Spatiotemporal formation of a single liquid-like condensate and amyloid fibrils of α-synuclein by optical trapping at solution surface.

Liquid-like protein condensates have recently attracted much attention due to their critical roles in biological phenomena. They typically show high fluidity and reversibility for exhibiting biological functions, while occasionally serving as sites for the formation of amyloid fibrils. To comprehend the properties of protein condensates that underlie biological function and pathogenesis, it is crucial to study them at the single-condensate level; however, this is currently challenging due to a lack of applicable methods. Here, we demonstrate that optical trapping is capable of inducing the formation of a single liquid-like condensate of α-synuclein in a spatiotemporally controlled manner. The irradiation of tightly focused near-infrared laser at an air/solution interface formed a condensate under conditions coexisting with polyethylene glycol. The fluorescent dye-labeled imaging showed that the optically induced condensate has a gradient of protein concentration from the center to the edge, suggesting that it is fabricated through optical pumping-up of the α-synuclein clusters and the expansion along the interface. Furthermore, Raman spectroscopy and thioflavin T fluorescence analysis revealed that continuous laser irradiation induces structural transition of protein molecules inside the condensate to ß-sheet rich structure, ultimately leading to the condensate deformation and furthermore, the formation of amyloid fibrils. These observations indicate that optical trapping is a powerful technique for examining the microscopic mechanisms of condensate appearance and growth, and furthermore, subsequent aging leading to amyloid fibril formation.

Di-Calcium Phosphate and Phytosphingosine as an Innovative Acid-Resistant Treatment to Occlude Dentine Tubules.

The present investigation evaluated the ability of an experimental di-calcium phosphate (DCP) desensitising agent used alone or combined with phytosphingosine (PHS) to occlude dentine tubules and resist a citric acid (CA) or artificial saliva (AS) challenge. Three groups of human dentine specimens (DS) were treated with the following: (1) PHS alone, (2) DCP or (3) a combination of PHS and DCP. Dentine hydraulic conductance of DS was evaluated using a digital flow sensor at 6.9 kPa. The average fluid volume for each of the treated DS was used to calculate the total dentine permeability reduction (%P) prior to and following CA immersion for 1 min or AS immersion for 4 weeks. The treated DS were subjected to both scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy analysis. Statistically significant differences (%P) were identified between the groups by ANOVA and Fisher's multiple comparison test (p < 0.05), respectively. Interestingly, both PHS and DCP appeared to work synergistically. DS treated with DCP or PHS/DCP demonstrated a significant reduction (%P) prior to and following CA or AS challenge (p < 0.05). Both the SEM and FTIR analyses showed consistent brushite crystals occluding the dentine tubules. Conversely, the application of PHS alone failed to demonstrate any significant reduction of dentine permeability (p > 0.05) or show any evidence of occlusion of the dentine tubules. DCP can be used alone or combined with PHS to decrease the dentine permeability as well as to resist a CA and AS challenge. These results would, therefore, suggest that DCP may be a suitable treatment option for dentine hypersensitivity.

Overexpression of urokinase-type plasminogen activator in pterygia and pterygium fibroblasts.

PURPOSE: Urokinase-type plasminogen activator (uPA) is a protease involved in tissue remodeling and cell migration. Little is known about the expression of uPA in pterygium. The purpose of this study was to investigate the expression of uPA mRNA and activities in various stages of surgically excised pterygia specimens and cultured pterygium fibroblasts and to compare them with normal conjunctival tissues and fibroblasts. METHODS: The expression of uPA mRNA and activity in 15 pterygium tissues and cultured fibroblasts from pterygium were measured using quantitative RT-PCR and zymography. Five normal conjunctiva specimens and cultured conjunctival fibroblasts were tested as the controls. RESULTS: The expression of uPA mRNA and activities in pterygia and pterygium fibroblasts were significantly greater than those of the normal samples (p<0.05) and were closely related to the progression of pterygium. The amounts of uPA mRNA and activities in early, moderate, and advanced pterygia were 100%, 208%, and 311% and 100%, 157%, and 280% of the early stage specimens, respectively. The amounts of uPA mRNA and the activities in cultured pterygium fibroblasts isolated from early, moderate, and advanced pterygium specimens were 100%, 219%, and 457% and 100%, 198% and 355% of early stage fibroblasts, respectively. CONCLUSIONS: Overexpression of uPA was present in pterygium and their fibroblasts. The expression of uPA by pterygium increased significantly following the progression of the pterygium. The increased expression of uPA may covert plasminogen to plasmin, degrade extracellular matrixes, stimulate cell migration, induce angiogenesis, and plays an important role in the development and progression of pterygium.

