A one-pot transcriptional assay method that detects the tumor biomarker FEN1 based on its flap cleavage activity.

BACKGROUND: Detection of tumor biomarkers in body fluids is a significant advancement in cancer treatment because it allows diagnosis without invasive tissue biopsies. Nucleases have long been regarded as a potential class of biomarkers that can indicate the occurrence and progression of cancers. Among these, flap endonuclease 1 (FEN1) plays an important role in DNA replication and repair, and also overexpressed in abnormally proliferating cells such as cancer cells. FEN1 is thus considered to be a potential biomarker as well as a target for cancer therapy. RESULTS: We developed a novel method for detecting FEN1 based on its specific endonuclease activity which incises bifurcated nucleic acids (flaps), in combination with in vitro transcription. Developed method uses a simple DNA structure (substrate DNA) carrying a short 5'-flap sequence, and a single-stranded sensor DNA encoding the Broccoli light-up aptamer. When the assay mixture was supplied with a FEN1-containing sample, the flap sequence encoding the sense sequence of T7 promoter was cleaved and released from the substrate DNA. Because the sensor DNA was designed to carry the Broccoli RNA aptamer under the antisense sequence of T7 promoter, hybridization of the excised flap onto the sensor DNA initiated the transcription of the Broccoli RNA aptamer, enabling determination of the FEN1 titer based on the fluorescence of transcribed Broccoli aptamer. By using a combination of FEN1-mediated generation of a short oligonucleotide and subsequent oligonucleotide-dependent in vitro transcription, this method could detect FEN1 in biological samples within 1 h. SIGNIFICANCE AND NOVELTY: Developed method enables the detection of FEN1 by a simple one-pot reaction. It can detect sub-nanomolar concentrations of FEN1 within an hour, and has the potential to be used for cancer diagnosis, prognosis, and drug screening. It also enables easy identification of compounds that inhibit FEN1 activity and is thus a versatile platform for screening anti-cancer drugs. We anticipate that the basic principles of this assay can be applied to detect other biomolecules, such as nucleic acids.

Genome-wide evidences of bisphenol a toxicity using Schizosaccharomyces pombe.

To clarify reliable toxic mechanisms of bisphenol A (BPA), an endocrine disrupting chemical, we approached an alternative animal and whole genome analyses with the yeast knockout library (YKO) of Schizosaccharomyces pombe. As results, the 50% growth inhibition concentrations (GI50) of BPA was approximately 600 µM and the YKO-three step screening revealed the top 10 target candidate genes including dbp2, utp18, srs1, tif224, use1, qcr1, etc. The screening results were confirmed in human embryonic stem cell (hES)-derived hepatic cells and HepG2 human liver cancer cells. We found BPA down-regulated UQCRC, the human orthlog of S. pombe- qcr1, as a part of the mitochondrial respiratory chain, in HepG2 cells and hESs during cell differentiation into hepatic cells. Therefore, BPA may induce mitochondrial dysfunction and disruption of differentiation by suppressing UQCRC1.

Characterization of the Two Methylation Steps Involved in the Biosynthesis of Mycinose in Tylosin.

The S-adenosyl-l-methionine-dependent O-methyltransferases TylE and TylF catalyze the last two methylation reactions in the tylosin biosynthetic pathway of Streptomyces fradiae. It has long been known that the TylE-catalyzed C2‴-O-methylation of the 6-deoxy-d-allose bound to demethylmacrocin or demethyllactenocin precedes the TylF-catalyzed C3‴-O-methylation of the d-javose (C2‴-O-methylated 6-deoxy-d-allose) attached to macrocin or lactenocin. This study reveals the unexpected substrate promiscuity of TylE and TylF responsible for the biosynthesis of d-mycinose (C3‴-O-methylated d-javose) in tylosin through the identification of a new minor intermediate 2‴-O-demethyldesmycosin (2; 3‴-methyl-demethyllactenocin), which lacks a 2‴-O-methyl group on the mycinose moiety of desmycosin, along with 2‴-O-demethyltylosin (1; 3‴-methyl-demethylmacrocin) that was previously detected from the S. fradiae mutant containing a mutation in the tylE gene. These results unveil the unique substrate flexibility of TylE and TylF and demonstrate their potential for the engineered biosynthesis of novel glycosylated macrolide derivatives.

