Cloning and Organelle Expression of Bamboo Mitochondrial Complex I Subunits Nad1, Nad2, Nad4, and Nad5 in the Yeast Saccharomyces cerevisiae.

Mitochondrial respiratory complex I catalyzes electron transfer from NADH to ubiquinone and pumps protons from the matrix into the intermembrane space. In particular, the complex I subunits Nad1, Nad2, Nad4, and Nad5, which are encoded by the nad1, nad2, nad4, and nad5 genes, reside at the mitochondrial inner membrane and possibly function as proton (H+) and ion translocators. To understand the individual functional roles of the Nad1, Nad2, Nad4, and Nad5 subunits in bamboo, each cDNA of these four genes was cloned into the pYES2 vector and expressed in the mitochondria of the yeast Saccharomyces cerevisiae. The mitochondrial targeting peptide mt gene (encoding MT) and the egfp marker gene (encoding enhanced green fluorescent protein, EGFP) were fused at the 5'-terminal and 3'-terminal ends, respectively. The constructed plasmids were then transformed into yeast. RNA transcripts and fusion protein expression were observed in the yeast transformants. Mitochondrial localizations of the MT-Nad1-EGFP, MT-Nad2-EGFP, MT-Nad4-EGFP, and MT-Nad5-EGFP fusion proteins were confirmed by fluorescence microscopy. The ectopically expressed bamboo subunits Nad1, Nad2, Nad4, and Nad5 may function in ion translocation, which was confirmed by growth phenotype assays with the addition of different concentrations of K+, Na+, or H+.

Prediction for major adverse outcomes in cardiac surgery: comparison of three prediction models.

BACKGROUND/PURPOSE: Recent advances in medical treatment have altered the profile of patients referred for cardiac surgery. The proportion of high risk patients has increased dramatically. Numerous multifactorial risk scores have been developed to predict outcomes after cardiac surgery. However, these additive risk models were all developed outside of Asia and have never been validated in Taiwan. We applied the Parsonnet score, Tu score and logistic regression to a population in Taiwan who received cardiac surgery to predict the mortality, morbidity and likelihood of prolonged stay in the intensive care unit (ICU). METHODS: This retrospective study included 622 adult patients who received cardiac surgery during a 2-year period at Taichung Veterans General Hospital. The patients were randomly divided into a reference set (n = 423) and a validation set (n = 199). The Parsonnet score and Tu score were calibrated separately with the reference set to determine mortality, morbidity and likelihood of prolonged ICU stay. We developed a separate logistic regression model for each of the three outcomes by using the reference set. The validation set was used to test these models. RESULTS: The area under the receiver operating characteristic (ROC) curve (AUC) of the Parsonnet score, Tu score and logistic regression for predicting in-hospital mortality were 0.843, 0.714 and 0.867, respectively. The AUC of the Parsonnet score, Tu score and logistic regression for predicting major morbidity were 0.784, 0.736 and 0.808, respectively. The AUC of the Parsonnet score, Tu score and logistic regression for predicting likelihood of prolonged ICU stay were 0.701, 0.689 and 0.764, respectively. CONCLUSION: The Parsonnet score performed as well as the logistic regression models in predicting major adverse outcomes. The Parsonnet score appears to be a very suitable model for clinicians to use in risk stratification of cardiac surgery.

Study of the inhibitory effects on TNF-α-induced NF-κB activation of IMD0354 analogs.

Nuclear factor-κB (NF-κB) is an important nuclear transcription factor which regulates pro-inflammatory cytokines such as TNF-α, IL-6. Its role as immunoregulatory mediator makes it an attractive target in the development of treatments for inflammatory and autoimmune diseases. In this study, we synthesized derivatives of IMD0354, a known inhibitor for NF-κB, in attempt to understand the effect of benzanilide substitutions on its activity. The inhibition of these analogs on NF-κB activation was analyzed by luciferase assay. The inhibition of IKKß phosphorylation and pro-inflammatory cytokines was determined by Western blot and real-time PCR. The structure activity relationships showed that the hydroxyl group on IMD0354 is a critical moiety that resulting in the inhibition of NF-κB. Derivatives 1m, 2b, and 2c were shown to inhibit pro-inflammatory cytokine production at low concentration. These newly synthesized compounds may be useful for the treatment of chronic inflammatory disorders or for cancer prevention.