Numerical suppression of zero-order image in digital holography.

This work describes a novel approach that adopts numerical operation to suppress the zero-order images of reconstruction in digital holography. The entire process needs only one digital hologram and keeps under control the intensity ratio of the object wave to reference wave in recording procedure. Also the performance of numerical suppression is simple and effective by subtracting the numerical generated intensity of the object and reference waves from the digital hologram. The experimental results demonstrate that the zero-order images of reconstruction can be suppressed completely and represents the satisfactory reconstructed image even if the distribution of the object wave is not uniform. Therefore this approach can simplify the procedure of phase-shifting digital holographic-based scheme involving multiple exposures. Moreover, the investigation of performance using the novel suppression approach is presented for proving the practical feasibility.

Wave-front reconstruction without twin-image blurring by two arbitrary step digital holograms.

We discuss a novel approach for numerical wave-front reconstruction which utilizes arbitrary phase step digital holography. Our experimental results demonstrate that only two digital holograms and a simple estimation procedure are required for twin-image suppression, and for numerical reconstruction. One advantage of this approach is its simplicity. Only one estimate equation needs be applied. In addition the optical system can be constructed from inexpensive, generally available elements. Another advantage is the effectiveness of the method. The tolerance of the estimated value is less than 1% different than the actual value. This means that the quality of the reconstructed image is superior. This novel approach should make the application of digital holography easier and more widely available.

Massive expansion of EBV+ monoclonal T cells with CD5 down regulation in EBV-associated haemophagocytic lymphohistiocytosis.

Haemophagocytic lymphohistiocytosis (HLH) comprises primary and secondary forms; the secondary form is most commonly triggered by the Epstein-Barr virus (EBV; EBV-HLH). Patients with EBV-HLH usually exhibit oligoclonal or monoclonal T cell proliferation, which may mimic T cell lymphoproliferative disorder (T-LPD). This article reports on EBV-HLH in a 17-month-old girl with an extreme surge of reactive T lymphocytosis (absolute count 167x10(9)/l) with CD5 down regulation. Bone marrow aspirate and trephine contained florid haemophagocytosis and massive infiltration of CD3+ Epstein-Barr virus-encoded RNA+ lymphocytes, as seen by double labelling. These lymphocytes were monoclonal for EBV and T cell receptor gamma chain gene rearrangement. The patient responded dramatically to intravenous immunoglobulin, interferon alpha2b, ganciclovir and prednisolone, suggesting restoration of her immune system and eradication of the clonal T cells through these immunoregulatory agents. Thus, careful clinicopathological correlation is warranted in the interpretation of immunophenotyping and clonality data in T cell proliferation in association with EBV-HLH to avoid erroneous diagnosis of T-LPD.

Incidence of Non-Traumatic Subconjunctival Hemorrhage in a Nationwide Study in Taiwan from 2000 to 2011.

OBJECTIVE: To determine the nationwide incidence of non-traumatic subconjunctival hemorrhage (NTSCH) in Taiwan from 2000-2011 and to analyze the risk factors for NTSCH using a case-control analysis. METHODS: This is a population-based cohort administrative database study. Randomly selected 1,000,000 residents from the Taiwan National Health Insurance Research Database in 2000 and followed for 12 years to determine the population incidence of NTSCH. Individuals with the first diagnosis of subconjunctival hemorrhage were identified by the corresponding International Classification of Diseases code (ICD) 372.72. Traumatic subconjunctival hemorrhages (ICD-9 codes 921, 871, 850-854 and 959.01) were excluded. The association of NTSCH with various demographic factors, comorbidities and use of medications was studied by a population based case-control analysis using data of 2008-2011. RESULTS: A total of 67,720 patients with a first-time diagnosis of NTSCH were identified during the 12 years period. The mean annual incidence was 65 per 10,000 individuals (crude incidence) and 60 per 10,000 individuals (age- and sex-standardized incidence). The incidence rate of NTSCH was higher in women than in men, [men-women ratio: 0.80 (95% confidence interval: 0.78-0.81)]. The age-specific incidence decreased from childhood to the group of teenagers, after which it increased to a maximal value of 136.2 per 10,000 people in the age group of 60-69 years. Case-control analysis showed that comorbidities of hypertension, purpura and thrombocytopenia, and the use of aspirin were significantly associated with the risk of NTSCH. CONCLUSIONS: This study indicates that NTSCH is a common eye disease that occurs once in 167 individuals in a general East Asian population per year. It occurs more often in women than in men and the age-specific incidence peaked in the age group of 60-69 years. Hypertension, purpura and thrombocytopenia, and the use of aspirin are the major risk factors for NTSCH.