Identification of a Mitochondrial DNA Polymerase Affecting Cardiotoxicity of Sunitinib Using a Genome-Wide Screening on S. pombe Deletion Library.

Drug toxicity is a key issue for drug R&D, a fundamental challenge of which is to screen for the targets genome-wide. The anticancer tyrosine kinase inhibitor sunitinib is known to induce cardiotoxicity. Here, to understand the molecular insights of cardiotoxicity by sunitinib at the genome level, we used a genome-wide drug target screening technology (GPScreen) that measures drug-induced haploinsufficiency (DIH) in the fission yeast Schizosaccharomyces pombe genome-wide deletion library and found a mitochondrial DNA polymerase (POG1). In the results, sunitinib induced more severe cytotoxicity and mitochondrial damage in POG1-deleted heterozygous mutants compared to wild type (WT) of S. pombe. Furthermore, knockdown of the human ortholog POLG of S. pombe POG1 in human cells significantly increased the cytotoxicity of sunitinib. Notably, sunitinib dramatically decreased the levels of POLG mRNAs and proteins, of which downregulation was already known to induce mitochondrial damage of cardiomyocytes, causing cardiotoxicity. These results indicate that POLG might play a crucial role in mitochondrial damage as a gene of which expressional pathway is targeted by sunitinib for cardiotoxicity, and that genome-wide drug target screening with GPScreen can be applied to drug toxicity target discovery to understand the molecular insights regarding drug toxicity.

Synthesis and Evaluation of Neuroprotective Selenoflavanones.

The physicochemical properties and antioxidant activity of a molecule could be improved by the substitution of an oxygen atom in a molecule with selenium. We synthesized selenoflavanones and flavanones to evaluate their neuroprotective effects. The selenoflavanones showed improved physicochemical properties, suggestive of the ability to pass through the blood-brain barrier (BBB). They showed in vitro antioxidant effects against hydrogen peroxide, and did not result in severe cytotoxicity. Moreover, infarction volumes in a transient ischemia mouse model were significantly reduced by the selenoflavanone treatments.

Implementing bacterial acid resistance into cell-free protein synthesis for buffer-free expression and screening of enzymes.

Cell-free protein synthesis utilizes translational machinery isolated from the cells for in vitro expression of template genes. Because it produces proteins without gene cloning and cell cultivation steps, cell-free protein synthesis can be used as a versatile platform for high-throughput expression of enzyme libraries. Furthermore, the open nature of cell-free protein synthesis allows direct integration of enzyme synthesis with subsequent screening steps. However, the presence of high concentration of chemical buffers in the conventional reaction mixture makes it difficult to streamline cell-free protein synthesis with pH-based assay of the synthesized enzymes. In this study, we have implemented an enzyme-assisted bacterial acid resistance mechanism into an Escherichia coli (E.coli) extract-based cell-free protein synthesis system in place of chemical buffers. When deployed in the reaction mixture for cell-free synthesis of enzymes, through proton-consuming conversion of glutamate into γ-aminobutyric acid (GABA), an engineered glutamate decarboxylase (GADß) was able to maintain the pH of reaction mixture during enzyme synthesis. Because the reaction mixture becomes free of buffering capacity upon the depletion of glutamate, synthesized enzyme could be directly assayed without purification steps. The designed method was successfully applied to the screening of mutant library of sialyltransferase genes to identify mutants with improved enzymatic activity.

Five-year incidence of primary open-angle glaucoma and rate of progression in health center-based Korean population: the Gangnam Eye Study.

