Survey of entomopathogenic nematodes and associate bacteria in Thailand and their potential to control Aedes aegypti

Aedes aegypti is an insect vector that transmits several viruses affecting humans worldwide. Entomopathogenic nematodes (EPNs) and their symbiotic bacteria are organisms with the potential to control many insects. In this study, we did a survey aimed to identify EPNs and their symbiotic bacteria and evaluate the larvicidal activity of bacteria against Ae. aegypti. We collected 540 soil samples from 108 sites in Phitsanulok Province, lower northern Thailand. Baiting techniques and White traps were used to isolate EPNs from soil samples. By sequencing of 28S rDNA and internal transcribed spacer regions, 51 EPN isolates were identified as Steinernema surkhetense (35 isolates), Heterorhabditis indica (14 isolates) and Heterorhabditis sp. SGmg3 (two isolates). Based on sequencing of a partial region of the recA gene, 35 isolates of Xenorhabdus were identified as Xenorhabdus stockiae, and 20 Photorhabdus isolates were identified as Photorhabdus luminescens subsp. akhurstii (10 isolates), P. luminescens subsp. hainanensis (seven isolates) and P. asymbiotica subsp. australis (three isolates). Screening for larvicidal activity of bacteria against Ae. aegypti was performed in the laboratory. Xenorhabdus WB5.4 and Xenorhabdus WB12.5, which were closely related to X. stockiae, resulted in high mortality of Ae. aegypti (99.99% and 70%, respectively) at 96 hr after exposure. Comparing with control groups, mortality of Ae. aegypti larvae was low (1.11%–6.67%) after exposure for 24–96 hr. Our findings showed the potential of X. stockiae for controlling Ae. aegypti. Further studies are needed to elucidate the mechanisms through which these bacteria kill Ae. aegypti larvae.     

Construction and Physiological Analysis of a Xanthomonas Mutant To Examine the Role of the oxyR Gene in Oxidant-Induced Protection against Peroxide Killing

We constructed and characterized a Xanthomonas campestris pv. phaseoli oxyR mutant. The mutant was hypersensitive to H₂O₂ and menadione killing and had reduced aerobic plating efficiency. The oxidants’ induction of the catalase and ahpC genes was also abolished in the mutant. Analysis of the adaptive responses showed that hydrogen peroxide-induced protection against hydrogen peroxide was lost, while menadione-induced protection against hydrogen peroxide was retained in the oxyR mutant. These results show that X. campestris pv. phaseoli oxyR is essential to peroxide adaptation and revealed the existence of a novel superoxide-inducible peroxide protection system that is independent of OxyR.     

Mass transfer of terpenes through a silicone rubber membrane in a liquid-liquid contacting system

Terpenoids are important compounds for the fragrance industry, and recently, biocatalytic methods have been developed to produce them from cheap monoterpenes, such as alpha-pinene oxide. The biotransformation of alpha-pinene oxide using resting cells of Pseudomonas fluorescens NCIMB 11671 produces isonovalal (cis-2-methyl-5-isopropylhexa-2,5-dienal), which is a fragrance. However, this biotransformation has technical problems including the following: alpha-pinene oxide undergoes autoxidation in water and light; it is hydrophobic and relatively toxic to the biocatalyst; and it suffers from product inhibition. Therefore, removal of isonovalal as it is formed should reduce its toxicity and increase volumetric productivity and production yield. Aqueous-organic two-phase (AOTP) systems can be used in the biotransformation of hydrophobic substrates and can protect biocatalysts from toxic substrates and products. However, the formation of stable emulsions makes further downstream processing and continuous operation difficult. One solution to these problems is to use a solid-phase membrane between the aqueous and organic phases in a Membrane Bioreactor for Biotransformation (MBB). Since there are no data in the literature on the behavior of terpenes and terpenoids with solid-phase membranes, or their mass transfer behavior, the objective of this work was to measure these parameters for a wide range of compounds so as to be able to design an MBB, and to gain a greater understanding of their behavior in these types of systems. Organic/aqueous (P (org) (aq)) and membrane/aqueous (P (mem) (aq)) partition coefficients were measured first, and subsequently used to quantify the overall mass transfer coefficients (k(ov)). The overall mass transfer coefficient (k(ov)) of alpha-pinene oxide through the membrane was found to be 2.5 x 10(-5) m x s(-1) using thicknesses of both 250 and 1,000 microm. Extraction kinetics were successfully described using a resistance-in-series model and were controlled by the aqueous boundary layer and/or membrane resistance (k(mem)), while organic film resistance played an insignificant role. Aqueous film resistance (k(aq)) was found to be the limiting step for mono- and diterpenes, and the effect of the hydrodynamics on k(ov) was successfully predicted using a Wilson plot. However, the extraction kinetics of larger terpenes, such as steroids, were influenced solely by the k(mem), suggesting that membrane diffusivity also depended on the size of the permeating molecule. Finally, the influence of other terpene byproducts on the flux of alpha-pinene oxide was investigated and found to decrease the flux into the organic phase by up to 10%.     

