Thick Fibrils Research Articles

Comment on "Extracellular Vesicles Slow Down Aβ(1-42) Aggregation by Interfering with the Amyloid Fibril Elongation Step".

Halipi et al. explored the impact of extracellular vesicles (EVs) on amyloid-β (Aβ) aggregation. They concluded that EVs reduce Aβ aggregation, as seen by shorter and thicker fibrils. While we agree with the complex role of EVs in Alzheimer's disease, we are sceptical of the claim that EVs slow down Aβ aggregation, noting missing key references. Previous literature rather suggests that EVs (derived from neuronal cell lines) accelerate the process of Aβ fibrillation and plaque formation. Halipi et al.'s findings may be skewed due to the lack of essential neuronally expressed Aβ-binding partners, like the prion protein (PrPC) in their EV samples. The commentary, in the light of included original experiments and cited literature, suggests that membrane proteins like PrPC are crucial to fully understand the role of EVs in Aβ aggregation, and Halipi et al.'s conclusions should be reexamined in light of these factors.

Effect of Quadriceps Tendon Autograft Preparation and Fixation on Graft Laxity During Suspensory Anterior Cruciate Ligament Reconstruction: A Biomechanical Analysis.

Favorable collagen fibril density and thickness combined with advances in graft preparation and fixation have significantly increased interest in the quadriceps tendon (QT) autograft for anterior cruciate ligament (ACL) reconstruction. While various suspensory techniques have been described, the biomechanical profile of these constructs is largely undefined. To compare the biomechanics of suspensory techniques for soft tissue QT autograft fixation in an in vitro model of ACL reconstruction. Controlled laboratory study. Full-thickness QT grafts were harvested using a 9-mm graft blade. Adjustable-loop devices (ALDs) were secured to the graft (n = 6 per group) with a combination implant containing the ALD and suture tape-reinforced whipstitching (tape-reinforced [TR] group), tethered superficially to the graft with a whipstitch (onlay [OL] group), luggage-tagged through and around the graft (luggage tag [LT] group), or staggered behind superficial suturing (staggered [SG] group). Grafts were tested on an electromechanical testing machine following a validated in vitro reconstruction model of intraoperative workflow and postoperative ACL kinematics, cyclic loading, and load to failure. The TR group had significantly less postcyclic tension loss (mean, 24%) compared with the OL (56%; P = .002), LT (69%; P < .001), and SG (90%; P < .001) constructs. Cyclic elongation was below the 3.0-mm threshold defined as clinical failure for TR (1.6 mm), but not for OL (3.3 mm), LT (7.9 mm), and SG (11.3 mm). All constructs were within native ACL stiffness limits (220 ± 72 N/mm) without significant differences. Ultimate loads significantly exceeded a normal ACL loading limit of 454 N for TR (739 N; P = .023), OL (547 N; P = .020), and LT (769 N; P = .001), but not for SG (346 N; P = .236). The TR ALD construct demonstrated the most favorable time-zero biomechanical properties of modern soft tissue QT suspensory constructs, with 32% less tension loss and 52% less cyclic elongation versus the closest construct. Failure loading of all constructs was acceptable with respect to the native ACL except for the SG group, which had suboptimal ultimate load. TR ALD implants may protect soft tissue QT autografts before graft-bone healing in ACL reconstruction by minimizing time-zero laxity and fixation failure.

Abstract A054: Collagen fiber architecture modulates PDAC cell and organoid phenotype