OBJECTIVE: To investigate the 5-year incidence and progression rate of primary open-angle glaucoma (POAG) in a health-center-based Korean population. METHODS: The study population comprised 5,021 subjects who participated in standardized health screening (including non-contact tonometry and fundus photography) at the Gangnam Healthcare Center during the period from January 2005 to December 2006 and again from January 2010 to December 2011. Among these subjects, 948 (18.9%) with findings suggestive of glaucoma were subjected to a comprehensive glaucoma evaluation, which included applanation tonometry and standard automated perimetry. Based on the results, the subjects were diagnosed as POAG suspect or definite POAG. RESULTS: The 5-year incidences of POAG suspect and definite POAG were 0.84% (42 subjects) and 0.72% (36 subjects), respectively. The rate of progression from POAG suspect to definite POAG was 4.75% per year. In subjects with a baseline intraocular pressure (IOP) >21 mmHg, the incidence of POAG suspect or definite POAG was significantly higher than in those with a baseline IOP ≤ 21 mmHg (32% vs. 1.05%; P<0.001). A multivariate analysis showed that the progression from POAG suspect to definite POAG was significantly associated with older age (odds ratio [OR], 1.07; 95% confidence interval [CI], 1.03-1.10), higher baseline IOP (OR, 1.10; 95% CI, 1.01-1.24), higher body mass index (BMI) (OR, 1.15; 95% CI, 1.03-1.31), higher education level (OR, 1.57; 95% CI, 1.05-2.17), and higher hematocrit level (OR, 1.22; 95% CI, 1.08-1.43). CONCLUSIONS: In the health-center-based Korean population, the 5-year incidence of POAG was 0.72%, and the rate of progression from POAG suspect to definite POAG was 4.75% per year. This study identified old age, high baseline IOP, high BMI, high level of education, and high hematocrit level as significant risk factors for incident POAG.

The natural anticancer agent plumbagin induces potent cytotoxicity in MCF-7 human breast cancer cells by inhibiting a PI-5 kinase for ROS generation.

Drug-induced haploinsufficiency (DIH) in yeast has been considered a valuable tool for drug target identification. A plant metabolite, plumbagin, has potent anticancer activity via reactive oxygen species (ROS) generation. However, the detailed molecular targets of plumbagin for ROS generation are not understood. Here, using DIH and heterozygous deletion mutants of the fission yeast Schizosaccharomyces pombe, we identified 1, 4-phopshatidylinositol 5-kinase (PI5K) its3 as a new molecular target of plumbagin for ROS generation. Plumbagin showed potent anti-proliferative activity (GI(50); 10 µM) and induced cell elongation and septum formation in wild-type S. pombe. Furthermore, plumbagin dramatically increased the intracellular ROS level, and pretreatment with the ROS scavenger, N-acetyl cysteine (NAC), protected against growth inhibition by plumbagin, suggesting that ROS play a crucial role in the anti-proliferative activity in S. pombe. Interestingly, significant DIH was observed in an its3-deleted heterozygous mutant, in which ROS generation by plumbagin was higher than that in wild-type cells, implying that its3 contributes to ROS generation by plumbagin in this yeast. In MCF7 human breast cancer cells, plumbagin significantly decreased the level of a human ortholog, 1, 4-phopshatidylinositol 5-kinase (PI5K)-1B, of yeast its3, and knockdown of PI5K-1B using siPI5K-1B increased the ROS level and decreased cell viability. Taken together, these results clearly show that PI5K-1B plays a crucial role in ROS generation as a new molecular target of plumbagin. Moreover, drug target screening using DIH in S. pombe deletion mutants is a valuable tool for identifying molecular targets of anticancer agents.

Anterior chamber configuration changes after cataract surgery in eyes with glaucoma.

