Increasing xylanase activity of Bacillus subtilis by atmospheric pressure plasma jet for biomass hydrolysis.

Xylanase producing bacteria, Bacillus subtilis, was bombarded by an atmospheric pressure plasma jet (APPJ) and screened for higher catalytic activity. The bacteria were bombarded with plasma of argon or helium with energy of 120 W for a duration of 1-5 min. A mutant with higher xylanase activity was observed under argon plasma treatment at 1 min on media containing xylan as substrate. Subsequently, the xylanase gene from the mutant was sequenced and named MxynA. Sequence analysis revealed only a single missense mutation on the MxynA gene causing amino acid substitution from threonine to serine at position 162 (T162S) within the xylanase protein of the mutant. Consequently, MxynA was subcloned into expression vector, pETDuet-1 under T7 promoter and expressed in E. coli BL21 (DE3). The optimum temperature and pH of MxynA and its parent expressed in E. coli, named CxynA were 60 °C and pH 5, respectively. Moreover, MxynA showed higher xylanase activity approximately 4 fold higher than that of the control upon a wide range of pH and temperature conditions. From kinetic parameters analysis, the mutant showed higher enzyme turnover (k cat) than the control. The hydrolysis ability of the MxynA enzyme on lignocellulosic wastes, such as rice straw, corncob and para grass was investigated using the released reducing sugar as an indicator. The MxynA enzyme showed a greater amount of reducing sugar released from all lignocellulosic wastes other than the control, particularly from para grass. This study demonstrated that the T162S mutation possibly improved the catalytic efficiency of MxynA.

Developmental and Phylogenetic Characteristics of Stellantchasmus falcatus (Trematoda: Heterophyidae) from Thailand.

This study aimed to investigate the infection status, worm development, and phylogenetic characteristics of the intestinal trematode, Stellantchasmus falcatus. The metacercariae of S. falcatus were detected only in the half-beak (Dermogenus pusillus) out of the 4 fish species examined. Their prevalence was 90.0%, and the intensity of infection was 919 metacercariae on average. Worms were recovered from 33 (97.1%) of 34 chicks that were experimentally infected with 200 S. falcatus metacercariae each, and the average recovery rate was 43.0%. The body size and inner organs of S. falcatus quickly increased in the experimental chicks over days 1-2 post-infection (PI). In addition, ITS2 sequence data of this parasite were analyzed to examine the phylogenetic relationships with other trematodes using the UPGMA method. The results indicated that the ITS2 sequence data recorded from trematodes in the family Heterophyidae appeared to be monophyletic. This study concluded that D. pusillus serves as a compatible second intermediate host of S. falcatus in Thailand and that S. falcatus can develop rapidly in the experimental chicks. Data collected from this study can help to close the gap in knowledge regarding the epidemiology, biology, and phylogenetic characteristics of S. falcatus in Thailand.

Chemical composition and antibacterial activity of rhizome oils from five Hedychium species.

The essential oils from rhizomes of five Hedychium species, H. coronarium, H. neocarneum, H. flavescens, H. speciosum and H. stenopetalum (Zingiberaceae), were obtained by hydrodistillation and analyzed by capillary GC and GC/MS. Sixty components were identified and percentage oil yields from the fresh plants ranged from 0.06-0.17 % (v/w). All rhizome oils were rich in terpenes, especially monoterpenes (75.0-95.9 %). The most common compounds in the rhizome oils of Hedychium were beta-pinene, linalool and 1,8-cineole. The essential oils were tested against four bacterial strains. They showed moderate to weak activity against Gram-positive bacteria (inhibition zone 25-13 mm, MIC 0.3-8.3 mg/mL, MBC 0.6-8.3 mg/mL).

Overexpression of OsRab7B3, a small GTP-binding protein gene, enhances leaf senescence in transgenic rice.

Rab family proteins are small GTP-binding proteins involved in intracellular trafficking. They play critical roles in several plant development processes. Different expression patterns of 46 Rabs in the rice genome were examined in various rice tissues and in leaves treated with plant growth regulators and under senescence conditions. One of the OsRab genes, OsRab7B3, closely associated with senescence in expression pattern, was chosen for functional analysis. Expression of sGFP under the control of the OsRab7B3 promoter increased in leaves when ABA and NaCl were applied or when kept in dark. In transgenic rice overexpressing OsRab7B3, the senescence-related genes were upregulated and leaf senescence was significantly enhanced under dark conditions. Moreover, leaf yellowing occurred earlier in the transgenic plants than in the wild type at the ripening stage. Hence it is suggested that OsRab7B3 act as a stress-inducible gene that plays an important role in the leaf senescence process.