Abstract Introduction Collagen plays a significant role in the development of pancreatic ductal adenocarcinoma (PDAC). Disease progression is mediated by a dense collagenous stroma, which acts as a barrier to drug delivery and immune cell access. Current in vitro models do not well represent the collagenous stroma seen in PDAC. The aim of this study is to develop a fibrillar collagen-based hydrogel system which mimics the tumour microenvironment seen in PDAC and assess cell-mediated collagen remodeling. Methods Metastatic SUIT2-007 pancreatic cancer cells, non-metastatic BxPC3 pancreatic cancer cells, and human pancreatic stellate cells (hPSCs) were encapsulated in bovine type I collagen gels of various concentrations. KPC mouse derived tumour organoids were also encapsulated in bovine type I collagen gels of 2 mg/ml and 6 mg/ml concentrations, basement membrane extract (BME), and a combination of BME and collagen gel. Cell viability and proliferation was assessed using Live/Dead imaging and CellTitre-Glo 3D respectively. Cell mediated collagen remodeling was assessed via monitoring of gel contraction on the mm scale, and confocal reflectance microscopy on the cellular scale. Collagen contraction around cells was assessed by measuring the pixel intensity of collagen fibrils within the vicinity of the cell. Cell phenotype was assessed via immunofluorescent staining and qPCR. Results Collagen concentration alters the hydrogel structure and stiffness, with 3 mg/ml collagen resulting in straighter, shorter and thicker fibrils. Metastatic SUIT-007 cells contracted collagen gels more rapidly than non-metastatic BxPC3 cells, with both achieving similar contraction by day 7. SUIT2-007 cells remodel and align soft 1 mg/ml gels while 3 mg/ml gels proved to be sufficiently stiff to resist remodeling. Cellular collagen association was increased in more elongated SUIT-007 cells than in BxPC3 cells. Organoid proliferation varies with hydrogel collagen concentration. Conclusion Metastatic SUIT2-007 cells were able to remodel collagen more rapidly and to a greater extent than non-metastatic BxPC3 cells. Collagen supports an invasive organoid phenotype. Collagen gels combined with confocal reflectance microscopy provide a platform for the quantitative assessment of cell-matrix interactions in PDAC. Acknowledgements This project was funded by a Medical Traineeship from UCD School of Medicine, and summer student scholarships from Irish Cancer Society and Breakthrough Cancer Research. We acknowledge the Conway Imaging Core Facility for assistance with microscopy. Citation Format: Ciara L Doyle, Shanu Xavier, Jarren Y.S Lu, Leah Fallon, Anwesha Sarkar, Jan J Baguyo, Maela Gars, Stephen D Thorpe. Collagen fiber architecture modulates PDAC cell and organoid phenotype [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A054.

Development of Tissue-Engineered Model of Fibrotic Scarring after Spinal Cord Injury to Study Astrocyte Activation and Neurite Outgrowth In Vitro.

Traumatic spinal cord injuries (SCI) are debilitating injuries affecting twenty-seven million people worldwide and cause functional impairments. Despite decades of research and medical advancements, current treatment options for SCI remain limited, in part due to the complex pathophysiology of spinal cord lesions including cellular transformation and extracellular matrix (ECM) remodeling. Recent studies have increased focus on fibrotic scarring after SCI, and yet much remains unclear about the impact of fibrotic scarring on SCI lesion progression. Here, using collagen and decellularized spinal cord-based composite hydrogels, a three-dimensional (3D) cell culture model mimicking the fibrous core of spinal cord lesions was implemented to investigate its influence on the surrounding astrocytes. To mimic the fibrotic milieu, collagen fibril thickness was tuned using previously established temperature-controlled casting methods. In our platforms, astrocytes in fibro-mimetic hydrogels exhibited increased levels of activation markers such as glial fibrillary acidic protein and N-cadherin. Furthermore, astrocytes in fibro-mimetic hydrogels deposited more fibronectin and laminin, further hinting that astrocytes may also contribute to fibrotic scarring. These markers were decreased when Rho-ROCK and integrin β1 were inhibited via pharmacological inhibitors. Mechanistic analysis of Yes-associated protein reveals that blocking integrin β1 prevents mechanosensing of astrocytes, contributing to altered phenotypes in variable culture conditions. In the presence of these inhibitors, astrocytes increased the secretion of brain-derived neurotrophic factor, and a greater degree of dorsal root ganglia neurite infiltration into the underlying hydrogels was observed. Altogether, this study presents a novel tissue-engineered platform to study fibrotic scarring after SCI and may be a useful platform to advance our understanding of SCI lesion aggravation.

