Fibril Density Research Articles

A unique and biocompatible corneal collagen crosslinking in vivo

Corneal crosslinking (CXL) is a widely applied technique to halt the progression of ectatic diseases through increasing the thickness and mechanical stiffness of the cornea. This study investigated the biocompatibility and efficiency of a novel CXL procedure using ruthenium and blue light in rat corneas and evaluated parameters important for clinical application. To perform the CXL procedure, the corneal epithelium of rats was removed under anaesthesia, followed by the application of a solution containing ruthenium and sodium persulfate (SPS). The corneas were then exposed to blue light at 430 nm at 3 mW/cm2 for 5 min. Rat corneas were examined and evaluated for corneal opacity, corneal and limbal neovascularization, and corneal epithelial regeneration on days 0, 1, 3, 6, 8, and 14. On day 28, the corneas were isolated for subsequent tissue follow-up and analysis. CXL with ruthenium and blue light showed rapid epithelial healing, with 100% regeneration of the corneal epithelium and no corneal opacity on day 6. The ruthenium group also exhibited significantly reduced corneal (p < 0.01) and limbal neovascularization (p < 0.001). Histological analysis revealed no signs of cellular damage or apoptosis, which further confirms the biocompatibility and nontoxicity of our method. Confocal and scanning electron microscopy (SEM) images confirmed high density of collagen fibrils, indicating efficient crosslinking and enhanced structural integrity. This study is unique that demonstrates in vivo safety, biocompatibility, and functionality of ruthenium and blue light CXL. This approach can prevent toxicity caused by UV-A light and can be an immediate alternative compared to the existing crosslinking procedures that have side effects and clinical risks for the patients.

Matrix stiffening from collagen fibril density and alignment modulates YAP-mediated T-cell immune suppression

T-cells are essential components of the immune system, adapting their behavior in response to the mechanical environments they encounter within the body. In pathological conditions like cancer, the extracellular matrix (ECM) often becomes stiffer due to increased density and alignment of collagen fibrils, which can have a significant impact on T-cell function. In this study, we explored how these ECM properties—density and fibrillar alignment—affect T-cell behavior using three-dimensional (3D) collagen matrices that mimic these conditions. Our results show that increased matrix stiffness, whether due to higher density or alignment, significantly suppresses T-cell activation, reduces cytokine production, and limits proliferation, largely through enhanced YAP signaling. Individually, matrix alignment appears to lower actin levels in activated T-cells and changes migration behavior in both resting and activated T-cells, an effect not observed in matrices with randomly oriented fibrils. Notably, inhibiting YAP signaling was able to restore T-cell activation and improve immune responses, suggesting a potential strategy to boost the effectiveness of immunotherapy in stiff ECM environments. Overall, this study provides new insights into how ECM characteristics influence T-cell function, offering potential avenues for overcoming ECM-induced immunosuppression in diseases such as cancer.

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.

Neural network auto-segmentation of serial-block-face scanning electron microscopy images exhibit collagen fibril structural differences with tendon type and health.

A U-Net machine learning algorithm was adapted to automatically segment tendon collagen fibril cross-sections from serial block face scanning electron microscopy (SBF-SEM) and create three-dimensional (3D) renderings. We compared the performance of routine Otsu thresholding and U-Net for a positional tendon that has low fibril density (rat tail tendon), an energy-storing tendon that has high fibril density (rat plantaris tendon), and a high fibril density tendon hypothesized to have disorganized 3D ultrastructure (degenerated rat plantaris tendon). The area segmentation of the tail and healthy plantaris tendon had excellent accuracy for both the Otsu and U-Net, with an Intersection over Union (IoU) of 0.8. With degeneration, only the U-Net could accurately segment the area, whereas Otsu IoU was only 0.45. For boundary validation, the U-Net outperformed Otsu segmentation for all tendons. The fibril diameter from U-Net was within 10% of the manual segmentation, however, the Otsu underestimated the fibril diameter by 39% in healthy plantaris and by 84% in the degenerated plantaris. Fibril geometry was averaged across the entire image stack and compared across tendon types. The tail had a lower fibril area fraction (58%) and larger fibril diameter (0.31 µm) than the healthy plantaris (67% and 0.21 µm) and degenerated plantaris tendon (66% and 0.19 µm). This method can be applied to a large variety of tissues to quantify 3D collagen fibril structure.