Expression of OsSPY and 14-3-3 genes involved in plant height variations of ion-beam-induced KDML 105 rice mutants.

The culm length of two semidwarf rice mutants (PKOS1, HyKOS1) obtained from low-energy N-ion beam bombardments of dehusked Thai jasmine rice (Oryza sativa L. cv. KDML 105) seeds showed 25.7% and 21.5% height reductions and one spindly rice mutant (TKOS4) showed 21.4% increase in comparison with that of the KDML 105 control. A cDNA-RAPD analysis identified differential gene expression in internode tissues of the rice mutants. Two genes identified from the cDNA-RAPD were OsSPY and 14-3-3, possibly associated with stem height variations of the semidwarf and spindly mutants, respectively. The OsSPY gene encoded the SPY protein which is considered to be a negative regulator of gibberellin (GA). On the other hand, the 14-3-3 encoded a signaling protein which can bind and prevent the RSG (repression of shoot growth) protein function as a transcriptional repressor of the kaurene oxidase (KO) gene in the GA biosynthetic pathway. Expression analysis of OsSPY, 14-3-3, RSG, KO, and SLR1 was confirmed in rice internode tissues during the reproductive stage of the plants by semi-quantitative RT-PCR technique. The expression analysis showed a clear increase of the levels of OsSPY transcripts in PKOS1 and HyKOS1 tissue samples compared to that of the KDML 105 and TKOS4 samples at the age of 50-60 days which were at the ages of internode elongation. The 14-3-3 expression had the highest increase in the TKOS4 samples compared to those in KDML 105, PKOS1 and HyKOS1 samples. The expression analysis of RSG and KO showed an increase in TKOS4 samples compared to that of the KDML 105 and that of the two semidwarf mutants. These results indicate that changes of OsSPY and 14-3-3 expression could affect internode elongation and cause the phenotypic changes of semidwarf and spindly rice mutants, respectively.

Molecular simulations of ultra-low-energy nitrogen ion bombardment of A-DNA in vacuum.

For investigating mechanisms involved in low-energy ion beam induced mutation, besides experiments using low-energy and low-fluence ions to bombard naked DNA, molecular simulations were carried out as an effort towards the insight in molecular interactions between ions and DNA. In the current study, Monte Carlo (MC) and molecular dynamics (MD) simulations were applied. The results of MC simulations provide some clues about the interaction energies and sites of preference of N-ion bombardment on an A-DNA short duplex strand. MD simulations of a single N-ion moving towards the same DNA strand with different linear velocities corresponding to bombardment energies of 0.1, 1, 10 and 100 eV revealed information about changes in bond lengths and visibly distorted structures of bombarded nucleotides. The simulations demonstrated that ion-bombardment-induced DNA change in structure was not a random but preferential effect.

Copro-diagnosis of Haplorchis taichui infection using sedimentation and PCR-based methods.

Fifty-one human fecal specimens were collected from villagers inhabiting along Mae Kuang River, Ban Sob Tha, Pa Sang District, Lamphun Province, Thailand. By the formalin-ether sedimentation technique (FEST) under a light microscope, eggs of 3 helminth species, Haplorchis taichui, Ascaris lumbricoides and unidentified hookworm species were detected with prevalences of 22, 14 and 4%, respectively. PCR amplification with H. taichui specific primers showed that H. taichui specific amplicon 260 bp was generated in all FEST-positive specimens, and also in some FEST negative specimens. This H. taichui specific PCR method can be used to detect this parasite in all developmental stages and in both definitive and intermediate hosts, which should be useful in prevention and control programs.

Haplorchis taichui, Witenberg, 1930: Development of a HAT-RAPD marker for the detection of minute intestinal fluke infection.

