Comparison of outcomes between modified double-flanged sutureless scleral fixation and conventional sutured scleral fixation.

This retrospective study aimed to compare the outcomes of modified double-flanged sutureless scleral fixation versus sutured scleral fixation. Medical records of 65 eyes from 65 patients who underwent double-flanged scleral fixation (flange group) or conventional scleral fixation (suture group) between 2021 and 2022 were reviewed. Visual and refractive outcomes, as well as postoperative complications, were compared 1, 2, and 6 months after surgery. We included 31 eyes in the flange group and 34 eyes in the suture group. At 6 months postoperatively, the flange group showed better uncorrected visual acuity (0.251 ± 0.328 vs. 0.418 ± 0.339 logMAR, P = 0.041) and a smaller myopic shift (- 0.74 ± 0.93 vs. - 1.33 ± 1.15 diopter, P = 0.007) compared to the suture group. The flange group did not experience any instances of iris capture, while the suture group had iris capture in 10 eyes (29.4%; P < 0.001). In the flange group, all intraocular lenses remained centered, whereas in the suture group, they were decentered in 8 eyes (23.5%; P = 0.005). The double-flanged technique not only prevented iris capture and decentration of the intraocular lens but also reduced myopic shift by enhancing the stability of the intraocular lens.

Dimerization of pro-oncogenic protein Anterior Gradient 2 is required for the interaction with BiP/GRP78.

Anterior Gradient 2 (AGR2), an ER stress-inducible protein, has been reported to be localized in endoplasmic reticulum (ER) and its level is elevated in numerous metastatic cancers. Recently, it has been demonstrated that AGR2 is involved in the control of ER homeostasis. However, the molecular mechanism how AGR2 regulates ER stress response remains unclear. Herein we show that AGR2 homo-dimerizes through an intermolecular disulfide bond. Moreover, dimerization of AGR2 attenuates ER stress-induced cell death through the association with BiP/GRP78. Thus, these results suggest that dimerization of AGR2 is crucial in mediating the ER stress signaling pathway.

Confirmation of Frm2 as a novel nitroreductase in Saccharomyces cerevisiae.

Nitroreductases comprise a group of FMN- or FAD-dependent enzymes that reduce nitrosubstituted compounds by using NAD(P)H, and are found in bacterial species and yeast. Although there is little information on the biological functions of nitroreductases, some studies suggest their possible involvement in oxidative stress responses. In the yeast Saccharomyces cerevisiae, a putative nitroreductase protein, Frm2, has been identified based on its sequence similarity with known bacterial nitroreductases. Frm2 has been reported to function in the lipid signaling pathway. To study the functions of Frm2, we measured the nitroreductase activity of purified Frm2 on 4-nitroquinoline-N-oxide (4-NQO) using NADH. LC-MS analysis of the reaction products revealed that Frm2 reduced NQO into 4-aminoquinoline-N-oxide (4-AQO) via 4-hydroxyaminoquinoline (4-HAQO). An Frm2 deletion mutant exhibited growth inhibition in the presence of 4-NQO. Thus, in this study, we demonstrate a novel nitroreductase activity of Frm2 and its involvement in the oxidative stress defense system.

Proteomic analysis of pancreata from mini-pigs treated with streptozotocin as a type I diabetes models.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by extreme insulin deficiency due to an overall reduction in the mass of functional pancreatic beta-cells. Several animal models have been used to study T1DM. Among them, mini-pig seems to be a useful model of diabetes because of its similarities in anatomy and physiology to humans. The purpose of this study is to analyze differentially expressed pancreatic proteins in streptozotocin (STZ)-induced mini-pig T1DM model. The pancreas proteins from mini-pigs treated with STZ were separated by 2-dimensional gel electrophoresis and eleven protein spots were found to be altered significantly compared with control mini-pigs. The data in this study from proteomic analysis provide a valuable resource for the further understanding of T1DM pathomechanism.

Proteomic analysis of liver tissue from HBx-transgenic mice at early stages of hepatocarcinogenesis.