A potential therapeutic strategy of an innovative probiotic formulation toward topical treatment of diabetic ulcer: an in vivo study

BackgroundThe probiotic potential of Lacticacid bacteria has been studied in various medical complications, from gastrointestinal diseases to antibiotic resistance infections recently. Moreover, diabetic ulcer (DU) is known as one of the most significant global healthcare concerns, which comprehensively impacts the quality of life for these patients. Given that the conventional treatments of DUs have failed to prevent later complications completely, developing alternative therapies seems to be crucial.MethodsWe designed the stable oleogel-based formulation of viable probiotic cells, including Lactobacillus rhamnosus (L. rhamnosus), Lactobacillus casei (L. casei), Lactobacillus fermentum (L. fermentum), and Lactobacillus acidophilus (L. acidophilus) individually to investigate their effect on wound healing process as an in vivo study. The wound repair process was closely monitored regarding morphology, biochemical, and histopathological changes over two weeks and compared it with the effects of topical tetracycline as an antibiotic approach. Furthermore, the antibiofilm activity of probiotic bacteria was assessed against some common pathogens.ResultsThe findings indicated that all tested lactobacillus groups (excluded L. casei) included in the oleogel-based formulation revealed a high potential for repairing damaged skin due to the considerably more levels of hydroxyproline content of tissue samples along with the higher numerical density of mature fibroblasts cell and volume density of hair follicles, collagen fibrils, and neovascularization in comparison with antibiotic and control groups. L. acidophilus and L. rhamnosus showed the best potential of wound healing among all lactobacillus species, groups treated by tetracycline and control groups. Besides, L. rhamnosus showed a significant biofilm inhibition activity against tested pathogens.ConclusionsThis experiment demonstrated that the designed formulations containing probiotics, particularly L. acidophilus and L. rhamnosus, play a central role in manipulating diabetic wound healing. It could be suggested as an encouraging nominee for diabetic wound-healing alternative approaches, though further studies in detailed clinical trials are needed.

Reversed Corneal Fibroblasts Therapy Restores Transparency of Mouse Cornea after Injury.

Cell-based therapies using corneal stromal stem cells (CSSC), corneal keratocytes, or a combination of both suppress corneal scarring. The number of quiescent keratocytes in the cornea is small; it is difficult to expand them in vitro in quantities suitable for transplantation. This study examined the therapeutic effect of corneal fibroblasts reversed into keratocytes (rCF) in a mouse model of mechanical corneal injury. The therapeutic effect of rCF was studied in vivo (slit lamp, optical coherence tomography) and ex vivo (transmission electron microscopy and immunofluorescence staining). Injection of rCF into the injured cornea was accompanied by recovery of corneal thickness, improvement of corneal transparency, reduction of type III collagen in the stroma, absence of myofibroblasts, and the improvement in the structural organization of collagen fibers. TEM results showed that 2 months after intrastromal injection of cells, there was a decrease in the fibril density and an increase in the fibril diameter and the average distance between collagen fibrils. The fibrils were well ordered and maintained the short-range order and the number of nearest-neighbor fibrils, although the averaged distance between them increased. Our results demonstrated that the cell therapy of rCF from ReLEx SMILe lenticules promotes the recovery of transparent corneal stroma after injury.

