Lih-Chiann Wang

Singlet oxygen is the major species participating in the induction of DNA strand breakage and 8-hydroxydeoxyguanosine adduct by lead acetate

To investigate DNA damage induced by Pb2+ and its prevention by scavengers, we determined DNA strand breakage and the formation of 8‐hydroxydeoxyguanosine (8‐OHdG) in DNA using plasmid relaxation assay and HPLC with electrochemical detection, respectively. Lead acetate induced DNA strand breakage in 10 mM of Hepes buffer, pH 6.8, in a time‐ and dose‐dependent manner. Compared with lead, zinc acetate did not significantly induce DNA breakage. The singlet oxygen scavengers NaN3 and 2,2,6,6‐tetramethyl‐4‐piperidone (TEMP) inhibited lead‐induced DNA breakage more efficiently than the hydroxyl radical scavengers mannitol and DMPO. Deuterium oxide (D2O), a singlet oxygen enhancer, potentiated lead‐induced DNA breakage. At low ratios to Pb2+, NADPH, glutathione, and 2‐mercaptoethanol enhanced lead‐induced DNA breakage, whereas high ratios of these agents protected it. Catalase and superoxide dismutase (SOD) did not protect DNA breaks induced by Pb2+. Lead‐induced DNA breakage was markedly enhanced by H2O2, and this induction was inhibited by NaN3, TEMP, EDTA, catalase, BSA, and glutathione. In contrast, mannitol and SOD potentiated Pb2+/H2O2‐induced DNA breaks. The results indicate that singlet oxygen, lead, and H2O2 are all involved in the reaction system, whereas hydroxyl radical and superoxide did not. Lead could cause a small amount of 8‐OHdG formation in calf thymus DNA and dose‐dependently induced the formation of this adduct in the presence of H2O2. Singlet oxygen scavengers were more effective than hydroxyl radical scavengers in protection from lead/H2O2‐induced 8‐OHdG adducts. Taken together, these results suggest that lead may induce DNA damage through a Fenton‐like reaction and that singlet oxygen is the principal species involved. Environ. Mol. Mutagen. 33:194–201, 1999

A novel strategy for avian species and gender identification using the CHD gene

We report on a novel and rapid strategy for the simultaneous identification of both avian species and gender by analyzing a section of the CHD gene. The CHD gene is carried by the avian sex determining chromosomes where a female bird carries both a W and Z chromosome but a cock bird carries two copies of the Z chromosome. Two primer pairs, CHD1F/CHD1R and P2/P8, were used to amplify a part of the CHD gene from 144 samples corresponding to 58 avian species. For all species tested, two fragments were observed at least in one amplification for female samples. All tested species produced species specific size fragments allowing both sex determination and species identification using these primer pairs. However, special care is still warranted as so few samples have been characterised. This novel strategy for avian species and gender identification using the CHD gene was developed for a number of applications from ecology to forensic science.

A novel strategy for avian species identification by cytochrome b gene

We report a DNA‐based test that can be applied to any avian species so that the amplicon can be used in species identification. The need for the test arose from the requirement to enforce the Wildlife Conservation Act in Taiwan where over 150 avian species are protected. It is difficult to enforce the law if no gross morphology is present and hence there is a requirement to develop a DNA test. This study uses a novel strategy for avian species identification by the cytochrome b gene where a series of primer pairs producing amplicons of decreasing size was designed. The test is designed to produce the largest possible amplicon based upon the quality of the DNA in the sample. A total of 331 avian samples were tested representing 40 species. Sequencing of the amplicons revealed limited intraspecies variation and that no DNA sequence was shared by samples from two different avian species. The closest genetic distance among the 40 species was 0.059 which was between Lonchura punctulata and Estrilda melpoda based upon data from the smallest amplicon. A DNA databank including 138 sequence types from 331 samples tested, representing 40 different species, was constructed in this study. A blind test was used to determine the value for this system for forensic applications that successfully identified the species.

Establishing the pangolin mitochondrial D-loop sequences from the confiscated scales

Pangolin scales are encountered in traditional East Asian medicines (TEAM) and the ever increasing demand for these scales has escalated the decline in the numbers of these mammals. The identification of protected pangolin species is necessary to enforce international and national legislation as well as assist with conservation measures. There is limited morphological feature on a pangolin scale thus requiring DNA analysis as a means of identification. We report on the isolation of DNA from pangolin scales and a strategy for obtaining the full length of the mitochondrial D-loop, being 1159 bp. Primer sets creating five overlapping amplicons were designed to amplify sections of this mitochondrial DNA locus. DNA from the blood stain of nineteen Formosan pangolins (Manis pentadactyla pentadactyla) along with 145 scale samples that were suspected to have come from pangolins, was amplified and sequenced; leading to a total of 91 D-loop sequences being obtained. The 19 Formosan pangolin sequences produced 5 haplotypes and 72 of the 145 seized scales provided useable sequence classified as a further 38 haplotypes. The D-loop sequences from those scales suspected to be from a pangolin had a higher similarity to any of the 19 samples taken from M. p. pentadactyla compared to a D-loop sequence from Manis tetradactyla (the only pangolin D-loop sequence in GenBank, NC_004027). These 43 haplotypes were used to establish a local database for the D-loop sequence of pangolins and add to the data of Manis sp. held on GenBank. The PCR amplification strategy development in this study could be used in forensic DNA identification of scales suspected to be from protected pangolin species.

