclic pyrone named CP2 is found to prevent cell death associated with Aβ oligomers. We studied the possible mechanisms of neuroprotection by CP2. Surface plasmon resonance spectroscopy shows a direct binding of CP2 with Aβ42 oligomer. Circular dichroism spectroscopy reveals monomeric Aβ42 peptide remains as a random coil/α-helix structure in the presence of CP2 over 48 h. Atomic force microscopy (AFM) studies show CP2 exhibits similar ability to inhibit Aβ42 aggregation as that of Congo Red and curcumin. AFM closed-fluid cell study demonstrates that CP2 disaggregates Aβ42 oligomers and protofibrils. CP2 also blocks Aβ fibrillations using a protein quantification method. Treatment of 5x FAD mice, a robust Aβ42-producing animal model of AD, with a two-week course of CP2 resulted in 40% and 50% decreases in non-fibrillar and fibrillar Aβ species, respectively. Our results suggest that CP2 might be beneficial to AD patients by preventing Aβ aggregation and disaggregating existing Aβ oligomers and protofibrils. PMID:19141069

[The 3-dimensional organization of the nucleolus and nucleolus-organizer regions of differentiated cells. IV. The structural and functional heterogeneity of the nucleoli in the epithelium of the proximal nephron in the mouse].

PubMed

Chelidze, P V; Dzidziguri, D V; Zarandiia, M A; Georgobiani, N M; Tumanishvili, G D

1993-01-01

By means of stereological and morphometrical analysis, the ultrastructure of nucleoli in epitheliocytes of mouse kidney cortex proximal tubuli has been studied. In accordance to the nucleolar composition, three main groups of nephrocytes with different levels of rRNA and protein synthesis were defined. Functional heterogeneity of proximal tubuli epithelium was established by correlation between different variants of ultrastructural organization of nucleoli and the total RNA synthesis activity, determined by 3H-uridine incorporation intensity. It has been shown that a greater part of cells (about 52%) in the nephron proximal section, which is characterized by slow RNA synthesis, causing a low functional activity of these cells, presumably represents a reparative cellular reserve. Such cells, defined as the 1st group cells, have resting, ring-shaped nucleoli with one fibrillar centre, and nucleoli similar to the ring-shaped ones but containing 2-3 fibrillar centres. Nucleoli of the 2nd group of nephrocytes (about 37%), most actively incorporating labeled precursor, contain 4-6 fibrillar centres. Their structural organization is closer to the reticular type of nucleoli. The 3rd most actively labeled group of nephrocytes includes cells with typical reticulated nucleoli. The number of fibrillar centres in the reticulated nucleoli is much higher (18-22) than in the 1st and 2nd groups of nephrocytes. Structural and functional polymorphism of nephrocytes was revealed not only in the proximal part of one nephron. During the increase in functional activity of nephrocytes, caused by unilateral nephrectomy, the quantitative correlation between cells related to these different groups was seen to change. The number of cells of the 1st group decreased by 24%, whereas that in the 2nd and 3rd groups increased by 9 and 15%, respectively. Nucleoli with 2-3 fibrillar centres are considered as transitional forms between the inactive ring-shaped nucleoli and the active reticulated nucleoli. Differences in the ultrastructure of nucleoli may be considered as an evidence of functional heterogeneity of nephrocytes within the proximal segment of nephron.

Improved power for characterizing longitudinal amyloid-β PET changes and evaluating amyloid-modifying treatments with a cerebral white matter reference region.

PubMed

Chen, Kewei; Roontiva, Auttawut; Thiyyagura, Pradeep; Lee, Wendy; Liu, Xiaofen; Ayutyanont, Napatkamon; Protas, Hillary; Luo, Ji Luo; Bauer, Robert; Reschke, Cole; Bandy, Daniel; Koeppe, Robert A; Fleisher, Adam S; Caselli, Richard J; Landau, Susan; Jagust, William J; Weiner, Michael W; Reiman, Eric M

2015-04-01

In this article, we describe an image analysis strategy with improved power for tracking longitudinal amyloid-β (Aβ) PET changes and evaluating Aβ-modifying treatments. Our aims were to compare the power of template-based cerebellar, pontine, and cerebral white matter reference regions to track 24-mo florbetapir standardized uptake value (SUV) ratio (SUVR) changes; to relate those changes to 24-mo clinical declines; and to evaluate Aβ-modifying treatments in Aβ-positive (Aβ+) and Aβ-negative (Aβ-) patients with probable Alzheimer dementia (pAD), in patients with mild cognitive impairment (MCI), in cognitively normal controls (NCs), and in cognitively normal apolipoprotein E4 (APOE4) carriers and noncarriers. We used baseline and follow-up (∼24 mo) florbetapir PET scans from 332 Aβ+ and Aβ- subjects participating in the multicenter Alzheimer's Disease Neuroimaging Initiative. Each of the proposed analyses included 31 pAD patients, 187 MCI patients, and 114 NCs. Cerebral-to-white matter, cerebellar, and pontine SUVRs were characterized in terms of their longitudinal variability; their power to track longitudinal fibrillar Aβ increases in Aβ+ and Aβ- subgroups and cognitively normal APOE4 carriers and noncarriers; the sample sizes needed to detect attenuated accumulation of or clearance of fibrillar Aβ accumulation in randomized clinical trials; and their ability to relate 24-mo fibrillar Aβ increases to clinical declines. As predicted, cerebral-to-white matter SUVR changes were significantly less variable and had significantly greater power to detect 24-mo fibrillar Aβ increases and evaluate Aβ-modifying treatment effects in Aβ+ pAD, MCI, and NC subjects and cognitively normal APOE4 carriers. They were also distinguished by the ability to detect significant associations between 24-mo Aβ increases and clinical declines. A cerebral white matter reference region may improve the power to track longitudinal fibrillar Aβ increases, to characterize their relationship to longitudinal clinical declines, and to evaluate Aβ-modifying treatments in randomized clinical trials. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Dietary DHA supplementation in an APP/PS1 transgenic rat model of AD reduces behavioral and Aβ pathology and modulates Aβ oligomerization.

PubMed

Teng, Edmond; Taylor, Karen; Bilousova, Tina; Weiland, David; Pham, Thaidan; Zuo, Xiaohong; Yang, Fusheng; Chen, Ping-Ping; Glabe, Charles G; Takacs, Alison; Hoffman, Dennis R; Frautschy, Sally A; Cole, Gregory M

2015-10-01

Increased dietary consumption of docosahexaenoic acid (DHA) is associated with decreased risk for Alzheimer's disease (AD). These effects have been postulated to arise from DHA's pleiotropic effects on AD pathophysiology, including its effects on β-amyloid (Aβ) production, aggregation, and toxicity. While in vitro studies suggest that DHA may inhibit and reverse the formation of toxic Aβ oligomers, it remains uncertain whether these mechanisms operate in vivo at the physiological concentrations of DHA attainable through dietary supplementation. We sought to clarify the effects of dietary DHA supplementation on Aβ indices in a transgenic APP/PS1 rat model of AD. Animals maintained on a DHA-supplemented diet exhibited reductions in hippocampal Aβ plaque density and modest improvements on behavioral testing relative to those maintained on a DHA-depleted diet. However, DHA supplementation also increased overall soluble Aβ oligomer levels in the hippocampus. Further quantification of specific conformational populations of Aβ oligomers indicated that DHA supplementation increased fibrillar (i.e. putatively less toxic) Aβ oligomers and decreased prefibrillar (i.e. putatively more toxic) Aβ oligomers. These results provide in vivo evidence suggesting that DHA can modulate Aβ aggregation by stabilizing soluble fibrillar Aβ oligomers and thus reduce the formation of both Aβ plaques and prefibrillar Aβ oligomers. However, since fibrillar Aβ oligomers still retain inherent neurotoxicity, DHA may need to be combined with other interventions that can additionally reduce fibrillar Aβ oligomer levels for more effective prevention of AD in clinical settings. Published by Elsevier Inc.

Zinc-Amyloid Interactions on a Millisecond Time-Scale Stabilize Non-Fibrillar Alzheimer Related Species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Noy,D.; Solomonov, I.; Sinkevich, O.

2008-01-01

The role of zinc, an essential element for normal brain function, in the pathology of Alzheimer's disease (AD) is poorly understood. On one hand, physiological and genetic evidence from transgenic mouse models supports its pathogenic role in promoting the deposition of the amyloid {beta}-protein (A{beta}) in senile plaques. On the other hand, levels of extracellular ('free') zinc in the brain, as inferred by the levels of zinc in cerebrospinal fluid, were found to be too low for inducing A{beta} aggregation. Remarkably, the release of transient high local concentrations of zinc during rapid synaptic events was reported. The role of suchmore » free zinc pulses in promoting A{beta} aggregation has never been established. Using a range of time-resolved structural and spectroscopic techniques, we found that zinc, when introduced in millisecond pulses of micromolar concentrations, immediately interacts with A{beta} 1-40 and promotes its aggregation. These interactions specifically stabilize non-fibrillar pathogenic related aggregate forms and prevent the formation of A{beta} fibrils (more benign species) presumably by interfering with the self-assembly process of A{beta}. These in vitro results strongly suggest a significant role for zinc pulses in A{beta} pathology. We further propose that by interfering with A{beta} self-assembly, which leads to insoluble, non-pathological fibrillar forms, zinc stabilizes transient, harmful amyloid forms. This report argues that zinc represents a class of molecular pathogens that effectively perturb the self-assembly of benign A{beta} fibrils, and stabilize harmful non-fibrillar forms.« less

Changes of the surface structure of corn stalk in the cooking process with active oxygen and MgO-based solid alkali as a pretreatment of its biomass conversion.

PubMed

Pang, Chunsheng; Xie, Tujun; Lin, Lu; Zhuang, Junping; Liu, Ying; Shi, Jianbin; Yang, Qiulin

2012-01-01

This study presents a novel, efficient and environmentally friendly process for the cooking of corn stalk that uses active oxygen (O2 and H2O2) and a recoverable solid alkali (MgO). The structural changes on the surface of corn stalk before and after cooking were characterized by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) techniques. The results showed that lignin and extractives were effectively removed, especially those on the surface of corn stalk. Additionally, the changes included becoming fibrillar, the exposure of cellulose and hemi-cellulose and the pitting corrosion on the surface, etc. The results also showed that the removal reaction is from outside to inside, but the main reaction is possibly on the surface. Furthermore, the results of active oxygen cooking with a solid alkali are compared with those of alkaline cooking in the paper. Copyright © 2011 Elsevier Ltd. All rights reserved.

Controllable load sharing for soft adhesive interfaces on three-dimensional surfaces.

PubMed

Song, Sukho; Drotlef, Dirk-Michael; Majidi, Carmel; Sitti, Metin

2017-05-30

For adhering to three-dimensional (3D) surfaces or objects, current adhesion systems are limited by a fundamental trade-off between 3D surface conformability and high adhesion strength. This limitation arises from the need for a soft, mechanically compliant interface, which enables conformability to nonflat and irregularly shaped surfaces but significantly reduces the interfacial fracture strength. In this work, we overcome this trade-off with an adhesion-based soft-gripping system that exhibits enhanced fracture strength without sacrificing conformability to nonplanar 3D surfaces. Composed of a gecko-inspired elastomeric microfibrillar adhesive membrane supported by a pressure-controlled deformable gripper body, the proposed soft-gripping system controls the bonding strength by changing its internal pressure and exploiting the mechanics of interfacial equal load sharing. The soft adhesion system can use up to ∼26% of the maximum adhesion of the fibrillar membrane, which is 14× higher than the adhering membrane without load sharing. Our proposed load-sharing method suggests a paradigm for soft adhesion-based gripping and transfer-printing systems that achieves area scaling similar to that of a natural gecko footpad.

Controllable load sharing for soft adhesive interfaces on three-dimensional surfaces

NASA Astrophysics Data System (ADS)

Song, Sukho; Drotlef, Dirk-Michael; Majidi, Carmel; Sitti, Metin

2017-05-01

For adhering to three-dimensional (3D) surfaces or objects, current adhesion systems are limited by a fundamental trade-off between 3D surface conformability and high adhesion strength. This limitation arises from the need for a soft, mechanically compliant interface, which enables conformability to nonflat and irregularly shaped surfaces but significantly reduces the interfacial fracture strength. In this work, we overcome this trade-off with an adhesion-based soft-gripping system that exhibits enhanced fracture strength without sacrificing conformability to nonplanar 3D surfaces. Composed of a gecko-inspired elastomeric microfibrillar adhesive membrane supported by a pressure-controlled deformable gripper body, the proposed soft-gripping system controls the bonding strength by changing its internal pressure and exploiting the mechanics of interfacial equal load sharing. The soft adhesion system can use up to ˜26% of the maximum adhesion of the fibrillar membrane, which is 14× higher than the adhering membrane without load sharing. Our proposed load-sharing method suggests a paradigm for soft adhesion-based gripping and transfer-printing systems that achieves area scaling similar to that of a natural gecko footpad.

Controllable load sharing for soft adhesive interfaces on three-dimensional surfaces

PubMed Central

Song, Sukho; Drotlef, Dirk-Michael; Majidi, Carmel; Sitti, Metin

2017-01-01

For adhering to three-dimensional (3D) surfaces or objects, current adhesion systems are limited by a fundamental trade-off between 3D surface conformability and high adhesion strength. This limitation arises from the need for a soft, mechanically compliant interface, which enables conformability to nonflat and irregularly shaped surfaces but significantly reduces the interfacial fracture strength. In this work, we overcome this trade-off with an adhesion-based soft-gripping system that exhibits enhanced fracture strength without sacrificing conformability to nonplanar 3D surfaces. Composed of a gecko-inspired elastomeric microfibrillar adhesive membrane supported by a pressure-controlled deformable gripper body, the proposed soft-gripping system controls the bonding strength by changing its internal pressure and exploiting the mechanics of interfacial equal load sharing. The soft adhesion system can use up to ∼26% of the maximum adhesion of the fibrillar membrane, which is 14× higher than the adhering membrane without load sharing. Our proposed load-sharing method suggests a paradigm for soft adhesion-based gripping and transfer-printing systems that achieves area scaling similar to that of a natural gecko footpad. PMID:28507143

Intrinsic Determinants of Neurotoxic Aggregate Formation by the Amyloid β Peptide

PubMed Central

Brorsson, Ann-Christin; Bolognesi, Benedetta; Tartaglia, Gian Gaetano; Shammas, Sarah L.; Favrin, Giorgio; Watson, Ian; Lomas, David A.; Chiti, Fabrizio; Vendruscolo, Michele; Dobson, Christopher M.; Crowther, Damian C.; Luheshi, Leila M.

2010-01-01

Abstract The extent to which proteins aggregate into distinct structures ranging from prefibrillar oligomers to amyloid fibrils is key to the pathogenesis of many age-related degenerative diseases. We describe here for the Alzheimer's disease-related amyloid β peptide (Aβ) an investigation of the sequence-based determinants of the balance between the formation of prefibrillar aggregates and amyloid fibrils. We show that by introducing single-point mutations, it is possible to convert the normally harmless Aβ40 peptide into a pathogenic species by increasing its relative propensity to form prefibrillar but not fibrillar aggregates, and, conversely, to abolish the pathogenicity of the highly neurotoxic E22G Aβ42 peptide by reducing its relative propensity to form prefibrillar species rather than mature fibrillar ones. This observation can be rationalized by the demonstration that whereas regions of the sequence of high aggregation propensity dominate the overall tendency to aggregate, regions with low intrinsic aggregation propensities exert significant control over the balance of the prefibrillar and fibrillar species formed, and therefore play a major role in determining the neurotoxicity of the Aβ peptide. PMID:20409489

Collagen and the myocardium: fibrillar structure, biosynthesis and degradation in relation to hypertrophy and its regression.

PubMed

Eghbali, M; Weber, K T

1990-07-17

The extracellular matrix of the myocardium contains an elaborate structural matrix composed mainly of fibrillar types I and III collagen. This matrix is responsible for the support and alignment of myocytes and capillaries. Because of its alignment, location, configuration and tensile strength, relative to cardiac myocytes, the collagen matrix represents a major determinant of myocardial stiffness. Cardiac fibroblasts, not myocytes, contain the mRNA for these fibrillar collagens. In the hypertrophic remodeling of the myocardium that accompanies arterial hypertension, a progressive structural and biochemical remodeling of the matrix follows enhanced collagen gene expression. The resultant significant accumulation of collagen in the interstitium and around intramyocardial coronary arteries, or interstitial and perivascular fibrosis, represents a pathologic remodeling of the myocardium that compromises this normally efficient pump. This report reviews the structural nature, biosynthesis and degradation of collagen in the normal and hypertrophied myocardium. It suggests that interstitial heart disease, or the disproportionate growth of the extracellular matrix relative to myocyte hypertrophy, is an entity that merits greater understanding, particularly the factors regulating types I and III collagen gene expression and their degradation.

Amyloid-like aggregation of provasopressin in diabetes insipidus and secretory granule sorting.

PubMed

Beuret, Nicole; Hasler, Franziska; Prescianotto-Baschong, Cristina; Birk, Julia; Rutishauser, Jonas; Spiess, Martin

2017-01-26

Aggregation of peptide hormone precursors in the trans-Golgi network is an essential process in the biogenesis of secretory granules in endocrine cells. It has recently been proposed that this aggregation corresponds to the formation of functional amyloids. Our previous finding that dominant mutations in provasopressin, which cause cell degeneration and diabetes insipidus, prevent native folding and produce fibrillar aggregates in the endoplasmic reticulum (ER) might thus reflect mislocalized amyloid formation by sequences that evolved to mediate granule sorting. Here we identified two sequences responsible for fibrillar aggregation of mutant precursors in the ER: the N-terminal vasopressin nonapeptide and the C-terminal glycopeptide. To test their role in granule sorting, the glycopeptide was deleted and/or vasopressin mutated to inactivate ER aggregation while still permitting precursor folding and ER exit. These mutations strongly reduced sorting into granules and regulated secretion in endocrine AtT20 cells. The same sequences - vasopressin and the glycopeptide - mediate physiological aggregation of the wild-type hormone precursor into secretory granules and the pathological fibrillar aggregation of disease mutants in the ER. These findings support the amyloid hypothesis for secretory granule biogenesis.

Brain propagation of transduced α-synuclein involves non-fibrillar protein species and is enhanced in α-synuclein null mice.

PubMed

Helwig, Michael; Klinkenberg, Michael; Rusconi, Raffaella; Musgrove, Ruth E; Majbour, Nour K; El-Agnaf, Omar M A; Ulusoy, Ayse; Di Monte, Donato A

2016-03-01

Aggregation and neuron-to-neuron transmission are attributes of α-synuclein relevant to its pathogenetic role in human synucleinopathies such as Parkinson's disease. Intraparenchymal injections of fibrillar α-synuclein trigger widespread propagation of amyloidogenic protein species via mechanisms that require expression of endogenous α-synuclein and, possibly, its structural corruption by misfolded conformers acting as pathological seeds. Here we describe another paradigm of long-distance brain diffusion of α-synuclein that involves inter-neuronal transfer of monomeric and/or oligomeric species and is independent of recruitment of the endogenous protein. Targeted expression of human α-synuclein was induced in the mouse medulla oblongata through an injection of viral vectors into the vagus nerve. Enhanced levels of intra-neuronal α-synuclein were sufficient to initiate its caudo-rostral diffusion that likely involved at least one synaptic transfer and progressively reached specific brain regions such as the locus coeruleus, dorsal raphae and amygdala in the pons, midbrain and forebrain. Transfer of human α-synuclein was compared in two separate lines of α-synuclein-deficient mice versus their respective wild-type controls and, interestingly, lack of endogenous α-synuclein expression did not counteract diffusion but actually resulted in a more pronounced and advanced propagation of exogenous α-synuclein. Self-interaction of adjacent molecules of human α-synuclein was detected in both wild-type and mutant mice. In the former, interaction of human α-synuclein with mouse α-synuclein was also observed and might have contributed to differences in protein transmission. In wild-type and α-synuclein-deficient mice, accumulation of human α-synuclein within recipient axons in the pons, midbrain and forebrain caused morphological evidence of neuritic pathology. Tissue sections from the medulla oblongata and pons were stained with different antibodies recognizing oligomeric, fibrillar and/or total (monomeric and aggregated) α-synuclein. Following viral vector transduction, monomeric, oligomeric and fibrillar protein was detected within donor neurons in the medulla oblongata. In contrast, recipient axons in the pons were devoid of immunoreactivity for fibrillar α-synuclein, indicating that non-fibrillar forms of α-synuclein were primarily transferred from one neuron to the other, diffused within the brain and led to initial neuronal injury. This study elucidates a paradigm of α-synuclein propagation that may play a particularly important role under pathophysiological conditions associated with enhanced α-synuclein expression. Rapid long-distance diffusion and accumulation of monomeric and oligomeric α-synuclein does not necessarily involve pathological seeding but could still result in a significant neuronal burden during the pathogenesis of neurodegenerative diseases. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Insight into the Structure of Amyloid Fibrils from the Analysis of Globular Proteins

PubMed Central

Trovato, Antonio; Chiti, Fabrizio; Maritan, Amos; Seno, Flavio

2006-01-01

The conversion from soluble states into cross-β fibrillar aggregates is a property shared by many different proteins and peptides and was hence conjectured to be a generic feature of polypeptide chains. Increasing evidence is now accumulating that such fibrillar assemblies are generally characterized by a parallel in-register alignment of β-strands contributed by distinct protein molecules. Here we assume a universal mechanism is responsible for β-structure formation and deduce sequence-specific interaction energies between pairs of protein fragments from a statistical analysis of the native folds of globular proteins. The derived fragment–fragment interaction was implemented within a novel algorithm, prediction of amyloid structure aggregation (PASTA), to investigate the role of sequence heterogeneity in driving specific aggregation into ordered self-propagating cross-β structures. The algorithm predicts that the parallel in-register arrangement of sequence portions that participate in the fibril cross-β core is favoured in most cases. However, the antiparallel arrangement is correctly discriminated when present in fibrils formed by short peptides. The predictions of the most aggregation-prone portions of initially unfolded polypeptide chains are also in excellent agreement with available experimental observations. These results corroborate the recent hypothesis that the amyloid structure is stabilised by the same physicochemical determinants as those operating in folded proteins. They also suggest that side chain–side chain interaction across neighbouring β-strands is a key determinant of amyloid fibril formation and of their self-propagating ability. PMID:17173479

Fluorescence molecular probes for sensitive point detection of amyloid fibrils and protofibrils

NASA Astrophysics Data System (ADS)

Lindgren, Mikael; Jonsson, Per; Sörgjerd, Karin; Hammarström, Per

2005-10-01

Protein based infections such as prion diseases have lately attracted a large amount of interest, primarily due to the Mad Cow Epidemic in Great Britain, and the increase of Alzheimer's disease and related diseases in the ageing Western society. Infective proteins are very stable and almost untraceable prior to infection making them ideal as biological weapons. Particularly if the used agent is of human origin, the immunoresponse can be avoided, leaving no trace of the infectious agent. The transient nature of infectious oligomeric intermediates of misfolded proteins or peptide fragments that later matures into fibrillar aggregates makes them hard to study, and methods to detect and study these species are sparse. There exist a number of fluorescent probes that bind specifically to protein amyloidic structures. Thioflavins (ThT, ThS), Congo and Nile red, 4-(dicyanovinyl)-julolidine (DCVJ), as well as derivatives amino-8-naphtalene sulphonate (ANS, Bis-ANS) which are known to bind to the fibrillar or pre-fibrillar states with dissociation constants of typically 1 - 20 μM. Here, transthyretin (TTR), insulin and lysozyme were used as model proteins to detect different amyloid precursor states for diseases such as senile systemic amyloidosis, familial amyloidotic polyneuropathy (FAP) and iatrogenic amyloidosis. Specifically, the probes were employed in static assays to characterize protofibrillar and mature amyloid fibrillar states using steady state and time-resolved fluorescence techniques. Particularly, we investigate and report on the possibility to detect protofibrillar states at low concentration levels using modern fluorescence array detector systems in conjunction with lasers operating in the blue or ultraviolett wavelengths as excitation source. Results of ANS, ThT and a ThT analogue (abbreviated ThC) are discussed.

Chitosan bio-based organic-inorganic hybrid aerogel microspheres.

PubMed

El Kadib, Abdelkrim; Bousmina, Mosto

2012-07-02

Recently, organic-inorganic hybrid materials have attracted tremendous attention thanks to their outstanding properties, their efficiency, versatility and their promising applications in a broad range of areas at the interface of chemistry and biology. This article deals with a new family of surface-reactive organic-inorganic hybrid materials built from chitosan microspheres. The gelation of chitosan (a renewable amino carbohydrate obtained by deacetylation of chitin) by pH inversion affords highly dispersed fibrillar networks shaped as self-standing microspheres. Nanocasting of sol-gel processable monomeric alkoxides inside these natural hydrocolloids and their subsequent CO(2) supercritical drying provide high-surface-area organic-inorganic hybrid materials. Examples including chitosan-SiO(2), chitosan-TiO(2), chitosan-redox-clusters and chitosan-clay-aerogel microspheres are described and discussed on the basis of their textural and structural properties, thermal and chemical stability and their performance in catalysis and adsorption. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Viscoelastic properties of pressure overload hypertrophied myocardium: effect of serine protease treatment

NASA Technical Reports Server (NTRS)

Stroud, Jason D.; Baicu, Catalin F.; Barnes, Mary A.; Spinale, Francis G.; Zile, Michael R.

2002-01-01

To determine whether and to what extent one component of the extracellular matrix, fibrillar collagen, contributes causally to abnormalities in viscoelasticity, collagen was acutely degraded by activation of endogenous matrix metalloproteinases (MMPs) with the serine protease plasmin. Papillary muscles were isolated from normal cats and cats with right ventricular pressure overload hypertrophy (POH) induced by pulmonary artery banding. Plasmin treatment caused MMP activation, collagen degradation, decreased the elastic stiffness constant, and decreased the viscosity constant in both normal and POH muscles. Thus, whereas many mechanisms may contribute to the abnormalities in myocardial viscoelasticity in the POH myocardium, changes in fibrillar collagen appear to play a predominant role.

New fluorescent probes for detection and characterization of amyloid fibrils

NASA Astrophysics Data System (ADS)

Gorbenko, Galyna; Trusova, Valeriya; Kirilova, Elena; Kirilov, Georgiy; Kalnina, Inta; Vasilev, Aleksey; Kaloyanova, Stefka; Deligeorgiev, Todor

2010-08-01

The applicability of the novel fluorescent probes, aminoderivative of benzanthrone ABM, squaraine dye SQ-1 and polymethine dye V2 to identification and structural analysis of amyloid fibrils has been evaluated using the lysozyme model system in which fibrillar aggregates have been formed in concentrated ethanol solution. The association constant, binding stoichiometry and molar fluorescence of the bound dye have been determined. ABM was found to surpass classical amyloid marker ThT in the sensitivity to the presence of fibrillar aggregates. Resonance energy transfer measurements involving ABM-SQ-1 and SQ-1-V2 donor-acceptor pairs yielded the limits for fractal-like dimension of lysozyme fibrils.

The echinoderm collagen fibril: a hero in the connective tissue research of the 1990s.

PubMed

Szulgit, Greg

2007-07-01

Collagen fibrils are some of the most-abundant and important extracellular structures in our bodies, yet we are unsure of their shape and size. This is largely due to an inherent difficulty in isolating them from their surrounding tissues. Echinoderms have collagenous tissues that are similar to ours in many ways, yet they can be manipulated to easily relinquish their collagen fibrils, providing an excellent opportunity to study native fibrillar structure. In the early 1990s, they were found to defy the commonly accepted fibrillar model of the time in that they were much shorter, they were shaped like double-ended spindles, and their centers exhibited a reversal in molecular polarity. Realization of these features helped to reform the questions that were being asked about vertebrate fibrils, shifting the focus toward shape and size. Since then, researchers working with both groups (echinoderms and vertebrates) have worked together to find the structure of native fibrils. This information will be fundamental in understanding what holds collagenous tissues together at the fibrillar level, and could have important implications for people with Ehlers-Danlos syndrome. (c) 2007 Wiley Periodicals, Inc.

Investigation of copper(II) binding to the protein precursor of Non-Amyloid-Beta Component of Alzheimer Disease Amyloid Plaque

NASA Astrophysics Data System (ADS)

Rose, Francis; Hodak, Miroslav; Bernholc, Jerry

2007-03-01

The Non-Amyloid-Beta Component Precursor (NACP) is a natively unfolded synaptic protein that is implicated in Alzheimers and Parkinsons diseases. Its aggregation into fibrillar structures is accelerated by the binding of copper(II). Experimental studies suggest that the dominant copper binding site is located at the histidine residue in NACP. Based on this evidence we assembled a model fragment of the binding site and used DFT to analyze the conformational details of the most probable binding motifs. We investigated the overall conformational effects with classical MD by constraining the copper binding site to the most energetically favorable geometry obtained from the DFT calculations. These results are compared and contrasted with those of the unbound NACP.

Breakout character of islet amyloid polypeptide hydrophobic mutations at the onset of type-2 diabetes

NASA Astrophysics Data System (ADS)

Frigori, Rafael B.

2014-11-01

Toxic fibrillar aggregates of islet amyloid polypeptide (IAPP) appear as the physical outcome of a peptidic phase transition signaling the onset of type-2 diabetes mellitus in different mammalian species. In particular, experimentally verified mutations on the amyloidogenic segment 20-29 in humans, cats, and rats are highly correlated with the molecular aggregation propensities. Through a microcanonical analysis of the aggregation of IAPP20 -29 isoforms, we show that a minimalist one-bead hydrophobic-polar continuum model for protein interactions properly quantifies those propensities from free-energy barriers. Our results highlight the central role of sequence-dependent hydrophobic mutations on hot spots for stabilization, and thus for the engineering, of such biological peptides.

Coherent X-Ray Imaging of Collagen Fibril Distributions within Intact Tendons

PubMed Central

Berenguer, Felisa; Bean, Richard J.; Bozec, Laurent; Vila-Comamala, Joan; Zhang, Fucai; Kewish, Cameron M.; Bunk, Oliver; Rodenburg, John M.; Robinson, Ian K.

2014-01-01

The characterization of the structure of highly hierarchical biosamples such as collagen-based tissues at the scale of tens of nanometers is essential to correlate the tissue structure with its growth processes. Coherent x-ray Bragg ptychography is an innovative imaging technique that gives high resolution images of the ordered parts of such samples. Herein, we report how we used this method to image the collagen fibrillar ultrastructure of intact rat tail tendons. The images show ordered fibrils extending over 10–20 μm in length, with a quantifiable D-banding spacing variation of 0.2%. Occasional defects in the fibrils distribution have also been observed, likely indicating fibrillar fusion events. PMID:24461021

Uniform spatial distribution of collagen fibril radii within tendon implies local activation of pC-collagen at individual fibrils

NASA Astrophysics Data System (ADS)

Rutenberg, Andrew D.; Brown, Aidan I.; Kreplak, Laurent

2016-08-01

Collagen fibril cross-sectional radii show no systematic variation between the interior and the periphery of fibril bundles, indicating an effectively constant rate of collagen incorporation into fibrils throughout the bundle. Such spatially homogeneous incorporation constrains the extracellular diffusion of collagen precursors from sources at the bundle boundary to sinks at the growing fibrils. With a coarse-grained diffusion equation we determine stringent bounds, using parameters extracted from published experimental measurements of tendon development. From the lack of new fibril formation after birth, we further require that the concentration of diffusing precursors stays below the critical concentration for fibril nucleation. We find that the combination of the diffusive bound, which requires larger concentrations to ensure homogeneous fibril radii, and lack of nucleation, which requires lower concentrations, is only marginally consistent with fully processed collagen using conservative bounds. More realistic bounds may leave no consistent concentrations. Therefore, we propose that unprocessed pC-collagen diffuses from the bundle periphery followed by local C-proteinase activity and subsequent collagen incorporation at each fibril. We suggest that C-proteinase is localized within bundles, at fibril surfaces, during radial fibrillar growth. The much greater critical concentration of pC-collagen, as compared to fully processed collagen, then provides broad consistency between homogeneous fibril radii and the lack of fibril nucleation during fibril growth.

Kinetics of the neuroinflammation-oxidative stress correlation in rat brain following the injection of fibrillar amyloid-beta onto the hippocampus in vivo.

PubMed

Rosales-Corral, Sergio; Tan, Dun-Xian; Reiter, Russel J; Valdivia-Velázquez, Miguel; Acosta-Martínez, J Pablo; Ortiz, Genaro G

2004-05-01

The purpose of this study was to describe-following the injection of a single intracerebral dose of fibrillar amyloid-beta(1-40) in vivo-some correlations between proinflammatory cytokines and oxidative stress indicators in function of time, as well as how these variables fit in a regression model. We found a positive, significant correlation between interleukin (IL)-1beta or IL-6 and the activity of the glutathione peroxidase enzyme (GSH-Px), but IL-1beta or IL-6 maintained a strong, negative correlation with the lipid peroxidation (LPO). The first 12 h marked a positive correlation between IL-6 and tumor necrosis factor-alpha (TNF-alpha), but starting from the 36 h, this relationship became negative. We found also particular patterns of behavior through the time for IL-1beta, nitrites and IL-6, with parallel or sequential interrelationships. Results shows clearly that, in vivo, the fibrillar amyloid-beta (Abeta) disrupts the oxidative balance and initiate a proinflammatory response, which in turn feeds the oxidative imbalance in a coordinated, sequential way. This work contributes to our understanding of the positive feedbacks, focusing the "cytokine cycle" along with the oxidative stress mediators in a complex, multicellular, and interactive environment.

Quantitative analysis of nucleolar chromatin distribution in the complex convoluted nucleoli of Didinium nasutum (Ciliophora).

PubMed

Leonova, Olga G; Karajan, Bella P; Ivlev, Yuri F; Ivanova, Julia L; Skarlato, Sergei O; Popenko, Vladimir I

2013-01-01

We have earlier shown that the typical Didinium nasutum nucleolus is a complex convoluted branched domain, comprising a dense fibrillar component located at the periphery of the nucleolus and a granular component located in the central part. Here our main interest was to study quantitatively the spatial distribution of nucleolar chromatin structures in these convoluted nucleoli. There are no "classical" fibrillar centers in D.nasutum nucleoli. The spatial distribution of nucleolar chromatin bodies, which play the role of nucleolar organizers in the macronucleus of D.nasutum, was studied using 3D reconstructions based on serial ultrathin sections. The relative number of nucleolar chromatin bodies was determined in macronuclei of recently fed, starved D.nasutum cells and in resting cysts. This parameter is shown to correlate with the activity of the nucleolus. However, the relative number of nucleolar chromatin bodies in different regions of the same convoluted nucleolus is approximately the same. This finding suggests equal activity in different parts of the nucleolar domain and indicates the existence of some molecular mechanism enabling it to synchronize this activity in D. nasutum nucleoli. Our data show that D. nasutum nucleoli display bipartite structure. All nucleolar chromatin bodies are shown to be located outside of nucleoli, at the periphery of the fibrillar component.

Tumor-associated macrophages and stromal TNF-α regulate collagen structure in a breast tumor model as visualized by second harmonic generation

NASA Astrophysics Data System (ADS)

Burke, Ryan M.; Madden, Kelley S.; Perry, Seth W.; Zettel, Martha L.; Brown, Edward B.

2013-08-01

Collagen fibers can be imaged with second harmonic generation (SHG) and are associated with efficient tumor cell locomotion. Preferential locomotion along these fibers correlates with a more aggressively metastatic phenotype, and changes in SHG emission properties accompany changes in metastatic outcome. We therefore attempted to elucidate the cellular and molecular machinery that influences SHG in order to understand how the microstructure of tumor collagen fibers is regulated. By quantifying SHG and immunofluorescence (IF) from tumors grown in mice with and without stromal tumor necrosis factor (TNF)-α and in the presence or absence of tumor-associated macrophages (TAMs), we determined that depletion of TAMs alters tumor collagen fibrillar microstructure as quantified by SHG and IF. Furthermore, we determined that abrogation of TNF-α expression by tumor stromal cells also alters fibrillar microstructure and that subsequent depletion of TAMs has no further effect. In each case, metastatic burden correlated with optical readouts of collagen microstructure. Our results implicate TAMs and stromal TNF-α as regulators of breast tumor collagen microstructure and suggest that this regulation plays a role in tumor metastasis. Furthermore, these results indicate that quantification of SHG represents a useful strategy for evaluating the cells and molecular pathways responsible for manipulating fibrillar collagen in breast tumor models.

In Situ D-periodic Molecular Structure of Type II Collagen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antipova, Olga; Orgel, Joseph P.R.O.

Collagens are essential components of extracellular matrices in multicellular animals. Fibrillar type II collagen is the most prominent component of articular cartilage and other cartilage-like tissues such as notochord. Its in situ macromolecular and packing structures have not been fully characterized, but an understanding of these attributes may help reveal mechanisms of tissue assembly and degradation (as in osteo- and rheumatoid arthritis). In some tissues such as lamprey notochord, the collagen fibrillar organization is naturally crystalline and may be studied by x-ray diffraction. We used diffraction data from native and derivative notochord tissue samples to solve the axial, D-periodic structuremore » of type II collagen via multiple isomorphous replacement. The electron density maps and heavy atom data revealed the conformation of the nonhelical telopeptides and the overall D-periodic structure of collagen type II in native tissues, data that were further supported by structure prediction and transmission electron microscopy. These results help to explain the observed differences in collagen type I and type II fibrillar architecture and indicate the collagen type II cross-link organization, which is crucial for fibrillogenesis. Transmission electron microscopy data show the close relationship between lamprey and mammalian collagen fibrils, even though the respective larger scale tissue architecture differs.« less

Oligomerization and neurotoxicity of the amyloid ADan peptide implicated in familial Danish dementia.

PubMed

Gibson, Gillian; Gunasekera, Nicola; Lee, Maria; Lelyveld, Victor; El-Agnaf, Omar M A; Wright, Andrew; Austen, Brian

2004-01-01

Familial Danish dementia (FDD) is a rare neurodegenerative disorder, which is pathologically characterized by widespread cerebral amyloid angiopathy, parenchymal protein deposits and neurofibrillary degeneration. FDD is associated with mutation in the BRI gene. In FDD a decamer duplication between codons 265 and 266 in the 3' region of the BRI gene originates an amyloid peptide named ADan, 11 residues longer than the wild-type peptide produced from the normal BRI gene. ADan deposits have been found widely distributed in the CNS of FDD cases. The deposits of ADan are predominantly non-fibrillar aggregates. We show here that synthetic ADan forms oligomers in vitro, seen by Tricine-PAGE and gel filtration, and higher aggregates, which are seen by atomic force spectroscopy and electron microscopy as carrot-shaped objects that bunch together. Here we report that oligomeric ADan is toxic to neuronal cell lines. We find that the soluble non-fibrillar oligomeric species of both the reduced and oxidized forms of ADan are toxic. These results support the idea that the non-fibrillar soluble aggregates are the pathogenic species, which may play a central role in the pathogenesis of FDD, and imply that similar mechanism may also be involved in other neurodegenerative diseases associated with amyloid deposits.

Alpha-helix to beta-sheet transition in long-chain poly-l-lysine: Formation of alpha-helical fibrils by poly-l-lysine.

PubMed

Cieślik-Boczula, Katarzyna

2017-06-01

The temperature-induced α-helix to β-sheet transition in long-chain poly-l-lysine (PLL), accompanied by the gauche-to-trans isomerization of CH 2 groups in the hydrocarbon side chains of Lys amino acid residues, and formation of β-sheet as well as α-helix fibrillar aggregates of PLL have been studied using Fourier-transform infrared (FT-IR) and vibrational circular dichroism (VCD) spectroscopy, and transmission electron microscopy (TEM). In a low-temperature alkaline water solution or in a methanol-rich water mixture, the secondary structure of PLL is represented by α-helical conformations with unordered and gauche-rich hydrocarbon side chains. Under these conditions, PLL molecules aggregate into α-helical fibrils. PLLs dominated by extended antiparallel β-sheet structures with highly ordered trans-rich hydrocarbon side chains are formed in a high-temperature range at alkaline pD and aggregate into fibrillar, protofibrillar, and spherical forms. Presented data support the idea that fibrillar aggregation is a varied phenomenon possible in repetitive structural elements with not only a β-sheet-rich conformation, but also an α-helical-rich conformation. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Second harmonic generation microscopy differentiates collagen type I and type III in COPD

NASA Astrophysics Data System (ADS)

Suzuki, Masaru; Kayra, Damian; Elliott, W. Mark; Hogg, James C.; Abraham, Thomas

2012-03-01

The structural remodeling of extracellular matrix proteins in peripheral lung region is an important feature in chronic obstructive pulmonary disease (COPD). Multiphoton microscopy is capable of inducing specific second harmonic generation (SHG) signal from non-centrosymmetric structural proteins such as fibrillar collagens. In this study, SHG microscopy was used to examine structural remodeling of the fibrillar collagens in human lungs undergoing emphysematous destruction (n=2). The SHG signals originating from these diseased lung thin sections from base to apex (n=16) were captured simultaneously in both forward and backward directions. We found that the SHG images detected in the forward direction showed well-developed and well-structured thick collagen fibers while the SHG images detected in the backward direction showed striking different morphological features which included the diffused pattern of forward detected structures plus other forms of collagen structures. Comparison of these images with the wellestablished immunohistochemical staining indicated that the structures detected in the forward direction are primarily the thick collagen type I fibers and the structures identified in the backward direction are diffusive structures of forward detected collagen type I plus collagen type III. In conclusion, we here demonstrate the feasibility of SHG microscopy in differentiating fibrillar collagen subtypes and understanding their remodeling in diseased lung tissues.

Immunotherapy alleviates amyloid-associated synaptic pathology in an Alzheimer’s disease mouse model

PubMed Central

Dorostkar, Mario M.; Burgold, Steffen; Filser, Severin; Barghorn, Stefan; Schmidt, Boris; Anumala, Upendra Rao; Hillen, Heinz; Klein, Corinna

2014-01-01

Cognitive decline in Alzheimer’s disease is attributed to loss of functional synapses, most likely caused by synaptotoxic, oligomeric forms of amyloid-β. Many treatment options aim at reducing amyloid-β levels in the brain, either by decreasing its production or by increasing its clearance. We quantified the effects of immunotherapy directed against oligomeric amyloid-β in Tg2576 mice, a mouse model of familial Alzheimer’s disease. Treatment of 12-month-old mice with oligomer-specific (A-887755) or conformation-unspecific (6G1) antibodies for 8 weeks did not affect fibrillar plaque density or growth. We also quantified densities of DLG4 (previously known as PSD95) expressing post-synapses and synapsin expressing presynapses immunohistochemically. We found that both pre- and post-synapses were strongly reduced in the vicinity of plaques, whereas distant from plaques, in the cortex and hippocampal CA1 field, only post-synapses were reduced. Immunotherapy alleviated this synapse loss. Synapse loss was completely abolished distant from plaques, whereas it was only attenuated in the vicinity of plaques. These results suggest that fibrillar plaques may act as reservoirs for synaptotoxic, oligomeric amyloid-β and that sequestering oligomers suffices to counteract synaptic pathology. Therefore, cognitive function may be improved by immunotherapy even when the load of fibrillar amyloid remains unchanged. PMID:25281869

Development of diagnostic and treatment strategies for glaucoma through understanding and modification of scleral and lamina cribrosa connective tissue

PubMed Central

Quigley, Harry A.; Cone, Frances E.

2013-01-01

There is considerable evidence that the state of ocular connective tissues and their response in glaucomatous disease affects the degree of glaucoma damage. Both experimental and clinical data suggest that improved diagnostic and prognostic information could be derived from assessment of the mechanical responsiveness of the sclera and lamina cribrosa to intraocular pressure (IOP). Controlled mutagenesis of the sclera has produced a mouse strain that is relatively resistant to increased IOP. Alteration of the baseline scleral state could be accomplished through either increased cross-linking of fibrillar components or their reduction. The sclera is a dynamic structure, altering its structure and behavior in response to IOP change. The biochemical pathways that control these responses are fertile areas for new glaucoma treatments. PMID:23535950

Development and characterization of amorphous acrylate networks for use as switchable adhesives inspired from shapememory behavior

NASA Astrophysics Data System (ADS)

Lakhera, Nishant

Several types of insects and animals such as spiders and geckos are inherently able to climb along vertical walls and ceilings. This remarkable switchable adhesive behavior has been attributed to the fibrillar structures on their feet, with size ranging from few nanometers to a few micrometers depending on the species. Several studies have attempted to create synthetic micro-patterned surfaces trying to imitate this adhesive behavior seen in nature. The experimental procedures are scattered, with sole purpose of trying to increase adhesion, thereby making direct comparison between studies very difficult. There is a lack of fundamental understanding on adhesion of patterned surfaces. The influence of critical parameters like material modulus, glass transition temperature, viscoelastic effects, temperature and water absorption on adhesion is not fully explored and characterized. These parameters are expected to have a decisive influence on adhesion behavior of the polymer. Previous studies have utilized conventional "off-the-shelf" materials like epoxy, polyurethanes etc. It is however, impossible to change the material modulus, glass transition temperature etc. of these polymer systems without changing the base constituents itself, thereby explaining the gaps in the current research landscape. The purpose of this study was to use acrylate shape-memory polymers (SMPs) for their ability to be tailored to specific mechanical properties by control of polymer chemistry, without changing the base constituents. Polymer networks with tailorable glass transition, material modulus, water absorption etc. were developed and adhesion studies were performed to investigate the influence of temperature, viscoelastic effects, material modulus on the adhesion behavior of flat acrylate polymer surfaces. The knowledge base gained from these studies was utilized to better understand the fundamental mechanisms associated with adhesion behavior of patterned acrylate surfaces. Thermally induced switchable adhesion and water induced switchable adhesion of patterned acrylate surfaces was investigated. The viscoelastic energy dissipation occurring during the detachment phase was shown to dramatically increase adhesion under both thermally induced and water induced conditions. This effect was most pre-dominant at the glass transition temperature of the material. Increase in pre-load force and unloading velocity were also shown to increase the adhesive capability of the patterned acrylate SMPs.

Biofouling of marbles by oxygenic photosynthetic microorganisms.

PubMed

Karaca, Zeki; Öztürk, Ayten; Çolak, Emel

2015-08-01

Phototrophic microorganisms disfigure the surfaces of different types of stone. Stone structure is damaged by the activity of photoautotrophic and other microorganisms. However, to date few, investigations have been undertaken into the relationship between microorganisms and the properties of different types of marble. In this study, biological activity of photoautotrophic microorganisms on three types of marble (Yatagan White, Giallo Anticato and Afyon White) was investigated under laboratory conditions over a short period of time. The three types of marble supported the growth of phototrophic microbial communities on their outer and inner layers, turning their original colour from white to a yellowish green colour. The porosity of the marble types facilitated filamentous microbial growth in the presence of water. Scanning electron microscope analysis revealed the accumulation of aggregates such as small spherical, fibrillar, calcified globular bodies on the inner surfaces of the marbles. This suggests that the microscopic characteristics of particular marble types may stimulate the growth of certain types of microorganisms.

Microcanonical thermostatistics of coarse-grained proteins with amyloidogenic propensity

NASA Astrophysics Data System (ADS)

Frigori, Rafael B.; Rizzi, Leandro G.; Alves, Nelson A.

2013-01-01

The formation of fibrillar aggregates seems to be a common characteristic of polypeptide chains, although the observation of these aggregates may depend on appropriate experimental conditions. Partially folded intermediates seem to have an important role in the generation of protein aggregates, and a mechanism for this fibril formation considers that these intermediates also correspond to metastable states with respect to the fibrillar ones. Here, using a coarse-grained (CG) off-lattice model, we carry out a comparative analysis of the thermodynamic aspects characterizing the folding transition with respect to the propensity for aggregation of four different systems: two isoforms of the amyloid β-protein, the Src SH3 domain, and the human prion proteins (hPrP). Microcanonical analysis of the data obtained from replica exchange method is conducted to evaluate the free-energy barrier and latent heat in these models. The simulations of the amyloid β isoforms and Src SH3 domain indicated that the folding process described by this CG model is related to a negative specific heat, a phenomenon that can only be verified in the microcanonical ensemble in first-order phase transitions. The CG simulation of the hPrP heteropolymer yielded a continuous folding transition. The absence of a free-energy barrier and latent heat favors the presence of partially unfolded conformations, and in this context, this thermodynamic aspect could explain the reason why the hPrP heteropolymer is more aggregation-prone than the other heteropolymers considered in this study. We introduced the hydrophobic radius of gyration as an order parameter and found that it can be used to obtain reliable information about the hydrophobic packing and the transition temperatures in the folding process.

High-resolution molecular structure of a peptide in an amyloid fibril determined by magic angle spinning NMR spectroscopy

NASA Astrophysics Data System (ADS)

Jaroniec, Christopher P.; Macphee, Cait E.; Bajaj, Vikram S.; McMahon, Michael T.; Dobson, Christopher M.; Griffin, Robert G.

2004-01-01

Amyloid fibrils are self-assembled filamentous structures associated with protein deposition conditions including Alzheimer's disease and the transmissible spongiform encephalopathies. Despite the immense medical importance of amyloid fibrils, no atomic-resolution structures are available for these materials, because the intact fibrils are insoluble and do not form diffraction-quality 3D crystals. Here we report the high-resolution structure of a peptide fragment of the amyloidogenic protein transthyretin, TTR(105-115), in its fibrillar form, determined by magic angle spinning NMR spectroscopy. The structure resolves not only the backbone fold but also the precise conformation of the side chains. Nearly complete 13C and 15N resonance assignments for TTR(105-115) formed the basis for the extraction of a set of distance and dihedral angle restraints. A total of 76 self-consistent experimental measurements, including 41 restraints on 19 backbone dihedral angles and 35 13C-15N distances between 3 and 6 Å were obtained from 2D and 3D NMR spectra recorded on three fibril samples uniformly 13C, 15N-labeled in consecutive stretches of four amino acids and used to calculate an ensemble of peptide structures. Our results indicate that TTR(105-115) adopts an extended -strand conformation in the amyloid fibrils such that both the main- and side-chain torsion angles are close to their optimal values. Moreover, the structure of this peptide in the fibrillar form has a degree of long-range order that is generally associated only with crystalline materials. These findings provide an explanation of the unusual stability and characteristic properties of this form of polypeptide assembly.

Isolation, Characterization and Biological Evaluation of Jellyfish Collagen for Use in Biomedical Applications

PubMed Central

Addad, Sourour; Exposito, Jean-Yves; Faye, Clément; Ricard-Blum, Sylvie; Lethias, Claire

2011-01-01

Fibrillar collagens are the more abundant extracellular proteins. They form a metazoan-specific family, and are highly conserved from sponge to human. Their structural and physiological properties have been successfully used in the food, cosmetic, and pharmaceutical industries. On the other hand, the increase of jellyfish has led us to consider this marine animal as a natural product for food and medicine. Here, we have tested different Mediterranean jellyfish species in order to investigate the economic potential of their collagens. We have studied different methods of collagen purification (tissues and experimental procedures). The best collagen yield was obtained using Rhizostoma pulmo oral arms and the pepsin extraction method (2–10 mg collagen/g of wet tissue). Although a significant yield was obtained with Cotylorhiza tuberculata (0.45 mg/g), R. pulmo was used for further experiments, this jellyfish being considered as harmless to humans and being an abundant source of material. Then, we compared the biological properties of R. pulmo collagen with mammalian fibrillar collagens in cell cytotoxicity assays and cell adhesion. There was no statistical difference in cytotoxicity (p > 0.05) between R. pulmo collagen and rat type I collagen. However, since heparin inhibits cell adhesion to jellyfish-native collagen by 55%, the main difference is that heparan sulfate proteoglycans could be preferentially involved in fibroblast and osteoblast adhesion to jellyfish collagens. Our data confirm the broad harmlessness of jellyfish collagens, and their biological effect on human cells that are similar to that of mammalian type I collagen. Given the bioavailability of jellyfish collagen and its biological properties, this marine material is thus a good candidate for replacing bovine or human collagens in selected biomedical applications. PMID:21747742

Isolation, characterization and biological evaluation of jellyfish collagen for use in biomedical applications.

PubMed

Addad, Sourour; Exposito, Jean-Yves; Faye, Clément; Ricard-Blum, Sylvie; Lethias, Claire

2011-01-01

Fibrillar collagens are the more abundant extracellular proteins. They form a metazoan-specific family, and are highly conserved from sponge to human. Their structural and physiological properties have been successfully used in the food, cosmetic, and pharmaceutical industries. On the other hand, the increase of jellyfish has led us to consider this marine animal as a natural product for food and medicine. Here, we have tested different Mediterranean jellyfish species in order to investigate the economic potential of their collagens. We have studied different methods of collagen purification (tissues and experimental procedures). The best collagen yield was obtained using Rhizostoma pulmo oral arms and the pepsin extraction method (2-10 mg collagen/g of wet tissue). Although a significant yield was obtained with Cotylorhiza tuberculata (0.45 mg/g), R. pulmo was used for further experiments, this jellyfish being considered as harmless to humans and being an abundant source of material. Then, we compared the biological properties of R. pulmo collagen with mammalian fibrillar collagens in cell cytotoxicity assays and cell adhesion. There was no statistical difference in cytotoxicity (p > 0.05) between R. pulmo collagen and rat type I collagen. However, since heparin inhibits cell adhesion to jellyfish-native collagen by 55%, the main difference is that heparan sulfate proteoglycans could be preferentially involved in fibroblast and osteoblast adhesion to jellyfish collagens. Our data confirm the broad harmlessness of jellyfish collagens, and their biological effect on human cells that are similar to that of mammalian type I collagen. Given the bioavailability of jellyfish collagen and its biological properties, this marine material is thus a good candidate for replacing bovine or human collagens in selected biomedical applications.

Data in support of the identification of neuronal and astrocyte proteins interacting with extracellularly applied oligomeric and fibrillar α-synuclein assemblies by mass spectrometry

PubMed Central

Shrivastava, Amulya Nidhi; Redeker, Virginie; Fritz, Nicolas; Pieri, Laura; Almeida, Leandro G.; Spolidoro, Maria; Liebmann, Thomas; Bousset, Luc; Renner, Marianne; Léna, Clément; Aperia, Anita; Melki, Ronald; Triller, Antoine

2016-01-01

α-Synuclein (α-syn) is the principal component of Lewy bodies, the pathophysiological hallmark of individuals affected by Parkinson disease (PD). This neuropathologic form of α-syn contributes to PD progression and propagation of α-syn assemblies between neurons. The data we present here support the proteomic analysis used to identify neuronal proteins that specifically interact with extracellularly applied oligomeric or fibrillar α-syn assemblies (conditions 1 and 2, respectively) (doi: 10.15252/embj.201591397[1]). α-syn assemblies and their cellular partner proteins were pulled down from neuronal cell lysed shortly after exposure to exogenous α-syn assemblies and the associated proteins were identified by mass spectrometry using a shotgun proteomic-based approach. We also performed experiments on pure cultures of astrocytes to identify astrocyte-specific proteins interacting with oligomeric or fibrillar α-syn (conditions 3 and 4, respectively). For each condition, proteins interacting selectively with α-syn assemblies were identified by comparison to proteins pulled-down from untreated cells used as controls. The mass spectrometry data, the database search and the peak lists have been deposited to the ProteomeXchange Consortium database via the PRIDE partner repository with the dataset identifiers PRIDE: PXD002256 to PRIDE: PXD002263 and doi: 10.6019/PXD002256 to 10.6019/PXD002263. PMID:26958642

Data in support of the identification of neuronal and astrocyte proteins interacting with extracellularly applied oligomeric and fibrillar α-synuclein assemblies by mass spectrometry.

PubMed

Shrivastava, Amulya Nidhi; Redeker, Virginie; Fritz, Nicolas; Pieri, Laura; Almeida, Leandro G; Spolidoro, Maria; Liebmann, Thomas; Bousset, Luc; Renner, Marianne; Léna, Clément; Aperia, Anita; Melki, Ronald; Triller, Antoine

2016-06-01

α-Synuclein (α-syn) is the principal component of Lewy bodies, the pathophysiological hallmark of individuals affected by Parkinson disease (PD). This neuropathologic form of α-syn contributes to PD progression and propagation of α-syn assemblies between neurons. The data we present here support the proteomic analysis used to identify neuronal proteins that specifically interact with extracellularly applied oligomeric or fibrillar α-syn assemblies (conditions 1 and 2, respectively) (doi: 10.15252/embj.201591397[1]). α-syn assemblies and their cellular partner proteins were pulled down from neuronal cell lysed shortly after exposure to exogenous α-syn assemblies and the associated proteins were identified by mass spectrometry using a shotgun proteomic-based approach. We also performed experiments on pure cultures of astrocytes to identify astrocyte-specific proteins interacting with oligomeric or fibrillar α-syn (conditions 3 and 4, respectively). For each condition, proteins interacting selectively with α-syn assemblies were identified by comparison to proteins pulled-down from untreated cells used as controls. The mass spectrometry data, the database search and the peak lists have been deposited to the ProteomeXchange Consortium database via the PRIDE partner repository with the dataset identifiers PRIDE: PXD002256 to PRIDE: PXD002263 and doi: 10.6019/PXD002256 to 10.6019/PXD002263.

Dynamics and three-dimensional localization of ribosomal RNA within the nucleolus.

PubMed Central

Thiry, M; Cheutin, T; O'Donohue, M F; Kaplan, H; Ploton, D

2000-01-01

Although rRNA synthesis, maturation, and assembly into preribosomal particles occur within the nucleolus, the route taken by pre-rRNAs from their synthetic sites toward the cytoplasm remains largely unexplored. Here, we employed a nondestructive method for the incorporation of BrUTP into the RNA of living cells. By using pulse-chase experiments, three-dimensional image reconstructions of confocal optical sections, and electron microscopy analysis of ultrathin sections, we were able to describe topological and spatial dynamics of rRNAs within the nucleolus. We identified the precise location and the volumic organization of four typical subdomains, in which rRNAs are successively moving towards the nucleolar periphery during their synthesis and processing steps. The incorporation of BrUTP takes place simultaneously within several tiny spheres, centered on the fibrillar centers. Then, the structures containing the newly synthesized RNAs enlarge and appear as compact ringlets disposed around the fibrillar centers. Later, they form hollow spheres surrounding the latter components and begin to fuse together. Finally, these structures widen and form large rings reaching the limits of the nucleoli. These results clearly show that the transport of pre-rRNAs within the nucleolus does not occur randomly, but appears as a radial flow starting from the fibrillar centers that form concentric rings, which finally fuse together as they progress toward the nucleolar periphery. PMID:11142375

Nonlinear optical response of the collagen triple helix and second harmonic microscopy of collagen liquid crystals

NASA Astrophysics Data System (ADS)

Deniset-Besseau, A.; De Sa Peixoto, P.; Duboisset, J.; Loison, C.; Hache, F.; Benichou, E.; Brevet, P.-F.; Mosser, G.; Schanne-Klein, M.-C.

2010-02-01

Collagen is characterized by triple helical domains and plays a central role in the formation of fibrillar and microfibrillar networks, basement membranes, as well as other structures of the connective tissue. Remarkably, fibrillar collagen exhibits efficient Second Harmonic Generation (SHG) and SHG microscopy proved to be a sensitive tool to score fibrotic pathologies. However, the nonlinear optical response of fibrillar collagen is not fully characterized yet and quantitative data are required to further process SHG images. We therefore performed Hyper-Rayleigh Scattering (HRS) experiments and measured a second order hyperpolarisability of 1.25 10-27 esu for rat-tail type I collagen. This value is surprisingly large considering that collagen presents no strong harmonophore in its amino-acid sequence. In order to get insight into the physical origin of this nonlinear process, we performed HRS measurements after denaturation of the collagen triple helix and for a collagen-like short model peptide [(Pro-Pro-Gly)10]3. It showed that the collagen large nonlinear response originates in the tight alignment of a large number of weakly efficient harmonophores, presumably the peptide bonds, resulting in a coherent amplification of the nonlinear signal along the triple helix. To illustrate this mechanism, we successfully recorded SHG images in collagen liquid solutions by achieving liquid crystalline ordering of the collagen triple helices.

The mesangial matrix in the normal and sclerotic glomerulus.

PubMed

Rosenblum, N D

1994-02-01

Mesangial sclerosis is a final common pathway to glomerular destruction in a variety of glomerular diseases. The expression of several classes of extracellular matrix (ECM) molecules has been defined in the normal and diseased mesangial matrix (MM). However, the manner in which these ECM components determine the three dimensional structure and function of the MM remains to be defined. Structural studies of the MM suggest that its constituent molecules are regionally organized into subcompartments with different three dimensional structures. The diversity of matrix molecules expressed within the MM as well as the organization of these components in nonrenal ECM's, such as the cornea, provides further support for this organizational model. The study of the cornea has also revealed that novel short chain collagenous proteins partially determine the three dimensional structure of the matrix. Recently, a novel collagen, type VIII collagen, has been described in mesangial cells and in the intact glomerulus. It is hypothesized that type VIII collagen is expressed both as a polymer and as a monomer within the glomerulus, and depending on its conformation, may serve unique functions. In the chronically diseased MM, normal MM components are overexpressed and fibrillar collagens are expressed de novo in a delayed fashion. Enhanced proteoglycan expression, observed early in disease, may determine increased volume of the mesangium. This, in turn, may stimulate the production of fibrillar collagens by mesangial cells resulting in a fibrillar noncompliant mesangial matrix.

REFORMATION OF NUCLEOLI AFTER ETHIONE-INDUCED FRAGMENTATION IN THE ABSENCE OF SIGNIFICANT PROTEIN SYNTHESIS

PubMed Central

Shinozuka, Hisashi; Farber, Emmanuel

1969-01-01

The rat liver nucleolus, after fragmentation induced by ethionine treatment, has been found to undergo complete reformation by adenine in the presence of a dose of cycloheximide sufficient to cause inhibition of protein synthesis by 90–95%. In contrast, actinomycin D given along with adenine was followed by the appearance of a small compact mass containing only the fibrillar component with no evident granules. This structure resembled pseudonucleoli seen in the anucleolate mutant of Xenopus laevis or in certain early stages of amphibian oocytes. Actinomycin D administered 2 hr after adenine induced a segregation of the fibrillar and granular components of nucleoli similar to that induced in the normal nucleolus. The implications of these findings in relation to nucleolar organization are briefly discussed. PMID:5775789

Preparation of thick silica coatings on carbon fibers with fine-structured silica nanotubes induced by a self-assembly process

PubMed Central

Baumgärtner, Benjamin; Möller, Hendrik; Neumann, Thomas

2017-01-01

A facile method to coat carbon fibers with a silica shell is presented in this work. By immobilizing linear polyamines on the carbon fiber surface, the high catalytic activity of polyamines in the sol–gel-processing of silica precursors is used to deposit a silica coating directly on the fiber’s surface. The surface localization of the catalyst is achieved either by attaching short-chain polyamines (e.g., tetraethylenepentamine) via covalent bonds to the carbon fiber surface or by depositing long-chain polyamines (e.g., linear poly(ethylenimine)) on the carbon fiber by weak non-covalent bonding. The long-chain polyamine self-assembles onto the carbon fiber substrate in the form of nanoscopic crystallites, which serve as a template for the subsequent silica deposition. The silicification at close to neutral pH is spatially restricted to the localized polyamine and consequently to the fiber surface. In case of the linear poly(ethylenimine), silica shells of several micrometers in thickness can be obtained and their morphology is easily controlled by a considerable number of synthesis parameters. A unique feature is the hierarchical biomimetic structure of the silica coating which surrounds the embedded carbon fiber by fibrillar and interconnected silica fine-structures. The high surface area of the nanostructured composite fiber may be exploited for catalytic applications and adsorption purposes. PMID:28685115

Preparation of thick silica coatings on carbon fibers with fine-structured silica nanotubes induced by a self-assembly process.

PubMed

Baumgärtner, Benjamin; Möller, Hendrik; Neumann, Thomas; Volkmer, Dirk

2017-01-01

A facile method to coat carbon fibers with a silica shell is presented in this work. By immobilizing linear polyamines on the carbon fiber surface, the high catalytic activity of polyamines in the sol-gel-processing of silica precursors is used to deposit a silica coating directly on the fiber's surface. The surface localization of the catalyst is achieved either by attaching short-chain polyamines (e.g., tetraethylenepentamine) via covalent bonds to the carbon fiber surface or by depositing long-chain polyamines (e.g., linear poly(ethylenimine)) on the carbon fiber by weak non-covalent bonding. The long-chain polyamine self-assembles onto the carbon fiber substrate in the form of nanoscopic crystallites, which serve as a template for the subsequent silica deposition. The silicification at close to neutral pH is spatially restricted to the localized polyamine and consequently to the fiber surface. In case of the linear poly(ethylenimine), silica shells of several micrometers in thickness can be obtained and their morphology is easily controlled by a considerable number of synthesis parameters. A unique feature is the hierarchical biomimetic structure of the silica coating which surrounds the embedded carbon fiber by fibrillar and interconnected silica fine-structures. The high surface area of the nanostructured composite fiber may be exploited for catalytic applications and adsorption purposes.