Central versus peripheral thickness in the human cornea explained

PurposeThe human cornea is thicker in the periphery than the center and it has been suggested that this must be due to greater numbers of lamellae in the peripheral corneal stroma. The purpose of this study was to use high-resolution ultrastructural imaging to determine if the greater thickness of the peripheral cornea is due to the presence of more lamellae or if there is some other anatomical explanation. MethodsIn this study, full thickness corneas from three human donors were processed for light microscopy (LM) and transmission electron microscopy (TEM). Images were taken in three distinct stromal regions (anterior, middle, and posterior) from the central and peripheral cornea. Stromal thickness was evaluated by LM while TEM was used to evaluate numbers and thicknesses of lamellae, mean collagen fibril diameter, and mean collagen fibril density. ResultsMean stromal thickness was significantly thinner in the central (415 ± 34 µm) compared to the peripheral (536 ± 29 µm) cornea (P = 0.009). Numbers of lamellae were not significantly different between central (246 ± 14) and peripheral (251 ± 14) cornea. Average lamellar thickness was not different across all regions of the cornea, except for the peripheral posterior where the lamellae were approximately 50 % thicker (P < 0.05). Collagen fibril diameters were larger in the peripheral cornea by approximately 30 % when compared to the central cornea, in all regions (P < 0.01). ConclusionsThis study shows that it is an increase peripheral posterior lamellar thickness, rather than an increase in the number of lamellae, that accounts for the increase in corneal stromal thickness in the periphery of the human cornea. While collagen fibril diameters are greater throughout the peripheral stroma, the lamellae in the mid and anterior peripheral stroma are not thicker than centrally.

Insight into the Solar Plage Chromosphere with DKIST

The strongly coupled hydrodynamic, magnetic, and radiation properties of the plasma in the solar chromosphere make it a region of the Sun's atmosphere that is poorly understood. We use data obtained with the high-resolution Visible Broadband Imager (VBI) equipped with an Hβ filter and the Visible Spectro-Polarimeter (ViSP) on the Daniel K. Inouye Solar Telescope to investigate the fine-scale structure of the plage chromosphere. To aid in the interpretation of the VBI imaging data, we also analyze spectra from the CHROMospheric Imaging Spectrometer on the Swedish Solar Telescope. The analysis of spectral properties, such as enhanced line widths and line depths, explains the high contrast of the fibrils relative to the background atmosphere demonstrating that Hβ is an excellent diagnostic for the enigmatic fine-scale structure of the chromosphere. A correlation between the parameters of the Hβ line indicates that opacity broadening created by overdense fibrils could be the main reason for the spectral line broadening frequently observed in chromospheric fine-scale structures. Spectropolarimetric inversions of the ViSP data in the Ca ii 8542 Å and Fe i 6301/6302 Å lines are used to construct semiempirical models of the plage atmosphere. Inversion outputs indicate the existence of dense fibrils in the Ca ii 8542 Å line. The analyses of the ViSP data show that the morphological characteristics, such as orientation, inclination, and length of fibrils, are defined by the topology of the magnetic field in the photosphere. Chromospheric maps reveal a prominent magnetic canopy in the area where fibrils are directed toward the observer.

Efficient fabrication of anisotropic regenerated cellulose films from bamboo via a facile wet extrusion strategy

Bamboo, featuring fast growth rate and high cellulose content, is considered to be one of the most attractive feedstocks for degradable bio-materials as a substitute for plastics. However, those was limited to the fields of bamboo structural materials mainly by physical processes. Herein, we report a facile continuous wet extrusion strategy for scalable manufacturing of anisotropic regenerated cellulose films in alkali/urea aqueous solution for the first time. The bamboo cellulose solution was regenerated in H2SO4/Na2SO4/ZnSO4 aqueous solution to facilitate the construction of dense fibrils networks. Moreover, under the synergistic effect of shear orientations and stretching processes in wet extrusion molding, the cellulose networks promoted further orientated assembly into aligned fibrils. Therefore, these anisotropic cellulose hydrogels exhibited good mechanical properties, and the tensile strength was increased from 1.67 MPa of anisotropic cellulose hydrogel with 1.0 of stretching ration (ACH-1.0) to 2.13 MPa of ACH-1.4 with increasing stretching ratio from 1.0 to 1.4, which was about 1.34 times higher than that of the isotropic hydrogel fabricated by tape-casting. Moreover, ACH-1.4 exhibited commendable thermal stability and air barrier properties. This work demonstrated a simple and continuous bottom-up approach for fabrication of anisotropic bamboo-based cellulose hydrogels and films with excellent mechanical properties.