Sex Identification of Owls (Family Strigidae) Using Oligonucleotide Microarrays

Molecular sexing of the diversified avian family Strigidae is difficult. Sex identification using the intron length difference between W and Z chromosomal CHD1 genes, as visualized by agarose gel electrophoreses, often produces ambiguous results. Here we describe a simple method for sexing a variety of Strigidae species using oligonucleotide microarrays, on which several sex-specific probes operated complementarily or in concert. The sex of 8 owl species was identified clearly on the microarrays through sequence recognition. This sequence-directed method can be easily applied to a wider range of Strigidae species.

Influenza A(H6N1) Virus in Dogs, Taiwan.

We determined the prevalence of influenza A virus in dogs in Taiwan and isolated A/canine/Taiwan/E01/2014. Molecular analysis indicated that this isolate was closely related to influenza A(H6N1) viruses circulating in Taiwan and harbored the E627K substitution in the polymerase basic 2 protein, which indicated its ability to replicate in mammalian species.

A novel method for the preparation of uranium metal, oxide and carbide via electrolytic amalgamation

Abstract A solid uranium amalgam was prepared electrolytically using a two-compartment cell separated with an ion exchange membrane for the purpose of regulating pH value within a narrowly restricted region of 2 to 3. The mercury cathode was kept at −1.8 vs SCE during electrolysis. The thereby obtained amalgam containing as high as 1.9 gm U/ml Hg is easily converted into uranium metal by heating in vacuo above 1300°C. Uranium dioxide and uranium monocarbide could be easily obtained at relatively low temperature by reacting the amalgam with water vapor and methane.

Avian oncogenic virus differential diagnosis in chickens using oligonucleotide microarray

Avian oncogenic viruses include the avian leukosis virus (ALV), reticuloendotheliosis virus (REV) and Mareks disease virus (MDV). Multiple oncogenic viral infections are frequently seen, with even Mareks disease vaccines reported to be contaminated with ALV and REV. The gross lesions caused by avian oncogenic viruses often overlap, making differentiation diagnosis based on histopathology difficult. The objective of this study is to develop a rapid approach to simultaneously differentiate, subgroup and pathotype the avian oncogenic viruses. The oligonucleotide microarray was employed in this study. Particular DNA sequences were recognized using specific hybridization between the DNA target and probe on the microarray, followed with colorimetric development through enzyme reaction. With 10 designed probes, ALV-A, ALV-E, ALV-J, REV, MDV pathogenic and vaccine strains were clearly discriminated on the microarray with the naked eyes. The detection limit was 27 copy numbers, which was 10-100 times lower than multiplex PCR. Of 102 field samples screened using the oligonucleotide microarray, 32 samples were positive for ALV-E, 17 samples were positive for ALV-J, 6 samples were positive for REV, 4 samples were positive for MDV, 7 samples were positive for both ALV-A and ALV-E, 5 samples were positive for ALV-A, ALV-E and ALV-J, one sample was positive for both ALV-J and MDV, and 3 samples were positive for both REV and MDV. The oligonucleotide microarray, an easy-to-use, high-specificity, high-sensitivity and extendable assay, presents a potent technique for rapid differential diagnosis of avian oncogenic viruses and the detection of multiple avian oncogenic viral infections under field conditions.

Development of an antigen-capture enzyme-linked immunosorbent assay using monoclonal antibodies for detecting H6 avian influenza viruses

The H6 subtype of avian influenza virus (AIV) infection occurs frequently in wild and domestic birds. AIV antigen detection is preferred for controlling AIV as birds are infected before they produce antibodies. The purpose of this study was to develop an early diagnostic method for AIV detection. Six monoclonal antibodies (mAbs) developed from a field H6N1 AIV strain were tested for their ability to bind to viruses. The two that showed the greatest binding ability to AIVs were used for antigen detection. An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect H6 AIVs was developed using these mAbs. One mAb was coated onto an ELISA plate as the capture antibody. The other mAb was used as the detector antibody after labeling with horseradish peroxidase. The antigen-capture ELISA detected H6N1 AIVs but not H5 AIVs, human H1N1, H3N2 influenza or other viruses. This antigen-capture ELISA could be used to specifically detect H6N1 AIV.

H5 avian influenza virus pathotyping using oligonucleotide microarray.

The H5 avian influenza virus subtype has huge impact on the poultry industry. Rapid diagnosis and accurate identification of the highly pathogenic avian influenza virus and low-pathogenicity avian influenza virus is essential, especially during H5 outbreaks and surveillance. To this end, a novel and rapid strategy for H5 virus molecular pathotyping is presented. The specific hemagglutinin gene of the H5 virus and the basic amino acid number of the motif at the hemagglutinin precursor protein cleavage site were detected using oligonucleotide microarray. Highly pathogenic and low-pathogenicity avian influenza viruses in Taiwan were differentiated using 13 microarray probes with the naked eye. The detection limit reached 3.4 viral RNA copies, 1000 times more sensitive than reverse transcription polymerase chain reaction. Thus, the oligonucleotide microarray would provide an alternative H5 pathogenicity determination using the naked eye for laboratories lacking facilities.