Further characterization of Dothistromin genes in the fungal forest pathogen Dothistroma septosporum : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Molecular Genetics at Massey University, Palmerston North, New Zealand

Dothistroma septosporum is a forest pathogen that causes a disease called Dothistroma needle blight. The symptoms are thought to be due to the accumulation of dothistromin toxin produced by D. septosporum. Dothistromin is characterized as a difuranoanthraquinone and shows remarkable similarity to the aflatoxin (AF) and sterigmatocystin (ST) precursor versicolorin B. The similar structure to AF/ST suggests that dothistromin biosynthesis shares biosynthetic steps with the AF/ST pathway. The AF gene cluster in Aspergillus parasiticus and ST gene cluster in A. nidulans have been well characterized. Nine putative dothistromin biosynthetic genes have been identified. One of them, dotA was previously characterized by gene disruption and shown to have a similar function to homologous genes in AF/ST biosynthesis. Two additional putative dothistromin biosynthetic genes, pksA and epoA, were characterized by gene disruption in this study. The inability of the pksA mutants to produce dothistromin indicated that the pksA is a key gene in dothistromin biosynthesis. The feeding of intermediates confirmed that pksA gene product is required for a very early step of dothistromin biosynthesis. The pksA mutants also showed reduced sporulation compared to wildtype, suggesting a relationship between dothistromin production and sporulation. The epoA gene replacements were also obtained successfully by homologous recombination. Both Southern blot and northern hybridization confirmed that the epoA gene was disrupted. However, the epoA mutants did not show any difference to the wild type in three analyses (growth rate, sporulation rate, dothistromin biosynthesis). However it was not possible to rule out a role for EpoA at a very late stage of dothistromin biosynthesis. RACE analysis of the nine identified dothistromin genes characterized the transcription start and stop sites of the genes. Analyzing the putative regulatory protein binding motifs in the untranscribed region of the genes provided clues about the regulation of dothistromin biosynthesis and suggested there might be an aflR-like gene that governs dothistromin biosynthesis. Both the pksA gene disruption and the RACE results suggested that the dothistromin biosynthetic pathway is homologous to that of AF/ST biosynthesis. Further work on the dothistromin gene cluster will help us to understand the evolution of fungal toxin gene clusters

Determination of metabolites of phloretin in rats using UHPLC-LTQ-Orbitrap mass spectrometry

Purpose: To study the metabolites of phloretin in vivo using ultra-high performance liquid chromatography linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap). Methods: After administration of phloretin (50 mg/kg; oral route) to six rats, blood samples were taken from each animal. Each sample was then subjected to solid-phase extraction to prepare it for chromatographic/spectroscopic analysis. Finally, each sample was analyzed using UHPLC-LTQOrbitrap with a negative-mode electrospray ionization source. Results: Based on mass measurements, chromatographic retention times, and MS2 fragmentation ions, we detected and identified phloretin and 16 metabolites of the drug in vivo in rats. Metabolic reactions of phloretin included glucosylation and glucuronide conjugation, diglucuronide conjugation, glucosylation and sulfate conjugation, sulfate conjugation, glucuronide conjugation, and glucosylation and hydroxylation. Conclusion: The findings provide a better understanding of phloretin metabolism and metabolites, and new information about their effective forms, pharmacological actions, metabolic fate, and toxic actions in vivo

The Design and Realization for a Multiplex Time Sequence Controller

AbstractIn order to meet the demand of activating several devices at different moments, a multiplex time sequence controller is developed in this paper. When the controller receives the trigger signal for starting, the time sequential control circuit module, consisting of the microcontroller and the FPGA, it can generate a delay trigging signal according to the preset delay value, which will activate the testing device after being driven. The delay value is import by the computer or the dial on the panel. The real firing results show that the time sequence controller can realize the delay of 20-channel independently, one of which is able to be adjustable within 0∼10s, the maximum amplitude of output delay trigging signal is 12V, the width of the signal is 5ms and the error of the delay time is less than 2colons. The multiplex time sequence controller can satisfy the requirements of technical specifications of testing system in conventional shooting range, and it can meet the demand of activating several testing devices operating at different moments

Profiling and Identification of the Metabolites of Evodiamine in Rats using Ultra–Performance Liquid Chromatography with Linear Ion Trap Orbitrap Mass Spectrometer

Purpose: To develop a highly sensitive and specific ultra-performance liquid chromatography with linear ion trap Orbitrap mass spectrometer (UPLC-LTQ-Orbitrap) method to profile and identify the metabolites of evodiamine in rats.Methods: First, blood samples were collected after oral administration of evodiamine to rats (50 mg/kg). Next, the plasma samples were pretreated using a solid-phase extraction (SPE) method. Finally, all the samples were analyzed by ultra-performance liquid chromatography LTQ-Orbitrap mass spectrometry (UPLC-LTQ- Orbitrap) coupled with electrospray ionization source (ESI) in negative mode.Results: A total of 7 metabolites (2 phase I and 4 phase II metabolites, including 4 new metabolites, viz, 10-hydroxyevodiamine sulfate, 10-hydroxyevodiamine sulfate, 10-hydroxyevodiamine glucuronide and 3-hydroxyevodiamine glucuronide) as well as the parent drug itself , were detected and identified based on accurate mass measurements, fragmentation patterns, and chromatographic retention times. The in vivo metabolic reactions of evodiamine in rats were hydroxylation, hydroxylation + sulfate conjugation, and hydroxylation + glucuronidation.Conclusion: These results provide better understanding of the metabolism of evodiamine as well as strong indications of the effective forms of the drug in vivo.Keywords: Evodiamine, Ultra–performance liquid chromatography with linear ion trap-Orbitrap, Hydroxyevodiamine sulfate, Hydroxyevodiamine glucuronide, Metabolite

