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.

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.

Identification of the novel substrates for caspase-6 in apoptosis using proteomic approaches.

Apoptosis, programmed cell death, is a process involved in the development and maintenance of cell homeostasis in multicellular organisms. It is typically accompanied by the activation of a class of cysteine proteases called caspases. Apoptotic caspases are classified into the initiator caspases and the executioner caspases, according to the stage of their action in apoptotic processes. Although caspase-3, a typical executioner caspase, has been studied for its mechanism and substrates, little is known of caspase-6, one of the executioner caspases. To understand the biological functions of caspase-6, we performed proteomics analyses, to seek for novel caspase-6 substrates, using recombinant caspase-6 and HepG2 extract. Consequently, 34 different candidate proteins were identified, through 2-dimensional electrophoresis/MALDI-TOF analyses. Of these identified proteins, 8 proteins were validated with in vitro and in vivo cleavage assay. Herein, we report that HAUSP, Kinesin5B, GEP100, SDCCAG3 and PARD3 are novel substrates for caspase-6 during 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.

Candidate target genes for the Saccharomyces cerevisiae transcription factor, Yap2.

In Saccharomyces cerevisiae, the Yap family of basic leucine zipper (bZip) proteins contains eight members. The Yap family proteins are implicated in a variety of stress responses; among these proteins, Yap1 acts as a major regulator of oxidative stress responses. However, the functional roles of the remaining Yap family members are poorly understood. To elucidate the function of Yap2, we mined candidate target genes of Yap2 by proteomic analysis. Among the identified genes, FRM2 was previously identified as a target gene of Yap2, which confirmed the validity of our screening method. YNL134C and YDL124W were also identified as candidate Yap2 target genes. These genes were upregulated in strains overexpressing Yap2 and possess Yap2 target sequences in their promoter regions. Furthermore, chromatin immunoprecipitation assays showed that YNL134C and YDL124W have Yap2 binding motif. These data will help to elucidate the functional role of Yap2.

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.

The S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase 2 is reduced by interaction with glutathione peroxidase 3 in Saccharomyces cerevisiae.

Glutathione peroxidases (Gpxs) are the key anti-oxidant enzymes found in Saccharomyces cerevisiae. Among the three Gpx isoforms, glutathione peroxidase 3 (Gpx3) is ubiquitously expressed and modulates the activities of redox-sensitive thiol proteins involved in various biological reactions. By using a proteomic approach, glyceraldehyde-3-phosphate dehydrogenase 2 (GAPDH2; EC 1.2.1.12) was found as a candidate protein for interaction with Gpx3. GAPDH, a key enzyme in glycolysis, is a multi-functional protein with multiple intracellular localizations and diverse activities. To validate the interaction between Gpx3 and GAPDH2, immunoprecipitation and a pull-down assay were carried out. The results clearly showed that GAPDH2 interacts with Gpx3 through its carboxyl-terminal domain both in vitro and in vivo. Additionally, Gpx3 helps to reduce the S-nitrosylation of GAPDH upon nitric oxide (NO) stress; this subsequently increases cellular viability. On the basis of our findings, we suggest that Gpx3 protects GAPDH from NO stress and thereby contributes to the maintenance of homeostasis during exposure to NO stress.

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.

Severe choroidal ischemia following photodynamic therapy for pigment epithelial detachment and chronic central serous chorioretinopathy.

BACKGROUND: Photodynamic therapy (PDT) has proven beneficial in the treatment of central serous chorioretinopathy (CSC). CASES: Two patients with serous pigment epithelial detachments (PEDs) and one patient with chronic CSC developed an abrupt visual loss caused by severe choroidal ischemia following PDT. Serous PEDs in the two patients were regarded as a latent variant of CSC. PDT was performed in accordance with the Treatment of Age-Related Macular Degeneration with Photodynamic Therapy guidelines. OBSERVATIONS: Although following PDT, PEDs and serous sensory retinal detachments resolved in all patients, the laser-treated areas of the patients revealed severe choroidal ischemia and their visual acuity decreased. Only in case 1 did visual acuity recover to the pretreatment level 2 months after treatment. In case 2, visual acuity was still reduced 7 months after treatment. In case 3, secondary choroidal neovascularization developed in the treatment area 1 month after treatment. CONCLUSIONS: In the CSC variant associated with PED only or in chronic CSC, PDT performed by the standard procedure can increase the risk of severe choroidal ischemia. Further studies are needed to find safe and optimal PDT parameters.

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.

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.