Synergistic apoptosis by combination of metformin and an O-GlcNAcylation inhibitor in colon cancer cells.

BACKGROUND: Although autophagy is an important mediator of metformin antitumor activity, the role of metformin in the crosstalk between autophagy and apoptosis remains unclear. The aim was to confirm the anticancer effect by inducing apoptosis by co-treatment with metformin and OSMI-1, an inhibitor of O-GlcNAcylation, in colon cancer cells. METHODS: Cell viability was measured by MTT in colon cancer cell lines HCT116 and SW620 cells. Co-treatment with metformin and OSMI-1 induced autophagy and apoptosis, which was analyzed using western blot, reverse transcription-polymerase chain reaction (RT-PCR) analysis, and fluorescence-activated cell sorting (FACS). Combined treatment with metformin and OSMI-1 synergistically inhibit the growth of HCT116 was confirmed by xenograft tumors. RESULTS: We showed that metformin inhibited mammalian target of rapamycin (mTOR) activity by inducing high levels of C/EBP homologous protein (CHOP) expression through endoplasmic reticulum (ER) stress and activating adenosine monophosphate-activated protein kinase (AMPK) to induce autophagy in HCT116 cells. Interestingly, metformin increased O-GlcNAcylation and glutamine:fructose-6-phosphate amidotransferase (GFAT) levels in HCT116 cells. Thus, metformin also blocks autophagy by enhancing O-GlcNAcylation, whereas OSMI-1 increases autophagy via ER stress. In contrast, combined metformin and OSMI-1 treatment resulted in continuous induction of autophagy and disruption of O-GlcNAcylation homeostasis, resulting in excessive autophagic flux, which synergistically induced apoptosis. Downregulation of Bcl2 promoted apoptosis via the activation of c-Jun N-terminal kinase (JNK) and CHOP overexpression, synergistically inducing apoptosis. The activation of IRE1α/JNK signaling by OSMI-1 and PERK/CHOP signaling by metformin combined to inhibit Bcl2 activity, ultimately leading to the upregulation of cytochrome c release and activation of caspase-3. CONCLUSIONS: In conclusion, combinatorial treatment of HCT116 cells with metformin and OSMI-1 resulted in more synergistic apoptosis being induced by enhancement of signal activation through ER stress-induced signaling rather than the cell protective autophagy function. These results in HCT116 cells were also confirmed in xenograft models, suggesting that this combination strategy could be utilized for colon cancer treatment.

Biosensor-Linked Immunosorbent Assay for the Quantification of Methionine Oxidation in Target Proteins.

Methionine oxidation is involved in regulating the protein activity and often leads to protein malfunction. However, tools for quantitative analyses of protein-specific methionine oxidation are currently unavailable. In this work, we developed a biological sensor that quantifies oxidized methionine in the form of methionine-R-sulfoxide in target proteins. The biosensor "tpMetROG" consists of methionine sulfoxide reductase B (MsrB), circularly permuted yellow fluorescent protein (cpYFP), thioredoxin, and protein G. Protein G binds to the constant region of antibodies against target proteins, specifically capturing them. Then, MsrB reduces the oxidized methionine in these proteins, leading to cpYFP fluorescence changes. We assessed this biosensor for quantitative analysis of methionine-R-sulfoxide in various proteins, such as calmodulin, IDLO, LegP, Sacde, and actin. We further developed an immunosorbent assay using the biosensor to quantify methionine oxidation in specific proteins such as calmodulin in animal tissues. The biosensor-linked immunosorbent assay proves to be an indispensable tool for detecting methionine oxidation in a protein-specific manner. This is a versatile tool for studying the redox biology of methionine oxidation in proteins.

Development of a novel fluorescent biosensor for dynamic monitoring of metabolic methionine redox status in cells and tissues.