Microscopic and nanoscale mechanical properties of tonkin cane bamboo measured by advanced AFM methods

Bamboo is a natural composite material with a high strength-to-weight ratio traditionally used in various consumer products as well as in building and modern advanced and sustainable manufacturing technology. While its macroscopic mechanical properties are well known, nanoscale studies of the mechanical properties of the hierarchical structure of bamboo at the level of individual cells and cell walls are lacking. Here we use different AFM-based methods to analyse the mechanical properties of individual bamboo fibres that are the foundation of the hierarchical structure at the nanoscale. The elastic modulus of the fiber in the outer region varies from 7.5 to 8.9 GPa, while that of the inner fiber ranges between 4.1 and 6.1 GPa. The results demonstrate that the fibre stiffness varies in nanoscale regions between fibre interior and the fibre wall and strongly depends on the position of the individual bamboo fibre within the culm. Outer fibres with high cellulose fibril density and low lignin level show low adhesion interaction force (13.5%) in the interface between cellulose and lignin/hemicellulose matrix, hence, resulting in low plastic deformation energy release during mechanical deformation. The implications of the interrelation of plasticity, lignin level, and adhesion force are discussed for the application of bamboo as a high-performance, renewable, and sustainable material.

Characterizing cannabis-prevalent terpenes for neuroprotection reveal a role for α and β-pinenes in mitigating amyloid β-evoked neurotoxicity and aggregation in vitro

BackgroundCannabis sativa L. (C. sativa) can efficiently synthesize of over 200 terpenes, including monoterpenes, sesquiterpenes and triterpenes that may contribute to the known biological activities of phytocannabinoids of relevance for the burgeoning access to medicinal cannabis formulations globally; however, to date have been uncharacterized. We assessed twelve predominant terpenes in C. sativa for neuroprotective and anti-aggregative properties in semi-differentiated PC12 neuronal cell line that is robust and validated as a cell model responsive to amyloid β (Aβ1–42) protein exposure and oxidative stress. MethodsCell viability was assessed biochemically using the MTT assay in the presence of myrcene, β-caryophyllene, terpinolene, limonene, linalool, humulene, α-pinene, nerolidol, β-pinene, terpineol, citronellol and friedelin (1–200 μM) for 24 hr. Sub-toxic threshold test concentrations of each terpene were then applied to cells, alone or with concomitant incubation with the lipid peroxidant tert-butyl hyrdroperoxide (t-BHP; 0–250 μM) or amyloid β (Aβ1–42; 0–1 μM) to assess neuroprotective effects. Direct effects of each terpene on Aβ fibril formation and aggregation were also evaluated using the Thioflavin T (ThT) fluorometric kinetic assay and transmission electron microscopy (TEM) to visualize fibril and aggregate morphology. ResultsTerpenes were intrinsically benign to PC12 cells up to 50 μM, with higher concentrations of β-caryophyllene, humulene and nerolidol inducing some loss of PC12 cell viability. No significant protective effects of terpenes were observed following t-BHP (0–200 µM) administration, with some enhanced toxicity instead demonstrated from both β-caryophyllene and humulene treatment (each at 50 µM). α-pinene and β-pinene demonstrated a significant neuroprotective effect against amyloid β exposure. α-pinene, β-pinene, terpineol, terpinolene and friedelin were associated with a variable inhibition of Aβ1–42 fibril and aggregate density. ConclusionsThe outcomes of this study underline a neuroprotective role of α-pinene and β-pinene against Aβ-mediated neurotoxicity associated with an inhibition of Aβ1–42 fibrilization and density. This demonstrates the bioactive potential of selected terpenes for consideration in the development of medicinal cannabis formulations targeting neurodegenerative diseases.