Modeling collagen fibril degradation as a function of matrix microarchitecture.

Collagenolytic degradation is a process fundamental to tissue remodeling. The microarchitecture of collagen fibril networks changes during development, aging, and disease. Such changes to microarchitecture are often accompanied by changes in matrix degradability. In vitro, collagen matrices of the same concentration but different microarchitectures also vary in degradation rate. How do different microarchitectures affect matrix degradation? To answer this question, we developed a computational model of collagen degradation. We first developed a lattice model that describes collagen degradation at the scale of a single fibril. We then extended this model to investigate the role of microarchitecture using Brownian dynamics simulation of enzymes in a multi-fibril three dimensional matrix to predict its degradability. Our simulations predict that the distribution of enzymes around the fibrils is non-uniform and depends on the microarchitecture of the matrix. This non-uniformity in enzyme distribution can lead to different extents of degradability for matrices of different microarchitectures. Our model predictions were tested using in vitro experiments with synthesized collagen gels of different microarchitectures. Experiments showed that indeed degradation of collagen depends on the matrix architecture and fibril thickness. In summary, our study shows that the microarchitecture of the collagen matrix is an important determinant of its degradability.

Lutein, a carotenoid found in numerous plants and the human eye, demonstrates the capacity to bundle collagen fibrils

Collagen fibrils serve as the building blocks of the extracellular matrix, providing a resilient and structural framework for tissues. However, the bundling of collagen fibrils is of paramount importance in maintaining the structural integrity and functionality of various tissues in the human body. In this scenario, there is limited exploration of molecules that promote the bundling of collagen fibrils. Investigating the interactions of well-known carotenoids, commonly associated with ocular health, particularly in the retina, with collagen presents a novel and significant area of study. Here, we studied the influence of lutein, a well-known carotenoid present in many plant tissues and has several biological properties, on the structure, thermal stability, self-assembly, and fibrillation of collagen. Fibrillation kinetics and electron microscopic analyses indicated that lutein did not interfere with fibrillation process of collagen, whereas it enhances the lateral fusion of collagen fibrils leading to the formation of compact bundles of thick fibrils under physiological conditions. The hydrophobic and hydrogen bonding interactions between lutein and collagen fibrils are most likely the cause of the bundling of the fibrils. This study establishes the first investigation of collagen-carotenoid interactions, showcasing the unique property of lutein in bundling collagen fibrils, which may find potential application in tissue engineering.

Modeling the deterioration of the stiffness and of the collagen fibril distribution in a discrete model of the cornea microstructure

We present a stochastic approach to model the mechanical deterioration of the reinforcing microstructure of the human cornea. The fundamental structural micro-components of the stroma, collagen and crosslinks, are assembled deterministically into an elementary trusswork cell, multiply repeated and distorted to form a three-dimensional shell with the shape of a cornea. The spatial orientation of the collagen-like elements of each cell is thus characterized stochastically with a non correlated random angle field, obeying an assigned probability density function, leading to a non-deterministic structural stiffness. It follows that the mechanical response of the model to the action of deterministic forces equivalent to the intraocular pressure is stochastic due to the random spatial orientation of collagen fibers. The deterioration of the mechanical stiffness of the collagen components is described through a scalar variable field, evolving in space and in time, representative of a progressive damage which causes heterogeneity and asymmetric behavior. The damage variable acts in two ways on the global stiffness: (i) by reducing the stiffness of the collagen components; (ii) by modifying the dispersion coefficient of the probability density function. The equilibrium equations of the damaging model are solved at discrete time steps, with a fully explicit solution scheme, by means of the stochastic finite element improved perturbation method. The results show that when the collagen fibril stiffness reduces to 10% of the healthy value, as expected in the case of the vision-impairing condition known as keratoconus, the displacement field due to intraocular pressure is significantly affected in terms of both average and variance distributions. This effect confers a typical conical shape to the cornea. In particular, the analysis shows that high values of the response variances are confined in the keratoconus area, which agrees with a high level of uncertainties due to loss of fibril organization and thickness reduction under pathologic conditions.