Specific primers to determine the presence of an intestinal fluke, Haplorchis taichui, were investigated using the high annealing temperature random amplified polymorphic DNA (HAT-RAPD) PCR, and 18 arbitrary primers (Operon Technologies), to generate different polymorphic DNA profiles. Thirteen kinds of parasites were used to compare fingerprints. A 256bp HAT-RAPD marker, generated from the OPP-11 primer, was found to be H. taichui-specific, and this marker was cloned, transformed, and sequenced. From the sequence data, a pair of primers were designed with Genetyx-MAC ver.11 and indicated as: Hap-t F 5'-GGC CAA CGC AAT CGT CAT CC-3' and Hap-t R 5'-GCG TCG GGT TTC AGA CAT GG-3'. These specific primers were tested for efficacy and specificity by amplifying them with all 13 parasites DNAs in PCR reaction. A 256bp amplicon was generated, which was shown to have a positive result, only for H. taichui DNA. It revealed no cross-reaction with any of the other tested parasite species. The minimum DNA template, needed for detection by PCR, was 0.1picogram (pg). The successful development of H. taichui-specific primers is expected to be beneficial for epidemiological studies and for prevention and control of these parasitic infections.

High annealing temperature-random amplified polymorphic DNA (HAT-RAPD) analysis of three paramphistome flukes from Thailand.

The rumen flukes of 37 cows (Bos indicus) from Chiang Mai and Lamphun provinces were investigated, and the overall prevalence of infection was 78.38% (29/37). Three species were found: Paramphistomum epiclitum, Orthocoelium streptocoelium, and Fischoederius elongatus with prevalences of infection of 75.68%, 48.65%, and 40.54%, respectively. Genomic DNA was amplified by polymerase chain reaction based on the high annealing temperature-random amplification of polymorphic DNA (HAT-RAPD) technique. Five random 10-mer oligonucleotide primers (OPA2, OPA4, OPB18, OPC9, and OPH11) produced distinct banding patterns in three species. No genetic variations in these three species were identified using 10 arbitary primers.

Dna quantities and qualities from various stages of some trematodes using optical and HAT-RAPD methods.

The aim of this experiment was to minimize DNA quantity and quality for detection by optical (spectrophotometer at 260 nm and 280 nm) and HAT-RAPD methods. Total DNA from different stages, adult, metacercaria and eggs of 6 trematode species were isolated for analysis. In this experiment, the adult trematodes were classified into 3 groups by size: small, Haplorchis taichui and Stellantchasmus falcatus; medium, Opisthorchis viverrini and Ganeo tigrinus; and large, Paramphistomum epiclitum and Fischoederius elongatus. The adult minimal DNA quantities and qualities of all specimen samples detected by optical method were 97.22,72.28, 3,167.00, 1,490.62, 21,382.66, and 27,321.77 ng; eggs were 3.92, 3.57, 3.72, 6.23, 17.53, and 14.01 ng, respectively; and metacercarial stages 50.70 and 40.98 ng in H. taichui and S. falcatus. In addition, the HAT-RAPD technique was chosen to amplify the minimal DNA qualities and quantities of all trematode specimens. Total DNA was 1-1 x 10(-12) ng; DNA templates in each dilution were used for amplification by primer OPA-09. DNA concentrations ranging between 1 x 10(-8) and 1 x 10(-11) ng were amplified with high polymorphism. Our experiment concluded that only a single specimen of each egg, metacercaria, or adult stage could be amplified with distinct bands.

Investigation of Stellantchasmus falcatus metacercariae in half-beaked fish, Dermogenus pusillus from four districts of Chiang Mai Province, Thailand.

Half-beaked fish, Dermogenus pusillus, collected from Mueang, Hang Dong, Doi Saket and Saraphi Districts, Chiang Mai Province, were examined for their infection status with Stellantchasmus falcatus (Digenea: Heterophyidae) metacercariae. The infection rate of the fish was 100%. Fish in three of four districts were found only to have metacercariae of S. falcatus, whereas those in Saraphi District had mixed infections with metacercariae of Posthodiplostomum sp. The intensity of S. falcatus infection per fish varied; 652-1,342 (mean 999.5), 562-2,422 (1,323.1), 185-2,492 (502.6), and 22-550 (210.4) in Mueang, Hang Dong, Saraphi, and Doi Saket Districts, respectively. The body portions of the fish with the heaviest metacercarial infection were the muscles, in all districts. The present study confirms that half-beaked fish in Chiang Mai Province are heavily infected with S. falcatus metacercariae.