Correlations between [18F]flutemetamol PET and region‐matched postmortem integrated density measures of cyano‐flutemetamol signal in Aβ plaques

AbstractBackgroundThe binding substrates of [18F]flutemetamol and [11C]PiB PET consist of fibrillar amyloid‐ß (Aß) aggregates in morphologically and structurally distinct diffuse plaques (DP) and cored plaques (CP) which are present at different proportions across brain regions in Alzheimer’s disease (AD). To account for this heterogeneity, an unbiased quantitative measure integrating plaque size and density of amyloid‐ß (Aß) fibrils in plaques could help avoid false positive results, improve imaging‐to‐autopsy correlations, and further advance our understanding of pathological substrates for binding of amyloid PET tracers.MethodSixteen subjects underwent [18F]flutemetamol PET imaging in the pivotal Phase III end‐of‐life study (Study GE067‐007 and GE067‐026) and later came to autopsy (mean imaging‐to‐autopsy interval was 111 ± 87.6 days). [18F]flutemetamol SUVRpons values in four neocortical regions were correlated with region‐matched postmortem measures of cyano‐flutemetamol labeling of all plaques, as well as CP and DP separately, using percent area coverage, mean grayscale intensity of fluorescence, or a combination of the two measures referred to as integrated density.ResultIn the analysis of all cyano‐flutemetamol‐labeled plaques, [18F]flutemetamol SUVRpons values were more closely associated with integrated density measures than percent area coverage and mean grayscale values. Similarly, when CP and DP were analyzed separately, the integrated density values showed the closest association with neocortical region‐matched [18F]flutemetamol SUVRpons values. When area coverage of CP and DP were each expressed as a fraction of total plaque area coverage with or without integrating fluorescence intensity values, [18F]flutemetamol SUVRpons values within each region of interest were associated with the DP, but not the CP, area coverage measure.ConclusionThe association of antemortem [18F]flutemetamol PET and region‐matched postmortem integrated density measurements of cyano‐flutemetamol labeled plaques further supports the hypothesis that amyloid PET signal is influenced both by plaque size and Aß fibril density within DP and CP. Further elucidating the relative contributions of each plaque type to the retention of amyloid PET radioligands could influence how a positive amyloid PET signal is interpreted in relation to postmortem pathology data.

Correlations between [18F]flutemetamol PET and region‐matched postmortem integrated density measures of cyano‐flutemetamol signal in Aβ plaques

AbstractBackgroundThe binding substrates of [18F]flutemetamol and [11C]PiB PET consist of fibrillar amyloid‐β (Aβ) aggregates in morphologically and structurally distinct diffuse plaques (DP) and cored plaques (CP) which are present at different proportions across brain regions in Alzheimer’s disease (AD). To account for this heterogeneity, an unbiased quantitative measure integrating plaque size and density of amyloid‐β (Aβ) fibrils in plaques could help avoid false positive results, improve imaging‐to‐autopsy correlations, and further advance our understanding of pathological substrates for binding of amyloid PET tracers.MethodSixteen subjects underwent [18F]flutemetamol PET imaging in the pivotal Phase III end‐of‐life study (Study GE067‐007 and GE067‐026) and later came to autopsy (mean imaging‐to‐autopsy interval was 111 ± 87.6 days). [18F]flutemetamol SUVRpons values in four neocortical regions were correlated with region‐matched postmortem measures of cyano‐flutemetamol labeling of all plaques, as well as CP and DP separately, using percent area coverage, mean grayscale intensity of fluorescence, or a combination of the two measures referred to as integrated density.ResultIn the analysis of all cyano‐flutemetamol‐labeled plaques, [18F]flutemetamol SUVRpons values were more closely associated with integrated density measures than percent area coverage and mean grayscale values. Similarly, when CP and DP were analyzed separately, the integrated density values showed the closest association with neocortical region‐matched [18F]flutemetamol SUVRpons values. When area coverage of CP and DP were each expressed as a fraction of total plaque area coverage with or without integrating fluorescence intensity values, [18F]flutemetamol SUVRpons values within each region of interest were associated with the DP, but not the CP, area coverage measure.ConclusionThe association of antemortem [18F]flutemetamol PET and region‐matched postmortem integrated density measurements of cyano‐flutemetamol labeled plaques further supports the hypothesis that amyloid PET signal is influenced both by plaque size and Aβ fibril density within DP and CP. Further elucidating the relative contributions of each plaque type to the retention of amyloid PET radioligands could influence how a positive amyloid PET signal is interpreted in relation to postmortem pathology data.