Nanoscale Morphology to Macroscopic Performance in Ultra High Molecular Weight Polyethylene Fibers

NASA Astrophysics Data System (ADS)

McDaniel, Preston B.

Ultra high molecular weight polyethylene (UHMWPE) fibers are increasingly used in high -performance applications where strength, stiffness, and the ability to dissipate energy are of critical importance. Despite their use in a variety of applications, the influence of morphological features at the meso/nanoscale on the macroscopic performance of the fibers has not been well understood. There is particular interest in gaining a better understanding of the nanoscale structure-property relationships in UHMWPE fibers used in ballistics applications. In order to accurately model and predict failure in the fiber, a more complete understanding of the complex load pathways that dictate the ways in which load is transferred through the fiber, across interfaces and length scales is required. The goal of the work discussed herein is to identify key meso/nanostructural features evolved in high performance fibers and determine how these features influence the performance of the fiber through a variety of different loading mechanisms. The important structural features in high-performance UHMWPE fibers are first identified through examination of the meso/nanostructure of a series of fibers with different processing conditions. This is achieved primarily through the use of wide-angle x-ray diffraction (WAXD) and atomic force microscopy (AFM). Analysis of AFM images and WAXD data allows identification and quantifications of important structural features at these length scales. Key meso/nanostructural features are then examined with respect to their influence on the transverse compression behavior of single fibers. Through post-mortem AFM analysis of samples at incremental compressive strains, the evolution of damage is examined and compared with macroscopic fiber mechanical response. It was found that collapse of mesoscale voids, followed by nanoscale fibrillation and reorganization of a fibrillar network has a significant influence on the mechanical response of the fiber. Through this work, the importance of nanoscale fibril adhesive interactions is highlighted. However, very little information exists in the literature as to the nature and magnitude of these interactions. Examination of nanoscale fibrillar adhesive interactions is experimentally difficult, and necessitated the development of an AFM based nanoscale splitting technique to quantify the interactions between fibrils. Through analysis of split geometry and careful partitioning of energies, the adhesive energy between fibrils in UHMWPE fibers are determined. The calculated average adhesive energies are significantly larger than the estimated energy due to van der Waals interactions, suggesting that there are physical connections (e.g., tie chains, tie fibrils, and lamellar crystalline bridges) that influence the interactions between fibrils. The interactions identified through this work are believed to be responsible for the creation of load pathways across fibril interfaces where load may be translated through the fiber in tension, compression, and shear. Finally, the nature of the mesoscale fibrillar network is explored through the development of a variable angle, single fiber peel test. This peel test enables the quantification of Mode I and Mode II peel energies. The modes of deformation observed in the peel test are representative of the mechanisms experienced during tensile and transverse compression loading. The quantification of peel energies in both Mode I and Mode II failure highlight the importance of the fibrillar network as a key mechanism for the translation of load through the fiber. In both modes of failure, the fibril network acts as a framework for the orientation and subsequent failure of nanoscale fibrils.

Low temperature corneal laser welding investigated by atomic force microscopy

NASA Astrophysics Data System (ADS)

Matteini, Paolo; Sbrana, Francesca; Tiribilli, Bruno; Pini, Roberto

2009-02-01

The structural modifications in the stromal matrix induced by low-temperature corneal laser welding were investigated by atomic force microscopy (AFM). This procedure consists of staining the wound with Indocyanine Green (ICG), followed by irradiation with a near-infrared laser operated at low-power densities. This induces a local heating in the 55-65 °C range. In welded tissue, extracellular components undergo heat-induced structural modifications, resulting in a joining effect between the cut edges. However, the exact mechanism generating the welding, to date, is not completely understood. Full-thickness cuts, 3.5 mm in length, were made in fresh porcine cornea samples, and these were then subjected to laser welding operated at 16.7 W/cm2 power density. AFM imaging was performed on resin-embedded semi-thin slices once they had been cleared by chemical etching, in order to expose the stromal bulk of the tissue within the section. We then carried out a morphological analysis of characteristic fibrillar features in the laser-treated and control samples. AFM images of control stromal regions highlighted well-organized collagen fibrils (36.2 +/- 8.7 nm in size) running parallel to each other as in a typical lamellar domain. The fibrils exhibited a beaded pattern with a 22-39 nm axial periodicity. Laser-treated corneal regions were characterized by a significant disorganization of the intralamellar architecture. At the weld site, groups of interwoven fibrils joined the cut edges, showing structural properties that were fully comparable with those of control regions. This suggested that fibrillar collagen is not denatured by low-temperature laser welding, confirming previous transmission electron microscopy (TEM) observations, and thus it is probably not involved in the closure mechanism of corneal cuts. The loss of fibrillar organization may be related to some structural modifications in some interfibrillar substance as proteoglycans or collagen VI. Furthermore, AFM imaging was demonstrated to be a suitable tool for attaining three-dimensional information on the fibrillar assembly of corneal stroma. The results suggested that AFM analyses of resin-embedded histological sections subjected to chemical etching provide a rapid and cost-effective response, with an imaging resolution that is quite similar to that of TEM.

Fabrication and Evaluation of Bis-GMA/TEGDMA Dental Resins/Composites Containing Nano Fibrillar Silicate

PubMed Central

Tian, Ming; Gao, Yi; Liu, Yi; Liao, Yiliang; Hedin, Nyle E.; Fong, Hao

2008-01-01

Objective To investigate the reinforcement of Bis-GMA/TEGDMA dental resins (without conventional glass filler) and composites (with conventional glass filler) with various mass fractions of nano fibrillar silicate (FS). Methods Three dispersion methods were studied to separate the silanized FS as nano-scaled single crystals and uniformly distribute them into dental matrices. The photo-curing behaviors of the Bis-GMA/TEGDMA/FS resins were monitored in situ by RT-NIR to study the photopolymerization rate and the vinyl double bond conversion. Mechanical properties (flexural strength, elastic modulus and work of fracture) of the nano FS reinforced resins/composites were tested, and Analysis of Variance (ANOVA) was used for the statistical analysis of the acquired data. The morphology of nano FS and the representative fracture surfaces of its reinforced resins/composites were examined by SEM/TEM. Results Impregnation of small mass fractions (1 % and 2.5 %) of nano FS into Bis-GMA/TEGDMA (50/50 mass ratio) dental resins/composites improved the mechanical properties substantially. Larger mass fraction of impregnation (7.5 %), however, did not further improve the mechanical properties (one way ANOVA, P > 0.05) and may even reduce the mechanical properties. The high degree of separation and uniform distribution of nano FS into dental resins/composites was a challenge. Impregnation of nano FS into dental resins/composites could result in two opposite effects: a reinforcing effect due to the highly separated and uniformly distributed nano FS single crystals, or a weakening effect due to the formation of FS agglomerates/particles. Significance Uniform distribution of highly separated nano FS single crystals into dental resins/composites could significantly improve the mechanical properties of the resins/composites. PMID:17572485

The Properties of Amyloid-β Fibrils Are Determined by their Path of Formation.

PubMed

Brännström, Kristoffer; Islam, Tohidul; Gharibyan, Anna L; Iakovleva, Irina; Nilsson, Lina; Lee, Cheng Choo; Sandblad, Linda; Pamrén, Annelie; Olofsson, Anders

2018-06-22

Fibril formation of the amyloid-β peptide (Aβ) follows a nucleation-dependent polymerization process and is associated with Alzheimer's disease. Several different lengths of Aβ are observed in vivo, but Aβ1-40 and Aβ1-42 are the dominant forms. The fibril architectures of Aβ1-40 and Aβ1-42 differ and Aβ1-42 assemblies are generally considered more pathogenic. We show here that monomeric Aβ1-42 can be cross-templated and incorporated into the ends of Aβ1-40 fibrils, while incorporation of Aβ1-40 monomers into Aβ1-42 fibrils is very poor. We also show that via cross-templating incorporated Aβ monomers acquire the properties of the parental fibrils. The suppressed ability of Aβ1-40 to incorporate into the ends of Aβ1-42 fibrils and the capacity of Aβ1-42 monomers to adopt the properties of Aβ1-40 fibrils may thus represent two mechanisms reducing the total load of fibrils having the intrinsic, and possibly pathogenic, features of Aβ1-42 fibrils in vivo. We also show that the transfer of fibrillar properties is restricted to fibril-end templating and does not apply to cross-nucleation via the recently described path of surface-catalyzed secondary nucleation, which instead generates similar structures to those acquired via de novo primary nucleation in the absence of catalyzing seeds. Taken together these results uncover an intrinsic barrier that prevents Aβ1-40 from adopting the fibrillar properties of Aβ1-42 and exposes that the transfer of properties between amyloid-β fibrils are determined by their path of formation. Copyright © 2018. Published by Elsevier Ltd.

Fabrication and evaluation of Bis-GMA/TEGDMA dental resins/composites containing nano fibrillar silicate.

PubMed

Tian, Ming; Gao, Yi; Liu, Yi; Liao, Yiliang; Hedin, Nyle E; Fong, Hao

2008-02-01

To investigate the reinforcement of Bis-GMA/TEGDMA dental resins (without conventional glass filler) and composites (with conventional glass filler) with various mass fractions of nano fibrillar silicate (FS). Three dispersion methods were studied to separate the silanized FS as nano-scaled single crystals and uniformly distribute them into dental matrices. The photo-curing behaviors of the Bis-GMA/TEGDMA/FS resins were monitored in situ by RT-NIR to study the photopolymerization rate and the vinyl double bond conversion. Mechanical properties (flexural strength, elastic modulus and work-of-fracture) of the nano FS reinforced resins/composites were tested, and analysis of variance (ANOVA) was used for the statistical analysis of the acquired data. The morphology of nano FS and the representative fracture surfaces of its reinforced resins/composites were examined by SEM/TEM. Impregnation of small mass fractions (1% and 2.5%) of nano FS into Bis-GMA/TEGDMA (50/50 mass ratio) dental resins/composites improved the mechanical properties substantially. Larger mass fraction of impregnation (7.5%), however, did not further improve the mechanical properties (one way ANOVA, P>0.05) and may even reduce the mechanical properties. The high degree of separation and uniform distribution of nano FS into dental resins/composites was a challenge. Impregnation of nano FS into dental resins/composites could result in two opposite effects: a reinforcing effect due to the highly separated and uniformly distributed nano FS single crystals, or a weakening effect due to the formation of FS agglomerates/particles. Uniform distribution of highly separated nano FS single crystals into dental resins/composites could significantly improve the mechanical properties of the resins/composites.

Assembly and remodeling of the fibrillar fibronectin extracellular matrix during gastrulation and neurulation in Xenopus laevis.

PubMed

Davidson, Lance A; Keller, Raymond; DeSimone, Douglas W

2004-12-01

Fibronectin, a major component of the extracellular matrix is critical for processes of cell traction and cell motility. Whole-mount confocal imaging of the three-dimensional architecture of the extracellular matrix is used to describe dynamic assembly and remodeling of fibronectin fibrils during gastrulation and neurulation in the early frog embryo. As previously reported, fibrils first appear under the prospective ectoderm. We describe here the first evidence for regulated assembly of fibrils along the somitic mesoderm/endoderm boundary as well as at the notochord/somitic mesoderm boundary and clearing of fibrils from the dorsal and ventral surfaces of the notochord that occurs over the course of a few hours. As gastrulation proceeds, fibrils are restored to the dorsal surface of the notochord, where the notochord contacts the prospective floor plate. As the neural folds form, fibrils are again remodeled as deep neural plate cells move medially. The process of neural tube closure leaves a region of the ectoderm overlying the neural crest transiently bare of fibrils. Fibrils are assembled surrounding the dorsal surface of the neural tube as the neural tube lumen is restored. Copyright (c) 2004 Wiley-Liss, Inc.

Protection of SK-N-MC cells against β-amyloid peptide-induced degeneration using neuron growth factor-loaded liposomes with surface lactoferrin.

PubMed

Kuo, Yung-Chih; Wang, Cheng-Ting

2014-07-01

A liposomal system with surface lactoferrin (Lf) was developed for delivering neuron growth factor (NGF) across the blood-brain barrier (BBB) and improving the viability of neuron-like SK-N-MC cells with deposited β-amyloid peptide (Aβ). The Lf-grafted liposomes carrying NGF (Lf/NGF-liposomes) were applied to a monolayer of human brain-microvascular endothelial cells (HBMECs) regulated by human astrocytes (HAs) and to fibrillar Aβ1-42-insulted SK-N-MC cells. An increase in cholesterol mole percentage enhanced the particle size, absolute value of zeta potential, and physical stability, however, reduced the entrapment efficiency and release rate of NGF. In addition, an increase in Lf concentration increased the particle size, surface nitrogen percentage, NGF permeability across the BBB, and viability of HBMECs, HAs, and SK-N-MC cells, however, decreased the absolute value of zeta potential, surface phosphorus percentage, and loading efficiency of Lf. After treating with Lf/NGF-liposomes, a higher Aβ concentration yielded a lower survival of SK-N-MC cells. The current Lf/NGF-liposomes are efficacious drug carriers to target the BBB and inhibit the Aβ-induced neurotoxicity as potential pharmacotherapy for Alzheimer's disease. Copyright © 2014 Elsevier Ltd. All rights reserved.

Nanomechanical properties of α-synuclein amyloid fibrils: a comparative study by nanoindentation, harmonic force microscopy, and Peakforce QNM

PubMed Central

2011-01-01

We report on the use of three different atomic force spectroscopy modalities to determine the nanomechanical properties of amyloid fibrils of the human α-synuclein protein. α-Synuclein forms fibrillar nanostructures of approximately 10 nm diameter and lengths ranging from 100 nm to several microns, which have been associated with Parkinson's disease. Atomic force microscopy (AFM) has been used to image the morphology of these protein fibrils deposited on a flat surface. For nanomechanical measurements, we used single-point nanoindentation, in which the AFM tip as the indenter is moved vertically to the fibril surface and back while the force is being recorded. We also used two recently developed AFM surface property mapping techniques: Harmonic force microscopy (HarmoniX) and Peakforce QNM. These modalities allow extraction of mechanical parameters of the surface with a lateral resolution and speed comparable to tapping-mode AFM imaging. Based on this phenomenological study, the elastic moduli of the α-synuclein fibrils determined using these three different modalities are within the range 1.3-2.1 GPa. We discuss the relative merits of these three methods for the determination of the elastic properties of protein fibrils, particularly considering the differences and difficulties of each method. PMID:21711775

Mechanical behaviour of staggered array of mineralised collagen fibrils in protein matrix: Effects of fibril dimensions and failure energy in protein matrix.

PubMed

Lai, Zheng Bo; Yan, Cheng

2017-01-01

Many biological composite materials such as bone have demonstrated unique mechanical performance, i.e., a combination of superior stiffness and toughness. It has become increasingly clear that the constituents at the nano- and micro-length scales play a critical role in determining the mechanical performance of these biological composites. In this study, the underlying mechanisms governing the mechanical behaviour of the staggered array of mineralised collagen fibrils (MCF) embedded in extra-fibrillar protein matrix were numerically investigated. The evolution of damage zone in protein was estimated using cohesive zone models (CZM). The results indicate that the mechanisms and mechanical behaviour of MCF array are largely dependent on the MCF dimensions and the intrinsic failure energy in extra-fibrillar protein matrix. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fibrillar Organic Phases And Their Roles In Rigid Biological Composites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arey, Bruce W.; Park, John J.; Mayer, George

2015-06-01

This study focused on determining the presence of organic phases in the siliceous components of rigid marine composites ("glass" sponge spicules), and thereby to clarify how those composites dissipate significant mechanical energy. Through the use of imaging by helium ion microscopy in the examination of the spicules, the organic phase that is present between the layers of hydrated silica was also detected within the silica cylinders of the composite, indicating the existence therein of a network, scaffolding, or other pattern that has not yet been determined. It was concluded that the presence of an interpenetrating network of some kind, andmore » tenacious fibrillar interfaces are responsible for the large energy dissipation in these siliceous composites by viscoelastic processes. This discovery means that future mechanics analyses of such composites, extending to large deformations must consider such interpenetrating phases.« less

Electron Microscopy Analysis of the Nucleolus of Trypanosoma cruzi

NASA Astrophysics Data System (ADS)

López-Velázquez, Gabriel; Hernández, Roberto; López-Villaseñor, Imelda; Reyes-Vivas, Horacio; Segura-Valdez, María De L.; Jiménez-García, Luis F.

2005-08-01

The nucleolus is the main site for synthesis and processing of ribosomal RNA in eukaryotes. In mammals, plants, and yeast the nucleolus has been extensively characterized by electron microscopy, but in the majority of the unicellular eukaryotes no such studies have been performed. Here we used ultrastructural cytochemical and immunocytochemical techniques as well as three-dimensional reconstruction to analyze the nucleolus of Trypanosoma cruzi, which is an early divergent eukaryote of medical importance. In T. cruzi epimastigotes the nucleolus is a spherical intranuclear ribonucleoprotein organelle localized in a relatively central position within the nucleus. Dense fibrillar and granular components but not fibrillar centers were observed. In addition, nuclear bodies resembling Cajal bodies were observed associated to the nucleolus in the surrounding nucleoplasm. Our results provide additional morphological data to better understand the synthesis and processing of the ribosomal RNA in kinetoplastids.

Preparation of Amyloid Fibrils Seeded from Brain and Meninges.

PubMed

Scherpelz, Kathryn P; Lu, Jun-Xia; Tycko, Robert; Meredith, Stephen C

2016-01-01

Seeding of amyloid fibrils into fresh solutions of the same peptide or protein in disaggregated form leads to the formation of replicate fibrils, with close structural similarity or identity to the original fibrillar seeds. Here we describe procedures for isolating fibrils composed mainly of β-amyloid (Aβ) from human brain and from leptomeninges, a source of cerebral blood vessels, for investigating Alzheimer's disease and cerebral amyloid angiopathy. We also describe methods for seeding isotopically labeled, disaggregated Aβ peptide solutions for study using solid-state NMR and other techniques. These methods should be applicable to other types of amyloid fibrils, to Aβ fibrils from mice or other species, tissues other than brain, and to some non-fibrillar aggregates. These procedures allow for the examination of authentic amyloid fibrils and other protein aggregates from biological tissues without the need for labeling the tissue.

Structural transition of glucagon in the concentrated solution observed by electrophoretic and spectroscopic techniques.

PubMed

Onoue, Satomi; Iwasa, Sumiko; Kojima, Takashi; Katoh, Fumie; Debari, Kazuhiro; Koh, Keitatsu; Matsuda, Yoshihisa; Yajima, Takehiko

2006-03-24

Glucagon, a polypeptide hormone consisting of 29 amino acid residues, tends to form gel-like fibrillar aggregates, and the glucagon fibril, as well as other pathologically related fibrils including prion, amylin, and beta-amyloid, have been found to be cytotoxic through the activation of apoptotic signaling pathways. To understand the aggregation properties of glucagon fibril, we have characterized and compared the physicochemical properties of glucagon, secretin, a member of the glucagon superfamily, and amylin using analytical techniques including capillary electrophoresis (CE), circular dichroism (CD), FT-IR, FT-Raman, transmission electron microscopy (TEM), and beta-sheet-imaging probe. Aging treatment of glucagon resulted in the formation of fibrillar aggregates in time- and concentration-dependent manner, and FT-IR and FT-Raman analyses showed the spectral shift of amide I band, suggesting the conformational changes from alpha-helix to beta-sheet structure. Interestingly, secretin, having high sequential and secondary structural homology with glucagon, did not generate the fibrillar aggregates at the conditions tested. In addition, we evaluated the association state of glucagon at various pHs raging from pH 2.0 to 3.5 using CE. Based on the CE data, the rate constants of glucagon aggregation were calculated to be 0.002 +/- 0.004/h and 0.080 +/- 0.011/h for aging at pH 2.0 and 3.5, respectively, suggesting the pH dependence of self-association. CE showed the potential to separate and detect the glucagon aggregates and intermediates during aging process.

Measurement of the quadratic hyperpolarizability of the collagen triple helix and application to second harmonic imaging of natural and biomimetic collagenous tissues

NASA Astrophysics Data System (ADS)

Deniset-Besseau, A.; Strupler, M.; Duboisset, J.; De Sa Peixoto, P.; Benichou, E.; Fligny, C.; Tharaux, P.-L.; Mosser, G.; Brevet, P.-F.; Schanne-Klein, M.-C.

2009-09-01

Collagen is a major protein of the extracellular matrix that is characterized by triple helical domains. It plays a central role in the formation of fibrillar and microfibrillar networks, basement membranes, as well as other structures of the connective tissue. Remarkably, fibrillar collagen exhibits efficient Second Harmonic Generation (SHG) so that SHG microscopy proved to be a sensitive tool to probe the three-dimensional architecture of fibrillar collagen and to assess the progression of fibrotic pathologies. We obtained sensitive and reproducible measurements of the fibrosis extent, but we needed quantitative data at the molecular level to further process SHG images. We therefore performed Hyper- Rayleigh Scattering (HRS) experiments and measured a second order hyperpolarisability of 1.25 10-27 esu for rat-tail type I collagen. This value is surprisingly large considering that collagen presents no strong harmonophore in its aminoacid sequence. In order to get insight into the physical origin of this nonlinear process, we performed HRS measurements after denaturation of the collagen triple helix and for a collagen-like short model peptide [(Pro-Pro- Gly)10]3. It showed that the collagen large nonlinear response originates in the tight alignment of a large number of weakly efficient harmonophores, presumably the peptide bonds, resulting in a coherent amplification of the nonlinear signal along the triple helix. To illustrate this mechanism, we successfully recorded SHG images in collagenous biomimetic matrices.

Microrheological Characterization of Collagen Systems: From Molecular Solutions to Fibrillar Gels

PubMed Central

Shayegan, Marjan; Forde, Nancy R.

2013-01-01

Collagen is the most abundant protein in the extracellular matrix (ECM), where its structural organization conveys mechanical information to cells. Using optical-tweezers-based microrheology, we investigated mechanical properties both of collagen molecules at a range of concentrations in acidic solution where fibrils cannot form and of gels of collagen fibrils formed at neutral pH, as well as the development of microscale mechanical heterogeneity during the self-assembly process. The frequency scaling of the complex shear modulus even at frequencies of ∼10 kHz was not able to resolve the flexibility of collagen molecules in acidic solution. In these solutions, molecular interactions cause significant transient elasticity, as we observed for 5 mg/ml solutions at frequencies above ∼200 Hz. We found the viscoelasticity of solutions of collagen molecules to be spatially homogeneous, in sharp contrast to the heterogeneity of self-assembled fibrillar collagen systems, whose elasticity varied by more than an order of magnitude and in power-law behavior at different locations within the sample. By probing changes in the complex shear modulus over 100-minute timescales as collagen self-assembled into fibrils, we conclude that microscale heterogeneity appears during early phases of fibrillar growth and continues to develop further during this growth phase. Experiments in which growing fibrils dislodge microspheres from an optical trap suggest that fibril growth is a force-generating process. These data contribute to understanding how heterogeneities develop during self-assembly, which in turn can help synthesis of new materials for cellular engineering. PMID:23936454

Tensile properties in collagen-rich tissues of Quarter Horses with hereditary equine regional dermal asthenia (HERDA).

PubMed

Bowser, J E; Elder, S H; Pasquali, M; Grady, J G; Rashmir-Raven, A M; Wills, R; Swiderski, C E

2014-03-01

Hereditary equine regional dermal asthenia (HERDA) is an autosomal recessive disorder of Quarter Horses characterised by skin fragility. Horses with HERDA have a missense mutation in peptidyl-prolyl cis-trans isomerase B (PPIB), which encodes cyclophilin B and alters folding and post translational modifications of fibrillar collagen. The study aimed to test the hypothesis that tendons, ligaments and great vessels, which, like skin, are rich in fibrillar collagen, will also have abnormal biomechanical properties in horses with HERDA. Ex vivo biomechanical study comparing horses with and without a diagnosis of HERDA. Forelimb suspensory ligament, superficial and deep digital flexor tendons; withers, forelimb and abdominal skin; the main pulmonary artery and the aortic arch were harvested from 6 horses with HERDA and 6 control horses without the HERDA allele. Tissues were distracted to failure. Tensile strength (TS), elastic modulus (EM) and energy to failure (ETF) were compared. Horses with HERDA had significantly lower TS and EM in tendinoligamentous tissues and great vessels, respectively. The TS, EM and ETF were significantly lower in skin from horses with HERDA. Differences in TS and ETF were more extreme at the withers than at the forelimb or abdomen. Tendinoligamentous tissue, great vessels and skin are significantly weaker in horses with HERDA than in horses lacking the PPIB mutation, substantiating that diverse tissues with high fibrillar collagen content are abnormal in HERDA and that the HERDA phenotype is not limited to the integument. © 2013 EVJ Ltd.

Exploring the aggregation free energy landscape of the amyloid-β protein (1–40)

PubMed Central

Zheng, Weihua; Tsai, Min-Yeh; Chen, Mingchen; Wolynes, Peter G.

2016-01-01

A predictive coarse-grained protein force field [associative memory, water-mediated, structure, and energy model for molecular dynamics (AWSEM)-MD] is used to study the energy landscapes and relative stabilities of amyloid-β protein (1–40) in the monomer and all of its oligomeric forms up to an octamer. We find that an isolated monomer is mainly disordered with a short α-helix formed at the central hydrophobic core region (L17-D23). A less stable hairpin structure, however, becomes increasingly more stable in oligomers, where hydrogen bonds can form between neighboring monomers. We explore the structure and stability of both prefibrillar oligomers that consist of mainly antiparallel β-sheets and fibrillar oligomers with only parallel β-sheets. Prefibrillar oligomers are polymorphic but typically take on a cylindrin-like shape composed of mostly antiparallel β-strands. At the concentration of the simulation, the aggregation free energy landscape is nearly downhill. We use umbrella sampling along a structural progress coordinate for interconversion between prefibrillar and fibrillar forms to identify a conversion pathway between these forms. The fibrillar oligomer only becomes favored over its prefibrillar counterpart in the pentamer where an interconversion bottleneck appears. The structural characterization of the pathway along with statistical mechanical perturbation theory allow us to evaluate the effects of concentration on the free energy landscape of aggregation as well as the effects of the Dutch and Arctic mutations associated with early onset of Alzheimer’s disease. PMID:27698130

Fibrillar Collagen Organization Associated with Broiler Wooden Breast Fibrotic Myopathy.

PubMed

Velleman, Sandra G; Clark, Daniel L; Tonniges, Jeffrey R

2017-12-01

Wooden breast (WB) is a fibrotic myopathy affecting the pectoralis major (p. major) muscle in fast-growing commercial broiler lines. Birds with WB are phenotypically detected by the palpation of a hard p. major muscle. A primary feature of WB is the fibrosis of muscle with the replacement of muscle fibers with extracellular matrix proteins, such as collagen. The ability of a tissue to be pliable and stretch is associated with the organization of collagen fibrils in the connective tissue areas surrounding muscle fiber bundles (perimysium) and around individual muscle fibers (endomysium). The objective of this study was to compare the structure and organization of fibrillar collagen by using transmission electron microscopy in two fast-growing broiler lines (Lines A and B) with incidence of WB to a slower growing broiler Line C with no phenotypically detectable WB. In Line A, the collagen fibrils were tightly packed in a parallel organization, whereas in Line B, the collagen fibrils were randomly aligned. Tightly packed collagen fibrils arranged in parallel are associated with nonpliable collagen that is highly cross-linked. This will lead to a phenotypically hard p. major muscle. In Line C, the fibrillar collagen was sparse in its distribution. Furthermore, the average collagen fibril diameter and banding D-period length were altered in Line A p. major muscles affected with WB. Taken together, these data are suggestive of different fibrotic myopathies beyond just what is classified as WB in fast-growing broiler lines.

Structural change of human hair induced by mercury exposure.

PubMed

Xing, Xueqing; Du, Rong; Li, Yufeng; Li, Bai; Cai, Quan; Mo, Guang; Gong, Yu; Chen, Zhongjun; Wu, Zhonghua

2013-10-01

Mercury is one of the most hazardous pollutants in the environment. In this paper, the structural change of human hair induced by mercury exposure was studied. Human hair samples were, respectively, collected from the normal Beijing area and the Hg-contaminated Wanshan area of the Guizhou Province, China. Inductively coupled plasma mass spectroscopy was used to detect the element contents. A small angle X-ray scattering technique was used to probe the structural change. Three reflections with 8.8, 6.7, and 4.5 nm spacing were compared between the normal and the Hg-contaminated hair samples. The results confirm that the 4.5 nm reflection is from the ordered fibrillar structure of glycosaminoglycan (GAG) in proteoglycan (PG) that composes the matrix around the intermediate filaments. The increase of Ca content makes the regular oriented fibrillar structure of GAG transform to a random oriented one, broadening the angular extent of the reflection with 4.5 nm spacing. However, overdose Hg makes the core proteins where the ordered fibrils of GAG are attached become coiled, which destroys the ordered arrangements of fibrillar GAG in PG, resulting in the disappearance of the reflections with 4.5 nm spacing. The disappearance of the 4.5 nm reflection can be used as a bioindicator of overdose Hg contamination to the human body. A supercoiled-coil model of hair nanoscale structure and a possible mechanism of mercury effect in human hair are proposed in this paper.

LASER METHODS IN BIOLOGY: Optical anisotropy of fibrous biological tissues: analysis of the influence of structural properties

NASA Astrophysics Data System (ADS)

Zimnyakov, D. A.; Sinichkin, Yu P.; Ushakova, O. V.

2007-08-01

The results of theoretical analysis of the optical anisotropy of multiply scattering fibrillar biological tissues based on the model of an effective anisotropic medium are compared with the experimental in vivo birefringence data for the rat derma obtained earlier in spectral polarisation measurements of rat skin samples in the visible region. The disordered system of parallel dielectric cylinders embedded into an isotropic dielectric medium was considered as a model medium. Simulations were performed taking into account the influence of a partial mutual disordering of the bundles of collagen and elastin fibres in derma on birefringence in samples. The theoretical optical anisotropy averaged over the spectral interval 550-650 nm for the model medium with parameters corresponding to the structural parameters of derma is in good agreement with the results of spectral polarisation measurements of skin samples in the corresponding wavelength range.

Molecular, biochemical and functional analysis of a novel and developmentally important fibrillar collagen (Hcol-I) in hydra.

PubMed

Deutzmann, R; Fowler, S; Zhang, X; Boone, K; Dexter, S; Boot-Handford, R P; Rachel, R; Sarras, M P

2000-11-01

The body wall of hydra (a member of the phylum Cnidaria) is structurally reduced to an epithelial bilayer with an intervening extracellular matrix (ECM). Previous studies have established that cell-ECM interactions are important for morphogenesis and cell differentiation in this simple metazoan. The ECM of hydra is particularly interesting because it represents a primordial form of matrix. Despite progress in our understanding of hydra ECM, we still know little about the nature of hydra collagens. In the current study we provide a molecular, biochemical and functional analysis of a hydra fibrillar collagen that has similarity to vertebrate type I and type II collagens. This fibrillar collagen has been named hydra collagen-I (Hcol-I) because of its structure and because it is the first ECM collagen to be identified in hydra. It represents a novel member of the collagen family. Similar to vertebrate type I and II collagens, Hcol-I contains an N-terminal propeptide-like domain, a triple helical domain containing typical Gly-X-Y repeats and a C-terminal propeptide domain. The overall identity to vertebrate fibrillar collagens is about 30%, while the identity of the C-terminal propeptide domain is 50%. Because the N-terminal propeptide domain is retained after post-translational processing, Hcol-I does not form thick fibers as seen in vertebrates. This was confirmed using transmission electron microscopy to study rotary shadow images of purified Hcol-I. In addition, absence of crucial lysine residues and an overall reduction in proline content, results in reduced crosslinking of fibrils and increased flexibility of the molecule, respectively. These structural changes in Hcol-I help to explain the flexible properties of hydra ECM. Immunocytochemical studies indicate that Hcol-I forms the 10 nm fibrils that comprise the majority of molecules in the central fibrous zone of hydra ECM. The central fibrous zone resides between the two subepithelial zones where hydra laminin is localized. While previous studies have shown that basal lamina components like laminin are expressed by the endoderm, in situ hybridisation studies show that Hcol-I mRNA expression is restricted to the ectoderm. Hcol-I expression is upregulated during head regeneration, and antisense studies using thio-oligonucleotides demonstrated that blocking the translation of Hcol-I leads to a reversible inhibition of head morphogenesis during this regenerative process. Taken in total, the data presented in this study indicate that Hcol-I is required for morphogensis in hydra and represents a novel fibrillar collagen whose structural characteristics help to explain the unique biophysical properties of hydra ECM. Interestingly, the structure of Hcol-I mimics what is seen in Ehlers-Danlos syndrome type VII in humans; an inherited pathological condition that leads to joint and skin abnormalities. Hcol-I therefore illustrates an adaptive trait in which the normal physiological situation in hydra translates into a pathological condition in humans.

Fluorescent nanonetworks: A novel bioalley for collagen scaffolds and Tissue Engineering

PubMed Central

Nidhin, Marimuthu; Vedhanayagam, Mohan; Sangeetha, Selvam; Kiran, Manikantan Syamala; Nazeer, Shaiju S.; Jayasree, Ramapurath S.; Sreeram, Kalarical Janardhanan; Nair, Balachandran Unni

2014-01-01

Native collagen is arranged in bundles of aligned fibrils to withstand in vivo mechanical loads. Reproducing such a process under in vitro conditions has not met with major success. Our approach has been to induce nanolinks, during the self-assembly process, leading to delayed rather than inhibited fibrillogenesis. For this, a designed synthesis of nanoparticles - using starch as a template and a reflux process, which would provide a highly anisotropic (star shaped) nanoparticle, with large surface area was adopted. Anisotropy associated decrease in Morin temperature and superparamagnetic behavior was observed. Polysaccharide on the nanoparticle surface provided aqueous stability and low cytotoxicity. Starch coated nanoparticles was utilized to build polysaccharide - collagen crosslinks, which supplemented natural crosslinks in collagen, without disturbing the conformation of collagen. The resulting fibrillar lamellae showed a striking resemblance to native lamellae, but had a melting and denaturation temperature higher than native collagen. The biocompatibility and superparamagnetism of the nanoparticles also come handy in the development of stable collagen constructs for various biomedical applications, including that of MRI contrast agents. PMID:25095810

Ultrastructural analysis of v-myb oncogene product cooperation with components of avian cell nuclear matrix.

PubMed

Korb, J; Stokrová, J; Karafiát, V

2000-01-01

The cooperation of the v-Myb oncoprotein with extracted nuclear matrix of avian haematopoietic cells expressing the v-myb oncogene was studied by means of immunoelectron microscopy. The nuclear matrix was extracted by a gentle method of detergent treatment at moderate ionic strength and visualized either in ultrathin LR White sections, in unembedded resin-free sections, and in addition by the aqueous spreading technique. Using anti-Myb polyclonal antibody we have shown interaction of the v-Myb protein product with extracted nuclear matrix. This oncoprotein, however, was easily released from the structure by a detergent as well as by DNAase treatment and ammonium sulphate extraction. Prefixation of structures before detergent treatment prevented this extraction. The v-Myb protein marker was distributed in clusters or associated with fibrillar structures in most cases. Single markers decorating these fibrillar or less dense structures were also detected.

Fibrillar dimer formation of islet amyloid polypeptides

DOE PAGES

Chiu, Chi -cheng; de Pablo, Juan J.

2015-05-08

Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimentalmore » and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.« less

Further exploration of the conformational space of α-synuclein fibrils: solid-state NMR assignment of a high-pH polymorph.

PubMed

Verasdonck, Joeri; Bousset, Luc; Gath, Julia; Melki, Ronald; Böckmann, Anja; Meier, Beat H

2016-04-01

Polymorphism is a common and important phenomenon for protein fibrils which has been linked to the appearance of strains in prion and other neurodegenerative diseases. Parkinson disease is a frequently occurring neurodegenerative pathology, tightly associated with the formation of Lewy bodies. These deposits mainly consist of α-synuclein in fibrillar, β-sheet-rich form. α-synuclein is known to form numerous different polymorphs, which show distinct structural features. Here, we describe the chemical shift assignments, and derive the secondary structure, of a polymorph that was fibrillized at higher-than-physiological pH conditions. The fibrillar core contains residues 40-95, with both the C- and N-terminus not showing any ordered, rigid parts. The chemical shifts are similar to those recorded previously for an assigned polymorph that was fibrillized at neutral pH.

Immunoelectron microscopy of RNA combined with nucleic acid cytochemistry in plant nucleoli.

PubMed

Mena, C G; Testillano, P S; González-Melendi, P; Gorab, E; Risueño, M C

1994-06-01

The immunoelectron microscopy detection of RNA using anti-RNA monoclonal antibodies has been performed for the first time over different plant cells. The use of the methylation-acetylation (MA) method permits clear distinction among the nuclear and nucleolar compartments and can be combined with the immunogold approach. Cytochemical methods for nucleic acids were performed together with the immunoassays, providing additional data about the different composition of the various nucleolar components. Anti-RNA antibodies highly labeled the ribosome-rich areas of the cytoplasm and the nucleolus. The interchromatin region also is labeled. The labeling was intense in the granular component, lower in the dense fibrillar component, and very scarce in the fibrillar centers. The MA method made possible the statistical evaluation of the labeling density in the various nuclear compartments by permitting the clear assignment of the particles to precise nuclear structures.

Clusterin promotes amyloid plaque formation and is critical for neuritic toxicity in a mouse model of Alzheimer's disease

PubMed Central

DeMattos, Ronald B.; O'dell, Mark A.; Parsadanian, Maia; Taylor, Jennie W.; Harmony, Judith A. K.; Bales, Kelly R.; Paul, Steven M.; Aronow, Bruce J.; Holtzman, David M.

2002-01-01

Studies have shown that clusterin (also called apolipoprotein J) can influence the structure and toxicity of amyloid-β (Aβ) in vitro. To determine whether endogenous clusterin plays a role in influencing Aβ deposition, structure, and toxicity in vivo, we bred PDAPP mice, a transgenic mouse model of Alzheimer's disease, to clusterin−/− mice. By 12 months of age, PDAPP, clusterin−/− mice had similar levels of brain Aβ deposition as did PDAPP, clusterin+/+ mice. Although Aβ deposition was similar, PDAPP, clusterin−/− mice had significantly fewer fibrillar Aβ (amyloid) deposits than PDAPP mice expressing clusterin. In the absence of clusterin, neuritic dystrophy associated with the deposited amyloid was markedly reduced, resulting in a dissociation between fibrillar amyloid formation and neuritic dystrophy. These findings demonstrate that clusterin markedly influences Aβ structure and neuritic toxicity in vivo and is likely to play an important role in Alzheimer's disease pathogenesis. PMID:12145324

Internal strain drives spontaneous periodic buckling in collagen and regulates remodeling.

PubMed

Dittmore, Andrew; Silver, Jonathan; Sarkar, Susanta K; Marmer, Barry; Goldberg, Gregory I; Neuman, Keir C

2016-07-26

Fibrillar collagen, an essential structural component of the extracellular matrix, is remarkably resistant to proteolysis, requiring specialized matrix metalloproteinases (MMPs) to initiate its remodeling. In the context of native fibrils, remodeling is poorly understood; MMPs have limited access to cleavage sites and are inhibited by tension on the fibril. Here, single-molecule recordings of fluorescently labeled MMPs reveal cleavage-vulnerable binding regions arrayed periodically at ∼1-µm intervals along collagen fibrils. Binding regions remain periodic even as they migrate on the fibril, indicating a collective process of thermally activated and self-healing defect formation. An internal strain relief model involving reversible structural rearrangements quantitatively reproduces the observed spatial patterning and fluctuations of defects and provides a mechanism for tension-dependent stabilization of fibrillar collagen. This work identifies internal-strain-driven defects that may have general and widespread regulatory functions in self-assembled biological filaments.

Collagens--structure, function, and biosynthesis.

PubMed

Gelse, K; Pöschl, E; Aigner, T

2003-11-28

The extracellular matrix represents a complex alloy of variable members of diverse protein families defining structural integrity and various physiological functions. The most abundant family is the collagens with more than 20 different collagen types identified so far. Collagens are centrally involved in the formation of fibrillar and microfibrillar networks of the extracellular matrix, basement membranes as well as other structures of the extracellular matrix. This review focuses on the distribution and function of various collagen types in different tissues. It introduces their basic structural subunits and points out major steps in the biosynthesis and supramolecular processing of fibrillar collagens as prototypical members of this protein family. A final outlook indicates the importance of different collagen types not only for the understanding of collagen-related diseases, but also as a basis for the therapeutical use of members of this protein family discussed in other chapters of this issue.

Cellulose fibres, nanofibrils and microfibrils: The morphological sequence of MFC components from a plant physiology and fibre technology point of view

NASA Astrophysics Data System (ADS)

Chinga-Carrasco, Gary

2011-06-01

During the last decade, major efforts have been made to develop adequate and commercially viable processes for disintegrating cellulose fibres into their structural components. Homogenisation of cellulose fibres has been one of the principal applied procedures. Homogenisation has produced materials which may be inhomogeneous, containing fibres, fibres fragments, fibrillar fines and nanofibrils. The material has been denominated microfibrillated cellulose (MFC). In addition, terms relating to the nano-scale have been given to the MFC material. Several modern and high-tech nano-applications have been envisaged for MFC. However, is MFC a nano-structure? It is concluded that MFC materials may be composed of (1) nanofibrils, (2) fibrillar fines, (3) fibre fragments and (4) fibres. This implies that MFC is not necessarily synonymous with nanofibrils, microfibrils or any other cellulose nano-structure. However, properly produced MFC materials contain nano-structures as a main component, i.e. nanofibrils.

Hydrogen-bonding A(LS)2-type low-molecular-mass gelator and its thermotropic mesomorphic behavior.

PubMed

Hou, Qiufei; Wang, Shichao; Zang, Libin; Wang, Xiaoliang; Jiang, Shimei

2009-10-15

A unique cholesterol-based A(LS)2-type gelator, which is a hydrogen-bonding complex based on an ALS-type non-gelator molecule 3-cholesteryl 4-(trans-2-(4-pyridinyl)vinyl)phenyl succinate and a counterpart 3-cholesteryloxycarbonylpropanoic acid, shows strong gelation ability in alcohol and aromatic solvents. The formed gel has a high Tg at low gelation concentration, and its xerogel shows fibrillar microstructure revealed by scanning electron microscopy (SEM). FTIR confirms the existence of intermolecular hydrogen bond in the gelator, and X-ray diffraction (XRD) analysis reveals that the gelator possesses a folded conformation in gel and self-assembles into the fibrillar structure mainly by van der Waals interaction between cholesteryl moieties of the gelator. Further more, the thermotropic behavior of the xerogel is studied by differential scanning calorimetry (DSC) and polarized optical microscopy (POM), which shows typical optical textures of liquid crystals.

Ultrastructure of the synovial membrane in seronegative inflammatory arthropathies.

PubMed Central

Morris, C J; Farr, M; Hollywell, C A; Hawkins, C F; Scott, D L; Walton, K W

1983-01-01

The ultrastructure of the synovial membrane has been studied in 6 patients with seronegative inflammatory arthropathies: Reiter's (2), Crohn's (2), Whipple's (1) and Behçet's disease (1). The most striking changes were found in the synovial B cells, many containing abnormally large mitochondria with altered cristae surrounded by fibrillar material. Similar material was present in dilated endoplasmic reticulum which was the probable source of groups of extracellular fibrillar spheroidal bodies. The B cells also contained electron dense granular lysosomes of very variable size which, in common with the abnormal mitochondria, were often associated with bundles of orientated microfilaments and large golgi complexes. Light microscopy of the synovial membrane was consistent with an inflammatory arthritis, as were the high white cell counts in the synovial fluid. Systemic activity in the patients was indicated by raised ESR and C-reactive protein (CRP). Images Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. A Figure 5. B PMID:6186810

Interfibrillar stiffening of echinoderm mutable collagenous tissue demonstrated at the nanoscale

PubMed Central

Mo, Jingyi; Blowes, Liisa M.; Egertová, Michaela; Terrill, Nicholas J.; Wang, Wen; Elphick, Maurice R.; Gupta, Himadri S.

2016-01-01

The mutable collagenous tissue (MCT) of echinoderms (e.g., sea cucumbers and starfish) is a remarkable example of a biological material that has the unique attribute, among collagenous tissues, of being able to rapidly change its stiffness and extensibility under neural control. However, the mechanisms of MCT have not been characterized at the nanoscale. Using synchrotron small-angle X-ray diffraction to probe time-dependent changes in fibrillar structure during in situ tensile testing of sea cucumber dermis, we investigate the ultrastructural mechanics of MCT by measuring fibril strain at different chemically induced mechanical states. By measuring a variable interfibrillar stiffness (EIF), the mechanism of mutability at the nanoscale can be demonstrated directly. A model of stiffness modulation via enhanced fibrillar recruitment is developed to explain the biophysical mechanisms of MCT. Understanding the mechanisms of MCT quantitatively may have applications in development of new types of mechanically tunable biomaterials. PMID:27708167

Interfibrillar stiffening of echinoderm mutable collagenous tissue demonstrated at the nanoscale.

PubMed

Mo, Jingyi; Prévost, Sylvain F; Blowes, Liisa M; Egertová, Michaela; Terrill, Nicholas J; Wang, Wen; Elphick, Maurice R; Gupta, Himadri S

2016-10-18

The mutable collagenous tissue (MCT) of echinoderms (e.g., sea cucumbers and starfish) is a remarkable example of a biological material that has the unique attribute, among collagenous tissues, of being able to rapidly change its stiffness and extensibility under neural control. However, the mechanisms of MCT have not been characterized at the nanoscale. Using synchrotron small-angle X-ray diffraction to probe time-dependent changes in fibrillar structure during in situ tensile testing of sea cucumber dermis, we investigate the ultrastructural mechanics of MCT by measuring fibril strain at different chemically induced mechanical states. By measuring a variable interfibrillar stiffness (E IF ), the mechanism of mutability at the nanoscale can be demonstrated directly. A model of stiffness modulation via enhanced fibrillar recruitment is developed to explain the biophysical mechanisms of MCT. Understanding the mechanisms of MCT quantitatively may have applications in development of new types of mechanically tunable biomaterials.

Internal strain drives spontaneous periodic buckling in collagen and regulates remodeling

PubMed Central

Dittmore, Andrew; Silver, Jonathan; Sarkar, Susanta K.; Marmer, Barry; Goldberg, Gregory I.; Neuman, Keir C.

2016-01-01

Fibrillar collagen, an essential structural component of the extracellular matrix, is remarkably resistant to proteolysis, requiring specialized matrix metalloproteinases (MMPs) to initiate its remodeling. In the context of native fibrils, remodeling is poorly understood; MMPs have limited access to cleavage sites and are inhibited by tension on the fibril. Here, single-molecule recordings of fluorescently labeled MMPs reveal cleavage-vulnerable binding regions arrayed periodically at ∼1-µm intervals along collagen fibrils. Binding regions remain periodic even as they migrate on the fibril, indicating a collective process of thermally activated and self-healing defect formation. An internal strain relief model involving reversible structural rearrangements quantitatively reproduces the observed spatial patterning and fluctuations of defects and provides a mechanism for tension-dependent stabilization of fibrillar collagen. This work identifies internal–strain-driven defects that may have general and widespread regulatory functions in self-assembled biological filaments. PMID:27402741

Red cell membrane skeleton: structure-function relationships.

PubMed

Palek, J; Liu, S C

1980-01-01

This papaer reviews our present understanding of ultrastructure, organization, and functional characteristics of the erythrocyte membrane cytoskeleton. This two-dimensional fibrillar network of submembrane proteins can be visualized after extraction of lipids and integral membrane proteins by Triton X-100. Current data suggest that the major structural components of the cytoskeleton are heterodimers of double-stranded spectrin that form tetramers by head-to-head associations. The tetramers may be connected into a fibrillar meshwork by oligomers of actin. The control of membrane integrity by this network is illustrated by examples of two hemolyotic anemias characterized by marked membrane instability and vesiculation: 1) hereditary spherocytic anemia of the house mouse associated with spectrin deficiency and 2) hereditary pyropoikilocytosis, a hemolytic anemia in man characterized by thermal instability of the membrane and the presence of abnormal spectrin, which exhibits an increased propensity to thermal denaturation. Stabilization of the cytoskeletal network by covalent cross-links between the nearest cytoskeletal and integral membrane proteins results in a decrease of membrane deformability and a fixation of erythrocytes in their abnormal shape. Such cross-linkings include: 1) transamidative cross-links produced by introduction of Ca2+ (>0.5 mM) into fresh erythrocytes, and 2) intermolecular disulfide couplings, which are formed after extensive oxidation of fresh erythrocytes or after mild oxidation of ATP-depleted, but not fresh, erythrocytes. The significance of these cross-links in stabilization of shape of abnormal erythrocytes such as schistocytes remains to be determined. We conclude that spectrin and actin form a fibrillar submembrane network that plays an important role in control of membrane integrity, erythrocyte deformability, and stabilization of cells in abnormal shapes.

Enhanced neuroinvasion by smaller, soluble prions.

PubMed

Bett, Cyrus; Lawrence, Jessica; Kurt, Timothy D; Orru, Christina; Aguilar-Calvo, Patricia; Kincaid, Anthony E; Surewicz, Witold K; Caughey, Byron; Wu, Chengbiao; Sigurdson, Christina J

2017-04-21

Infectious prion aggregates can propagate from extraneural sites into the brain with remarkable efficiency, likely transported via peripheral nerves. Yet not all prions spread into the brain, and the physical properties of a prion that is capable of transit within neurons remain unclear. We hypothesized that small, diffusible aggregates spread into the CNS via peripheral nerves. Here we used a structurally diverse panel of prion strains to analyze how the prion conformation impacts transit into the brain. Two prion strains form fibrils visible ultrastructurally in the brain in situ, whereas three strains form diffuse, subfibrillar prion deposits and no visible fibrils. The subfibrillar strains had significantly higher levels of soluble prion aggregates than the fibrillar strains. Primary neurons internalized both the subfibrillar and fibril-forming prion strains by macropinocytosis, and both strain types were transported from the axon terminal to the cell body in vitro. However in mice, only the predominantly soluble, subfibrillar prions, and not the fibrillar prions, were efficiently transported from the tongue to the brain. Sonicating a fibrillar prion strain increased the solubility and enabled prions to spread into the brain in mice, as evident by a 40% increase in the attack rate, indicating that an increase in smaller particles enhances prion neuroinvasion. Our data suggest that the small, highly soluble prion particles have a higher capacity for transport via nerves. These findings help explain how prions that predominantly assemble into subfibrillar states can more effectively traverse into and out of the CNS, and suggest that promoting fibril assembly may slow the neuron-to-neuron spread of protein aggregates.

Impact of Ionic Liquids on the Structure and Dynamics of Collagen.

PubMed

Tarannum, Aafiya; Adams, Alina; Blümich, Bernhard; Fathima, Nishter Nishad

2018-01-25

The changes in the structure and dynamics of collagen treated with two different classes of ionic liquids, bis-choline sulfate (CS) and 1-butyl-3-methyl imidazolium dimethyl phosphate (IDP), have been studied at the molecular and fibrillar levels. At the molecular level, circular dichroic studies revealed an increase in molar ellipticity values for CS when compared with native collagen, indicating cross-linking, albeit pronounced conformational changes for IDP were witnessed indicating denaturation. The impedance was analyzed to correlate the conformational changes with the hydration dynamics of protein. Changes in the dielectric properties of collagen observed upon treatment with CS and IDP reported molecular reorientation in the surrounding water milieu, suggesting compactness or destabilization of the collagen. This was further confirmed by proton transverse NMR relaxation time measurements, which demonstrated that the water mobility changes in the presence of the ILs. At the fibrillar level, differential scanning calorimetry thermograms for rat tail tendon collagen fibers treated with CS show a 5 °C increase in denaturation temperature, suggesting imparted stability. On the contrary, a significant temperature decrease was noticed for IDP, indicating the destabilization of collagen fibers. The obtained results clearly indicate that the changes in the secondary structure of protein are due to the changes in the hydration dynamics of collagen upon interaction with ILs. Thus, this study on the interaction of collagen with ionic liquids unfolds the propensity of ILs to stabilize or destabilize collagen depending on the changes invoked at the molecular level in terms of structure and dynamics of protein, which also got manifested at the fibrillar level.

DEVELOPMENT OF RADIATION PNEUMONITIS TIME AND DOSE FACTORS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jennings, F.L.; Arden, A.

1962-10-01

Histologic evaluation of the lungs was done at autopsy in 215 patients who had received thoracic radiation. The presence or absence of tissue changes noted were: edema, congestion, atelectasis, fibrin exudate in alveoli, epithelial changes, fibrillar thickening of alveolar septa, increased cellularity of alveolar septa, fibrosis of alveolar septa, and proliferative changes in blood vessels. Some or all of the tissue changes listed were found in 165 cases. Two changes were particularly frequent: accumulation of a fibrin-rich exudate within alveoli, often forming a membrane in the lation of fibrillar material, by cellular proliferation, or by increase of fibrous tissue. Suchmore » fibrin accumulations were found at radiation doses below 500 r and in excess of 6000 r, and at postirradiation intervals of less than 30 days and over 5 yr. Fibrin accumulations formed within 90 days after less than 1500-r x-ray exposure were similar to those seen almost 9 yr after 5000 r. Three separate types of proliferative changes in the connective tissue of the alveolar septa appeared to be secondary to radiation; 27% showed a fibrillar deposit in the septa which tended to thicken the septa markedly. The time interval appeared to make little difference in the development of this lesion. Increased septal cellularity, due primarily to accumulation of histiocytes and fibroblasts, was seen in 18% of the cases, most commonly at doses between 2000 r and 5000 r. Fibrosis of alveolar septa was the third type of proliferative change and was seen in 42% of the cases. (P.C.H.)« less

Emergence of the interplay between hierarchy and contact splitting in biological adhesion highlighted through a hierarchical shear lag model.

PubMed

Brely, Lucas; Bosia, Federico; Pugno, Nicola M

2018-06-20

Contact unit size reduction is a widely studied mechanism as a means to improve adhesion in natural fibrillar systems, such as those observed in beetles or geckos. However, these animals also display complex structural features in the way the contact is subdivided in a hierarchical manner. Here, we study the influence of hierarchical fibrillar architectures on the load distribution over the contact elements of the adhesive system, and the corresponding delamination behaviour. We present an analytical model to derive the load distribution in a fibrillar system loaded in shear, including hierarchical splitting of contacts, i.e. a "hierarchical shear-lag" model that generalizes the well-known shear-lag model used in mechanics. The influence on the detachment process is investigated introducing a numerical procedure that allows the derivation of the maximum delamination force as a function of the considered geometry, including statistical variability of local adhesive energy. Our study suggests that contact splitting generates improved adhesion only in the ideal case of extremely compliant contacts. In real cases, to produce efficient adhesive performance, contact splitting needs to be coupled with hierarchical architectures to counterbalance high load concentrations resulting from contact unit size reduction, generating multiple delamination fronts and helping to avoid detrimental non-uniform load distributions. We show that these results can be summarized in a generalized adhesion scaling scheme for hierarchical structures, proving the beneficial effect of multiple hierarchical levels. The model can thus be used to predict the adhesive performance of hierarchical adhesive structures, as well as the mechanical behaviour of composite materials with hierarchical reinforcements.

Statistical physics approaches to Alzheimer's disease

NASA Astrophysics Data System (ADS)

Peng, Shouyong

Alzheimer's disease (AD) is the most common cause of late life dementia. In the brain of an AD patient, neurons are lost and spatial neuronal organizations (microcolumns) are disrupted. An adequate quantitative analysis of microcolumns requires that we automate the neuron recognition stage in the analysis of microscopic images of human brain tissue. We propose a recognition method based on statistical physics. Specifically, Monte Carlo simulations of an inhomogeneous Potts model are applied for image segmentation. Unlike most traditional methods, this method improves the recognition of overlapped neurons, and thus improves the overall recognition percentage. Although the exact causes of AD are unknown, as experimental advances have revealed the molecular origin of AD, they have continued to support the amyloid cascade hypothesis, which states that early stages of aggregation of amyloid beta (Abeta) peptides lead to neurodegeneration and death. X-ray diffraction studies reveal the common cross-beta structural features of the final stable aggregates-amyloid fibrils. Solid-state NMR studies also reveal structural features for some well-ordered fibrils. But currently there is no feasible experimental technique that can reveal the exact structure or the precise dynamics of assembly and thus help us understand the aggregation mechanism. Computer simulation offers a way to understand the aggregation mechanism on the molecular level. Because traditional all-atom continuous molecular dynamics simulations are not fast enough to investigate the whole aggregation process, we apply coarse-grained models and discrete molecular dynamics methods to increase the simulation speed. First we use a coarse-grained two-bead (two beads per amino acid) model. Simulations show that peptides can aggregate into multilayer beta-sheet structures, which agree with X-ray diffraction experiments. To better represent the secondary structure transition happening during aggregation, we refine the model to four beads per amino acid. Typical essential interactions, such as backbone hydrogen bond, hydrophobic and electrostatic interactions, are incorporated into our model. We study the aggregation of Abeta16-22, a peptide that can aggregate into a well-ordered fibrillar structure in experiments. Our results show that randomly-oriented monomers can aggregate into fibrillar subunits, which agree not only with X-ray diffraction experiments but also with solid-state NMR studies. Our findings demonstrate that coarse-grained models and discrete molecular dynamics simulations can help researchers understand the aggregation mechanism of amyloid peptides.

Study of the Elasto-plastic Properties of Mineralized Biomaterials via Synchrotron High-energy X-ray Diffraction

NASA Astrophysics Data System (ADS)

Deymier-Black, Alix Christine

Synchrotron high-energy X-ray diffraction was employed to investigate the strains in the hydroxyapatite (HAP) platelets and mineralized collagen fibrils in bovine dentin and cortical bone. The HAP and the fibrillar apparent moduli, defined as the applied stress divided by the phase strain, in dentin were measured as 27+/-7.2 and 16+/-4.9 GPa. The HAP apparent modulus ( EHAPapp ) is less than the lower bound calculated for EHAPapp from the Voigt model. This discrepancy is probably due to stress concentrators or decreases in the HAP Young's modulus due to size or composition effects. EHAPapp and Efibapp in dentin vary significantly within a single tooth in both the apical-cervical direction and the buccal-lingual direction. However, the variation between teeth is minimal. The HAP and fibrillar apparent moduli are not affected by freezing in dentin or by X-ray irradiation in bone and dentin. X-ray irradiation causes a decrease in HAP residual strain in bone. This decrease suggests the presence of HAP-collagen interfacial damage. It was determined from the HAP 00.2 peak broadening that irradiation damage mostly affects the HAP unit cells which are under the highest strain. From this it was theorized that irradiation may damage highly-strained bonds at stress concentrators and/or calcium-mediated electrostatic bonds. The fact that the apparent modulus does not change with irradiation suggests that the interfacial damage must be reversible. Bone and dentin both undergo creep when loaded to high stresses. At low irradiation doses, both the fibrillar and HAP strains increase with creep time indicating that load is being transferred from the matrix to the HAP. However, at high doses, the strain on the HAP decreases with creep time. This supports the interfacial damage theory which would allow the HAP to release its elastic load upon interfacial debonding. At -80 MPa, beyond a dose of 50 kGy, the rate of change in HAP strain with time begins to increase, becoming positive at ˜115 kGy. After 300 kGy the HAP strain rate decreases and plateaus probably due to stiffening of the matrix through cross-linking. The HAP and fibrillar strain rate in irradiated bone and dentin samples increase with increased temperature and applied load.

Structural changes of TasA in biofilm formation of Bacillus subtilis.

PubMed

Diehl, Anne; Roske, Yvette; Ball, Linda; Chowdhury, Anup; Hiller, Matthias; Molière, Noel; Kramer, Regina; Stöppler, Daniel; Worth, Catherine L; Schlegel, Brigitte; Leidert, Martina; Cremer, Nils; Erdmann, Natalja; Lopez, Daniel; Stephanowitz, Heike; Krause, Eberhard; van Rossum, Barth-Jan; Schmieder, Peter; Heinemann, Udo; Turgay, Kürşad; Akbey, Ümit; Oschkinat, Hartmut

2018-03-27

Microorganisms form surface-attached communities, termed biofilms, which can serve as protection against host immune reactions or antibiotics. Bacillus subtilis biofilms contain TasA as major proteinaceous component in addition to exopolysaccharides. In stark contrast to the initially unfolded biofilm proteins of other bacteria, TasA is a soluble, stably folded monomer, whose structure we have determined by X-ray crystallography. Subsequently, we characterized in vitro different oligomeric forms of TasA by NMR, EM, X-ray diffraction, and analytical ultracentrifugation (AUC) experiments. However, by magic-angle spinning (MAS) NMR on live biofilms, a swift structural change toward only one of these forms, consisting of homogeneous and protease-resistant, β-sheet-rich fibrils, was observed in vivo. Thereby, we characterize a structural change from a globular state to a fibrillar form in a functional prokaryotic system on the molecular level. Copyright © 2018 the Author(s). Published by PNAS.

Structural changes of TasA in biofilm formation of Bacillus subtilis

PubMed Central

Diehl, Anne; Roske, Yvette; Ball, Linda; Chowdhury, Anup; Hiller, Matthias; Molière, Noel; Kramer, Regina; Stöppler, Daniel; Worth, Catherine L.; Schlegel, Brigitte; Leidert, Martina; Cremer, Nils; Erdmann, Natalja; Lopez, Daniel; Stephanowitz, Heike; Krause, Eberhard; Schmieder, Peter; Akbey, Ümit; Oschkinat, Hartmut

2018-01-01

Microorganisms form surface-attached communities, termed biofilms, which can serve as protection against host immune reactions or antibiotics. Bacillus subtilis biofilms contain TasA as major proteinaceous component in addition to exopolysaccharides. In stark contrast to the initially unfolded biofilm proteins of other bacteria, TasA is a soluble, stably folded monomer, whose structure we have determined by X-ray crystallography. Subsequently, we characterized in vitro different oligomeric forms of TasA by NMR, EM, X-ray diffraction, and analytical ultracentrifugation (AUC) experiments. However, by magic-angle spinning (MAS) NMR on live biofilms, a swift structural change toward only one of these forms, consisting of homogeneous and protease-resistant, β-sheet–rich fibrils, was observed in vivo. Thereby, we characterize a structural change from a globular state to a fibrillar form in a functional prokaryotic system on the molecular level. PMID:29531041

Hierarchical and non-hierarchical mineralisation of collagen

PubMed Central

Liu, Yan; Kim, Young-Kyung; Dai, Lin; Li, Nan; Khan, Sara; Pashley, David H.; Tay, Franklin R.

2010-01-01

Biomineralisation of collagen involves functional motifs incorporated in extracellular matrix protein molecules to accomplish the objectives of stabilising amorphous calcium phosphate into nanoprecursors and directing the nucleation and growth of apatite within collagen fibrils. Here we report the use of small inorganic polyphosphate molecules to template hierarchical intrafibrillar apatite assembly in reconstituted collagen in the presence of polyacrylic acid to sequester calcium and phosphate into transient amorphous nanophases. The use of polyphosphate without a sequestration analogue resulted only in randomly-oriented extrafibrillar precipitations along the fibrillar surface. Conversely, the use of polyacrylic acid without a templating analogue resulted only in non-hierarchical intrafibrillar mineralisation with continuous apatite strands instead of discrete crystallites. The ability of using simple non-protein molecules to recapitulate different levels of structural hierarchy in mineralised collagen signifies the ultimate simplicity in Nature’s biomineralisation design principles and challenges the need for using more complex recombinant matrix proteins in bioengineering applications. PMID:21040969

Longitudinal cognitive decline is associated with fibrillar amyloid-beta measured by [11C]PiB.