Aberrant production of reactive oxygen species (ROS) leads to tissue damage accumulation, which is associated with a myriad of human pathologies. Although several sensors have been developed for ROS quantification, their applications for ROS-related human physiologies and pathologies still remain problematic due to the unstable nature of ROS. Herein, we developed Trx1-cpYFP-fRMsr (TYfR), a genetically-encoded fluorescent biosensor with the remarkable specificity and sensitivity toward fMetRO (free Methionine-R-sulfoxide), allowing for dynamic quantification of physiological levels of fMetRO, a novel indicator of ROS and methionine redox status in vitro and in vivo. Moreover, using the sensor, we observed a significant fMetRO enrichment in serum from patients with acute coronary syndrome, one of the most severe cardiovascular diseases, which becomes more evident following percutaneous coronary intervention. Collectively, this study proposes that fMetRO is a novel biomarker of tissue damage accumulation in ROS-associated human pathologies, and that TYfR is a promising tool for quantifying fMetRO with potentials in versatile applications.

Development of novel extraction reagents for analyzing dried blood spots from crime scenes.

Evidence of dried blood is very valuable in forensic science. Since the discovery of luminescence with Luminol and dried blood spots (DBSs) in 1928, interest and research on blood have continued to date. One of the most important factor that DBSs have is genes. However, the current use of distilled water (DDW) to collect and extract blood samples has disadvantages related to DNA stability. Therefore, this study aimed to develop an extraction reagent that is most suitable for gene extraction from DBSs. Blood was collected from 45 healthy adult men and women in vacuum blood containers without coagulants or anticoagulants. The collected blood was dried in various settings to check the performance of the extraction reagent. Extraction with Tris-EDTA (TE) and phosphate-buffered saline (PBS) was found more suitable in terms of gene interference effects compared with DDW; their performance was also compared with those of the newly developed extraction reagents. Upon comparing the results of polymerase chain reaction for human genomic DNA samples using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as the target, the performance of the newly developed extraction reagents, modified TE and PBS, was found to be relatively good. To determine the optimal composition of the developed extraction reagents, 12 new extraction reagents were developed with different pH and sodium concentrations. Among them, the best results were found when the DNA was extracted using extraction reagent No. 3 with pH 8.0 and containing 1 M NaCl. Next, the four extraction reagents, DDW, TE, PBS, and No. 3 were compared under nine different temperature and humidity conditions. Similarly, under various environmental conditions, extraction reagent No. 3 performed better than other reagents. It is proposed that modified TE and PBS mixed extraction reagents are the most suitable for collecting and preserving crime site samples. The proposed composition for a DNA extraction reagent can contribute greatly to crime scene reconstruction.

The Selenoprotein MsrB1 Instructs Dendritic Cells to Induce T-Helper 1 Immune Responses.

Immune activation associates with the intracellular generation of reactive oxygen species(ROS). To elicit effective immune responses, ROS levels must be balanced. Emerging evidenceshows that ROS-mediated signal transduction can be regulated by selenoproteins such asmethionine sulfoxide reductase B1 (MsrB1). However, how the selenoprotein shapes immunityremains poorly understood. Here, we demonstrated that MsrB1 plays a crucial role in the ability ofdendritic cells (DCs) to provide the antigen presentation and costimulation that are needed forcluster of differentiation antigen four (CD4) T-cell priming in mice. We found that MsrB1 regulatedsignal transducer and activator of transcription-6 (STAT6) phosphorylation in DCs. Moreover, bothin vitro and in vivo, MsrB1 potentiated the lipopolysaccharide (LPS)-induced Interleukin-12 (IL-12)production by DCs and drove T-helper 1 (Th1) differentiation after immunization. We propose thatMsrB1 activates the STAT6 pathway in DCs, thereby inducing the DC maturation and IL-12production that promotes Th1 differentiation. Additionally, we showed that MsrB1 promotedfollicular helper T-cell (Tfh) differentiation when mice were immunized with sheep red blood cells.This study unveils as yet unappreciated roles of the MsrB1 selenoprotein in the innate control ofadaptive immunity. Targeting MsrB1 may have therapeutic potential in terms of controllingimmune reactions.

Role of Selenoproteins in Redox Regulation of Signaling and the Antioxidant System: A Review.

