High-throughput genotyping by DHPLC of the dihydropyrimidine dehydrogenase gene implicated in (fluoro)pyrimidine catabolism

Dihydropyrimidine dehydrogenase (DPD) is the first and rate-limiting enzyme in the degradation of pyrimidines and pyrimidine base analogs including the anticancer drugs 5-fluorouracil (5-FU) and Xeloda. A decreased DPD enzyme activity has been described in cancer patients experiencing severe and life-threatening toxicity after 5-FU treatment and distinct sequence variants in the DPD gene (DPYD) have been associated with impaired enzyme function. The most prominent mutation in the DPD deficient patient group, a mutation in the splicing donor consensus sequence of intron 14, IVS14+1g>a, resulting in a truncated protein, has been observed in the Caucasian population at frequencies as high as 0.91%-0.94%. This underlines the need for a test system for DPYD mutations in patients undergoing chemotherapy with 5-FU or with Xeloda. To set up a fast and sensitive method to identify variant DPYD alleles, we analyzed 50 healthy individuals by denaturing high performance liquid chromatography (DHPLC). A primer set spanning the whole coding region and the exon-intron boundaries of DPYD was used. In addition, a cDNA-based assay was developed to rapidly identify the 165 base pair deletion in the corresponding RNA of IVS14+1g>a mutation carriers. The optimal mutation detection was elaborated for each of the PCR fragments. DHPLC analysis detected 5 different genetic alterations occurring in the coding region of the gene, as well as 10 intronic sequence variants, respectively. In conclusion, high-throughput screening for DPYD variants by DHPLC may be a reliable tool in the investigation of the molecular basis of DPD deficiency. Furthermore, it will help to identify patients at risk for toxic side effects upon chemotherapy using 5-FU and will facilitate individual treatment of patients.     

Three Dimensional Transient Multifield Analysis of a Piezoelectric Micropump for Drug Delivery System for Treatment of Hemodynamic Dysfunctions

In this paper, we present design of a transdermal drug delivery system for treatment of cardiovascular or hemodynamic disorders such as hypertension. The system comprises of integrated control electronics and microelectromechanical system devices such as micropump, micro blood pressure sensor and microneedle array. The objective is to overcome the limitations of oral therapy such as variable absorption profile and the need for frequent dosing, by fabricating a safe, reliable and cost effective transdermal drug delivery system to dispense various pharmacological agents through the skin for treatment of hemodynamic dysfunction such as hypertension. Moreover, design optimization of a piezoelectrically actuated valveless micropump is presented for the drug delivery system. Because of the complexity in analysis of piezoelectric micropump, which involves structural and fluid field couplings in a complicated geometrical arrangement, finite element (FE) numerical simulation rather than an analytical system has been used. The behavior of the piezoelectric actuator with biocompatible polydimethylsiloxane membrane is first studied by conducting piezoelectric analysis. Then the performance of the valveless micropump is analyzed by building a three dimensional electric-solid-fluid model of the micropump. The effect of geometrical dimensions on micropump characteristics and efficiency of nozzle/diffuser elements of a valveless micropump is investigated in the transient analysis using multiple code coupling method. The deformation results of the membrane using multifield code coupling analysis are in good agreement with analytical as well as results of single code coupling analysis of a piezoelectric micropump. The analysis predicts that to enhance the performance of the micropump, diffuser geometrical dimensions such as diffuser length, diffuser neck width and diffuser angle need to be optimized. Micropump flow rate is not strongly affected at low excitation frequencies from 10 to 200 Hz. The excitation voltage is the more dominant factor that affects the flow rate of the micropump as compared with the excitation frequency. However, at extremely high excitation frequencies beyond 8,000 Hz, the flow rate drops as the membrane exhibits multiple bending peaks which is not desirable for fluid flow. Following the extensive numerical analysis, actual fabrication and performance characterization of the micropump is presented. The performance of the micropump is characterized in terms of piezoelectric actuator deflection and micropump flow rate at different operational parameters. The set of multifield simulations and experimental measurement of deflection and flow rate at varying voltage and excitation frequency is a significant advance in the study of the electric-solid-fluid coupled field effects as it allows transient, three dimensional piezoelectric and fluid analysis of the micropump thereby facilitating a more realistic multifield analysis. The results of the present study will also help to conduct relevant strength duration tests of integrated drug delivery device with micropump and microneedle array in future.     