PURPOSE: To evaluate changes in anterior chamber depth (ACD) and angle width induced by phacoemulsification and intraocular lens (IOL) implantation in eyes with glaucoma, using anterior segment optical coherence tomography (AS-OCT). METHODS: Eleven eyes of 11 patients with angle-closure glaucoma (ACG) and 12 eyes of 12 patients with open-angle glaucoma (OAG) underwent phacoemulsification and IOL implantation. Using AS-OCT, ACD and angle parameters were measured before and 2 days after surgery. Change in intraocular pressure (IOP) and number of ocular hypotensive drugs were evaluated. RESULTS: After surgery, central ACD and angle parameters increased significantly in eyes with glaucoma (p < 0.05). Prior to surgery, mean central ACD in the ACG group was approximately 1.0 mm smaller than that in the OAG group (p < 0.001). Post surgery, mean ACD of the ACG group was still significantly smaller than that of the OAG group. No significant differences were found in angle parameters between the ACG and OAG groups. In the ACG group, postoperative IOP at the final visit was significantly lower than preoperative IOP (p = 0.018) and there was no significant change in the number of ocular hypotensive medications used, although clinically, patients required fewer medications. In the OAG group, the IOP and number of ocular hypotensive drugs were almost unchanged after surgery. CONCLUSIONS: The ACD and angle width in eyes with glaucoma increased significantly after phacoemulsification and IOL implantation. Postoperative ACD significantly differed between the ACG and OAG groups, whereas angle parameters did not differ.

Correlation between retinal nerve fiber layer thickness and visual field sensitivity: diffuse atrophy imaging study.

BACKGROUND AND OBJECTIVE: To identify the correlation between retinal nerve fiber layer (RNFL) thickness and visual field sensitivity in healthy eyes with preperimetric and perimetric glaucoma and to estimate the functional RNFL loss in eyes with pre-perimetric glaucoma. PATIENTS AND METHODS: One hundred and two eyes with glaucoma and diffuse RNFL atrophy and 102 healthy eyes were enrolled. The correlation between optical coherence tomography (OCT)-measured RNFL thickness of the superior (clock-hour segments 10, 11, 12, 1, 2, and 3) and inferior (clock-hour segments 5, 6, 7, and 8) area and the average total deviations of the inferior and superior hemifields in standard automated perimetry (SAP) were evaluated using the simple linear model, respectively. The OCT-measured RNFL thickness was assumed to comprise functional and residual thicknesses; the residual thickness was calculated from the simple linear model and the eyes with severe diffuse RNFL atrophy. Functional RNFL thickness was compared between groups. RESULTS: Twenty-seven eyes had preperimetric and 75 eyes had perimetric glaucoma. The coefficient of determination (R(2)) of the simple linear model was 0.71 to 0.77 for the correlation between RNFL thickness and total deviation of SAP. The estimated residual thickness was 50.4 to 56.5 µm. On comparison with normal eyes, eyes with preperimetric glaucoma were estimated to have 37% to 41% functional RNFL loss. CONCLUSION: The correlation between RNFL thickness and SAP sensitivity was well explained by the simple linear model. Approximately 40% loss of the functional RNFL was found in preperimetric glaucoma.

Changes in anterior chamber configuration after cataract surgery as measured by anterior segment optical coherence tomography.

PURPOSE: To evaluate the changes in anterior chamber depth (ACD) and angle width induced by phacoemulsification and intraocular lens (IOL) implantation in normal eyes using anterior segment optical coherence tomography (AS-OCT). METHODS: Forty-five eyes (45 patients) underwent AS-OCT imaging to evaluate anterior chamber configuration before and 2 days after phacoemulsification and IOL implantation. We analyzed the central ACD and angle width using different methods: anterior chamber angle (ACA), trabecular-iris angle (TIA), angle opening distance (AOD), and trabecular iris surface area (TISA) in the nasal and temporal quadrants. Comparison between preoperative and postoperative measurement was done using paired t-tests and each of the angle parameters was analyzed with Pearson correlation testing. Subgroup analyses according to the IOL and axial length were performed with a general multivariate linear model adjusted for age. RESULTS: Before surgery, the mean anterior chamber angle widths were 23.21 ± 6.70° in the nasal quadrant and 24.89 ± 7.66° in the temporal quadrant. The mean central ACD was 2.75 ± 0.43 mm. After phacoemulsification and IOL implantation, the anterior chamber angle width increased significantly to 35.16 ± 4.65° in the nasal quadrant (p = 0.001) and 36.03 ± 4.86° in the temporal quadrant (p = 0.001). Also, central ACD increased to 4.14 ± 0.31 mm (p = 0.001). AOD, TISA, and TIA increased significantly after cataract surgery and showed positive correlation with ACA. CONCLUSIONS: After cataract surgery, the ACD and angle width significantly increased in eyes with cataract. AS-OCT is a good method for obtaining quantitative data regarding anterior chamber configuration.