PubMed

Resnick, S M; Sojkova, J; Zhou, Y; An, Y; Ye, W; Holt, D P; Dannals, R F; Mathis, C A; Klunk, W E; Ferrucci, L; Kraut, M A; Wong, D F

2010-03-09

To investigate whether longitudinal declines in cognition are associated with higher fibrillar amyloid-beta (Abeta) deposition in vivo in individuals without dementia. [(11)C]PiB images were obtained to measure fibrillar Abeta burden in 57 participants without dementia from the Baltimore Longitudinal Study of Aging. Participants (33 men, 24 women) had a mean (SD) age of 78.7 (6.2) years. Six participants (4 men, 2 women) had mild cognitive impairment defined as Clinical Dementia Rating = 0.5. To measure [(11)C]PiB retention, distribution volume ratios (DVR) for 15 regions of interest were estimated by fitting a simplified reference tissue model to the measured time activity curves. Mixed effects regression was used to predict cognitive trajectories over time using data before and including time of PiB (mean follow-up 10.8 years), with mean cortical DVR, age at baseline, sex, and education as independent predictors. Voxel-based analysis identified local associations. [(11)C]PiB retention was higher in older individuals. Greater declines over time in mental status and verbal learning and memory, but not visual memory, were associated significantly with higher PiB retention. Voxel-based analysis showed significant associations in frontal and lateral temporal regions. Higher Abeta deposition is associated with greater longitudinal decline in mental status and verbal memory in the preceding years. The differential association for verbal but not visual memory may reflect the greater reliance of verbal word list learning on prefrontal regions, which show early Abeta deposition. Prospective imaging may help distinguish between individuals with evolving neuropathology who develop accelerated cognitive decline vs those with normal aging.

Micro-scale and meso-scale architectural cues cooperate and compete to direct aligned tissue formation

PubMed Central

Gilchrist, Christopher L.; Ruch, David S.; Little, Dianne; Guilak, Farshid

2014-01-01

Tissue and biomaterial microenvironments provide architectural cues that direct important cell behaviors including cell shape, alignment, migration, and resulting tissue formation. These architectural features may be presented to cells across multiple length scales, from nanometers to millimeters in size. In this study, we examined how architectural cues at two distinctly different length scales, “micro-scale” cues on the order of ~1–2 μm, and “meso-scale” cues several orders of magnitude larger (>100 μm), interact to direct aligned neo-tissue formation. Utilizing a micro-photopatterning (μPP) model system to precisely arrange cell-adhesive patterns, we examined the effects of substrate architecture at these length scales on human mesenchymal stem cell (hMSC) organization, gene expression, and fibrillar collagen deposition. Both micro- and meso-scale architectures directed cell alignment and resulting tissue organization, and when combined, meso cues could enhance or compete against micro-scale cues. As meso boundary aspect ratios were increased, meso-scale cues overrode micro-scale cues and controlled tissue alignment, with a characteristic critical width (~500 μm) similar to boundary dimensions that exist in vivo in highly aligned tissues. Meso-scale cues acted via both lateral confinement (in a cell-density-dependent manner) and by permitting end-to-end cell arrangements that yielded greater fibrillar collagen deposition. Despite large differences in fibrillar collagen content and organization between μPP architectural conditions, these changes did not correspond with changes in gene expression of key matrix or tendon-related genes. These findings highlight the complex interplay between geometric cues at multiple length scales and may have implications for tissue engineering strategies, where scaffold designs that incorporate cues at multiple length scales could improve neo-tissue organization and resulting functional outcomes. PMID:25263687

Using In situ Dynamic Cultures to Rapidly Biofabricate Fabric-Reinforced Composites of Chitosan/Bacterial Nanocellulose for Antibacterial Wound Dressings

PubMed Central

Zhang, Peng; Chen, Lin; Zhang, Qingsong; Hong, Feng F.

2016-01-01

Bacterial nano-cellulose (BNC) is considered to possess incredible potential in biomedical applications due to its innate unrivaled nano-fibrillar structure and versatile properties. However, its use is largely restricted by inefficient production and by insufficient strength when it is in a highly swollen state. In this study, a fabric skeleton reinforced chitosan (CS)/BNC hydrogel with high mechanical reliability and antibacterial activity was fabricated by using an efficient dynamic culture that could reserve the nano-fibrillar structure. By adding CS in culture media to 0.25–0.75% (w/v) during bacterial cultivation, the CS/BNC composite hydrogel was biosynthesized in situ on a rotating drum composed of fabrics. With the proposed method, BNC biosynthesis became less sensitive to the adverse antibacterial effects of CS and the production time of the composite hydrogel with desirable thickness could be halved from 10 to 5 days as compared to the conventional static cultures. Although, its concentration was low in the medium, CS accounted for more than 38% of the CS/BNC dry weight. FE-SEM observation confirmed conservation of the nano-fibrillar networks and covering of CS on BNC. ATR-FTIR showed a decrease in the degree of intra-molecular hydrogen bonding and water absorption capacity was improved after compositing with CS. The fabric-reinforced CS/BNC composite exhibited bacteriostatic properties against Escherichia coli and Staphylococcus aureus and significantly improved mechanical properties as compared to the BNC sheets from static culture. In summary, the fabric-reinforced CS/BNC composite constitutes a desired candidate for advanced wound dressings. From another perspective, coating of BNC or CS/BNC could upgrade the conventional wound dressings made of cotton gauze to reduce pain during wound healing, especially for burn patients. PMID:26973634

Time course of right ventricular pressure-overload induced myocardial fibrosis: relationship to changes in fibroblast postsynthetic procollagen processing.

PubMed

Baicu, Catalin F; Li, Jiayu; Zhang, Yuhua; Kasiganesan, Harinath; Cooper, George; Zile, Michael R; Bradshaw, Amy D

2012-11-01

Myocardial fibrillar collagen is considered an important determinant of increased ventricular stiffness in pressure-overload (PO)-induced cardiac hypertrophy. Chronic PO was created in feline right ventricles (RV) by pulmonary artery banding (PAB) to define the time course of changes in fibrillar collagen content after PO using a nonrodent model and to determine whether this time course was dependent on changes in fibroblast function. Total, soluble, and insoluble collagen (hydroxyproline), collagen volume fraction (CVF), and RV end-diastolic pressure were assessed 2 days and 1, 2, 4, and 10 wk following PAB. Fibroblast function was assessed by quantitating the product of postsynthetic processing, insoluble collagen, and levels of SPARC (secreted protein acidic and rich in cysteine), a protein that affects procollagen processing. RV hypertrophic growth was complete 2 wk after PAB. Changes in RV collagen content did not follow the same time course. Two weeks after PAB, there were elevations in total collagen (control RV: 8.84 ± 1.03 mg/g vs. 2-wk PAB: 11.50 ± 0.78 mg/g); however, increased insoluble fibrillar collagen, as measured by CVF, was not detected until 4 wk after PAB (control RV CVF: 1.39 ± 0.25% vs. 4-wk PAB: 4.18 ± 0.87%). RV end-diastolic pressure was unchanged at 2 wk, but increased until 4 wk after PAB. RV fibroblasts isolated after 2-wk PAB had no changes in either insoluble collagen or SPARC expression; however, increases in insoluble collagen and in levels of SPARC were detected in RV fibroblasts from 4-wk PAB. Therefore, the time course of PO-induced RV hypertrophy differs significantly from myocardial fibrosis and diastolic dysfunction. These temporal differences appear dependent on changes in fibroblast function.

Time course of right ventricular pressure-overload induced myocardial fibrosis: relationship to changes in fibroblast postsynthetic procollagen processing

PubMed Central

Baicu, Catalin F.; Li, Jiayu; Zhang, Yuhua; Kasiganesan, Harinath; Cooper, George; Zile, Michael R.

2012-01-01

Myocardial fibrillar collagen is considered an important determinant of increased ventricular stiffness in pressure-overload (PO)-induced cardiac hypertrophy. Chronic PO was created in feline right ventricles (RV) by pulmonary artery banding (PAB) to define the time course of changes in fibrillar collagen content after PO using a nonrodent model and to determine whether this time course was dependent on changes in fibroblast function. Total, soluble, and insoluble collagen (hydroxyproline), collagen volume fraction (CVF), and RV end-diastolic pressure were assessed 2 days and 1, 2, 4, and 10 wk following PAB. Fibroblast function was assessed by quantitating the product of postsynthetic processing, insoluble collagen, and levels of SPARC (secreted protein acidic and rich in cysteine), a protein that affects procollagen processing. RV hypertrophic growth was complete 2 wk after PAB. Changes in RV collagen content did not follow the same time course. Two weeks after PAB, there were elevations in total collagen (control RV: 8.84 ± 1.03 mg/g vs. 2-wk PAB: 11.50 ± 0.78 mg/g); however, increased insoluble fibrillar collagen, as measured by CVF, was not detected until 4 wk after PAB (control RV CVF: 1.39 ± 0.25% vs. 4-wk PAB: 4.18 ± 0.87%). RV end-diastolic pressure was unchanged at 2 wk, but increased until 4 wk after PAB. RV fibroblasts isolated after 2-wk PAB had no changes in either insoluble collagen or SPARC expression; however, increases in insoluble collagen and in levels of SPARC were detected in RV fibroblasts from 4-wk PAB. Therefore, the time course of PO-induced RV hypertrophy differs significantly from myocardial fibrosis and diastolic dysfunction. These temporal differences appear dependent on changes in fibroblast function. PMID:22942178

Analysis of the frequency and nature of hyaline ring granulomas in inflammatory odontogenic cysts.

PubMed

Henriques, A C G; Pereira, J S; Nonaka, C F W; Freitas, R A; Pinto, L P; Miguel, M C C

2013-01-01

To determine the prevalence of hyaline ring granulomas (HRGs) in a large case series of inflammatory odontogenic cysts, and to investigate the nature of these structures. All records from the patients diagnosed with inflammatory odontogenic cysts between January 1970 and April 2009 were reviewed. Histologic sections were evaluated by light microscopy and cases with HRGs for which sufficient biological material was available were submitted to histochemical analysis (Masson's trichrome) and immunohistochemistry (CD34, CD68 and collagen IV). Twenty-two (3.3%) of the 661 cases of inflammatory odontogenic cysts diagnosed during the study period presented HRGs. The relative frequency of HRGs was higher amongst residual radicular cysts (6.1%), followed by paradental cysts (5.6%) and radicular cysts (3.0%). HRGs appeared as roughly circular homogeneous/fibrillar masses in 14 (63.6%) cases and as round structures enclosing amorphous material in 3 (13.6%) cases. Most (77.8%) roughly circular homogeneous/fibrillar masses were positive for collagen, whereas all (100.0%) round structures enclosing amorphous material were negative for this protein. Immunohistochemistry showed that most mononucleated cells and all multinucleated giant cells were positive for CD68, but negative for CD34, in all cases. In addition, collagen IV immunostaining was negative in amorphous structures and weakly positive in homogeneous/fibrillar masses. The present results suggest a very low frequency of HRGs in inflammatory odontogenic cysts and support the hypothesis that these structures arise from the implantation of foreign material, most likely food particles of plant or vegetable origin. The diverse microscopic features of HRG possibly represent different developmental stages of this structure. © 2012 International Endodontic Journal.

In vivo laser confocal microscopy of Bowman's layer of the cornea.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2006-12-01

To investigate in vivo microstructures of Bowman's layer in normal human subjects using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT2-RCM). Single-center, prospective, observational case series. Nineteen normal volunteers (10 male, 9 female; mean age, 46.2+/-21.7 years [range, 18-77]). The central and peripheral cornea, specifically the epithelium, Bowman's layer, and its subjacent stroma, were examined using the HRT2-RCM. Selected images of the corneal layers were evaluated qualitatively for the shape and degree of light reflection of the microstructures. In all subjects, normal epithelial (superficial, wing, basal) cells, subbasal nerve plexus, Bowman's layer, and its subjacent stoma were observed clearly. However, in all subjects, polymorphic structures composed of fibrillar materials with less reflectivity than corneal nerves were observed beneath Bowman's layer. After application of pressure by a Tomo-cap, we observed numerous ridges that protruded into the epithelial basal and wing cell layers. Superficial stromal striae were also observed. These ridges and striae corresponded exactly to the orientation of the fibrous structures located beneath the epithelial cells. We report for the first time, the presence of polymorphic structures composed of fibrillar materials (K-structures) beneath Bowman's layer in normal human subjects, detected by HRT2-RCM. We surmise that these microstructures may correspond to the modified and condensed anterior stromal collagen fibers/lamellae that merge into Bowman's layer and that these fibrillar materials may be responsible for the formation of the anterior corneal mosaic. Further investigation of these microstructures in diseased eyes may provide insights into their pathophysiologic role in Bowman's layer.

Cellular Inclusion Bodies of Mutant Huntingtin Exon 1 Obscure Small Fibrillar Aggregate Species

PubMed Central

Sahl, Steffen J.; Weiss, Lucien E.; Duim, Whitney C.; Frydman, Judith; Moerner, W. E.

2012-01-01

The identities of toxic aggregate species in Huntington's disease pathogenesis remain ambiguous. While polyQ-expanded huntingtin (Htt) is known to accumulate in compact inclusion bodies inside neurons, this is widely thought to be a protective coping response that sequesters misfolded conformations or aggregated states of the mutated protein. To define the spatial distributions of fluorescently-labeled Htt-exon1 species in the cell model PC12m, we employed highly sensitive single-molecule super-resolution fluorescence imaging. In addition to inclusion bodies and the diffuse pool of monomers and oligomers, fibrillar aggregates ~100 nm in diameter and up to ~1–2 µm in length were observed for pathogenic polyQ tracts (46 and 97 repeats) after targeted photo-bleaching of the inclusion bodies. These short structures bear a striking resemblance to fibers described in vitro. Definition of the diverse Htt structures in cells will provide an avenue to link the impact of therapeutic agents to aggregate populations and morphologies. PMID:23193437

Choline-Based Amino Acid ILs-Collagen Interaction: Enunciating Its Role in Stabilization/Destabilization Phenomena.

PubMed

Tarannum, Aafiya; Rao, J Raghava; Fathima, N Nishad

2018-01-25

Given the potential of productive interaction between choline-based amino acid ionic liquids (CAAILs) and collagen, we investigated the role of four CAAILs, viz., choline serinate, threoninate, lysinate, and phenylalaninate, and the changes mediated by them in the structure of collagen at different hierarchical orderings, that is, at molecular and fibrillar levels. The rheological, dielectric behavior and the secondary structural changes signify the alteration in the triple helical structure of collagen at higher concentrations of CAAILs. A marginal swelling and slight decrease in the thermal stability of rat tail tendon collagen fibers were observed for choline serinate and threoninate, albeit distortions in banding patterns were noticed for choline lysinate and phenylalaninate, suggesting chaotropicity of the ions at the fibrillar level. This signifies the changes in the hydrogen-bonding environment of collagen with increasing concentrations of CAAILs, which could be due to competitive hydrogen bonding between the carbonyl group of amino acid ionic liquids and the hydroxyl groups of collagen.

Ultra-Fast Supercritical Hydrothermal Synthesis of Tobermorite under Thermodynamically Metastable Conditions.

PubMed

Diez-Garcia, Marta; Gaitero, Juan J; Dolado, Jorge S; Aymonier, Cyril

2017-03-13

Tobermorite is a fibrillar mineral of the family of calcium silicates. In spite of not being abundant in nature, its structure and properties are reasonably well known because of its interest in the construction industry. Currently, tobermorite is synthesized by hydrothermal methods at mild temperatures. The problem is that such processes are very slow (>5 h) and temperature cannot be increased to speed them up because tobermorite is metastable over 130 °C. Furthermore the product obtained is generally foil-like and not very crystalline. Herein we propose an alternative synthesis method based on the use of a continuous flow reactor and supercritical water. In spite of the high temperature, the transformation of tobermorite to more stable phases can be prevented by accurately controlling the reaction time. As a result, highly crystalline fibrillar tobermorite can be obtained in just a few seconds under thermodynamically metastable conditions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function

PubMed Central

Murakami, Tetsuro; Qamar, Seema; Lin, Julie Qiaojin; Schierle, Gabriele S. Kaminski; Rees, Eric; Miyashita, Akinori; Costa, Ana R.; Dodd, Roger B.; Chan, Fiona T.S.; Michel, Claire H.; Kronenberg-Versteeg, Deborah; Li, Yi; Yang, Seung-Pil; Wakutani, Yosuke; Meadows, William; Ferry, Rodylyn Rose; Dong, Liang; Tartaglia, Gian Gaetano; Favrin, Giorgio; Lin, Wen-Lang; Dickson, Dennis W.; Zhen, Mei; Ron, David; Schmitt-Ulms, Gerold; Fraser, Paul E.; Shneider, Neil A.; Holt, Christine; Vendruscolo, Michele; Kaminski, Clemens F.; St George-Hyslop, Peter

2015-01-01

Summary The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins. PMID:26526393

Structure, Function, and Assembly of Type 1 Fimbriae

NASA Astrophysics Data System (ADS)

Knight, Stefan D.; Bouckaert, Julie

Bacterial infections constitute a major global health problem, acutely accentuated by the rapid spread of antibiotic resistant bacterial strains. The widespread need for bacteria to attach - adhere - to target cells before they can initiate an infection may be used to advantage by targeting the bacterial adhesion tools such as pili and fimbriae for development of novel anti-bacterial vaccines and drugs. Type 1 fimbriae are widely expressed by Escherichia coli. and are used by uropathogenic strains to mediate attachment to specific niches in the urinary tract. These fimbriae belong to a class of fibrillar adhesion organelles assembled through the chaperone/usher pathway, one of the terminal branches of the general secretion pathway in Gram-negative bacteria. Our understanding of the assembly, structure and function of these structures has evolved significantly over the last decade. Here, we summarize current understanding of the function and biogenesis of fibrillar adhesion organelles, and provide some examples of recent progress towards interfering with bacterial adhesion as a means to prevent infection.

Hierarchical macroscopic fibrillar adhesives: in situ study of buckling and adhesion mechanisms on wavy substrates.

PubMed

Bauer, Christina T; Kroner, Elmar; Fleck, Norman A; Arzt, Eduard

2015-10-23

Nature uses hierarchical fibrillar structures to mediate temporary adhesion to arbitrary substrates. Such structures provide high compliance such that the flat fibril tips can be better positioned with respect to asperities of a wavy rough substrate. We investigated the buckling and adhesion of hierarchically structured adhesives in contact with flat smooth, flat rough and wavy rough substrates. A macroscopic model for the structural adhesive was fabricated by molding polydimethylsiloxane into pillars of diameter in the range of 0.3-4.8 mm, with up to three different hierarchy levels. Both flat-ended and mushroom-shaped hierarchical samples buckled at preloads one quarter that of the single level structures. We explain this behavior by a change in the buckling mode; buckling leads to a loss of contact and diminishes adhesion. Our results indicate that hierarchical structures can have a strong influence on the degree of adhesion on both flat and wavy substrates. Strategies are discussed that achieve highly compliant substrates which adhere to rough substrates.

Impact of thermal annealing on wettability and antifouling characteristics of alginate poly-l-lysine polyelectrolyte multilayer films.

PubMed

Diamanti, Eleftheria; Muzzio, Nicolas; Gregurec, Danijela; Irigoyen, Joseba; Pasquale, Miguel; Azzaroni, Omar; Brinkmann, Martin; Moya, Sergio Enrique

2016-09-01

Polyelectrolyte multilayers (PEMs) of poly-l-lysine (PLL) and alginic acid sodium salt (Alg) are fabricated applying the layer by layer technique and annealed at a constant temperature; 37, 50 and 80°C, for 72h. Atomic force microscopy reveals changes in the topography of the PEM, which is changing from a fibrillar to a smooth surface. Advancing contact angle in water varies from 36° before annealing to 93°, 77° and 95° after annealing at 37, 50 and 80°C, respectively. Surface energy changes after annealing were calculated from contact angle measurements performed with organic solvents. Quartz crystal microbalance with dissipation, contact angle and fluorescence spectroscopy measurements show a significant decrease in the adsorption of the bovine serum albumin protein to the PEMs after annealing. Changes in the physical properties of the PEMs are interpreted as a result of the reorganization of the polyelectrolytes in the PEMs from a layered structure into complexes where the interaction of polycations and polyanions is enhanced. This work proposes a simple method to endow bio-PEMs with antifouling characteristics and tune their wettability. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of water chemistry and surface contact on the toxicity of silver nanoparticles to Bacillus subtilis.

PubMed

Yi, Jun; Cheng, Jinping

2017-07-01

The growing use of silver nanoparticles (AgNPs) has created concerns about its potential impacts on natural microbial communities. In this study, the physicochemical properties of AgNPs and its toxicity on natural bacteria Bacillus subtilis (B. subtilis) were investigated in aqueous conditions. The characterization data showed that AgNPs highly aggregated in aqueous conditions, and the hydrodynamic diameter of AgNPs in aqueous conditions was larger than its primary size. The studied AgNPs was less toxic to B. subtilis in estuarine water as compared to that in Milli-Q water and artificial seawater, which might be due to the observed enhanced aggregation of AgNPs in estuarine water. The toxicity of AgNPs to B. subtilis was greatly reduced when their surface contact was blocked by a dialysis membrane. Scanning electron microscope images showed that exposure contact to AgNPs resulted in damage of the microbial cell wall and enhanced formation of fibrillar structures. These results suggest that particle-cell contact is largely responsible for the observed toxicity of AgNPs in B. subtilis. This study can help to understand the potential impacts of AgNPs to natural microbes, especially in the complex aquatic environments.

Gelsolin as therapeutic target in Alzheimer's disease.

PubMed

Carro, Eva

2010-06-01

Fibrillar amyloid beta-protein (Abeta) is a major component of amyloid plaques in the brains of individuals with Alzheimer's disease (AD). However, a comprehensive explanation of the mechanisms leading to brain amyloidosis is still pending. Previous studies have identified the anti-amyloidogenic role of gelsolin in AD. Gelsolin can reduce amyloid burden by acting as an inhibitor of Abeta fibrillization, and as an antioxidant and anti-apoptotic protein. Recent evidence indicates reduced brain gelsolin levels in AD. Therefore, a better understanding of the roles of gelsolin in AD pathology, particularly those related with cognition, is required. Most of the information reviewed here relates to experimental studies. However, gelsolin may progress from the present evidence to preclinical and clinical applications. In addition, a greater insight into the environmental factors contributing to abnormally reduced gelsolin function in AD brains may become crucial for the development of much needed disease-modifying strategies. Because, the efficacy of available medicines is still poor, there is an urgent need for novel AD treatments. In this sense, gelsolin could play an important role.

Role of prion protein aggregation in neurotoxicity.

PubMed

Corsaro, Alessandro; Thellung, Stefano; Villa, Valentina; Nizzari, Mario; Florio, Tullio

2012-01-01

In several neurodegenerative diseases, such as Parkinson, Alzheimer's, Huntington, and prion diseases, the deposition of aggregated misfolded proteins is believed to be responsible for the neurotoxicity that characterizes these diseases. Prion protein (PrP), the protein responsible of prion diseases, has been deeply studied for the peculiar feature of its misfolded oligomers that are able to propagate within affected brains, inducing the conversion of the natively folded PrP into the pathological conformation. In this review, we summarize the available experimental evidence concerning the relationship between aggregation status of misfolded PrP and neuronal death in the course of prion diseases. In particular, we describe the main findings resulting from the use of different synthetic (mainly PrP106-126) and recombinant PrP-derived peptides, as far as mechanisms of aggregation and amyloid formation, and how these different spatial conformations can affect neuronal death. In particular, most data support the involvement of non-fibrillar oligomers rather than actual amyloid fibers as the determinant of neuronal death.

Role of Prion Protein Aggregation in Neurotoxicity

PubMed Central

Corsaro, Alessandro; Thellung, Stefano; Villa, Valentina; Nizzari, Mario; Florio, Tullio

2012-01-01

In several neurodegenerative diseases, such as Parkinson, Alzheimer’s, Huntington, and prion diseases, the deposition of aggregated misfolded proteins is believed to be responsible for the neurotoxicity that characterizes these diseases. Prion protein (PrP), the protein responsible of prion diseases, has been deeply studied for the peculiar feature of its misfolded oligomers that are able to propagate within affected brains, inducing the conversion of the natively folded PrP into the pathological conformation. In this review, we summarize the available experimental evidence concerning the relationship between aggregation status of misfolded PrP and neuronal death in the course of prion diseases. In particular, we describe the main findings resulting from the use of different synthetic (mainly PrP106-126) and recombinant PrP-derived peptides, as far as mechanisms of aggregation and amyloid formation, and how these different spatial conformations can affect neuronal death. In particular, most data support the involvement of non-fibrillar oligomers rather than actual amyloid fibers as the determinant of neuronal death. PMID:22942726

Optical fiber-based full Mueller polarimeter for endoscopic imaging using a two-wavelength simultaneous measurement method

NASA Astrophysics Data System (ADS)

Vizet, Jérémy; Manhas, Sandeep; Tran, Jacqueline; Validire, Pierre; Benali, Abdelali; Garcia-Caurel, Enric; Pierangelo, Angelo; Martino, Antonello De; Pagnoux, Dominique

2016-07-01

This paper reports a technique based on spectrally differential measurement for determining the full Mueller matrix of a biological sample through an optical fiber. In this technique, two close wavelengths were used simultaneously, one for characterizing the fiber and the other for characterizing the assembly of fiber and sample. The characteristics of the fiber measured at one wavelength were used to decouple its contribution from the measurement on the assembly of fiber and sample and then to extract sample Mueller matrix at the second wavelength. The proof of concept was experimentally validated by measuring polarimetric parameters of various calibrated optical components through the optical fiber. Then, polarimetric images of histological cuts of human colon tissues were measured, and retardance, diattenuation, and orientation of the main axes of fibrillar regions were displayed. Finally, these images were successfully compared with images obtained by a free space Mueller microscope. As the reported method does not use any moving component, it offers attractive integration possibilities with an endoscopic probe.

Electrostatic Effects in Filamentous Protein Aggregation

PubMed Central

Buell, Alexander K.; Hung, Peter; Salvatella, Xavier; Welland, Mark E.; Dobson, Christopher M.; Knowles, Tuomas P.J.

2013-01-01

Electrostatic forces play a key role in mediating interactions between proteins. However, gaining quantitative insights into the complex effects of electrostatics on protein behavior has proved challenging, due to the wide palette of scenarios through which both cations and anions can interact with polypeptide molecules in a specific manner or can result in screening in solution. In this article, we have used a variety of biophysical methods to probe the steady-state kinetics of fibrillar protein self-assembly in a highly quantitative manner to detect how it is modulated by changes in solution ionic strength. Due to the exponential modulation of the reaction rate by electrostatic forces, this reaction represents an exquisitely sensitive probe of these effects in protein-protein interactions. Our approach, which involves a combination of experimental kinetic measurements and theoretical analysis, reveals a hierarchy of electrostatic effects that control protein aggregation. Furthermore, our results provide a highly sensitive method for the estimation of the magnitude of binding of a variety of ions to protein molecules. PMID:23473495

Signature of an aggregation-prone conformation of tau

NASA Astrophysics Data System (ADS)

Eschmann, Neil A.; Georgieva, Elka R.; Ganguly, Pritam; Borbat, Peter P.; Rappaport, Maxime D.; Akdogan, Yasar; Freed, Jack H.; Shea, Joan-Emma; Han, Songi

2017-03-01

The self-assembly of the microtubule associated tau protein into fibrillar cell inclusions is linked to a number of devastating neurodegenerative disorders collectively known as tauopathies. The mechanism by which tau self-assembles into pathological entities is a matter of much debate, largely due to the lack of direct experimental insights into the earliest stages of aggregation. We present pulsed double electron-electron resonance measurements of two key fibril-forming regions of tau, PHF6 and PHF6*, in transient as aggregation happens. By monitoring the end-to-end distance distribution of these segments as a function of aggregation time, we show that the PHF6(*) regions dramatically extend to distances commensurate with extended β-strand structures within the earliest stages of aggregation, well before fibril formation. Combined with simulations, our experiments show that the extended β-strand conformational state of PHF6(*) is readily populated under aggregating conditions, constituting a defining signature of aggregation-prone tau, and as such, a possible target for therapeutic interventions.

Optical fiber-based full Mueller polarimeter for endoscopic imaging using a two-wavelength simultaneous measurement method.

PubMed

Vizet, Jérémy; Manhas, Sandeep; Tran, Jacqueline; Validire, Pierre; Benali, Abdelali; Garcia-Caurel, Enric; Pierangelo, Angelo; De Martino, Antonello; Pagnoux, Dominique

2016-07-01

This paper reports a technique based on spectrally differential measurement for determining the full Mueller matrix of a biological sample through an optical fiber. In this technique, two close wavelengths were used simultaneously, one for characterizing the fiber and the other for characterizing the assembly of fiber and sample. The characteristics of the fiber measured at one wavelength were used to decouple its contribution from the measurement on the assembly of fiber and sample and then to extract sample Mueller matrix at the second wavelength. The proof of concept was experimentally validated by measuring polarimetric parameters of various calibrated optical components through the optical fiber. Then, polarimetric images of histological cuts of human colon tissues were measured, and retardance, diattenuation, and orientation of the main axes of fibrillar regions were displayed. Finally, these images were successfully compared with images obtained by a free space Mueller microscope. As the reported method does not use any moving component, it offers attractive integration possibilities with an endoscopic probe.

Fluorescent 'two-faced' polymer wafers with embedded pyrene-functionalised gelator nanofibres.

PubMed

Moffat, Jamie R; Smith, David K

2011-11-21

Pyrene-functionalised gelators self-assemble into nano-fibrillar organogels in DMSO/styrene/divinylbenzene mixtures, which when polymerised yield polymer wafers with two distinct faces, only one of which is fluorescent and has embedded gelator nanofibres. This journal is © The Royal Society of Chemistry 2011

[Stratification in the central nervous system].

PubMed

Menkes, B; Alexandru, C; Checiu, I

1978-01-01

In the authors' opinion the neuroblasts (glioblasts respectively) are moving along an oxigen-gradient, out of the proliferative layers toward the source of O2 (the vascular plexi). Stratigenesis is influenced by "guiding structures" (fibrillar plate in the tectum opticum), by the elongations of ependymal cells and by the similarly oriented vascular-connectives.

Synthesis of TiO2-poly(3-hexylthiophene) hybrid particles through surface-initiated Kumada catalyst-transfer polycondensation.

PubMed

Boon, Florian; Moerman, David; Laurencin, Danielle; Richeter, Sébastien; Guari, Yannick; Mehdi, Ahmad; Dubois, Philippe; Lazzaroni, Roberto; Clément, Sébastien

2014-09-30

TiO2/conjugated polymers are promising materials in solar energy conversion where efficient photoinduced charge transfers are required. Here, a "grafting-from" approach for the synthesis of TiO2 nanoparticles supported with conjugated polymer brushes is presented. Poly(3-hexylthiophene) (P3HT), a benchmark material for organic electronics, was selectively grown from TiO2 nanoparticles by surface-initiated Kumada catalyst-transfer polycondensation. The grafting of the polymer onto the surface of the TiO2 nanoparticles by this method was demonstrated by (1)H and (13)C solid-state NMR, X-ray photoelectron spectrometry, thermogravimetric analysis, transmission electron microscopy, and UV-visible spectroscopy. Sedimentation tests in tetrahydrofuran revealed improved dispersion stability for the TiO2@P3HT hybrid material. Films were produced by solvent casting, and the quality of the dispersion of the modified TiO2 nanoparticles was evaluated by atomic force microscopy. The dispersion of the P3HT-coated TiO2 NPs in the P3HT matrix was found to be homogeneous, and the fibrillar structure of the P3HT matrix was maintained which is favorable for charge transport. Fluorescence quenching measurements on these hybrid materials in CHCl3 indicated improved photoinduced electron-transfer efficiency. All in all, better physicochemical properties for P3HT/TiO2 hybrid material were reached via the surface-initiated "grafted-from" approach compared to the "grafting-onto" approach.

Shaping the micromechanical behavior of multi-phase composites for bone tissue engineering.

PubMed

Ranganathan, Shivakumar I; Yoon, Diana M; Henslee, Allan M; Nair, Manitha B; Smid, Christine; Kasper, F Kurtis; Tasciotti, Ennio; Mikos, Antonios G; Decuzzi, Paolo; Ferrari, Mauro

2010-09-01

Mechanical stiffness is a fundamental parameter in the rational design of composites for bone tissue engineering in that it affects both the mechanical stability and the osteo-regeneration process at the fracture site. A mathematical model is presented for predicting the effective Young's modulus (E) and shear modulus (G) of a multi-phase biocomposite as a function of the geometry, material properties and volume concentration of each individual phase. It is demonstrated that the shape of the reinforcing particles may dramatically affect the mechanical stiffness: E and G can be maximized by employing particles with large geometrical anisotropy, such as thin platelet-like or long fibrillar-like particles. For a porous poly(propylene fumarate) (60% porosity) scaffold reinforced with silicon particles (10% volume concentration) the Young's (shear) modulus could be increased by more than 10 times by just using thin platelet-like as opposed to classical spherical particles, achieving an effective modulus E approximately 8 GPa (G approximately 3.5 GPa). The mathematical model proposed provides results in good agreement with several experimental test cases and could help in identifying the proper formulation of bone scaffolds, reducing the development time and guiding the experimental testing. 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Self-assembly of short amyloidogenic peptides at the air-water interface.

PubMed

Chaudhary, Nitin; Nagaraj, Ramakrishnan

2011-08-01

Short peptide stretches in amyloidogenic proteins can form amyloid fibrils in vitro and have served as good models for studying amyloid fibril formation. Recently, these amyloidogenic peptides have gained considerable attention, as non-amyloid ordered structures can be obtained from these peptides by carefully tuning the conditions of self-assembly, especially pH, temperature and presence of organic solvents. We have examined the effect of surface pressure on the self-assembled structures of two amyloidogenic peptides, Pβ(2)m (Ac-DWSFYLLYYTEFT-am) and AcPHF6 (Ac-VQIVYK-am) at the air-water interface when deposited from different solvents. Both the peptides are surface-active and form Thioflavin T (ThT) positive structures at the air-water interface. There is considerable hysteresis in the compression and expansion isotherms, suggesting the occurrence of structural rearrangements during compression. Preformed Pβ(2)m fibrillar structures at the air-water interface are disrupted as peptide is compressed to lower molecular areas but restored if the film is expanded, suggesting that the process is reversible. AcPHF6, on the other hand, shows largely sheet-like structures at lower molecular areas. The solvents used for dissolution of the peptides appear to influence the nature of the aggregates formed. Our results show that like hydrostatic pressure, surface pressure can also be utilized for modulating the self-assembly of the amyloidogenic and self-assembling peptides. Copyright © 2011 Elsevier Inc. All rights reserved.

Orgyia pseudotsugata baculovirus p10 and polyhedron envelope protein genes: analysis of their relative expression levels and role in polyhedron structure.

PubMed

Gross, C H; Russell, R L; Rohrmann, G F

1994-05-01

To investigate the regulation of p10 and polyhedron envelope protein (PEP) gene expression and their role in polyhedron development, Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis viruses lacking these genes were constructed. Recombinant viruses were produced, in which the p10 gene, the PEP gene or both genes were disrupted with the beta-glucuronidase (GUS) or beta-galactosidase (lacZ) genes. GUS activity under the control of the PEP protein promoter was observed later in infection and its maximal expression was less than 10% the level for p10 promoter-GUS constructs. Tissues from O. pseudotsugata larvae infected with these recombinants were examined by electron microscopy. Cells from insects infected with the p10- viruses lacked p10-associated fibrillar structures, but fragments of polyhedron envelope-like structures were observed on the surface of some polyhedra. Immunogold labelling of cells infected with the p10-GUS+ virus with an antibody directed against PEP showed that the PEP was concentrated at the surface of polyhedra. Although polyhedra produced by p10 and PEP gene deletion mutants demonstrated what appeared to be a polyhedron envelope by transmission electron microscopy, scanning electron microscopy showed that they had irregular, pitted surfaces that were different from wild-type polyhedra. These data suggested that both p10 and PEP are important for the proper formation of the periphery of polyhedra.

Annulus fibrosus functional extrafibrillar and fibrous mechanical behaviour: experimental and computational characterisation.

PubMed

Mengoni, Marlène; Kayode, Oluwasegun; Sikora, Sebastien N F; Zapata-Cornelio, Fernando Y; Gregory, Diane E; Wilcox, Ruth K

2017-08-01

The development of current surgical treatments for intervertebral disc damage could benefit from virtual environment accounting for population variations. For such models to be reliable, a relevant description of the mechanical properties of the different tissues and their role in the functional mechanics of the disc is of major importance. The aims of this work were first to assess the physiological hoop strain in the annulus fibrosus in fresh conditions ( n  = 5) in order to extract a functional behaviour of the extrafibrillar matrix; then to reverse-engineer the annulus fibrosus fibrillar behaviour ( n  = 6). This was achieved by performing both direct and global controlled calibration of material parameters, accounting for the whole process of experimental design and in silico model methodology. Direct-controlled models are specimen-specific models representing controlled experimental conditions that can be replicated and directly comparing measurements. Validation was performed on another six specimens and a sensitivity study was performed. Hoop strains were measured as 17 ± 3% after 10 min relaxation and 21 ± 4% after 20-25 min relaxation, with no significant difference between the two measurements. The extrafibrillar matrix functional moduli were measured as 1.5 ± 0.7 MPa. Fibre-related material parameters showed large variability, with a variance above 0.28. Direct-controlled calibration and validation provides confidence that the model development methodology can capture the measurable variation within the population of tested specimens.

Annulus fibrosus functional extrafibrillar and fibrous mechanical behaviour: experimental and computational characterisation

PubMed Central

Kayode, Oluwasegun; Sikora, Sebastien N. F.; Zapata-Cornelio, Fernando Y.; Gregory, Diane E.; Wilcox, Ruth K.

2017-01-01

The development of current surgical treatments for intervertebral disc damage could benefit from virtual environment accounting for population variations. For such models to be reliable, a relevant description of the mechanical properties of the different tissues and their role in the functional mechanics of the disc is of major importance. The aims of this work were first to assess the physiological hoop strain in the annulus fibrosus in fresh conditions (n = 5) in order to extract a functional behaviour of the extrafibrillar matrix; then to reverse-engineer the annulus fibrosus fibrillar behaviour (n = 6). This was achieved by performing both direct and global controlled calibration of material parameters, accounting for the whole process of experimental design and in silico model methodology. Direct-controlled models are specimen-specific models representing controlled experimental conditions that can be replicated and directly comparing measurements. Validation was performed on another six specimens and a sensitivity study was performed. Hoop strains were measured as 17 ± 3% after 10 min relaxation and 21 ± 4% after 20–25 min relaxation, with no significant difference between the two measurements. The extrafibrillar matrix functional moduli were measured as 1.5 ± 0.7 MPa. Fibre-related material parameters showed large variability, with a variance above 0.28. Direct-controlled calibration and validation provides confidence that the model development methodology can capture the measurable variation within the population of tested specimens. PMID:28879014

Bovine Respiratory Syncytial Virus and Histophilus somni Interaction at the Alveolar Barrier

PubMed Central

Agnes, J. T.; Zekarias, B.; Shao, M.; Anderson, M. L.; Gershwin, L. J.

2013-01-01

Our previous studies showed that Histophilus somni and bovine respiratory syncytial virus (BRSV) act synergistically in vivo to cause more severe bovine respiratory disease than either agent alone causes. Since H. somni surface and secreted immunoglobulin binding protein A (IbpA) causes retraction of bovine alveolar type 2 (BAT2) cells and invasion between BAT2 cells in vitro, we investigated mechanisms of BRSV-plus-H. somni infection at the alveolar barrier. BRSV treatment of BAT2 cells prior to treatment with IbpA-rich H. somni concentrated culture supernatant (CCS) resulted in increased BAT2 cell rounding and retraction compared to those with either treatment alone. This mimicked the increased alveolar cell thickening in calves experimentally infected with BRSV followed by H. somni compared to that in calves infected with BRSV or H. somni alone. BRSV-plus-H. somni CCS treatment of BAT2 cells also enhanced paracellular migration. The effect of matrix metalloproteinases (MMPs) was investigated as well because microarray analysis revealed that treatment with BRSV plus H. somni synergistically upregulated BAT2 cell expression of mmp1 and mmp3 compared to that in cells treated with either agent alone. Enzyme-linked immunosorbent assay (ELISA) confirmed that MMP1 and MMP3 protein levels were similarly upregulated. In collagen I and collagen IV (targets for MMP1 and MMP3, respectively) substrate zymography, digestion was increased with supernatants from dually treated BAT2 cells compared with those from singly treated cells. Enhanced breakdown of collagen IV in the basal lamina and of fibrillar collagen I in the adjacent interstitium in the dual infection may facilitate dissemination of H. somni infection. PMID:23649093

Bovine respiratory syncytial virus and Histophilus somni interaction at the alveolar barrier.

PubMed

Agnes, J T; Zekarias, B; Shao, M; Anderson, M L; Gershwin, L J; Corbeil, L B

2013-07-01

Our previous studies showed that Histophilus somni and bovine respiratory syncytial virus (BRSV) act synergistically in vivo to cause more severe bovine respiratory disease than either agent alone causes. Since H. somni surface and secreted immunoglobulin binding protein A (IbpA) causes retraction of bovine alveolar type 2 (BAT2) cells and invasion between BAT2 cells in vitro, we investigated mechanisms of BRSV-plus-H. somni infection at the alveolar barrier. BRSV treatment of BAT2 cells prior to treatment with IbpA-rich H. somni concentrated culture supernatant (CCS) resulted in increased BAT2 cell rounding and retraction compared to those with either treatment alone. This mimicked the increased alveolar cell thickening in calves experimentally infected with BRSV followed by H. somni compared to that in calves infected with BRSV or H. somni alone. BRSV-plus-H. somni CCS treatment of BAT2 cells also enhanced paracellular migration. The effect of matrix metalloproteinases (MMPs) was investigated as well because microarray analysis revealed that treatment with BRSV plus H. somni synergistically upregulated BAT2 cell expression of mmp1 and mmp3 compared to that in cells treated with either agent alone. Enzyme-linked immunosorbent assay (ELISA) confirmed that MMP1 and MMP3 protein levels were similarly upregulated. In collagen I and collagen IV (targets for MMP1 and MMP3, respectively) substrate zymography, digestion was increased with supernatants from dually treated BAT2 cells compared with those from singly treated cells. Enhanced breakdown of collagen IV in the basal lamina and of fibrillar collagen I in the adjacent interstitium in the dual infection may facilitate dissemination of H. somni infection.

Cytochemical study of the nucleolus of the slime mold Dictyostelium discoideum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benichou, J.C.; Quiviger, B.; Ryter, A.

1983-07-01

The nucleus of the slime mold Dictyostelium discoideum is characterized by the presence of several large dense masses which are all in tight contact with the nuclear membrane. These dense masses, considered as nucleoli, present a rather homogeneous texture, in which dense chromatin, fibrillar, and granular material are not easily detected. The autoradiographic study of (/sup 3/H)uridine pulse-labeled cells showed that the majority of the silver grains were located inside these masses. The use of EDTA regressive-staining, acetylation and enzymatic digestion indicated that they are mostly composed of RNP and are totally devoid of dense chromatin as the rest ofmore » the nucleus is. After treatment with actinomycin D, fibrillar and granular material segregated but no chromatin could be found. All these observations confirmed that the dense masses correspond to nucleoli despite their peculiar ultrastructure. It can also be concluded that this type of nucleoli cannot be considered as a taxonomic character of the slime molds because it does not exist in all slime molds and was observed in some dinoflagellates, and ascomycetes.« less

Structural and functional properties of prefibrillar α-synuclein oligomers.

PubMed

Pieri, Laura; Madiona, Karine; Melki, Ronald

2016-04-14

The deposition of fibrillar alpha-synuclein (α-syn) within inclusions (Lewy bodies and Lewy neurites) in neurons and glial cells is a hallmark of synucleinopathies. α-syn populates a variety of assemblies ranging from prefibrillar oligomeric species to fibrils whose specific contribution to neurodegeneration is still unclear. Here, we compare the specific structural and biological properties of distinct soluble prefibrillar α-syn oligomers formed either spontaneously or in the presence of dopamine and glutaraldehyde. We show that both on-fibrillar assembly pathway and distinct dopamine-mediated and glutaraldehyde-cross-linked α-syn oligomers are only slightly effective in perturbing cell membrane integrity and inducing cytotoxicity, while mature fibrils exhibit the highest toxicity. In contrast to low-molecular weight and unstable oligomers, large stable α-syn oligomers seed the aggregation of soluble α-syn within reporter cells although to a lesser extent than mature α-syn fibrils. These oligomers appear elongated in shape. Our findings suggest that α-syn oligomers represent a continuum of species ranging from unstable low molecular weight particles to mature fibrils via stable elongated oligomers composed of more than 15 α-syn monomers that possess seeding capacity.

Structure of interphase chromosomes in the nuclei of Drosophila cells.

PubMed

Banfalvi, Gaspar

2006-10-01

Fluorescent images of interphase chromatin structures and chromosome structures isolated from reversibly permeable Drosophila cells were analyzed. Decondensed chromatin in early S phase (2.0-2.5 C-value) consisted of a veil-like fibrillary network. Fibrillar chromatin formed rodlets later in the early S phase (2.5-2.75 C). Drosophila chromosomes contain several smaller subunits called rodlets. Fibrillar chromatin turned to chromatin ribbon and the early mid-S-phase globular chromosomes (2.75-3.0 C), then to opened fibrous globular forms later in the mid-S-phase (3.0-3.25 C), to late-S-phase supercoiled ribbons (3.25-3.5 C), end-S-phase elongated prechromosomes (3.5-3.75 C), bent and linear chromosomes (3.75-4.0 C). Early-S phase chromatin fibrils in the nuclei of Drosophila cells are thinner than the veil-like structures in mammalian cells. The connectivity of chromosomes shows linear arrangement (3, 1, 2, 4), with larger chromosomes (1 and 2) inside and smaller chromosomes (3, 4) at the two ends in the chromosomal chain.

Proteins containing expanded polyglutamine tracts and neurodegenerative disease

PubMed Central

Adegbuyiro, Adewale; Sedighi, Faezeh; Pilkington, Albert W.; Groover, Sharon; Legleiter, Justin

2017-01-01

Several hereditary neurological and neuromuscular diseases are caused by an abnormal expansion of trinucleotide repeats. To date, there have been ten of these trinucleotide repeat disorders associated with an expansion of the codon CAG encoding glutamine (Q). For these polyglutamine (polyQ) diseases, there is a critical threshold length of the CAG repeat required for disease, and further expansion beyond this threshold is correlated with age of onset and symptom severity. PolyQ expansion in the translated proteins promotes their self-assembly into a variety of oligomeric and fibrillar aggregate species that accumulate into the hallmark proteinaceous inclusion bodies associated with each disease. Here, we review aggregation mechanisms of proteins with expanded polyQ-tracts, structural consequences of expanded polyQ ranging from monomers to fibrillar aggregates, the impact of protein context and post translational modifications on aggregation, and a potential role for lipids membranes in aggregation. As the pathogenic mechanisms that underlie these disorders are often classified as either a gain of toxic function or loss of normal protein function, some toxic mechanisms associated with mutant polyQ tracts will also be discussed. PMID:28170216

Identification of an Electrostatic Ruler Motif for Sequence-Specific Binding of Collagenase to Collagen.

PubMed

Subramanian, Sundar Raman; Singam, Ettayapuram Ramaprasad Azhagiya; Berinski, Michael; Subramanian, Venkatesan; Wade, Rebecca C

2016-08-25

Sequence-specific cleavage of collagen by mammalian collagenase plays a pivotal role in cell function. Collagenases are matrix metalloproteinases that cleave the peptide bond at a specific position on fibrillar collagen. The collagenase Hemopexin-like (HPX) domain has been proposed to be responsible for substrate recognition, but the mechanism by which collagenases identify the cleavage site on fibrillar collagen is not clearly understood. In this study, Brownian dynamics simulations coupled with atomic-detail and coarse-grained molecular dynamics simulations were performed to dock matrix metalloproteinase-1 (MMP-1) on a collagen IIIα1 triple helical peptide. We find that the HPX domain recognizes the collagen triple helix at a conserved R-X11-R motif C-terminal to the cleavage site to which the HPX domain of collagen is guided electrostatically. The binding of the HPX domain between the two arginine residues is energetically stabilized by hydrophobic contacts with collagen. From the simulations and analysis of the sequences and structural flexibility of collagen and collagenase, a mechanistic scheme by which MMP-1 can recognize and bind collagen for proteolysis is proposed.

Spatiotemporal behavior of nuclear cyclophilin B indicates a role in RNA transcription.

PubMed

Dieriks, Birger; Van Oostveldt, Patrick

2012-06-01

Cyclophilin B (CypB) is an ubiquitously expressed protein, which performs several intra- and extracellular functions. Despite its abundant use as a household protein, little is known about its exact cellular localization and dynamics. In the present study we show that endogenous CypB localizes in one of two distinct compartments, either within the endoplasmic reticulum (ER) or inside the nucleus, accumulating in the fibrillar centers of the nucleoli. By means of a genetic deletion screen, we identified a minimal nucleolar localization signal for efficient relocation to the nucleoli. Within the fibrillar centers, CypB colocalized with RNA polymerase, upstream binding factor-1 (UBF), fibrillarin and dyskerin (DCK1). Even after chemical disruption of the nucleoli, a strong interaction with these proteins remained. Using live cell imaging, we showed a persistent colocalization of CypB with proteins involved in the ribosome biogenesis during the transcriptionally more active phases of the cell cycle. Supported by in silico data, our observations suggest that CypB interacts with these proteins and is involved in ribosome biogenesis and RNA transcription.

The PKD domain distinguishes the trafficking and amyloidogenic properties of the pigment cell protein PMEL and its homologue GPNMB

PubMed Central

Theos, Alexander C.; Watt, Brenda; Harper, Dawn C.; Janczura, Karolina J.; Theos, Sarah C.; Herman, Kathryn E.; Marks, Michael S.

2013-01-01

SUMMARY Proteolytic fragments of the pigment cell-specific glycoprotein, PMEL, form the amyloid fibrillar matrix underlying melanins in melanosomes. The fibrils form within multivesicular endosomes to which PMEL is selectively sorted and that serve as melanosome precursors. GPNMB is a tissue-restricted glycoprotein with substantial sequence homology to PMEL but no known function, and was proposed to localize to non-fibrillar domains of distinct melanosome subcompartments in melanocytes. Here we confirm that GPNMB localizes to compartments distinct from the PMEL-containing multivesicular premelanosomes or late endosomes in melanocytes and HeLa cells, respectively, and is largely absent from fibrils. Using domain swapping, the unique PMEL localization is ascribed to its PKD domain, whereas the homologous PKD domain of GPNMB lacks apparent sorting function. The difference likely reflects extensive modification of the GPNMB PKD domain by N-glycosylation, nullifying its sorting function. These results reveal the molecular basis for the distinct trafficking and morphogenetic properties of PMEL and GPNMB, and support a deterministic function of the PMEL PKD domain in both protein sorting and amyloidogenesis. PMID:23452376

Investigation of Fibril Forming Mechanisms of l-Phenylalanine and l-Tyrosine: Microscopic Insight toward Phenylketonuria and Tyrosinemia Type II.

PubMed

Banik, Debasis; Kundu, Sangita; Banerjee, Pavel; Dutta, Rupam; Sarkar, Nilmoni

2017-02-23

Phenylketonuria and tyrosinemia type II, the two metabolic disorders, are originated due to the complications in metabolism of phenylalanine (Phe) and tyrosine (Tyr), respectively. Several neurological injuries, involving microcephaly, mental retardation, epilepsy, motor disease, and skin problems etc., are the symptoms of these two diseases. It has been reported that toxic amyloid fibrils are formed at high concentrations of Phe and Tyr. Our study indicates that the fibril forming mechanisms of Phe and Tyr are completely different. In the case of Phe, -NH 3 + and -COO - groups of neighboring molecules interact via hydrogen bonding and polar interactions. On the other hand, there is no role of - NH 3 + group in the fibril forming mechanism of Tyr. In Tyr fibril, the two hydrogen bonding partners are -OH and -COO - groups. In addition, we have also investigated the effect of three lanthanide cations on the fibrillar assemblies of Phe. It has been observed that the efficiencies of three lanthanides to inhibit the fibrillar assemblies of Phe follow the order Tb 3+ < Sm 3+ < Eu 3+ .

Nonlinear microscopy of collagen fibers

NASA Astrophysics Data System (ADS)

Strupler, M.; Pena, A.-M.; Hernest, M.; Tharaux, P.-L.; Fabre, A.; Marchal-Somme, J.; Crestani, B.; Débarre, D.; Martin, J.-L.; Beaurepaire, E.; Schanne-Klein, M.-C.

2007-02-01

We used intrinsic Second Harmonic Generation (SHG) by fibrillar collagen to visualize the three-dimensional architecture of collagen fibrosis at the micrometer scale using laser scanning nonlinear microscopy. We showed that SHG signals are highly specific to fibrillar collagen and provide a sensitive probe of the micrometer-scale structural organization of collagen in tissues. Moreover, recording simultaneously other nonlinear optical signals in a multimodal setup, we visualized the tissue morphology using Two-Photon Excited Fluorescence (2PEF) signals from endogenous chromophores such as NADH or elastin. We then compared different methods to determine accurate indexes of collagen fibrosis using nonlinear microscopy, given that most collagen fibrils are smaller than the microscope resolution and that second harmonic generation is a coherent process. In order to define a robust method to process our three-dimensional images, we either calculated the fraction of the images occupied by a significant SHG signal, or averaged SHG signal intensities. We showed that these scores provide an estimation of the extension of renal and pulmonary fibrosis in murine models, and that they clearly sort out the fibrotic mice.

The shape modulation of osteoblast-osteocyte transformation and its correlation with the fibrillar organization in secondary osteons: a SEM study employing the graded osmic maceration technique.

PubMed

Pazzaglia, Ugo E; Congiu, Terenzio; Marchese, Marcella; Dell'Orbo, Carlo

2010-06-01

Cortex fractured surface and graded osmic maceration techniques were used to study the secretory activity of osteoblasts, the transformation of osteoblast to osteocytes, and the structural organization of the matrix around the cells with scanning electron microscopy (SEM). A specialized membrane differentiation at the base of the cell was observed with finger-like, flattened processes which formed a diffuse meshwork. These findings suggested that this membrane differentiation below the cells had not only functioned in transporting collagen through the membrane but also in orienting the fibrils once assembled. Thin ramifications arose from the large and flat membrane foldings oriented perpendicular to the plane of the osteoblasts. This meshwork of fine filaments could not be visualized with SEM because they were obscured within the matrix substance. Their 3-D structure, however, should be similar to the canalicular system. The meshwork of large, flattened processes was no more evident in the cells which had completed their transformation into osteocytes.

Core sequence of PAPf39 amyloid fibrils and mechanism of pH-dependent fibril formation: the role of monomer conformation.

PubMed

French, Kinsley C; Makhatadze, George I

2012-12-21

PAPf39, a 39-residue peptide fragment from human prostatic acidic phosphatase, has been shown to form amyloid fibrils in semen (SEVI), which increase HIV infectivity by up to 5 orders of magnitude. The sequence of the PAPf39 fibrillar core was identified using hydrogen-deuterium exchange (HDX) mass spectrometry and protease protection assays. The central and C-terminal regions are highly protected from HDX and proteolytic cleavage and, thus, are part of the fibrillar core. Conversely, the N-terminal region is unprotected from HDX and proteolytic cleavage, suggesting that it is exposed and not part of the fibrillar core. This finding was tested using two N-terminal truncated variants, PAPf39Δ1-8 and PAPf39Δ1-13. Both variants formed amyloid fibrils at neutral pH. However, these variants showed a markedly different pH dependence of fibril formation versus that of PAPf39. PAPf39 fibrils can form at pH 7.7, but not at pH 5.5 or 2.5, while both N-terminally truncated variants can form fibrils at these pH values. Thus, the N-terminal region is not necessary for fibril formation but modulates the pH dependence of PAPf39 fibril formation. PAPf39Δ1-8 and PAPf39Δ1-13 are capable of seeding PAPf39 fibril formation at neutral pH, suggesting that these variants are structurally compatible with PAPf39, yet no mixed fibril formation occurs between the truncated variants and PAPf39 at low pH. This suggests that pH affects the PAPf39 monomer conformational ensemble, which is supported by far-UV circular dichroism spectroscopy. A conceptual model describing the pH dependence of PAPf39 aggregation is proposed and provides potential biological implications.

Characteristics of liver fibrosis with different etiologies using a fully quantitative fibrosis assessment tool.

PubMed

Wu, Q; Zhao, X; You, H

2017-05-18

This study aimed to test the diagnostic performance of a fully quantitative fibrosis assessment tool for liver fibrosis in patients with chronic hepatitis B (CHB), primary biliary cirrhosis (PBC) and non-alcoholic steatohepatitis (NASH). A total of 117 patients with liver fibrosis were included in this study, including 50 patients with CHB, 49 patients with PBC and 18 patients with NASH. All patients underwent liver biopsy (LB). Fibrosis stages were assessed by two experienced pathologists. Histopathological images of LB slices were processed by second harmonic generation (SHG)/two-photon excited fluorescence (TPEF) microscopy without staining, a system called qFibrosis (quantitative fibrosis) system. Altogether 101 quantitative features of the SHG/TPEF images were acquired. The parameters of aggregated collagen in portal, septal and fibrillar areas increased significantly with stages of liver fibrosis in PBC and CHB (P<0.05), but the same was not found for parameters of distributed collagen (P>0.05). There was a significant correlation between parameters of aggregated collagen in portal, septal and fibrillar areas and stages of liver fibrosis from CHB and PBC (P<0.05), but no correlation was found between the distributed collagen parameters and the stages of liver fibrosis from those patients (P>0.05). There was no significant correlation between NASH parameters and stages of fibrosis (P>0.05). For CHB and PBC patients, the highest correlation was between septal parameters and fibrosis stages, the second highest was between portal parameters and fibrosis stages and the lowest correlation was between fibrillar parameters and fibrosis stages. The correlation between the septal parameters of the PBC and stages is significantly higher than the parameters of the other two areas (P<0.05). The qFibrosis candidate parameters based on CHB were also applicable for quantitative analysis of liver fibrosis in PBC patients. Different parameters should be selected for liver fibrosis assessment in different stages of PBC compared with CHB.

Characteristics of liver fibrosis with different etiologies using a fully quantitative fibrosis assessment tool

PubMed Central

Wu, Q.; Zhao, X.; You, H.

2017-01-01

This study aimed to test the diagnostic performance of a fully quantitative fibrosis assessment tool for liver fibrosis in patients with chronic hepatitis B (CHB), primary biliary cirrhosis (PBC) and non-alcoholic steatohepatitis (NASH). A total of 117 patients with liver fibrosis were included in this study, including 50 patients with CHB, 49 patients with PBC and 18 patients with NASH. All patients underwent liver biopsy (LB). Fibrosis stages were assessed by two experienced pathologists. Histopathological images of LB slices were processed by second harmonic generation (SHG)/two-photon excited fluorescence (TPEF) microscopy without staining, a system called qFibrosis (quantitative fibrosis) system. Altogether 101 quantitative features of the SHG/TPEF images were acquired. The parameters of aggregated collagen in portal, septal and fibrillar areas increased significantly with stages of liver fibrosis in PBC and CHB (P<0.05), but the same was not found for parameters of distributed collagen (P>0.05). There was a significant correlation between parameters of aggregated collagen in portal, septal and fibrillar areas and stages of liver fibrosis from CHB and PBC (P<0.05), but no correlation was found between the distributed collagen parameters and the stages of liver fibrosis from those patients (P>0.05). There was no significant correlation between NASH parameters and stages of fibrosis (P>0.05). For CHB and PBC patients, the highest correlation was between septal parameters and fibrosis stages, the second highest was between portal parameters and fibrosis stages and the lowest correlation was between fibrillar parameters and fibrosis stages. The correlation between the septal parameters of the PBC and stages is significantly higher than the parameters of the other two areas (P<0.05). The qFibrosis candidate parameters based on CHB were also applicable for quantitative analysis of liver fibrosis in PBC patients. Different parameters should be selected for liver fibrosis assessment in different stages of PBC compared with CHB. PMID:28538834

Thrombi produced in stagnation point flows have a core-shell structure.

PubMed

Herbig, Bradley A; Diamond, Scott L

2017-12-01

In regions of flow separation/reattachment within diseased arteries, the local hemodynamics can result in stagnation point flow that provides an atypical environment in atherosclerosis. Impinging flows occur with recirculation eddies distal of coronary stenosis or diseased carotid bifurcations. By perfusing whole blood directly perpendicular to a fibrillar collagen thrombotic surface, a microfluidic device produced a stagnation point flow. Side view visualization of thrombosis in this assay allowed for observation of clot structure and composition at various flow rates and blood biochemistry conditions. For clotting over collagen/tissue factor surfaces, platelet thrombi formed in this device displayed a core-shell architecture with a fibrin-rich, platelet P-selectin-positive core and an outer platelet P-selectin-negative shell. VWF was detected in clots at low and high shear, but when N-acetylcysteine was added to the whole blood, both platelet and VWF deposition were markedly decreased at either low or high flow. To further examine the source of clot stability, 1 mM GPRP was added to prevent fibrin formation while allowing the PAR1/4-cleaving activity of thrombin to progress. The inhibition of fibrin polymerization did not change the overall structure of the clots, demonstrating the stability of these clots without fibrin. Impinging flow microfluidics generate thrombi with a core-shell structure.

An Efficient Procedure for Removal and Inactivation of Alpha-Synuclein Assemblies from Laboratory Materials.

PubMed

Bousset, Luc; Brundin, Patrik; Böckmann, Anja; Meier, Beat; Melki, Ronald

2016-01-01

Preformed α-synuclein fibrils seed the aggregation of soluble α-synuclein in cultured cells and in vivo. This, and other findings, has kindled the idea that α-synuclein fibrils possess prion-like properties. As α-synuclein fibrils should not be considered as innocuous, there is a need for decontamination and inactivation procedures for laboratory benches and non-disposable laboratory material. We assessed the effectiveness of different procedures designed to disassemble α-synuclein fibrils and reduce their infectivity. We examined different commercially available detergents to remove α-synuclein assemblies adsorbed on materials that are not disposable and that are most found in laboratories (e.g. plastic, glass, aluminum or stainless steel surfaces). We show that methods designed to decrease PrP prion infectivity neither effectively remove α-synuclein assemblies adsorbed to different materials commonly used in the laboratory nor disassemble the fibrillar form of the protein with efficiency. In contrast, both commercial detergents and SDS detached α-synuclein assemblies from contaminated surfaces and disassembled the fibrils. We describe three cleaning procedures that effectively remove and disassemble α-synuclein seeds. The methods rely on the use of detergents that are compatible with most non-disposable tools in a laboratory. The procedures are easy to implement and significantly decrease any potential risks associated to handling α-synuclein assemblies.

Collagenous microstructure of the glenoid labrum and biceps anchor

PubMed Central

Hill, A M; Hoerning, E J; Brook, K; Smith, C D; Moss, J; Ryder, T; Wallace, A L; Bull, A M J

2008-01-01

The glenoid labrum is a significant passive stabilizer of the shoulder joint. However, its microstructural form remains largely unappreciated, particularly in the context of its variety of functions. The focus of labral microscopy has often been histology and, as such, there is very little appreciation of collagen composition and arrangement of the labrum, and hence the micromechanics of the structure. On transmission electron microscopy, significant differences in diameter, area and perimeter were noted in the two gross histological groups of collagen fibril visualized; this suggests a heterogeneous collagenous composition with potentially distinct mechanical function. Scanning electron microscopy demonstrated three distinct zones of interest: a superficial mesh, a dense circumferential braided core potentially able to accommodate hoop stresses, and a loosely packed peri-core zone. Confocal microscopy revealed an articular surface fine fibrillar mesh potentially able to reduce surface friction, bundles of circumferential encapsulated fibres in the bulk of the tissue, and bone anchoring fibres at the osseous interface. Varying microstructure throughout the depth of the labrum suggests a role in accommodating different types of loading. An understanding of the labral microstructure can lead to development of hypotheses based upon an appreciation of this component of material property. This may aid an educated approach to surgical timing and repair. PMID:18429974

Polyolefin Nanocomposites with Enhanced Photostability Weathering Effect on Morphology and Mechanical Properties

NASA Astrophysics Data System (ADS)

Panda, Bishnu P.; Mohanty, Smita; Nayak, Sanjay K.