The hepatitis B virus X-protein (HBx), a multifunctional viral regulator, participates in the viral life cycle and in the development of hepatocellular carcinoma (HCC). We previously reported a high incidence of HCC in transgenic mice expressing HBx. In this study, proteomic analysis was performed to identify proteins that may be involved in hepatocarcinogenesis and/or that could be utilized as early detection biomarkers for HCC. Proteins from the liver tissue of HBx-transgenic mice at early stages of carcinogenesis (dysplasia and hepatocellular adenoma) were separated by 2-DE, and quantitative changes were analyzed. A total of 22 spots displaying significant quantitative changes were identified using LC-MS/MS. In particular, several proteins involved in glucose and fatty acid metabolism, such as mitochondrial 3-ketoacyl-CoA thiolase, intestinal fatty acid-binding protein 2 and cytoplasmic malate dehydrogenase, were differentially expressed, implying that significant metabolic alterations occurred during the early stages of hepatocarcinogenesis. The results of this proteomic analysis provide insights into the mechanism of HBx-mediated hepatocarcinogenesis. Additionally, this study identifies possible therapeutic targets for HCC diagnosis and novel drug development for treatment of the disease.

Reduced formation of advanced glycation endproducts via interactions between glutathione peroxidase 3 and dihydroxyacetone kinase 1.

Dihydroxyacetone (DHA) induces the formation of advanced glycation endproducts (AGEs), which are involved in several diseases. Earlier, we identified dihydroxyacetone kinase 1 (Dak1) as a candidate glutathione peroxidase 3 (Gpx3)-interacting protein in Saccharomyces cerevisiae. This finding is noteworthy, as no clear evidence on the involvement of oxidative stress systems in DHA-induced AGE formation has been found to date. Here, we demonstrate that Gpx3 interacts with Dak1, alleviates DHA-mediated stress by upregulating Dak activity, and consequently suppresses AGE formation. Based on these results, we propose that defense systems against oxidative stress and DHA-induced AGE formation are related via interactions between Gpx3 and Dak1.

Differential expression of two glutamate transporters, GLAST and GLT-1, in an experimental rat model of glaucoma.

Glutamate is the major excitatory neurotransmitter of the mammalian retina, and excessive glutamate has been implicated in the pathogenesis of glaucoma. It is well known that glutamate transport, mainly via GLAST and GLT-1, is cardinal mechanism for maintaining glutamate homeostasis in normal and pathological conditions, including ischemia in the brain. In an effort to understand the role of glutamate and the glutamate regulation system of the retina in the pathogenesis of glaucoma, we examined changes in the expression of two glutamate transporters, GLAST and GLT-1, by Western blot analysis and immunocytochemistry in a rat glaucoma model. GLT-1 was expressed in cone photoreceptors and some cone bipolar cells and the levels of expression were significantly increased in the cauterized eyes throughout the entire experimental period. In contrast, GLAST expression, which occurred in Müller cells, the main retinal glial cells, remained stable during the experimental period. These results suggest that GLT-1 may be a prerequisite for the maintenance of glutamate homeostasis in the retina undergoing glaucoma.

Glycoproteomic analysis of plasma from patients with atopic dermatitis: CD5L and ApoE as potential biomarkers.

Atopic dermatitis (AD) is an inflammatory skin disorder that is both uncomfortable and distressing to patients, and its prevalence has been steadily increasing. It is obvious that the identification of efficient markers of AD in plasma would offer the possibility of effective diagnosis, prevention, and treatment strategies. In this study, a proteomic approach was used to analyze plasma glycoproteins from both children with AD and healthy child donors. Several protein spots showing significant quantitative changes in the AD patients were identified. Through sequential studies, it was confirmed that CD5L and ApoE were significantly up-regulated or down-regulated, respectively, in the plasma from AD patients compared with that from healthy donors. In addition, we suggest that the up-regulated CD5L in AD patients causes eosinophilia by inhibiting apoptosis or promoting the proliferation of eosinophils either in combination with or without IL-5. The glycoproteomic data in this study provides clues to understanding the mechanism of atopic alterations in plasma and suggests AD-related proteins can be used as candidate markers for AD.