A new cloning strategy for generating multiple repeats of a repetitive polypeptide based on non-template PCR.

A new cloning method for generating multiple repeats of amino acids is described which can be used as biomaterials, protein polymers and biomedical applications. Although several traditional methods for cloning multiple repeats are still exploited, these are laborious and complicated because they must go through several consecutive cloning steps. To solve these problems, synthetic gene libraries encoding repetitive patterns were constructed by using non-template PCR. As a result, a 'length library' with fourteen different ELP repeating genes was constructed and expressed in a cell-free protein synthesis system. These results showed our novel cloning method is efficient, and has the potential capacity for synthesizing repetitive genes by PCR to be cloned in any commercial expression vectors.

Ahmed glaucoma valve implantation for neovascular glaucoma after vitrectomy for proliferative diabetic retinopathy.

PURPOSE: To evaluate the safety and efficacy of Ahmed Glaucoma Valve implantation (AGVI) for the management of neovascular glaucoma (NVG) associated with proliferative diabetic retinopathy (PDR) in the vitrectomized eyes. PATIENTS AND METHODS: We reviewed the medical records of patients with NVG associated with PDR who underwent AGVI for intraocular pressure (IOP) control and compared the surgical outcome according to vitrectomy history. The main outcome measures were: postoperative IOP control, visual acuity, and complications. Success was defined as an IOP of ≤21 mm Hg and ≥6 mm Hg, without further glaucoma surgery or loss of light perception and devastating complications. RESULTS: A total of 73 patients (73 eyes) were included: 42 patients with vitrectomy history before AGVI (vitrectomized group) and 31 patients without vitrectomy history (nonvitrectomized group). The cumulative probabilities of success after AGVI were 89.9% and 83.8% after 1 year, 74.8% and 74.7% after 2 years, and 62.5% and 68.5% after 3 years for the vitrectomized group and the nonvitrectomized group, respectively (P=0.9309). Cox proportional hazards regression showed the intraocular silicone oil tamponade as a risk factor for the surgical failure (odds ratio=4.543, P=0.047). Final visual acuity improved or stabilized in 33 patients (78.6%) in the vitrectomized group and 18 patients (58.1%) in the nonvitrectomized group. Complications were comparable between the groups, but surgical interventions were needed for 5 patients (11.9%) in the vitrectomized group. CONCLUSION: Despite some complications that necessitate surgical intervention, the AGVI is a safe and effective procedure that enables successful IOP control and vision preservation in patients with NVG associated with vitrectomy for the PDR.

NSC126188, a piperazine alkyl derivative, induces apoptosis via upregulation of RhoB in HeLa cells.

We describe here a piperazine alkyl derivative, NSC126188, which induced apoptosis of HeLa cells by upregulating RhoB expression. NSC126188 caused multi-septation of fission yeast and hypersensitized a ∆rho3 mutant, which implicates the involvement of functional human homolog RhoB. The treatment of cells with NSC126188 induced apoptosis and a dramatic increase in RhoB expression. In addition, RhoB knockdown using siRNA rescued cells from apoptosis, indicating a crucial role of RhoB in NSC126188-induced apoptosis. In a reporter assay using luciferase and EGFP under control of the RhoB promoter, NSC126188 increased both luciferase activity and the expression of EGFP, implicating transcriptional activation of RhoB by NSC126188. Furthermore, NSC126188 demonstrated in vivo anti-tumor activity, inhibiting tumor growth by 66.8% in a nude mouse xenograft using PC-3 human prostate cancer cells. These results suggest that NSC126188 is a potential lead compound and that upregulation of RhoB is associated with NSC126188-induced apoptosis.

Upregulation of RhoB via c-Jun N-terminal kinase signaling induces apoptosis of the human gastric carcinoma NUGC-3 cells treated with NSC12618.