2014-09-01

This research aims to study the effect of accelerated weathering conditions on the photodegradation characteristics for fibrillar silicate clay-filled Polypropylene (PP) nanocomposites in the presence of metallocene linear low density polyethylene (m-LLDPE). Silane-treated attapulgite (ATP) clay along with ethylene octene elastomer-grafted maleic anhydride (POE-g-MAH) was used to compatibilize both blend and nanocomposite system. The result showed that developed PP/m-LLDPE nanocomposites displayed good UV resistance with little change in retained stress-at-break and elongation-at-break values. Balanced loss of toughness values noted maintaining higher fracture toughness values for nanocomposites containing 5 phr ATP clay. Infrared analysis was used to detect progress of degradation followed by change in carbonyl index revealed predominated chain scission in late irradiation, while crosslinking was dominant for initial irradiation period. An increase in crystallinity during UV exposure (chemi-crystallization) was detected with exposure time for all compositions and virtually independent of initial structure of the polymer. The highest value of crystallization observed for PP and the lowest one for nanocomposites containing 5 phr of ATP clay revealed good oxidation stability. Surface morphology revealed induced degradation throughout cross-section of PP, while severity of the surface degradation was significantly reduced for developed nanocomposites.

Nucleation and Growth of Insulin Fibrils in Bulk Solution and at Hydrophobic Polystyrene Surfaces

PubMed Central

Smith, M. I.; Sharp, J. S.; Roberts, C. J.

2007-01-01

A technique was developed for studying the nucleation and growth of fibrillar protein aggregates. Fourier transform infrared and attenuated total reflection spectroscopy were used to measure changes in the intermolecular β-sheet content of bovine pancreatic insulin in bulk solution and on model polystyrene (PS) surfaces at pH 1. The kinetics of β-sheet formation were shown to evolve in two stages. Combined Fourier transform infrared, dynamic light scattering, atomic force microscopy, and thioflavin-T fluorescence measurements confirmed that the first stage in the kinetics was related to the formation of nonfibrillar aggregates that have a radius of 13 ± 1 nm. The second stage was found to be associated with the growth of insulin fibrils. The β-sheet kinetics in this second stage were used to determine the nucleation and growth rates of fibrils over a range of temperatures between 60°C and 80°C. The nucleation and growth rates were shown to display Arrhenius kinetics, and the associated energy barriers were extracted for fibrils formed in bulk solution and at PS surfaces. These experiments showed that fibrils are nucleated more quickly in the presence of hydrophobic PS surfaces but that the corresponding fibril growth rates decrease. These observations are interpreted in terms of the differences in the attempt frequencies and energy barriers associated with the nucleation and growth of fibrils. They are also discussed in the context of differences in protein concentration, mobility, and conformational and colloidal stability that exist between insulin molecules in bulk solution and those that are localized at hydrophobic PS interfaces. PMID:17496011

Alignment of human cardiomyocytes on laser patterned biphasic core/shell nanowire assemblies

NASA Astrophysics Data System (ADS)

Kiefer, Karin; Lee, Juseok; Haidar, Ayman; Martinez Miró, Marina; Akkan, Cagri Kaan; Veith, Michael; Cenk Aktas, Oral; Abdul-Khaliq, Hashim

2014-12-01

The management of end stage heart failure patients is only possible by heart transplantation or by the implantation of artificial hearts as a bridge for later transplantation. However, these therapeutic strategies are limited by a lack of donor hearts and by the associated complications, such as coagulation and infection, due to the used artificial mechanical circulatory assist devices. Therefore, new strategies for myocardial regenerative approaches are under extensive research to produce contractile myocardial tissue in the future to replace non-contractile myocardial ischemic and scarred tissue. Different approaches, such as cell transplantation, have been studied intensively. Although successful approaches have been observed, there are still limitations to the application. It is envisaged that myocardial tissue engineering can be used to help replace infarcted non-contractile tissue. The developed tissue should later mimic the aligned fibrillar structure of the extracellular matrix and provide important guidance cues for the survival, function and the needed orientation of cardiomyocytes. Nanostructured surfaces have been tested to provide a guided direction that cells can follow. In the present study, the cellular adhesion/alignment of human cardiomyocytes and the biocompatibility have been investigated after cultivation on different laser-patterned nanowires compared with unmodified nanowires. As a result, the nanostructured surfaces possessed good biocompatibility before and after laser modification. The laser-induced scalability of the pattern enabled the growth and orientation of the adhered myocardial tissue. Such approaches may be used to modify the surface of potential scaffolds to develop myocardial contractile tissue in the future.

Aging and the cardiac collagen matrix: Novel mediators of fibrotic remodelling.

PubMed

Horn, Margaux A; Trafford, Andrew W

2016-04-01

Cardiovascular disease is a leading cause of death worldwide and there is a pressing need for new therapeutic strategies to treat such conditions. The risk of developing cardiovascular disease increases dramatically with age, yet the majority of experimental research is executed using young animals. The cardiac extracellular matrix (ECM), consisting predominantly of fibrillar collagen, preserves myocardial integrity, provides a means of force transmission and supports myocyte geometry. Disruptions to the finely balanced control of collagen synthesis, post-synthetic deposition, post-translational modification and degradation may have detrimental effects on myocardial functionality. It is now well established that the aged heart is characterized by fibrotic remodelling, but the mechanisms responsible for this are incompletely understood. Furthermore, studies using aged animal models suggest that interstitial remodelling with disease may be age-dependent. Thus with the identification of new therapeutic strategies targeting fibrotic remodelling, it may be necessary to consider age-dependent mechanisms. In this review, we discuss remodelling of the cardiac collagen matrix as a function of age, whilst highlighting potential novel mediators of age-dependent fibrotic pathways. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Phenytoin accelerates tendon healing in a rat model of Achilles tendon rupture.

PubMed

Hajipour, B; Navali, A M; Mohammad, S Ali; Mousavi, G; Akbari, M Gahvechi; Miyandoab, T Maleki; Roshangar, L; Saleh, B Mohammadi; Kermani, T Asvadi; Laleh, F Moutab; Ghabili, M

2016-01-01

Tendons are vulnerable to various types of acute or chronic injures. Different methods have been investigated to achieve better healing. Phenytoin is a drug which could stimulate fibroblasts to produce collagen. This experimental study was performed to assess the effect of phenytoin on tendon healing in a rat model of tendon rupture. Thirty healthy rats were divided into 3 groups, 1) Sham group; 2) Tendon rupture; 3) Tendon rupture+phenytoin (100 mg/kg intraperitoneally) for 21 days. On 21st day after tendon injury, the rats were anesthetized and tendon tissue was sampled for studying by light and electron microscopy. Qualitative and quantitative microscopic comparisons of the repair tissues of both groups were made on the 21st day. The results obtained from light and electron microscopy studies showed that tendon tissue healing was significantly better in phenytoin group compared to the control group (p < 0.05). Systemic administration of phenytoin may have a positive effect on tendon healing by increasing fibroblast quantity, fibrillar collagen synthesis, vascularity, and suppressing inflammation (Tab. 2, Ref. 25).

Texture analysis applied to second harmonic generation image data for ovarian cancer classification

NASA Astrophysics Data System (ADS)

Wen, Bruce L.; Brewer, Molly A.; Nadiarnykh, Oleg; Hocker, James; Singh, Vikas; Mackie, Thomas R.; Campagnola, Paul J.

2014-09-01

Remodeling of the extracellular matrix has been implicated in ovarian cancer. To quantitate the remodeling, we implement a form of texture analysis to delineate the collagen fibrillar morphology observed in second harmonic generation microscopy images of human normal and high grade malignant ovarian tissues. In the learning stage, a dictionary of "textons"-frequently occurring texture features that are identified by measuring the image response to a filter bank of various shapes, sizes, and orientations-is created. By calculating a representative model based on the texton distribution for each tissue type using a training set of respective second harmonic generation images, we then perform classification between images of normal and high grade malignant ovarian tissues. By optimizing the number of textons and nearest neighbors, we achieved classification accuracy up to 97% based on the area under receiver operating characteristic curves (true positives versus false positives). The local analysis algorithm is a more general method to probe rapidly changing fibrillar morphologies than global analyses such as FFT. It is also more versatile than other texture approaches as the filter bank can be highly tailored to specific applications (e.g., different disease states) by creating customized libraries based on common image features.

Distribution of DNA in human Sertoli cell nucleoli.

PubMed

Mosgöller, W; Schöfer, C; Derenzini, M; Steiner, M; Maier, U; Wachtler, F

1993-10-01

For better understanding of nucleolar architecture, different techniques have been used to localize DNA within the dense fibrillar component (DF) or within the fibrillar centers (FC) by electron microscopy (EM). Since it still remains controversial which components contain DNA, we investigated the distribution of DNA in human Sertoli cells using various approaches. In situ hybridization (ISH) with human total genomic DNA as probe and the use of anti-DNA antibody were followed by immunogold detection. This allowed statistical evaluation of the signal density over individual components. The Feulgen-like osmium-ammine (OA) technique for the selective visualization of DNA was also applied. The anti-DNA antibodies detected DNA in mitochondria, in chromatin, and in the DF of the nucleolus. ISH using human total genomic DNA showed similar labeling patterns. The OA technique revealed DNA filaments in the FC and focal agglomerates of decondensed DNA within the DF. We conclude that (a) EM staining techniques that utilize colloidal gold appear to be less sensitive for DNA detection than the OA method, (b) the DF consists of different domains with different molecular composition, and (c) decondensed DNA is not necessarily confined to one particular nucleolar component.

A Nonribosomal Landscape in the Nucleolus Revealed by the Stem Cell Protein Nucleostemin

PubMed Central

Politz, Joan C. Ritland; Polena, Ilvin; Trask, Ian; Bazett-Jones, David P.; Pederson, Thoru

2005-01-01

Nucleostemin is a p53-interactive cell cycle progression factor that shuttles between the nucleolus and nucleoplasm, but it has no known involvement in ribosome synthesis. We found the dynamic properties of nucleostemin differed strikingly from fibrillarin (a protein directly involved in rRNA processing) both in response to rRNA transcription inhibition and in the schedule of reentry into daughter nuclei and the nucleolus during late telophase/early G1. Furthermore, nucleostemin was excluded from the nucleolar domains in which ribosomes are born—the fibrillar centers and dense fibrillar component. Instead it was concentrated in rRNA-deficient sites within the nucleolar granular component. This finding suggests that the nucleolus may be more subcompartmentalized than previously thought. In support of this concept, electron spectroscopic imaging studies of the nitrogen and phosphorus distribution in the nucleolar granular component revealed regions that are very rich in protein and yet devoid of nucleic acid. Together, these results suggest that the ultrastructural texture of the nucleolar granular component represents not only ribosomal particles but also RNA-free zones populated by proteins or protein complexes that likely serve other functions. PMID:15857956

Reproduction of the FC/DFC units in nucleoli.

PubMed

Smirnov, Evgeny; Hornáček, Matúš; Kováčik, Lubomír; Mazel, Tomáš; Schröfel, Adam; Svidenská, Silvie; Skalníková, Magdalena; Bartová, Eva; Cmarko, Dušan; Raška, Ivan

2016-04-25

The essential structural components of the nucleoli, Fibrillar Centers (FC) and Dense Fibrillar Components (DFC), together compose FC/DFC units, loci of rDNA transcription and early RNA processing. In the present study we followed cell cycle related changes of these units in 2 human sarcoma derived cell lines with stable expression of RFP-PCNA (the sliding clamp protein) and GFP-RPA43 (a subunit of RNA polymerase I, pol I) or GFP-fibrillarin. Correlative light and electron microscopy analysis showed that the pol I and fibrillarin positive nucleolar beads correspond to individual FC/DFC units. In vivo observations showed that at early S phase, when transcriptionally active ribosomal genes were replicated, the number of the units in each cell increased by 60-80%. During that period the units transiently lost pol I, but not fibrillarin. Then, until the end of interphase, number of the units did not change, and their duplication was completed only after the cell division, by mid G1 phase. This peculiar mode of reproduction suggests that a considerable subset of ribosomal genes remain transcriptionally silent from mid S phase to mitosis, but become again active in the postmitotic daughter cells.

Structural Insights into Amyloid Oligomers of the Parkinson Disease-related Protein α-Synuclein*

PubMed Central

Gallea, J. Ignacio; Celej, M. Soledad

2014-01-01

The presence of intraneuronal deposits mainly formed by amyloid fibrils of the presynaptic protein α-synuclein (AS) is a hallmark of Parkinson disease. Currently, neurotoxicity is attributed to prefibrillar oligomeric species rather than the insoluble aggregates, although their mechanisms of toxicity remain elusive. Structural details of the supramolecular organization of AS oligomers are critically needed to decipher the structure-toxicity relationship underlying their pathogenicity. In this study, we employed site-specific fluorescence to get a deeper insight into the internal architecture of AS oligomeric intermediates. We demonstrate that AS oligomers are ordered assemblies possessing a well defined pattern of intermolecular contacts. Some of these contacts involve regions that form the β-sheet core in the fibrillar state, although their spatial arrangement may differ in the two aggregated forms. However, even though the two termini are excluded from the fibrillar core, they are engaged in a number of intermolecular interactions within the oligomer. Therefore, substantial structural remodeling of early oligomeric interactions is essential for fibril growth. The intermolecular contacts identified in AS oligomers can serve as targets for the rational design of anti-amyloid compounds directed at preventing oligomeric interactions/reorganizations. PMID:25143382

Solid-state NMR Study Reveals Collagen I Structural Modifications of Amino Acid Side Chains upon Fibrillogenesis*

PubMed Central

De Sa Peixoto, Paulo; Laurent, Guillaume; Azaïs, Thierry; Mosser, Gervaise

2013-01-01

In vivo, collagen I, the major structural protein in human body, is found assembled into fibrils. In the present work, we study a high concentrated collagen sample in its soluble, fibrillar, and denatured states using one and two dimensional {1H}-13C solid-state NMR spectroscopy. We interpret 13C chemical shift variations in terms of dihedral angle conformation changes. Our data show that fibrillogenesis increases the side chain and backbone structural complexity. Nevertheless, only three to five rotameric equilibria are found for each amino acid residue, indicating a relatively low structural heterogeneity of collagen upon fibrillogenesis. Using side chain statistical data, we calculate equilibrium constants for a great number of amino acid residues. Moreover, based on a 13C quantitative spectrum, we estimate the percentage of residues implicated in each equilibrium. Our data indicate that fibril formation greatly affects hydroxyproline and proline prolyl pucker ring conformation. Finally, we discuss the implication of these structural data and propose a model in which the attractive force of fibrillogenesis comes from a structural reorganization of 10 to 15% of the amino acids. These results allow us to further understand the self-assembling process and fibrillar structure of collagen. PMID:23341452

Formation of vesicles through solvent assisted self-assembly of hydrophobic pentapeptides: encapsulation and pH responsive release of dyes by the vesicles.

PubMed

Kar, Sudeshna; Drew, Michael G B; Pramanik, Animesh

2011-09-01

In the biomimetic design two hydrophobic pentapetides Boc-Ile-Aib-Leu-Phe-Ala-OMe (I) and Boc-Gly-Ile-Aib-Leu-Phe-OMe (II) (Aib: α-aminoisobutyric acid) containing one Aib each are found to undergo solvent assisted self-assembly in methanol/water to form vesicular structures, which can be disrupted by simple addition of acid. The nanovesicles are found to encapsulate dye molecules that can be released by the addition of acid as confirmed by fluorescence microscopy and UV studies. The influence of solvent polarity on the morphology of the materials generated from the peptides has been examined systematically, and shows that fibrillar structures are formed in less polar chloroform/petroleum ether mixture and vesicular structures are formed in more polar methanol/water. Single crystal X-ray diffraction studies reveal that while β-sheet mediated self-assembly leads to the formation of fibrillar structures, the solvated β-sheet structure leads to the formation of vesicular structures. The results demonstrate that even hydrophobic peptides can generate vesicular structures from polar solvent which may be employed in model studies of complex biological phenomena.

Contact compliance effects in the frictional response of bioinspired fibrillar adhesives

PubMed Central

Piccardo, Marco; Chateauminois, Antoine; Fretigny, Christian; Pugno, Nicola M.; Sitti, Metin

2013-01-01

The shear failure and friction mechanisms of bioinspired adhesives consisting of elastomer arrays of microfibres terminated by mushroom-shaped tips are investigated in contact with a rigid lens. In order to reveal the interplay between the vertical and lateral loading directions, experiments are carried out using a custom friction set-up in which normal stiffness can be made either high or low when compared with the stiffness of the contact between the fibrillar adhesive and the lens. Using in situ contact imaging, the shear failure of the adhesive is found to involve two successive mechanisms: (i) cavitation and peeling at the contact interface between the mushroom-shaped fibre tip endings and the lens; and (ii) side re-adhesion of the fibre's stem to the lens. The extent of these mechanisms and their implications regarding static friction forces is found to depend on the crosstalk between the normal and lateral loading directions that can result in contact instabilities associated with fibre buckling. In addition, the effects of the viscoelastic behaviour of the polyurethane material on the rate dependence of the shear response of the adhesive are accounted for. PMID:23554349

[Cadmium citotoxicity in mice hepatocytes and implications on tropical environments].

PubMed

Marcano, Letty; Faría, Clarisa de R; Carruyo, Ingrid; Montiel, Xiomara

2006-06-01

We analyzed phenotypic, structural and ultrastructural alterations induced by Cd+2 in hepatocytes extracted from Swiss Albino mice. Cadmium was given orally in watery solution of CdCl2 during 100 days at concentrations of 50 ppm, 100 ppm and 150 ppm. In controls, distilled water alone was used. The samples were processed with the paraffin inclusion and hematoxilin-eosin coloration techniques for light microscopy. For transmission electron microscopy we used the conventional technique. We found phenotypic (size and weight differences) and physiologic changes (muscular weakness, unrest); at the structural level we noticed loss of trabecular disposition and of lobulillar architecture, lymphocyte agglomeration, vacuolization, dilatation of sinusoid and central vein, among others. The ultrastructural study evidenced alterations coincident with those seen with light microscopy, which were accentuated with the increase of metal concentration: nucleolus with a high number of fibrillar centers (50 ppm); voluminous lipidic drops in the cytoplasm, loose endoplasmic rough reticulum, citoplasmatic vacuolization, altered lisosomes and peroxisomes (100 ppm); contracted nuclei with condensed cromatine, dilatation of intracellular space and mitochondria, and loss of fibrillar areas (150 ppm). Cadmium produces a toxic effect in the hepatic cells; the effect is more severe at higher concentration, leading to cellular necrosis.

A common pathway for p10 and calyx proteins in progressive stages of polyhedron envelope assembly in AcMNPV-infected Spodoptera frugiperda larvae.

PubMed

Lee, S Y; Poloumienko, A; Belfry, S; Qu, X; Chen, W; MacAfee, N; Morin, B; Lucarotti, C; Krause, M

1996-01-01

The assembly of the polyhedron envelope in baculovirus-infected cells has been the subject of several studies, yet it is still poorly understood. We have used immunogold-labelled antibodies to two baculovirus proteins, p10 and calyx (also referred to as polyhedron envelope protein or PEP), to follow envelope assembly in AcMNPV-infected tissues of Spodoptera frugiperda larvae. We show that, in wild type virus, both proteins colocalize in fibrillar structures and associated electron-dense spacers which progress to encircle the polyhedra, as well as in completed polyhedron envelopes. In cells infected with polyhedrin-negative (PH-) viruses, an unusual proliferation of these spacers was observed suggesting a deregulatory event in the envelope assembly process. Results of Northern and Western blot analysis revealed that synthesis of P10 and calyx mRNA and proteins in PH- AcMNPV is unaffected as compared to wild type virus. Taken together, the observed physical and compositional connection between fibrillar structures, spacers and polyhedron envelopes, as well as the abnormal appearance of the spacers in PH- mutants, provide further evidence in support of a cooperative role of these structures in the assembly of the polyhedron envelope.

Structural and functional properties of prefibrillar α-synuclein oligomers

PubMed Central

Pieri, Laura; Madiona, Karine; Melki, Ronald

2016-01-01

The deposition of fibrillar alpha-synuclein (α-syn) within inclusions (Lewy bodies and Lewy neurites) in neurons and glial cells is a hallmark of synucleinopathies. α-syn populates a variety of assemblies ranging from prefibrillar oligomeric species to fibrils whose specific contribution to neurodegeneration is still unclear. Here, we compare the specific structural and biological properties of distinct soluble prefibrillar α-syn oligomers formed either spontaneously or in the presence of dopamine and glutaraldehyde. We show that both on-fibrillar assembly pathway and distinct dopamine-mediated and glutaraldehyde-cross-linked α-syn oligomers are only slightly effective in perturbing cell membrane integrity and inducing cytotoxicity, while mature fibrils exhibit the highest toxicity. In contrast to low-molecular weight and unstable oligomers, large stable α-syn oligomers seed the aggregation of soluble α-syn within reporter cells although to a lesser extent than mature α-syn fibrils. These oligomers appear elongated in shape. Our findings suggest that α-syn oligomers represent a continuum of species ranging from unstable low molecular weight particles to mature fibrils via stable elongated oligomers composed of more than 15 α-syn monomers that possess seeding capacity. PMID:27075649

Molecular characterization and expression analysis of the first Porifera tumor necrosis factor superfamily member and of its putative receptor in the marine sponge Chondrosia reniformis.

PubMed

Pozzolini, Marina; Scarfì, Sonia; Ghignone, Stefano; Mussino, Francesca; Vezzulli, Luigi; Cerrano, Carlo; Giovine, Marco

2016-04-01

Here we report the molecular cloning and characterization of the first Tumor Necrosis Factor homologous and of its putative receptor in the marine sponge Chondrosia reniformis: chTNF and chTNFR, respectively. The deduced chTNF amino acid sequence is a type II transmembrane protein containing the typical TNFSF domain. Phylogenetic analysis reveals that chTNF is more related to Chordata TNFs rather than to other invertebrates. chTNF and chTNFR are constitutively expressed both in the ectosome and in the choanosome of the sponge, with higher levels in the ectosome. chTNF and chTNFR mRNAs were monitored in sponge fragmorphs treated with Gram(+) or Gram(-) bacteria. chTNF was significantly upregulated in Gram(+)-treated fragmorphs as compared to controls, while chTNFR was upregulated by both treatments. Finally, the possible chTNF fibrogenic role in sponge fragmorphs was studied by TNF inhibitor treatment measuring fibrillar and non fibrillar collagen gene expression; results indicate that the cytokine is involved in sponge collagen deposition and homeostasis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Florbetapir PET, FDG PET, and MRI in Down syndrome individuals with and without Alzheimer's dementia.

PubMed

Sabbagh, Marwan N; Chen, Kewei; Rogers, Joseph; Fleisher, Adam S; Liebsack, Carolyn; Bandy, Dan; Belden, Christine; Protas, Hillary; Thiyyagura, Pradeep; Liu, Xiaofen; Roontiva, Auttawut; Luo, Ji; Jacobson, Sandra; Malek-Ahmadi, Michael; Powell, Jessica; Reiman, Eric M

2015-08-01

Down syndrome (DS) is associated with amyloid b (Ab) deposition. We characterized imaging measurements of regional fibrillar Ab burden, cerebral metabolic rate for glucose (rCMRgl), gray matter volumes (rGMVs), and age associations in 5 DS with dementia (DS/AD1), 12 DS without dementia (DS/AD2), and 9 normal controls (NCs). There were significant group differences in mean standard uptake value ratios (SUVRs) for florbetapir with DS/AD1 having the highest, followed by DS/AD2, followed by NC. For [18F]-fluorodeoxyglucose positron emission tomography, posterior cingulate rCMRgl in DS/AD1 was significantly reduced compared with DS/AD2 and NC. For volumetric magnetic resonance imaging (vMRI), hippocampal volumes were significantly reduced for the DS/AD1 compared with DS/AD2 and NC. Age-related SUVR increases and rCMRgl reductions were greater in DS participants than in NCs. DS is associated with fibrillar Ab, rCMRgl, and rGMV alterations in the dementia stage and before the presence of clinical decline. This study provides a foundation for the studies needed to inform treatment and prevention in DS. Copyright © 2015 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.

Nonlinear optical microscopy: use of second harmonic generation and two-photon microscopy for automated quantitative liver fibrosis studies.

PubMed

Sun, Wanxin; Chang, Shi; Tai, Dean C S; Tan, Nancy; Xiao, Guangfa; Tang, Huihuan; Yu, Hanry

2008-01-01

Liver fibrosis is associated with an abnormal increase in an extracellular matrix in chronic liver diseases. Quantitative characterization of fibrillar collagen in intact tissue is essential for both fibrosis studies and clinical applications. Commonly used methods, histological staining followed by either semiquantitative or computerized image analysis, have limited sensitivity, accuracy, and operator-dependent variations. The fibrillar collagen in sinusoids of normal livers could be observed through second-harmonic generation (SHG) microscopy. The two-photon excited fluorescence (TPEF) images, recorded simultaneously with SHG, clearly revealed the hepatocyte morphology. We have systematically optimized the parameters for the quantitative SHG/TPEF imaging of liver tissue and developed fully automated image analysis algorithms to extract the information of collagen changes and cell necrosis. Subtle changes in the distribution and amount of collagen and cell morphology are quantitatively characterized in SHG/TPEF images. By comparing to traditional staining, such as Masson's trichrome and Sirius red, SHG/TPEF is a sensitive quantitative tool for automated collagen characterization in liver tissue. Our system allows for enhanced detection and quantification of sinusoidal collagen fibers in fibrosis research and clinical diagnostics.

Apoferritin fibers: a new template for 1D fluorescent hybrid nanostructures

NASA Astrophysics Data System (ADS)

Jurado, Rocío; Castello, Fabio; Bondia, Patricia; Casado, Santiago; Flors, Cristina; Cuesta, Rafael; Domínguez-Vera, José M.; Orte, Angel; Gálvez, Natividad

2016-05-01

Recently, research in the field of protein amyloid fibers has gained great attention due to the use of these materials as nanoscale templates for the construction of functional hybrid materials. The formation of apoferritin amyloid-like protein fibers is demonstrated herein for the first time. The morphology, size and stiffness of these one-dimensional structures are comparable to the fibers formed by β-lactoglobulin, a protein frequently used as a model in the study of amyloid-like fibrillar proteins. Nanometer-sized globular apoferritin is capable of self-assembling to form 1D micrometer-sized structures after being subjected to a heating process. Depending on the experimental conditions, fibers with different morphologies and sizes are obtained. The wire-like protein structure is rich in functional groups and allows chemical functionalization with diverse quantum dots (QD), as well as with different Alexa Fluor (AF) dyes, leading to hybrid fluorescent fibers with variable emission wavelengths, from green to near infrared, depending on the QD and AFs coupled. For fibers containing the pair AF488 and AF647, efficient fluorescence energy transfer from the covalently coupled donor (AF488) to acceptor tags (AF647) takes place. Apoferritin fibers are proposed here as a new promising template for obtaining hybrid functional materials.Recently, research in the field of protein amyloid fibers has gained great attention due to the use of these materials as nanoscale templates for the construction of functional hybrid materials. The formation of apoferritin amyloid-like protein fibers is demonstrated herein for the first time. The morphology, size and stiffness of these one-dimensional structures are comparable to the fibers formed by β-lactoglobulin, a protein frequently used as a model in the study of amyloid-like fibrillar proteins. Nanometer-sized globular apoferritin is capable of self-assembling to form 1D micrometer-sized structures after being subjected to a heating process. Depending on the experimental conditions, fibers with different morphologies and sizes are obtained. The wire-like protein structure is rich in functional groups and allows chemical functionalization with diverse quantum dots (QD), as well as with different Alexa Fluor (AF) dyes, leading to hybrid fluorescent fibers with variable emission wavelengths, from green to near infrared, depending on the QD and AFs coupled. For fibers containing the pair AF488 and AF647, efficient fluorescence energy transfer from the covalently coupled donor (AF488) to acceptor tags (AF647) takes place. Apoferritin fibers are proposed here as a new promising template for obtaining hybrid functional materials. Electronic supplementary information (ESI) available: TEM images of ferritin protein fiber formation, and apoferritin after 18 days of heat treatment; FLIM-PIE technique details; fluorescence emission spectra of apoferritin and β-lactoglobulin fibers functionalized with different QDs. See DOI: 10.1039/c6nr01044j

Altered gravity influences rDNA and NopA100 localization in nucleoli

NASA Astrophysics Data System (ADS)

Sobol, M. A.; Kordyum, E. L.

Fundamental discovery of gravisensitivity of cells no specified to gravity perception focused increasing attention on an elucidation of the mechanisms involved in altered gravity effects at the cellular and subcellular levels. The nucleolus is the transcription site of rRNA genes as well as the site of processing and initial packaging of their transcripts with ribosomal and nonribosomal proteins. The mechanisms inducing the changes in the subcomponents of the nucleolus that is morphologically defined yet highly dynamic structure are still unknown in detail. To understand the functional organization of the nucleolus as in the control as under altered gravity conditions it is essential to determine both the precise location of rDNA and the proteins playing the key role in rRNA processing. Lepidium sativum seeds were germinated in 1% agar medium on the slow horizontal clinostat (2 rpm) and in the stationary conditions. We investigated the root meristematic cells dissected from the seedlings grown in darkness for two days. The investigations were carried out with anti-DNA and anti-NopA100 antibodies labeling as well as with TdT procedure, and immunogold electron microscopy. In the stationary growth conditions, the anti-DNA antibody as well TdT procedure were capable of detecting fibrillar centers (FCs) and the dense fibrillar component (DFC) in the nucleolus. In FCs, gold particles were revealed on the condensed chromatin inclusions, internal fibrils of decondensed rDNA and the transition zone FC-DFC. Quantitatively, FCs appeared 1,5 times more densely labeled than DFC. NopA100 was localized in FCs and in DFC. In FCs, the most of protein was revealed in the transition zone FC-DFC. After a quantitative study, FCs and the transition zone FC-DFC appeared to contain NopA100 1,7 times more than DFC. Under the conditions of altered gravity, quantitative data clearly showed a redistribution of nucleolar DNA and NopA100 between FCs and DFC in comparison with the control. In labeling both with anti-DNA antibody and by TdT method, 1,5 times more gold particles were localized on FCs, and 1,5 times less in DFC. Unlike the control, condensed r-chromatin blocks and inner rDNA were labeled much more than the transition zone FC-DFC in fibrillar centers. The content of NopA100 in FCs and the transition zone FC-DFC was 2,4 times more than in the control. Twice less quantity of the protein was revealed in DFC as compared to the control. In fibrillar centers, the majority of NopA100 was localized in the inner space of FCs than in the transition zone FC-DFC. Re-localization of rDNA and NopA100 in the nucleolar subcomponents indicates lowering the level of rDNA transcription as well as middle and late processing of rRNA that let us to propose lowering the functional activity of the nucleolus under the influence of altered gravity.

Florbetapir PET analysis of amyloid-β deposition in the presenilin 1 E280A autosomal dominant Alzheimer’s disease kindred: a cross-sectional study

PubMed Central

Fleisher, Adam S; Chen, Kewei; Quiroz, Yakeel T; Jakimovich, Laura J; Gomez, Madelyn Gutierrez; Langois, Carolyn M; Langbaum, Jessica B S; Ayutyanont, Napatkamon; Roontiva, Auttawut; Thiyyagura, Pradeep; Lee, Wendy; Mo, Hua; Lopez, Liliana; Moreno, Sonia; Acosta-Baena, Natalia; Giraldo, Margarita; Garcia, Gloria; Reiman, Rebecca A; Huentelman, Matthew J; Kosik, Kenneth S; Tariot, Pierre N; Lopera, Francisco; Reiman, Eric M

2012-01-01

Summary Background Fibrillar amyloid-β (Aβ) is thought to begin accumulating in the brain many years before the onset of clinical impairment in patients with Alzheimer’s disease. By assessing the accumulation of Aβ in people at risk of genetic forms of Alzheimer’s disease, we can identify how early preclinical changes start in individuals certain to develop dementia later in life. We sought to characterise the age-related accumulation of Aβ deposition in presenilin 1 (PSEN1) E280A mutation carriers across the spectrum of preclinical disease. Methods Between Aug 1 and Dec 6, 2011, members of the familial Alzheimer’s disease Colombian kindred aged 18–60 years were recruited from the Alzheimer’s Prevention Initiative’s registry at the University of Antioquia, Medellín, Colombia. Cross-sectional assessment using florbetapir PET was done in symptomatic mutation carriers with mild cognitive impairment or mild dementia, asymptomatic carriers, and asymptomatic non-carriers. These assessments were done at the Banner Alzheimer’s Institute in Phoenix, AZ, USA. A cortical grey matter mask consisting of six predefined regions. was used to measure mean cortical florbetapir PET binding. Cortical-to-pontine standard-uptake value ratios were used to characterise the cross-sectional accumulation of fibrillar Aβ deposition in carriers and non-carriers with regression analysis and to estimate the trajectories of fibrillar Aβ deposition. Findings We enrolled a cohort of 11 symptomatic individuals, 19 presymptomatic mutation carriers, and 20 asymptomatic non-carriers, ranging in age from 20 to 56 years. There was greater florbetapir binding in asymptomatic PSEN1 E280A mutation carriers than in age matched non-carriers. Fibrillar Aβ began to accumulate in PSEN 1E280A mutation carriers at a mean age of 28·2 years (95% CI 27·3–33·4), about 16 years and 21 years before the predicted median ages at mild cognitive impairment and dementia onset, respectively. 18F florbetapir binding rose steeply over the next 9·4 years and plateaued at a mean age of 37·6 years (95% CI 35·3–40·2), about 6 and 11 years before the expected respective median ages at mild cognitive impairment and dementia onset. Prominent florbetapir binding was seen in the anterior and posterior cingulate, precuneus, and parietotemporal and frontal grey matter, as well as in the basal ganglia. Binding in the basal ganglia was not seen earlier or more prominently than in other regions. Interpretation These findings contribute to the understanding of preclinical familial Alzheimer’s disease and help set the stage for assessment of amyloid-modifying treatments in the prevention of familial Alzheimer’s disease. Funding Avid Radiopharmaceuticals, Banner Alzheimer’s Foundation, Nomis Foundation, Anonymous Foundation, Forget Me Not Initiative, Colciencias, National Institute on Aging, and the State of Arizona. PMID:23137949

The use of biomaterials for cell function enhancement: acceleration of fibroblast migration and promotion of stem cell proliferation

NASA Astrophysics Data System (ADS)

Qin, Sisi

Wound healing and tissue regeneration proceed via fibroblast migration along three dimensional scaffolds composed of fibers with different diameters, spacing, and junction angles. In order to understand how each of these factors influences fibroblast migration, a technique for preparation of three dimensional fibrillar scaffolds was developed where the fiber diameters and the angles between adjacent fiber layers could be precisely controlled. In order to study the en-mass migration process, the agarose droplet method was chosen since it enabled accurate determinations of the dependence of the migration speed, focal adhesion distribution, and nuclear deformation on the fiber structures. Results showed that on oriented single fiber layers, if the fiber diameters exceeded 1microm, large focal adhesion complexes formed in a linear arrangement along the fiber axis and cell motion was highly correlated. For fibers 1microm or less, some cell alignment along the fiber direction was measured, but no correlation between the distribution of focal adhesion points and fiber orientation was found. On multi layered scaffolds the focal adhesion sites were found to concentrate at the junction points and the migration speed followed a parabolic function with a distinct minimum at 35°. When compared to fibroblasts plated on 90° fibers, fibroblasts plated on 30° fibers showed a decrease of 25% in the degree of nuclear deformation and an increase of 25% in the number of focal adhesion sites, indicating that cell migration speed was correlated to the angle and distance of approach to the junction point. The time dependence of the migration velocity on oriented fibers was measured for four days and compared to the value measured on flat surfaces. After the initial 24 hour incubation period, the cells on both the 8microm fibers and flat surfaces migrated with a similar speed. During the next three days the migration speed for the cells on the fibrillar surfaces doubled in magnitude, while remained constant for the cells on the flat surfaces. The increased speed on the 8microm fiber surfaces could be correlated with a 20% increase in the nuclear deformation, and a decrease around 30% in the number of focal adhesion during the same observation period. RNA expression of Myosin IIA, a protein which complexes to the actin and is responsible for exertion of traction forces during migration was not upregulated during this process. On the other hand, histochemical staining of Myosin IIA showed that the protein had re-organized into large fibers which spanned the length of the cells. Observation of the cell morphology indicated that a new mode of motion had been established. Rather than the classical retraction of the cytoplasm followed by center of mass translation, which was observed on the flat surfaces, the cells were now moving by a contractile motion around the nucleus similar to that found in muscular motion. This mode was found to be more efficient when undergoing oriented motion. In addition to orientation, surface mechanics are also important in the tissue regeneration process. This study demonstrated that mechanical factors are important for the maintenance of pluripotency and control of proliferation rates. CD34+ hematopoietic stem cells (HSCs) were transduced with ICD (intracellular domain)-Notch and plated on gelatin hydrogels, whose moduli were controlled by the crosslinking ratio. On the softer hydrogel, a synergy was achieved which resulted in more than a five-fold increase in proliferation and a four-fold increase in the preservation of stemness, while HSCs maintained their ability to differentiate into multiple blood cell lineages. These results point the way for achieving clinically significant expansion of human HSCs.

The nanostructures of native celluloses, their transformations upon isolation, and their implications for production of nanocelluloses

Treesearch

Rajai H. Atalla; Rowan S Atalla; Umesh P. Agarwal

2018-01-01

Native celluloses in plant cell walls occur in a variety of highly periodic fibrillar forms that have curvature and varying degrees of twist about their longitudinal axes. Though X-ray measurements reveal diffraction patterns, the celluloses are not crystalline in the traditional sense. The diffraction patterns rather are a consequence of the high degree of spatial...

Combined enzymatic and physical deinking methodology for efficient eco-friendly recycling of old newsprint.

PubMed

Virk, Antar Puneet; Puri, Minakshi; Gupta, Vijaya; Capalash, Neena; Sharma, Prince

2013-01-01

The development in the deinking process has made recycled fiber a major part of the raw material for pulp and paper industry. Enzymes have revolutionized the deinking process obtaining brightness levels surpassing conventional deinking processes. This study explores the deinking efficiencies of bacterial alkalophilic laccase (L) and xylanase (X) enzymes along with physical deinking methods of microwaving (MW) and sonication (S) for recycling of old newsprint (ONP). The operational parameters viz. enzyme dose, pH and treatment time for X and L deinking were optimized statistically using Response Surface Methodology. Laccase did not require any mediator supplementation for deinking. Deinking of ONP pulp with a combination of xylanase and laccase enzymes was investigated, and fiber surface composition and morphological changes were studied using X-ray diffraction, fourier transform infrared spectroscopy and scanning electron microscopy. Compared to the pulp deinked with xylanase (47.9%) or laccase (62.2%) individually, the percentage reduction of effective residual ink concentration (ERIC) was higher for the combined xylanase/laccase-deinked pulp (65.8%). An increase in brightness (21.6%), breaking length (16.5%), burst factor (4.2%) tear factor (6.9%), viscosity (13%) and cellulose crystallinity (10.3%) along with decrease in kappa number (22%) and chemical consumption (50%) were also observed. Surface appeared more fibrillar along with changes in surface functional groups. A combination of physical and enzymatic processes (S-MW-XL) for deinking further improved brightness (28.8%) and decreased ERIC (73.9%) substantially. This is the first report on deinking of ONP with laccase without any mediator supplementation. XL pretreatment resulted in marked improvement in paper quality and a new sequence being reported for deinking (S-MW-XL) will contribute further in decreasing chemical consumption and making the process commercially feasible.

Combined Enzymatic and Physical Deinking Methodology for Efficient Eco-Friendly Recycling of Old Newsprint

PubMed Central

Virk, Antar Puneet; Puri, Minakshi; Gupta, Vijaya; Capalash, Neena; Sharma, Prince

2013-01-01

Background The development in the deinking process has made recycled fiber a major part of the raw material for pulp and paper industry. Enzymes have revolutionized the deinking process obtaining brightness levels surpassing conventional deinking processes. This study explores the deinking efficiencies of bacterial alkalophilic laccase (L) and xylanase (X) enzymes along with physical deinking methods of microwaving (MW) and sonication (S) for recycling of old newsprint (ONP). Methods and Results The operational parameters viz. enzyme dose, pH and treatment time for X and L deinking were optimized statistically using Response Surface Methodology. Laccase did not require any mediator supplementation for deinking. Deinking of ONP pulp with a combination of xylanase and laccase enzymes was investigated, and fiber surface composition and morphological changes were studied using X-ray diffraction, fourier transform infrared spectroscopy and scanning electron microscopy. Compared to the pulp deinked with xylanase (47.9%) or laccase (62.2%) individually, the percentage reduction of effective residual ink concentration (ERIC) was higher for the combined xylanase/laccase-deinked pulp (65.8%). An increase in brightness (21.6%), breaking length (16.5%), burst factor (4.2%) tear factor (6.9%), viscosity (13%) and cellulose crystallinity (10.3%) along with decrease in kappa number (22%) and chemical consumption (50%) were also observed. Surface appeared more fibrillar along with changes in surface functional groups. A combination of physical and enzymatic processes (S-MW-XL) for deinking further improved brightness (28.8%) and decreased ERIC (73.9%) substantially. Conclusion This is the first report on deinking of ONP with laccase without any mediator supplementation. XL pretreatment resulted in marked improvement in paper quality and a new sequence being reported for deinking (S-MW-XL) will contribute further in decreasing chemical consumption and making the process commercially feasible. PMID:23977287

Generating standardized image data for testing and calibrating quantification of volumes, surfaces, lengths, and object counts in fibrous and porous materials using X-ray microtomography.

PubMed

Jiřík, Miroslav; Bartoš, Martin; Tomášek, Petr; Malečková, Anna; Kural, Tomáš; Horáková, Jana; Lukáš, David; Suchý, Tomáš; Kochová, Petra; Hubálek Kalbáčová, Marie; Králíčková, Milena; Tonar, Zbyněk

2018-06-01

Quantification of the structure and composition of biomaterials using micro-CT requires image segmentation due to the low contrast and overlapping radioopacity of biological materials. The amount of bias introduced by segmentation procedures is generally unknown. We aim to develop software that generates three-dimensional models of fibrous and porous structures with known volumes, surfaces, lengths, and object counts in fibrous materials and to provide a software tool that calibrates quantitative micro-CT assessments. Virtual image stacks were generated using the newly developed software TeIGen, enabling the simulation of micro-CT scans of unconnected tubes, connected tubes, and porosities. A realistic noise generator was incorporated. Forty image stacks were evaluated using micro-CT, and the error between the true known and estimated data was quantified. Starting with geometric primitives, the error of the numerical estimation of surfaces and volumes was eliminated, thereby enabling the quantification of volumes and surfaces of colliding objects. Analysis of the sensitivity of the thresholding upon parameters of generated testing image sets revealed the effects of decreasing resolution and increasing noise on the accuracy of the micro-CT quantification. The size of the error increased with decreasing resolution when the voxel size exceeded 1/10 of the typical object size, which simulated the effect of the smallest details that could still be reliably quantified. Open-source software for calibrating quantitative micro-CT assessments by producing and saving virtually generated image data sets with known morphometric data was made freely available to researchers involved in morphometry of three-dimensional fibrillar and porous structures in micro-CT scans. © 2018 Wiley Periodicals, Inc.

Evaluation of protease resistance and toxicity of amyloid-like food fibrils from whey, soy, kidney bean, and egg white.

PubMed

Lassé, Moritz; Ulluwishewa, Dulantha; Healy, Jackie; Thompson, Dion; Miller, Antonia; Roy, Nicole; Chitcholtan, Kenny; Gerrard, Juliet A

2016-02-01

The structural properties of amyloid fibrils combined with their highly functional surface chemistry make them an attractive new food ingredient, for example as highly effective gelling agents. However, the toxic role of amyloid fibrils in disease may cause some concern about their food safety because it has not been established unequivocally if consumption of food fibrils poses a health risk to consumers. Here we present a study of amyloid-like fibrils from whey, kidney bean, soy bean, and egg white to partially address this concern. Fibrils showed varied resistance to proteolytic digestion in vitro by either Proteinase K, pepsin or pancreatin. The toxicity of mature fibrils was measured in vitro and compared to native protein, early-stage-fibrillar protein, and sonicated fibrils in two immortalised human cancer cell lines, Caco-2 and Hec-1a. There was no reduction in the viability of either Caco-2 or Hec-1a cells after treatment with a fibril concentration of up to 0.25 mg/mL. Copyright © 2015 Elsevier Ltd. All rights reserved.

Extra-domain B in oncofetal fibronectin structurally promotes fibrillar head-to-tail dimerization of extracellular matrix protein.

PubMed

Schiefner, André; Gebauer, Michaela; Skerra, Arne

2012-05-18

The type III extra-domain B (ED-B) is specifically spliced into fibronectin (Fn) during embryogenesis and neoangiogenesis, including many cancers. The x-ray structure of the recombinant four-domain fragment Fn(III)7B89 reveals a tightly associated, extended head-to-tail dimer, which is stabilized via pair-wise shape and charge complementarity. A tendency toward ED-B-dependent dimer formation in solution was supported by size exclusion chromatography and analytical ultracentrifugation. When amending the model with the known three-dimensional structure of the Fn(III)10 domain, its RGD loop as well as the adhesion synergy region in Fn(III)9-10 become displayed on the same face of the dimer; this should allow simultaneous binding of at least two integrins and, thus, receptor clustering on the cell surface and intracellular signaling. Insertion of ED-B appears to stabilize overall head-to-tail dimerization of two separate Fn chains, which, together with alternating homodimer formation via disulfide bridges at the C-terminal Fn tail, should lead to the known macromolecular fibril formation.

An Efficient Procedure for Removal and Inactivation of Alpha-Synuclein Assemblies from Laboratory Materials

PubMed Central

Bousset, Luc; Brundin, Patrik; Böckmann, Anja; Meier, Beat; Melki, Ronald

2015-01-01

Background: Preformed α-synuclein fibrils seed the aggregation of soluble α-synuclein in cultured cells and in vivo. This, and other findings, has kindled the idea that α-synuclein fibrils possess prion-like properties. Objective: As α-synuclein fibrils should not be considered as innocuous, there is a need for decontamination and inactivation procedures for laboratory benches and non-disposable laboratory material. Methods: We assessed the effectiveness of different procedures designed to disassemble α-synuclein fibrils and reduce their infectivity. We examined different commercially available detergents to remove α-synuclein assemblies adsorbed on materials that are not disposable and that are most found in laboratories (e.g. plastic, glass, aluminum or stainless steel surfaces). Results: We show that methods designed to decrease PrP prion infectivity neither effectively remove α-synuclein assemblies adsorbed to different materials commonly used in the laboratory nor disassemble the fibrillar form of the protein with efficiency. In contrast, both commercial detergents and SDS detached α-synuclein assemblies from contaminated surfaces and disassembled the fibrils. Conclusions: We describe three cleaning procedures that effectively remove and disassemble α-synuclein seeds. The methods rely on the use of detergents that are compatible with most non-disposable tools in a laboratory. The procedures are easy to implement and significantly decrease any potential risks associated to handling α-synuclein assemblies. PMID:26639448

Structural Characterization of IgG1 mAb Aggregates and Particles Generated under Various Stress Conditions

PubMed Central

Telikepalli, Srivalli N.; Kumru, Ozan S.; Kalonia, Cavan; Esfandiary, Reza; Joshi, Sangeeta B.; Middaugh, C. Russell; Volkin, David B.

2014-01-01

IgG1 mAb solutions were prepared with and without sodium chloride and subjected to different environmental stresses. Formation of aggregates and particles of varying size was monitored by a combination of size exclusion chromatography (SEC), Nanosight Tracking Analysis (NTA), Micro-flow Imaging (MFI), turbidity, and visual assessments. Stirring and heating induced the highest concentration of particles. In general, the presence of NaCl enhanced this effect. The morphology of the particles formed from mAb samples exposed to different stresses was analyzed from TEM and MFI images. Shaking samples without NaCl generated the most fibrillar particles, while stirring created largely spherical particles. The composition of the particles was evaluated for covalent cross-linking by SDS-PAGE, overall secondary structure by FTIR microscopy, and surface apolarity by extrinsic fluorescence spectroscopy. Freeze-thaw and shaking led to particles containing protein with native-like secondary structure. Heating and stirring produced IgG1 containing aggregates and particles with some non-native disulfide crosslinks, varying levels of intermolecular beta sheet content, and increased surface hydrophobicity. These results highlight the importance of evaluating protein particle morphology and composition, in addition to particle number and size distributions, to better understand the effect of solution conditions and environmental stresses on the formation of protein particles in mAb solutions. PMID:24452866

Structural characterization of IgG1 mAb aggregates and particles generated under various stress conditions.

PubMed

Telikepalli, Srivalli N; Kumru, Ozan S; Kalonia, Cavan; Esfandiary, Reza; Joshi, Sangeeta B; Middaugh, C Russell; Volkin, David B

2014-03-01

IgG1 mAb solutions were prepared with and without sodium chloride and subjected to different environmental stresses. Formation of aggregates and particles of varying size was monitored by a combination of size-exclusion chromatography, Nanoparticle Tracking Analysis, Micro-flow Imaging (MFI), turbidity, and visual assessments. Stirring and heating induced the highest concentration of particles. In general, the presence of NaCl enhanced this effect. The morphology of the particles formed from mAb samples exposed to different stresses was analyzed from transmission electron microscopy and MFI images. Shaking samples without NaCl generated the most fibrillar particles, whereas stirring created largely spherical particles. The composition of the particles was evaluated for covalent cross-linking by SDS-PAGE, overall secondary structure by FTIR microscopy, and surface apolarity by extrinsic fluorescence spectroscopy. Freeze-thaw and shaking led to particles containing protein with native-like secondary structure. Heating and stirring produced IgG1-containing aggregates and particles with some non-native disulfide cross-links, varying levels of intermolecular beta sheet content, and increased surface hydrophobicity. These results highlight the importance of evaluating protein particle morphology and composition, in addition to particle number and size distributions, to better understand the effect of solution conditions and environmental stresses on the formation of protein particles in mAb solutions. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Modulation of haemostatic function and prevention of experimental thrombosis by red wine in rats: a role for increased nitric oxide production

PubMed Central

Wollny, Tomasz; Aiello, Luca; Di Tommaso, Donata; Bellavia, Vincenzo; Rotilio, Domenico; Donati, Maria Benedetta; de Gaetano, Giovanni; Iacoviello, Licia

1999-01-01

The effects of ethyl alcohol and wine (red and white) on haemostatic parameters and experimental thrombosis were studied in rats; NO was evaluated as a possible mediator of these effects. We found that red wine (12% alcohol) supplementation (8.4±0.4 ml d−1 in drinking water, for 10 days) induced a marked prolongation of ‘template' bleeding time (BT) (258±13 vs 132±13 s in controls; P<0.001), a decrease in platelet adhesion to fibrillar collagen (11.6±1.0 vs 32.2±1.3%; P<0.01) and a reduction in thrombus weight (1.45±0.33 vs 3.27±0.39 mg; P<0.01). Alcohol-free red wine showed an effect similar to red wine. In contrast, neither ethyl alcohol (12%) nor white wine (12% alcohol) affected these systems. All these effects were also observed after red wine i.v. injection (1 ml kg−1 of 1 : 4 dilution) 15 min before the experiments. The effects of red wine were prevented by the NO inhibitor, Nωnitro-L-arginine-methyl ester (L-NAME). L-arginine, not D-arginine, reversed the effect of L-NAME on red wine infusion. Red wine injection induced a 3 fold increase in total radical-trapping antioxidant parameter values of rat plasma with respect to controls, while white wine and alcohol did not show any effect. Our study provides evidence that red wine modulates primary haemostasis and prevents experimental thrombosis in rats, independently of its alcohol content, by a NO-mediated mechanism. PMID:10401566

Synthesis of Polyhydroxybutyrate Particles with Micro-to-Nanosized Structures and Application as Protective Coating for Packaging Papers

PubMed Central

Rastogi, Vibhore Kumar; Samyn, Pieter

2016-01-01

This study reports on the development of bio-based hydrophobic coatings for packaging papers through deposition of polyhydroxybutyrate (PHB) particles in combination with nanofibrillated cellulose (NFC) and plant wax. In the first approach, PHB particles in the micrometer range (PHB-MP) were prepared through a phase-separation technique providing internally-nanosized structures. The particles were transferred as a coating by dip-coating filter papers in the particle suspension, followed by sizing with a carnauba wax solution. This approach allowed partial to almost full surface coverage of PHB-MP over the paper surface, resulting in static water contact angles of 105°–122° and 129°–144° after additional wax coating. In the second approach, PHB particles with submicron sizes (PHB-SP) were synthesized by an oil-in-water emulsion (o/w) solvent evaporation method and mixed in aqueous suspensions with 0–7 wt % NFC. After dip-coating filter papers in PHB-SP/NFC suspensions and sizing with a carnauba wax solution, static water contact angles of 112°–152° were obtained. The intrinsic properties of the particles were analyzed by scanning electron microscopy, thermal analysis and infrared spectroscopy, indicating higher crystallinity for PHB-SP than PHB-MP. The chemical interactions between the more amorphous PHB-MP particles and paper fibers were identified as an esterification reaction, while the morphology of the NFC fibrillar network was playing a key role as the binding agent in the retention of more crystalline PHB-SP at the paper surface, hence contributing to higher hydrophobicity. PMID:28336839

Synthesis of Polyhydroxybutyrate Particles with Micro-to-Nanosized Structures and Application as Protective Coating for Packaging Papers.

PubMed

Rastogi, Vibhore Kumar; Samyn, Pieter

2016-12-30

This study reports on the development of bio-based hydrophobic coatings for packaging papers through deposition of polyhydroxybutyrate (PHB) particles in combination with nanofibrillated cellulose (NFC) and plant wax. In the first approach, PHB particles in the micrometer range (PHB-MP) were prepared through a phase-separation technique providing internally-nanosized structures. The particles were transferred as a coating by dip-coating filter papers in the particle suspension, followed by sizing with a carnauba wax solution. This approach allowed partial to almost full surface coverage of PHB-MP over the paper surface, resulting in static water contact angles of 105°-122° and 129°-144° after additional wax coating. In the second approach, PHB particles with submicron sizes (PHB-SP) were synthesized by an oil-in-water emulsion (o/w) solvent evaporation method and mixed in aqueous suspensions with 0-7 wt % NFC. After dip-coating filter papers in PHB-SP/NFC suspensions and sizing with a carnauba wax solution, static water contact angles of 112°-152° were obtained. The intrinsic properties of the particles were analyzed by scanning electron microscopy, thermal analysis and infrared spectroscopy, indicating higher crystallinity for PHB-SP than PHB-MP. The chemical interactions between the more amorphous PHB-MP particles and paper fibers were identified as an esterification reaction, while the morphology of the NFC fibrillar network was playing a key role as the binding agent in the retention of more crystalline PHB-SP at the paper surface, hence contributing to higher hydrophobicity.

ARO STIR: Defining Peptide Nanostructures By Engineering Assembly Interfaces

DTIC Science & Technology

2013-10-16

geometrically ‘flat’ valine faces of a pair of peptides.11-14 The non-specific hydrophobic interactions have a significant influence on the fibrillar...alternating hydrophobic valine and hydrophilic lysine residues, with a –VDPPT- turn sequence in the middle. At neutral to low pH, due to repulsion...the hydrophobic interactions between the hydrophobic side chains of the valine residues and serves as another factor that affects folding and

Comparative ultrastructure of CRM1-Nucleolar bodies (CNoBs), Intranucleolar bodies (INBs) and hybrid PML/p62 bodies uncovers new facets of nuclear body dynamic and diversity

PubMed Central

Souquere, Sylvie; Weil, Dominique; Pierron, Gérard

2015-01-01

In order to gain insights on the nuclear organization in mammalian cells, we characterized ultrastructurally nuclear bodies (NBs) previously described as fluorescent foci. Using high resolution immunoelectron microscopy (I-EM), we provide evidence that CNoBs (CRM1-Nucleolar bodies) and INBs (Intranucleolar bodies) are distinct genuine nucleolar structures in untreated HeLa cells. INBs are fibrillar and concentrate the post-translational modifiers SUMO1 and SUMO-2/3 as strongly as PML bodies. In contrast, the smallest CRM1-labeled CNoBs are vitreous, preferentially located at the periphery of the nucleolus and, intricately linked to the chromatin network. Upon blockage of the CRM1-dependent nuclear export by leptomycin B (LMB), CNoBs disappear while p62/SQSTM1-containing fibrillar nuclear bodies are induced. These p62 bodies are enriched in ubiquitinated proteins. They progressively associate with PML bodies to form hybrid bodies of which PML decorates the periphery while p62/SQSTM1 is centrally-located. Our study is expanding the repertoire of nuclear bodies; revealing a previously unrecognized composite nucleolar landscape and a new mode of interactions between ubiquitous (PML) and stress-induced (p62) nuclear bodies, resulting in the formation of hybrid bodies. PMID:26275159

Organization of the nucleoli of soybean root meristematic cells at different states of their activity.

PubMed

Stepiński, Dariusz

2010-06-01

Internal organization of a nucleolus changes along with rRNA transcriptional activity. These changes mainly concern qualitative and quantitative alternations of three main nucleolar components: fibrillar centres (FC), dense fibrillar component (DFC) and granular component (GC). In the present work quantitative measurements of the number and sizes of FCs and DFCs in nucleoli of root meristematic cells of soybean seedlings grown at (1) chilling conditions that reduce transcriptional activity of soybean nucleoli (temp. of 10 degrees C) and at (2) conditions that increase this activity (recovery at optimal temp. of 25 degrees C after previous chilling), even more than (3) the control, have been carried out. Morphometric measurements showed that the highest number of FCs and DFCs was in the most active nucleoli, while the smallest number - in those with the lowest activity. The average size of an individual FC was similar in all nucleoli regardless of their transcriptional activity, that of the individual DFC varied, being bigger in the nucleoli of the chilled plants and smallest in those of the recovered plants. The numbers of FCs and DFCs seem to be indicators of transcriptional activity of plant nucleoli - the higher number of FCs and DFCs the more active nucleoli. Copyright 2009 Elsevier Ltd. All rights reserved.

Differential Relationships of Reactive Astrocytes and Microglia to Fibrillar Amyloid Deposits in Alzheimer Disease

PubMed Central

Serrano-Pozo, Alberto; Muzikansky, Alona; Gómez-Isla, Teresa; Growdon, John H.; Betensky, Rebecca A.; Frosch, Matthew P.; Hyman, Bradley T.

2013-01-01

While it is clear that astrocytes and microglia cluster around dense-core amyloid plaques in Alzheimer disease (AD), whether they are primarily attracted to amyloid deposits or are just reacting to plaque-associated neuritic damage remains elusive. We postulate that astrocytes and microglia may differentially respond to fibrillar amyloid β (Aβ). Therefore, we quantified the size distribution of dense-core Thioflavin-S (ThioS)-positive plaques in the temporal neocortex of 40 AD patients and the microglial and astrocyte responses in their vicinity (≤50 μm), and performed correlations between both measures. As expected, both astrocytes and microglia were clearly spatially associated with ThioS-positive plaques (p = 0.0001, ≤50 μm vs. >50 μm from their edge), but their relationship to ThioS-positive plaque size differed; larger ThioS-positive plaques were associated with more surrounding activated microglia (p = 0.0026), but this effect was not observed with reactive astrocytes. Microglial response to dense-core plaques appears to be proportional to their size, which we postulate reflects a chemotactic effect of Aβ. By contrast, plaque-associated astrocytic response does not correlate with plaque size and seems to parallel the behavior of plaque-associated neuritic damage. PMID:23656989

Persistence of an intact endometrial matrix and vessels structure in women exposed to VA-2914, a selective progesterone receptor modulator.

PubMed

Ravet, S; Munaut, C; Blacher, S; Brichant, G; Labied, S; Beliard, A; Chabbert-Buffet, N; Bouchard, P; Foidart, J-M; Pintiaux, A

2008-11-01

VA-2914 is a selective progesterone receptor modulator with potential contraceptive activity that induces amenorrhea, whereas progestins cause endometrial spotting and bleeding. This abnormal bleeding due to progestins is a consequence of focal stromal proteolysis by an increase in naked vessel size and density. Our objective was to quantify the effects of VA-2914 on endometrial vascularization, fibrillar matrix, and vascular endothelial growth factor (VEGF)-A expression in endometrial biopsies from 41 women before and after 12 wk daily treatment with a placebo, or 2.5, 5, or 10 mg VA-2914. Collagen fibrillar network was stained by silver impregnation. Vessel area, density, and structure were quantified with a computer-assisted image analysis system after double immunostaining using an anti-von Willebrand factor (endothelial cells) and an anti-alpha smooth muscle actin (vascular smooth muscle cells) marker antibody. VEGF-A mRNAs were quantified by RT-PCR and localized by immunohistochemistry. The endometrial vessels, collagen network, and mRNA levels of VEGF-A were identical during the luteal phase at baseline and in VA-2914 treated women. VEGF-A distribution was unchanged. VA-2914 does not alter the endometrial matrix and cells, and does not modify the endometrial vessel morphology as compared with baseline biopsies.

Some Structural Observations of Self-Assembling, Fibrillar Gels Composed of Two-Directional Bolaform Arborols

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, J.

2005-01-12

Arborols are dumbbell shaped molecules (bolaform amphiphiles) in which a hydrophobic spacer separates two hydrophilic end groups. They are a valuable model for naturally occurring fibers, such as actin or amyloid. Applications to materials science can be envisioned. On cooling from warm aqueous or methanolic solutions, arborols spontaneously assemble into long fibers. When the solutions are above a certain concentration that depends on the hydrophilic/hydrophobic balance, this leads to thermally reversible gels stabilized by a mechanism that is poorly understood. With the help of wide angle X-ray scattering, details of the arborol fiber and gel structure were obtained on wetmore » gels. The characteristic dimensions of the fibers vary in a sensible fashion with the molecular specifics. Solvent character appears to affect the average domain length of arborols stacked into fibers. Fluorescently labeled arborols were prepared. The label does not prevent incorporation into the fibrillar structure, rendering fibril bundles visible in wet gels. Bundles are visible in concentrated gels, but not in less concentrated sols. These results are consistent with observations of dried arborols using atomic force microscopy and with previously published freeze-fracture electron microscopy and small angle X-ray scattering experiments on dried gels.« less

Preventing α-synuclein aggregation: the role of the small heat-shock molecular chaperone proteins.

PubMed

Cox, Dezerae; Carver, John A; Ecroyd, Heath

2014-09-01

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. The failure of proteostasis can result in the accumulation of non-native proteins leading to their aggregation and deposition in cells and in tissues. The amyloid fibrillar aggregation of the protein α-synuclein into Lewy bodies and Lewy neuritis is associated with neurodegenerative diseases classified as α-synucleinopathies, which include Parkinson's disease and dementia with Lewy bodies. The small heat-shock proteins (sHsps) are molecular chaperones that are one of the cell's first lines of defence against protein aggregation. They act to stabilise partially folded protein intermediates, in an ATP-independent manner, to maintain cellular proteostasis under stress conditions. Thus, the sHsps appear ideally suited to protect against α-synuclein aggregation, yet these fail to do so in the context of the α-synucleinopathies. This review discusses how sHsps interact with α-synuclein to prevent its aggregation and, in doing so, highlights the multi-faceted nature of the mechanisms used by sHsps to prevent the fibrillar aggregation of proteins. It also examines what factors may contribute to α-synuclein escaping the sHsp chaperones in the context of the α-synucleinopathies. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

Ammonium hydroxide treatment of Aβ produces an aggregate free solution suitable for biophysical and cell culture characterization

PubMed Central

Ryan, Timothy M.; Caine, Joanne; Mertens, Haydyn D.T.; Kirby, Nigel; Nigro, Julie; Breheney, Kerry; Waddington, Lynne J.; Streltsov, Victor A.; Curtain, Cyril; Masters, Colin L.

2013-01-01

Alzheimer’s disease is the leading cause of dementia in the elderly. Pathologically it is characterized by the presence of amyloid plaques and neuronal loss within the brain tissue of affected individuals. It is now widely hypothesised that fibrillar structures represent an inert structure. Biophysical and toxicity assays attempting to characterize the formation of both the fibrillar and the intermediate oligomeric structures of Aβ typically involves preparing samples which are largely monomeric; the most common method by which this is achieved is to use the fluorinated organic solvent 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Recent evidence has suggested that this method is not 100% effective in producing an aggregate free solution. We show, using dynamic light scattering, size exclusion chromatography and small angle X-ray scattering that this is indeed the case, with HFIP pretreated Aβ peptide solutions displaying an increased proportion of oligomeric and aggregated material and an increased propensity to aggregate. Furthermore we show that an alternative technique, involving treatment with strong alkali results in a much more homogenous solution that is largely monomeric. These techniques for solubilising and controlling the oligomeric state of Aβ are valuable starting points for future biophysical and toxicity assays. PMID:23678397

PMEL: A PIGMENT CELL-SPECIFIC MODEL FOR FUNCTIONAL AMYLOID FORMATION

PubMed Central

Watt, Brenda; van Niel, Guillaume; Raposo, Graça; Marks, Michael S.