Tyramine detection using PEDOT:PSS/AuNPs/1-methyl-4-mercaptopyridine modified screen-printed carbon electrode with molecularly imprinted polymer solid phase extraction.

Tyramine (4-hydroxyphenethylamine), which is a monoamine metabolized by monoamine oxidase (MAO), exists widely in plants, animals, fermented foods, and salted foods. The incidence of hypertension, or "cheese effect", which is associated with a large dietary intake of tyramine while taking MAO inhibitors has been reported; therefore, the measurement of tyramine is an urgent concern. Herein, an efficient approach that integrates a molecular imprinting polymer for solid phase extraction (MISPE) technique with a sensitive electrochemical sensing platform (SPCE/PEDOT: PSS/AuNP/1-m-4-MP) for the quantification of tyramine is presented. Enhanced electrode conductivity was achieved sequentially by constructing a conductive polymer (PEDOT: PSS) on a screen-printed carbon electrode (SPCE), followed by electrodeposition with gold nanoparticles (AuNPs) and, finally, by modification with positively charged 1-methyl-4-mercaptopyridine (1-m-4-MP) using an Au-S bond. Tyramine was isolated selectively and pre-concentrated by the MISPE technique; electroanalysis that used differential pulse voltammetry (DPV) in NaOH (0.1M, pH 13) was conducted successively. Experimental parameters (such as modes of electrode modification, ratio of PEDOT: PSS, pH of electrolyte, time required for AuNP deposition, and 1-m-4-MP concentrations) that were associated with optimal detection conditions were evaluated also. We obtained a linear concentration range (5-100nM, R2=0.9939) with LOD and sensitivity at 2.31nM, and 3.11µAnM-1cm-2, respectively. The applicability of our technique was demonstrated by analyzing tyramine in spiked serum and milk. The feature of our newly developed analytical methods that coupled sample pre-treatment (sample clean-up and pre-concentration) with sensitive detection makes it a promising tool for quantifying of tyramine.

Upregulation of actin-like gene expression in giant freshwater prawns Macrobrachium rosenbergii infected with Metschnikowia bicuspidata.

The giant freshwater prawn Macrobrachium rosenbergii is commercially cultured throughout the world including Taiwan. From 1992 to 1995, Taiwanese production decreased by approximately 50% due to disease. The yeast Metschnikowia bicuspidata is considered to be one of the major causes of white muscle disease, but the molecular mechanism of its pathogenesis is not known. Using RNA differential display (DD) with muscle and hepatopancreatic tissue, we identified a 324 nucleotide (nt) message specifically expressed by M. rosenbergii infected with M. bicuspidata but not in the controls. A ribonuclease protection assay (RPA) confirmed expression in both tissues. RPA data also revealed an additional 230 bp mRNA message that was not identified by DD. Using RNA ligase-mediated rapid amplification of 5' cDNA ends (5'-RACE), we successfully isolated a 1357 bp full-length gene (c57) that showed 92 and 87% sequence identity to the actin gene of the Kuruma shrimp Marsupenaeus japonicus (also called Penaeus japonicus) (GenBank accession number AB055975) and the beta-actin gene of the white shrimp Litopenaeus vannamei (also called Penaeus vannamei) (GenBank accession number AF300705), respectively. The deduced amino acid sequence of c57 showed 83 % sequence similarity to M. japonicus and L. vannamei actin proteins. Based on this high homology, we suggest that upregulation of actin expression in the muscle and hepatopancreas is part of the shrimp response to M. bicuspidata infection. Increased expression may be related to repair of tissues damaged by yeast infection.

Determination of retardation parameters of multiple-order wave plate using a phase-sensitive heterodyne ellipsometer.

To characterize the linear birefringence of a multiple-order wave plate (MWP), an oblique incidence is one of the methods available. Multiple reflections in the MWP are produced, and oscillations in the phase retardation measurement versus the oblique incident angle are then measured. Therefore, an antireflection coated MWP is required to avoid oscillation of the phase retardation measurement. In this study, we set up a phase-sensitive heterodyne ellipsometer to measure the phase retardations of an uncoated MWP versus the oblique incident angle, which was scanned in the x-z plane and y-z plane independently. Thus, the effect on multiple reflections by the MWP is reduced by means of subtracting the two measured phase retardations from each other. As a result, a highly sensitive and accurate measurement of retardation parameters (RPs), which includes the refractive indices of the extraordinary ray n(e) and ordinary ray n(o), is obtained by this method. On measurement, a sensitivity (n(e),n(o)) of 10(-6) was achieved by this experiment setup. At the same time, the spatial shifting of the P and S waves emerging from the MWP introduced a deviation between experimental results and the theoretical calculation.