Ultraviolet-A irradiation upregulated urokinase-type plasminogen activator in pterygium fibroblasts through ERK and JNK pathways.

PURPOSE: Effects of ultraviolet (UV) B on the pterygium and its cells have been studied previously, whereas little is known on the effects of UVA. Urokinase-type plasminogen activator (uPA) is a protease involved in tissue remodeling and cell migration, and its levels are increased in pterygium. The purpose of our study was to investigate the effects of UVA on the expression of uPA in cultured pterygium fibroblasts. METHODS: Cultured fibroblasts from early-stage pterygia and normal conjunctiva were irradiated with different dosages of UVA and compared to nonirradiated cells. uPA activities in the medium and uPA mRNA in the cells were measured by casein zymography and RT-PCR, respectively. Total and phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal-related kinase (ERK), and c-Jun N-terminal kinase (JNK) levels of cells treated with and without UVA were measured by Western blotting. Inhibitors of p38 (SB203580), ERK (UO1026), and JNK (SP600125) were added before the irradiation of UVA to test their effects. RESULTS: UVA irradiation increased the uPA mRNA levels in pterygium fibroblasts and the uPA activities in cultured medium, which was accompanied with an increase in phosphorylated ERK and JNK. The ERK and JNK inhibitor, but not p38 MAPK inhibitors, significantly decreased the UVA-induced expression of uPA by pterygium fibroblasts. Normal conjunctival fibroblasts were less sensitive to UVA irradiation compared to the pterygium fibroblasts. CONCLUSIONS: UVA stimulated the production of uPA, a key factor in the modulation of extracellular matrixes, inflammatory processes, and angiogenesis. This may have a role in the development and progression of pterygium.

Increased expression of gelatinase (MMP-2 and MMP-9) in pterygia and pterygium fibroblasts with disease progression and activation of protein kinase C.

PURPOSE: To study the expression of matrix metalloprotease (MMP)-2 and MMP-9 mRNA and activities in various stages of surgically excised pterygium specimens and cultured pterygium fibroblasts and to study the effects of activation of protein kinase C (PKC) on the expression of these MMPs in pterygium fibroblasts. METHODS: MMP-2 and MMP-9 mRNA expression and activities in 15 pterygium tissues and cultured pterygium fibroblasts were measured by RT-PCR and zymography. Five normal conjunctiva specimens and fibroblasts were tested as the controls. Changes of expression of MMP-2 and MMP-9 of fibroblasts after the simulation of a standard PKC activator, 2-O-tetradecanoyl-phorbol-13-acetate (TPA), were studied. RESULTS: MMP-2 and MMP-9 expression in pterygium tissues and fibroblasts was greater than those of normal tissues and fibroblasts and was closely relevant to the progression of pterygium. In early-stage pterygium tissues and cultured fibroblasts, MMP-9 was not expressed, activated MMP-2 could not be detected, and only a small amount of latent MMP-2 was present. In advanced-stage pterygium (pterygium head passed the papillary region), MMP-9 was expressed; activated MMP-2 and a large amount of latent MMP-2 could be detected in pterygium tissues and fibroblasts. TPA stimulated the expression of MMP-2 and MMP-9 by pterygium fibroblasts isolated from early-stage specimens in a dose-dependent manner. CONCLUSIONS: MMP-2 and MMP-9 expression by pterygium fibroblasts is significantly increased after the progression of pterygium. Activation of the PKC signaling pathway, aside from other previously reported signaling pathways, may play a role in the development and progression of pterygium.