2013-01-01

PMEL is a pigment cell-specific protein responsible for the formation of fibrillar sheets within the pigment organelle, the melanosome. The fibrillar sheets serve as a template upon which melanins polymerize as they are synthesized. The PMEL fibrils are required for optimal pigment cell function, as animals that either lack PMEL expression or express mutant PMEL variants show varying degrees of hypopigmentation and pigment cell inviability. The PMEL fibrils have biophysical properties of amyloid, a protein fold that is frequently associated with neurodegenerative and other diseases. However, PMEL is one of a growing number of non-pathogenic amyloid proteins that contribute to the function of the cell and/or organism that produces them. Understanding how PMEL generates amyloid in a non-pathogenic manner might provide insights into how to avoid toxicity due to pathological amyloid formation. In this review we summarize and reconcile data concerning the fate of PMEL from its site of synthesis in the endoplasmic reticulum to newly formed melanosomes and the role of distinct PMEL subdomains in trafficking and amyloid fibril formation. We then discuss how its progression through the secretory pathway into the endosomal system might allow for the regulated and non-toxic conversion of PMEL to an ordered amyloid polymer. PMID:23350640

Amyloid-like aggregates formation by bovine apo-carbonic anhydrase in various alcohols: A comparative study.

PubMed

Es-Haghi, Ali; Ebrahim-Habibi, Azadeh; Sabbaghian, Marjan; Nemat-Gorgani, Mohsen

2016-11-01

Peptides and proteins convert from their native states to amyloid fibrillar aggregates in a number of pathological conditions. Characterizing these species could provide useful information on their pathogenicity and the key factors involved in their generation. In this study, we have observed the ability of the model protein apo-bovine carbonic anhydrase (apo-BCA) to form amyloid-like aggregates in the presence of halogenated and non-halogenated alcohols. Far-UV circular dichroism, ThT fluorescence, atomic force microscopy and dynamic light scattering were used to characterize these structures. The concentration required for effective protein aggregation varied between the solvents, with non-halogenated alcohols acting in a wider range. These aggregates show amyloid-like structures as determined by specific techniques used for characterizing amyloid structures. Oligomers were obtained with various size distributions, but fibrillar structures were not observed. Use of halogenated alcohols resulted into smaller hydrodynamic radii, and most stable oligomers were formed in hexafluoropropan-2-ol (HFIP). At optimal concentrations used to generate these structures, the non-halogenated alcohols showed higher hydrophobicity, which may be related to the lower stability of the generated oligomers. These oligomers have the potential to be used as models in the search for effective treatments in proteinopathies. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of the fibrillar layer at the epithelial-mesenchymal junction in tooth germs.

PubMed

Sawada, T; Inoue, S

1994-12-01

A characteristic layer containing numerous fibrils is associated with the basement membrane of the inner enamel epithelium during the early stages of odontogenesis. However, its nature is not well understood. In this study, the layer was examined with high-resolution electron microscopy and immuno-histochemical staining. Tooth germs of monkeys (Macaca fuscata) were studied and each fibril in the layer was found to be a tubular structure, 8-9 nm in width, resembling a "basotubule", the tubular structure previously observed in various basement membranes. The space between the fibrils was filled with a network formed by irregular anastomosing strands with an average thickness of 4 nm; these strands resembled the "cords" forming the network in the lamina densa of basement membranes. After immunoperoxidase staining, fine threads immunoreactive for laminin staining were seen winding along the strands of the network, and 1.5-nm wide filaments, immunoreactive for type IV collagen, took the form of a network arrangement. The 5-nm-wide ribbon-like structures associated with the strands were identified as heparan sulfate proteoglycan by immunostaining. These results are similar to those obtained for the cord network of the lamina densa. The "fibrillar layer" therefore represents a highly specialized lamina fibroreticularis of the basement membrane of the inner enamel epithelium, and rich in basotubules.

Hemin as a generic and potent protein misfolding inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Yanqin; Carver, John A.; Ho, Lam H.

2014-11-14

Highlights: • Hemin prevents Aβ42, α-synuclein and RCM-κ-casein forming amyloid fibrils. • Hemin inhibits the β-sheet structure formation of Aβ42. • Hemin reduces the cell toxicity caused by fibrillar Aβ42. • Hemin dissociates partially formed Aβ42 fibrils. • Hemin prevents amorphous aggregation by ADH, catalase and γs-crystallin. - Abstract: Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer’s disease, Parkinson’s disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin preventsmore » amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.« less

Skeletal muscle weakness in osteogenesis imperfecta mice.

PubMed

Gentry, Bettina A; Ferreira, J Andries; McCambridge, Amanda J; Brown, Marybeth; Phillips, Charlotte L

2010-09-01

Exercise intolerance, muscle fatigue and weakness are often-reported, little-investigated concerns of patients with osteogenesis imperfecta (OI). OI is a heritable connective tissue disorder hallmarked by bone fragility resulting primarily from dominant mutations in the proα1(I) or proα2(I) collagen genes and the recently discovered recessive mutations in post-translational modifying proteins of type I collagen. In this study we examined the soleus (S), plantaris (P), gastrocnemius (G), tibialis anterior (TA) and quadriceps (Q) muscles of mice expressing mild (+/oim) and moderately severe (oim/oim) OI for evidence of inherent muscle pathology. In particular, muscle weight, fiber cross-sectional area (CSA), fiber type, fiber histomorphology, fibrillar collagen content, absolute, relative and specific peak tetanic force (P(o), P(o)/mg and P(o)/CSA respectively) of individual muscles were evaluated. Oim/oim mouse muscles were generally smaller, contained less fibrillar collagen, had decreased P(o) and an inability to sustain P(o) for the 300-ms testing duration for specific muscles; +/oim mice had a similar but milder skeletal muscle phenotype. +/oim mice had mild weakness of specific muscles but were less affected than their oim/oim counterparts which demonstrated readily apparent skeletal muscle pathology. Therefore muscle weakness in oim mice reflects inherent skeletal muscle pathology. Copyright © 2010 Elsevier B.V. All rights reserved.

Interactions between lipid-free apolipoprotein-AI and a lipopeptide incorporating the RGDS cell adhesion motif

NASA Astrophysics Data System (ADS)

Castelletto, V.; Hamley, I. W.; Reza, M.; Ruokolainen, J.

2014-11-01

The interaction of a designed bioactive lipopeptide C16-GGGRGDS, comprising a hexadecyl lipid chain attached to a functional heptapeptide, with the lipid-free apoliprotein, Apo-AI, is examined. This apolipoprotein is a major component of high density lipoprotein and it is involved in lipid metabolism and may serve as a biomarker for cardiovascular disease and Alzheimers' disease. We find via isothermal titration calorimetry that binding between the lipopeptide and Apo-AI occurs up to a saturation condition, just above equimolar for a 10.7 μM concentration of Apo-AI. A similar value is obtained from circular dichroism spectroscopy, which probes the reduction in α-helical secondary structure of Apo-AI upon addition of C16-GGGRGDS. Electron microscopy images show a persistence of fibrillar structures due to self-assembly of C16-GGGRGDS in mixtures with Apo-AI above the saturation binding condition. A small fraction of spheroidal or possibly ``nanodisc'' structures was observed. Small-angle X-ray scattering (SAXS) data for Apo-AI can be fitted using a published crystal structure of the Apo-AI dimer. The SAXS data for the lipopeptide/Apo-AI mixtures above the saturation binding conditions can be fitted to the contribution from fibrillar structures coexisting with flat discs corresponding to Apo-AI/lipopeptide aggregates.

Curcumin Promotes A-beta Fibrillation and Reduces Neurotoxicity in Transgenic Drosophila

PubMed Central

Caesar, Ina; Jonson, Maria; Nilsson, K. Peter R.; Thor, Stefan; Hammarström, Per

2012-01-01

The pathology of Alzheimer's disease (AD) is characterized by the presence of extracellular deposits of misfolded and aggregated amyloid-β (Aβ) peptide and intraneuronal accumulation of tangles comprised of hyperphosphorylated Tau protein. For several years, the natural compound curcumin has been proposed to be a candidate for enhanced clearance of toxic Aβ amyloid. In this study we have studied the potency of feeding curcumin as a drug candidate to alleviate Aβ toxicity in transgenic Drosophila. The longevity as well as the locomotor activity of five different AD model genotypes, measured relative to a control line, showed up to 75% improved lifespan and activity for curcumin fed flies. In contrast to the majority of studies of curcumin effects on amyloid we did not observe any decrease in the amount of Aβ deposition following curcumin treatment. Conformation-dependent spectra from p-FTAA, a luminescent conjugated oligothiophene bound to Aβ deposits in different Drosophila genotypes over time, indicated accelerated pre-fibrillar to fibril conversion of Aβ1–42 in curcumin treated flies. This finding was supported by in vitro fibrillation assays of recombinant Aβ1–42. Our study shows that curcumin promotes amyloid fibril conversion by reducing the pre-fibrillar/oligomeric species of Aβ, resulting in a reduced neurotoxicity in Drosophila. PMID:22348084

Structure and dynamics of human vimentin intermediate filament dimer and tetramer in explicit and implicit solvent models.

PubMed

Qin, Zhao; Buehler, Markus J

2011-01-01

Intermediate filaments, in addition to microtubules and microfilaments, are one of the three major components of the cytoskeleton in eukaryotic cells, and play an important role in mechanotransduction as well as in providing mechanical stability to cells at large stretch. The molecular structures, mechanical and dynamical properties of the intermediate filament basic building blocks, the dimer and the tetramer, however, have remained elusive due to persistent experimental challenges owing to the large size and fibrillar geometry of this protein. We have recently reported an atomistic-level model of the human vimentin dimer and tetramer, obtained through a bottom-up approach based on structural optimization via molecular simulation based on an implicit solvent model (Qin et al. in PLoS ONE 2009 4(10):e7294, 9). Here we present extensive simulations and structural analyses of the model based on ultra large-scale atomistic-level simulations in an explicit solvent model, with system sizes exceeding 500,000 atoms and simulations carried out at 20 ns time-scales. We report a detailed comparison of the structural and dynamical behavior of this large biomolecular model with implicit and explicit solvent models. Our simulations confirm the stability of the molecular model and provide insight into the dynamical properties of the dimer and tetramer. Specifically, our simulations reveal a heterogeneous distribution of the bending stiffness along the molecular axis with the formation of rather soft and highly flexible hinge-like regions defined by non-alpha-helical linker domains. We report a comparison of Ramachandran maps and the solvent accessible surface area between implicit and explicit solvent models, and compute the persistence length of the dimer and tetramer structure of vimentin intermediate filaments for various subdomains of the protein. Our simulations provide detailed insight into the dynamical properties of the vimentin dimer and tetramer intermediate filament building blocks, which may guide the development of novel coarse-grained models of intermediate filaments, and could also help in understanding assembly mechanisms.

Early Events in Insulin Fibrillization Studied by Time-Lapse Atomic Force Microscopy

PubMed Central

Podestà, Alessandro; Tiana, Guido; Milani, Paolo; Manno, Mauro

2006-01-01

The importance of understanding the mechanism of protein aggregation into insoluble amyloid fibrils lies not only in its medical consequences, but also in its more basic properties of self-organization. The discovery that a large number of uncorrelated proteins can form, under proper conditions, structurally similar fibrils has suggested that the underlying mechanism is a general feature of polypeptide chains. In this work, we address the early events preceding amyloid fibril formation in solutions of zinc-free human insulin incubated at low pH and high temperature. Here, we show by time-lapse atomic force microscopy that a steady-state distribution of protein oligomers with a quasiexponential tail is reached within a few minutes after heating. This metastable phase lasts for a few hours, until fibrillar aggregates are observable. Although for such complex systems different aggregation mechanisms can occur simultaneously, our results indicate that the prefibrillar phase is mainly controlled by a simple coagulation-evaporation kinetic mechanism, in which concentration acts as a critical parameter. These experimental facts, along with the kinetic model used, suggest a critical role for thermal concentration fluctuations in the process of fibril nucleation. PMID:16239333

Fibrillar structure and elasticity of hydrating collagen: a quantitative multiscale approach.

PubMed

Morin, Claire; Hellmich, Christian; Henits, Peter

2013-01-21

It is well known that hydration of collagenous tissues leads to their swelling, as well as to softening of their elastic behavior. However, it is much less clear which microstructural and micromechanical "rules" are involved in this process. Here, we develop a theoretical approach cast in analytical mathematical formulations, which is experimentally validated by a wealth of independent tests on collagenous tissues, such as X-ray diffraction, vacuum drying, mass measurements, and Brillouin light scattering. The overall emerging picture is the following: air-drying leaves water only in the gap zones between the triple-helical collagen molecules; upon re-hydration, the extrafibrillar space is established at volumes directly proportional to the hydration-induced swelling of the (micro) fibrils, until the maximum equatorial distance between the long collagen molecules is reached. Thereafter, the volume of the fibrils stays constant, and only the extrafibrillar volume continues to grow. At all these hydration stages, the elastic behavior is governed by the same, hydration-invariant mechanical interaction pattern of only two, interpenetrating mechanical phases: transversely isotropic molecular collagen and isotropic water (or empty pores in the vacuum-dried case). Copyright © 2012 Elsevier Ltd. All rights reserved.

Ultrastructural sinusoidal changes in extrahepatic cholestasis. Light and electron microscopic immunohistochemical localization of collagen type III and type IV.

PubMed

Gulubova, M V

1996-07-01

Extrahepatic cholestasis causes excessive extracellular matrix formation perisinusoidally. Ito cells, transitional and endothelial cells are considered to be a source of extracellular matrix proteins in experimental cholestasis. The localization of collagens type III and type IV in human liver in extrahepatic cholestasis was investigated immunohistochemically in the present study. Immersion fixation was used after modification to be applied to surgical biopsies with commercially available kits. Sinusoidal changes were observed that indicated excessive collagen and matrix formation. Light microscopically, increased immunostaining with the two collagen antibodies was found perisinusoidally and portally. Ultrastructurally, collagen type III positive fibres were found beneath basement membranes of vessels, in collagen bundles and as a fibrillar network in the space of Disse. Collagen type IV immunostaining was located in portal tracts and near hepatocyte microvilli. Intracellular staining with collagen type IV was detected in the rough endoplasmic reticulum of some transitional cells. Immunostaining was located around transitional cells, Ito cells or endothelial cells mainly. Our study indicates that Ito cells, transitional and endothelial cells are the main source of collagens type III and IV in the space of Disse in extrahepatic cholestasis in humans.

Molecular insights into early stage aggregation of di-Fmoc-L-lysine in binary mixture of organic solvent and water

NASA Astrophysics Data System (ADS)

Huda, Md Masrul; Rai, Neeraj

Molecular gels are relatively new class of soft materials, which are formed by the supramolecular aggregation of low molecular weight gelators (LMWGs) in organic solvents and/or water. Hierarchical self-assembly of small gelator molecules lead to three-dimensional complex fibrillar networks, which restricts the flow of solvents and results in viscous solid like materials or gels. These gels have drawn significant attentions for their potential applications for drug delivery, tissue engineering, materials for sensors etc. As of now, self-assembly of gelator molecules into one-dimensional fibers is not well understood, although that is very important to design new gelators for desired applications. Here, we present molecular dynamics study that provides molecular level insight into early stage aggregation of selected gelator, di-Fmoc-L-lysine in binary mixture of organic solvent and water. We will present the role of different functional groups of gelator molecule such as aromatic ring, amide, and carboxylic group on aggregation. We will also present the effect of concentrations of gelator and solvent on self-assembly of gelators. This study has captured helical fiber growth and branching of fiber, which is in good agreement with experimental observations.

An energy landscape approach to protein aggregation

NASA Astrophysics Data System (ADS)

Buell, Alexander; Knowles, Tuomas

2012-02-01

Protein aggregation into ordered fibrillar structures is the hallmark of a class of diseases, the most prominent examples of which are Alzheimer's and Parkinson's disease. Recent results (e.g. Baldwin et al. J. Am. Chem. Soc. 2011) suggest that the aggregated state of a protein is in many cases thermodynamically more stable than the soluble state. Therefore the solubility of proteins in a cellular context appears to be to a large extent under kinetic control. Here, we first present a conceptual framework for the description of protein aggregation ( see AK Buell et al., Phys. Rev. Lett. 2010) that is an extension to the generally accepted energy landscape model for protein folding. Then we apply this model to analyse and interpret a large set of experimental data on the kinetics of protein aggregation, acquired mainly with a novel biosensing approach (see TPJK Knowles et al, Proc. Nat. Acad. Sc. 2007). We show how for example the effect of sequence modifications on the kinetics and thermodynamics of human lysozyme aggregation can be understood and quantified (see AK Buell et al., J. Am. Chem. Soc. 2011). These results have important implications for therapeutic strategies against protein aggregation disorders, in this case lysozyme systemic amyloidosis.

Procoagulant expression in platelets and defects leading to clinical disorders.

PubMed

Solum, N O

1999-12-01

Hemostasis is a result of interactions between fibrillar structures in the damaged vessel wall, soluble components in plasma, and cellular elements in blood represented mainly by platelets and platelet-derived material. During formation of a platelet plug at the damaged vessel wall, factors IXa and VIIIa form the "tenase" complex, leading to activation of factor X on the surface of activated platelets. Subsequently, factors Xa and Va form the "prothrombinase" complex, which catalyzes the formation of thrombin from prothrombin, leading to fibrin formation. An enhanced expression of negatively charged phosphatidylserine in the outer membrane leaflet resulting from a breakdown of the phospholipid asymmetry is essential for the formation of the procoagulant surface. An ATP-driven and inward-acting aminophospholipid "translocase" and a "floppase" counterbalancing this have been postulated to maintain the dynamic state of phospholipid asymmetry. A phospholipid-nonspecific "scramblase," believed to be responsible for the fast breakdown of the asymmetry during cell activation, has recently been isolated from erythrocytes, cloned, and characterized. An intracellular calcium-binding segment and one or more thioesterified fatty acids are probably of importance for calcium-induced activation of this transporter protein. Cytosolic calcium ions also activate the calcium-dependent protease calpain associated with shedding of microvesicles from the transformed platelet membrane. These are shed with a procoagulant surface and with surface-exposed P-selectin from the alpha-granules. Theoretically, therefore, microvesicles can be involved in both coagulation and inflammation. Scott syndrome is probably caused by a defect in the activation of an otherwise normal scramblase, resulting in a relatively severe bleeding tendency. In Stormorken syndrome, the patients demonstrate a spontaneous surface expression of aminophospholipids. Activated platelets and the presence of procoagulant microvesicles have been demonstrated in several clinical conditions, such as thrombotic and idiopathic thrombocytopenia, disseminated intravascular coagulation, and HIV-1 infection, and have been found to be associated with fibrin in thrombosis. Procoagulant microvesicles may also be formed from other cells as a result of apoptosis.

Transmission electron microscopy of amyloid fibrils.

PubMed

Gras, Sally L; Waddington, Lynne J; Goldie, Kenneth N

2011-01-01

Transmission Electron Microscopy of negatively stained and cryo-prepared specimens allows amyloid fibrils to be visualised at high resolution in a dried or a hydrated state, and is an essential method for characterising the morphology of fibrils and pre-fibrillar species. We outline the key steps involved in the preparation and observation of samples using negative staining and cryo-electron preservation. We also discuss methods to measure fibril characteristics, such as fibril width, from electron micrographs.

Chromatin fibers: from classical descriptions to modern interpretation.

PubMed

Kuznetsova, Maria A; Sheval, Eugene V

2016-11-01

The first description of intrachromosomal fibers was made by Baranetzky in 1880. Since that time, a plethora of fibrillar substructures have been described inside the mitotic chromosomes, and published data indicate that chromosomes may be formed as a result of the hierarchical folding of chromatin fibers. In this review, we examine the evolution and the current state of research on the morphological organization of mitotic chromosomes. © 2016 International Federation for Cell Biology.

Moderate severity heart failure does not involve a downregulation of myocardial fatty acid oxidation

DTIC Science & Technology

2004-10-01

malonyl-CoA-sensitive form of carnitine palmitoyltransferase is not local - ized exclusively in the outer membrane of rat liver mitochondria . J Biol...for the isolation of fresh mitochondria , both subsarcolemmal and interfibrillar. Analytic methods. Detailed analytic methods have been previously cited...populations of mitochondria , the subsarcolemmal and inter- fibrillar, were isolated from hearts of normal and HF dogs using the procedure of Palmer et al

Large-scale isolation and fractionation of organs of Drosophila melanogaster larvae.

PubMed

Zweidler, A; Cohen, L H

1971-10-01

Methods for the mass isolation of diverse organs from small animals are described. They involve novel devices: a mechanical dissecting system, a centrifugal agitator for the separation of fibrillar from globular particles, and a settling chamber for the fractionation at unit gravity of particles with sedimentation velocities above the useful range for centrifugation. The application of these methods to the isolation of polytene and nonpolytene nuclei from Drosophila melanogaster larvae is described.

Acceleration of Ligament Healing with Cellular Attractants

DTIC Science & Technology

2008-07-01

major cause of morbidity in the armed forces. type VI collagen is a haptotactic cell attractant. We have shown that type VI collagen with bound...heparin/FGF-2 or hyaluronan or fibronectin promotes migration of canine ACL and DET cells. Insertion of type VI collagen into a wound in the canine...1984). Type I collagen is known to be the predominant fibrillar collagen in the meniscus. Smaller amounts of type II collagen are also present. In

Formation of continuous activated carbon fibers for barrier fabrics

NASA Astrophysics Data System (ADS)

Liang, Ying

1997-08-01

Commercial protective suits made of active carbon granules or nonwoven fabrics are heavy, have low moisture vapor transport rate, and are uncomfortable. Inherent problems due to construction of barrier fabrics lead to severe heat stress when worn for even short time in warm environments. One proposed method to eliminate these problems is to facilitate the construction of a fabric made of continuous activated carbon fibers (CACF). This study is directed toward investigating the possibility of developing CAFC from two precursors: aramid and fibrillated PAN fiber. It was shown in this study that Kevlar-29 fibers could be quickly carbonized and activated to CACF with high adsorptivity and relatively low weight loss. CACF with high surface area (>500 msp2/g) and reasonable tenacity (≈1g/denier) were successfully prepared from Kevlar fibers through a three-step process: pretreatment, carbonization, and activation. X-ray diffraction, Fourier Transform Infrared Spectroscopy (FTIR), and thermal analysis were conducted to understand the evolution of physical and chemical properties during pretreatment. The influence of temperature, heating rate, and pyrolysis environment on the thermal behavior was determined by DSC and TGA/DTA and used as an indicator for optimizing the pyrolysis conditions. Surface analysis by nitrogen isotherms indicated that the resultant fibers had micropores and mesopores on the surface of CACF. This was also inferred by studies on the surface morphology through Scanning Electron Microscopy (SEM) and Scanning Tunneling Microscopy (STM). An investigation of the surface chemical structure by X-ray photoelectron spectroscopy (XPS) before and after activation and elemental analysis confirmed that adsorption of Kevlar based CACF mainly arises due to the physisorption instead of chemisorption. A multistep stabilization along with carbonization and activation was used to prepare active carbon fiber from fibrillated PAN fiber. The resultant fiber retained its fibrillar structure and provided a very high surface area, up to 1400 msp2/g, but was brittle. The characterization of the thermal behavior, mechanical properties, and surface structure of the pyrolyzed fiber at each processing step was also carried out by using various techniques, such as DSC and TGA, Instron, and SEM. These studies provide directions for preparation of CACF from novel precursors.

Preparation and characterization of injectable fibrillar type I collagen and evaluation for pseudoaneurysm treatment in a pig model.

PubMed

Geutjes, Paul J; van der Vliet, J Adam; Faraj, Kaeuis A; de Vries, Noes; van Moerkerk, Herman T B; Wismans, Ronnie G; Hendriks, Thijs; Daamen, Willeke F; van Kuppevelt, Toin H

2010-11-01

Despite the efficacy of collagen in femoral artery pseudoaneurysm treatment, as reported in one patient study, its use has not yet gained wide acceptance in clinical practice. In this particular study, the collagen was not described in detail. To further investigate the potential of collagen preparations, we prepared and characterized highly purified injectable fibrillar type I collagen and evaluated its use for femoral artery pseudoaneurysm (PSA) treatment in vivo using a pig model. Purified fibrillar type I collagen was characterized using electron microscopy. The effect of three different sterilization procedures, ie, hydrogen peroxide gas plasma (H2O2), ethylene oxide gas (EtO), and gamma irradiation, was studied on both SDS-PAGE and platelet aggregation. Different collagen injectables were prepared (3%, 4%, and 5%) and tested using an injection force test applying a 21-gauge needle. To evaluate the network characteristics of the injectable collagen, the collagen was suspended in phosphate buffered saline (PBS) at 37°C and studied both macroscopically and electron microscopically. To determine whether the collagen induced hemostasis in vivo, a pig PSA model was used applying a 4% EtO sterilized collagen injectable, and evaluation by angiography and routine histology. Electron microscopy of the purified type I collagen revealed intact fibrils with a distinct striated pattern and a length<300 μm. Both SDS-PAGE and platelet aggregation analysis of the sterilized collagen indicated no major differences between EtO and H2O2 sterilization, although gamma-irradiated collagen showed degradation products. Both 3% and 4% (w/v) collagen suspensions were acceptable with respect to the force used (<50 N). The 4% suspension was selected as the preferred injectable collagen, which formed a dense network under physiologic conditions. Testing the collagen in vivo (n=5), the angiograms revealed that the PSA partly or completely coagulated. Histology confirmed the network formation, which was surrounded by thrombus. Collagen injectables were prepared and EtO sterilized without major loss of structural integrity and platelet activity. In vivo, the injectable collagen formed a dense network and triggered (partial) local hemostasis. Although optimization is needed, an injectable collagen may be used as a therapeutic agent for femoral PSA treatment. Copyright © 2010 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.

Multiphoton microscopy based cryo-imaging of inflated frozen human lung sections at -60°C in healthy and COPD lungs

NASA Astrophysics Data System (ADS)

Abraham, Thomas; Kayra, Damian; Zhang, Angela; Suzuki, Masaru; McDonough, John; Elliott, W. M.; Cooper, Joel D.; Hogg, James C.

2013-02-01

Lung is a complex gas exchanger with interfacial area (where the gas exchange takes place) is about the size of a tennis court. Respiratory function is linked to the biomechanical stability of the gas exchange or alveolar regions which directly depends on the spatial distributions of the extracellular matrix fibers such fibrillar collagens and elastin fibers. It is very important to visualize and quantify these fibers at their native and inflated conditions to have correct morphometric information on differences between control and diseased states. This can be only achieved in the ex vivo states by imaging directly frozen lung specimens inflated to total lung capacity. Multiphoton microscopy, which uses ultra-short infrared laser pulses as the excitation source, produces multiphoton excitation fluorescence (MPEF) signals from endogenously fluorescent proteins (e.g. elastin) and induces specific second harmonic generation (SHG) signals from non-centrosymmetric proteins such as fibrillar collagens in fresh human lung tissues [J. Struct. Biol. (2010)171,189-196]. Here we report for the first time 3D image data obtained directly from thick frozen inflated lung specimens (~0.7- 1.0 millimeter thick) visualized at -60°C without prior fixation or staining in healthy and diseased states. Lung specimens donated for transplantation and released for research when no appropriate recipient was identified served as controls, and diseased lung specimens donated for research by patients receiving lung transplantation for very severe COPD (n=4) were prepared as previously described [N. Engl. J. Med. (2011) 201, 1567]. Lung slices evenly spaced between apex and base were examined using multiphoton microscopy while maintained at -60°C using a temperature controlled cold stage with a temperature resolution of 0.1°C. Infrared femto-second laser pulses tuned to 880nm, dry microscopic objectives, and non-de-scanned detectors/spectrophotometer located in the reflection geometry were used for generating the 3D images/spectral information. We found that this novel imaging approach can provide spatially resolved 3D images with spectral specificities from frozen inflated lungs that are sensitive enough to identity the micro-structural details of fibrillar collagens and elastin fibers in alveolar walls in both healthy and diseased tissues.

The Influence of Drying on the Structures and Mechanics of Poly (P- Phenylene Benzobisthiazole) Fibers

DTIC Science & Technology

1986-09-01

since the first fibers based on modified cellulose were developed at the end of the 19th century. Recent advances in fiber science have focused on high...investigations to date have focused on the wet spinning of such flexible extended chain polymers as cellulosic materials (30), proteins (31,32), and...instabilities. Materials such as coagulated cellulose , PAN, poly (amino acids), and wet wood possess an interconnected fibrillar structure (30,32,35

LARGE-SCALE ISOLATION AND FRACTIONATION OF ORGANS OF DROSOPHILA MELANOGASTER LARVAE

PubMed Central

Zweidler, Alfred; Cohen, Leonard H.

1971-01-01

Methods for the mass isolation of diverse organs from small animals are described. They involve novel devices: a mechanical dissecting system, a centrifugal agitator for the separation of fibrillar from globular particles, and a settling chamber for the fractionation at unit gravity of particles with sedimentation velocities above the useful range for centrifugation. The application of these methods to the isolation of polytene and nonpolytene nuclei from Drosophila melanogaster larvae is described. PMID:5000070

Biological coating of EPDM-membranes of fine bubble diffusers.

PubMed

Wagner, M; von Hoessle, R

2004-01-01

Biological coatings on EPDM-membranes are a problem on many large wastewater treatment plants, as the oxygen supply of the micro-organisms is no longer guaranteed. Investigations prove that the pressure loss and the Shore A-hardness of the EPDM-membranes increase while on the other hand their softener content decreases accordingly. The detected coatings on the membrane surfaces and in the slits or holes of the membranes show extra-cellular organic substances (EPS), which, compared with fibrillar/filamented EPS usually found on surfaces in wastewater treatment plants, are viscous to a much greater extent. As, besides primary organic parts (carbon), the coatings on the membranes as well as in the slits or holes also consist of inorganic constituents (magnesium, silicon, and others), the authors assume that, the separating agent (and also inactive filler) talcum (magnesium silicate), used when producing the membranes, supports at least a first beginning of the coating. Superfine dust constituents and fibres, input via the compressed air, will build up inside the coating and consequently lead to a gradual clogging of the holes or slits. Besides chemical cleaning measures, the exchange of the EPDM-membranes against membranes of silicone would also be a possible measure to solve this problem. The market will decide, if, in the future, a cleaning or an exchange of the EPDM-membranes against membranes of silicone will be applied, but it has to be considered that the loss of softener is irreversible.

Antibacterial activity of silver nanoparticles synthesized In-situ by solution spraying onto cellulose.

PubMed

Yan, Jinhua; Abdelgawad, Abdelrahman M; El-Naggar, Mehrez E; Rojas, Orlando J

2016-08-20

Spray technique was used for the adsorption of in-situ silver nanoparticles (AgNPs) onto and inside the surface of nano- and micro- fibrillar cellulose (NFC and MFC) as well as filter paper. The abundance of hydroxyl and carboxyl groups located in NFC and MFC are used to stabilize Ag ions (Ag(+)) which were then in-situ reduced to (AgNPs) by chemical or UV reduction. The surface characteristic features, elemental analysis, particle size as well as size distribution of the obtained MFC, NFC and filter paper loaded with AgNPs were characterized via field emission scanning electron microscopy connected to energy dispersive X-ray spectroscopy (FESEM- EDX) and transmission electron microscopy (TEM). The associated chemical changes after growth of AgNPs onto the cellulose substrates were assessed by fourier transform infra-red (FT-IR) while the thermal stability of such systems were investigated by thermogravimetrical analyses (TGA). The antibacterial properties of AgNPs loaded NFC, MFC and filter paper as well was investigated against Escherichia Coli. The resulted data indicate that the particle size was found to be 11 and 26nm for AgNPs nucleated on NFC and MFC-based papers respectively. The antibacterial activity of AgNPs loaded MFC exhibited higher antibacterial activity than that of AgNPs loaded NFC. Overall, the present research demonstrates facile and fast method for in-situ antibacterial AgNPs loading on cellulose substrates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structural properties and adsorption capacity of holocellulose aerogels synthesized from an alkali hydroxide-urea solution

NASA Astrophysics Data System (ADS)

Kwon, Gu-Joong; Kim, Dae-Young; Hwang, Jae-Hyun; Kang, Joo-Hyon

2014-05-01

A tulip tree was used to synthesize a holocellulose aerogel from an aqueous alkali hydroxide-urea solution with the substitution of an organic solvent followed by freeze-drying. For comparison, the synthesized holocellulose aerogels were divided into two groups according to the source of the hydrogel, an upper suspended layer and a bottom concentrated layer of the centrifuged solution of cellulose and NaOH/urea solvents. We investigated the effects of the temperature of the pre-cooled NaOH/urea solution ( i.e., dissolution temperature) on the pore structure and the adsorption capacity of the holocellulose aerogel. A nano-fibrillar network structure of the holocellulose aerogel was observed, with little morphological difference in pore structure for different dissolution temperatures. Both micropores and mesopores were observed in the holocellulose aerogel. The specific surface area of the holocellulose aerogel was generally greater at lower dissolution temperatures. In a series of adsorption tests using methylene blue, the holocellulose aerogel showed the greatest adsorption capacity at the lowest dissolution temperature tested (-2°C). However, the dissolution temperature generally had little effect on the adsorption capacity. The holocellulose aerogel produced from the upper suspended layer of the centrifuged hydrogel solution showed a greater porosity and adsorption capacity than the one produced from the bottom concentrated layer. Overall, the aerogel made by utilizing a delignified tulip tree display a high surface area and a high adsorption property, indicating its possible application in eco-friendly adsorption materials.

Synthesis and Self-Assembly of Cellulose Microfibrils from Reconstituted Cellulose Synthase.

PubMed

Cho, Sung Hyun; Purushotham, Pallinti; Fang, Chao; Maranas, Cassandra; Díaz-Moreno, Sara M; Bulone, Vincent; Zimmer, Jochen; Kumar, Manish; Nixon, B Tracy

2017-09-01

Cellulose, the major component of plant cell walls, can be converted to bioethanol and is thus highly studied. In plants, cellulose is produced by cellulose synthase, a processive family-2 glycosyltransferase. In plant cell walls, individual β-1,4-glucan chains polymerized by CesA are assembled into microfibrils that are frequently bundled into macrofibrils. An in vitro system in which cellulose is synthesized and assembled into fibrils would facilitate detailed study of this process. Here, we report the heterologous expression and partial purification of His-tagged CesA5 from Physcomitrella patens Immunoblot analysis and mass spectrometry confirmed enrichment of PpCesA5. The recombinant protein was functional when reconstituted into liposomes made from yeast total lipid extract. The functional studies included incorporation of radiolabeled Glc, linkage analysis, and imaging of cellulose microfibril formation using transmission electron microscopy. Several microfibrils were observed either inside or on the outer surface of proteoliposomes, and strikingly, several thinner fibrils formed ordered bundles that either covered the surfaces of proteoliposomes or were spawned from liposome surfaces. We also report this arrangement of fibrils made by proteoliposomes bearing CesA8 from hybrid aspen. These observations describe minimal systems of membrane-reconstituted CesAs that polymerize β-1,4-glucan chains that coalesce to form microfibrils and higher-ordered macrofibrils. How these micro- and macrofibrils relate to those found in primary and secondary plant cell walls is uncertain, but their presence enables further study of the mechanisms that govern the formation and assembly of fibrillar cellulosic structures and cell wall composites during or after the polymerization process controlled by CesA proteins. © 2017 American Society of Plant Biologists. All Rights Reserved.

Synthesis and Self-Assembly of Cellulose Microfibrils from Reconstituted Cellulose Synthase1[OPEN

PubMed Central

Purushotham, Pallinti; Fang, Chao; Maranas, Cassandra; Bulone, Vincent

2017-01-01

Cellulose, the major component of plant cell walls, can be converted to bioethanol and is thus highly studied. In plants, cellulose is produced by cellulose synthase, a processive family-2 glycosyltransferase. In plant cell walls, individual β-1,4-glucan chains polymerized by CesA are assembled into microfibrils that are frequently bundled into macrofibrils. An in vitro system in which cellulose is synthesized and assembled into fibrils would facilitate detailed study of this process. Here, we report the heterologous expression and partial purification of His-tagged CesA5 from Physcomitrella patens. Immunoblot analysis and mass spectrometry confirmed enrichment of PpCesA5. The recombinant protein was functional when reconstituted into liposomes made from yeast total lipid extract. The functional studies included incorporation of radiolabeled Glc, linkage analysis, and imaging of cellulose microfibril formation using transmission electron microscopy. Several microfibrils were observed either inside or on the outer surface of proteoliposomes, and strikingly, several thinner fibrils formed ordered bundles that either covered the surfaces of proteoliposomes or were spawned from liposome surfaces. We also report this arrangement of fibrils made by proteoliposomes bearing CesA8 from hybrid aspen. These observations describe minimal systems of membrane-reconstituted CesAs that polymerize β-1,4-glucan chains that coalesce to form microfibrils and higher-ordered macrofibrils. How these micro- and macrofibrils relate to those found in primary and secondary plant cell walls is uncertain, but their presence enables further study of the mechanisms that govern the formation and assembly of fibrillar cellulosic structures and cell wall composites during or after the polymerization process controlled by CesA proteins. PMID:28768815

Carbon nanotubes exhibit fibrillar pharmacology in primates

DOE PAGES

Alidori, Simone; Thorek, Daniel L. J.; Beattie, Bradley J.; ...

2017-08-28

Nanomedicine rests at the nexus of medicine, bioengineering, and biology with great potential for improving health through innovation and development of new drugs and devices. Carbon nanotubes are an example of a fibrillar nanomaterial poised to translate into medical practice. The leading candidate material in this class is ammonium-functionalized carbon nanotubes (fCNT) that exhibits unexpected pharmacological behavior in vivo with important biotechnology applications. Here, we provide a multi-organ evaluation of the distribution, uptake and processing of fCNT in nonhuman primates using quantitative whole body positron emission tomography (PET), compartmental modeling of pharmacokinetic data, serum biomarkers and ex vivo pathology investigation.more » Kidney and liver are the two major organ systems that accumulate and excrete [ 86Y]fCNT in nonhuman primates and accumulation is cell specific as described by compartmental modeling analyses of the quantitative PET data. A serial two-compartment model explains renal processing of tracer-labeled fCNT; hepatic data fits a parallel two-compartment model. These modeling data also reveal significant elimination of the injected activity (>99.8%) from the primate within 3 days (t 1/2 = 11.9 hours). Thus, these favorable results in nonhuman primates provide important insight to the fate of fCNT in vivo and pave the way to further engineering design considerations for sophisticated nanomedicines to aid late stage development and clinical use in man.« less

Nucleolar evolution and coiled bodies during meiotic prophase in Olea europaea: differential localization of nucleic acids.

PubMed

Olmedilla, A; de Dios Alché, J; Rodríguez-García, M I

1997-10-01

We studied the ultrastructural evolution of the nucleolus during meiotic prophase in olive microsporocytes. During prophase, nuclear bodies morphologically similar to coiled bodies were observed. The nucleic acid composition of these bodies was examined in microsporocytes using electron microscopic techniques with EDTA preferential ribonucleoprotein staining, anti-DNA immunolabeling, the in situ terminal deoxynucleotidyl transferase-immunogold technique, and in situ hybridization with 18S rRNA and U3 snoRNA digoxigenin-labeled probes. The ultrastructural appearance of the meiocyte nucleolus indicated a low level of activity from the early prophase stage: the granular component was practically absent and nucleoli were constituted almost exclusively by dense fibrillar component containing large fibrillar centers that lacked chromatin inclusions. However, the appearance of reactivation vacuoles in the nucleolus during zygotene and high levels of rRNA in the nucleoplasm during pachytene support the presence of a peak in rRNA synthesis. Our results also show that the nuclear bodies that appear during prophase I are ribonucleoproteinaceous in nature; neither DNA nor ribosomal RNA were detected. The presence of U3 snoRNA, as shown by in situ hybridization in nuclear bodies from plant material, is also evidence that these structures are coiled bodies. We suggest that coiled bodies are involved not only in pre- and post-splicing events but also in the storage, transport or recycling of rRNA maturation elements.

Fluctuations of pol I and fibrillarin contents of the nucleoli.

PubMed

Hornáček, M; Kováčik, L; Mazel, T; Cmarko, D; Bártová, E; Raška, I; Smirnov, E

2017-07-04

Nucleoli are formed on the basis of ribosomal DNA (rDNA) clusters called Nucleolus Organizer Regions (NORs). Each NOR contains multiple genes coding for RNAs of the ribosomal particles. The prominent components of the nucleolar ultrastructure, fibrillar centers (FC) and dense fibrillar components (DFC), together compose FC/DFC units. These units are centers of rDNA transcription by RNA polymerase I (pol I), as well as the early processing events, in which an essential role belongs to fibrillarin. Each FC/DFC unit probably corresponds to a single transcriptionally active gene. In this work, we transfected human-derived cells with GFP-RPA43 (subunit of pol I) and RFP-fibrillarin. Following changes of the fluorescent signals in individual FC/DFC units, we found two kinds of kinetics: 1) the rapid fluctuations with periods of 2-3 min, when the pol I and fibrillarin signals oscillated in anti-phase manner, and the intensities of pol I in the neighboring FC/DFC units did not correlate. 2) fluctuations with periods of 10 to 60 min, in which pol I and fibrillarin signals measured in the same unit did not correlate, but pol I signals in the units belonging to different nucleoli were synchronized. Our data indicate that a complex pulsing activity of transcription as well as early processing is common for ribosomal genes.

Nucleoli in human early erythroblasts (K2, K1, K1/2 cells).

PubMed

Smetana, K; Jirásková, I; Klamová, H

2005-01-01

Human early erythroid precursors classified according to the nuclear size were studied to provide information on nucleoli in these cells using simple cytochemical procedures for demonstration of RNA and proteins of silver-stained nucleolar organizers. K2 cells with nuclear diameter larger than 13 microm and K1 cells with nuclear diameter larger than 9 microm corresponding to proerythroblasts and macroblasts (large basophilic erythroblasts) mostly possessed large irregularly shaped nucleoli with multiple fibrillar centres representing "active nucleoli". K1/2 cells with nuclear diameter smaller than 9 microm corresponding to small basophilic erythroblasts were usually characterized by the presence of micronucleoli representing "inactive nucleolar types". On the other hand, a few K1/2 cells contained large nucleoli with multiple fibrillar centres similar to those present in K2 cells and thus appeared as "microproerythroblasts". The nucleolar asynchrony expressed by the presence of large irregularly shaped nucleoli with multiple nucleoli (active nucleoli) and ring-shaped nucleoli (resting nucleoli) in one and the same nucleus of K2 or K1 cells was not exceptional and might reflect a larger resistance of these cells to negative factors influencing the erythropoiesis. The intranucleolar translocation of silver-stained nucleolus organized regions was noted in K2 cells and might indicate the premature aging of these cells without further differentiation. More studies, however, are required in this direction.

Evaluation of Small Intestine Grafts Decellularization Methods for Corneal Tissue Engineering

PubMed Central

Oliveira, Ana Celeste; Garzón, Ingrid; Ionescu, Ana Maria; Carriel, Victor; Cardona, Juan de la Cruz; González-Andrades, Miguel; Pérez, María del Mar; Alaminos, Miguel; Campos, Antonio

2013-01-01

Advances in the development of cornea substitutes by tissue engineering techniques have focused on the use of decellularized tissue scaffolds. In this work, we evaluated different chemical and physical decellularization methods on small intestine tissues to determine the most appropriate decellularization protocols for corneal applications. Our results revealed that the most efficient decellularization agents were the SDS and triton X-100 detergents, which were able to efficiently remove most cell nuclei and residual DNA. Histological and histochemical analyses revealed that collagen fibers were preserved upon decellularization with triton X-100, NaCl and sonication, whereas reticular fibers were properly preserved by decellularization with UV exposure. Extracellular matrix glycoproteins were preserved after decellularization with SDS, triton X-100 and sonication, whereas proteoglycans were not affected by any of the decellularization protocols. Tissue transparency was significantly higher than control non-decellularized tissues for all protocols, although the best light transmittance results were found in tissues decellularized with SDS and triton X-100. In conclusion, our results suggest that decellularized intestinal grafts could be used as biological scaffolds for cornea tissue engineering. Decellularization with triton X-100 was able to efficiently remove all cells from the tissues while preserving tissue structure and most fibrillar and non-fibrillar extracellular matrix components, suggesting that this specific decellularization agent could be safely used for efficient decellularization of SI tissues for cornea TE applications. PMID:23799114

Carbon nanotubes exhibit fibrillar pharmacology in primates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alidori, Simone; Thorek, Daniel L. J.; Beattie, Bradley J.

Nanomedicine rests at the nexus of medicine, bioengineering, and biology with great potential for improving health through innovation and development of new drugs and devices. Carbon nanotubes are an example of a fibrillar nanomaterial poised to translate into medical practice. The leading candidate material in this class is ammonium-functionalized carbon nanotubes (fCNT) that exhibits unexpected pharmacological behavior in vivo with important biotechnology applications. Here, we provide a multi-organ evaluation of the distribution, uptake and processing of fCNT in nonhuman primates using quantitative whole body positron emission tomography (PET), compartmental modeling of pharmacokinetic data, serum biomarkers and ex vivo pathology investigation.more » Kidney and liver are the two major organ systems that accumulate and excrete [ 86Y]fCNT in nonhuman primates and accumulation is cell specific as described by compartmental modeling analyses of the quantitative PET data. A serial two-compartment model explains renal processing of tracer-labeled fCNT; hepatic data fits a parallel two-compartment model. These modeling data also reveal significant elimination of the injected activity (>99.8%) from the primate within 3 days (t 1/2 = 11.9 hours). Thus, these favorable results in nonhuman primates provide important insight to the fate of fCNT in vivo and pave the way to further engineering design considerations for sophisticated nanomedicines to aid late stage development and clinical use in man.« less

Microcin Amyloid Fibrils A Are Reservoir of Toxic Oligomeric Species

PubMed Central

Shahnawaz, Mohammad; Soto, Claudio

2012-01-01

Microcin E492 (Mcc), a low molecular weight bacteriocin produced by Klebsiella pneumoniae RYC492, has been shown to exist in two forms: soluble forms that are believed to be toxic to the bacterial cell by forming pores and non-toxic fibrillar forms that share similar biochemical and biophysical properties with amyloids associated with several human diseases. Here we report that fibrils polymerized in vitro from soluble forms sequester toxic species that can be released upon changing environmental conditions such as pH, ionic strength, and upon dilution. Our results indicate that basic pH (≥8.5), low NaCl concentrations (≤50 mm), and dilution (>10-fold) destabilize Mcc fibrils into more soluble species that are found to be toxic to the target cells. Additionally, we also found a similar conversion of non-toxic fibrils into highly toxic oligomers using Mcc aggregates produced in vivo. Moreover, the soluble protein released from fibrils is able to rapidly polymerize into amyloid fibrils under fibril-forming conditions and to efficiently seed aggregation of monomeric Mcc. Our findings indicate that fibrillar forms of Mcc constitute a reservoir of toxic oligomeric species that is released into the medium upon changing the environmental conditions. These findings may have substantial implications to understand the dynamic process of interconversion between toxic and non-toxic aggregated species implicated in protein misfolding diseases. PMID:22337880

Critical early roles for col27a1a and col27a1b in zebrafish notochord morphogenesis, vertebral mineralization and post-embryonic axial growth.

PubMed

Christiansen, Helena E; Lang, Michael R; Pace, James M; Parichy, David M

2009-12-29

Fibrillar collagens are well known for their links to human diseases, with which all have been associated except for the two most recently identified fibrillar collagens, type XXIV collagen and type XXVII collagen. To assess functions and potential disease phenotypes of type XXVII collagen, we examined its roles in zebrafish embryonic and post-embryonic development. We identified two type XXVII collagen genes in zebrafish, col27a1a and col27a1b. Both col27a1a and col27a1b were expressed in notochord and cartilage in the embryo and early larva. To determine sites of type XXVII collagen function, col27a1a and col27a1b were knocked down using morpholino antisense oligonucleotides. Knockdown of col27a1a singly or in conjunction with col27a1b resulted in curvature of the notochord at early stages and formation of scoliotic curves as well as dysmorphic vertebrae at later stages. These defects were accompanied by abnormal distributions of cells and protein localization in the notochord, as visualized by transmission electron microscopy, as well as delayed vertebral mineralization as detected histologically. Together, our findings indicate a key role for type XXVII collagen in notochord morphogenesis and axial skeletogenesis and suggest a possible human disease phenotype.

Triple helix-forming oligonucleotide corresponding to the polypyrimidine sequence in the rat alpha 1(I) collagen promoter specifically inhibits factor binding and transcription.

PubMed

Kovacs, A; Kandala, J C; Weber, K T; Guntaka, R V

1996-01-19

Type I and III fibrillar collagens are the major structural proteins of the extracellular matrix found in various organs including the myocardium. Abnormal and progressive accumulation of fibrillar type I collagen in the interstitial spaces compromises organ function and therefore, the study of transcriptional regulation of this gene and specific targeting of its expression is of major interest. Transient transfection of adult cardiac fibroblasts indicate that the polypurine-polypyrimidine sequence of alpha 1(I) collagen promoter between nucleotides - 200 and -140 represents an overall positive regulatory element. DNase I footprinting and electrophoretic mobility shift assays suggest that multiple factors bind to different elements of this promoter region. We further demonstrate that the unique polypyrimidine sequence between -172 and -138 of the promoter represents a suitable target for a single-stranded polypurine oligonucleotide (TFO) to form a triple helix DNA structure. Modified electrophoretic mobility shift assays show that this TFO specifically inhibits the protein-DNA interaction within the target region. In vitro transcription assays and transient transfection experiments demonstrate that the transcriptional activity of the promoter is inhibited by this oligonucleotide. We propose that TFOs represent a therapeutic potential to specifically influence the expression of alpha 1(I) collagen gene in various disease states where abnormal type I collagen accumulation is known to occur.

Tenascin-x deficiency mimics ehlers-danlos syndrome in mice through alteration of collagen deposition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mao, J.R.; Taylor, G.; Dean, W.B.

2002-03-01

Tenascin-X is a large extracellular matrix protein of unknown function1-3. Tenascin-X deficiency in humans is associated with Ehlers-Danlos syndrome4,5, a generalized connective tissue disorder resulting from altered metabolism of the fibrillar collagens6. Because TNXB is the first Ehlers-Danlos syndrome gene that does not encode a fibrillar collagen or collagen-modifying enzyme7-14, we suggested that tenascin-X might regulate collagen synthesis or deposition15. To test this hypothesis, we inactivated Tnxb in mice. Tnxb-/- mice showed progressive skin hyperextensibility, similar to individuals with Ehlers-Danlos syndrome. Biomechanical testing confirmed increased deformability and reduced tensile strength of their skin. The skin of Tnxb-/- mice was histologicallymore » normal, but its collagen content was significantly reduced. At the ultrastructural level, collagen fibrils of Tnxb-/- mice were of normal size and shape, but the density of fibrils in their skin was reduced, commensurate with the reduction in collagen content. Studies of cultured dermal fibroblasts showed that although synthesis of collagen I by Tnxb-/- and wildtype cells was similar, Tnxb-/- fibroblasts failed to deposit collagen I into cell-associated matrix. This study confirms a causative role for TNXB in human Ehlers-Danlos syndrome and suggests that tenascin-X is an essential regulator of collagen deposition by dermal fibroblasts.« less

SPARC Regulates Transforming Growth Factor Beta Induced (TGFBI) Extracellular Matrix Deposition and Paclitaxel Response in Ovarian Cancer Cells.

PubMed

Tumbarello, David A; Andrews, Melissa R; Brenton, James D

2016-01-01

TGFBI has been shown to sensitize ovarian cancer cells to the cytotoxic effects of paclitaxel via an integrin receptor-mediated mechanism that modulates microtubule stability. Herein, we determine that TGFBI localizes within organized fibrillar structures in mesothelial-derived ECM. We determined that suppression of SPARC expression by shRNA decreased the deposition of TGFBI in mesothelial-derived ECM, without affecting its overall protein expression or secretion. Conversely, overexpression of SPARC increased TGFBI deposition. A SPARC-YFP fusion construct expressed by the Met5a cell line co-localized with TGFBI in the cell-derived ECM. Interestingly, in vitro produced SPARC was capable of precipitating TGFBI from cell lysates dependent on an intact SPARC carboxy-terminus with in vitro binding assays verifying a direct interaction. The last 37 amino acids of SPARC were shown to be required for the TGFBI interaction while expression of a SPARC-YFP construct lacking this region (aa 1-256) did not interact and co-localize with TGFBI in the ECM. Furthermore, ovarian cancer cells have a reduced motility and decreased response to the chemotherapeutic agent paclitaxel when plated on ECM derived from mesothelial cells lacking SPARC compared to control mesothelial-derived ECM. In conclusion, SPARC regulates the fibrillar ECM deposition of TGFBI through a novel interaction, subsequently influencing cancer cell behavior.

SPARC Regulates Transforming Growth Factor Beta Induced (TGFBI) Extracellular Matrix Deposition and Paclitaxel Response in Ovarian Cancer Cells

PubMed Central

Andrews, Melissa R.; Brenton, James D.

2016-01-01

TGFBI has been shown to sensitize ovarian cancer cells to the cytotoxic effects of paclitaxel via an integrin receptor-mediated mechanism that modulates microtubule stability. Herein, we determine that TGFBI localizes within organized fibrillar structures in mesothelial-derived ECM. We determined that suppression of SPARC expression by shRNA decreased the deposition of TGFBI in mesothelial-derived ECM, without affecting its overall protein expression or secretion. Conversely, overexpression of SPARC increased TGFBI deposition. A SPARC-YFP fusion construct expressed by the Met5a cell line co-localized with TGFBI in the cell-derived ECM. Interestingly, in vitro produced SPARC was capable of precipitating TGFBI from cell lysates dependent on an intact SPARC carboxy-terminus with in vitro binding assays verifying a direct interaction. The last 37 amino acids of SPARC were shown to be required for the TGFBI interaction while expression of a SPARC-YFP construct lacking this region (aa 1–256) did not interact and co-localize with TGFBI in the ECM. Furthermore, ovarian cancer cells have a reduced motility and decreased response to the chemotherapeutic agent paclitaxel when plated on ECM derived from mesothelial cells lacking SPARC compared to control mesothelial-derived ECM. In conclusion, SPARC regulates the fibrillar ECM deposition of TGFBI through a novel interaction, subsequently influencing cancer cell behavior. PMID:27622658

Characterisation of rheology and microstructures of κ-carrageenan in ethanol-water mixtures.

PubMed

Yang, Zhi; Yang, Huijuan; Yang, Hongshun

2018-05-01

The effects of ethanol (up to 20 wt%) on the rheological properties and structural characteristics of κ-carrageenan gel were investigated by Field Emission Scanning Electron Microscopy (FESEM) and Small Angle X-ray Scattering (SAXS). Both the sol-gel and gel-sol transition temperatures shifted to higher degree (from 36.8 ± 0.5 to 52.5 ± 1.4 °C and from 51.2 ± 0.6 to 67.0 ± 0.5 °C, respectively) upon 20 wt% ethanol addition (P < 0.05). The critical relaxation exponent n and the critical gel strength S g obtained from Winter-Chambon criterion decreased and increased, respectively as the ethanol concentration increased. The κ-carrageenan gel was formed due to the formation of fibrillar networks, and the fibrillar density increased upon ethanol addition via FESEM. Moreover, upon 20 wt% ethanol addition, the average radius of gyration of κ-carrageenan strand increased from 1.18 ± 0.03 of control to 1.55 ± 0.02 nm by SAXS. A mechanism underlying the effect of ethanol on the κ-carrageenan gelation was proposed based on coil to double helix transition followed by the helix aggregation. Copyright © 2018 Elsevier Ltd. All rights reserved.

An Atomistic View of Amyloidogenic Self-assembly: Structure and Dynamics of Heterogeneous Conformational States in the Pre-nucleation Phase

PubMed Central

Matthes, Dirk; Gapsys, Vytautas; Brennecke, Julian T.; de Groot, Bert L.

2016-01-01

The formation of well-defined filamentous amyloid structures involves a polydisperse collection of oligomeric states for which relatively little is known in terms of structural organization. Here we use extensive, unbiased explicit solvent molecular dynamics (MD) simulations to investigate the structural and dynamical features of oligomeric aggregates formed by a number of highly amyloidogenic peptides at atomistic resolution on the μs time scale. A consensus approach has been adopted to analyse the simulations in multiple force fields, yielding an in-depth characterization of pre-fibrillar oligomers and their global and local structure properties. A collision cross section analysis revealed structurally heterogeneous aggregate ensembles for the individual oligomeric states that lack a single defined quaternary structure during the pre-nucleation phase. To gain insight into the conformational space sampled in early aggregates, we probed their substructure and found emerging β-sheet subunit layers and a multitude of ordered intermolecular β-structure motifs with growing aggregate size. Among those, anti-parallel out-of-register β-strands compatible with toxic β-barrel oligomers were particularly prevalent already in smaller aggregates and formed prior to ordered fibrillar structure elements. Notably, also distinct fibril-like conformations emerged in the oligomeric state and underscore the notion that pre-nucleated oligomers serve as a critical intermediate step on-pathway to fibrils. PMID:27616019

Nucleolar structure across evolution: the transition between bi- and tri-compartmentalized nucleoli lies within the class Reptilia.

PubMed

Lamaye, Françoise; Galliot, Sonia; Alibardi, Lorenzo; Lafontaine, Denis L J; Thiry, Marc

2011-05-01

Two types of nucleolus can be distinguished among eukaryotic cells: a tri-compartmentalized nucleolus in amniotes and a bi-compartmentalized nucleolus in all the others. However, though the nucleolus' ultrastructure is well characterized in mammals and birds, it has been so far much less studied in reptiles. In this work, we examined the ultrastructural organization of the nucleolus in various tissues from different reptilian species (three turtles, three lizards, two crocodiles, and three snakes). Using cytochemical and immunocytological methods, we showed that in reptiles both types of nucleolus are present: a bi-compartmentalized nucleolus in turtles and a tri-compartmentalized nucleolus in the other species examined in this study. Furthermore, in a given species, the same type of nucleolus is present in all the tissues, however, the importance and the repartition of those nucleolar components could vary from one tissue to another. We also reveal that, contrary to the mammalian nucleolus, the reptilian fibrillar centers contain small clumps of condensed chromatin and that their surrounding dense fibrillar component is thicker. Finally, we also report that Cajal bodies are detected in reptiles. Altogether, we believe that these results have profound evolutionarily implications since they indicate that the point of transition between bipartite and tripartite nucleoli lies at the emergence of the amniotes within the class Reptilia. Copyright © 2011 Elsevier Inc. All rights reserved.

α-Synuclein aggregation, seeding and inhibition by scyllo-inositol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ibrahim, Tarek; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, M4N 3M5, ON; McLaurin, JoAnne, E-mail: jmclaurin@sri.utoronto.ca

2016-01-15

Recent literature demonstrates the accelerated aggregation of α-synuclein, a protein implicated in the pathogenesis of Parkinson's disease (PD), by the presence of preformed fibrillar conformers in vitro. Furthermore, these preformed fibrillar seeds are suggested to accelerate pathological induction in vivo when injected into the brains of mice. Variation in the results of in vivo studies is proposed to be caused by α-synuclein conformational variants. To investigate the impact of amino acid sequence on seeding efficiency, human and mouse α-synuclein seeds, which vary at 7 amino acid residues, were generated and cross-seeding kinetics studied. Using transmission electron microscopy (TEM), we confirmed that mouse α-synucleinmore » aggregated more rapidly than human α-synuclein. Subsequently, we determined that seeding of human and mouse α-synuclein was more rapid in the presence of seeds generated from the same species. In addition, an established amyloid inhibitor, scyllo-inositol, was examined for potential inhibitory effects on α-synuclein aggregation. TEM analysis of protein:inhibitor assays demonstrated that scyllo-inositol inhibits the aggregation of α-synuclein, suggesting the therapeutic potential of the small molecule in PD. - Highlights: • Mouse α-syn fibrillizes in a significantly shorter timeframe than human α-syn. • Seeding of monomers is more efficient when seeds originate from the same species. • scyllo-Inositol has anti-aggregation effects on mouse and human α-syn.« less

WtF‐Nano: One‐Pot Dewatering and Water‐Free Topochemical Modification of Nanocellulose in Ionic Liquids or γ‐Valerolactone

PubMed Central

Laaksonen, Tiina; Helminen, Jussi K. J.; Lemetti, Laura; Långbacka, Jesper; Rico del Cerro, Daniel; Hummel, Michael; Rantamäki, Antti H.; Kakko, Tia; Kemell, Marianna L.; Wiedmer, Susanne K.; Heikkinen, Sami; Kilpeläinen, Ilkka

2017-01-01

Abstract Ionic liquids are used to dewater a suspension of birch Kraft pulp cellulose nanofibrils (CNF) and as a medium for water‐free topochemical modification of the nanocellulose (a process denoted as “WtF‐Nano”). Acetylation was applied as a model reaction to investigate the degree of modification and scope of effective ionic liquid structures. Little difference in reactivity was observed when water was removed, after introduction of an ionic liquid or molecular co‐solvent. However, the viscoelastic properties of the CNF suspended in two ionic liquids show that the more basic, but non‐dissolving ionic liquid, allows for better solvation of the CNF. Vibrio fischeri bacterial tests show that all ionic liquids in this study were harmless. Scanning electron microscopy and wide‐angle X‐ray scattering on regenerated samples show that the acetylated CNF is still in a fibrillar form. 1 D and 2 D NMR analyses, after direct dissolution in a novel ionic liquid electrolyte solution, indicate that both cellulose and residual xylan on the surface of the nanofibrils reacts to give acetate esters. PMID:29112334

Carbon aerogels by pyrolysis of TEMPO-oxidized cellulose

NASA Astrophysics Data System (ADS)

Zhang, Sizhao; Feng, Jian; Feng, Junzong; Jiang, Yonggang; Ding, Feng

2018-05-01

Although carbon aerogels derived from naturally occurring materials have been developed extensively, a reasonable synthetic approach using cellulose-resource remains unclear. Here, we report a strategy to prepare carbon aerogels originated from cellulose position-selectively oxidized by TEMPO-oxidized process. Contrary to non-TEMPO-oxidized cellulose-derived carbon aerogels (NCCA) with relative loose structure, TEMPO-oxidized cellulose-derived carbon aerogels (TCCA) with tight fibrillar-continuous network are monitored, suggesting the importance of TEMPO-oxidized modification towards creating the architecture of subsequently produced carbon aerogels. TCCA endows a higher BET area despite owning slightly dense bulk density comparing with that of NCCA. The structural texture of TCCA could be maintained in a way in comparison to TEMPO-oxidized cellulose-derived aerogel, due to the integration and aggregation effect by losing the electric double layer repulsion via ionization of the surface carboxyl groups. FTIR and XPS analyses signify the evidence of non-functionalized carbon-skeleton network formation in terms of TCCA. Further, the mechanism concerning the creation of carbon aerogels is also established. These findings not only provide new insights into the production of carbon aerogels but also open up a new opportunity in the field of functional carbon materials.

Histophilus somni host-parasite relationships.

PubMed

Corbeil, Lynette B

2007-12-01

Histophilus somni (Haemophilus somnus) is one of the key bacterial pathogens involved in the multifactorial etiology of the Bovine Respiratory Disease Complex. This Gram negative pleomorphic rod also causes bovine septicemia, thrombotic meningencephalitis, myocarditis, arthritis, abortion and infertility, as well as disease in sheep, bison and bighorn sheep. Virulence factors include lipooligosaccharide, immunoglobulin binding proteins (as a surface fibrillar network), a major outer membrane protein (MOMP), other outer membrane proteins (OMPs) and exopolysaccharide. Histamine production, biofilm formation and quorum sensing may also contribute to pathogenesis. Antibodies are very important in protection as shown in passive protection studies. The lack of long-term survival of the organism in macrophages, unlike facultative intracellular bacteria, also suggests that antibodies should be critical in protection. Of the immunoglobulin classes, IgG2 antibodies are most implicated in protection and IgE antibodies in immunopathogenesis. The immunodominant antigen recognized by IgE is the MOMP and by IgG2 is a 40 kDa OMP. Pathogenetic synergy of bovine respiratory syncytial virus (BRSV) and H. somni in calves can be attributed, in part at least, to the higher IgE anti-MOMP antibody responses in dually infected calves. Other antigens are probably involved in stimulating host defense or immunopathology as well.

In vivo comparative property study of the bioactivity of coated Mg-3Zn-0.8Zr alloy.