A facile one-pot green synthesis of gold nanoparticle-graphene-PEDOT:PSS nanocomposite for selective electrochemical detection of dopamine

A facile one-pot and green method was developed to prepare a nanocomposite of gold nanoparticle (AuNP), graphene (GP) and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS). Graphene was first electro-exfoliated in a polystyrene sulfonate solution, followed by a one-step simultaneous in situ formation of gold nanoparticle and PEDOT. The as-synthesized aqueous dispersion of AuNP-GP-PEDOT:PSS was thereafter used to modify the glassy carbon electrode (GCE). For the first time, the quaternary composite between AuNP, GP, PEDOT and PSS was used for selective determination of dopamine (DA) and uric acid (UA) in the presence of ascorbic acid (AA). In comparison to a bare GCE, the nanocomposite electrode shows considerably higher electrocatalytic activities toward the oxidation of DA and UA due to a synergistic effect between AuNP, GP, PEDOT and PSS. Using differential pulse voltammetry (DPV), selective determination of DA and UA in the presence of AA could be achieved with a peak potential separation of 110 mV between DA and UA. The sensor exhibits wide linear responses for DA and UA in the ranges of 1 nM to 300 μM and 10 μM to 1 mM with detection limits (S/N = 3) of 100 pM and 10 μM, respectively. Furthermore, the proposed sensor was also successfully used to determine DA in a real pharmaceutical injection sample as well as DA and UA in human serum with satisfactory recovery results.

A facile one-pot green synthesis of gold nanoparticle-graphene-PEDOT:PSS nanocomposite was successfully demonstrated.     

Clinical, laboratory findings and microbiologic characterization of bronchiectasis in Thai patients

OBJECTIVE: The aim of the present study was to characterize the clinical features, underlying disease states, laboratory findings and microbiological characterization of bronchiectasis in Thai patients. METHODOLOGY: For a 2-year period all consecutive patients diagnosed with bronchiectasis at Phramongkutklao Hospital, Bangkok, Thailand, were recruited. Data including history, physical examination, underlying disease and laboratory studies were carefully reviewed and recorded. RESULTS: Fifty patients diagnosed with bronchiectasis were enrolled. Their mean age was 58 years. The most common background aetiology was tuberculosis. Six per cent of the patients were diagnosed as having diffuse panbronchiolitis. Normal chest radiographs were found in 10%. The common organisms isolated were Pseudomonas aeruginosa (20%), Haemophilus influenzae (14%), Klebsiella pneumoniae (14%) and Streptococcus pneumoniae (6%). Non-tuberculous mycobacteria which included Mycobacterium kansasii and Mycobacterium chelonae were found in 6%. CONCLUSION: We report the characteristics of bronchiectasis in Thai patients. The most common identifiable aetiology was tuberculosis.     

Malondialdehyde–Deoxyguanosine Adducts among Workers of a Thai Industrial Estate and Nearby Residents

Background

Humans living near industrial point emissions can experience high levels of exposures to air pollutants. Map Ta Phut Industrial Estate in Thailand is the location of the largest steel, oil refinery, and petrochemical factory complexes in Southeast Asia. Air pollution is an important source of oxidative stress and reactive oxygen species, which interact with DNA and lipids, leading to oxidative damage and lipid peroxidation, respectively.

Objective

We measured the levels of malondialdehyde–deoxyguanosine (dG) adducts, a biomarker of oxidative stress and lipid peroxidation, in petrochemical workers, nearby residents, and subjects living in a control district without proximity to industrial sources.