In silico and microarray-based genomic approaches to identifying potential vaccine candidates against Leptospira interrogans

BACKGROUND: Currently available vaccines against leptospirosis are of low efficacy, have an unacceptable side-effect profile, do not induce long-term protection, and provide no cross-protection against the different serovars of pathogenic leptospira. The current major focus in leptospirosis research is to discover conserved protective antigens that may elicit longer-term protection against a broad range of Leptospira. There is a need to screen vaccine candidate genes in the genome of Leptospira interrogans. RESULTS: Bioinformatics, comparative genomic hybridization (CGH) analysis and transcriptional analysis were used to identify vaccine candidates in the genome of L. interrogans serovar Lai strain #56601. Of a total of 4727 open reading frames (ORFs), 616 genes were predicted to encode surface-exposed proteins by P-CLASSIFIER combined with signal peptide prediction, α-helix transmembrane topology prediction, integral β-barrel outer membrane protein and lipoprotein prediction, as well as by retaining the genes shared by the two sequenced L. interrogans genomes and by subtracting genes with human homologues. A DNA microarray of L. interrogans strain #56601 was constructed for CGH analysis and transcriptome analysis in vitro. Three hundred and seven differential genes were identified in ten pathogenic serovars by CGH; 1427 genes had high transcriptional levels (Cy3 signal ≥ 342 and Cy5 signal ≥ 363.5, respectively). There were 565 genes in the intersection between the set encoding surface-exposed proteins and the set of 307 differential genes. The number of genes in the intersection between this set of 565 and the set of 1427 highly transcriptionally active genes was 226. These 226 genes were thus identified as putative vaccine candidates. The proteins encoded by these genes are not only potentially surface-exposed in the bacterium, but also conserved in two sequenced L. interrogans. Moreover, these genes are conserved among ten epidemic serovars in China and have high transcriptional levels in vitro. CONCLUSION: Of the 4727 ORFs in the genome of L. interrogans, 226 genes were identified as vaccine candidates by bioinformatics, CGH and transcriptional analysis on the basis of the theory of reverse vaccinology. The proteins encoded by these genes might be useful as vaccine candidates as well as for diagnosis of leptospirosis

ZnO Nanorods Grown on p-GaN Using Hydrothermal Synthesis and Its Optoelectronic Devices Application

The ZnO nanorods with the length of 1-1.5 μm were deposited on p-GaN by hydrothermal synthesis at low temperature 100°C. The structural and optical properties of the as-grown ZnO rods were investigated by X-Ray diffraction (XRD) and photoluminescence (PL) spectra. After annealing treatment the as-grown films in air at 600°C, 30min, and the ZnO rods showed good crystallinity and optical properties with strong UV emission at 378 nm. In addition, a sharp UV emission peak at 369.45 nm with the FWHM 20 meV, which attributed to the bound exciton recombination, was also observed from the ZnO rods at 80K. Next, the e-beam evaporation method was used to deposit metal contact on n-ZnO and p-GaN. Here, we use Au and Ni/Au as metal contacts for n-ZnO and p-GaN, respectively. The current-voltage characteristics of the fabricated n-ZnO/p-GaN heterojunction revealed rectifying behavior with a leakage current of 10⁻⁸ A at -10V, a forward current 4x10⁻⁶ A at 10V bias. The heterojunction also showed a good photoresponse, with the change of the current – voltage characteristics under ultraviolet illumination. Under UV illumination, the forward turn on voltage changed to 7.5V. This result showed the ability to manipulate the electron transport in the ZnO based heterojunction devices.Singapore-MIT Alliance (SMA

Production and uptake of dissolved carbon, nitrogen, and phosphorus in overlying water of aquaculture shrimp ponds in subtropical estuaries, China

Water quality deterioration can adversely affect the long-term sustainability of aquaculture industry. Understanding the processes of nutrient regeneration and uptake is important for improving water quality and the overall ecosystem health of aquaculture system. In spite of the importance of dissolved nutrients (DOC, DIC, N-NO , N-NH , and P-PO ) in governing water quality and ecosystem functioning, the spatiotemporal variations in the production and uptake of dissolved nutrients in aquaculture ponds is still poorly understood. In this study, the nutrient production and uptake rates in the overlying water were quantified among different shrimp growth stages in the aquaculture ponds in the Min River Estuary (MRE) and Jiulong River Estuary (JRE), southeast China. Significant differences in the nutrient production and uptake rates in the overlying water were observed among the three growth stages and two estuaries. The temporal variations of DOC and DIC production rates in both estuarine ponds closely followed the seasonal cycle of temperature, while the difference in DOC and DIC production rates between the two estuaries was likely caused by differences in water salinity. The changes in the production and uptake rates of dissolved inorganic nitrogen (N-NO and N-NH ) and P-PO in the water column over time were partly related to the interactions between thermal conditions and phytoplankton biomass (e.g., chlorophyll a concentrations) in the ponds. Our results demonstrate the complex dynamics and environmental risk of dissolved nutrients in subtropical shrimp ponds, and call for a more effective management of nutrient-laden wastewater in safeguarding the long-term sustainability of aquaculture production