PubMed

Sun, Jin'e; Wang, Jingbo; Jiang, Hongfeng; Chen, Minfang; Bi, Yanze; Liu, Debao

2013-08-01

In this in vivo study, degradable Mg-3Zn-0.8Zr cylinders were coated with a calcium phosphorus compound (Ca-P) layer or a magnesium fluoride (MgF2) layer; uncoated Mg-3Zn-0.8Zr alloy was used as a control. These were then implanted intramedullary into the femora of nine Japanese big-ear white rabbits for implantation periods of 1, 2 and 3 months. During the postoperative observation period with radiographic examination, the results showed that the MgF2-coated implants were tolerated well compared to the Ca-P-coated implants and uncoated implants. Moreover, large amounts of cells, rich fibrillar collagen and calcium and phosphorus products were found on the surface of the MgF2-coated implants using scanning electron microscopy. Micro-computed tomography further showed a slight decrease in volume (23.85%) and a greater increase in new bone mass (new bone volume fraction=11.56%, tissue mineral density=248.81 mg/cm(3)) for the MgF2-coated implants in comparison to uncoated and Ca-P compound-coated implants after 3 months of implantation. Copyright © 2013 Elsevier B.V. All rights reserved.

Visualization of Neutrophil Extracellular Traps and Fibrin Meshwork in Human Fibrinopurulent Inflammatory Lesions: II. Ultrastructural Study

PubMed Central

Onouchi, Takanori; Shiogama, Kazuya; Matsui, Takahiro; Mizutani, Yasuyoshi; Sakurai, Kouhei; Inada, Ken-ichi; Tsutsumi, Yutaka

2016-01-01

Neutrophil extracellular traps (NETs) represent an extracellular, spider’s web-like structure resulting from cell death of neutrophils. NETs play an important role in innate immunity against microbial infection, but their roles in human pathological processes remain largely unknown. NETs and fibrin meshwork both showing fibrillar structures are observed at the site of fibrinopurulent inflammation, as described in our sister paper [Acta Histochem. Cytochem. 49; 109–116, 2016]. In the present study, immunoelectron microscopic study was performed for visualizing NETs and fibrin fibrils (thick fibrils in our tongue) in formalin-fixed, paraffin-embedded sections of autopsied lung tissue of legionnaire’s pneumonia. Lactoferrin and fibrinogen gamma chain were utilized as markers of NETs and fibrin, respectively. Analysis of immuno-scanning electron microscopy indicated that NETs constructed thin fibrils and granular materials were attached onto the NETs fibrils. The smooth-surfaced fibrin fibrils were much thicker than the NETs fibrils. Pre-embedding immunoelectron microscopy demonstrated that lactoferrin immunoreactivities were visible as dots on the fibrils, whereas fibrinogen gamma chain immunoreactivities were homogeneously observed throughout the fibrils. Usefulness of immunoelectron microscopic analysis of NETs and fibrin fibrils should be emphasized. PMID:27682015

Isolation of integrin-based adhesion complexes.

PubMed

Jones, Matthew C; Humphries, Jonathan D; Byron, Adam; Millon-Frémillon, Angélique; Robertson, Joseph; Paul, Nikki R; Ng, Daniel H J; Askari, Janet A; Humphries, Martin J

2015-03-02

The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter-receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions, and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signaling across the plasma membrane, and, as such, help to coordinate and/or modulate the multitude of physical and chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin-associated adhesion complexes, together with their local plasma membrane/cytosolic environments, from cells in culture. In the first protocol, integrin-associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components via subsequent downstream analysis by western blotting or mass spectrometry. Copyright © 2015 John Wiley & Sons, Inc.

Immunogold localisation of laminin in normal and exfoliative iris.

PubMed Central

Konstas, A. G.; Marshall, G. E.; Lee, W. R.

1990-01-01

Immunoelectron microscopic studies of exfoliative iris tissue (seven specimens) revealed the presence of laminin in the fibrillar component of exfoliation material. The immunogold label was uniformly distributed on the exfoliation fibres. Deposition of laminin labelled exfoliation material in the dilator muscle was a noteworthy feature, as was an apparent depletion of laminin in the basement membranes of ostensibly unaffected vessels. In control iris tissue (five enucleated eyes) laminin was identified in the basement membrane round vascular contractile cells, but not beneath the endothelium. Images PMID:2390517

Nucleolar chromatin organization at different activities of soybean root meristematic cell nucleoli.

PubMed

Stępiński, Dariusz

2013-06-01

Nucleolar chromatin, including nucleolus-associated chromatin as well as active and inactive condensed ribosomal DNA (rDNA) chromatin, derives mostly from secondary constrictions known as nucleolus organizer regions containing rDNA genes on nucleolus-forming chromosomes. This chromatin may occupy different nucleolar positions being in various condensation states which may imply different rDNA transcriptional competence. Sections of nucleoli originating from root meristematic cells of soybean seedlings grown at 25 °C (the control), then subjected to chilling stress (10 °C), and next transferred again to 25 °C (the recovery) were used to measure profile areas occupied by nucleolar condensed chromatin disclosed with sodium hydroxide methylation-acetylation plus uranyl acetate technique. The biggest total area of condensed chromatin was found in the nucleoli of chilled plants, while the smallest was found in those of recovered plants in relation to the amounts of chromatin in the control nucleoli. The condensed nucleolar chromatin, in the form of different-sized and different-shaped clumps, was mainly located in fibrillar centers. One can suppose that changes of condensed rDNA chromatin amounts might be a mechanism controlling the number of transcriptionally active rDNA genes as the nucleoli of plants grown under these experimental conditions show different transcriptional activity and morphology.

Structure-mechanics relationships in mineralized tendons.

PubMed

Spiesz, Ewa M; Zysset, Philippe K

2015-12-01

In this paper, we review the hierarchical structure and the resulting elastic properties of mineralized tendons as obtained by various multiscale experimental and computational methods spanning from nano- to macroscale. The mechanical properties of mineralized collagen fibres are important to understand the mechanics of hard tissues constituted by complex arrangements of these fibres, like in human lamellar bone. The uniaxial mineralized collagen fibre array naturally occurring in avian tendons is a well studied model tissue for investigating various stages of tissue mineralization and the corresponding elastic properties. Some avian tendons mineralize with maturation, which results in a graded structure containing two zones of distinct morphology, circumferential and interstitial. These zones exhibit different amounts of mineral, collagen, pores and a different mineral distribution between collagen fibrillar and extrafibrillar space that lead to distinct elastic properties. Mineralized tendon cells have two phenotypes: elongated tenocytes placed between fibres in the circumferential zone and cuboidal cells with lower aspect ratios in the interstitial zone. Interestingly some regions of avian tendons seem to be predestined to mineralization, which is exhibited as specific collagen cross-linking patterns as well as distribution of minor tendon constituents (like proteoglycans) and loss of collagen crimp. Results of investigations in naturally mineralizing avian tendons may be useful in understanding the pathological mineralization occurring in some human tendons. Copyright © 2015 Elsevier Ltd. All rights reserved.

New archetypes in self-assembled Phe-Phe motif induced nanostructures from nucleoside conjugated-diphenylalanines.

PubMed

Datta, Dhrubajyoti; Tiwari, Omshanker; Ganesh, Krishna N

2018-02-15

During the last two decades, the molecular self-assembly of the short peptide diphenylalanine (Phe-Phe) motif has attracted increasing focus due to its unique morphological structure and utility for potential applications in biomaterial chemistry, sensors and bioelectronics. Due to the ease of their synthetic modifications and a plethora of available experimental tools, the self-assembly of free and protected diphenylalanine scaffolds (H-Phe-Phe-OH, Boc-Phe-Phe-OH and Boc-Phe-Phe-OMe) has unfurled interesting tubular, vesicular or fibrillar morphologies. Developing on this theme, here we attempt to examine the effect of structure and properties (hydrophobic and H-bonding) modifying the functional C-terminus conjugated substituents on Boc-Phe-Phe on its self-assembly process. The consequent self-sorting due to H-bonding, van der Waals force and π-π interactions, generates monodisperse nano-vesicles from these peptides characterized via their SEM, HRTEM, AFM pictures and DLS experiments. The stability of these vesicles to different external stimuli such as pH and temperature, encapsulation of fluorescent probes inside the vesicles and their release by external trigger are reported. The results point to a new direction in the study and applications of the Phe-Phe motif to rationally engineer new functional nano-architectures.

The chaperonin CCT promotes the formation of fibrillar aggregates of γ-tubulin.

PubMed

Pouchucq, Luis; Lobos-Ruiz, Pablo; Araya, Gissela; Valpuesta, José María; Monasterio, Octavio

2018-04-01

The type II chaperonin CCT is involved in the prevention of the pathogenesis of numerous human misfolding disorders, as it sequesters misfolded proteins, blocks their aggregation and helps them to achieve their native state. In addition, it has been reported that CCT can prevent the toxicity of non-client amyloidogenic proteins by the induction of non-toxic aggregates, leading to new insight in chaperonin function as an aggregate remodeling factor. Here we add experimental evidence to this alternative mechanism by which CCT actively promotes the formation of conformationally different aggregates of γ-tubulin, a non-amyloidogenic CCT client protein, which are mediated by specific CCT-γ-tubulin interactions. The in vitro-induced aggregates were in some cases long fiber polymers, which compete with the amorphous aggregates. Direct injection of unfolded purified γ-tubulin into single-cell zebra fish embryos allowed us to relate this in vitro activity with the in vivo formation of intracellular aggregates. Injection of a CCT-binding deficient γ-tubulin mutant dramatically diminished the size of the intracellular aggregates, increasing the toxicity of the misfolded protein. These results point to CCT having a role in the remodeling of aggregates, constituting one of its many functions in cellular proteostasis. Copyright © 2018. Published by Elsevier B.V.

A sequential assignment procedure for proteins that have intermediate line widths in MAS NMR spectra: amyloid fibrils of human CA150.WW2.

PubMed

Becker, Johanna; Ferguson, Neil; Flinders, Jeremy; van Rossum, Barth-Jan; Fersht, Alan R; Oschkinat, Hartmut

2008-08-11

The second WW domain (WW2) of CA150, a human transcriptional activator, forms amyloid fibrils in vitro under physiological conditions. Based on experimental constraints from MAS NMR spectroscopy experiments, alanine scanning and electron microscopy, a structural model of CA150.WW2 amyloid fibrils was calculated earlier. Here, the assignment strategy is presented and suggested as a general approach for proteins that show intermediate line width. The (13)C,(13)C correlation experiments were recorded on fully or partially (13)C-labelled fibrils. The earlier (13)C assignment (26 residues) was extended to 34 of the 40 residues by direct (13)C-excitation experiments by using a deuterated sample that showed strongly improved line width. A 3D HNC-TEDOR (transferred-echo double-resonance) experiment with deuterated CA150.WW2 fibrils yielded 14 amide nitrogen and proton resonance assignments. The obtained chemical shifts were compared with the chemical shifts determined with the natively folded WW domain. TALOS (Torsion angle likelihood obtained from shift and sequence similarity) predictions confirmed that, under physiological conditions, the fibrillar form of CA150.WW2 adopts a significantly different beta structure than the native WW-domain fold.

Protein Polymerization into Fibrils from the Viewpoint of Nucleation Theory.

PubMed

Kashchiev, Dimo

2015-11-17

The assembly of various proteins into fibrillar aggregates is an important phenomenon with wide implications ranging from human disease to nanoscience. Using general kinetic results of nucleation theory, we analyze the polymerization of protein into linear or helical fibrils in the framework of the Oosawa-Kasai (OK) model. We show that while within the original OK model of linear polymerization the process does not involve nucleation, within a modified OK model it is nucleation-mediated. Expressions are derived for the size of the fibril nucleus, the work for fibril formation, the nucleation barrier, the equilibrium and stationary fibril size distributions, and the stationary fibril nucleation rate. Under otherwise equal conditions, this rate decreases considerably when the short (subnucleus) fibrils lose monomers much more frequently than the long (supernucleus) fibrils, a feature that should be born in mind when designing a strategy for stymying or stimulating fibril nucleation. The obtained dependence of the nucleation rate on the concentration of monomeric protein is convenient for experimental verification and for use in rate equations accounting for nucleation-mediated fibril formation. The analysis and the results obtained for linear fibrils are fully applicable to helical fibrils whose formation is describable by a simplified OK model. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Simultaneous determination of the second-harmonic generation emission directionality and reduced scattering coefficient from three-dimensional imaging of thick tissues

PubMed Central

Hall, Gunnsteinn; Eliceiri, Kevin W.

2013-01-01

Abstract. Second-harmonic generation (SHG) microscopy has intrinsic contrast for imaging fibrillar collagen and has shown great promise for disease characterization and diagnostics. In addition to morphology, additional information is achievable as the initially emitted SHG radiation directionality is related to subresolution fibril size and distribution. We show that by two parameter fittings, both the emission pattern (FSHG/BSHG)creation and the reduced scattering coefficient μs′, can be obtained from the best fits between three-dimensional experimental data and Monte Carlo simulations. The improved simulation framework accounts for collection apertures for the detected forward and backward components. We apply the new simulation framework to mouse tail tendon for validation and show that the spectral slope of μs′ obtained is similar to that from bulk optical measurements and that the (FSHG/BSHG)creation values are also similar to previous results. Additionally, we find that the SHG emission becomes increasingly forward directed at longer wavelengths, which is consistent with decreased dispersion in refractive index between the laser and SHG wavelengths. As both the spectral slope of μs′ and (FSHG/BSHG)creation have been linked to the underlying tissue structure, simultaneously obtaining these parameters on a microscope platform from the same tissue provides a powerful method for tissue characterization. PMID:24220726

Neuron-to-neuron transmission of α-synuclein fibrils through axonal transport

PubMed Central

Freundt, Eric C.; Maynard, Nate; Clancy, Eileen K.; Roy, Shyamali; Bousset, Luc; Sourigues, Yannick; Covert, Markus; Melki, Ronald; Kirkegaard, Karla; Brahic, Michel

2012-01-01

Objective The lesions of Parkinson's disease spread through the brain in a characteristic pattern that corresponds to axonal projections. Previous observations suggest that misfolded α-synuclein could behave as a prion, moving from neuron to neuron and causing endogenous α-synuclein to misfold. Here, we characterized and quantified the axonal transport of α-synuclein fibrils and showed that fibrils could be transferred from axons to second-order neurons following anterograde transport. Methods We grew primary cortical mouse neurons in microfluidic devices to separate soma from axonal projections in fluidically isolated microenvironments. We used live-cell imaging and immunofluorescence to characterize the transport of fluorescent α-synuclein fibrils and their transfer to second-order neurons. Results Fibrillar α-synuclein was internalized by primary neurons and transported in axons with kinetics consistent with slow component-b of axonal transport (fast axonal transport with saltatory movement). Fibrillar α-synuclein was readily observed in the cell bodies of second-order neurons following anterograde axonal transport. Axon-to-soma transfer appeared not to require synaptic contacts. Interpretation These results support the hypothesis that the progression of Parkinson's disease can be caused by neuron-to-neuron spread of α-synuclein aggregates and that the anatomical pattern of progression of lesions between axonally connected areas results from the axonal transport of such aggregates. That the transfer did not appear to be transsynaptic gives hope that α-synuclein fibrils could be intercepted by drugs during the extra-cellular phase of their journey. PMID:23109146

Cellular response of human neuroblastoma cells to α-synuclein fibrils, the main constituent of Lewy bodies.

PubMed

Pieri, Laura; Chafey, Philippe; Le Gall, Morgane; Clary, Guilhem; Melki, Ronald; Redeker, Virginie

2016-01-01

α-Synuclein (α-Syn) fibrils are the main constituent of Lewy bodies and a neuropathological hallmark of Parkinson's disease (PD). The propagation of α-Syn assemblies from cell to cell suggests that they are involved in PD progression. We previously showed that α-Syn fibrils are toxic because of their ability to bind and permeabilize cell membranes. Here, we document the cellular response in terms of proteome changes of SH-SY5Y cells exposed to exogenous α-Syn fibrils. We compare the proteomes of cells of neuronal origin exposed or not either to oligomeric or fibrillar α-Syn using two dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Only α-Syn fibrils induce significant changes in the proteome of SH-SY5Y cells. In addition to proteins associated to apoptosis and toxicity, or proteins previously linked to neurodegenerative diseases, we report an overexpression of proteins involved in intracellular vesicle trafficking. We also report a remarkable increase in fibrillar α-Syn heterogeneity, mainly due to C-terminal truncations. Our results show that cells of neuronal origin adapt their proteome to exogenous α-Syn fibrils and actively modify those assemblies. Cells of neuronal origin adapt their proteome to exogenous toxic α-Syn fibrils and actively modify those assemblies. Our results bring insights into the cellular response and clearance events the cells implement to face the propagation of α-Syn assemblies associated to pathology.

Separation of replication and transcription domains in nucleoli.

PubMed

Smirnov, E; Borkovec, J; Kováčik, L; Svidenská, S; Schröfel, A; Skalníková, M; Švindrych, Z; Křížek, P; Ovesný, M; Hagen, G M; Juda, P; Michalová, K; Cardoso, M C; Cmarko, D; Raška, I

2014-12-01

In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate this problem, we measured the correlation of various replication and transcription signals in the nucleoli of HeLa, HT-1080 and NIH 3T3 cells using a specially devised software for analysis of confocal images. Additionally, to follow the relationship between nucleolar replication and transcription in living cells, we produced a stable cell line expressing GFP-RPA43 (subunit of RNA polymerase I, pol I) and RFP-PCNA (the sliding clamp protein) based on human fibrosarcoma HT-1080 cells. We found that replication and transcription signals are more efficiently separated in nucleoli than in the nucleoplasm. In the course of S phase, separation of PCNA and pol I signals gradually increased. During the same period, separation of pol I and incorporated Cy5-dUTP signals decreased. Analysis of single molecule localization microscopy (SMLM) images indicated that transcriptionally active FC/DFC units (i.e. fibrillar centers with adjacent dense fibrillar components) did not incorporate DNA nucleotides. Taken together, our data show that replication of the ribosomal genes is spatially separated from their transcription, and FC/DFC units may provide a structural basis for that separation. Copyright © 2014 Elsevier Inc. All rights reserved.

High-Throughput Image Analysis of Fibrillar Materials: A Case Study on Polymer Nanofiber Packing, Alignment, and Defects in Organic Field Effect Transistors.

PubMed

Persson, Nils E; Rafshoon, Joshua; Naghshpour, Kaylie; Fast, Tony; Chu, Ping-Hsun; McBride, Michael; Risteen, Bailey; Grover, Martha; Reichmanis, Elsa

2017-10-18

High-throughput discovery of process-structure-property relationships in materials through an informatics-enabled empirical approach is an increasingly utilized technique in materials research due to the rapidly expanding availability of data. Here, process-structure-property relationships are extracted for the nucleation, growth, and deposition of semiconducting poly(3-hexylthiophene) (P3HT) nanofibers used in organic field effect transistors, via high-throughput image analysis. This study is performed using an automated image analysis pipeline combining existing open-source software and new algorithms, enabling the rapid evaluation of structural metrics for images of fibrillar materials, including local orientational order, fiber length density, and fiber length distributions. We observe that microfluidic processing leads to fibers that pack with unusually high density, while sonication yields fibers that pack sparsely with low alignment. This is attributed to differences in their crystallization mechanisms. P3HT nanofiber packing during thin film deposition exhibits behavior suggesting that fibers are confined to packing in two-dimensional layers. We find that fiber alignment, a feature correlated with charge carrier mobility, is driven by increasing fiber length, and that shorter fibers tend to segregate to the buried dielectric interface during deposition, creating potentially performance-limiting defects in alignment. Another barrier to perfect alignment is the curvature of P3HT fibers; we propose a mechanistic simulation of fiber growth that reconciles both this curvature and the log-normal distribution of fiber lengths inherent to the fiber populations under consideration.

Intracellular accumulation of a mild-denatured monomer of the human PrP fragment 90-231, as possible mechanism of its neurotoxic effects.

PubMed

Chiovitti, Katia; Corsaro, Alessandro; Thellung, Stefano; Villa, Valentina; Paludi, Domenico; D'Arrigo, Cristina; Russo, Claudio; Perico, Angelo; Ianieri, Adriana; Di Cola, Domenico; Vergara, Alberto; Aceto, Antonio; Florio, Tullio

2007-12-01

Because of high tendency of the prion protein (PrP) to aggregate, the exact PrP isoform responsible for prion diseases as well as the pathological mechanism that it activates remains still controversial. In this study, we show that a pre-fibrillar, monomeric or small oligomeric conformation of the human PrP fragment 90-231 (hPrP90-231), rather than soluble or fibrillar large aggregates, represents the neurotoxic species. In particular, we demonstrate that monomeric mild-denatured hPrP90-231 (incubated for 1 h at 53 degrees C) induces SH-SY5Y neuroblastoma cell death, while, when structured in large aggregates, it is ineffective. Using spectroscopic and cellular techniques we demonstrate that this toxic conformer is characterized by a high exposure of hydrophobic regions that favors the intracellular accumulation of the protein. Inside the cells hPrP90-231 is mainly compartmentalized into the lysosomes where it may trigger pro-apoptotic 'cell death' signals. The PrP toxic conformation, which we have obtained inducing a controlled in vitro conformational change of the protein, might mimic mild-unfolding events occurring in vivo, in the presence of specific mutations, oxidative reactions or proteolysis. Thus, in light of this model, we propose that novel therapeutic strategies, designed to inhibit the interaction of the toxic PrP with the plasmamembrane, could be beneficial to prevent the formation of intracellular neurotoxic aggregates and ultimately the neuronal death.

Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.

PubMed

Soikkeli, Johanna; Podlasz, Piotr; Yin, Miao; Nummela, Pirjo; Jahkola, Tiina; Virolainen, Susanna; Krogerus, Leena; Heikkilä, Päivi; von Smitten, Karl; Saksela, Olli; Hölttä, Erkki

2010-07-01

Although the outgrowth of micrometastases into macrometastases is the rate-limiting step in metastatic progression and the main determinant of cancer fatality, the molecular mechanisms involved have been little studied. Here, we compared the gene expression profiles of melanoma lymph node micro- and macrometastases and unexpectedly found no common up-regulation of any single growth factor/cytokine, except for the cytokine-like SPP1. Importantly, metastatic outgrowth was found to be consistently associated with activation of the transforming growth factor-beta signaling pathway (confirmed by phospho-SMAD2 staining) and concerted up-regulation of POSTN, FN1, COL-I, and VCAN genes-all inducible by transforming growth factor-beta. The encoded extracellular matrix proteins were found to together form intricate fibrillar networks around tumor cell nests in melanoma and breast cancer metastases from various organs. Functional analyses suggested that these newly synthesized protein networks regulate adhesion, migration, and growth of tumor cells, fibroblasts, and endothelial cells. POSTN acted as an anti-adhesive molecule counteracting the adhesive functions of FN1 and COL-I. Further, cellular FN and POSTN were specifically overexpressed in the newly forming/formed tumor blood vessels. Transforming growth factor-beta receptors and the metastasis-related matrix proteins, POSTN and FN1, in particular, may thus provide attractive targets for development of new therapies against disseminated melanoma, breast cancer, and possibly other tumors, by affecting key processes of metastasis: tumor/stromal cell migration, growth, and angiogenesis.

Cell wall of pathogenic yeasts and implications for antimycotic therapy.

PubMed

Cassone, A

1986-01-01

Yeast cell wall is a complex, multilayered structure where amorphous, granular and fibrillar components interact with each other to confer both the specific cell shape and osmotic protection against lysis. Thus it is widely recognized that as is the case with bacteria, yeast cell wall is a major potential target for selective chemotherapeutic drugs. Despite intensive research, very few such drugs have been discovered and none has found substantial application in human diseases to date. Among the different cell wall components, beta-glucan and chitin are the fibrillar materials playing a fundamental role in the overall rigidity and resistance of the wall. Inhibition of the metabolism of these polymers, therefore, should promptly lead to lysis. This indeed occurs and aculeacin, echinocandin and polyoxins are examples of agents producing such an action. Particular attention should be focused on chitin synthesis. Although quantitatively a minor cell wall component, chitin is important in the mechanism of dimorphic transition, especially in Candida albicans, a major human opportunistic pathogen. This transition is associated with increased invasiveness and general virulence of the fungus. Yeast cell wall may also limit the effect of antifungals which owe their action to disturbance of the cytoplasmic membrane or of cell metabolism. Indeed, the cell wall may hinder access to the cell interior both under growing conditions and, particularly, during cell ageing in the stationary phase, when important structural changes occur in the cell wall due to unbalanced wall growth (phenotypic drug resistance).

Treatment of meats with ionising radiations. VIII.—pH, water-binding capacity and proteolysis of irradiated raw beef and pork during storage, and the ATP-ase activity of irradiated rabbit muscle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lawrie, R. A.; Sharp, J. G.; Bendall, J. R.

1961-11-01

The immediate effects of 5-Mrad ionizing radiation on beef and pork longissimus dorsi muscles were an increase in pH, a decrease in water-holding capacity, in increment in gel-volume for a given pH rise, and in soluble protein, and increased resistance to low- and high-speed homogenization. The indications of cross-binding induced by irradiation were supported by studies of isolated myofibrils from rabbit psoas muscle. Irradiation markedly reduced the syneresis (18 deg , mu = 0.04) and the swelling (0 deg , mu = 0.25) induced by ATP and, to a lesser extent, over-all fibrillar ATP-ase activity (the initial fast phase beingmore » depressed more than the slower second phase of the reaction). On storage (at -20 deg +37 deg pH and water-binding capacity increased generally with increase of temperature. Changes in pH occurred earlier with pork and to a greater extent than with beef. In sterile beef longissimus dorsi (irradiated or unirradiated) there was a decrease in soluble protein during storage for 60-90 days at 37- (indicating denaturation) and lncreases in TCA-soluble nitrogen and tyrosine (indicating proteolysis, which was more marked in unirradiated samples). The absence of soluble hydroxyproline and the presence of clearly marked cross- striations indicated that the autolysis must have involved sarcoplasmic and not fibrillar or connective tissue protein.« less

An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated β-sheet structure for amyloid

PubMed Central

Balbirnie, Melinda; Grothe, Robert; Eisenberg, David S.

2001-01-01

X-ray diffraction and other biophysical tools reveal features of the atomic structure of an amyloid-like crystal. Sup35, a prion-like protein in yeast, forms fibrillar amyloid assemblies intrinsic to its prion function. We have identified a polar peptide from the N-terminal prion-determining domain of Sup35 that exhibits the amyloid properties of full-length Sup35, including cooperative kinetics of aggregation, fibril formation, binding of the dye Congo red, and the characteristic cross-β x-ray diffraction pattern. Microcrystals of this peptide also share the principal properties of the fibrillar amyloid, including a highly stable, β-sheet-rich structure and the binding of Congo red. The x-ray powder pattern of the microcrystals, extending to 0.9-Å resolution, yields the unit cell dimensions of the well-ordered structure. These dimensions restrict possible atomic models of this amyloid-like structure and demonstrate that it forms packed, parallel-stranded β-sheets. The unusually high density of the crystals shows that the packed β-sheets are dehydrated, despite the polar character of the side chains. These results suggest that amyloid is a highly intermolecularly bonded, dehydrated array of densely packed β-sheets. This dry β-sheet could form as Sup35 partially unfolds to expose the peptide, permitting it to hydrogen-bond to the same peptide of other Sup35 molecules. The implication is that amyloid-forming units may be short segments of proteins, exposed for interactions by partial unfolding. PMID:11226247

Texture analysis applied to second harmonic generation image data for disease classification and development of a multi-view second harmonic generation imaging platform

NASA Astrophysics Data System (ADS)

Wen, Lianggong

Many diseases, e.g. ovarian cancer, breast cancer and pulmonary fibrosis, are commonly associated with drastic alterations in surrounding connective tissue, and changes in the extracellular matrix (ECM) are associated with the vast majority of cellular processes in disease progression and carcinogenesis: cell differentiation, proliferation, biosynthetic ability, polarity, and motility. We use second harmonic generation (SHG) microscopy for imaging the ECM because it is a non-invasive, non-linear laser scanning technique with high sensitivity and specificity for visualizing fibrillar collagen. In this thesis, we are interested in developing imaging techniques to understand how the ECM, especially the collagen architecture, is remodeled in diseases. To quantitate remodeling, we implement a 3D texture analysis to delineate the collagen fibrillar morphology observed in SHG microscopy images of human normal and high grade malignant ovarian tissues. In the learning stage, a dictionary of "textons"---frequently occurring texture features that are identified by measuring the image response to a filter bank of various shapes, sizes, and orientations---is created. By calculating a representative model based on the texton distribution for each tissue type using a training set of respective mages, we then perform classification between normal and high grade malignant ovarian tissues classification based on the area under receiver operating characteristic curves (true positives versus false positives). The local analysis algorithm is a more general method to probe rapidly changing fibrillar morphologies than global analyses such as FFT. It is also more versatile than other texture approaches as the filter bank can be highly tailored to specific applications (e.g., different disease states) by creating customized libraries based on common image features. Further, we describe the development of a multi-view 3D SHG imaging platform. Unlike fluorescence microscopy, SHG excites intrinsic characteristics of collagen, bypassing the need for additional primary and secondary imaging labels. However, single view image collection from endogenous SHG contrast of collagen molecules is not "a true 3D technique", because collagen fibers oriented along the plane of the lasers used to excite them are invisible to the excitation The loss of information means that researchers cannot resolve the 3D structure of the ECM using this technique. We are developing a new, multi-view approach that involves rotation of agarose embedded sample in FEP tubing, so that the excitation beam path travels to from multiple angles, to reveal new insight in understanding the 3D collagen structure and its role in normal and diseased tissue.

Milk kefir: ultrastructure, antimicrobial activity and efficacy on aflatoxin B1 production by Aspergillus flavus.

PubMed

Ismaiel, Ahmed A; Ghaly, Mohamed F; El-Naggar, Ayman K

2011-05-01

The association of kefir microbiota was observed by electron microscopic examination. Scanning electron microscopic (SEM) observations revealed that kefir grain surface is very rough and the inner portions had scattered irregular holes on its surface. The interior of the grain comprised fibrillar materials which were interpreted as protein, lipid and a soluble polysaccharide, the kefiran complex that surrounds yeast and bacteria in the grain. Yeast was observed more clearly than bacteria on the outer portion of the grain. Transmission electron microscopic (TEM) observations of kefir revealed that the grain comprised a mixed culture of yeast and bacteria growing in close association with each other. Microbiota is dominated by budded and long-flattened yeast cells growing together with lactobacilli and lactococci bacteria. Bacterial cells with rounded ends were also observed in this mixed culture. Kefir grains, kefir suspensions, and kefiran were tested for antimicrobial activities against several bacterial and fungal species. The highest activity was obtained against Streptococcus faecalis KR6 and Fusarium graminearum CZ1. Growth of Aspergillus flavus AH3 producing for aflatoxin B1 for 10 days in broth medium supplemented with varying concentrations of kefir filtrate (%, v/v) showed that sporulation was completely inhibited at the higher concentrations of kefir filtrate (7-10%, v/v). The average values of both mycelial dry weights and aflatoxin B1 were completely inhibited at 10% (v/v). This is the first in vitro study about the antifungal characteristics of kefir against filamentous fungi which was manifested by applying its inhibitory effect on the productivity of aflatoxin B1 by A. flavus AH3.

Structure and function of the elastic organ in the tibia of a tenebrionid beetle

NASA Astrophysics Data System (ADS)

Ichikawa, Toshio; Toh, Yoshihiro; Sakamoto, Hirofumi

2016-06-01

Many insects have a pair of claws on the tip of each foot (tarsus and pretarsus). The movement of the pretarsal claws is mediated by a long apodeme that originates from the claw retractor muscles in the femur. It is generally accepted that the pulling of the apodeme by the muscles flexes the claws to engage with a rough surface of a substrate, and the flexed claws return to their initial position by passive elastic forces within the tarso-pretarsal joint. We found that each tibia of the tenebrionid beetle Zophobas atratus had a chordal elastic organ that tied the apodeme to the distal end of the tibia and assisted the pulled apodeme to return smoothly. The elastic body of the elastic organ consists of a bundle of more than 1000 thin fibrils (0.3-1.5 μm in diameter) with a hairy yarn-shaped structure made by assemblies of intricately interwoven microfibers. Both ends of the fibrillar elastic body were supported by clusters of columnar cells. Ablation of the elastic organ often disturbed the rapid and smooth return of claws from a flexed position when the tarsal segments were forced to curve in order to increase the friction between the apodeme and surrounding tissues in the segments. The result suggests that rapid claw disengagement is an important step in each cycle of leg movements, and the elastic organ may have evolved to assist the reliable detachment of claws that engage tightly with the substrate when climbing or traversing inverted surfaces.

[Collagens: why such a structural complexity?].

PubMed

Borel, J P; Monboisse, J C

1993-01-01

The collagens are a family of extracellular fibrillar proteins, characterized by the presence of one or several domains termed "triple helix", that are made of three polypeptide chains folded around each other. They elicit a huge worldwide research activity, marked every year by the publishing of dozens of books and thousands of papers. This family is presently represented by more than 16 individualized types, all differing by their molecular structure and by the way helical and globular domains are arranged. In any case, however, at least one triple helical domain exists. It is formed by the association of three polypeptide chains, each of them containing a glycine every three residues and many proline or hydroxyproline residues, and attests for the belonging of the protein to the collagen group. These multiple molecular forms and their specific architecture raise questions that remain unsolved. Why is this triple helix structure adopted in the case of collagens? Is it because the simple alpha helix of protein cannot extend over more than a few nanometers and is not solid enough? Why not a double helix like that of DNA? It would probably not be rigid enough. Why are there many globular domains interspersed between fibrillar ones? Probably these domains are useful for the association of peptide chains in register prior to their folding, then they participate in the transport of the elementary molecules from the synthesizing cells to their final place in the connective tissue and, finally, they insert the molecules into their specific place inside the growing fibrils. Collagen fibres as they are evidenced by histological methods, for instance in tendons, are of complex structure. Most of their constituting sub-units are type I tropocollagen molecules but they also contain in their center a filament of type V collagen that seems to serve as a guide during their edification. On the surface of the fibres are molecules of type III collagen that limit the growth in diameter and also type XII molecules that serve to bind the fibres to the surrounding substances. The collagen type multiplicity is explained by their various functions (mechanical role for tendons and ligaments, functions of wrapping around muscle cells, basement membrane role as a support for endothelial cells, function of glomerular filter, etc.). The fact that every collagen type contains several different polypeptide chains remains poorly explained. It may serve for the orientation of every elementary molecule inside the complex array of the polymer.(ABSTRACT TRUNCATED AT 400 WORDS)

Acidic pH retards the fibrillization of human Islet Amyloid Polypeptide due to electrostatic repulsion of histidines.

PubMed

Li, Yang; Xu, Weixin; Mu, Yuguang; Zhang, John Z H

2013-08-07

The human Islet Amyloid Polypeptide (hIAPP) is the major constituent of amyloid deposits in pancreatic islets of type-II diabetes. IAPP is secreted together with insulin from the acidic secretory granules at a low pH of approximately 5.5 to the extracellular environment at a neutral pH. The increased accumulation of extracellular hIAPP in diabetes indicates that changes in pH may promote amyloid formation. To gain insights and underlying mechanisms of the pH effect on hIAPP fibrillogenesis, all-atom molecular dynamics simulations in explicit solvent model were performed to study the structural properties of five hIAPP protofibrillar oligomers, under acidic and neutral pH, respectively. In consistent with experimental findings, simulation results show that acidic pH is not conducive to the structural stability of these oligomers. This provides a direct evidence for a recent experiment [L. Khemtemourian, E. Domenech, J. P. F. Doux, M. C. Koorengevel, and J. A. Killian, J. Am. Chem. Soc. 133, 15598 (2011)], which suggests that acidic pH inhibits the fibril formation of hIAPP. In addition, a complementary coarse-grained simulation shows the repulsive electrostatic interactions among charged His18 residues slow down the dimerization process of hIAPP by twofold. Besides, our all-atom simulations reveal acidic pH mainly affects the local structure around residue His18 by destroying the surrounding hydrogen-bonding network, due to the repulsive interactions between protonated interchain His18 residues at acidic pH. It is also disclosed that the local interactions nearby His18 operating between adjacent β-strands trigger the structural transition, which gives hints to the experimental findings that the rate of hIAPP fibril formation and the morphologies of the fibrillar structures are strongly pH-dependent.

Near Net-Shape, Ultra High Melting, Erosion Resistant Carbide/Metal Composites with Tailored Fibrillar Microstructures via the Displacive Compensation of Porosity Process

DTIC Science & Technology

2006-11-26

vapor species, formed over tungsten trioxide powder, is 1.25xl0Ŗ atm at 1400°C and 1 atm total pressure (assuming an oxygen partial pressure greater...with CO(g). ■19- These hollow tungsten fibers were then carburized via reaction with CO(g) to generate the polycrystalline WC-based fibers shown in...of tungsten carbide via reaction with a hafnium-copper melt," Ada Mater., 57(13), 3924-3931 (2009).) The kinetic mechanism of incongruent reduction

Fabrication of ZnS nanoparticle chains on a protein template

PubMed Central

Hulleman, J.; Kim, S. M.; Tumkur, T.; Rochet, J.-C.; Stach, E.; Stanciu, L.

2011-01-01

In the present study, we have exploited the properties of a fibrillar protein for the template synthesis of zinc sulfide (ZnS) nanoparticle chains. The diameter of the ZnS nanoparticle chains was tuned in range of ~30 to ~165 nm by varying the process variables. The nanoparticle chains were characterized by field emission scanning electron microscopy, UV–Visible spectroscopy, transmission electron microscopy, electron energy loss spectroscopy, and high-resolution transmission electron microscopy. The effect of incubation temperature on the morphology of the nanoparticle chains was also studied. PMID:21804765

Major review: Exfoliation syndrome; advances in disease genetics, molecular biology, and epidemiology.

PubMed

Aboobakar, Inas F; Johnson, William M; Stamer, W Daniel; Hauser, Michael A; Allingham, R Rand

2017-01-01

Exfoliation syndrome (XFS) is a common age-related disorder that leads to deposition of extracellular fibrillar material throughout the body. The most recognized disease manifestation is exfoliation glaucoma (XFG), which is a common cause of blindness worldwide. Recent developments in XFS genetics, cell biology and epidemiology have greatly improved our understanding of the etiology of this complex inherited disease. This review summarizes current knowledge of XFS pathogenesis, identifies gaps in knowledge, and discusses areas for future research. Copyright © 2016. Published by Elsevier Ltd.

Role of PAMAM-OH dendrimers against the fibrillation pathway of biomolecules.

PubMed

Sekar, Gajalakshmi; Florance, Ida; Sivakumar, A; Mukherjee, Amitava; Chandrasekaran, Natarajan

2016-12-01

The binding behavior of nanoparticle with proteins determines its biocompatibility. This study reports the interaction of ten different biomolecules (proteins-BSA, HSA, haemoglobin, gamma globulin, transferrin and enzymes-hog and bacillus amylase, lysozyme from chicken and human and laccases from Tramates versicolor) with a surface group hydroxylated Poly AMido AMide dendrimer (PAMAM) of generation 5. The study has utilized various spectroscopic methods like UV-vis spectroscopy, Fluorescence emission, Synchronous, 3-D spectroscopy and Circular Dichroism to detect the binding induced structural changes in biomolecules that occur upon interaction with mounting concentration of the dendrimers. Aggregation of proteins results in the formation of amyloid fibrils causing several human diseases. In this study, fibrillar samples of all ten biomolecules formed in the absence and the presence of dendrimers were investigated with Congo Red absorbance and ThT Assay to detect fibril formation, Trp Emission and 3-D scan to evaluate the effect of fibrillation on aromatic environment of biomolecules, and CD spectroscopy to measure the conformational changes in a quantitative manner. These assays have generated useful information on the role of dendrimers in amyloid fibril formation of biomolecules. The outcomes of the study remain valuable in evaluating the biological safety of PAMAM-OH dendrimers for their biomedical application in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

Ectodermal fragments from normal frog gastrulae condition substrata to support normal and hybrid mesodermal cell migration in vitro.

PubMed

Nakatsuji, N; Johnson, K E

1984-06-01

Using time-lapse cinemicrography and scanning electron microscopy, we have shown that normal Rana embryos and gastrulating hybrid embryos have extracellular fibrils on the inner surface of the ectodermal layer. These fibrils are absent prior to gastrulation and appear in increasing numbers during gastrulation. They can also be deposited in vitro where they condition substrata in such a way that normal presumptive mesodermal cells placed on them show extensive attachment and unoriented cell movement. These fibrils are also present in some arrested hybrid embryos, but in reduced numbers, or are lacking in other arrested hybrid embryos. Explanted ectodermal fragments from arrested hybrid embryos fail both to condition culture substrata by the deposition of fibrils and to promote cell attachment and translocation. In contrast, ectodermal fragments from normal embryos can condition culture substrata so as to promote moderate cell attachment and, for one particular gamete combination, even cell translocation of presumptive mesodermal cells taken from arrested hybrid embryos. These results provide new evidence to support the hypothesis that extracellular fibrils represent a system that promotes mesodermal cell migration in amphibian embryos. Differences in the fibrillar system in urodele and anuran embryos are discussed in relation to fundamental differences in the mode of mesodermal cell migration in these two classes of Amphibia.

Functionality-Oriented Derivatization of Naphthalene Diimide: A Molecular Gel Strategy-Based Fluorescent Film for Aniline Vapor Detection.

PubMed

Fan, Jiayun; Chang, Xingmao; He, Meixia; Shang, Congdi; Wang, Gang; Yin, Shiwei; Peng, Haonan; Fang, Yu

2016-07-20

Modification of naphthalene diimide (NDI) resulted in a photochemically stable, fluorescent 3,4,5-tris(dodecyloxy)benzamide derivative of NDI (TDBNDI), and introduction of the long alkyl chains endowed the compound with good compatibility with commonly found organic solvents and in particular superior self-assembly in the solution state. Further studies revealed that TDBNDI forms gels with nine of the 18 solvents tested at a concentration of 2.0% (w/v), and the critical gelation concentrations of five of the eight gels are lower than 1.0% (w/v), indicating the high efficiency of the compound as a low-molecular mass gelator (LMMG). Transmission electron microscopy, scanning electron microscopy, and confocal laser scanning microscopy studies revealed the networked fibrillar structure of the TDBNDI/methylcyclohexane (MCH) gel. On the basis of these findings, a fluorescent film was developed via simple spin-coating of the TDBNDI/MCH gel on a glass substrate surface. Fluorescence behavior and sensing performance studies demonstrated that this film is photochemically stable, and sensitive and selective to the presence of aniline vapor. Notably, the response is instantaneous, and the sensing process is fully and quickly reversible. This case study demonstrates that derivatization of photochemically stable fluorophores into LMMGs is a good strategy for developing high-performance fluorescent sensing films.

Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro

NASA Astrophysics Data System (ADS)

Tamaddon, M.; Burrows, M.; Ferreira, S. A.; Dazzi, F.; Apperley, J. F.; Bradshaw, A.; Brand, D. D.; Czernuszka, J.; Gentleman, E.

2017-03-01

Osteoarthritis (OA) is a common cause of pain and disability and is often associated with the degeneration of articular cartilage. Lesions to the articular surface, which are thought to progress to OA, have the potential to be repaired using tissue engineering strategies; however, it remains challenging to instruct cell differentiation within a scaffold to produce tissue with appropriate structural, chemical and mechanical properties. We aimed to address this by driving progenitor cells to adopt a chondrogenic phenotype through the tailoring of scaffold composition and physical properties. Monomeric type-I and type-II collagen scaffolds, which avoid potential immunogenicity associated with fibrillar collagens, were fabricated with and without chondroitin sulfate (CS) and their ability to stimulate the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells was assessed. Immunohistochemical analyses showed that cells produced abundant collagen type-II on type-II scaffolds and collagen type-I on type-I scaffolds. Gene expression analyses indicated that the addition of CS - which was released from scaffolds quickly - significantly upregulated expression of type II collagen, compared to type-I and pure type-II scaffolds. We conclude that collagen type-II and CS can be used to promote a more chondrogenic phenotype in the absence of growth factors, potentially providing an eventual therapy to prevent OA.

Cells Recognize and Prefer Bone-like Hydroxyapatite: Biochemical Understanding of Ultrathin Mineral Platelets in Bone.

PubMed

Liu, Cuilian; Zhai, Halei; Zhang, Zhisen; Li, Yaling; Xu, Xurong; Tang, Ruikang

2016-11-09

Hydroxyapatite (HAP) nanocrystallites in all types of bones are distinguished by their ultrathin characteristics, which are uniaxially oriented with fibrillar collagen to uniquely expose the (100) faces. We speculate that living organisms prefer the specific crystal morphology and orientation of HAP because of the interactions between cells and crystals at the mineral-cell interface. Here, bone-like platy HAP (p-HAP) and two different rod-like HAPs were synthesized to investigate the ultrathin mineral modulating effect on cell bioactivity and bone generation. Cell viability and osteogenic differentiation of mesenchymal stem cells (MSCs) were significantly promoted by the platy HAP with (100) faces compared to rod-like HAPs with (001) faces as the dominant crystal orientation, which indicated that MSCs can recognize the crystal face and prefer the (100) HAP faces. This face-specific preference is dependent on the selective adsorption of fibronectin (FN), a plasma protein that plays a central role in cell adhesion, on the HAP surface. This selective adsorption is further confirmed by molecule dynamics (MD) simulation. Our results demonstrate that it is an intelligent choice for cells to use ultrathin HAP with a large (100) face as a basic building block in the hierarchical structure of bone, which is crucial to the promotion of MSCs osteoinductions during bone formation.

Advanced gecko-foot-mimetic dry adhesives based on carbon nanotubes.

PubMed

Hu, Shihao; Xia, Zhenhai; Dai, Liming

2013-01-21

Geckos can run freely on vertical walls and even ceilings. Recent studies have discovered that gecko's extraordinary climbing ability comes from a remarkable design of nature with nanoscale beta-keratin elastic hairs on their feet and toes, which collectively generate sufficiently strong van der Waals force to hold the animal onto an opposing surface while at the same time disengaging at will. Vertically aligned carbon nanotube (VA-CNT) arrays, resembling gecko's adhesive foot hairs with additional superior mechanical, chemical and electrical properties, have been demonstrated to be a promising candidate for advanced fibrillar dry adhesives. The VA-CNT arrays with tailor-made hierarchical structures can be patterned and/or transferred onto various flexible substrates, including responsive polymers. This, together with recent advances in nanofabrication techniques, could offer 'smart' dry adhesives for various potential applications, even where traditional adhesives cannot be used. A detailed understanding of the underlying mechanisms governing the material properties and adhesion performances is critical to the design and fabrication of gecko inspired CNT dry adhesives of practical significance. In this feature article, we present an overview of recent progress in both fundamental and applied frontiers for the development of CNT-based adhesives by summarizing important studies in this exciting field, including our own work.

Vitronectin as a Micromanager of Cell Response in Material-Driven Fibronectin Nanonetworks.

PubMed

Cantini, Marco; Gomide, Karina; Moulisova, Vladimira; González-García, Cristina; Salmerón-Sánchez, Manuel

2017-09-01

Surface functionalization strategies of synthetic materials for regenerative medicine applications comprise the development of microenvironments that recapitulate the physical and biochemical cues of physiological extracellular matrices. In this context, material-driven fibronectin (FN) nanonetworks obtained from the adsorption of the protein on poly(ethyl acrylate) provide a robust system to control cell behavior, particularly to enhance differentiation. This study aims at augmenting the complexity of these fibrillar matrices by introducing vitronectin, a lower-molecular-weight multifunctional glycoprotein and main adhesive component of serum. A cooperative effect during co-adsorption of the proteins is observed, as the addition of vitronectin leads to increased fibronectin adsorption, improved fibril formation, and enhanced vitronectin exposure. The mobility of the protein at the material interface increases, and this, in turn, facilitates the reorganization of the adsorbed FN by cells. Furthermore, the interplay between interface mobility and engagement of vitronectin receptors controls the level of cell fusion and the degree of cell differentiation. Ultimately, this work reveals that substrate-induced protein interfaces resulting from the cooperative adsorption of fibronectin and vitronectin fine-tune cell behavior, as vitronectin micromanages the local properties of the microenvironment and consequently short-term cell response to the protein interface and higher order cellular functions such as differentiation.

Advanced gecko-foot-mimetic dry adhesives based on carbon nanotubes

NASA Astrophysics Data System (ADS)

Hu, Shihao; Xia, Zhenhai; Dai, Liming

2012-12-01

Geckos can run freely on vertical walls and even ceilings. Recent studies have discovered that gecko's extraordinary climbing ability comes from a remarkable design of nature with nanoscale beta-keratin elastic hairs on their feet and toes, which collectively generate sufficiently strong van der Waals force to hold the animal onto an opposing surface while at the same time disengaging at will. Vertically aligned carbon nanotube (VA-CNT) arrays, resembling gecko's adhesive foot hairs with additional superior mechanical, chemical and electrical properties, have been demonstrated to be a promising candidate for advanced fibrillar dry adhesives. The VA-CNT arrays with tailor-made hierarchical structures can be patterned and/or transferred onto various flexible substrates, including responsive polymers. This, together with recent advances in nanofabrication techniques, could offer `smart' dry adhesives for various potential applications, even where traditional adhesives cannot be used. A detailed understanding of the underlying mechanisms governing the material properties and adhesion performances is critical to the design and fabrication of gecko inspired CNT dry adhesives of practical significance. In this feature article, we present an overview of recent progress in both fundamental and applied frontiers for the development of CNT-based adhesives by summarizing important studies in this exciting field, including our own work.

Fine structure of the pecten oculi in the great horned owl (Bubo virginianus).

PubMed

Braekevelt, C R

1993-01-01

The pecten oculi of the great horned owl (Bubo virginianus) has been examined by light and electron microscopy. The pecten in this species is of the pleated type and is small in comparison to the size of the eyeball. It consists of 7-8 accordion folds which are joined apically by a pigmented bridge of tissue. Within each fold are numerous capillaries, larger supply and drainage vessels and plentiful pleomorphic melanocytes. The capillaries are extremely specialized vessels, most of which display plentiful microfolds on both their luminal and abluminal surfaces although some capillaries show but a few microfolds. The endothelial cell bodies are extremely thin with most organelles located near the nucleus. All capillaries are surrounded by a thick fibrillar basal lamina which is felt to be structurally important. Pericytes are a common feature within these thickened basal laminae. The numerous melanocytes form an incomplete sheath around the capillaries and are also presumed to be fulfilling a structural role. While the morphology of the pecten in the great horned owl is certainly indicative of a heavy involvement in transport, when compared to the pecten in species that are more visually oriented it is smaller, displays fewer folds and a reduced number of microfolds within the capillaries.

Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis.

PubMed

Chapman, Mark A; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David; Lieber, Richard L

2017-02-01

Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173-183, 2009; Kjaer M. Physiol Rev 84: 649-98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins-fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell. Copyright © 2017 the American Physiological Society.

Carotid artery mechanical properties and stresses quantified using in vivo data from normotensive and hypertensive humans.

PubMed

Masson, Ingrid; Beaussier, Hélène; Boutouyrie, Pierre; Laurent, Stéphane; Humphrey, Jay D; Zidi, Mustapha

2011-12-01

The goal of this study was to model the in vivo non-linear mechanical behavior of human common carotid arteries (CCAs) and then to compare wall stresses and associated contributions of micro-constituents in normotensive (NT) and treated hypertensive (HT) subjects. We used an established theoretical model of 3D arterial mechanics that assumes a hyperelastic, anisotropic, active-passive, and residually stressed wall. In vivo data were obtained non-invasively from CCAs in 16 NT (21-64 years old) and 25 treated HT (44-69 years old) subjects. The associated quasi-static boundary value problem was solved semi-analytically over a cardiac cycle while accounting for surrounding perivascular tissue. Best-fit values of model parameters, including those describing contributions by intramural elastin, fibrillar collagen, and vascular smooth muscle, were estimated by a non-linear least-squares method. The model (1) captured temporal changes in intraluminal pressure, (2) estimated wall stress fields that appeared to reflect the presence or absence of age and disease, and (3) suggested changes in mechanical characteristics of wall micro-constituents despite medical treatment of hypertension. For example, age was positively correlated with residual stresses and altered fibrillar collagen in NT subjects, which indirectly validated the modeling, and HT subjects had higher levels of stresses, increased smooth muscle tone, and a stiffer elastin-dominated matrix despite treatment. These results are consistent with prior reports on effects of age and hypertension, but provide increased insight into evolving contributions of cell and matrix mechanics to arterial behavior in vivo.

Pathogenesis of rhegmatogenous retinal detachment: predisposing anatomy and cell biology.

PubMed

Mitry, Danny; Fleck, Brian W; Wright, Alan F; Campbell, Harry; Charteris, David G

2010-01-01

The pathogenesis of rhegmatogenous retinal detachment is complex, and our knowledge of the exact mechanism of vitreoretinal attachment and detachment remains incomplete. We performed a Medline, Ovid, and EMBASE search using search words rhegmatogenous, retinal detachment, vitreous, and retinal adhesion. All appropriate articles were reviewed, and the evidence was compiled. Cortical vitreous contains fibrillar collagens type II, V/XI, and IX. The inner limiting membrane of the retina contains collagens type I, IV, VI, and XVIII as well as numerous other glycoproteins and potential adhesion molecules. The distribution and age-related changes in the structure of these molecules play an important role in the formation of a retinal break, which may compromise and disrupt the normal mechanisms of neurosensory retinal adhesion. Rhegmatogenous retinal detachment development is intimately related to changes in the fibrillar structure of the aging vitreous culminating in posterior vitreous detachment with regions of persistent and tangential vitreoretinal traction predisposing to retinal tear formation. A complex interplay of factors such as weakening of vitreoretinal adhesion, posterior migration of the vitreous base, and molecular changes at the vitreoretinal interface are important in predisposing to focal areas of vitreoretinal traction precipitating rhegmatogenous retinal detachment. Once formed, the passage of liquefied vitreous through a retinal break may overwhelm normal neurosensory-retinal pigment epithelium adhesion perpetuating and extending detachment and causing visual loss. To understand the molecular events underlying rhegmatogenous retinal detachment so that new therapies can be developed, it is important to appreciate the structural organization of the vitreous, the biology underlying vitreous liquefaction and posterior vitreous detachment, and the mechanisms of vitreoretinal attachment and detachment.

Blueberry Opposes β-Amyloid Peptide-Induced Microglial Activation Via Inhibition of p44/42 Mitogen-Activation Protein Kinase

PubMed Central

Zhu, Yuyan; Bickford, Paula C.; Sanberg, Paul; Giunta, Brian

2008-01-01

Abstract Alzheimer's Disease (AD) is the most common age-related dementia, with a current prevalence in excess of five million individuals in the United States. The aggregation of amyloid-beta (Aβ) into fibrillar amyloid plaques is a key pathological event in the development of the disease. Microglial proinflammatory activation is widely known to cause neuronal and synaptic damage that correlates with cognitive impairment in AD. However, current pharmacological attempts at reducing neuroinflammation mediated via microglial activation have been largely negative in terms of slowing AD progression. Previously, we have shown that microglia express proinflammatory cytokines and a reduced capacity to phagocytose Aβ in the context of CD40, Aβ peptides and/or lipopolysaccharide (LPS) stimulation, a phenomenon that can be opposed by attenuation of p44/42 mitogen-activated protein kinase (MAPK) signaling. Other groups have found that blueberry (BB) extract both inhibits phosphorylation of this MAPK module and also improves cognitive deficits in AD model mice. Given these considerations and the lack of reduced Aβ quantities in behaviorally improved BB-fed mice, we wished to determine whether BB supplementation would alter the microglial proinflammatory activation state in response to Aβ. We found that BB significantly enhances microglial clearance of Aβ, inhibits aggregation of Aβ1–42, and suppresses microglial activation, all via suppression of the p44/42 MAPK module. Thus, these data may explain the previously observed behavioral recovery in PSAPP mice and suggest a means by which dietary supplementation could mitigate an undesirable microglial response toward fibrillar Aβ. PMID:18789000

Second harmonic generation quantitative measurements on collagen fibrils through correlation to electron microscopy

NASA Astrophysics Data System (ADS)

Bancelin, S.; Aimé, C.; Gusachenko, I.; Kowalczuk, L.; Latour, G.; Coradin, T.; Schanne-Klein, M.-C.

2015-03-01

Type I collagen is a major structural protein in mammals that shows highly structured macromolecular organizations specific to each tissue. This biopolymer is synthesized as triple helices, which self-assemble into fibrils (Ø =10-300 nm) and further form various 3D organization. In recent years, Second Harmonic Generation (SHG) microscopy has emerged as a powerful technique to probe in situ the fibrillar collagenous network within tissues. However, this optical technique cannot resolve most of the fibrils and is a coherent process, which has impeded quantitative measurements of the fibril diameter so far. In this study, we correlated SHG microscopy with Transmission Electron Microscopy to determine the sensitivity of SHG microscopy and to calibrate SHG signals as a function of the fibril diameter in reconstructed collagen gels. To that end, we synthetized isolated fibrils with various diameters and successfully imaged the very same fibrils with both techniques, down to 30 nm diameter. We observed that SHG signals scaled as the fourth power of the fibril diameter, as expected from analytical and numerical calculations. This calibration was then applied to diabetic rat cornea in which we successfully recovered the diameter of hyperglycemia-induced fibrils in the Descemet's membrane without having to resolve them. Finally we derived the first hyperpolarizability from a single collagen triple helix which validates the bottom-up approach used to calculate the non-linear response at the fibrillar scale and denotes a parallel alignment of triple helices within the fibrils. These results represent a major step towards quantitative SHG imaging of nm-sized collagen fibrils.

Identification of Histological Patterns in Clinically Affected and Unaffected Palm Regions in Dupuytren's Disease

PubMed Central

Alfonso-Rodríguez, Camilo-Andrés; Garzón, Ingrid; Garrido-Gómez, Juan; Oliveira, Ana-Celeste-Ximenes; Martín-Piedra, Miguel-Ángel; Scionti, Giuseppe; Carriel, Víctor; Hernández-Cortés, Pedro; Campos, Antonio; Alaminos, Miguel

2014-01-01

Dupuytren's disease is a fibro-proliferative disease characterized by a disorder of the extracellular matrix (ECM) and high myofibroblast proliferation. However, studies failed to determine if the whole palm fascia is affected by the disease. The objective of this study was to analyze several components of the extracellular matrix of three types of tissues—Dupuytren's diseased contracture cords (DDC), palmar fascia clinically unaffected by Dupuytren's disease contracture (NPF), and normal forehand fascia (NFF). Histological analysis, quantification of cells recultured from each type of tissue, mRNA microarrays and immunohistochemistry for smooth muscle actin (SMA), fibrillar ECM components and non-fibrillar ECM components were carried out. The results showed that DDC samples had abundant fibrosis with reticular fibers and few elastic fibers, high cell proliferation and myofibroblasts, laminin and glycoproteins, whereas NFF did not show any of these findings. Interestingly, NPF tissues had more cells showing myofibroblasts differentiation and more collagen and reticular fibers, laminin and glycoproteins than NFF, although at lower level than DDC, with similar elastic fibers than DDC. Immunohistochemical expression of decorin was high in DDC, whereas versican was highly expressed NFF, with no differences for aggrecan. Cluster analysis revealed that the global expression profile of NPF was very similar to DDC, and reculturing methods showed that cells corresponding to DDC tissues proliferated more actively than NPF, and NPF more actively than NFF. All these results suggest that NPF tissues may be affected, and that a modification of the therapeutic approach used for the treatment of Dupuytren's disease should be considered. PMID:25379672

Identification of histological patterns in clinically affected and unaffected palm regions in dupuytren's disease.

PubMed

Alfonso-Rodríguez, Camilo-Andrés; Garzón, Ingrid; Garrido-Gómez, Juan; Oliveira, Ana-Celeste-Ximenes; Martín-Piedra, Miguel-Ángel; Scionti, Giuseppe; Carriel, Víctor; Hernández-Cortés, Pedro; Campos, Antonio; Alaminos, Miguel

2014-01-01

Dupuytren's disease is a fibro-proliferative disease characterized by a disorder of the extracellular matrix (ECM) and high myofibroblast proliferation. However, studies failed to determine if the whole palm fascia is affected by the disease. The objective of this study was to analyze several components of the extracellular matrix of three types of tissues-Dupuytren's diseased contracture cords (DDC), palmar fascia clinically unaffected by Dupuytren's disease contracture (NPF), and normal forehand fascia (NFF). Histological analysis, quantification of cells recultured from each type of tissue, mRNA microarrays and immunohistochemistry for smooth muscle actin (SMA), fibrillar ECM components and non-fibrillar ECM components were carried out. The results showed that DDC samples had abundant fibrosis with reticular fibers and few elastic fibers, high cell proliferation and myofibroblasts, laminin and glycoproteins, whereas NFF did not show any of these findings. Interestingly, NPF tissues had more cells showing myofibroblasts differentiation and more collagen and reticular fibers, laminin and glycoproteins than NFF, although at lower level than DDC, with similar elastic fibers than DDC. Immunohistochemical expression of decorin was high in DDC, whereas versican was highly expressed NFF, with no differences for aggrecan. Cluster analysis revealed that the global expression profile of NPF was very similar to DDC, and reculturing methods showed that cells corresponding to DDC tissues proliferated more actively than NPF, and NPF more actively than NFF. All these results suggest that NPF tissues may be affected, and that a modification of the therapeutic approach used for the treatment of Dupuytren's disease should be considered.

Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis

PubMed Central

Chapman, Mark A.; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David

2017-01-01

Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173–183, 2009; Kjaer M. Physiol Rev 84: 649–98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins—fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell. PMID:27881411

The C-terminus hot spot region helps in the fibril formation of bacteriophage-associated hyaluronate lyase (HylP2).

PubMed

Shukla, Harish; Singh, Sudhir Kumar; Singh, Amit Kumar; Mitra, Kalyan; Akhtar, Md Sohail

2015-09-23

The bacteriophage encoded hyaluronate lyases (HylP and HylP2) degrade hyaluronan and other glycosaminoglycans. HylP2 forms a functional fibril under acidic conditions in which its N-terminus is proposed to form the fibrillar core, leading to nucleation and acceleration of fibril formation. Here we report the presence of a hot spot region (A144GVVVY149) towards the carboxy terminus of HylP2, essential for the acceleration of fibril formation. The 'hot spot' is observed to be inherently mutated for valines (A178AMVMY183) in case of HylP. The N- terminal swapped chimeras between these phage HLs ((N)HylP2(C)HylP and (N)HylP(C)HylP2) or HylP did not form fibrils at acidic pH. However, seeding of prefibrils of HylP2 recompensed nucleation and led to fibrillation in (N)HylP(C)HylP2. The V147A mutation in the 'hot spot' region abolished fibril formation in HylP2. The M179V and M181V double mutations in the 'hot spot' region of HylP led to fibrillation with the seeding of prefibrils. It appears that fibrillation in HylP2 even though is initiated by the N-terminus, is accelerated by the conserved 'hot spot' region in the C-terminus. A collagenous (Gly-X-Y)10 motif in the N-terminus and a mutated 'hot spot' region in the C-terminus of HylP affect fibrillar nucleation and acceleration respectively.

The C-terminus hot spot region helps in the fibril formation of bacteriophage-associated hyaluronate lyase (HylP2)

PubMed Central

Shukla, Harish; Singh, Sudhir Kumar; Singh, Amit Kumar; Mitra, Kalyan; Akhtar, Md. Sohail

2015-01-01

The bacteriophage encoded hyaluronate lyases (HylP and HylP2) degrade hyaluronan and other glycosaminoglycans. HylP2 forms a functional fibril under acidic conditions in which its N-terminus is proposed to form the fibrillar core, leading to nucleation and acceleration of fibril formation. Here we report the presence of a hot spot region (A144GVVVY149) towards the carboxy terminus of HylP2, essential for the acceleration of fibril formation. The ‘hot spot’ is observed to be inherently mutated for valines (A178AMVMY183) in case of HylP. The N- terminal swapped chimeras between these phage HLs (NHylP2CHylP and NHylPCHylP2) or HylP did not form fibrils at acidic pH. However, seeding of prefibrils of HylP2 recompensed nucleation and led to fibrillation in NHylPCHylP2. The V147A mutation in the ‘hot spot’ region abolished fibril formation in HylP2. The M179V and M181V double mutations in the ‘hot spot’ region of HylP led to fibrillation with the seeding of prefibrils. It appears that fibrillation in HylP2 even though is initiated by the N-terminus, is accelerated by the conserved ‘hot spot’ region in the C-terminus. A collagenous (Gly-X-Y)10 motif in the N-terminus and a mutated ‘hot spot’ region in the C-terminus of HylP affect fibrillar nucleation and acceleration respectively. PMID:26395159

Components, structure, biogenesis and function of the Hydra extracellular matrix in regeneration, pattern formation and cell differentiation.