Outward migration of Gnathostoma spinigerum in interferon alpha treated hepatitis C patient

After the first dose injection of pegylated interferon alpha-2b (Peg-IFN alpha-2b) to a HCV infected Thai woman, she developed cyclic painful swelling nodules on right upper quadrant of abdomen and right anterior lower chest wall. The nodules subsided spontaneously within 1-2 days but were recurrent after every Peg-IFN alpha-2b injection. She also experienced acute urticaria. After nine months of therapy, an immature male of G. spinigerum migrated out from the skin nodule shortly after a Peg-IFN alpha-2b injection as scheduled. The worm showed a head-bulb bearing 8 transverse rows of spines which indicated immature stage. It had well defined four pairs of caudal papillae on posterior body part which were used to identify male gender. Painful migratory swelling and urticaria disappeared after the parasite was removed. She was continually treated and had sustained both virological and biochemical responses to HCV treatment. This case demonstrates that the outward migration of G. spinigerum may be stimulated by the injection of Peg-IFN alpha-2b.     

Characterizing Protein Interactions Employing a Genome-Wide siRNA Cellular Phenotyping Screen

Characterizing the activating and inhibiting effect of protein-protein interactions (PPI) is fundamental to gain insight into the complex signaling system of a human cell. A plethora of methods has been suggested to infer PPI from data on a large scale, but none of them is able to characterize the effect of this interaction. Here, we present a novel computational development that employs mitotic phenotypes of a genome-wide RNAi knockdown screen and enables identifying the activating and inhibiting effects of PPIs. Exemplarily, we applied our technique to a knockdown screen of HeLa cells cultivated at standard conditions. Using a machine learning approach, we obtained high accuracy (82% AUC of the receiver operating characteristics) by cross-validation using 6,870 known activating and inhibiting PPIs as gold standard. We predicted de novo unknown activating and inhibiting effects for 1,954 PPIs in HeLa cells covering the ten major signaling pathways of the Kyoto Encyclopedia of Genes and Genomes, and made these predictions publicly available in a database. We finally demonstrate that the predicted effects can be used to cluster knockdown genes of similar biological processes in coherent subgroups. The characterization of the activating or inhibiting effect of individual PPIs opens up new perspectives for the interpretation of large datasets of PPIs and thus considerably increases the value of PPIs as an integrated resource for studying the detailed function of signaling pathways of the cellular system of interest.     

Purification of l-(+)-lactic acid from pre-treated fermentation broth using vapor permeation-assisted esterification

The vapor permeation-assisted esterification of l-(+)-lactic acid and ethanol was investigated using a zeolite membrane. Pervaporation and vapor permeation were initially investigated for dehydration performances, and the latter showed much better results. The molecular sieve property of the membrane resulted in a high separation factor of over 1000 for all conditions. The maximum flux was 10.24 kg/(m² h) at a feed temperature of 145 °C, a water feed concentration of 10 wt%, and a feed pressure of 4.0 bars, respectively. For vapor permeation-assisted esterification using synthetic solutions, the productivity and ethyl lactate yield strongly depended on the dehydration rate. Realistic purifications were performed with fermentation broths of Pediococcus pentosaceus as the lactic acid producer. Experimental results revealed that most of the lactic acid was converted into ethyl lactate at the final stage of the reaction. After distillation and hydrolysis, high purity l-(+)-lactic acid was obtained with more than 95% recovery yields.     