Risk factors affecting adverse outcomes of cardiac surgery in patients aged 70 years and older.

BACKGROUND: The elderly segment of the population in Taiwan is increasing rapidly. According to the latest information from the Directorate-General of Budget, Accounting and Statistics, Executive Yuan, Taiwan, 1,553,367 civilians of the total population of 22,879,510 (6.8%) are seventy years old and older in 2007. The proportion of high-risk patients has increased dramatically owing to a greater number of elderly patients and increased number of average patients with heart diseases presenting for cardiac surgery. We analyzed the preoperative risk factors for in-hospital mortality, morbidity and the likelihood of prolonged intensive care unit (ICU) stay in elderly patients after cardiac surgery. METHODS: We retrospectively studied 952 adult patients who received cardiac surgery during a three-year period (from August 1, 2004 to December 31, 2006) at Taichung Veterans General Hospital. Patients were divided into a control group and a study group. The study group (n=395) exclusively consisted of patients aged seventy or over while the rest of the patients under study served as reference group (n=557). Continuous variables were compared using Student's t test, and categorical variables were compared using Pearson chi-square test. Variables associated with in-hospital mortality, major morbidity and prolonged ICU stay in univariate analysis with P < 0.05 were entered into multivariate analysis using logistic regression with a stepwise forward selection procedure to determine independent variables and identify variables associated with major adverse outcomes. RESULTS: Fifty-six (14.2%) patients died during their hospitalization in the study group and 46 (8.3%) in the reference group. Major mortality in the study group was 58.0% versus 39.7% in the reference group. The patients of the study group spent more days in the ICU than did patients of the reference group (8.7 +/- 12.0 versus 6.1 +/- 10.0 days, P < 0.05). In addition, 114 (28.9%) patients of the study group and 85 (15.3%) of the reference group spent more than 7 days in the ICU. Using multiple logistic regression analysis, risk factors affecting in-hospital mortality in the study group include impairment of renal function, reoperation, congestive heart failure (CHF), catastrophic event. Impaired renal function, complexity of surgical procedure, CHF, chronic obstructive pulmonary disease (COPD) and catastrophic state were significant factors affecting morbidity in the study group. CHF, COPD and catastrophic event contributed to prolonged ICU stay in the study group. CONCLUSIONS: The perioperative risk of cardiac surgery increases in older patient groups, and some risk factors have different influences on mortality, morbidity and ICU stay. Risk assessment in older cardiosurgical patients with convenient risk factors helps the clinicians to apply rational and cost-effective treatment strategies into practice.

The use of rocuronium and sevoflurane in acute intermittent porphyria--a case report.

Acute intermittent porphyria (AIP) is an inherited metabolic disorder caused by deficiency of porphobilinogen deaminase, an enzyme found in the synthetic pathway of heme. Acute attack of AIP may be precipitated by many factors during operation and anesthesia, including fasting, dehydration, stress, infection, and drugs. Acute attack of AIP is likely fatal. Therefore, the drugs recommended as being safe in anesthesia for porphyria patients are up-to-dately refreshed and renovated and the identification of whether a drug is safe or not is based on cumulative anecdotal experiences. Here, we report the safe use of rocuronium and sevoflurane for long exposure in a patient affected with acute intermittent porphyria.

Determination of optical parameters of a twisted-nematic liquid crystal by phase-sensitive optical heterodyne interferometric ellipsometry.

What is believed to be a novel phase-sensitive optical heterodyne interferometric ellipsometer is set up to characterize a twisted-nematic liquid crystal (TN-LC) by the elliptical parameters of the output polarization state. This ellipsometer presents the advantages of both polarized optical heterodyne interferometry and optical photometry, which introduce a polarization modulation that is capable of performing with high-sensitivity on phase detection in real time. The twist angle phi and the untwisted phase retardation gamma of TN-LC are measured precisely. The experimental results verify that a TN-LC can be treated as identical to an elliptical retarder.