PubMed

Sarras, Michael P

2012-01-01

The body wall of Hydra is organized as an epithelial bilayer (ectoderm and endoderm) with an intervening extracellular matrix (ECM), termed mesoglea by early biologists. Morphological studies have determined that Hydra ECM is composed of two basal lamina layers positioned at the base of each epithelial layer with an intervening interstitial matrix. Molecular and biochemical analyses of Hydra ECM have established that it contains components similar to those seen in more complicated vertebrate species. These components include such macromolecules as laminin, type IV collagen, and various fibrillar collagens. These components are synthesized in a complicated manner involving cross-talk between the epithelial bilayer. Any perturbation to ECM biogenesis leads to a blockage in Hydra morphogenesis. Blockage in ECM/cell interactions in the adult polyp also leads to problems in epithelial transdifferentiation processes. In terms of biophysical parameters, Hydra ECM is highly flexible; a property that facilitates continuous movements along the organism's longitudinal and radial axis. This is in contrast to the more rigid matrices often found in vertebrates. The flexible nature of Hydra ECM can in part now be explained by the unique structure of the organism's type IV collagen and fibrillar collagens. This review will focus on Hydra ECM in regard to: 1) its general structure, 2) its molecular composition, 3) the biophysical basis for the flexible nature of Hydra's ECM, 4) the relationship of the biogenesis of Hydra ECM to regeneration of body form, and 5) the functional role of Hydra ECM during pattern formation and cell differentiation.

Advanced septa size quantitation determines the evaluation of histological fibrosis outcome in chronic hepatitis B patients.

PubMed

Wang, Bingqiong; Sun, Yameng; Zhou, Jialing; Wu, Xiaoning; Chen, Shuyan; Wu, Shanshan; Liu, Hui; Wang, Tailing; Ou, Xiaojuan; Jia, Jidong; You, Hong

2018-05-21

Hepatitis B (HBV)-related fibrosis can be reversed after effective antiviral therapy. However, detailed changes of collagen characteristics during fibrosis regression remain unclear. Paired biopsy samples obtained from chronic hepatitis B patients were imaged with second harmonic generation/two photon excitation fluorescence (SHG/TPEF)-based microscopy to identify and quantify collagen features in portal, septal, and fibrillar areas. According to the changes of Ishak stage and qFibrosis score, a total of 117 patients with paired liver biopsy appeared to have four different outcomes after 78-week antiviral therapy: fast reverse (9%), reverse (63%), stable (15%), or progress (13%) on fibrosis. Among 71 collagen features identified by SHG/TPEF analysis, the most prominent fibrosis reversion occurred in the "septal" area, followed by the "fibrillar" area, but not in the "portal" area (P < 0.001). Further analysis of 1060 individual septa identified four parameters that correlated with fibrosis reversion: average width, maximum width, number of fibers, and number of cross-link fibers (P < 0.001). Average septal width was independently associated with regressive septa (odds ratio (OR) = 5.22, 95% confidence interval (CI): 4.17-6.53; P < 0.001), with an AUROC of 0.96 (95% CI: 0.95-0.97). The threshold used to discriminate reversal of fibrosis was 30 μm. In conclusion, septal collagen was determined to be the most useful histological feature for evaluation of dynamic changes in liver fibrosis. Septal width was the most predictive indicator of prognosis in liver fibrosis.

Glycerolipid Headgroups Control Rate and Mechanism of Superoxide Dismutase-1 Aggregation and Accelerate Fibrillization of Slowly Aggregating Amyotrophic Lateral Sclerosis Mutants.

PubMed

Rasouli, Sanaz; Abdolvahabi, Alireza; Croom, Corbin M; Plewman, Devon L; Shi, Yunhua; Shaw, Bryan F

2018-04-20

Interactions between superoxide dismutase-1 (SOD1) and lipid membranes might be directly involved in the toxicity and intercellular propagation of aggregated SOD1 in amyotrophic lateral sclerosis (ALS), but the chemical details of lipid-SOD1 interactions and their effects on SOD1 aggregation remain unclear. This paper determined the rate and mechanism of nucleation of fibrillar apo-SOD1 catalyzed by liposomal surfaces with identical hydrophobic chains (RCH 2 (O 2 C 18 H 33 ) 2 ), but headgroups of different net charge and hydrophobicity (i.e., R(CH 2 )N + (CH 3 ) 3 , RPO 4 - (CH 2 ) 2 N + (CH 3 ) 3 , and RPO 4 - ). Under semiquiescent conditions (within a 96 well microplate, without a gyrating bead), the aggregation of apo-SOD1 into thioflavin-T-positive (ThT(+)) amyloid fibrils did not occur over 120 h in the absence of liposomal surfaces. Anionic liposomes triggered aggregation of apo-SOD1 into ThT(+) amyloid fibrils; cationic liposomes catalyzed fibrillization but at slower rates and across a narrower lipid concentration; zwitterionic liposomes produced nonfibrillar (amorphous) aggregates. The inability of zwitterionic liposomes to catalyze fibrillization and the dependence of fibrillization rate on anionic lipid concentration suggests that membranes catalyze SOD1 fibrillization by a primary nucleation mechanism. Membrane-catalyzed fibrillization was also examined for eight ALS variants of apo-SOD1, including G37R, G93R, D90A, and E100G apo-SOD1 that nucleate slower than or equal to WT SOD1 in lipid-free, nonquiescent amyloid assays. All ALS variants (with one exception) nucleated faster than WT SOD1 in the presence of anionic liposomes, wherein the greatest acceleratory effects were observed among variants with lower net negative surface charge (G37R, G93R, D90A, E100G). The exception was H46R apo-SOD1, which did not form ThT(+) species.

Computational Selection of Inhibitors of A-beta Aggregation and Neuronal Toxicity

PubMed Central

Chen, Deliang; Martin, Zane S.; Soto, Claudio; Schein, Catherine H.

2009-01-01

Alzheimer’s Disease (AD) is characterized by the cerebral accumulation of misfolded and aggregated amyloid-β protein (Aβ). Disease symptoms can be alleviated, in vitro and in vivo, by “β-sheet breaker” pentapeptides that reduce plaque volume. However the peptide nature of these compounds, made them biologically unstable and unable to penetrate membranes with high efficiency. The main goal of this study was to use computational methods to identify small molecule mimetics with better drug-like properties. For this purpose, the docked conformations of the active peptides were used to identify compounds with similar activities. A series of related β-sheet breaker peptides were docked to solid state NMR structures of a fibrillar form of Aβ. The lowest energy conformations of the active peptides were used to design three dimensional (3D)-pharmacophores, suitable for screening the NCI database with Unity. Small molecular weight compounds with physicochemical features in a conformation similar to the active peptides were selected, ranked by docking solubility parameters. Of 16 diverse compounds selected for experimental screening, 2 prevented and reversed Aβ aggregation at 2–3 μM concentration, as measured by Thioflavin T (ThT) fluorescence and ELISA assays. They also prevented the toxic effects of aggregated Aβ on neuroblastoma cells. Their low molecular weight and aqueous solubility makes them promising lead compounds for treating AD. PMID:19540126

Dynamics of blood flow in a microfluidic ladder network

NASA Astrophysics Data System (ADS)

Maddala, Jeevan; Zilberman-Rudenko, Jevgenia; McCarty, Owen

The dynamics of a complex mixture of cells and proteins, such as blood, in perturbed shear flow remains ill-defined. Microfluidics is a promising technology for improving the understanding of blood flow under complex conditions of shear; as found in stent implants and in tortuous blood vessels. We model the fluid dynamics of blood flow in a microfluidic ladder network with dimensions mimicking venules. Interaction of blood cells was modeled using multiagent framework, where cells of different diameters were treated as spheres. This model served as the basis for predicting transition regions, collision pathways, re-circulation zones and residence times of cells dependent on their diameters and device architecture. Based on these insights from the model, we were able to predict the clot formation configurations at various locations in the device. These predictions were supported by the experiments using whole blood. To facilitate platelet aggregation, the devices were coated with fibrillar collagen and tissue factor. Blood was perfused through the microfluidic device for 9 min at a physiologically relevant venous shear rate of 600 s-1. Using fluorescent microscopy, we observed flow transitions near the channel intersections and at the areas of blood flow obstruction, which promoted larger thrombus formation. This study of integrating model predictions with experimental design, aids in defining the dynamics of blood flow in microvasculature and in development of novel biomedical devices.

MALDI Mass Spectrometry Imaging: A Novel Tool for the Identification and Classification of Amyloidosis.

PubMed

Winter, Martin; Tholey, Andreas; Kristen, Arnt; Röcken, Christoph

2017-11-01

Amyloidosis is a group of diseases caused by extracellular accumulation of fibrillar polypeptide aggregates. So far, diagnosis is performed by Congo red staining of tissue sections in combination with polarization microscopy. Subsequent identification of the causative protein by immunohistochemistry harbors some difficulties regarding sensitivity and specificity. Mass spectrometry based approaches have been demonstrated to constitute a reliable method to supplement typing of amyloidosis, but still depend on Congo red staining. In the present study, we used matrix-assisted laser desorption/ionization mass spectrometry imaging coupled with ion mobility separation (MALDI-IMS MSI) to investigate amyloid deposits in formalin-fixed and paraffin-embedded tissue samples. Utilizing a novel peptide filter method, we found a universal peptide signature for amyloidoses. Furthermore, differences in the peptide composition of ALλ and ATTR amyloid were revealed and used to build a reliable classification model. Integrating the peptide filter in MALDI-IMS MSI analysis, we developed a bioinformatics workflow facilitating the identification and classification of amyloidosis in a less time and sample-consuming experimental setup. Our findings demonstrate also the feasibility to investigate the amyloid's protein composition, thus paving the way to establish classification models for the diverse types of amyloidoses and to shed further light on the complex process of amyloidogenesis. © 2017 The Authors, Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Morphological analysis of oligomeric vs. fibrillar forms of α-synuclein aggregates with super-resolution BALM imaging

NASA Astrophysics Data System (ADS)

Huh, Hyun; Lee, Jinwoo; Kim, Hyung Jun; Hohng, Sungchul; Kim, Seong Keun

2017-12-01

Application of BALM (binding activated localization microcopy) was shown to allow facile imaging of amyloid fibrils with a typical diameter of ∼14 nm FWHM. We also observed a twisted ribbon-like substructure of mutant amyloid fibrils and even what appear to be toxic amyloid oligomers with their characteristic morphological features consistent with TEM images. Use of an easily available staining dye in this method greatly enhances the prospect of addressing amyloid-related diseases in their diagnosis and drug tests by allowing facile in situ and in vivo detection by optical imaging.

Tumor Tension Induces Persistent Inflammation and Promotes Breast Cancer Aggression

DTIC Science & Technology

2015-10-01

assessing collagen crosslinking, fibrillar collagen I deposition, and macrophage infiltration in a pilot study using MMTV-PyMT; Col1a1 -tTA;TetO_mLOX model...where LOX was overexpressed in Col1a1 + activated fibroblasts (Figure 4B-C). The second model is an inducible LOX knockout model: MMTV-PyMT; Col1a1 ...cells were isolated and analyzed using flow cytometry. (B) MMTV-PyMT; Col1a1 -tTA;TetO_mLOX mice have been taken off DOX treatment at 6 weeks of age

Curcumin-derived pyrazoles and isoxazoles: Swiss army knives or blunt tools for Alzheimer's disease?

PubMed

Narlawar, Rajeshwar; Pickhardt, Marcus; Leuchtenberger, Stefanie; Baumann, Karlheinz; Krause, Sabine; Dyrks, Thomas; Weggen, Sascha; Mandelkow, Eckhard; Schmidt, Boris

2008-01-01

Curcumin binds to the amyloid beta peptide (Abeta) and inhibits or modulates amyloid precursor protein (APP) metabolism. Therefore, curcumin-derived isoxazoles and pyrazoles were synthesized to minimize the metal chelation properties of curcumin. The decreased rotational freedom and absence of stereoisomers was predicted to enhance affinity toward Abeta(42) aggregates. Accordingly, replacement of the 1,3-dicarbonyl moiety with isosteric heterocycles turned curcumin analogue isoxazoles and pyrazoles into potent ligands of fibrillar Abeta(42) aggregates. Additionally, several compounds are potent inhibitors of tau protein aggregation and depolymerized tau protein aggregates at low micromolar concentrations.

Chronic systemic pesticide exposure reproduces features of Parkinson's disease.

PubMed

Betarbet, R; Sherer, T B; MacKenzie, G; Garcia-Osuna, M; Panov, A V; Greenamyre, J T

2000-12-01

The cause of Parkinson's disease (PD) is unknown, but epidemiological studies suggest an association with pesticides and other environmental toxins, and biochemical studies implicate a systemic defect in mitochondrial complex I. We report that chronic, systemic inhibition of complex I by the lipophilic pesticide, rotenone, causes highly selective nigrostriatal dopaminergic degeneration that is associated behaviorally with hypokinesia and rigidity. Nigral neurons in rotenone-treated rats accumulate fibrillar cytoplasmic inclusions that contain ubiquitin and alpha-synuclein. These results indicate that chronic exposure to a common pesticide can reproduce the anatomical, neurochemical, behavioral and neuropathological features of PD.

Dynamics of water in the amphiphilic pore of amyloid β fibrils

NASA Astrophysics Data System (ADS)

GhattyVenkataKrishna, Pavan K.; Mostofian, Barmak

2013-09-01

Alzheimers disease related amyloid peptide, Aβ, forms a fibrillar structure through aggregation. The aggregate is stabilized by a salt bridge that is responsible for the formation of an amphiphilic pore that can accommodate water molecules. None of the reported structures of Aβ, however, contain water. We present results from molecular dynamics simulations on dimeric Aβ fibrils solvated in water. Water penetrates and fills the amphiphilic pore increasing its volume. We observe a thick wire of water that is translationally and rotationally stiff in comparison to bulk water and may be essential for the stabilization of the amyloid Aβ protein.

A combined Bodian-Nissl stain for improved network analysis in neuronal cell culture.

PubMed

Hightower, M; Gross, G W

1985-11-01

Bodian and Nissl procedures were combined to stain dissociated mouse spinal cord cells cultured on coverslips. The Bodian technique stains fine neuronal processes in great detail as well as an intracellular fibrillar network concentrated around the nucleus and in proximal neurites. The Nissl stain clearly delimits neuronal cytoplasm in somata and in large dendrites. A combination of these techniques allows the simultaneous depiction of neuronal perikarya and all afferent and efferent processes. Costaining with little background staining by either procedure suggests high specificity for neurons. This procedure could be exploited for routine network analysis of cultured neurons.

Contributions of chaperone/usher systems to cell binding, biofilm formation and Yersinia pestis virulence.

PubMed

Felek, Suleyman; Jeong, Jenny J; Runco, Lisa M; Murray, Susan; Thanassi, David G; Krukonis, Eric S

2011-03-01

Yersinia pestis genome sequencing projects have revealed six intact uncharacterized chaperone/usher systems with the potential to play roles in plague pathogenesis. We cloned each locus and expressed them in the Δfim Escherichia coli strain AAEC185 to test the assembled Y. pestis surface structures for various activities. Expression of each chaperone/usher locus gave rise to specific novel fibrillar structures on the surface of E. coli. One locus, y0561-0563, was able to mediate attachment to human epithelial cells (HEp-2) and human macrophages (THP-1) but not mouse macrophages (RAW264.7), while several loci were able to facilitate E. coli biofilm formation. When each chaperone/usher locus was deleted in Y. pestis, only deletion of the previously described pH 6 antigen (Psa) chaperone/usher system resulted in decreased adhesion and biofilm formation. Quantitative RT-PCR (qRT-PCR) revealed low expression levels for each novel chaperone/usher system in vitro as well as in mouse tissues following intravenous infection. However, a Y. pestis mutant in the chaperone/usher locus y1858-1862 was attenuated for virulence in mice via the intravenous route of infection, suggesting that expression of this locus is, at some stage, sufficient to affect the outcome of a plague infection. qRT-PCR experiments also indicated that expression of the chaperone/usher-dependent capsule locus, caf1, was influenced by oxygen availability and that the well-described chaperone/usher-dependent pilus, Psa, was strongly induced in minimal medium even at 28 °C rather than 37 °C, a temperature previously believed to be required for Psa expression. These data indicate several potential roles for the novel chaperone/usher systems of Y. pestis in pathogenesis and infection-related functions such as cell adhesion and biofilm formation.

The interaction of polyglutamine peptides with lipid membranes is regulated by flanking sequences associated with huntingtin.

PubMed

Burke, Kathleen A; Kauffman, Karlina J; Umbaugh, C Samuel; Frey, Shelli L; Legleiter, Justin

2013-05-24

Huntington disease (HD) is caused by an expanded polyglutamine (poly(Q)) repeat near the N terminus of the huntingtin (htt) protein. Expanded poly(Q) facilitates formation of htt aggregates, eventually leading to deposition of cytoplasmic and intranuclear inclusion bodies containing htt. Flanking sequences directly adjacent to the poly(Q) domain, such as the first 17 amino acids on the N terminus (Nt17) and the polyproline (poly(P)) domain on the C-terminal side of the poly(Q) domain, heavily influence aggregation. Additionally, htt interacts with a variety of membraneous structures within the cell, and Nt17 is implicated in lipid binding. To investigate the interaction between htt exon1 and lipid membranes, a combination of in situ atomic force microscopy, Langmuir trough techniques, and vesicle permeability assays were used to directly monitor the interaction of a variety of synthetic poly(Q) peptides with different combinations of flanking sequences (KK-Q35-KK, KK-Q35-P10-KK, Nt17-Q35-KK, and Nt17-Q35-P10-KK) on model membranes and surfaces. Each peptide aggregated on mica, predominately forming extended, fibrillar aggregates. In contrast, poly(Q) peptides that lacked the Nt17 domain did not appreciably aggregate on or insert into lipid membranes. Nt17 facilitated the interaction of peptides with lipid surfaces, whereas the poly(P) region enhanced this interaction. The aggregation of Nt17-Q35-P10-KK on the lipid bilayer closely resembled that of a htt exon1 construct containing 35 repeat glutamines. Collectively, this data suggests that the Nt17 domain plays a critical role in htt binding and aggregation on lipid membranes, and this lipid/htt interaction can be further modulated by the presence of the poly(P) domain.

A baculovirus polyhedron envelope protein: immunogold localization in infected cells and mature polyhedra.

PubMed

Russell, R L; Rohrmann, G F

1990-01-01

A polyclonal antiserum against a trpE fusion protein containing the complete open reading frame of the polyhedron envelope (PE) protein from the nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV) was used for immunogold staining and electron microscopic examination of polyhedra, isolated polyhedron envelopes, and infected insect cells at selected times postinfection. The antiserum specifically stained the peripheral envelope of mature polyhedra and also stained the envelope structure which remained after polyhedra were dissolved in dilute alkaline solutions. In OpMNPV-infected Lymantria dispar cells, the PE protein was detected by 48 hr postinfection (hr p.i.) but specific localization and staining of developing polyhedra were not evident. However, by 72 hr p.i. substantial and preferential staining of the periphery of developing polyhedra was evident even though a distinct polyhedron envelope was not yet observed. In addition, the periphery of fibrillar structures was stained by the PE antiserum. By 96 hr p.i., mature envelopes surrounded polyhedra and these polyhedron envelopes were stained with the PE antibody. The progression of PE protein staining during polyhedron morphogenesis indicates that the PE protein accumulates and becomes associated with developing polyhedra in the nucleus between 48 and 72 hr p.i. Very late in infection the mature polyhedron envelope forms on the polyhedron surface. The apparent affinity of the PE protein for the surface of maturing polyhedra suggests that it may be a major component of the polyhedron envelope or may form the matrix for the deposition of other components which contribute to the mature envelope. Immunogold staining and protease digestion experiments indicate that protein is an essential component of the polyhedron envelope.

Enhanced antiadhesive properties of chitosan/hyaluronic acid polyelectrolyte multilayers driven by thermal annealing: Low adherence for mammalian cells and selective decrease in adhesion for Gram-positive bacteria.

PubMed

Muzzio, Nicolás E; Pasquale, Miguel A; Diamanti, Eleftheria; Gregurec, Danijela; Moro, Marta Martinez; Azzaroni, Omar; Moya, Sergio E

2017-11-01

The development of antifouling coatings with restricted cell and bacteria adherence is fundamental for many biomedical applications. A strategy for the fabrication of antifouling coatings based on the layer-by-layer assembly and thermal annealing is presented. Polyelectrolyte multilayers (PEMs) assembled from chitosan and hyaluronic acid were thermally annealed in an oven at 37°C for 72h. The effect of annealing on the PEM properties and topography was studied by atomic force microscopy, ζ-potential, circular dichroism and contact angle measurements. Cell adherence on PEMs before and after annealing was evaluated by measuring the cell spreading area and aspect ratio for the A549 epithelial, BHK kidney fibroblast, C2C12 myoblast and MC-3T3-E1 osteoblast cell lines. Chitosan/hyaluronic acid PEMs show a low cell adherence that decreases with the thermal annealing, as observed from the reduction in the average cell spreading area and more rounded cell morphology. The adhesion of S. aureus (Gram-positive) and E. coli (Gram-negative) bacteria strains was quantified by optical microscopy, counting the number of colony-forming units and measuring the light scattering of bacteria suspension after detachment from the PEM surface. A 20% decrease in bacteria adhesion was selectively observed in the S. aureus strain after annealing. The changes in mammalian cell and bacteria adhesion correlate with the changes in topography of the chitosan/hyaluronic PEMs from a rough fibrillar 3D structure to a smoother and planar surface after thermal annealing. Copyright © 2017. Published by Elsevier B.V.

Comparison of analytical and experimental subsonic steady and unsteady pressure distributions for a high-aspect-ratio-supercritical wing model with oscillating control surfaces

NASA Technical Reports Server (NTRS)

Mccain, W. E.

1982-01-01

The results of a comparative study using the unsteady aerodynamic lifting surface theory, known as the Doublet Lattice method, and experimental subsonic steady- and unsteady-pressure measurements, are presented for a high-aspect-ratio supercritical wing model. Comparisons of pressure distributions due to wing angle of attack and control-surface deflections were made. In general, good correlation existed between experimental and theoretical data over most of the wing planform. The more significant deviations found between experimental and theoretical data were in the vicinity of control surfaces for both static and oscillatory control-surface deflections.

Joint surface modeling with thin-plate splines.

PubMed

Boyd, S K; Ronsky, J L; Lichti, D D; Salkauskas, K; Chapman, M A; Salkauskas, D

1999-10-01

Mathematical joint surface models based on experimentally determined data points can be used to investigate joint characteristics such as curvature, congruency, cartilage thickness, joint contact areas, as well as to provide geometric information well suited for finite element analysis. Commonly, surface modeling methods are based on B-splines, which involve tensor products. These methods have had success; however, they are limited due to the complex organizational aspect of working with surface patches, and modeling unordered, scattered experimental data points. An alternative method for mathematical joint surface modeling is presented based on the thin-plate spline (TPS). It has the advantage that it does not involve surface patches, and can model scattered data points without experimental data preparation. An analytical surface was developed and modeled with the TPS to quantify its interpolating and smoothing characteristics. Some limitations of the TPS include discontinuity of curvature at exactly the experimental surface data points, and numerical problems dealing with data sets in excess of 2000 points. However, suggestions for overcoming these limitations are presented. Testing the TPS with real experimental data, the patellofemoral joint of a cat was measured with multistation digital photogrammetry and modeled using the TPS to determine cartilage thicknesses and surface curvature. The cartilage thickness distribution ranged between 100 to 550 microns on the patella, and 100 to 300 microns on the femur. It was found that the TPS was an effective tool for modeling joint surfaces because no preparation of the experimental data points was necessary, and the resulting unique function representing the entire surface does not involve surface patches. A detailed algorithm is presented for implementation of the TPS.

Solvent induced modifications to fiber nanostructure and morphology for 12HSA molecular gels

NASA Astrophysics Data System (ADS)

Gao, Jie

Molecular organogels are thermo reversible quasi-solid materials, which are formed by low molecular weight organogelators (LMOGs) undergoing supramolecular aggregation via non-covalent interactions, forming a three-dimensional fibrillar network. Numerous applications of molecular organogels are been investigated as edible oils, drug release matrices and personal care products. The chemistry of the organic phase (i.e., solvent) influences every level of structure in organogels. Different solvents induce LMOG to assemble into "crystal like" fibers, which have more than one crystal form, lamellar arrangement and domain size. Differences in these solid states are known to affect the macroscopic properties of the gel, including critical gelator concentration (CGC), melting point, melting enthalpy and opacity.12-hydroxystearic acid (12HSA) was examined in several classes of organic solvents with different function groups. These gels, sols or precipitates were analyzed using a series of techniques including: powder x-ray diffraction (XRD), differential scanning calorimetry (DSC), fourier-transform infrared spectroscopy (FT-IR), pulsed nuclear magnetic resonance spectroscopy (pNMR) and microscopy. Specifically, certain solvents caused 12HSA to self-assemble into a triclinic parallel polymorphic form with subcell spacing of ~4.6, 3.9, and 3.8 A and an interdigitated unit cell with a lamellar arrangement (38~44 A). This polymorphic form corresponded to a less effective sphereultic supramolecular crystalline network, which immobilizes solvents at CGC greater than 1.5 wt %. The other group of solvents induce a hexagonal subcell spacing (i.e., unit sub cell spacing ~4.1 A) and are arranged in a multi lamellar fashion with a unit cell greater than the bimolecular length of 12HSA (~54 A).This polymorphic form corresponds to fibrillar aggregates with a CGC less than 1 wt %.

Anle138b and related compounds are aggregation specific fluorescence markers and reveal high affinity binding to α-synuclein aggregates.

PubMed

Deeg, Andreas A; Reiner, Anne M; Schmidt, Felix; Schueder, Florian; Ryazanov, Sergey; Ruf, Viktoria C; Giller, Karin; Becker, Stefan; Leonov, Andrei; Griesinger, Christian; Giese, Armin; Zinth, Wolfgang

2015-09-01

Special diphenyl-pyrazole compounds and in particular anle138b were found to reduce the progression of prion and Parkinson's disease in animal models. The therapeutic impact of these compounds was attributed to the modulation of α-synuclein and prion-protein aggregation related to these diseases. Photophysical and photochemical properties of the diphenyl-pyrazole compounds anle138b, anle186b and sery313b and their interaction with monomeric and aggregated α-synuclein were studied by fluorescence techniques. The fluorescence emission of diphenyl-pyrazole is strongly increased upon incubation with α-synuclein fibrils, while no change in fluorescence emission is found when brought in contact with monomeric α-synuclein. This points to a distinct interaction between diphenyl-pyrazole and the fibrillar structure with a high binding affinity (Kd=190±120nM) for anle138b. Several α-synuclein proteins form a hydrophobic binding pocket for the diphenyl-pyrazole compound. A UV-induced dehalogenation reaction was observed for anle138b which is modulated by the hydrophobic environment of the fibrils. Fluorescence of the investigated diphenyl-pyrazole compounds strongly increases upon binding to fibrillar α-synuclein structures. Binding at high affinity occurs to hydrophobic pockets in the fibrils. The observed particular fluorescence properties of the diphenyl-pyrazole molecules open new possibilities for the investigation of the mode of action of these compounds in neurodegenerative diseases. The high binding affinity to aggregates and the strong increase in fluorescence upon binding make the compounds promising fluorescence markers for the analysis of aggregation-dependent epitopes. Copyright © 2015 Elsevier B.V. All rights reserved.

Axonal transport and secretion of fibrillar forms of α-synuclein, Aβ42 peptide and HTTExon 1.

PubMed

Brahic, Michel; Bousset, Luc; Bieri, Gregor; Melki, Ronald; Gitler, Aaron D

2016-04-01

Accruing evidence suggests that prion-like behavior of fibrillar forms of α-synuclein, β-amyloid peptide and mutant huntingtin are responsible for the spread of the lesions that characterize Parkinson disease, Alzheimer disease and Huntington disease, respectively. It is unknown whether these distinct protein assemblies are transported within and between neurons by similar or distinct mechanisms. It is also unclear if neuronal death or injury is required for neuron-to-neuron transfer. To address these questions, we used mouse primary cortical neurons grown in microfluidic devices to measure the amounts of α-synuclein, Aβ42 and HTTExon1 fibrils transported by axons in both directions (anterograde and retrograde), as well as to examine the mechanism of their release from axons after anterograde transport. We observed that the three fibrils were transported in both anterograde and retrograde directions but with strikingly different efficiencies. The amount of Aβ42 fibrils transported was ten times higher than that of the other two fibrils. HTTExon1 was efficiently transported in the retrograde direction but only marginally in the anterograde direction. Finally, using neurons from two distinct mutant mouse strains whose axons are highly resistant to neurodegeneration (Wld(S) and Sarm1(-/-)), we found that the three different fibrils were secreted by axons after anterograde transport, in the absence of axonal lysis, indicating that trans-neuronal spread can occur in intact healthy neurons. In summary, fibrils of α-synuclein, Aβ42 and HTTExon1 are all transported in axons but in directions and amounts that are specific of each fibril. After anterograde transport, the three fibrils were secreted in the medium in the absence of axon lysis. Continuous secretion could play an important role in the spread of pathology between neurons but may be amenable to pharmacological intervention.

Bone sialoprotein-collagen interaction promotes hydroxyapatite nucleation.

PubMed

Baht, Gurpreet S; Hunter, Graeme K; Goldberg, Harvey A

2008-09-01

In bone, hydroxyapatite (HA) crystals are deposited onto the type I collagen scaffold by a mechanism that has yet to be elucidated. Bone sialoprotein (BSP) is an acidic phosphoprotein that is expressed at high levels in mineralized tissues, capable of binding type I collagen, and nucleating HA. Both bone-extracted and recombinant BSP (rBSP) bind with equal affinity to collagen. The nature of the BSP-collagen interaction and its role in HA nucleation are not known. We have used a solid-phase binding assay and affinity chromatography to characterize the BSP-collagen interaction. rBSP-binding affinities of triple-helical and fibrillar type I collagen were similar (K(D) approximately 13 nM), while that of heat-denatured type I collagen was lower (K(D) approximately 44 nM), indicating the importance of triple-helical structure in binding BSP. Pepsin treatment of collagen had no effect on rBSP binding, demonstrating that the telopeptides of collagen are not involved. The majority of collagen-bound rBSP was eluted by acetonitrile, indicating that hydrophobic interactions are principally responsible for binding. Using an HA-nucleation assay, it was shown that rBSP is ten-fold more potent in reconstituted fibrillar collagen gels than in agarose gels. Nucleating potency of a non-collagen-binding, HA-nucleating peptide [rBSP(134-206)] showed no difference in the two gel systems. The work here shows that optimal binding of rBSP requires collagen to be in a native, triple-helical structure, does not require the telopeptides, and is stabilized by hydrophobic interactions. Upon binding to collagen, rBSP displays an increase in nucleation potency, implying a co-operative effect of BSP and collagen in mineral formation.

Congenital Myasthenic Syndrome Type 19 Is Caused by Mutations in COL13A1, Encoding the Atypical Non-fibrillar Collagen Type XIII α1 Chain.

PubMed

Logan, Clare V; Cossins, Judith; Rodríguez Cruz, Pedro M; Parry, David A; Maxwell, Susan; Martínez-Martínez, Pilar; Riepsaame, Joey; Abdelhamed, Zakia A; Lake, Alice V R; Moran, Maria; Robb, Stephanie; Chow, Gabriel; Sewry, Caroline; Hopkins, Philip M; Sheridan, Eamonn; Jayawant, Sandeep; Palace, Jacqueline; Johnson, Colin A; Beeson, David

2015-12-03

The neuromuscular junction (NMJ) consists of a tripartite synapse with a presynaptic nerve terminal, Schwann cells that ensheathe the terminal bouton, and a highly specialized postsynaptic membrane. Synaptic structural integrity is crucial for efficient signal transmission. Congenital myasthenic syndromes (CMSs) are a heterogeneous group of inherited disorders that result from impaired neuromuscular transmission, caused by mutations in genes encoding proteins that are involved in synaptic transmission and in forming and maintaining the structural integrity of NMJs. To identify further causes of CMSs, we performed whole-exome sequencing (WES) in families without an identified mutation in known CMS-associated genes. In two families affected by a previously undefined CMS, we identified homozygous loss-of-function mutations in COL13A1, which encodes the alpha chain of an atypical non-fibrillar collagen with a single transmembrane domain. COL13A1 localized to the human muscle motor endplate. Using CRISPR-Cas9 genome editing, modeling of the COL13A1 c.1171delG (p.Leu392Sfs(∗)71) frameshift mutation in the C2C12 cell line reduced acetylcholine receptor (AChR) clustering during myotube differentiation. This highlights the crucial role of collagen XIII in the formation and maintenance of the NMJ. Our results therefore delineate a myasthenic disorder that is caused by loss-of-function mutations in COL13A1, encoding a protein involved in organization of the NMJ, and emphasize the importance of appropriate symptomatic treatment for these individuals. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

APP Regulates Microglial Phenotype in a Mouse Model of Alzheimer's Disease

PubMed Central

Manocha, Gunjan D.; Floden, Angela M.; Rausch, Keiko; Kulas, Joshua A.; McGregor, Brett A.; Rojanathammanee, Lalida; Puig, Kelley R.; Puig, Kendra L.; Karki, Sanjib; Nichols, Michael R.; Darland, Diane C.; Porter, James E.

2016-01-01

Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer's disease. Although fibrillar amyloid β (Aβ)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP−/−) microglial cultures, oligomeric Aβ was unable to stimulate increased secretion from mAPP−/− cells. This was consistent with an ability of oligomeric Aβ to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aβ produced less microgliosis in mAPP−/− mice compared with wild-type mice. The mAPP−/− mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aβ plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease. SIGNIFICANCE STATEMENT A hallmark of Alzheimer's disease (AD) brains is the accumulation of amyloid β (Aβ) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aβ stimulation of microglial activation is one source of brain inflammatory changes during disease. Aβ is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aβ are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aβ production to drive the microgliosis associated with AD brains. PMID:27511018

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosello-Lleti, Esther; Rivera, Miguel; Cortes, Raquel

Highlights: Black-Right-Pointing-Pointer Heart failure alters nucleolar morphology and organization. Black-Right-Pointing-Pointer Nucleolin expression is significant increased in ischemic and dilated cardiomyopathy. Black-Right-Pointing-Pointer Ventricular function of heart failure patients was related with nucleolin levels. -- Abstract: We investigate for the first time the influence of heart failure (HF) on nucleolar organization and proteins in patients with ischemic (ICM) or dilated cardiomyopathy (DCM). A total of 71 human hearts from ICM (n = 38) and DCM (n = 27) patients, undergoing heart transplantation and control donors (n = 6), were analysed by western-blotting, RT-PCR and cell biology methods. When we compared protein levelsmore » according to HF etiology, nucleolin was increased in both ICM (117%, p < 0.05) and DCM (141%, p < 0.01). Moreover, mRNA expression were also upregulated in ICM (1.46-fold, p < 0.05) and DCM (1.70-fold, p < 0.05. Immunofluorescence studies showed that the highest intensity of nucleolin was into nucleolus (p < 0.0001), and it was increased in pathological hearts (p < 0.0001). Ultrastructure analysis by electron microscopy showed an increase in the nucleus and nucleolus size in ICM (17%, p < 0.05 and 131%, p < 0.001) and DCM (56%, p < 0.01 and 69%, p < 0.01). Nucleolar organization was influenced by HF irrespective of etiology, increasing fibrillar centers (p < 0.001), perinucleolar chromatin (p < 0.01) and dense fibrillar components (p < 0.01). Finally, left ventricular function parameters were related with nucleolin levels in ischemic hearts (p < 0.0001). The present study demonstrates that HF influences on morphology and organization of nucleolar components, revealing changes in the expression and in the levels of nucleolin protein.« less

Supramolecularly engineered perylene bisimide assemblies exhibiting thermal transition from columnar to multilamellar structures.

PubMed

Yagai, Shiki; Usui, Mari; Seki, Tomohiro; Murayama, Haruno; Kikkawa, Yoshihiro; Uemura, Shinobu; Karatsu, Takashi; Kitamura, Akihide; Asano, Atsushi; Seki, Shu

2012-05-09

Perylene 3,4:9,10-tetracarboxylic acid bisimide (PBI) was functionalized with ditopic cyanuric acid to organize it into complex columnar architectures through the formation of hydrogen-bonded supermacrocycles (rosette) by complexing with ditopic melamines possessing solubilizing alkoxyphenyl substituents. The aggregation study in solution using UV-vis and NMR spectroscopies showed the formation of extended aggregates through hydrogen-bonding and π-π stacking interactions. The cylindrical fibrillar nanostructures were visualized by microscopic techniques (AFM, TEM), and the formation of lyotropic mesophase was confirmed by polarized optical microscopy and SEM. X-ray diffraction study revealed that a well-defined hexagonal columnar (Col(h)) structure was formed by solution-casting of fibrillar assemblies. All of these results are consistent with the formation of hydrogen-bonded PBI rosettes that spontaneously organize into the Col(h) structure. Upon heating the Col(h) structure in the bulk state, a structural transition to a highly ordered lamellar (Lam) structure was observed by variable-temperature X-ray diffraction, differential scanning calorimetry, and AFM studies. IR study showed that the rearrangement of the hydrogen-bonding motifs occurs during the structural transition. These results suggest that such a striking structural transition is aided by the reorganization in the lowest level of self-organization, i.e., the rearrangement of hydrogen-bonded motifs from rosette to linear tape. A remarkable increase in the transient photoconductivity was observed by the flash-photolysis time-resolved microwave conductivity (FP-TRMC) measurements upon converting the Col(h) structure to the Lam structure. Transient absorption spectroscopy revealed that electron transfer from electron-donating alkoxyphenyl groups of melamine components to electron-deficient PBI moieties takes place, resulting in a higher probability of charge carrier generation in the Lam structure compared to the Col(h) structure.

Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral amyloidoses

PubMed Central

Åslund, Andreas; Sigurdson, Christina J.; Klingstedt, Therése; Grathwohl, Stefan; Bolmont, Tristan; Dickstein, Dara L.; Glimsdal, Eirik; Prokop, Stefan; Lindgren, Mikael; Konradsson, Peter; Holtzman, David M.; Hof, Patrick R.; Heppner, Frank L.; Gandy, Samuel; Jucker, Mathias; Aguzzi, Adriano; Hammarström, Per; Nilsson, K. Peter R.

2010-01-01

Molecular probes for selective identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying cerebral amyloidoses. Here we report the chemical design of pentameric thiophene derivatives, denoted luminescent conjugated oligothiophenes (LCOs), which could be used for real-time visualization of cerebral protein aggregates in transgenic mouse models of neurodegenerative diseases by multiphoton microscopy. One of the LCOs, p-FTAA, showed conformation-dependent optical properties and could be utilized for ex vivo spectral assignment of distinct prion deposits from two mouse-adapted prion strains. p-FTAA also revealed staining of transient soluble pre-fibrillar non-thioflavinophilic Aβ- assemblies during in vitro fibrillation of Aβ peptides. In brain tissue samples, Aβ deposits and neurofibrillary tangles (NFTs) were readily identified by a strong fluorescence from p-FTAA and the LCO staining showed complete co-localization with conventional antibodies (6E10 and AT8), indicating that p-FTAA detects all the immuno-positive aggregated proteinaceous species in Alzheimer disease, but with significantly shorter imaging time (100 fold) compared to immunofluorescence. In addition, a patchy islet-like staining of individual Aβ plaque was unveiled by the anti-oligomer A11 antibody during co-staining with p-FTAA, suggesting that pre-fibrillar species are likely an intrinsic component of Aβ plaques in human brain. The major hallmarks of Alzheimer’s disease, namely Aβ aggregates versus NFTs could also be distinguished due to distinct emission spectra from p-FTAA. Overall, we demonstrate that LCOs can be utilized as powerful practical research tools for studying protein aggregation diseases and facilitate the study of amyloid origin, evolution and maturation, Aβ−tau interactions and pathogenesis both ex vivo and in vivo. PMID:19624097

The characean internodal cell as a model system for studying wound healing

PubMed Central

Foissner, I.; Wasteneys, G.O.

2012-01-01

Summary This work describes the characean internodal cell as a model system for the study of wound healing and compares wounds induced by certain chemicals and UV irradiation with wounds occurring in the natural environment. We review the existing literature and define three types of wound response: 1) cortical window formation characterized by disassembly of microtubules, transient inhibition of actin-dependent cytoplasmic streaming and chloroplast detachment, 2) fibrillar wound walls characterized by exocytosis of vesicles carrying wall polysaccharides and membrane-bound cellulose synthase complexes coupled with endocytosis of surplus membrane and 3) amorphous, callose- and membrane-containing wound walls characterized by exocytosis of vesicles and endoplasmic reticulum (ER) cisternae in the absence of membrane recycling. We hypothesize that these three wound responses reflect the extent of damage, probably Ca2+ influx, and that the secretion of Ca2+ - loaded ER cisternae is an emergency reaction in case of severe Ca2+ load. Microtubules are not required for wound healing but their disassembly could have a signalling function. Transient reorganization of the actin cytoskeleton into a meshwork of randomly oriented filaments is required for the migration of wound wall forming organelles, just as occurs in tip-growing plant cells. New data presented in this study show that during the deposition of an amorphous wound wall numerous actin rings are present, which may indicate specific ion fluxes and/or a storage form for actin. In addition, we present new evidence for the exocytosis of FM1-43-stained organelles, putative endosomes, required for plasma membrane repair during wound healing. Finally we show that quickly growing fibrillar wound walls, even when deposited in the absence of microtubules, have a highly ordered helical structure of consistent handedness comprised of cellulose microfibrils. PMID:22118365

Early accumulation of intracellular fibrillar oligomers and late congophilic amyloid angiopathy in mice expressing the Osaka intra-Aβ APP mutation

PubMed Central

Kulic, L; McAfoose, J; Welt, T; Tackenberg, C; Späni, C; Wirth, F; Finder, V; Konietzko, U; Giese, M; Eckert, A; Noriaki, K; Shimizu, T; Murakami, K; Irie, K; Rasool, S; Glabe, C; Hock, C; Nitsch, R M

2012-01-01

Pathogenic amyloid-β peptide precursor (APP) mutations clustered around position 693 of APP—position 22 of the Aβ sequence—are commonly associated with congophilic amyloid angiopathy (CAA) and intracerebral hemorrhages. In contrast, the Osaka (E693Δ) intra-Aβ APP mutation shows a recessive pattern of inheritance that leads to AD-like dementia despite low brain amyloid on in vivo positron emission tomography imaging. Here, we investigated the effects of the Osaka APP mutation on Aβ accumulation and deposition in vivo using a newly generated APP transgenic mouse model (E22ΔAβ) expressing the Osaka mutation together with the Swedish (K670N/M671L) double mutation. E22ΔAβ mice exhibited reduced α-processing of APP and early accumulation of intraneuronal fibrillar Aβ oligomers associated with cognitive deficits. In line with our in vitro findings that recombinant E22Δ-mutated Aβ peptides form amyloid fibrils, aged E22ΔAβ mice showed extracellular CAA deposits in leptomeningeal cerebellar and cortical vessels. In vitro results from thioflavin T aggregation assays with recombinant Aβ peptides revealed a yet unknown antiamyloidogenic property of the E693Δ mutation in the heterozygous state and an inhibitory effect of E22Δ Aβ42 on E22Δ Aβ40 fibrillogenesis. Moreover, E22Δ Aβ42 showed a unique aggregation kinetics lacking exponential fibril growth and poor seeding effects on wild-type Aβ aggregation. These results provide a possible explanation for the recessive trait of inheritance of the Osaka APP mutation and the apparent lack of amyloid deposition in E693Δ mutation carriers. PMID:23149447

Tau Pathology is Present In Vivo and Develops In Vitro in Sensory Neurons from Human P301S Tau Transgenic Mice: A System for Screening Drugs against Tauopathies

PubMed Central

Mellone, Manuela; Kestoras, Dimitra; Andrews, Melissa R.; Dassie, Elisa; Crowther, R. Anthony; Stokin, Gorazd B.; Tinsley, Jon; Horne, Graeme; Goedert, Michel

2013-01-01

Intracellular tau aggregates are the neuropathological hallmark of several neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, and cases of frontotemporal dementia, but the link between these aggregates and neurodegeneration remains unclear. Neuronal models recapitulating the main features of tau pathology are necessary to investigate the molecular mechanisms of tau malfunction, but current models show little and inconsistent spontaneous tau aggregation. We show that dorsal root ganglion (DRG) neurons in transgenic mice expressing human P301S tau (P301S-htau) develop tau pathology similar to that found in brain and spinal cord and a significant reduction in mechanosensation occurs before detectable fibrillar tau formation. DRG neuronal cultures established from adult P301S-htau mice at different ages retained the pattern of aberrant tau found in vivo. Moreover, htau became progressively hyperphosphorylated over 2 months in vitro beginning with nonsymptomatic neurons, while hyperphosphorylated P301S-htau-positive neurons from 5-month-old mice cultured for 2 months died preferentially. P301S-htau-positive neurons grew aberrant axons, including spheroids, typically found in human tauopathies. Neurons cultured at advanced stages of tau pathology showed a 60% decrease in the fraction of moving mitochondria. SEG28019, a novel O-GlcNAcase inhibitor, reduced steady-state pSer396/pSer404 phosphorylation over 7 weeks in a significant proportion of DRG neurons showing for the first time the possible beneficial effect of prolonged dosing of O-GlcNAcase inhibitor in vitro. Our system is unique in that fibrillar tau forms without external manipulation and provides an important new tool for understanding the mechanisms of tau dysfunction and for screening of compounds for treatment of tauopathies. PMID:24227726

Reconstitution of in vivo macrophage-tumor cell pairing and streaming motility on one-dimensional micro-patterned substrates

PubMed Central

Sharma, Ved P.; Beaty, Brian T.; Patsialou, Antonia; Liu, Huiping; Clarke, Michael; Cox, Dianne; Condeelis, John S.; Eddy, Robert J.

2014-01-01

In mammary tumors, intravital imaging techniques have uncovered an essential role for macrophages during tumor cell invasion and metastasis mediated by an epidermal growth factor (EGF)/colony stimulating factor-1 (CSF-1) paracrine loop. It was previously demonstrated that mammary tumors in mice derived from rat carcinoma cells (MTLn3) exhibited high velocity migration on extracellular matrix (ECM) fibers. These cells form paracrine loop-dependent linear assemblies of alternating host macrophages and tumor cells known as “streams.” Here, we confirm by intravital imaging that similar streams form in close association with ECM fibers in a highly metastatic patient-derived orthotopic mammary tumor (TN1). To understand the in vivo cell motility behaviors observed in streams, an in vitro model of fibrillar tumor ECM utilizing adhesive 1D micropatterned substrates was developed. MTLn3 cells on 1D fibronectin or type I collagen substrates migrated with higher velocity than on 2D substrates and displayed enhanced lamellipodial protrusion and increased motility upon local interaction and pairing with bone marrow-derived macrophages (BMMs). Inhibitors of EGF or CSF-1 signaling disrupted this interaction and reduced tumor cell velocity and protrusion, validating the requirement for an intact paracrine loop. Both TN1 and MTLn3 cells in the presence of BMMs were capable of co-assembling into linear arrays of alternating tumor cells and BMMs that resembled streams in vivo, suggesting the stream assembly is cell autonomous and can be reconstituted on 1D substrates. Our results validate the use of 1D micropatterned substrates as a simple and defined approach to study fibrillar ECM-dependent cell pairing, migration and relay chemotaxis as a complementary tool to intravital imaging. PMID:24634804

Lysyl oxidases regulate fibrillar collagen remodelling in idiopathic pulmonary fibrosis.

PubMed

Tjin, Gavin; White, Eric S; Faiz, Alen; Sicard, Delphine; Tschumperlin, Daniel J; Mahar, Annabelle; Kable, Eleanor P W; Burgess, Janette K

2017-11-01

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung with few effective therapeutic options. Structural remodelling of the extracellular matrix [i.e. collagen cross-linking mediated by the lysyl oxidase (LO) family of enzymes (LOX, LOXL1-4)] might contribute to disease pathogenesis and represent a therapeutic target. This study aimed to further our understanding of the mechanisms by which LO inhibitors might improve lung fibrosis. Lung tissues from IPF and non-IPF subjects were examined for collagen structure (second harmonic generation imaging) and LO gene (microarray analysis) and protein (immunohistochemistry and western blotting) levels. Functional effects (collagen structure and tissue stiffness using atomic force microscopy) of LO inhibitors on collagen remodelling were examined in two models, collagen hydrogels and decellularized human lung matrices. LOXL1 / LOXL2 gene expression and protein levels were increased in IPF versus non-IPF. Increased collagen fibril thickness in IPF versus non-IPF lung tissues correlated with increased LOXL1/LOXL2, and decreased LOX, protein expression. β-Aminoproprionitrile (β-APN; pan-LO inhibitor) but not Compound A (LOXL2-specific inhibitor) interfered with transforming growth factor-β-induced collagen remodelling in both models. The β-APN treatment group was tested further, and β-APN was found to interfere with stiffening in the decellularized matrix model. LOXL1 activity might drive collagen remodelling in IPF lungs. The interrelationship between collagen structural remodelling and LOs is disrupted in IPF lungs. Inhibition of LO activity alleviates fibrosis by limiting fibrillar collagen cross-linking, thereby potentially impeding the formation of a pathological microenvironment in IPF. © 2017. Published by The Company of Biologists Ltd.

Functional reconstitution of cellulose synthase in Escherichia coli.

PubMed

Imai, Tomoya; Sun, Shi-Jing; Horikawa, Yoshiki; Wada, Masahisa; Sugiyama, Junji

2014-11-10

Cellulose is a high molecular weight polysaccharide of β1 → 4-d-glucan widely distributed in nature-from plant cell walls to extracellular polysaccharide in bacteria. Cellulose synthase, together with other auxiliary subunit(s) in the cell membrane, facilitates the fibrillar assembly of cellulose polymer chains into a microfibril. The gene encoding the catalytic subunit of cellulose synthase is cesA and has been identified in many cellulose-producing organisms. Very few studies, however, have shown that recombinant CesA protein synthesizes cellulose polymer, but the mechanism by which CesA protein synthesizes cellulose microfibrils is not known. Here we show that cellulose-synthesizing activity is successfully reconstituted in Escherichia coli by expressing the bacterial cellulose synthase complex of Gluconacetobacter xylinus: CesA and CesB (formerly BcsA and BcsB, respectively). Cellulose synthase activity was, however, only detected when CesA and CesB were coexpressed with diguanyl cyclase (DGC), which synthesizes cyclic-di-GMP (c-di-GMP), which in turn activates cellulose-synthesizing activity in bacteria. Direct observation by electron microscopy revealed extremely thin fibrillar structures outside E. coli cells, which were removed by cellulase treatment. This fiber structure is not likely to be the native crystallographic form of cellulose I, given that it was converted to cellulose II by a chemical treatment milder than ever described. We thus putatively conclude that this fine fiber is an unprecedented structure of cellulose. Despite the inability of the recombinant enzyme to synthesize the native structure of cellulose, the system described in this study, named "CESEC (CEllulose-Synthesizing E. Coli)", represents a useful tool for functional analyses of cellulose synthase and for seeding new nanomaterials.

Reliability of sonographic assessment of tendinopathy in tennis elbow.

PubMed

Poltawski, Leon; Ali, Syed; Jayaram, Vijay; Watson, Tim

2012-01-01

To assess the reliability and compute the minimum detectable change using sonographic scales to quantify the extent of pathology and hyperaemia in the common extensor tendon in people with tennis elbow. The lateral elbows of 19 people with tennis elbow were assessed sonographically twice, 1-2 weeks apart. Greyscale and power Doppler images were recorded for subsequent rating of abnormalities. Tendon thickening, hypoechogenicity, fibrillar disruption and calcification were each rated on four-point scales, and scores were summed to provide an overall rating of structural abnormality; hyperaemia was scored on a five point scale. Inter-rater reliability was established using the intraclass correlation coefficient (ICC) to compare scores assigned independently to the same set of images by a radiologist and a physiotherapist with training in musculoskeletal imaging. Test-retest reliability was assessed by comparing scores assigned by the physiotherapist to images recorded at the two sessions. The minimum detectable change (MDC) was calculated from the test-retest reliability data. ICC values for inter-rater reliability ranged from 0.35 (95% CI: 0.05, 0.60) for fibrillar disruption to 0.77 (0.55, 0.88) for overall greyscale score, and 0.89 (0.79, 0.95) for hyperaemia. Test-retest reliability ranged from 0.70 (0.48, 0.84) for tendon thickening to 0.82 (0.66, 0.90) for overall greyscale score and 0.86 (0.73, 0.93) for calcification. The MDC for the greyscale total score was 2.0/12 and for the hyperaemia score was 1.1/5. The sonographic scoring system used in this study may be used reliably to quantify tendon abnormalities and change over time. A relatively inexperienced imager can conduct the assessment and use the rating scales reliably.

The second Cu(II)-binding site in a proton-rich environment interferes with the aggregation of amyloid-beta(1-40) into amyloid fibrils.

PubMed

Jun, Sangmi; Gillespie, Joel R; Shin, Byong-kyu; Saxena, Sunil

2009-11-17

The overall morphology and Cu(II) ion coordination for the aggregated amyloid-beta(1-40) [Abeta(1-40)] in N-ethylmorpholine (NEM) buffer are affected by Cu(II) ion concentration. This effect is investigated by transmission electron microscopy (TEM), atomic force microscopy (AFM), and electron spin echo envelope modulation (ESEEM) spectroscopy. At lower than equimolar concentrations of Cu(II) ions, fibrillar aggregates of Abeta(1-40) are observed. At these concentrations of Cu(II), the monomeric and fibrillar Abeta(1-40) ESEEM data indicate that the Cu(II) ion is coordinated by histidine residues. For aggregated Abeta(1-40) at a Cu(II):Abeta molar ratio of 2:1, TEM and AFM images show both linear fibrils and granular amorphous aggregates. The ESEEM spectra show that the multi-histidine coordination for Cu(II) ion partially breaks up and becomes exposed to water or exchangeable protons of the peptide at a higher Cu(II) concentration. Since the continuous-wave electron spin resonance results also suggest two copper-binding sites in Abeta(1-40), the proton ESEEM peak may arise from the second copper-binding site, which may be significantly involved in the formation of granular amorphous aggregates. Thioflavin T fluorescence and circular dichroism experiments also show that Cu(II) inhibits the formation of fibrils and induces a nonfibrillar beta-sheet conformation. Therefore, we propose that Abeta(1-40) has a second copper-binding site in a proton-rich environment and the second binding Cu(II) ion interferes with a conformational transition into amyloid fibrils, inducing the formation of granular amorphous aggregates.

The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pozo-Yauner, Luis del, E-mail: ldelpozo@inmegen.gob.mx; Wall, Jonathan S.; González Andrade, Martín

2014-01-10

Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stabilitymore » and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.« less

Phase matching considerations in second harmonic generation from tissues: Effects on emission directionality, conversion efficiency and observed morphology

NASA Astrophysics Data System (ADS)

LaComb, Ronald; Nadiarnykh, Oleg; Townsend, Sallie S.; Campagnola, Paul J.

2008-04-01

We present a heuristic treatment which relates SHG image intensities, signal directionality, and observed morphology to the physical structure of collagen and cellulose fibrillar tissues. The SHG creation model is based upon relaxed phase matching conditions which account for dispersion, randomness, and axial momentum contributions from the media, and includes a mathematical treatment which relates SHG conversion efficiency to fibril diameter and packing through the inclusion of potential intensity amplification resultant from quasi-phase matching (QPM). A direct consequence of this theory is that SHG in biological tissues is not strictly a coherent process, and that the forward directed SHG has a longer coherence length than the backward component, Through this treatment, we show that the emission directionality and also conversion efficiency do not arise solely from the fibril size but also depend on packing density and order of the inter-fibril structure. We demonstrate these principles in comparing the SHG response in normal and Osteogenesis Imperfecta (OI) skin. We show that the observed directionality and decreased relative intensity in the diseased state is consistent with phase matching conditions arising from the decreased fibril size and more random assembly. We further use this theory to explain the differences in morphology seen in forward and backward collected SHG in fibrillar tissues (e.g., collagenous and cellulosic). Specifically, we attribute segmented appearance to destructive interference between small fibrils separated by less than the coherence length. We suggest the approach based on relaxed phasematching conditions is general in predicting the SHG response in tissues and may be broadly applicable in interpreting the SHG contrast for diagnostic applications.

Genetic susceptibility for Alzheimer disease neuritic plaque pathology.

PubMed

Shulman, Joshua M; Chen, Kewei; Keenan, Brendan T; Chibnik, Lori B; Fleisher, Adam; Thiyyagura, Pradeep; Roontiva, Auttawut; McCabe, Cristin; Patsopoulos, Nikolaos A; Corneveaux, Jason J; Yu, Lei; Huentelman, Matthew J; Evans, Denis A; Schneider, Julie A; Reiman, Eric M; De Jager, Philip L; Bennett, David A

2013-09-01

While numerous genetic susceptibility loci have been identified for clinical Alzheimer disease (AD), it is important to establish whether these variants are risk factors for the underlying disease pathology, including neuritic plaques. To investigate whether AD susceptibility loci from genome-wide association studies affect neuritic plaque pathology and to additionally identify novel risk loci for this trait. Candidate analysis of single-nucleotide polymorphisms and genome-wide association study in a joint clinicopathologic cohort, including 725 deceased subjects from the Religious Orders Study and the Rush Memory and Aging Project (2 prospective, community-based studies), followed by targeted validation in an independent neuroimaging cohort, including 114 subjects from multiple clinical and research centers. A quantitative measure of neuritic plaque pathologic burden, based on assessments of silver-stained tissue averaged from multiple brain regions. Validation based on β-amyloid load by immunocytochemistry, and replication with fibrillar β-amyloid positron emission tomographic imaging with Pittsburgh Compound B or florbetapir. Besides the previously reported APOE and CR1 loci, we found that the ABCA7 (rs3764650; P = .02) and CD2AP (rs9349407; P = .03) AD susceptibility loci are associated with neuritic plaque burden. In addition, among the top results of our genome-wide association study, we discovered a novel variant near the amyloid precursor protein gene (APP, rs2829887) that is associated with neuritic plaques (P = 3.3 × 10-6). This polymorphism was associated with postmortem β-amyloid load as well as fibrillar β-amyloid in 2 independent cohorts of adults with normal cognition. These findings enhance understanding of AD risk factors by relating validated susceptibility alleles to increased neuritic plaque pathology and implicate common genetic variation at the APP locus in the earliest, presymptomatic stages of AD.

Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-beta (1-42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators.

PubMed

Tõugu, Vello; Karafin, Ann; Zovo, Kairit; Chung, Roger S; Howells, Claire; West, Adrian K; Palumaa, Peep

2009-09-01

Aggregation of amyloid-beta (Abeta) peptides is a central phenomenon in Alzheimer's disease. Zn(II) and Cu(II) have profound effects on Abeta aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Abeta(42) fibrillization and initiate formation of non-fibrillar Abeta(42) aggregates, and that the inhibitory effect of Zn(II) (IC(50) = 1.8 micromol/L) is three times stronger than that of Cu(II). Medium and high-affinity metal chelators including metallothioneins prevented metal-induced Abeta(42) aggregation. Moreover, their addition to preformed aggregates initiated fast Abeta(42) fibrillization. Upon prolonged incubation the metal-induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Abeta(42). H13A and H14A mutations in Abeta(42) reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross-beta core structure within region 10-23 of the amyloid fibril. Cu(II)-Abeta(42) aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)-Abeta(42) aggregates were non-toxic. Disturbed metal homeostasis in the vicinity of zinc-enriched neurons might pre-dispose formation of metal-induced Abeta aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal-chelating therapies for Alzheimer's disease is discussed in this light.

Investigation of cell cycle-associated structural reorganization in nucleolar FC/DFCs from mouse MFC cells by electron microscopy.

PubMed

Chen, Lingling; Jiao, Yang; Guan, Xin; Li, Xiliang; Feng, Yunpeng; Jiao, Mingda

2018-05-01

Nucleolus structure alters as the cell cycle is progressing. It is established in telophase, maintained throughout the entire interphase and disassembled in metaphase. Fibrillar centers (FCs), dense fibrillar components (DFCs) and granular components (GCs) are essential nucleolar organizations where rRNA transcription and processing and ribosome assembly take place. Hitherto, little is known about the cell cycle-dependent reorganization of these structures. In this study, we followed the nucleolus structure during the cell cycle by electron microscopy (EM). We found the nucleolus experienced multiple rounds of structural reorganization within a single cell cycle: (1) when nucleoli are formed during the transition from late M to G1 phase, FCs, DFCs and GCs are constructed, leading to the establishment of tripartite nucleolus; (2) as FC/DFCs are disrupted at mid-G1, tripartite nucleolus is gradually changed into a bipartite organization; (3) at late G1, the reassembly of FC/DFCs results in a structural transition from bipartite nucleolus towards tripartite nucleolus; (4) as cells enter S phase, FC/DFCs are disassembled again and tripartite nucleolus is thus changed into a bipartite organization. Of note, FC/DFCs were not observed until late S phase; (5) FC/DFCs experience structural disruption and restoration during G2 and (6) when cells are at mitotic stage, FC/DFCs disappear before nucleolus structure is disassembled. These results also suggest that bipartite nucleolus can exist in higher eukaryotes at certain period of the cell cycle. As structures are the fundamental basis of diverse cell activities, unveiling the structural reorganization of nucleolar FCs and DFCs may bring insights into the spatial-temporal compartmentalization of relevant cellular functions.

Progesterone and gravidity differentially regulate expression of extracellular matrix components in the pregnant rat myometrium.

PubMed

Shynlova, Oksana; Mitchell, Jennifer A; Tsampalieros, Anne; Langille, B Lowell; Lye, Stephen J

2004-04-01

Myometrial growth and remodeling during pregnancy depends on increased synthesis of interstitial matrix proteins. We hypothesize that the presence of mechanical tension in a specific hormonal environment regulates the expression of extracellular matrix (ECM) components in the uterus. Myometrial tissue was collected from pregnant rats on Gestational Days 0, 12, 15, 17, 19, 21, 22, 23 (labor), and 1 day postpartum and ECM expression was analyzed by Northern blotting. Expression of fibronectin, laminin beta2, and collagen IV mRNA was low during early gestation but increased dramatically on Day 23 during labor. Expression of fibrillar collagens (type I and III) peaked Day 19 and decreased near term. In contrast, elastin mRNA remained elevated from midgestation onward. Injection of progesterone (P4) on Days 20-23 (to maintain elevated plasma P4 levels) delayed the onset of labor, caused dramatic reductions in the levels of fibronectin and laminin mRNA, and prevented the fall of collagen III mRNA levels on Day 23. Treatment of pregnant rats with the progesterone receptor antagonist RU486 on Day 19 induced preterm labor on Day 20 and a premature increase in mRNA levels of collagen IV, fibronectin, and laminin. Analysis of the uterine tissue from unilaterally pregnant rats revealed that most of the changes in ECM gene expression occurred specifically in the gravid horn. Our results show a decrease in expression of fibrillar collagens and a coordinated temporal increase in expression of components of the basement membrane near term associated with decreased P4 and increased mechanical tension. These ECM changes contribute to myometrial growth and remodeling during late pregnancy and the preparation for the synchronized contractions of labor.

Spirostomum spp. (Ciliophora, Protista), a suitable system for endocytobiosis research.

PubMed

Fokin, S I; Schweikert, M; Brümmer, F; Görtz, H-D

2005-04-01

Among ciliate genera, only Paramecium and Euplotes species have been studied extensively as host organisms of bacterial endocytobionts. In this article, we show that members of the genus Spirostomum may also serve as a suitable system for endocytobiosis research. Two strains of Spirostomum minus (Heterotrichea, Ciliophora) collected in Germany and Italy, respectively, were found to harbor different types of bacterial infections. Bacteria of various sizes and shapes were observed in the cytoplasm or in the nuclei of the ciliates. The bacteria in the cytoplasm were either surrounded by a peribacterial membrane or lay naked. One of the bacterial species was found in the vicinity of the contractile fibrillar system (myonemes) of the ciliates. In rare cases, another type of bacteria was observed associated with mitochondria. The macronuclei of both the Italian and the German strains were crowded with endocytobionts. The endonuclear bacteria in the two S. minus strains differed with respect to their cytoplasmic structures but they were of similar size and both were rod shaped. According to the results of in situ hybridization, the endonuclear bacteria of the Italian strain belong to the subgroup of alphaproteobacteria, whereas the bacteria associated with the fibrillar system appeared to be gram-positive bacteria with high G+C content. While both the German and the Italian strains were found to permanently maintain their endocytobionts, they were at least partly colonized by different bacteria. This is taken as an indication that geographically separated populations of ciliates may be stably infected by different endocytobionts, possibly due to different ecological conditions. For S. minus and S. ambiguum a total of 7 different bacterial endocytobionts have now been recorded. We recommend the members of the genus Spirostomum as a suitable system for endocytobiosis research.