Abstract 18049: Mathematical Modeling Predicts Treatment Efficacy in Transthyretin Cardiac Amyloidosis

Introduction: Increased recognition of transthyretin cardiac amyloidosis (ATTR-CA) led to the development of treatments with different therapeutic targets. Optimal patient selection, treatment choice, timing and duration remain unclear. We evaluated the efficacy and timing of common and emerging ATTR-CA treatments with a mathematical model based on therapeutic mechanisms. Methods: We developed a system of ordinary differential equations describing transthyretin amyloid (ATTR) deposition and myocardial clearance by clusterin (Figure 1A,B). Published data and equilibrium analyses determined parameters for initial maximal ATTR density. Mechanisms of four therapies were modeled and simulated at varying levels of efficacy. Complete success of the mechanism defines 100% efficacy, i.e. full stabilization of tetramer. Expected clinical efficacy (ECE) is based on studies of clinical doses. Minimum effective clearance (MEC) is the theoretical efficacy required to reduce ATTR fibrils below the threshold for complete clearance. Results: All treatments reduced ATTR fibril density, correlating with efficacy (Fig 1C). At ECE, only NI006 demonstrated complete clearance of ATTR fibrils. With other current treatment strategies, amyloid density will be lowered but never cleared. The secondary mechanism of diflusinil did not change equilibrium behavior compared to tafamidis. MEC values and time to clearance at MEC were lowest for NI006 (14.7%; 65 days) and doxycycline (16.1%; 36 months). Tetramer stabilization required almost complete efficacy (99.3%) and about a decade (119 months) for tetramer clearance. Conclusion: This mathematical model predicts ATTR clearance by four distinct mechanisms of action. The novel anti-ATTR antibody NI006 is a promising treatment with predicted faster onset and higher effectiveness for clearance of ATTR fibrils from the myocardium. This model can evaluate future treatment strategies and therapeutic targets.

Transforming growth factor beta receptor 2 (Tgfbr2) deficiency in keratocytes results in corneal ectasia

PurposeWe hypothesized that Transforming growth factor beta receptor 2 (Tgfbr2) deletion in keratocyte (Tgfbr2kera-cko), the corneal stroma cell, can result in corneal thinning and generate a potential model for Cornea Ectasia (CE). MethodsCorneal thickness of Tgfbr2kera-cko and Tgfbr2Ctrl was examined with Optical Coherence Tomography (OCT) at post-natal (P) days 42 and 70, respectively. Histological H&E staining, transmission electron micrograph (TEM), and immunofluorescence staining (IFS) were harnessed to examine corneal cell morphology, proliferation, differentiation, and collagen fibrils. ResultsSlit-Lamp revealed that corneas were transparent in both Tgfbr2kera-cko and Tgfbr2Ctrl, however, Tgfbr2kera-cko cornea was 33.5% and 42.9% thinner as compared with those of Tgfbr2Ctrl at P42 and P70, respectively. H&E and semithin section staining with toluidine blue-O confirmed that Tgfbr2kera-cko cornea has a thinner stroma. In contrast, the epithelium in Tgfbr2kera-cko was substantially thicker. The cell proliferation marker Ki67 expression level increased ∼9% in Tgfbr2kera-cko corneal epithelium as compared with that in Tgfbr2Ctrl, however, the Krt14 and Krt12 expression pattern was not obviously changed in Tgfbr2kera-cko corneal epithelium. It was noticed that Col1a1 expression was substantially reduced in Tgfbr2kera-cko as compared with that in Tgfbr2Ctrl. TEM showed that keratocytes were unhealthy and stromal collagen fibril density was significantly reduced in Tgfbr2kera-cko as compared with that in Tgfbr2Ctrl cornea. Moreover, mechanical eye-rubbing on Tgfbr2kera-cko resulted in corneal hydrops and edema. ConclusionTgfbr2 in keratocytes is indispensable for the corneal stroma at postnatal homeostasis. The cornea phenotype manifested in these Tgfbr2kera-cko mice resembles corneal ectasia disease in humans.