Photodynamic therapy for polypoidal choroidal vasculopathy: vaso-occlusive effect on the branching vascular network and origin of recurrence.

PURPOSE: To determine whether photodynamic therapy (PDT) has a vaso-occlusive effect on the branching vascular network in polypoidal choroidal vasculopathy (PCV) and whether PDT can prevent future recurrence. METHODS: We analyzed pre-and post-PDT indocyanine green angiography (ICGA) results of 27 patients (27 eyes) who were diagnosed with PCV and who had shown clinical improvement accompanied by occlusion of polypoidal lesions after PDT. We also investigated the recurrent events in these patients and the origin of the recurrences. RESULTS: The branching vascular network persisted, at least in part, in 20 (87%) of 23 eyes undergoing one PDT session and in two (50%) of four eyes undergoing two PDT sessions. In the remaining five eyes, we could not determine definitively whether the branching vessels were occluded completely. Recurrent serous changes occurred in nine eyes (33%) 14-48 months after the last PDT. ICGA revealed that these changes were caused by new polypoidal lesions that originated from further-grown branches of the persistent branching vascular network. Diffuse leakage from undetermined sources seemed to coexist in two eyes. CONCLUSIONS: PDT cannot induce complete occlusion of the branching vascular network. PDT does not prevent future recurrence, because new active polypoidal lesions may develop from the persistent branching vessels in the network.

Gpx3-dependent responses against oxidative stress in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has defense mechanisms identical to higher eukaryotes. It offers the potential for genome-wide experimental approaches owing to its smaller genome size and the availability of the complete sequence. It therefore represents an ideal eukaryotic model for studying cellular redox control and oxidative stress responses. S. cerevisiae Yap1 is a well-known transcription factor that is required for H2O2-dependent stress responses. Yap1 is involved in various signaling pathways in an oxidative stress response. The Gpx3 (Orp1/PHGpx3) protein is one of the factors related to these signaling pathways. It plays the role of a transducer that transfers the hydroperoxide signal to Yap1. In this study, using extensive proteomic and bioinformatics analyses, the function of the Gpx3 protein in an adaptive response against oxidative stress was investigated in wild-type, gpx3-deletion mutant, and gpx3-deletion mutant overexpressing Gpx3 protein strains. We identified 30 proteins that are related to the Gpx3- dependent oxidative stress responses and 17 proteins that are changed in a Gpx3-dependent manner regardless of oxidative stress. As expected, H2O2-responsive Gpx3-dependent proteins include a number of antioxidants related with cell rescue and defense. In addition, they contain a variety of proteins related to energy and carbohydrate metabolism, transcription, and protein fate. Based upon the experimental results, it is suggested that Gpx3-dependent stress adaptive response includes the regulation of genes related to the capacity to detoxify oxidants and repair oxidative stress-induced damages affected by Yap1 as well as metabolism and protein fate independent from Yap1.

Interactome analysis of yeast glutathione peroxidase 3.

Oxidative stress damages all cellular constituents, and therefore, cell has to possess various defense mechanisms to cope. Saccharomyces cerevisiae, widely used as a model organism for studying cellular responses to oxidative stress, contains three glutathione peroxidase (Gpx) proteins. Among them, Gpx3 plays a major defense role against oxidative stress in S. cerevisiae. In this study, in order to identify the new interaction proteins of Gpx3, we carried out two-dimensional gel electrophoresis after immunoprecipitation (IP-2DE), and MALDI-TOF mass spectrometry. The results showed that several proteins including protein disulfide isomerase, glutaredoxin 2, and SSY protein 3 specifically interact with Gpx3. These findings led us to suggest the possibility that Gpx3, known as a redox sensor and ROS scavenger, has another functional role by interacting with several proteins with various cellular functions.

Phosphoproteomic analysis of AML14.3D10 cell line as a model system of eosinophilia.