Design

We conducted a cross-sectional study to compare the prevalence of malondialdehyde-dG adducts in groups of subjects experiencing various degrees of air pollution.

Results

The multivariate regression analysis shows that the adduct levels were associated with occupational and environmental exposures to air pollution. The highest adduct level was observed in the steel factory workers. In addition, the formation of DNA damage tended to be associated with tobacco smoking, but without reaching statistical significance. A nonsignificant increase in DNA adducts was observed after 4–6 years of employment among the petrochemical complexes.

Conclusions

Air pollution emitted from the Map Ta Phut Industrial Estate complexes was associated with increased adduct levels in petrochemical workers and nearby residents. Considering the mutagenic potential of DNA lesions in the carcinogenic process, we recommend measures aimed at reducing the levels of air pollution.     

DNA adducts and combinations of multiple lung cancer at‐risk alleles in environmentally exposed and smoking subjects

Interindividual variation in DNA adduct levels in individuals exposed to similar amounts of environmental carcinogens may be due to genetic variability. We analysed the influence of genes involved in determining/modifying DNA damage, including microsomal epoxide hydrolase1 (EPHX1) His139Arg, N‐acetyl‐transferase, NAD(P)H:quinone oxidoreductase1 (NQO1) Pro187Ser, manganese superoxide dismutase2 (MnSOD2) Val16Ala, and apurinic/apyrimidinic endonuclease1 (APE1) Asp148Glu polymorphisms in blood of 120 smokers. Subsequently, we examined the effects of the combinations of the variant alleles of EPHX, NQO1 and MnSOD2 together with the wild type allele of APE1 on DNA damage by calculating the “sum of at‐risk alleles.” We reviewed the studies examining the relationships of DNA adducts with at‐risk alleles in environmentally exposed subjects. Our findings showed that smokers carrying the EPHX1–139Arg and the NQO1–187Ser variants were significantly more likely to have higher adduct levels. Null associations were found with the other variants. Nevertheless, DNA adduct levels in smokers with ≥5 at‐risk alleles were significantly different from those with fewer than two alleles. A similar picture emerged from studies of DNA adducts and at‐risk alleles in environmentally exposed and smoking subjects. Certain at‐risk allele combinations may confer a greater likelihood of increased levels of adducts after environmental insults. The increase in DNA adduct levels in susceptible subjects exposed to environmental carcinogens may reflect changes in the mechanisms that protect cells from the accumulation of genetic damage. Alterations of the physiological processes designed to maintain homeostasis may reduce the individual “genotoxic tolerance” to environmental challenges and result in phenotypes characterized by high levels of DNA adducts. Environ. Mol. Mutagen. 54:375–383, 2013. © 2013 Wiley Periodicals, Inc.     

Detection of shrimp Taura syndrome virus by loop-mediated isothermal amplification using a designed portable multi-channel turbidimeter

In this study, a portable turbidimetric end-point detection method was devised and tested for the detection of Taura syndrome virus (TSV) using spectroscopic measurement of a loop-mediated isothermal amplification (LAMP) by-product: magnesium pyrophosphate (Mg₂P₂O₇). The device incorporated a heating block that maintained an optimal temperature of 63 °C for the duration of the RT-LAMP reaction. Turbidity of the RT-LAMP by-product was measured when light from a light-emitting diode (LED) passed through the tube to reach a light dependent resistance (LDR) detector. Results revealed that turbidity measurement of the RT-LAMP reactions using this device provided the same detection sensitivity as the agarose gel electrophoresis detection of RT-LAMP and nested RT-PCR (IQ2000™) products. Cross reactions with other shrimp viruses were not found, indicating that the RT-LAMP-turbidity measurement was highly specific to TSV. The combination of 10 min for rapid RNA preparation with 30 min for RT-LAMP amplification followed by turbidity measurement resulted in a total assay time of less than 1 h compared to 4-8 h for the nested RT-PCR method. RT-LAMP plus turbidity measurement constitutes a platform for the development of more rapid and user-friendly detection of TSV in the field.