Maturation-dependent changes in the size, structure and seeding capacity of Aβ42 amyloid fibrils

Many proteins self-assemble to form amyloid fibrils, which are highly organized structures stabilized by a characteristic cross-β network of hydrogen bonds. This process underlies a variety of human diseases and can be exploited to develop versatile functional biomaterials. Thus, protein self-assembly has been widely studied to shed light on the properties of fibrils and their intermediates. A still open question in the field concerns the microscopic processes that underlie the long-time behaviour and properties of amyloid fibrillar assemblies. Here, we use atomic force microscopy with angstrom-sensitivity to observe that amyloid fibrils undergo a maturation process, associated with an increase in both fibril length and thickness, leading to a decrease of their density, and to a change in their cross-β sheet content. These changes affect the ability of the fibrils to catalyse the formation of new aggregates. The identification of these changes helps us understand the fibril maturation processes, facilitate the targeting of amyloid fibrils in drug discovery, and offer insight into the development of biocompatible and sustainable protein-based materials.

Inhibition of cytotoxic self-assembly of HEWL through promoting fibrillation by new synthesized α-hydroxycarbamoylphosphinic acids.

The main objective of the present study is to investigate the potency of new synthesized hydroxycarbamoyl phosphinic acid derivatives in modulating cytotoxic fibrillogenesis of hen egg white lysozyme (HEWL), as a common model in protein aggregation studies. Hydroxycarbamoyl phosphinic acid derivatives were prepared by the reaction of α-hydroxyalkylphosphinic acids with isocyanates (or isothiocyanates) in the presence of trimethylsilyl chloride (TMSCl). The designed process involves the condensation reaction leading to formation of new C sp2-P bond formation. The synthesis and purity of novel designed compounds were confirmed by NMR, LC-MS, and HPLC techniques. A range of experiments, including thioflavin T (ThT) and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence assays, Congo red binding measurement, atomic force microscopy imaging, MTT-based cell viability and hemolysis assays were employed to investigate anti-amyloidogenic effects of tested compounds. The obtained results demonstrate that these compounds are able to significantly modulate the self-assembly process of HEWL via shortening of nucleation phase leading to the acceleration of fibrillation and appearance of very large and thick fibrils with decreased surface hydrophobicity and cytotoxicity. Based on ANS binding data, we suggest that increased exposure of hydrophobic patches of oligomeric species is the possible mechanism by which tested compounds promote self-assembly process of HEWL. Fluorescence anisotropy and molecular docking studies indicate the interaction of both synthesized compounds with HEWL, and more specifically with residues that are situated in the highly aggregation-prone β-domain region of protein. This study unveils the potential of hydroxyalkylphosphinic acids as modulators of amyloid fibrillation highlighting these compounds as a promising approach for targeting protein aggregates associated with neurodegenerative diseases.