Eosinophils act as effectors in the inflammatory reactions of allergic diseases including atopic dermatitis. Atopic dermatitis patients and others with allergic disorders suffer from eosinophilia, an accumulation of eosinophils due to increased survival or decreased apoptosis of eosinophils. In this study, a differential phosphoproteome analysis of AML14.3D10 eosinophil cell line after treatment with IL-5 or dexamethasone was conducted in an effort to identify the phosphoproteins involved in the proliferation or apoptosis of eosinophils. Proteins were separated by 2-DE and alterations in phosphoproteins were then detected by Pro-Q Diamond staining. The significant quantitative changes were shown in nineteen phosphoproteins including retinoblastoma binding protein 7, MTHSP75, and lymphocyte cytosolic protein 1. In addition, seven phosphoproteins including galactokinase I, and proapolipoprotein, were appeared after treatment with IL-5 or dexamethasone. Especially, the phospho-APOE protein was down-regulated in IL-5 treated AML14.3D10, while the more heavily phosphorylated APOE form was induced after dexamethasone treatment. These phosphoproteome data for the AML14.3D10 cell line may provide clues to understand the mechanism of eosinophilia as well as allergic disorders including atopic dermatitis.

Neovascularization associated with large retinal pigment epithelial detachment in elderly Korean patients: subdivision according to indocyanine green angiographic features.

PURPOSE: In Korean patients, to subdivide the neovascular forms of age-related macular degeneration (AMD) associated with large retinal pigment epithelial detachment (PED), according to the indocyanine green angiographic features. METHODS: Indocyanine green angiograms (ICGA) of 67 elderly patients (72 eyes) who presented with a PED of at least 1 disc diameter were evaluated retrospectively. RESULTS: Polypoidal choroidal vasculopathy (PCV) and typical choroidal neovascularization (CNV)-associated PEDs were identified in 18 eyes (25%) and 19 eyes (26%), respectively. In ten eyes (13.9%), the exact type of neovascularization, whether PCV or CNV, could not be determined. Pure serous PEDs were identified in seven eyes (10%). The remaining 18 eyes (25%) were classified as having retinal angiomatous proliferation (RAP)-associated PED based upon the angiographic findings of vascular connections between the retinal vasculature and the neovascular complex. CONCLUSIONS: Three subset groups of PCV, CNV, and RAP were present with similar frequency in neovascularized AMD with a large PED in these Korean patients. In particular, RAP, previously thought to be rare in Asian patients, was found to be present with considerable frequency.

Anticancer activity of a novel small molecule tubulin inhibitor STK899704.

We have identified the small molecule STK899704 as a structurally novel tubulin inhibitor. STK899704 suppressed the proliferation of cancer cell lines from various origins with IC50 values ranging from 0.2 to 1.0 µM. STK899704 prevented the polymerization of purified tubulin in vitro and also depolymerized microtubule in cultured cells leading to mitotic arrest, associated with increased Cdc25C phosphorylation and the accumulation of both cyclin B1 and polo-like kinase 1 (Plk1), and apoptosis. Unlike many anticancer drugs such as Taxol and doxorubicin, STK899704 effectively displayed antiproliferative activity against multidrug-resistant cancer cell lines. The proposed binding mode of STK899704 is at the interface between αß-tubulin heterodimer overlapping with the colchicine-binding site. Our in vivo carcinogenesis model further showed that STK 899704 is potent in both the prevention and regression of tumors, remarkably reducing the number and volume of skin tumor by STK899704 treatment. Moreover, it was significant to note that the efficacy of STK899704 was surprisingly comparable to 5-fluorouracil, a widely used anticancer therapeutic. Thus, our results demonstrate the potential of STK899704 to be developed as an anticancer chemotherapeutic and an alternative candidate for existing therapies.

Histone H4 is cleaved by granzyme A during staurosporine-induced cell death in B-lymphoid Raji cells.