Accumulation of Calcium and Phosphorus in the Coronary Arteries of Thai Subjects

To clarify the manner of accumulation of Ca and P in the coronary arteries, the authors divided the coronary arteries into many segments based on arterial ramification and investigated the element contents of the segments by direct chemical analysis. After ordinary dissection at Chiang Mai University was finished, the left coronary (LC) and the right coronary (RC) arteries were removed successively from the hearts of Thai subjects. The Thai subjects consisted of seven men and five women, ranging in age from 42 to 87 years (average age = 73.9 ± 13.5 years). The LC and the RC arteries were divided into 19 segments based on arterial ramification. After incineration with nitric acid and perchloric acid, element contents of the segments were analyzed by inductively coupled plasma–atomic emission spectrometry. In two cases, a significant content of Ca and P was contained only in the left anterior descending (LAD) artery (type I). In four cases, a significant content of Ca and P was contained in both the LAD and the RC arteries (type II). In five cases, a significant content of Ca and P was contained in all the LAD, the RC, and the circumflex (CF) arteries (type III). In the other one case, no significant content of Ca and P was contained in the coronary arteries. The manner of accumulation of Ca and P in the coronary arteries was classified into the three types, I, II, and III. Regarding the average content of elements in 12 cases, the average content of Ca was the highest in the segment of the LAD artery ramifying the first left diagonal artery and was higher in the proximal and distal adjacent segments of the LAD artery ramifying the first left diagonal artery, the proximal segment of the RC artery, and the proximal segment of the CF artery. To examine an effect of arterial ramification on accumulation of Ca and P, the differences in the Ca and P content between artery-ramifying and non-ramified proximal or distal segments of the coronary arteries were analyzed with Student’s t test. It was found that there were no significant differences in the Ca and P content between the artery-ramifying and non-ramified proximal or distal segments of the coronary arteries.     

Gender Differences in Elements of Human Anterior Commissure and Olfactory Bulb and Tract

To examine whether there were gender differences in the various brain regions, the authors investigated the gender differences in seven element contents of the anterior commissure, mammillary body, and olfactory bulb and tract by direct chemical analysis. After ordinary dissection at Nara Medical University was finished, the anterior commissures, mammillary bodies, and olfactory bulbs and tracts were resected from the cerebra cut at median line. The brain samples were treated with 99.5% ethanol three times to remove lipids. After ashing with nitric acid and perchloric acid, the seven element contents Ca, P, S, Mg, Zn, Fe, and Na were determined by inductively coupled plasma–atomic emission spectrometry. It was found that the Zn content was significantly higher in the anterior commissures of men than in those of women. In the olfactory bulbs and tracts, it was found that the Ca, P, and Zn contents were significantly higher in men than in women. In contrast, no significant difference was found between the mammillary bodies of men and women regarding the seven element contents.     

Age-dependent increase of magnesium in the cerebral arteries of Thai

To elucidate compositional changes of the cerebral arteries with aging, the authors investigated age-related changes of elements in the cerebral arteries by inductively coupled plasma-atomic emission spectrometry (ICP-AES). After an ordinary dissection by medical students at Chiang Mai University was finished, the anterior, middle, and posterior cerebral arteries and the anterior and posterior communicating arteries were resected from the subjects. The subjects consisted of six men and seven women, ranging in age from 34 to 86 yr. The element content was determined by ICP-AES. It was found that the Mg content increased progressively with aging in all of the anterior, middle, and posterior cerebral arteries, but the Ca and P content did not increase significantly with aging in the anterior, middle, and posterior cerebral arteries, with the one exception being that the P content increased significantly in the posterior cerebral arteries with aging. The average content of Ca was higher in the order of the anterior communicating, anterior cerebral, posterior communicating, posterior cerebral, and middle cerebral arteries.     

Relationships Among the Hippocampus,Dentate Gyrus,Mammillary Body,Fornix, and Anterior Commissure from a Viewpoint of Elements

To elucidate the relationships among the brain regions belonging to the limbic system, the authors investigated the relationships among the hippocampus, dentate gyrus, mammillary body, and fornix, using the anterior commissure as a control, from a viewpoint of elements. After ordinary dissections at Nara Medical University were finished, the hippocampi, dentate gyri, mammillary bodies, fornices, and anterior commissures were resected from identical cerebra of the subjects. The subjects consisted of 23 men and 23 women, ranging in age from 70 to 101 years (average age = 83.5 ± 7.5 years). After ashing with nitric acid and perchloric acid, element contents were determined by inductively coupled plasma-atomic emission spectrometry. With regard to seven elements of Ca, P, S, Mg, Zn, Fe, and Na, it was examined whether there were significant correlations among the hippocampus, dentate gyrus, mammillary body, fornix, and anterior commissure. It was found that there were extremely or very significant direct correlations among all of the five brain regions of the hippocampus, dentate gyrus, mammillary body, fornix, and anterior commissure in the P content. Likewise, with regard to the Fe content, there were significant direct correlations among the four brain regions belonging to the limbic system, except for the anterior commissure. In both the Ca and Zn contents, there were extremely or very significant direct correlations among the hippocampus, dentate gyrus, and mammillary body of the gray matter.