Selenium is a vital trace element present as selenocysteine (Sec) in proteins that are, thus, known as selenoproteins. Humans have 25 selenoproteins, most of which are functionally characterized as oxidoreductases, where the Sec residue plays a catalytic role in redox regulation and antioxidant activity. Glutathione peroxidase plays a pivotal role in scavenging and inactivating hydrogen and lipid peroxides, whereas thioredoxin reductase reduces oxidized thioredoxins as well as non-disulfide substrates, such as lipid hydroperoxides and hydrogen peroxide. Selenoprotein R protects the cell against oxidative damage by reducing methionine-R-sulfoxide back to methionine. Selenoprotein O regulates redox homeostasis with catalytic activity of protein AMPylation. Moreover, endoplasmic reticulum (ER) membrane selenoproteins (SelI, K, N, S, and Sel15) are involved in ER membrane stress regulation. Selenoproteins containing the CXXU motif (SelH, M, T, V, and W) are putative oxidoreductases that participate in various cellular processes depending on redox regulation. Herein, we review the recent studies on the role of selenoproteins in redox regulation and their physiological functions in humans, as well as their role in various diseases.

Microarray and proteome array in an atherosclerosis mouse model for identification of biomarkers in whole blood.

Cardiovascular disease (CVD) is highly fatal, and 80 percent of the mortality is attributed to heart attack and stroke. Atherosclerosis is a disease that increases a patient's risk to CVD and is characterized by atheroma formed by immune cells, lipids, and smooth muscle cells. When an atherosclerotic lesion grows and blocks blood vessels or when an atheroma ruptures and blocks blood vessels by embolism, sudden angina, or stroke can occur. It is therefore important to diagnose atherosclerosis early and prevent its progression to more severe disease. Although myeloperoxidase, plasma fibrinogen, cardiac troponin-I, and C-reactive protein have been considered as diagnostic markers for multiple cardiac risks, specific biomarkers for atherosclerosis have not been clearly determined yet. Particularly, reliable biomarkers for the diagnosis of atherosclerosis using whole blood are not yet available. In this study, we screened potential biomarker genes and proteins from whole blood of apolipoprotein E knockout (ApoE-/- ) mice maintained on a Western diet, by comparing them to ApoE+/+ mice. We used whole blood for microarray and proteome array. Candidate genes and proteins identified from each method were confirmed with quantitative real-time PCR and ELISA. Based on our data, we speculate that Lilrb4a, n-R5s136, and IL-5 are potential targets that can be developed into novel biomarkers of atherosclerosis. Our study contributes to the diagnosis of atherosclerosis using whole blood in clinical settings.

Sulfate assimilation regulates hydrogen sulfide production independent of lifespan and reactive oxygen species under methionine restriction condition in yeast.

Endogenously produced hydrogen sulfide was proposed to be an underlying mechanism of lifespan extension via methionine restriction. However, hydrogen sulfide regulation and its beneficial effects via methionine restriction remain elusive. Here, we identified the genes required to increase hydrogen sulfide production under methionine restriction condition using genome-wide high-throughput screening in yeast strains with single-gene deletions. Sulfate assimilation-related genes, such as MET1, MET3, MET5, and MET10, were found to be particularly crucial for hydrogen sulfide production. Interestingly, methionine restriction failed to increase hydrogen sulfide production in mutant strains; however, it successfully extended chronological lifespan and reduced reactive oxygen species levels. Altogether, our observations suggested that increased hydrogen sulfide production via methionine restriction is not the mechanism underlying extended yeast lifespan, even though increased hydrogen sulfide production occurred simultaneously with yeast lifespan extension under methionine restriction condition.

Expression of the methionine sulfoxide reductase lost during evolution extends Drosophila lifespan in a methionine-dependent manner.