Granzyme A (GzmA) was first identified as a cytotoxic T lymphocyte protease protein with limited tissue expression. A number of cellular proteins are known to be cleaved by GzmA, and its function is to induce apoptosis. Histones H1, H2B, and H3 were identified as GzmA substrates during apoptotic cell death. Here, we demonstrated that histone H4 was cleaved by GzmA during staurosporine-induced cell death; however, in the presence of caspase inhibitors, staurosporine-treated Raji cells underwent necroptosis instead of apoptosis. Furthermore, histone H4 cleavage was blocked by the GzmA inhibitor nafamostat mesylate and by GzmA knockdown using siRNA. These results suggest that histone H4 is a novel substrate for GzmA in staurosporine-induced cells. [BMB Reports 2016; 49(10): 560-565].

Histone H3 is Digested by Granzyme A During Compromised Cell Death in the Raji Cells.

Granzyme A (GzmA) was identified as a cytotoxic T lymphocyte protease protein expressed in the nucleus. A number of nuclear proteins are well known as GzmA substrates, and GzmA is related with caspase-independent apoptosis. Histones H1, H2B, and H3 were identified as GzmA substrates through in vitro experiment with purified nucleosome. Here, we demonstrated that histone H3 was cleaved by GzmA in vivo during staurosporine-induced cell death. Moreover, histone H3 cleavage was blocked by the GzmA inhibitor nafamostat mesylate and by GzmA knockdown using siRNA. Taken together, we verified that histone H3 is a real substrate for GzmA in vivo in the Raji cells treated by staurosporin.

Small heterodimer partner interacts with NLRP3 and negatively regulates activation of the NLRP3 inflammasome.

Excessive activation of the NLRP3 inflammasome results in damaging inflammation, yet the regulators of this process remain poorly defined. Herein, we show that the orphan nuclear receptor small heterodimer partner (SHP) is a negative regulator of NLRP3 inflammasome activation. NLRP3 inflammasome activation leads to an interaction between SHP and NLRP3, proteins that are both recruited to mitochondria. Overexpression of SHP competitively inhibits binding of NLRP3 to apoptosis-associated speck-like protein containing a CARD (ASC). SHP deficiency results in increased secretion of proinflammatory cytokines IL-1ß and IL-18, and excessive pathologic responses typically observed in mouse models of kidney tubular necrosis and peritoneal gout. Notably, the loss of SHP results in accumulation of damaged mitochondria and a sustained interaction between NLRP3 and ASC in the endoplasmic reticulum. These data are suggestive of a role for SHP in controlling NLRP3 inflammasome activation through a mechanism involving interaction with NLRP3 and maintenance of mitochondrial homeostasis.

Expression of the pro-domain-deleted active form of caspase-6 in Escherichia coli.

Caspases are a family of cysteine proteases that play an important role in the apoptotic pathway. Caspase-6 is an apoptosis effector that cleaves a variety of cellular substrates. The active form of the enzyme is required for use in research. However, it has been difficult to obtain sufficient quantities of active caspase-6 from Escherichia coli. In the present study, we constructed a caspase-6 with a 23-amino-acid deletion in the pro-domain. This engineered enzyme was expressed as a soluble protein in E. coli and was purified using affinity resin. In vitro enzyme assay and cleavage analysis revealed that the engineered active caspase-6 protein had characteristics similar to those of wild-type caspase-6. This novel method can be a valuable tool for obtaining active caspase-6 that can be used for screening caspase-6-specific substrates, which in turn can be used to elucidate the function of caspase-6 in apoptosis.

Ginsenoside Rb1 is transformed into Rd and Rh2 by Microbacterium trichothecenolyticum.

Ginsenosides are the most important ingredient of ginseng and are known to possess many pharmacological and biological effects. Rb1, a major protopanaxadiol ginsenoside, is the most abundant ginsenoside in Panax ginseng C.A Meyer and can be hydrolyzed into more pharmaceutically potent minor ginsenosides. To identify a microorganism that is capable of converting Rb1 into other ginsenosides, we screened 12 Microbacterium spp., and M. trichothecenolyticum was identified as a likely candidate. M. trichothecenolyticum converted Rb1 into Rd and then into Rh2 based on TLC and HPLC analyses of reaction products. This biotransformation method can be easily applied for mass production of Rd and Rh2 by using Rb1.