Evaluation of desmoplasia in pancreatic and colon tumors by morphometric analysis

Abstract: Objectives: This study aims to quantify collagen deposition in the desmoplasia of pancreatic and colonial tumors. Methods: Samples of colon and pancreas tumors were selected from the Hospital das Clínicas de Pernambuco and submitted to trichomy with Gomori's Trichrome. Photomicrographic analysis was performed using the integrated Panthera imaging analysis system L. Results: It was identified that 31 colon tumors and 22 pancreatic tumors presented desmoplasia with the visualization of fibrillar collagen distributed irregularly around the tumor. There was a statistically significant difference (p = 0.0031) between collagen fiber deposition in desmoplastic and non-desmoplastic pancreatic tumors, as well as for colonic tumors (p &lt; 0.001). Conclusion: It was observed that more than half of the tumors analyzed presented desmoplasia with a difference of density of collagen fibrils statistically significant.

Ultrastructural features of the body wall of the helminth Heterakis dispar (Schrank, 1790) (Nematoda, Heterakidae)

The structure of the body wall (cuticle, hypoderm, and muscle layer) of the nematode Heterakis dispar (Schrank, 1790) from the family Heterakidae was studied using light and electron microscopy methods and compared with other species of the same family. The cuticle of the adult nematode H. dispar consists of 8 layers: 1 - an outer membrane layer or epicuticle; 2, 3 - outer and inner cortical layers; 4, 5 - outer and inner homogeneous or middle layers; 6, 7 - outer and inner fibrous or fibrillar layers; 8 - basement membrane. The cortical, homogeneous and fibrillary layers constitute 12.4, 45.3 and 42.3% of the all cuticle, respectively. The homogeneous layer of the cuticle in the lateral ridges in both male and female and near the bursa of the male is several times as thick as other parts of the helminth cuticle. Unlike other species of the family, males of H. dispar possess 3 different forms of cuticular structure in different parts of the body. In the basal layer of the cuticle, sustaining structures consisting of dense fibrils and microtubules were found, which were not previously noted in other species of the family. It is likely that they provide strength to the body wall of the helminth. In the hypodermis of the nematode, dorsal, ventral, and 2 lateral ridges are traced, the lateral ridges being twice as large as others. Ultrastructural features of the excretory channels and nerve cords located in the hypodermal ridges, were also revealed. The nervous system of the helminth is orthogonal. The ventral nerve cord is wider than the dorsal one. Muscle layer is of the polymyar type, number of muscle cells arranged in groups varies from 17 to 26, depending on the sex and body part of the helminth.

Self-Assembly of Amyloid Fibrils into 3D Gel Clusters versus 2D Sheets.