Accumulation of oxidized amino acids, including methionine, has been implicated in aging. The ability to reduce one of the products of methionine oxidation, free methionine-R-sulfoxide (Met-R-SO), is widespread in microorganisms, but during evolution this function, conferred by the enzyme fRMsr, was lost in metazoa. We examined whether restoration of the fRMsr function in an animal can alleviate the consequences of methionine oxidation. Ectopic expression of yeast fRMsr supported the ability of Drosophila to catalyze free Met-R-SO reduction without affecting fecundity, food consumption, and response to starvation. fRMsr expression also increased resistance to oxidative stress. Moreover, it extended lifespan of flies in a methionine-dependent manner. Thus, expression of an oxidoreductase lost during evolution can enhance metabolic and redox functions and lead to an increase in lifespan in an animal model. More broadly, our study exposes the potential of a combination of genetic and nutritional strategies in lifespan control.

Selenoprotein MsrB1 promotes anti-inflammatory cytokine gene expression in macrophages and controls immune response in vivo.

Post-translational redox modification of methionine residues often triggers a change in protein function. Emerging evidence points to this reversible protein modification being an important regulatory mechanism under various physiological conditions. Reduction of oxidized methionine residues is catalyzed by methionine sulfoxide reductases (Msrs). Here, we show that one of these enzymes, a selenium-containing MsrB1, is highly expressed in immune-activated macrophages and contributes to shaping cellular and organismal immune responses. In particular, lipopolysaccharide (LPS) induces expression of MsrB1, but not other Msrs. Genetic ablation of MsrB1 did not preclude LPS-induced intracellular signaling in macrophages, but resulted in attenuated induction of anti-inflammatory cytokines, such as interleukin (IL)-10 and the IL-1 receptor antagonist. This anomaly was associated with excessive pro-inflammatory cytokine production as well as an increase in acute tissue inflammation in mice. Together, our findings suggest that MsrB1 controls immune responses by promoting anti-inflammatory cytokine expression in macrophages. MsrB1-dependent reduction of oxidized methionine in proteins may be a heretofore unrecognized regulatory event underlying immunity and inflammatory disease, and a novel target for clinical applications.

Antiviral Activities of Trichothecenes Isolated from Trichoderma albolutescens against Pepper Mottle Virus.

A bioassay-guided isolation using a green fluorescence protein (GFP)-tagged pepper mottle virus (PepMoV-GFP) based leaf-disk method to obtain new antiviral agents led to the isolation of trichodermin, 1, and a new compound trichoderminol, 2, from EtOAc extract of Trichoderma albolutescens culture medium. The structures of compounds 1 and 2 were determined by MS and NMR experiments, and the absolute configurations of the compounds were established by experimental and calculated vibrational circular dichroism spectra. Compounds 1 and 2 were evaluated for their anti-PepMoV potential in systemic host plants, such as tobacco and pepper, by PepMoV-GFP based systemic host method. All compounds exhibited inactivation effects against PepMoV. Furthermore, compound 1 showed protective effects against PepMoV.

The Natural Course of Biopsy-Proven Isolated Microscopic Hematuria: a Single Center Experience of 350 Patients.

The increasing interest in healthcare and health screening events is revealing additional cases of asymptomatic isolated microscopic hematuria (IMH). However, a consensus of the evaluation and explanation of the IMH prognosis is controversial among physicians. Here, we present the natural course of IMH together with the pathological diagnosis and features to provide supportive data when approaching patients with IMH. We retrospectively evaluated 350 patients with IMH who underwent a renal biopsy between 2002 and 2011, and the pathological diagnosis and chronic histopathological features (glomerulosclerosis, interstitial fibrosis, and tubular atrophy) were reviewed. Deterioration of renal function was examined during follow up. The patients with IMH were evaluated for a mean of 86 months. IgA nephropathy was the most common diagnosis in 164 patients (46.9%). Chronic histopathological changes were observed in 166 (47.4%) but was not correlated with proteinuria or a decline in renal function. Ten patients developed proteinuria, and all of them had IgA nephropathy. Three patients progressed to chronic kidney disease with an estimated glomerular filtration rate < 60 mL/min/1.73 m(2) but none progressed to end stage renal disease. In conclusion, IMH had a generally benign course during 7-years of observation, although IgA nephropathy should be monitored if it progresses to proteinuria. Future prospective randomized studies may help conclude the long-term prognosis and lead to a consensus for managing IMH.