TRANSMISSION ELECTRON MICROSCOPY FOR THE DIRECT ANALYSIS OF FIBRIN CLOT STRUCTURE

The aim of our study was to compare the structure of clots formed as a result of thrombin-induced fibrin polymerization in the presence or absence of monoclonal fibrin-specific antibodies fragments as factors that change the clot structure. We concentrated on the final stage of fibrin clot formation at maximal turbidity point for every sample. Methods. Fibrin polymerization was studied by transmission electron microscopy (TEM) of negatively contrasted samples on H-600 Transmission Electron Microscope (“Hitachi”,Japan); 1% water solution of uranyl acetate (“Merck”, Germany) was used as a negative contrast. For sample preparation, in sterile glass tubes were sequentially added 0.32 mg/mL human fibrinogen, 0.025 M CaCl2 in 0.05 M ammonium formiate buffer (pH 7.9), and a total sample volume was 0.22 mL. The polymerization of fibrin was initiated by the introduction of thrombin at a final concentration of 0.25 NIH/mL. After 180 s, aliquots were taken from the polymerization medium. Each aliquot was diluted to a final fibrinogen concentration of 0.07 mg/mL; 0.01 mL probes of fibrinogen solution were transferred to a carbon lattice, which was treated with a 1% uranyl acetate solution after 2 minutes. Investigations were per-formed using an H-600 electron microscope at 75 kV. Electron microscopic images were obtained at magnification of 20,000 -50,000. Results. Two monoclonal antibodies fragments were obtained towards the mixture of separated Aα-, Bβ- and γ-chains of fibrinogen. Antibodies fragments that were marked as III-1D and I-4A, had different epitopes within fragment Аα105-206 of D-region of fibrinogen. It was shown that addition of antibody fragment I-4A lead to formation of abnormal fibrils that were thinner than in the control sample and were organized in the dense network (Figure). Control sample exhibited the thick fibrils with well-structured classically organized network. The difference between control and I-4A samples demonstrated that antibody I-4A disrupted the structure of polymerized fibrin. In the same time the fibrils obtained in the presence of antibody fragment III-1D were closer to the control ones. Conclusions. TEM is an informative method for the study of the fibrin network formation. Its application allows to estimate the disruption in fi brin formation directly. In a combination with turbidity study and other functional tests TEM can provide important information about molecular mechanisms of clot formation.

Antitumor Effect of Iscador on Breast Cancer Cell Lines with Different Metastatic Potential

Studies were performed for the first time on the effect of Iscador Qu and Iscador M on phototoxicity, cytotoxicity, antiproliferative activity, changes in ξ-potential of cells, membrane lipid order, actin cytoskeleton organization and migration on three breast cancer lines with different metastatic potential: MCF10A (control), MCF-7 (low metastatic) and MDA-MB231 (high metastatic) cells. The tested Iscador Qu and M did not show any phototoxicity. The antiproliferative effect of Iscador species appeared to be dose-dependent and was related to the metastatic potential of the tested cell lines. A higher selectivity index was obtained for Iscador Qu and M towards the low metastatic MCF-7 cell line compared to the high metastatic MDA-MB-231. Iscador Qu demonstrated higher selectivity for both cancer cell lines compared to Iscador M. The malignant cell lines exhibited a decrease in fibril number and thickness regardless of the type of Iscador used. The strongest effect on migration potential was observed for the low metastatic cancer cell line MCF-7 after Iscador treatment. Both Iscador species induced a slight increase in the percentage of cells in early apoptosis for the low and high metastatic cell lines, MCF-7 and MDA-MB-231, unlike control cells. Changes in the zeta potential and membrane lipid order were observed for the low metastatic MCF-7 cell line in contrast to the high metastatic MDA-MB-231 cells. The presented results reveal a higher potential of Iscador as an antitumor agent for the low metastatic cancer cell line MCF-7 compared to the high metastatic one. Iscador Qu appears to be more potent compared to Iscador M, but at this point, the exact mechanism of action is still unclear and needs further investigations.

Time-Resolved In Situ AFM Measurement of Growth Rates of Aβ40 Fibrils.

We employ time-resolved in situ atomic force microcopy to monitor the growth of individual Aβ40 fibrils and thereby directly measure the fibril growth rates. We describe procedures to express and purify the Aβ peptide and verify its identity, prepare solutions and seeds, quantify the displacements of the growing tips of individual fibrils, and determine their respective growth rates. We discuss approaches to evaluate and minimize the impact of the scanning tip on the monitored processes. We use the distribution of fibril thickness to characterize approximately the fibril structure. The ability to quantify faithfully the growth kinetics of amyloid fibrils empowers exploration of the molecular-level processes of fibril growth that relate to behaviors of amyloid species of laboratory and clinical interest.

Properties of Grass Carp (Ctenopharyngodon idella) Collagen and Gel for Application in Biomaterials.