RhoB expression is reduced in most invasive tumors, with loss of RhoB expression correlating significantly with tumor stage. Here, we demonstrate that upregulation of RhoB by the potent anticancer agent NSC126188 induces apoptosis of NUGC-3 human gastric carcinoma cells. The crucial role of RhoB in NSC126188-induced apoptosis is indicated by the rescue of NUGC-3 cells from apoptosis by knockdown of RhoB. In the presence of NSC126188, c-Jun N-terminal kinase (JNK) signaling was activated, and the JNK inhibitor SP600125 reduced RhoB expression and suppressed the apoptosis of NUGC-3 cells. Knockdowns of mitogen-activated protein kinase kinase (MKK) 4/7, JNK1/2 and c-Jun downregulated RhoB expression and rescued cells from apoptotic death in the presence of NSC126188. The JNK inhibitor SP600125 suppressed transcriptional activation of RhoB in the presence of NSC126188, as indicated by a reporter assay that used luciferase under the RhoB promoter. The ability of NSC126188 to increase luciferase activity through both the p300-binding site and the inverted CCAAT sequence (iCCAAT box) suggests that JNK signaling to upregulate RhoB expression is mediated through both the p300-binding site and the iCCAAT box. However, the JNK inhibitor SP600125 did not inhibit the upregulation of RhoB by farnesyltransferase inhibitor (FTI)-277. The p300-binding site did not affect activation of the RhoB promoter by FTI-277 in NUGC-3 cells, suggesting that the transcriptional activation of RhoB by NSC126188 occurs by a different mechanism than that reported for FTIs. Our data indicate that NSC126188 increases RhoB expression via JNK-mediated signaling through a p300-binding site and iCCAAT box resulting in apoptosis of NUGC-3 cells.

Long-term treatment of farnesyltransferase inhibitor FTI-277 induces neurotoxicity of hippocampal neurons from rat embryo in a ROS-dependent manner.

Despite the well established anti-cancer effect of farnesyltransferase inhibitor FTI-277, the neurotoxic effects of the agent are not yet clearly defined at the molecular and cellular levels. Here, we report the neurotoxic effects of FTI-277 and the involvement of reactive oxygen species (ROS) in FTI-induced neurotoxicity. Although there is no significant effect of FTI-277 for 2 days, long-term treatment of FTI-277 for 4 days induced dramatic reduction in outgrowth, maturation and branching of neuritis and considerable cytoxicity in a dose- and time-dependent manner in primary cultured rat embryo hippocampal neurons. Interestingly, FTI-277 for 4 days dramatically decreased expression of synapsin I, a crucial molecule involved in the neuronal growth and plasticity, and increased a cytotoxic G-protein RhoB of which ectopic expression induced the neurotoxicity in hippocampal neurons. Moreover, treatment with FTI-277 dramatically increased intracellular levels of ROS, which was sustained for 4 days; while blockage of ROS rescued FTI-277-induced neurotoxicity as well as both decrease of synapsin I and increase of RhoB. Taken together, these results provide the molecular insights for the mechanisms which might be of use aiming for avoiding neurotoxic side effects by FTI agent for a drug development for a clinical use.

JNK-mediated transcriptional upregulation of RhoB is critical for apoptosis of HCT-116 colon cancer cells by a novel diarylsulfonylurea derivative.

Diarylsulfonylureas are potent antitumor agents that have been tested in clinical trials. However, detailed mechanisms of their apoptotic activity remain unclear. Here, we report a new diarylsulfonylurea derivative, LB2A, that upregulates RhoB, thereby inducing potent apoptosis in HCT-116 human colon cancer cells independently of p53 status. LB2A decreased procaspase-3, increased phospho-JNK, and cleaved PARP, leading to apoptosis of HCT-116 cells. Prior treatment of HCT-116 cells with the JNK inhibitor SP600125 and the RNA synthesis inhibitor DRB blocked apoptosis, implying that JNK activation and mRNA production are important for apoptosis by LB2A. Western blotting, RT-PCR, and RhoB-promoter luciferase reporter assays revealed that LB2A increased RhoB via JNK-mediated transcriptional activation. LB2A decreased HDAC1 and increased acetyl-H3, both of which activate the RhoB promoter and were blocked by SP600125. Ectopic expression of RhoB induced apoptosis of HCT-116 cells, suggesting that RhoB is critical for the anti-cancer activity of LB2A in human colon cancer cells. LB2A also exhibited potent tumor growth inhibition of HCT-116 cells in vivo using a mouse xenograft assay. Taken together, these results show that LB2A induces apoptosis of HCT-116 cells via JNK-mediated transcriptional upregulation of RhoB and may therefore provide a potential therapy for human colon cancer.