Management of portal hypertension derived from uncommon causes.

Portal hypertension can arise from any condition interfering with normal blood flow at any level within the portal system. Herein, we presented two uncommon cases of the portal hypertension and its treatment with brief literature review. A 71-year-old man who underwent right hemihepatectomy revealed a tumor recurrence adjacent to the inferior vena cava (IVC). After radiofrequency ablation (RFA) with lymph node dissection, he was referred for abdominal distension. The abdomen computed tomography scan showed severe ascites with a narrowing middle hepatic vein (MHV) and IVC around the RFA site. After insertion of two stents at the IVC and MHV, the ascites disappeared. Another 73-year-old man underwent right trisectionectomy of liver and segmental resection of the portal vein (PV). After operation, he underwent conservative management due to continuous abdominal ascites. The abdomen computed tomography scan showed severe ascites with obliteration of the left PV. After insertion of stent, the ascites disappeared. A decrease of the pressure gradient between the PV and IVC is one of the important treatment strategies for portal hypertension. Vascular stent is useful in the reduction of pressure gradient and thus, can be a treatment option for portal hypertension.

Robotic surgery for gastric tumor: current status and new approaches.

Surgical techniques have evolved tremendously over this past century. To maximize the efficacy and minimize the invasiveness of laparoscopic surgery, researchers have sought to implement wider application of robotics. Nevertheless, both optimism without sound evidence and fear of new technology obscure the appropriate uses of robotic surgery. In the present review, we attempted to provide a balanced perspective on the current state of robotic gastrectomy, outlining evidence and opportunities for the use thereof. Although evidence is limited, the use of robotics is feasible for gastric cancer surgery, and less than 10 cases of robotic surgery are needed to become proficient therein. Compared to the clinical impact of laparoscopy on gastric cancer surgery, the additional benefits of robotic surgery to patients seem to be limited. Despite additional costs and longer surgeries, robotic surgery reportedly does not offer surgical outcomes superior to those for laparoscopic surgery, according to a recent multicenter study. Meanwhile, however, our in-depth review of retrospective and prospective reports revealed that robots could expand the indications of minimally invasive gastrectomy for patients requiring total gastrectomy and D2 lymph node dissection. Moreover, we found that robotic gastrectomy is associated with a higher number of retrieved lymph nodes, less bleeding, fewer complications, and shorter hospital stay, compared to laparoscopic gastrectomy. Accordingly, new surgical approaches using advanced technologies, such as near infrared detectors, the Tilepro® multi-input display, dual consoles, and the Single-Site® system, are under investigation. In conclusion, measuring the additional benefits of robotic over laparoscopic surgery would be difficult and clinically insignificant. Thus, developing new surgical procedures that extend the benefits of conventional laparoscopic surgery to patients in whom minimally invasive surgery would not be possible is necessary to justify the greater use of robotic surgery.

Risk factors in the progression of BK virus-associated nephropathy in renal transplant recipients.

BACKGROUND/AIMS: BK virus-associated nephropathy (BKVAN) is an important cause of allograft dysfunction in kidney transplant recipients. It has an unfavorable clinical course, and no definite treatment guidelines have yet been established. Here, we report our center's experience with biopsy-proven BKVAN and investigate factors associated with its progression. METHODS: From January 2004 to April 2013, 25 patients with BKVAN were diagnosed by biopsy at Seoul St. Mary's Hospital. Of the 25 patients, 10 were deceased-donor transplant recipients and 15 were living-donor transplant recipients. Three of the patients underwent retransplantation. The primary immunosuppressant used was tacrolimus in 17 patients and cyclosporine in eight patients. RESULTS: BKVAN was observed at a mean duration of 22.8 ± 29.1 months after transplantation. The mean serum creatinine level at biopsy was 2.2 ± 0.7 mg/dL. BKVAN occurred with acute rejection in eight patients (28%). Immunosuppression modification was performed in 21 patients (84%). Additionally, leflunomide and intravenous immunoglobulin were administered to 13 patients (52%) and two (8%), respectively. Allograft loss occurred in five patients (27.8%) during the follow- up period at 0.7, 17.1, 21.8, 39.8, and 41.5 months after the BKVAN diagnosis. Advanced stages of BKVAN, increased creatinine levels, and accompanying acute rejection at the time of BKVAN diagnosis increased the risk of allograft failure. CONCLUSIONS: The clinical outcomes in patients with biopsy-proven BKVAN were unfavorable in the present study, especially in patients with advanced-stage BKVAN, poor renal function, and acute allograft rejection.