The deposition of dense fibril plaques represents the pathological hallmark for a multitude of human disorders, including many neurodegenerative diseases. Fibril plaques are predominately composed of amyloid fibrils, characterized by their underlying cross beta-sheet architecture. Research into the mechanisms of amyloid formation has mostly focused on characterizing and modeling the growth of individual fibrils and associated oligomers from their monomeric precursors. Much less is known about the mechanisms causing individual fibrils to assemble into ordered fibrillar suprastructures. Elucidating the mechanisms regulating this "secondary" self-assembly into distinct suprastructures is important for understanding how individual protein fibrils form the prominent macroscopic plaques observed in disease. Whether and how amyloid fibrils assemble into either 2D or 3D supramolecular structures also relates to ongoing efforts on using amyloid fibrils as substrates or scaffolds for self-assembling functional biomaterials. Here, we investigated the conditions under which preformed amyloid fibrils of a lysozyme assemble into larger superstructures as a function of charge screening or pH. Fibrils either assembled into three-dimensional gel clusters or two-dimensional fibril sheets. The latter displayed optical birefringence, diagnostic of amyloid plaques. We presume that pH and salt modulate fibril charge repulsion, which allows anisotropic fibril-fibril attraction to emerge and drive the transition from 3D to 2D fibril self-assembly.

Polyphenol extracts from Ascophyllum nodosum protected sea cucumber (Apostichopus japonicas) body wall against thermal degradation during tenderization

To retard the protein degradation during sea cucumber processing, polyphenol extracts from Ascophyllum nodosum (PhE) was used as a potential antioxidant to maintain the structural integrity of sea cucumber body wall. Accordingly, the protection effects of PhE (0, 0.5, 1.0 and 1.5 mg PhE/g SFBW) against thermal degradation of the solid fragments of body wall (SFBW) have been investigated in order to evaluate their impact on the oxidation level and structural changes. Electronic Spin Resonance results showed that PhE could significantly inhibit the occurrence of oxidation by scavenging the free radicals. The effect of PhE on chemical analysis of soluble matters in SFBW was characterized by SDS-PAGE and HPLC. Compared with thermally treated SFBW, samples with PhE presented a decrease in protein dissolution. Thermal treatment resulted in the disintegration of collagen fibrils and fibril bundles in SFBW samples, while the density of collagen fibrils was increased, and the porosity decreased in samples with PhE. The results of FTIR and intrinsic tryptophan fluorescence confirmed that the structures of SFBW were modified by PhE. Besides, the denaturing temperature and decomposition temperature were both improved with the addition of PhE. These results suggested that PhE appeared to have a positive effect on lowering oxidation and improving thermostability and structural stability of SFBW, which could provide a theoretical basis for protecting sea cucumber body wall against degradation during thermal tenderization.

Ex vivo Detection of Amyloid-β in Naturally Formed Oral Biofilm.

Oral infection has been implicated in the possible etiology of Alzheimer's disease. To detect amyloid-β (Aβ) within microbial biofilms. Freshly extracted teeth (N = 87) with periodontal disease were separated into Group A (N = 11), with primary root canal infection and Group B (N = 21) with failed endodontic treatment identified by the presence of, gutta percha root filling. Biofilm characteristics were observed by scanning electron microscopy (SEM). Demineralized paraffin wax embedded tooth sections and mineralized calculus biofilm were immunostained with the anti-Aβ antibody. The gutta perchas were processed either for on-section acrylic resin tissue immunocolloidal gold silver staining (IGSS) using the anti-Aβ antibody or in Araldite resin for ultrastructure. SEM demonstrated calculus and gutta percha in situ harboring a polymicrobial biofilm featuring extracellular polymeric substance (EPS) and water channels. Immunohistochemistry on rehydrated paraffin wax tooth sections from Group A, demonstrated Aβ staining on external (calculus and plaque) and all intracanal infected regions. In Group B, the gutta percha biofilm IGSS gave an inconclusive result for Aβ. Transmission electron microscopy of selected teeth with infected intra-canals (Group A) and 20% of gutta percha biofilm (Group B) EPS contained electron dense fibrils of variable sizes, some of which were typical of human Aβ fibrils. This study detected both soluble and insoluble Aβ fibrils within the EPS of periodontal and endodontic natural biofilm, strongly suggesting its role as an antimicrobial peptide in combatting local infection, with potential risk for cross-seeding into the brain for AD development.