The biochemical properties of collagens and gels from grass carp (Ctenopharyngodon idella) were studied to explore the feasibility of their application in biomaterials. The yields of skin collagen (SC) and swim bladder collagen (SBC) extracted from grass carp were 10.41 ± 0.67% and 6.11 ± 0.12% on a wet basis, respectively. Both collagens were characterized as type I collagen. Denaturation temperatures of SC and SBC were 37.41 ± 0.02 °C and 39.82 ± 0.06 °C, respectively. SC and SBC had high fibril formation ability in vitro, and higher values of salinity (NaCl, 0-280 mM) and pH (6-8) in formation solution were found to result in faster self-assembly of SC and SBC fibrils as well as thicker fibrils. Further tests of SC gels with regular morphology revealed that their texture properties and water content were affected by pH and NaCl concentration. The hardness, springiness, and cohesiveness of SC gels increased and the chewiness and water content decreased as pH increased from 7 to 8 and NaCl concentration increased from 140 to 280 mM. These properties suggest that collagens from grass carp may be useful in biomaterial applications in the future.

Length and Unsaturation of Fatty Acids of Phosphatidic Acid Determines the Aggregation Rate of Insulin and Modifies the Structure and Toxicity of Insulin Aggregates.

Phosphatidic acid (PA) is a unique plasma membrane lipid that contains fatty acids (FAs) with different lengths and degrees of unsaturation. Under physiological conditions, PA acts as a second messenger regulating a wide variety of cellular processes. At the same time, the role of PA under pathological conditions, which are caused by an abrupt aggregation of amyloid proteins, remains unclear. In this study, we investigated the effect of PA with different lengths and unsaturation of FAs on insulin aggregation. We found that PA with C16:0 FAs strongly inhibited insulin aggregation, whereas PA with C18:0 FAs accelerated it. Furthermore, PA with unsaturated (C18:1) FAs made the insulin form extremely long and thick fibrils that were not observed for PAs with saturated FAs. We also found that the presence of PA with C16:0 FAs resulted in the formation of aggregates that exerted significantly lower cell toxicity compared to the aggregates formed in the presence of PAs with C18:0 and C18:1 FAs. These results suggest that PA may play a key role in neurodegeneration.

Valproic acid modulates collagen architecture in the postoperative conjunctival scar.

Valproic acid (VPA), widely used for the treatment of neurological disorders, has anti-fibrotic activity by reducing collagen production in the postoperative conjunctiva. In this study, we investigated the capacity of VPA to modulate the postoperative collagen architecture. Histochemical examination revealed that VPA treatment was associated with the formation of thinner collagen fibers in the postoperative days 7 and 14 scars. At the micrometer scale, measurements by quantitative multiphoton microscopy indicated that VPA reduced mean collagen fiber thickness by 1.25-fold. At the nanometer scale, collagen fibril thickness and diameter measured by transmission electron microscopy were decreased by 1.08- and 1.20-fold, respectively. Moreover, delicate filamentous structures in random aggregates or closely associated with collagen fibrils were frequently observed in VPA-treated tissue. At the molecular level, VPA reduced Col1a1 but induced Matn2, Matn3, and Matn4 in the postoperative day 7 conjunctival tissue. Elevation of matrilin protein expression induced by VPA was sustained till at least postoperative day 14. In primary conjunctival fibroblasts, Matn2 expression was resistant to both VPA and TGF-β2, Matn3 was sensitive to both VPA and TGF-β2 individually and synergistically, while Matn4 was modulable by VPA but not TGF-β2. MATN2, MATN3, and MATN4 localized in close association with COL1A1 in the postoperative conjunctiva. These data indicate that VPA has the capacity to reduce collagen fiber thickness and potentially collagen assembly, in association with matrilin upregulation. These properties suggest potential VPA application for the prevention of fibrotic progression in the postoperative conjunctiva. KEY MESSAGES: VPA reduces collagen fiber and fibril thickness in the postoperative scar. VPA disrupts collagen fiber assembly in conjunctival wound healing. VPA induces MATN2, MATN3, and MATN4 in the postoperative scar.