ZrO2-SiO2 nanosheets with ultrasmall WO3 nanoparticles and their enhanced pseudocapacitance and stability.

We report on the first synthesis of porous ZrO2-SiO2 sheets with well-defined ultrasmall WO3 nanoparticles for energy storage performance. In our system, for improving the surface deterioration of electrode, we use the ZrO2-SiO2 sheets using graphene oxide as a template to access electrode substrate. The synthesized electrode with about 20 nm thickness and about 10 nm pores, has a maximum value of 313 F/g at current density of 1 A/g and a minimum value of 160 F/g at current density of 30 A/g in the specific capacitance. In addition, over 90% of its initial specific capacitance is retained when they are cycled up to 2500 cycles.

Electrodeposition of Manganese-Nickel Oxide Films on a Graphite Sheet for Electrochemical Capacitor Applications.

Manganese-nickel (Mn-Ni) oxide films were electrodeposited on a graphite sheet in a bath consisting of manganese acetate and nickel chloride, and the structural, morphological, and electrochemical properties of these films were investigated. The electrodeposited Mn-Ni oxide films had porous structures covered with nanofibers. The X-ray diffractometer pattern revealed the presence of separate manganese oxide (g-MnO2) and nickel oxide (NiO) in the films. The electrodeposited Mn-Ni oxide electrode exhibited a specific capacitance of 424 F/g in Na2SO4 electrolyte. This electrode maintained 86% of its initial specific capacitance over 2000 cycles of the charge-discharge operation, showing good cycling stability.

[A case of severe bevacizumab-induced ischemic pancolitis, treated with conservative management].

Bevacizumab (Avastin(Ⓡ)) is a monoclonal antibody against the vascular endothelial growth factor (VEGF) receptor that increases the overall survival rate when added to standard chemotherapy regimens in patients with metastatic colorectal cancer. The known toxicities of bevacizumab are hypertension, proteinuria, wound healing complications, arterial thrombosis, bleeding, and gastrointestinal complications. Especially ischemic colitis can rapidly develop into bowel perforation, so an emergency operation often is needed. Recently, a 65-year-old male patient developed ischemic pancolitis after FOLFOX (85 mg/m(2) Oxaliplatin, d1; 200 mg/m(2) Leucovorin, d1; 400 mg/m(2) 5-FU iv bolus, d1-2; and 600 mg/m(2) 5-FU, d1-2, every two wk) and Bevacizumab combination chemotherapy was administered. However, he recovered after early conservative care without surgery. We report this case with a review of literature.

[A case of pseudomembranous colitis in a juvenile rheumatoid arthritis patient taking methotrexate].

Pseudomembranous colitis is mainly caused by antibiotics and Clostridium difficile infection. But conditions such as gastrointestinal surgery, antacid medication, anti-neoplastic agent or immunosuppressive agent which influences the normal flora of colon can induce colitis without the administration of any antibiotics. We experienced a 13 year-old male who was taking low-dose methotrexate for juvenile rheumatoid arthritis complained diarrhea and abdominal pain for 3 weeks. Sigmoidoscopic findings revealed diffuse patch yellowish pseudomembranes on the rectum. Histologic finding was compatible to pseudomembranous colitis. His symptom was improved after stop taking methotrexate and the administration of metronidazole. If a patient treated with immunosuppressive agents or antineoplastic agents complains diarrhea, fever or abdominal pain and has not improved with conservative care, pseudomembranous colitis should be taken into account as a differential diagnosis and prompt treatment is required for better prognosis.