The effects of supplementing specific amino acids on the expression of elastin-like polypeptides (ELPs).

Elastin-like polypeptides (ELPs) made from the repeating pentapeptides (Val-Pro-Gly-Xaa-Gly) are protein based biopolymers that contain useful properties, including the ability to self-assemble, biocompatibility, and stimuli sensitivity. However, due to the repeated consumption of specific amino acids, long ELPs generally have low expression yields in in vitro and in vivo systems. This is because of the lack of specific amino acids during the translation process. In this study, ELP fusion proteins of various lengths were prepared by recursive directional ligation (RDL) and expressed in a cell-free protein synthesis system. By measuring TCA-precipitated radioactivity with a liquid scintillation counter, their expression profiles were investigated. The expression levels of an ELP fusion protein were improved by almost 2-fold by adding specific amino acids. Additionally, we determined that the amount of increase in expression levels depends on the length of the ELPs. This study suggests a useful strategy to improve the yield of longer repetitive polypeptides such as ELPs or silk-like polypeptides (SLPs).

Ribosomal synthesis and in situ isolation of peptide molecules in a cell-free translation system.

Although the cell-free translation system is now widely accepted as an efficient platform for production, engineering and screening of recombinant proteins, it has not been successfully used for the synthesis of peptide molecules mainly due to low expression yields and rapid proteolysis of the expressed peptides. In this study, we propose a novel strategy for rapid expression and recovery of peptide molecules which involves the rational design of template DNA and heterogenous cell-free translation reaction in the presence of affinity beads. Various peptide molecules which were not expressed in a detectable level were successfully expressed and recovered in situ in a substantial yield. We expect that the presented approach will be widely used as a versatile platform for the generation of a variety of peptide molecules.

Clinical outcomes of Ahmed glaucoma valve implantation using tube ligation and removable external stents.

PURPOSE: To investigate the immediate and long-term outcomes of Ahmed glaucoma valve (AGV) implantation with silicone tube ligation and removable external stents. METHODS: This retrospective non-comparative study investigated the outcomes of AGV implantation with silicone tube ligation and removable external stents in 95 eyes (90 patients) with at least 12 months of postoperative follow-up. Qualified success was defined as an intraocular pressure (IOP) of or=6 mmHg regardless of anti-glaucoma medication. Those who required additional glaucoma surgery, implant removal or who had phthisis bulbi were considered failures. Hypotony was defined as an IOP of <6 mmHg. RESULTS: Mean IOP reduced from 37.1+/-9.7 mmHg preoperatively to 15.2+/-5.6 mmHg at 12 months postoperatively (p<0.001). Qualified success was achieved in 84.2% at 1 year. Hypotony with an IOP of <6 mmHg was seen in 8.4% and an IOP of <5 mmHg in 3.2% on the first postoperative day. No case of hypotony required surgical intervention. Suprachoroidal hemorrhage did not occur in this study. When stents were removed on the first postoperative day because of an insufficient IOP decrease, the mean IOP decreased significantly from 42.0 mmHg to 14.1 mmHg (p<0.001) after 1 hour. The most common complication was hyphema, which occurred in 17.9%. CONCLUSIONS: Hypotony-related early complications requiring surgical intervention were reduced by ligation and external stents in the tube. In addition, early postoperative high IOPs were managed by removing external stents. The described method can prevent postoperative hypotony after AGV implantation and showed long-term success rates comparable to those reported previously.