Process-Induced Morphology of Poly(Butylene Adipate Terephthalate)/Poly(Lactic Acid) Blown Extrusion Films Modified with Chain-Extending Cross-Linkers.

Process-induced changes in the morphology of biodegradable polybutylene adipate terephthalate (PBAT) and polylactic acid (PLA) blends modified with various multifunctional chain-extending cross-linkers (CECLs) are presented. The morphology of unmodified and modified films produced with blown film extrusion is examined in an extrusion direction (ED) and a transverse direction (TD). While FTIR analysis showed only small peak shifts indicating that the CECLs modify the molecular weight of the PBAT/PLA blend, SEM investigations of the fracture surfaces of blown extrusion films revealed their significant effect on the morphology formed during the processing. Due to the combined shear and elongation deformation during blown film extrusion, rather spherical PLA islands were partly transformed into long fibrils, which tended to decay to chains of elliptical islands if cooled slowly. The CECL introduction into the blend changed the thickness of the PLA fibrils, modified the interface adhesion, and altered the deformation behavior of the PBAT matrix from brittle to ductile. The results proved that CECLs react selectively with PBAT, PLA, and their interface. Furthermore, the reactions of CECLs with PBAT/PLA induced by the processing depended on the deformation directions (ED and TD), thus resulting in further non-uniformities of blown extrusion films.

Cavitation Fibrillation of Cellulose Fiber.

Cellulose fibrils are the structural backbone of plants and, if carefully liberated from biomass, a promising building block for a bio-based society. The mechanism of the mechanical release—fibrillation—is not yet understood, which hinders efficient production with the required reliable quality. One promising process for fine fibrillation and total fibrillation of cellulose is cavitation. In this study, we investigate the cavitation treatment of dissolving, enzymatically pretreated, and derivatized (TEMPO oxidized and carboxymethylated) cellulose fiber pulp by hydrodynamic and acoustic (i.e., sonication) cavitation. The derivatized fibers exhibited significant damage from the cavitation treatment, and sonication efficiently fibrillated the fibers into nanocellulose with an elementary fibril thickness. The breakage of cellulose fibers and fibrils depends on the number of cavitation treatment events. In assessing the damage to the fiber, we presume that microstreaming in the vicinity of imploding cavities breaks the fiber into fibrils, most likely by bending. A simple model showed the correlation between the fibrillation of the carboxymethylated cellulose (CMCe) fibers, the sonication power and time, and the relative size of the active zone below the sonication horn.

Collagen mineralization with lepidocrocite via Fe(OH)2 addition.

The mineralization of collagen in vitro has been extensively investigated for hydroxyapatite, silica, calcium carbonate and lepidocrocite (γ-FeOOH). Henceforth, it is interesting to investigate whether collagen also could serve as a generic mineralization template for other minerals, like magnetite. To this end, and inspired by the partial oxidation approach, first a ferrous hydroxide (Fe(OH)2) intermediate is synthesized via the titration of base to a solution of Fe2+. Subsequently, the Fe(OH)2 is mixed with collagen fibrils and poly(aspartic acid) is added to promote the formation of intrafibrillar crystals. Platelet-shaped lepidocrocite crystals being present throughout the entire thickness of the collagen fibrils can be realized, as was confirmed with electron tomography. The formation of lepidocrocite, which is an Fe3+ compound, is hypothesized to be induced via oxidation of the Fe2+ species and, therefore, the oxygen concentration during titration, TEM sample preparation and drying of TEM samples are investigated. Although the reaction is sensitive to small changes in experimental conditions, highly mineralized collagen fibers can be realized.