Amyloid-β 11C-PiB-PET imaging results from 2 randomized bapineuzumab phase 3 AD trials.

PubMed

Liu, Enchi; Schmidt, Mark E; Margolin, Richard; Sperling, Reisa; Koeppe, Robert; Mason, Neale S; Klunk, William E; Mathis, Chester A; Salloway, Stephen; Fox, Nick C; Hill, Derek L; Les, Andrea S; Collins, Peter; Gregg, Keith M; Di, Jianing; Lu, Yuan; Tudor, I Cristina; Wyman, Bradley T; Booth, Kevin; Broome, Stephanie; Yuen, Eric; Grundman, Michael; Brashear, H Robert

2015-08-25

To evaluate the effects of bapineuzumab on brain β-amyloid (Aβ) burden using (11)C-Pittsburgh compound B ((11)C-PiB)-PET. Two phase 3 clinical trials, 1 each in apolipoprotein APOE ε4 carriers and noncarriers, were conducted in patients with mild to moderate Alzheimer disease dementia. Bapineuzumab, an anti-Aβ monoclonal antibody, or placebo, was administered by IV infusion every 13 weeks for 78 weeks. PET substudies assessed change in brain fibrillar Aβ over 71 weeks using an (11)C-PiB-PET standardized uptake value ratio (SUVr) global cortical average (GCA) comprising the average SUVr from 5 cortical regions of interest with cerebellar gray matter as the reference region. A total of 115 carriers and 39 noncarriers were analyzed. The difference (δ) in mean baseline to 71 week change in (11)C-PiB-PET GCA between bapineuzumab and placebo was significant in carriers (0.5 mg/kg vs placebo δ = -0.101; p = 0.004) and in pooled analyses of both carriers and noncarriers (0.5 mg/kg vs placebo δ = -0.068; p = 0.027; 1.0 mg/kg vs placebo δ = -0.133; p = 0.028) but not in the noncarrier trial separately. Analyses by individual region of interest and in mild disease yielded findings similar to the main trial results. The (11)C-PiB-PET imaging results demonstrated reduction of fibrillar Aβ accumulation in patients with Alzheimer disease treated with bapineuzumab; however, as no clinical benefit was observed, the findings are consistent with the hypotheses that bapineuzumab may not have been initiated early enough in the disease course, the doses were insufficient, or the most critical Aβ species were inadequately targeted. © 2015 American Academy of Neurology.

In Situ Soft X-ray Spectromicroscopy of Early Tricalcium Silicate Hydration

DOE PAGES

Bae, Sungchul; Kanematsu, Manabu; Hernandez-Cruz, Daniel; ...

2016-12-01

The understanding and control of early hydration of tricalcium silicate (C 3S) is of great importance to cement science and concrete technology. However, traditional characterization methods are incapable of providing morphological and spectroscopic information about in situ hydration at the nanoscale. Using soft X-ray spectromicroscopy, we report the changes in morphology and molecular structure of C 3S at an early stage of hydration. In situ C 3S hydration in a wet cell, beginning with induction (~1 h) and acceleration (~4 h) periods of up to ~8 h, was studied and compared with ex situ measurements in the deceleration period aftermore » 15 h of curing. Analysis of the near-edge X-ray absorption fine structure showed that the Ca binding energy and energy splitting of C 3S changed rapidly in the early age of hydration and exhibited values similar to calcium silicate hydrate (C–S–H). The formation of C–S–H nanoseeds in the C 3S solution and the development of a fibrillar C–S–H morphology on the C 3S surface were visualized. Following this, silicate polymerization accompanied by C–S–H precipitation produced chemical shifts in the peaks of the main Si K edge and in multiple scattering. However, the silicate polymerization process did not significantly affect the Ca binding energy of C–S–H.« less

Investigation of the Josephin Domain protein-protein interaction by molecular dynamics.

PubMed

Deriu, Marco A; Grasso, Gianvito; Licandro, Ginevra; Danani, Andrea; Gallo, Diego; Tuszynski, Jack A; Morbiducci, Umberto

2014-01-01

Spinocerebellar ataxia (SCA) 3, the most common form of SCA, is a neurodegenerative rare disease characterized by polyglutamine tract expansion and self-assembly of Ataxin3 (At3) misfolded proteins into highly organized fibrillar aggregates. The At3 N-terminal Josephin Domain (JD) has been suggested as being responsible for mediating the initial phase of the At3 double-step fibrillogenesis. Several issues concerning the residues involved in the JD's aggregation and, more generally, the JD clumping mechanism have not been clarified yet. In this paper we present an investigation focusing on the JD protein-protein interaction by means of molecular modeling. Our results suggest possible aminoacids involved in JD contact together with local and non-local effects following JD dimerization. Surprisingly, JD conformational changes following the binding may involve ubiquitin binding sites and hairpin region even though they do not pertain to the JD interaction surfaces. Moreover, the JD binding event has been found to alter the hairpin open-like conformation toward a closed-like arrangement over the simulated timescale. Finally, our results suggest that the JD aggregation might be a multi-step process, with an initial fast JD-JD binding mainly driven by Arg101, followed by slower structural global rearrangements involving the exposure to the solvent of Leu84-Trp87, which might play a role in a second step of JD aggregation.

In Situ Soft X-ray Spectromicroscopy of Early Tricalcium Silicate Hydration

PubMed Central

Bae, Sungchul; Kanematsu, Manabu; Hernández-Cruz, Daniel; Moon, Juhyuk; Kilcoyne, David; Monteiro, Paulo J. M.

2016-01-01

The understanding and control of early hydration of tricalcium silicate (C3S) is of great importance to cement science and concrete technology. However, traditional characterization methods are incapable of providing morphological and spectroscopic information about in situ hydration at the nanoscale. Using soft X-ray spectromicroscopy, we report the changes in morphology and molecular structure of C3S at an early stage of hydration. In situ C3S hydration in a wet cell, beginning with induction (~1 h) and acceleration (~4 h) periods of up to ~8 h, was studied and compared with ex situ measurements in the deceleration period after 15 h of curing. Analysis of the near-edge X-ray absorption fine structure showed that the Ca binding energy and energy splitting of C3S changed rapidly in the early age of hydration and exhibited values similar to calcium silicate hydrate (C–S–H). The formation of C–S–H nanoseeds in the C3S solution and the development of a fibrillar C–S–H morphology on the C3S surface were visualized. Following this, silicate polymerization accompanied by C–S–H precipitation produced chemical shifts in the peaks of the main Si K edge and in multiple scattering. However, the silicate polymerization process did not significantly affect the Ca binding energy of C–S–H. PMID:28774096

Supramolecular Gel-Templated In Situ Synthesis and Assembly of CdS Quantum Dots Gels

NASA Astrophysics Data System (ADS)

Zhu, Lili; He, Jie; Wang, Xiaoliang; Li, Dawei; He, Haibing; Ren, Lianbing; Jiang, Biwang; Wang, Yong; Teng, Chao; Xue, Gi; Tao, Huchun

2017-01-01

Although many studies have attempted to develop strategies for spontaneously organizing nanoparticles (NPs) into three-dimensional (3D) geometries, it remains a fascinating challenge. In this study, a method for in situ synthesis and self-assembly of a CdS quantum dots (QDs) gel using a Cd supramolecular gel as a scaffold was demonstrated. During the QDs formation process, the Cd ions that constituted the Cd gels served as the precursors of the CdS QDs, and the oleic acid (OA) that ligated with the Cd in the supramolecular gels was capped on the surface of the CdS QDs in the form of carboxylate. The OA-stabilized CdS QDs were in situ synthesized in the entangled self-assembled fibrillar networks (SAFIN) of the Cd gels through reactions between the gelator and H2S. As a result, the QDs exactly replicated the framework of the SAFIN in the CdS QD gels instead of simply assembling along the SAFIN of the supramolecular gels. Moreover, the CdS QDs showed extraordinary sensitivity in the fluorescence detection of IO4 - anions. The facile one-step method developed here is a new approach to assembling nanostructured materials into 3D architectures and has general implications for the design of low molecular mass gelators to bring desired functionality to the developed supramolecular gels.

Characterization of the class III collagen receptor, a phosphorylated, transmembrane glycoprotein expressed in nucleated human cells.

PubMed

Carter, W G; Wayner, E A

1988-03-25

We previously identified a 90-kDa cell surface glycoprotein, termed the class III collagen receptor (CRIII), that bound to collagen in affinity chromatography experiments (Wayner, E. A., and Carter, W. G. (1987) J. Cell Biol. 105, 1873-1884). Here, we utilize monoclonal antibodies to define three domains of the CRIII, hydrophobic transmembrane, phosphorylated cytoplasmic, and glycosylated extracellular. The domain designations are based on the following characteristics. (i) Differential extraction, phase partitioning with Triton X-114, and incorporation into liposomes all indicate that the CRIII is an intrinsic membrane receptor with a hydrophobic domain. After incorporation into liposomes the CRIII binds collagen. (ii) Immunofluorescence microscopy reveals that most nucleated cells express the CRIII and that after extraction with Triton X-100, the Triton-insoluble CRIII distributes in a fibrillar pattern at the cell periphery and in closed loops that partially co-distributed with vimentin. The CRIII contains phosphoserine residues which are located on a cytoplasmic domain that may interact with the cytoskeleton. (iii) The CRIII contains 25% carbohydrate in 8-10 asparagine-linked carbohydrate chains of 2800 daltons each bound to a 65-kDa core peptide in the extracellular domain. Peptide mapping with trypsin defined a glycosylated 27-kDa extracellular fragment and a phosphorylated and glycosylated 35-kDa transmembrane fragment. These data suggest a model for the CRIII that links the cytoskeleton with the extracellular matrix.

Effect of serotonin on platelet function in cocaine exposed blood

PubMed Central

Ziu, Endrit; Hadden, Coedy; Li, Yicong; Lowery, Curtis Lee; Singh, Preeti; Ucer, Serra S.; Mercado, Charles P.; Gu, Howard H.; Kilic, Fusun

2014-01-01

5-hydroxytryptamine (5-HT) reuptake inhibitors counteract the pro-thrombotic effect of elevated plasma 5-HT by down-regulating the 5-HT uptake rates of platelets. Cocaine also down-regulates the platelet 5-HT uptake rates but in contrast, the platelets of cocaine-injected mice show a much higher aggregation rate than the platelets of control mice. To examine the involvement of plasma 5-HT in cocaine-mediated platelet aggregation, we studied the function of platelets isolated from wild-type and transgenic, peripheral 5-HT knock-out (TPH1-KO) mice, and cocaine-insensitive dopamine transporter knock in (DAT-KI) mice. In cocaine-injected mice compared to the control mice, the plasma 5-HT level as well as the surface level of P-selectin was elevated; in vitro platelet aggregation in the presence of type I fibrillar collagen was enhanced. However, cocaine injection lowered the 5-HT uptake rates of platelets and increased the plasma 5-HT levels of the DAT-KI mice but did not change their platelets aggregation rates further which are already hyper-reactive. Furthermore, the in vitro studies supporting these in vivo findings suggest that cocaine mimics the effect of elevated plasma 5-HT level on platelets and in 5-HT receptor- and transporter-dependent pathways in a two-step process propagates platelet aggregation by an additive effect of 5-HT and nonserotonergic catecholamine. PMID:25091505

A microfabricated gecko-inspired controllable and reusable dry adhesive

NASA Astrophysics Data System (ADS)

Chary, Sathya; Tamelier, John; Turner, Kimberly

2013-02-01

Geckos utilize a robust reversible adhesive to repeatedly attach and detach from a variety of vertical and inverted surfaces, using structurally anisotropic micro- and nano-scale fibrillar structures. These fibers, when suitably articulated, are able to control the real area of contact and thereby generate high-to-low van der Waals forces. Key characteristics of the natural system include highly anisotropic adhesion and shear forces for controllable attachment, a high adhesion to initial preload force ratio (μ‧) of 8-16, lack of inter-fiber self-adhesion, and operation over more than 30 000 cycles without loss of adhesion performance. A highly reusable synthetic adhesive has been developed using tilted polydimethylsiloxane (PDMS) half-cylinder micron-scale fibers, retaining up to 77% of the initial value over 10 000 repeated test cycles against a flat glass puck. In comparison with other gecko-inspired adhesives tested over 10 000 cycles or more thus far, this paper reports the highest value of μ‧, along with a large shear force of ˜78 kPa, approaching the 88-226 kPa range of gecko toes. The anisotropic adhesion forces are close to theoretical estimates from the Kendall peel model, quantitatively showing how lateral shearing articulation in a manner similar to the gecko may be used to obtain adhesion anisotropy with synthetic fibers using a combination of tilt angle and anisotropic fiber geometry.

De Novo Design and Experimental Characterization of Ultrashort Self-Associating Peptides

PubMed Central

Xue, Bo; Robinson, Robert C.; Hauser, Charlotte A. E.; Floudas, Christodoulos A.

2014-01-01

Self-association is a common phenomenon in biology and one that can have positive and negative impacts, from the construction of the architectural cytoskeleton of cells to the formation of fibrils in amyloid diseases. Understanding the nature and mechanisms of self-association is important for modulating these systems and in creating biologically-inspired materials. Here, we present a two-stage de novo peptide design framework that can generate novel self-associating peptide systems. The first stage uses a simulated multimeric template structure as input into the optimization-based Sequence Selection to generate low potential energy sequences. The second stage is a computational validation procedure that calculates Fold Specificity and/or Approximate Association Affinity (K*association) based on metrics that we have devised for multimeric systems. This framework was applied to the design of self-associating tripeptides using the known self-associating tripeptide, Ac-IVD, as a structural template. Six computationally predicted tripeptides (Ac-LVE, Ac-YYD, Ac-LLE, Ac-YLD, Ac-MYD, Ac-VIE) were chosen for experimental validation in order to illustrate the self-association outcomes predicted by the three metrics. Self-association and electron microscopy studies revealed that Ac-LLE formed bead-like microstructures, Ac-LVE and Ac-YYD formed fibrillar aggregates, Ac-VIE and Ac-MYD formed hydrogels, and Ac-YLD crystallized under ambient conditions. An X-ray crystallographic study was carried out on a single crystal of Ac-YLD, which revealed that each molecule adopts a β-strand conformation that stack together to form parallel β-sheets. As an additional validation of the approach, the hydrogel-forming sequences of Ac-MYD and Ac-VIE were shuffled. The shuffled sequences were computationally predicted to have lower K*association values and were experimentally verified to not form hydrogels. This illustrates the robustness of the framework in predicting self-associating tripeptides. We expect that this enhanced multimeric de novo peptide design framework will find future application in creating novel self-associating peptides based on unnatural amino acids, and inhibitor peptides of detrimental self-aggregating biological proteins. PMID:25010703

Including Finite Surface Span Effects in Empirical Jet-Surface Interaction Noise Models

NASA Technical Reports Server (NTRS)

Brown, Clifford A.

2016-01-01

The effect of finite span on the jet-surface interaction noise source and the jet mixing noise shielding and reflection effects is considered using recently acquired experimental data. First, the experimental setup and resulting data are presented with particular attention to the role of surface span on far-field noise. These effects are then included in existing empirical models that have previously assumed that all surfaces are semi-infinite. This extended abstract briefly describes the experimental setup and data leaving the empirical modeling aspects for the final paper.

The Role of Functional Amyloids in Multicellular Growth and Development of Gram-Positive Bacteria.

PubMed

Dragoš, Anna; Kovács, Ákos T; Claessen, Dennis

2017-08-07

Amyloid fibrils play pivotal roles in all domains of life. In bacteria, these fibrillar structures are often part of an extracellular matrix that surrounds the producing organism and thereby provides protection to harsh environmental conditions. Here, we discuss the role of amyloid fibrils in the two distant Gram-positive bacteria, Streptomyces coelicolor and Bacillus subtilis . We describe how amyloid fibrils contribute to a multitude of developmental processes in each of these systems, including multicellular growth and community development. Despite this variety of tasks, we know surprisingly little about how their assembly is organized to fulfill all these roles.

New secondary batteries utilizing electronically conductive polymer cathodes

NASA Technical Reports Server (NTRS)

Martin, Charles R.; White, Ralph E.

1989-01-01

The objectives of this project are to characterize the transport properties in electronically conductive polymers and to assess the utility of these films as cathodes in lithium/polymer secondary batteries. During this research period, progress has been made in a literature survey of the historical background, methods of preparation, the physical and chemical properties, and potential technological applications of polythiophene. Progress has also been made in the characterization of polypyrrole flat films and fibrillar films. Cyclic voltammetry and potential step chronocoulometry were used to gain information on peak currents and potentials switching reaction rates, charge capacity, and charge retention. Battery charge/discharge studies were also performed.

Mechanical suitability of glycerol-preserved human dura mater for construction of prosthetic cardiac valves.

PubMed

McGarvey, K A; Lee, J M; Boughner, D R

1984-03-01

We have examined the tensile viscoelastic properties of fresh and glycerol-preserved human dura mater, and correlated the results with structural information from the scanning electron microscope. The interwoven laminar structure of dura produces rather high flexural stiffness, while the crossed-fibrillar laminae produce planar mechanical isotropy. Glycerol storage shifts the stress-strain curve to lower strain, reduces stress relaxation and creep, and lowers the ultimate tensile strength and strain at fracture. These changes may be due to glyceraldehyde crosslinking, or to increased interfibrillar friction. The latter hypothesis suggests that glycerol storage may reduce the fatigue lifetime of the tissue.

Nanostructured thick 3D nanofibrous scaffold can induce bone.

PubMed

Eap, Sandy; Morand, David; Clauss, François; Huck, Olivier; Stoltz, Jean-François; Lutz, Jean-Christophe; Gottenberg, Jacques-Eric; Benkirane-Jessel, Nadia; Keller, Laetitia; Fioretti, Florence

2015-01-01

Designing unique nanostructured biomimetic materials is a new challenge in modern regenerative medicine. In order to develop functional substitutes for damaged organs or tissues, several methods have been used to create implants able to regenerate robust and durable bone. Electrospinning produces nonwoven scaffolds based on polymer nanofibers mimicking the fibrillar organization of bone extracellular matrix. Here, we describe a biomimetic 3D thick nanofibrous scaffold obtained by electrospinning of the biodegradable, bioresorbable and FDA-approved polymer, poly(ε-caprolactone). Such scaffold presents a thickness reaching one centimeter. We report here the demonstration that the designed nanostructured implant is able to induce in vivo bone regeneration.

The self-assembling zwitterionic form of L-phenylalanine at neutral pH.

PubMed

Mossou, Estelle; Teixeira, Susana C M; Mitchell, Edward P; Mason, Sax A; Adler-Abramovich, Lihi; Gazit, Ehud; Forsyth, V Trevor

2014-03-01

The title zwitterion (2S)-2-azaniumyl-1-hydroxy-3-phenylpropan-1-olate, C9H11NO2, also known as L-phenylalanine, was characterized using synchrotron X-rays. It crystallized in the monoclinic space group P21 with four molecules in the asymmetric unit. The 0.62 Å resolution structure is assumed to be closely related to the fibrillar form of phenylalanine, as observed by electron microscopy and electron diffraction. The structure exists in a zwitterionic form in which π-π stacking and hydrogen-bonding interactions are believed to form the basis of the self-assembling properties.

Ultrastructure of the mink parotid gland.

PubMed Central

Tandler, B

1991-01-01

Acini in the parotid gland of the North American mink (Mustela vision) are composed of seromucous cells that contain secretory granules of peculiar morphology. Many of the granules consist of a light matrix in which is embedded an inclusion made up of dense, frequently parallel rodlets in a fibrillar material of moderate density. Like the submandibular gland of the same animal, the tall cells of the parotid striated ducts contain numerous polygonal, often rhomboidal, crystalloids in their apical cytoplasm. These crystalloids are present equally in both sexes and are as abundant in the parotid as in the submandibular gland. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:1769893

Detection of Isolated Cerebrovascular β-Amyloid with Pittsburgh Compound B

PubMed Central

Greenberg, SM; Grabowski; Gurol, ME; Skehan, ME; Nandigam, RNK; Becker, JA; Garcia-Alloza, M; Prada, C; Frosch, MP; Rosand, J; Viswanathan, A; Smith, EE; Johnson, KA

2008-01-01

Imaging of cerebrovascular β-amyloid (cerebral amyloid angiopathy, CAA) is complicated by this pathology’s nearly universal overlap with Alzheimer pathology. We performed PET imaging with Pittsburgh Compound B (PiB) on 42-year old man with early manifestations of Iowa-type hereditary CAA, a form of the disorder with little or no plaque deposits of fibrillar β-amyloid. The results demonstrated elevated PiB retention selectively in occipital cortex, sparing regions typically labeled in Alzheimer disease. These results offer compelling evidence that PiB-PET can noninvasively detect isolated CAA prior to overt signs of tissue damage such as hemorrhage or white matter lesions. PMID:19067370

3D second harmonic generation imaging tomography by multi-view excitation

PubMed Central

Campbell, Kirby R.; Wen, Bruce; Shelton, Emily M.; Swader, Robert; Cox, Benjamin L.; Eliceiri, Kevin; Campagnola, Paul J.

2018-01-01

Biological tissues have complex 3D collagen fiber architecture that cannot be fully visualized by conventional second harmonic generation (SHG) microscopy due to electric dipole considerations. We have developed a multi-view SHG imaging platform that successfully visualizes all orientations of collagen fibers. This is achieved by rotating tissues relative to the excitation laser plane of incidence, where the complete fibrillar structure is then visualized following registration and reconstruction. We evaluated high frequency and Gaussian weighted fusion reconstruction algorithms, and found the former approach performs better in terms of the resulting resolution. The new approach is a first step toward SHG tomography. PMID:29541654

Evaluation of experimental coating to improve the zirconia-veneering ceramic bond strength.

PubMed

Matani, Jay D; Kheur, Mohit; Jambhekar, Shantanu Subhashchandra; Bhargava, Parag; Londhe, Aditya

2014-12-01

To evaluate the shear bond strength (SBS) between zirconia and veneering ceramic following different surface treatments of zirconia. The efficacy of an experimental zirconia coating to improve the bond strength was also evaluated. Zirconia strips were fabricated and were divided into four groups as per their surface treatment: polished (control), airborne-particle abrasion, laser irradiation, and application of the experimental coating. The surface roughness and the residual monoclinic content were evaluated before and after the respective surface treatments. A scanning electron microscope (SEM) analysis of the experimental surfaces was performed. All specimens were subjected to shear force in a universal testing machine. The SBS values were analyzed with one-way ANOVA followed by Bonferroni post hoc for groupwise comparisons. The fractured specimens were examined to observe the failure mode. The SBS (29.17 MPa) and roughness values (0.80) of the experimental coating group were the highest among the groups. The residual monoclinic content was minimal (0.32) when compared to the remaining test groups. SEM analysis revealed a homogenous surface well adhered to an undamaged zirconia base. The other test groups showed destruction of the zirconia surface. The analysis of failure following bond strength testing showed entirely cohesive failures in the veneering ceramic in all study groups. The experimental zirconia surface coating is a simple technique to increase the microroughness of the zirconia surface, and thereby improve the SBS to the veneering ceramic. It results in the least monoclinic content and produces no structural damage to the zirconia substructure. © 2014 by the American College of Prosthodontists.

CO adsorption on the “29” Cu xO/Cu(111) surface: An integrated DFT, STM, and TPD study

DOE PAGES

Hensley, Alyssa J. R.; Therrien, Andrew J.; Zhang, Renqin; ...

2016-10-04

The elucidation of an accurate atomistic model of surface structures is crucial for the design and understanding of effective catalysts, a process requiring a close collaboration between experimental observations and theoretical models. Any developed surface theoretical model must agree with experimental results for the surface when both clean and adsorbate covered. Here, we present a detailed study of the adsorption of CO on the “29” Cu xO/ Cu(111) surface, which is important in the understanding of ubiquitous Cubased catalysis. This study uses scanning tunneling microscopy, temperatureprogrammed desorption, and density functional theory to analyze CO adsorption on the “29” Cu xO/Cu(111)more » surface. From the experimental scanning tunneling microscopy images, CO was found to form six different ordered structures on the “29” Cu xO/Cu(111) surface depending on the surface CO coverage. By modeling the adsorption of CO on our atomistic model of the “29” Cu xO/Cu(111) surface at different coverages, we were able to match the experimentally observed CO ordered structures to specific combinations of sites on the “29” Cu xO/Cu(111) surface. Lastly, the high degree of agreement seen here between experiment and theory for the adsorption of CO on the “29” Cu xO/Cu(111) surface at various CO coverages provides further support that our atomistic model of the “29” Cu xO/Cu(111) surface is experimentally accurate.« less

Efficacy of platelet-rich fibrin matrix on viability of diced cartilage grafts in a rabbit model.

PubMed

Güler, İsmail; Billur, Deniz; Aydin, Sevim; Kocatürk, Sinan

2015-03-01

The objective of this study was to compare the viability of cartilage grafts embedded in platelet-rich fibrin matrix (PRFM) wrapped with no material (bare diced cartilage grafts), oxidized methylcellulose (Surgicel), or acellular dermal tissue (AlloDerm). Experimental study. In this study, six New Zealand rabbits were used. Cartilage grafts including perichondrium were excised from each ear and diced into 2-mm-by 2-mm pieces. There were four comparison groups: 1) group A, diced cartilage (not wrapped with any material); 2) group B, diced cartilage wrapped with AlloDerm; 3) group C, diced cartilage grafts wrapped with Surgicel; and 4) group D, diced cartilage wrapped with PRFM. Four cartilage grafts were implanted under the skin at the back of each rabbit. All rabbits were sacrificed at the end of 10 weeks. The cartilages were stained with hematoxylin-eosin, Masson's Trichrome, and Orcein. After that, they were evaluated for the viability of chondrocytes, collagen content, fibrillar structure of matrix, and changes in peripheral tissues. When the viability of chondrocytes, the content of fiber in matrix, and changes in peripheral tissues were compared, the cartilage embedded in the PRFM group was statistically significantly higher than in the other groups (P < 0.05). We concluded that PRFM has significant advantages in ensuring the chondrocyte viability of diced cartilage grafts. It is also biocompatible, with relatively lesser inflammation and fibrosis. © 2014 The American Laryngological, Rhinological and Otological Society, Inc.

Free energy landscapes for initiation and branching of protein aggregation.

PubMed

Zheng, Weihua; Schafer, Nicholas P; Wolynes, Peter G

2013-12-17

Experiments on artificial multidomain protein constructs have probed the early stages of aggregation processes, but structural details of the species that initiate aggregation remain elusive. Using the associative-memory, water-mediated, structure and energy model known as AWSEM, a transferable coarse-grained protein model, we performed simulations of fused constructs composed of up to four copies of the Titin I27 domain or its mutant I27* (I59E). Free energy calculations enable us to quantify the conditions under which such multidomain constructs will spontaneously misfold. Consistent with experimental results, the dimer of I27 is found to be the smallest spontaneously misfolding construct. Our results show how structurally distinct misfolded states can be stabilized under different thermodynamic conditions, and this result provides a plausible link between the single-molecule misfolding experiments under native conditions and aggregation experiments under denaturing conditions. The conditions for spontaneous misfolding are determined by the interplay among temperature, effective local protein concentration, and the strength of the interdomain interactions. Above the folding temperature, fusing additional domains to the monomer destabilizes the native state, and the entropically stabilized amyloid-like state is favored. Because it is primarily energetically stabilized, the domain-swapped state is more likely to be important under native conditions. Both protofibril-like and branching structures are found in annealing simulations starting from extended structures, and these structures suggest a possible connection between the existence of multiple amyloidogenic segments in each domain and the formation of branched, amorphous aggregates as opposed to linear fibrillar structures.

A Parametric Rosetta Energy Function Analysis with LK Peptides on SAM Surfaces.

PubMed

Lubin, Joseph H; Pacella, Michael S; Gray, Jeffrey J

2018-05-08

Although structures have been determined for many soluble proteins and an increasing number of membrane proteins, experimental structure determination methods are limited for complexes of proteins and solid surfaces. An economical alternative or complement to experimental structure determination is molecular simulation. Rosetta is one software suite that models protein-surface interactions, but Rosetta is normally benchmarked on soluble proteins. For surface interactions, the validity of the energy function is uncertain because it is a combination of independent parameters from energy functions developed separately for solution proteins and mineral surfaces. Here, we assess the performance of the RosettaSurface algorithm and test the accuracy of its energy function by modeling the adsorption of leucine/lysine (LK)-repeat peptides on methyl- and carboxy-terminated self-assembled monolayers (SAMs). We investigated how RosettaSurface predictions for this system compare with the experimental results, which showed that on both surfaces, LK-α peptides folded into helices and LK-β peptides held extended structures. Utilizing this model system, we performed a parametric analysis of Rosetta's Talaris energy function and determined that adjusting solvation parameters offered improved predictive accuracy. Simultaneously increasing lysine carbon hydrophilicity and the hydrophobicity of the surface methyl head groups yielded computational predictions most closely matching the experimental results. De novo models still should be interpreted skeptically unless bolstered in an integrative approach with experimental data.

Acidic pH retards the fibrillization of human islet amyloid polypeptide due to electrostatic repulsion of histidines

NASA Astrophysics Data System (ADS)

Li, Yang; Xu, Weixin; Mu, Yuguang; Zhang, John Z. H.

2013-08-01

The human Islet Amyloid Polypeptide (hIAPP) is the major constituent of amyloid deposits in pancreatic islets of type-II diabetes. IAPP is secreted together with insulin from the acidic secretory granules at a low pH of approximately 5.5 to the extracellular environment at a neutral pH. The increased accumulation of extracellular hIAPP in diabetes indicates that changes in pH may promote amyloid formation. To gain insights and underlying mechanisms of the pH effect on hIAPP fibrillogenesis, all-atom molecular dynamics simulations in explicit solvent model were performed to study the structural properties of five hIAPP protofibrillar oligomers, under acidic and neutral pH, respectively. In consistent with experimental findings, simulation results show that acidic pH is not conducive to the structural stability of these oligomers. This provides a direct evidence for a recent experiment [L. Khemtemourian, E. Domenech, J. P. F. Doux, M. C. Koorengevel, and J. A. Killian, J. Am. Chem. Soc. 133, 15598 (2011)], 10.1021/ja205007j, which suggests that acidic pH inhibits the fibril formation of hIAPP. In addition, a complementary coarse-grained simulation shows the repulsive electrostatic interactions among charged His18 residues slow down the dimerization process of hIAPP by twofold. Besides, our all-atom simulations reveal acidic pH mainly affects the local structure around residue His18 by destroying the surrounding hydrogen-bonding network, due to the repulsive interactions between protonated interchain His18 residues at acidic pH. It is also disclosed that the local interactions nearby His18 operating between adjacent β-strands trigger the structural transition, which gives hints to the experimental findings that the rate of hIAPP fibril formation and the morphologies of the fibrillar structures are strongly pH-dependent.

Study on the Optimization and Process Modeling of the Rotary Ultrasonic Machining of Zerodur Glass-Ceramic

NASA Astrophysics Data System (ADS)

Pitts, James Daniel

Rotary ultrasonic machining (RUM), a hybrid process combining ultrasonic machining and diamond grinding, was created to increase material removal rates for the fabrication of hard and brittle workpieces. The objective of this research was to experimentally derive empirical equations for the prediction of multiple machined surface roughness parameters for helically pocketed rotary ultrasonic machined Zerodur glass-ceramic workpieces by means of a systematic statistical experimental approach. A Taguchi parametric screening design of experiments was employed to systematically determine the RUM process parameters with the largest effect on mean surface roughness. Next empirically determined equations for the seven common surface quality metrics were developed via Box-Behnken surface response experimental trials. Validation trials were conducted resulting in predicted and experimental surface roughness in varying levels of agreement. The reductions in cutting force and tool wear associated with RUM, reported by previous researchers, was experimentally verified to also extended to helical pocketing of Zerodur glass-ceramic.

Body wall structure in the starfish Asterias rubens.

PubMed

Blowes, Liisa M; Egertová, Michaela; Liu, Yankai; Davis, Graham R; Terrill, Nick J; Gupta, Himadri S; Elphick, Maurice R

2017-09-01

The body wall of starfish is composed of magnesium calcite ossicles connected by collagenous tissue and muscles and it exhibits remarkable variability in stiffness, which is attributed to the mechanical mutability of the collagenous component. Using the common European starfish Asterias rubens as an experimental animal, here we have employed a variety of techniques to gain new insights into the structure of the starfish body wall. The structure and organisation of muscular and collagenous components of the body wall were analysed using trichrome staining. The muscle system comprises interossicular muscles as well as muscle strands that connect ossicles with the circular muscle layer of the coelomic lining. The collagenous tissue surrounding the ossicle network contains collagen fibres that form loop-shaped straps that wrap around calcite struts near to the surface of ossicles. The 3D architecture of the calcareous endoskeleton was visualised for the first time using X-ray microtomography, revealing the shapes and interactions of different ossicle types. Furthermore, analysis of the anatomical organisation of the ossicles indicates how changes in body shape may be achieved by local contraction/relaxation of interossicular muscles. Scanning synchrotron small-angle X-ray diffraction (SAXD) scans of the starfish aboral body wall and ambulacrum were used to study the collagenous tissue component at the fibrillar level. Collagen fibrils in aboral body wall were found to exhibit variable degrees of alignment, with high levels of alignment probably corresponding to regions where collagenous tissue is under tension. Collagen fibrils in the ambulacrum had a uniformly low degree of orientation, attributed to macrocrimp of the fibrils and the presence of slanted as well as horizontal fibrils connecting antimeric ambulacral ossicles. Body wall collagen fibril D-period lengths were similar to previously reported mammalian D-periods, but were significantly different between the aboral and ambulacral samples. The overlap/D-period length ratio within fibrils was higher than reported for mammalian tissues. Collectively, the data reported here provide new insights into the anatomy of the body wall in A. rubens and a foundation for further studies investigating the structural basis of the mechanical properties of echinoderm body wall tissue composites. © 2017 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.

Study of Surface Wave Propagation in Fluid-Saturated Porous Solids.

NASA Astrophysics Data System (ADS)

Azcuaga, Valery Francisco Godinez

1995-01-01

This study addresses the surface wave propagation phenomena on fluid-saturated porous solids. The analytical method for calculation of surface wave velocities (Feng and Johnson, JASA, 74, 906, 1983) is extended to the case of a porous solid saturated with a wetting fluid in contact with a non-wetting fluid, in order to study a material combination suitable for experimental investigation. The analytical method is further extended to the case of a non-wetting fluid/wetting fluid-saturated porous solid interface with an arbitrary finite surface stiffness. These extensions of the analytical method allows to theoretically study surface wave propagation phenomena during the saturation process. A modification to the 2-D space-time reflection Green's function (Feng and Johnson, JASA, 74, 915, 1983) is introduced in order to simulate the behavior of surface wave signals detected during the experimental investigation of surface wave propagation on fluid-saturated porous solids (Nagy, Appl. Phys. Lett., 60, 2735, 1992). This modification, together with the introduction of an excess attenuation for the Rayleigh surface mode, makes it possible to explain the apparent velocity changes observed on the surface wave signals during saturation. Experimental results concerning the propagation of surface waves on an alcohol-saturated porous glass are presented. These experiments were performed at frequencies of 500 and 800 kHz and show the simultaneous propagation of the two surface modes predicted by the extended analytical method. Finally an analysis of the displacements associated with the different surface modes is presented. This analysis reveals that it is possible to favor the generation of the Rayleigh surface mode or of the slow surface mode, simply by changing the type of transducer used in the generation of surface waves. Calculations show that a shear transducer couples more energy into the Rayleigh mode, whereas a longitudinal transducer couples more energy into the slow surface mode. Experimental results obtained with the modified experimental system show a qualitative agreement with the theoretical predictions.

Ab initio study of the electron-phonon coupling at the Cr(001) surface

NASA Astrophysics Data System (ADS)

Peters, L.; Rudenko, A. N.; Katsnelson, M. I.

2018-04-01

It is experimentally well established that the Cr(001) surface exhibits a sharp resonance around the Fermi level. However, there is no consensus about its physical origin. It is proposed to be either due to a single particle dz2 surface state renormalized by electron-phonon coupling or the orbital Kondo effect involving the degenerate dx z/ dy z states. In this paper we examine the electron-phonon coupling of the Cr(001) surface by means of ab-initio calculations in the form of density functional perturbation theory. More precisely, the electron-phonon mass-enhancement factor of the surface layer is investigated for the 3d states. For the majority and minority spin dz2 surface states we find values of 0.19 and 0.16. We show that these calculated electron-phonon mass-enhancement factors are not in agreement with the experimental data even if we use realistic values for the temperature range and surface Debye frequency for the fit of the experimental data. More precisely, then experimentally an electron-phonon mass-enhancement factor of 0.70 ±0.10 is obtained, which is not in agreement with our calculated values of 0.19 and 0.16. Our findings suggest that the experimentally observed resonance at the Cr(001) surface is not due to electron-phonon effects but due to electron-electron correlation effects.

The effect of bridge exercise accompanied by the abdominal drawing-in maneuver on an unstable support surface on the lumbar stability of normal adults.

PubMed

Gong, Wontae

2015-01-01

[Purpose] The present study sought to investigate the influence on static and dynamic lumbar stability of bridge exercise accompanied by an abdominal drawing-in maneuver (ADIM) performed on an uneven support surface. [Subjects] A total of 30 participants were divided into an experimental group (15 participants) and a control group (15 participants). [Methods] The experimental group performed bridge exercise on an unstable surface, whereas the control group performed bridge exercise on a stable surface. The respective bridge exercises were performed for 30 minutes, 3 times per week, for 6 weeks. The static lumbar stability (SLS) and dynamic lumbar stability (DLS) of both the experimental group and the control group were measured using a pressure biofeedback unit. [Results] In the comparison of the initial and final results of the experimental and control groups, only the SLS and DLS of the experimental group were found to be statistically significant. [Conclusion] The results of the present study show that when using bridge exercise to improve SLS and DLS, performing the bridge exercise accompanied by ADIM on an uneven surface is more effective than performing the exercise on a stable surface.

Experimental Observation of Dark Solitons on Water Surface

DTIC Science & Technology

2016-06-13

Experimental observation of dark solitons on water surface A. Chabchoub1,∗, O. Kimmoun2, H. Branger3, N. Hoffmann1, D. Proment4, M. Onorato4,5, and N...The shape and width of the soliton depend on the water depth, carrier frequency and the amplitude of the background wave. The experimental data...partic- ular, the governing equation describing the dynamics of weakly nonlinear and quasi -monochromatic waves prop- agating on the surface of water with

Imaging Fourier Transform Spectroscopy of the Boundary Layer Plume from Laser Irradiated Polymers and Carbon Materials

DTIC Science & Technology

2014-06-16

with surface desorption of the monomer. For laser-irradiated porous graphite targets, experimental results indicated a dominant CO2 production at...global models [78, 79, 87-89]. For simplicity, established global kinetics are considered and compare with experimental results obtained from IFTS...investigated up to 3 mm away from the surface into the boundary layer. At 0.72 mm from the surface, experimental results indicated a dominant production of

The stomatopod dactyl club: a formidable damage-tolerant biological hammer.

PubMed

Weaver, James C; Milliron, Garrett W; Miserez, Ali; Evans-Lutterodt, Kenneth; Herrera, Steven; Gallana, Isaias; Mershon, William J; Swanson, Brook; Zavattieri, Pablo; DiMasi, Elaine; Kisailus, David

2012-06-08

Nature has evolved efficient strategies to synthesize complex mineralized structures that exhibit exceptional damage tolerance. One such example is found in the hypermineralized hammer-like dactyl clubs of the stomatopods, a group of highly aggressive marine crustaceans. The dactyl clubs from one species, Odontodactylus scyllarus, exhibit an impressive set of characteristics adapted for surviving high-velocity impacts on the heavily mineralized prey on which they feed. Consisting of a multiphase composite of oriented crystalline hydroxyapatite and amorphous calcium phosphate and carbonate, in conjunction with a highly expanded helicoidal organization of the fibrillar chitinous organic matrix, these structures display several effective lines of defense against catastrophic failure during repetitive high-energy loading events.

Trimerization and Triple Helix Stabilization of the Collagen XIX NC2 Domain*

PubMed Central

Boudko, Sergei P.; Engel, Jürgen; Bächinger, Hans Peter

2008-01-01

The mechanisms of chain selection and assembly of fibril-associated collagens with interrupted triple helices (FACITs) must differ from that of fibrillar collagens, since they lack the characteristic C-propeptide. We analyzed two carboxyl-terminal noncollagenous domains, NC2 and NC1, of collagen XIX as potential trimerization units and found that NC2 forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. In contrast, the NC1 domain requires formation of an adjacent collagen triple helix to form interchain disulfide bridges. The NC2 domain of collagen XIX and probably of other FACITs is responsible for chain selection and trimerization. PMID:18845531

Therapeutic approaches against common structural features of toxic oligomers shared by multiple amyloidogenic proteins.

PubMed

Guerrero-Muñoz, Marcos J; Castillo-Carranza, Diana L; Kayed, Rakez

2014-04-15

Impaired proteostasis is one of the main features of all amyloid diseases, which are associated with the formation of insoluble aggregates from amyloidogenic proteins. The aggregation process can be caused by overproduction or poor clearance of these proteins. However, numerous reports suggest that amyloid oligomers are the most toxic species, rather than insoluble fibrillar material, in Alzheimer's, Parkinson's, and Prion diseases, among others. Although the exact protein that aggregates varies between amyloid disorders, they all share common structural features that can be used as therapeutic targets. In this review, we focus on therapeutic approaches against shared features of toxic oligomeric structures and future directions. Copyright © 2014 Elsevier Inc. All rights reserved.

Supra-organization and optical anisotropies of the extracellular matrix in the amniotic membrane and limbal stroma before and after explant culture

PubMed Central

Valdetaro, Gisele P.; Aldrovani, Marcela; Padua, Ivan R. M.; Cristovam, Priscila C.; Gomes, José A. P.; Laus, José L.

2016-01-01

In this research we evaluated the supramolecular organizations and the optical anisotropical properties of the de-epithelialized human amniotic membrane and rabbit limbal stroma, before and after explant culture. Birefringence, monochromatic light spectral absorption and linear dichroism of the main extracellular matrix biopolymers, that is, the fibrillar collagens and proteoglycans, were investigated by polarized light microscopy combined with image analysis. Our results demonstrated that the culture procedure–induced stimuli altered the supra-organizational characteristics (in terms of collagens/proteoglycans spatial orientation and ordered-aggregational state) of the amniotic and limbal extracellular matrix, which led to changes in optical anisotropical properties. PMID:28018719

Inhibition of Fibrillar Assemblies of l-Phenylalanine by Crown Ethers: A Potential Approach toward Phenylketonuria.

PubMed

Banik, Debasis; Dutta, Rupam; Banerjee, Pavel; Kundu, Sangita; Sarkar, Nilmoni

2016-08-11

In this article, our aim is to investigate the interaction of l-phenylalanine (l-Phe) fibrils with crown ethers (CEs). For this purpose, two different CEs (15-Crown-5 (15C5) and 18-Crown-6 (18C6)) were used. Interestingly, we have observed that both CEs have the ability to arrest fibril formation. However, 18C6 was found to be a better candidate compared to 15C5. Field emission scanning electron microscopy and fluorescence lifetime imaging microscopy were used to monitor the fibril-arresting kinetics of CEs. The arresting process was further confirmed by fluorescence correlation spectroscopy and nuclear magnetic resonance studies.

The Stomatopod Dactyl Club: A Formidable Damage-Tolerant Biological Hammer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weaver J. C.; DiMasi E.; Milliron, G.W.

2012-06-08

Nature has evolved efficient strategies to synthesize complex mineralized structures that exhibit exceptional damage tolerance. One such example is found in the hypermineralized hammer-like dactyl clubs of the stomatopods, a group of highly aggressive marine crustaceans. The dactyl clubs from one species, Odontodactylus scyllarus, exhibit an impressive set of characteristics adapted for surviving high-velocity impacts on the heavily mineralized prey on which they feed. Consisting of a multiphase composite of oriented crystalline hydroxyapatite and amorphous calcium phosphate and carbonate, in conjunction with a highly expanded helicoidal organization of the fibrillar chitinous organic matrix, these structures display several effective lines ofmore » defense against catastrophic failure during repetitive high-energy loading events.« less

Detection of isolated cerebrovascular beta-amyloid with Pittsburgh compound B.

PubMed

Greenberg, Steven M; Grabowski, Thomas; Gurol, M Edip; Skehan, Maureen E; Nandigam, R N Kaveer; Becker, John A; Garcia-Alloza, Monica; Prada, Claudia; Frosch, Matthew P; Rosand, Jonathan; Viswanathan, Anand; Smith, Eric E; Johnson, Keith A

2008-11-01

Imaging of cerebrovascular beta-amyloid (cerebral amyloid angiopathy) is complicated by the nearly universal overlap of this pathology with Alzheimer's pathology. We performed positron emission tomographic imaging with Pittsburgh Compound B on 42-year-old man with early manifestations of Iowa-type hereditary cerebral amyloid angiopathy, a form of the disorder with little or no plaque deposits of fibrillar beta-amyloid. The results demonstrated increased Pittsburgh Compound B retention selectively in occipital cortex, sparing regions typically labeled in Alzheimer's disease. These results offer compelling evidence that Pittsburgh Compound B positron emission tomography can noninvasively detect isolated cerebral amyloid angiopathy before overt signs of tissue damage such as hemorrhage or white matter lesions.

Modification of silicon nitride surfaces with GOPES and APTES for antibody immobilization: computational and experimental studies

NASA Astrophysics Data System (ADS)

Dien To, Thien; Nguyen, Anh Tuan; Nhat Thanh Phan, Khoa; Thu Thi Truong, An; Doan, Tin Chanh Duc; Mau Dang, Chien

2015-12-01

Chemical modification of silicon nitride (SiN) surfaces by silanization has been widely studied especially with 3-(aminopropyl)triethoxysilane (APTES) and 3-(glycidyloxypropyl) dimethylethoxysilane (GOPES). However few reports performed the experimental and computational studies together. In this study, surface modification of SiN surfaces with GOPES and APTES covalently bound with glutaraldehyde (GTA) was investigated for antibody immobilization. The monoclonal anti-cytokeratin-FITC (MACF) antibody was immobilized on the modified SiN surfaces. The modified surfaces were characterized by water contact angle measurements, atomic force microscopy and fluorescence microscopy. The FITC-fluorescent label indicated the existence of MACF antibody on the SiN surfaces and the efficiency of the silanization reaction. Absorption of APTES and GOPES on the oxidized SiN surfaces was computationally modeled and calculated by Materials Studio software. The computational and experimental results showed that modification of the SiN surfaces with APTES and GTA was more effective than the modification with GOPES.

Transmission Electron Microscopy of Bombyx Mori Silk Fibers

NASA Astrophysics Data System (ADS)

Shen, Y.; Martin, D. C.

1997-03-01

The microstructure of B. Mori silk fibers before and after degumming was examined by TEM, selected area electron diffraction (SAED), WAXS and low voltage SEM. SEM micrographs of the neat cocoon revealed a network of pairs of twisting filaments. After degumming, there were only individual filaments showing a surface texture consistent with an oriented fibrillar structure in the fiber interior. WAXS patterns confirmed the oriented beta-sheet crystal structure common to silkworm and spider silks. Low dose SAED results were fully consistent with the WAXS data, and revealed that the crystallographic texture did not vary significantly across the fiber diameter. TEM observations of microtomed fiber cross sections indicated a somewhat irregular shape, and also revealed a 0.5-2 micron sericin coating which was removed by the degumming process. TEM observations of the degummed silk fiber showed banded features with a characteristic spacing of nominally 600 nm along the fiber axis. These bands were oriented in a roughly parabolic or V-shape pointing along one axis within a given fiber. We hypothesize that this orientation is induced by the extrusion during the spinning process. Equatorial DF images revealed that axial and lateral sizes of the β-sheet crystallites in silk fibroin ranged from 20 to 170 nm and from 1 to 24 nm, respectively. Crazes developed in the degummed silk fiber parallel to the fiber direction. The formation of these crazes suggests that there are significant lateral interactions between fibrils in silk fibers.

Depth-Dependent Anisotropies of Amides and Sugar in Perpendicular and Parallel Sections of Articular Cartilage by Fourier Transform Infrared Imaging (FTIRI)

PubMed Central

Xia, Yang; Mittelstaedt, Daniel; Ramakrishnan, Nagarajan; Szarko, Matthew; Bidthanapally, Aruna

2010-01-01

Full thickness blocks of canine humeral cartilage were microtomed into both perpendicular sections and a series of 100 parallel sections, each 6 μm thick. Fourier Transform Infrared Imaging (FTIRI) was used to image each tissue section eleven times under different infrared polarizations (from 0° to 180° polarization states in 20° increments and with an additional 90° polarization), at a spatial resolution of 6.25 μm and a wavenumber step of 8 cm−1. With increasing depth from the articular surface, amide anisotropies increased in the perpendicular sections and decreased in the parallel sections. Both types of tissue sectioning identified a 90° difference between amide I and amide II in the superficial zone of cartilage. The fibrillar distribution in the parallel sections from the superficial zone was shown to not be random. Sugar had the greatest anisotropy in the upper part of the radial zone in the perpendicular sections. The depth-dependent anisotropic data were fitted with a theoretical equation that contained three signature parameters, which illustrate the arcade structure of collagens with the aid of a fibril model. Infrared imaging of both perpendicular and parallel sections provides the possibility of determining the three-dimensional macromolecular structures in articular cartilage. Being sensitive to the orientation of the macromolecular structure in healthy articular cartilage aids the prospect of detecting the early onset of the tissue degradation that may lead to pathological conditions such as osteoarthritis. PMID:21274999

Amyloid precursor protein and Presenilin1 interact with the adaptor GRB2 and modulate ERK 1,2 signaling.

PubMed

Nizzari, Mario; Venezia, Valentina; Repetto, Emanuela; Caorsi, Valentina; Magrassi, Raffaella; Gagliani, Maria Cristina; Carlo, Pia; Florio, Tullio; Schettini, Gennaro; Tacchetti, Carlo; Russo, Tommaso; Diaspro, Alberto; Russo, Claudio

2007-05-04

The amyloid precursor protein (APP) and the presenilins 1 and 2 are genetically linked to the development of familial Alzheimer disease. APP is a single-pass transmembrane protein and precursor of fibrillar and toxic amyloid-beta peptides, which are considered responsible for Alzheimer disease neurodegeneration. Presenilins are multipass membrane proteins, involved in the enzymatic cleavage of APP and other signaling receptors and transducers. The role of APP and presenilins in Alzheimer disease development seems to be related to the formation of amyloid-beta peptides; however, their physiological function, reciprocal interaction, and molecular mechanisms leading to neurodegeneration are unclear. APP and presenilins are also involved in multiple interactions with intracellular proteins, the significance of which is under investigation. Among the different APP-interacting proteins, we focused our interest on the GRB2 adaptor protein, which connects cell surface receptors to intracellular signaling pathways. In this study we provide evidence by co-immunoprecipitation experiments, confocal and electron microscopy, and by fluorescence resonance energy transfer experiments that both APP and presenilin1 interact with GRB2 in vesicular structures at the centrosome of the cell. The final target for these interactions is ERK1,2, which is activated in mitotic centrosomes in a PS1- and APP-dependent manner. These data suggest that both APP and presenilin1 can be part of a common signaling pathway that regulates ERK1,2 and the cell cycle.

Involvement of human decidual cell-expressed tissue factor in uterine hemostasis and abruption.

PubMed

Lockwood, C J; Paidas, M; Murk, W K; Kayisli, U A; Gopinath, A; Huang, S J; Krikun, G; Schatz, F

2009-11-01

Vascular injury increases access and binding of plasma-derived factor VII to perivascular cell membrane-bound tissue factor (TF). The resulting TF/VIIa complex promotes hemostasis by cleaving pro-thrombin to thrombin leading to the fibrin clot. In human pregnancy, decidual cell-expressed TF prevents decidual hemorrhage (abruption). During placentation, trophoblasts remodel decidual spiral arteries into high conductance vessels. Shallow trophoblast invasion impedes decidual vascular conversion, producing an inadequate uteroplacental blood flow that elicits abruption-related placental ischemia. Thrombin induces several biological effects via cell surface protease activated receptors. In first trimester human DCs thrombin increases synthesis of sFlt-1, which elicits placental ischemia by impeding angiogenesis-related decidual vascular remodeling. During pregnacy, the fibrillar collagen-rich amnion and choriodecidua extracellular matrix (ECM) provides greater than additive tensile strength and structural integrity. Thrombin acts as an autocrine/paracrine mediator that degrades these ECMs by augmenting decidual cell expression of: 1) matrix metalloproteinases and 2) interleukin-8, a key mediator of abruption-associated decidual infiltration of neutrophils, which express several ECM degrading proteases. Among the cell types at the maternal fetal interface at term, TF expression is highest in decidual cells indicating that this TF meets the hemostatic demands of labor and delivery. TF expression in cultured term decidual cells is enhanced by progestin and thrombin suggesting that the maintenance of elevated circulating progesterone provides hemostatic protection and that abruption-generated thrombin acts in an autocrine/paracrine fashion on decidual cells to promote hemostasis via enhanced TF expression.

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

PubMed Central

Hunt, Geoffrey C.; Singh, Purva; Schwarzbauer, Jean E.

2012-01-01

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. PMID:22710062

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells.

PubMed

Hunt, Geoffrey C; Singh, Purva; Schwarzbauer, Jean E

2012-09-10

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. Copyright © 2012 Elsevier Inc. All rights reserved.

A Cytochemical Study of Extracellular Sheaths Associated with Rigidoporus lignosus during Wood Decay

PubMed Central

Nicole, M.; Chamberland, H.; Rioux, D.; Lecours, N.; Rio, B.; Geiger, J. P.; Ouellette, G. B.

1993-01-01

An ultrastructural and cytochemical investigation of the development of Rigidoporus lignosus, a white-rot fungus inoculated into wood blocks, was carried out to gain better insight into the structure and role of the extracellular sheaths produced by this fungus during wood degradation. Fungal sheaths had a dense or loose fibrillar appearance and were differentiated from the fungal cell wall early after wood inoculation. Close association between extracellular fibrils and wood cell walls was observed at both early and advanced stages of wood alteration. Fungal sheaths were often seen deep in host cell walls, sometimes enclosing residual wood fragments. Specific gold probes were used to investigate the chemical nature of R. lignosus sheaths. While labeling of chitin, pectin, β-1,4- and β-1,3-glucans, β-glucosides, galactosamine, mannose, sialic acid, RNA, fucose, and fimbrial proteins over fungal sheaths did not succeed, galactose residues and laccase (a fungal phenoloxidase) were found to be present. The positive reaction of sheaths with the PATAg test indicates that polysaccharides such as β-1,6-glucans are important components. Our data suggest that extracellular sheaths produced by R. lignosus during host cell colonization play an important role in wood degradation. Transportation of lignin-degrading enzymes by extracellular fibrils indicates that alteration of plant polymers may occur within fungal sheaths. It is also proposed that R. lignosus sheaths may be involved in recognition mechanisms in fungal cell-wood surface interactions. Images PMID:16349017

In Situ Evaluation of Calcium Phosphate Nucleation Kinetics and Pathways during Intra- and Extrafibrillar Mineralization of Collagen Matrices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Doyoon; Lee, Byeongdu; Thomopoulos, Stavros

Calcium phosphate (CaP) nanocrystals nucleate and grow in intrafibrillar and/or extrafibrillar spaces of collagen fibrils during the mineralization of bones and teeth. Little is known about the early stages of CaP nucleation and distribution in fibrillar matrices, despite their significant influence on the physical and chemical structures of tissue-level constructs. Using in situ small angle X-ray scattering (SAXS), we examined the nucleation and growth of CaP within collagen matrices and elucidated how a nucleation inhibitor, polyaspartic acid (pAsp), governs mineralization kinetics and pathways at multiple length scales. In situ SAXS analysis clearly revealed that nucleation sites, kinetically-controlled by the nucleationmore » inhibitor, determined the pathways of CaP morphological transformation. Mineralization with pAsp led to intrafibrillar CaP plates with a spatial distribution gradient through the depth of the matrix. Mineralization without pAsp led initially to spherical aggregates of CaP in the entire extrafibrillar spaces. With time, the spherical aggregates transformed into plates at the outermost surface of the collagen matrix, preventing intrafibrillar mineralization inside. The results illuminate mineral nucleation kinetics and real-time nanoparticle distributions within organic matrices in solutions containing body fluid components. Because the macroscale mechanical properties of collagen matrices depend on their mineral content, phase, and arrangement at the nanoscale, this study contributes to better design and fabrication of biomaterials for regenerative medicine.« less

VANGL2 interacts with integrin αv to regulate matrix metalloproteinase activity and cell adhesion to the extracellular matrix.

PubMed

Jessen, Tammy N; Jessen, Jason R

2017-12-15

Planar cell polarity (PCP) proteins are implicated in a variety of morphogenetic processes including embryonic cell migration and potentially cancer progression. During zebrafish gastrulation, the transmembrane protein Vang-like 2 (VANGL2) is required for PCP and directed cell migration. These cell behaviors occur in the context of a fibrillar extracellular matrix (ECM). While it is thought that interactions with the ECM regulate cell migration, it is unclear how PCP proteins such as VANGL2 influence these events. Using an in vitro cell culture model system, we previously showed that human VANGL2 negatively regulates membrane type-1 matrix metalloproteinase (MMP14) and activation of secreted matrix metalloproteinase 2 (MMP2). Here, we investigated the functional relationship between VANGL2, integrin αvβ3, and MMP2 activation. We provide evidence that VANGL2 regulates cell surface integrin αvβ3 expression and adhesion to fibronectin, laminin, and vitronectin. Inhibition of MMP14/MMP2 activity suppressed the cell adhesion defect in VANGL2 knockdown cells. Furthermore, our data show that MMP14 and integrin αv are required for increased proteolysis by VANGL2 knockdown cells. Lastly, we have identified integrin αvβ3 as a novel VANGL2 binding partner. Together, these findings begin to dissect the molecular underpinnings of how VANGL2 regulates MMP activity and cell adhesion to the ECM. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Fine structure of the pecten oculi of the barred owl (Strix varia).

PubMed

Smith, B J; Smith, S A; Braekevelt, C R

1996-01-01

The pecten oculi of the barred owl (Strix varia) has been examined by light and transmission electron microscopy. The pecten in this species is of the pleated type and is small in comparison to the size of the ocular globe. The pecten consists of 8-10 accordion-like folds that are linked apically by a pigmented tissue bridge. Each fold contains numerous capillaries, larger supply and drainage vessels, and abundant pleomorphic melanocytes. Most of these capillaries are extremely specialized vessels that possess plentiful microfolds on both the luminal and abluminal surfaces. Some capillaries however display only a few microfolds. The endothelial cell bodies are extremely attenuated, with most organelles located near the nucleus. All capillaries are surrounded by a very thick fibrillar basal lamina, which is thought to provide structural support to these small vessels. Pericytes are commonly found within these thickened basal laminae. Numerous melanocytes are also present, with processes that form an incomplete sheath around the capillaries. These processes are also presumed to provide structural support for the capillaries. As in other avian species, the morphology of the barred owl pecten is indicative of extensive involvement in substance transport. When compared to the pecten of more visually-oriented species, this pecten is smaller, has fewer folds, and displays a reduced number of microfolds within the capillaries. In these and other features, the barred owl pecten is similar to the pecten of the great horned owl (Bubo virginianus).

A Nonconventional Approach to Patterned Nanoarrays of DNA Strands for Template-Assisted Assembly of Polyfluorene Nanowires.

PubMed

Bae, Dong Geun; Jeong, Ji-Eun; Kang, Seok Hee; Byun, Myunghwan; Han, Dong-Wook; Lin, Zhiqun; Woo, Han Young; Hong, Suck Won

2016-08-01

DNA molecules have been widely recognized as promising building blocks for constructing functional nanostructures with two main features, that is, self-assembly and rich chemical functionality. The intrinsic feature size of DNA makes it attractive for creating versatile nanostructures. Moreover, the ease of access to tune the surface of DNA by chemical functionalization offers numerous opportunities for many applications. Herein, a simple yet robust strategy is developed to yield the self-assembly of DNA by exploiting controlled evaporative assembly of DNA solution in a unique confined geometry. Intriguingly, depending on the concentration of DNA solution, highly aligned nanostructured fibrillar-like arrays and well-positioned concentric ring-like superstructures composed of DNAs are formed. Subsequently, the ring-like negatively charged DNA superstructures are employed as template to produce conductive organic nanowires on a silicon substrate by complexing with a positively charged conjugated polyelectrolyte poly[9,9-bis(6'-N,N,N-trimethylammoniumhexyl)fluorene dibromide] (PF2) through the strong electrostatic interaction. Finally, a monolithic integration of aligned arrays of DNA-templated PF2 nanowires to yield two DNA/PF2-based devices is demonstrated. It is envisioned that this strategy can be readily extended to pattern other biomolecules and may render a broad range of potential applications from the nucleotide sequence and hybridization as recognition events to transducing elements in chemical sensors. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Entropic and Electrostatic Effects on the Folding Free Energy of a Surface-Attached Biomolecule: An Experimental and Theoretical Study

PubMed Central

Watkins, Herschel M.; Vallée-Bélisle, Alexis; Ricci, Francesco; Makarov, Dmitrii E.; Plaxco, Kevin W.

2012-01-01

Surface-tethered biomolecules play key roles in many biological processes and biotechnologies. However, while the physical consequences of such surface attachment have seen significant theoretical study, to date this issue has seen relatively little experimental investigation. In response we present here a quantitative experimental and theoretical study of the extent to which attachment to a charged –but otherwise apparently inert– surface alters the folding free energy of a simple biomolecule. Specifically, we have measured the folding free energy of a DNA stem loop both in solution and when site-specifically attached to a negatively charged, hydroxyl-alkane-coated gold surface. We find that, whereas surface attachment is destabilizing at low ionic strength it becomes stabilizing at ionic strengths above ~130 mM. This behavior presumably reflects two competing mechanisms: excluded volume effects, which stabilize the folded conformation by reducing the entropy of the unfolded state, and electrostatics, which, at lower ionic strengths, destabilizes the more compact folded state via repulsion from the negatively charged surface. To test this hypothesis we have employed existing theories of the electrostatics of surface-bound polyelectrolytes and the entropy of surface-bound polymers to model both effects. Despite lacking any fitted parameters, these theoretical models quantitatively fit our experimental results, suggesting that, for this system, current knowledge of both surface electrostatics and excluded volume effects is reasonably complete and accurate. PMID:22239220

Experimental and computational surface and flow-field results for an all-body hypersonic aircraft

NASA Technical Reports Server (NTRS)

Lockman, William K.; Lawrence, Scott L.; Cleary, Joseph W.

1990-01-01

The objective of the present investigation is to establish a benchmark experimental data base for a generic hypersonic vehicle shape for validation and/or calibration of advanced computational fluid dynamics computer codes. This paper includes results from the comprehensive test program conducted in the NASA/Ames 3.5-foot Hypersonic Wind Tunnel for a generic all-body hypersonic aircraft model. Experimental and computational results on flow visualization, surface pressures, surface convective heat transfer, and pitot-pressure flow-field surveys are presented. Comparisons of the experimental results with computational results from an upwind parabolized Navier-Stokes code developed at Ames demonstrate the capabilities of this code.

Experimental and computational studies of positron-stimulated ion desorption from TiO2(1 1 0) surface

NASA Astrophysics Data System (ADS)

Yamashita, T.; Hagiwara, S.; Tachibana, T.; Watanabe, K.; Nagashima, Y.

2017-11-01

Experimental and computational studies of the positron-stimulated O+ ion desorption process from a TiO2(1 1 0) surface are reported. The measured data indicate that the O+ ion yields depend on the positron incident energy in the energy range between 0.5 keV and 15 keV. This dependence is closely related to the fraction of positrons which diffuse back to the surface after thermalization in the bulk. Based on the experimental and computational results, we conclude that the ion desorption via positron-stimulation occurs dominantly by the annihilation of surface-trapped positrons with core electrons of the topmost surface atoms.