Abstract 9438: Unipolar Voltages Are Associated with Left Atrial Wall Thickness and Predict Atrial Fibrillation Recurrence After Pulmonary Vein Isolation

Introduction: This study was conducted to investigate the relationship between left atrial (LA) wall thickness and the corresponding local unipolar/bipolar voltages, and its impact on the atrial fibrillation (AF) recurrence. Methods: Two-hundred-forty-two AF patients (196 male; 62 ± 9 years) who underwent successful PVI were enrolled and classified into the 2 groups: AF-Free group (n = 172), AF did not recur during the mean follow-up periods of 18 ± 8 months; and AF-Rec group (n = 70), those who had AF recurrence. LA wall thickness along the pulmonary vein isolation (PVI) line was measured using computed tomography and the voltage heights were measured by 3D electroanatomical map. Results: Left-superior PV posterior, right-superior PV (RSPV) posterior and right-inferior PV posterior wall thickness in AF-Rec group was significantly thicker than that in AF-Fee group (P &lt; 0.001, respectively). RSPV posterior wall (RSPV-PW) thickness &gt; 1.5 mm was revealed as cut-off value for AF recurrence with the sensitivity (73%) and specificity (71%) (P &lt; 0.001), and was the independent predictor of AF recurrence (Hazard ratio (HR): 4.511, confidence interval: 2.637-7.717, P = 0.001). Although both of the bipolar and unipolar voltages were associated with RSPV-PW thickness, the latter had a stronger correlation (R 2 = 0.0519, P = 0.043 vs R 2 = 0.5099, P = 0.001, respectively). The unipolar voltages of 3.1 mV was defined as cut-off value for AF recurrence prediction (P = 0.001). Conclusions: The current study demonstrated that RSPV-PW hypertrophy had strong correlation with the local unipolar voltages, and that RSPV-PW thickness &gt; 1.5 mm and the unipolar voltage &gt; 3.1 mV can be predictor for AF recurrence, where we should intensively deliver radiofrequency energy for the durable PVI.

Declines in PDE4B activity promote myopia progression through downregulation of scleral collagen expression

Myopia is the most common cause of a visual refractive error worldwide. Cyclic adenosine monophosphate (cAMP)-linked signaling pathways contribute to the regulation of myopia development, and increases in cAMP accumulation promote myopia progression. To pinpoint the underlying mechanisms by which cAMP modulates myopia progression, we performed scleral transcriptome sequencing analysis in form-deprived mice, a well-established model of myopia development. Form deprivation significantly inhibited the expression levels of genes in the cAMP catabolic pathway. Quantitative real-time polymerase chain reaction analysis validated that the gene expression level of phosphodiesterase 4B (PDE4B), a cAMP hydrolase, was downregulated in form-deprived mouse eyes. Under visually unobstructed conditions, loss of PDE4B function in Pde4b-knockout mice increased the myopic shift in refraction, −3.661 ± 1.071 diopters, more than that in the Pde4b-wildtype littermates (P < 0.05). This suggests that downregulation and inhibition of PDE4B gives rise to myopia. In guinea pigs, subconjunctival injection of rolipram, a selective inhibitor of PDE4, led to myopia in normal eyes, and it also enhanced form-deprivation myopia (FDM). Subconjunctival injection of dibutyryl-cyclic adenosine monophosphate, a cAMP analog, induced only a myopic shift in the normal visually unobstructed eyes, but it did not enhance FDM. As myopia developed, axial elongation occurred during scleral remodeling that was correlated with changes in collagen fibril thickness and distribution. The median collagen fibril diameter in the FDM + rolipram group, 55.09 ± 1.83 nm, was thinner than in the FDM + vehicle group, 59.33 ± 2.06 nm (P = 0.011). Thus, inhibition of PDE4 activity with rolipram thinned the collagen fibril diameter relative to the vehicle treatment in form-deprived eyes. Rolipram also inhibited increases in collagen synthesis induced by TGF-β2 in cultured human scleral fibroblasts. The current results further support a role for PDE enzymes such as PDE4B in the regulation of normal refractive development and myopia because either loss or inhibition of PDE4B function increased myopia and FDM development through declines in the scleral collagen fibril diameter.