Phytoremediation of fuel oil and lead co-contaminated soil by Chromolaena odorata in association with Micrococcus luteus

Phytoremediation is widely promoted as a cost-effective technology for treating heavy metal and total petroleum hydrocarbon (TPH) co-contaminated soil. This study investigated the concurrent removal of TPHs and Pb in co-contaminated soil (27,000 mg kg^?1 TPHs, 780 mg kg^?1 Pb) by growing Siam weed (Chromolaena odorata) in a pot experiment for 90 days. There were four treatments: co-contaminated soil; co-contaminated soil with C. odorata only; co-contaminated soil with C. odorata and Micrococcus luteus inoculum; and co-contaminated soil with M. luteus only. C. odorata survived and grew well in the co-contaminated soil. C. odorata with M. luteus showed the highest Pb accumulation (513.7 mg kg^?1) and uptake (7.7 mg plant^?1), and the highest reduction percentage of TPHs (52.2%). The higher TPH degradation in vegetated soils indicated the interaction between the rhizosphere microorganisms and plants. The results suggested that C. odorata together with M. luteus and other rhizosphere microorganisms is a promising candidate for the removal of Pb and TPHs in co-contaminated soils.     

Evaluation of the impact of shielding materials in radiation protection in transgenic animals

We are using a plasmid-based transgenic mouse mutation model system to evaluate the effectiveness of aluminum or low-density polyethylene (LDPE) shielding after 250 MeV/u protons or 1 GeV/u iron ion irradiation. Transgenic mice, with multiple copies of the plasmid pUR288 lacZ transgene integrated into the genome of every cell of the animal, were either irradiated or sham-treated. Multiple endpoints, including early cytogenetic damage in erythrocytes at 48 h after exposure, chromosome aberrations in bone marrow lymphocytes, and lacZ mutant frequencies (MF) in brain and spleen tissues were measured in the same animals. The frequency of total circulating reticulocytes (fRET) dropped precipitously at 48 h after 2 Gy of proton irradiation. The average level of micronucleated reticulocytes (fMN–RET) was fivefold higher in the irradiated samples relative to the controls at the same time point. There was an increase in total chromosome aberrations in bone marrow lymphocytes at 8 weeks after proton irradiation but this increase was not statistically significant relative to the controls. Evaluation of the lacZ MF in the brain and spleen tissues showed that proton irradiation induced a twofold increase in MF in each tissue. Similar samples were collected from animals that were shielded from the proton beam by aluminum. Compared to the unshielded treatment group, we noted no difference in fRET, fMN–RET, chromosome aberrations in lymphocytes and lacZ MF in brain and spleen tissues obtained from these animals. In a separate study, animals were exposed to high-energy iron ions with or without 10 or 15 cm LDPE. Using the same approach, we noted a precipitous drop in fRET, and an elevation in fMN–RET within 48 h after 1 Gy of iron ions. Total chromosome aberrations in bone marrow lymphocytes were slightly elevated but not significant at 8 weeks after iron ion exposure. Shielding animals with 10 or 15 cm of polyethylene appeared to have no effect on the level of RET, MN–RET or chromosome aberrations in these animals. LacZ MF in brain and spleen tissues increased 1.5–2-fold above control levels after 1 Gy iron ions at 8 weeks after treatment. On the other hand, MF in tissues harvested from shielded animals appeared to be lower than their unshielded litermates, suggesting the polyethylene shielding was effective in reducing the iron-induced genomic damage in tissues. Although shielding may be effective, in some cases, in reducing the physical dose of particle radiation, our cytogenetic results showed that the biological impact of the particle beam remain unchanged. On the other hand, reduction in transgene MF in tissues from LDPE-shielded animals but not in the aluminum-shielded animals strongly suggests that careful consideration of the biological endpoints used is necessary in the evaluation of the efficacy of the selected shielding material.     

Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba delta-endotoxin complex revealed by electron crystallography: implications for toxin-pore formation

The insecticidal nature of Cry delta-endotoxins produced by Bacillus thuringiensis is generally believed to be caused by their ability to form lytic pores in the midgut cell membrane of susceptible insect larvae. Here we have analyzed membrane-associated structures of the 65-kDa dipteran-active Cry4Ba toxin by electron crystallography. The membrane-associated toxin complex was crystallized in the presence of DMPC via detergent dialysis. Depending upon the charge of the adsorbed surface, 2D crystals of the oligomeric toxin complex have been captured in two distinct conformations. The projection maps of those crystals have been generated at 17A resolution. Both complexes appeared to be trimeric; as in one crystal form, its projection structure revealed a symmetrical pinwheel-like shape with virtually no depression in the middle of the complex. The other form revealed a propeller-like conformation displaying an obvious hole in the center region, presumably representing the toxin-induced pore. These crystallographic data thus demonstrate for the first time that the 65-kDa activated Cry4Ba toxin in association with lipid membranes could exist in at least two different trimeric conformations, conceivably implying the closed and open states of the pore.     

Streptavidin crystals as nanostructured supports and image-calibration references for cryo-EM data collection

For cryo-EM structural studies, we seek to image membrane proteins as single particles embedded in proteoliposomes. One technical difficulty has been the low density of liposomes that can be trapped in the approximately 100nm ice layer that spans holes in the perforated carbon support film of EM grids. Inspired by the use of two-dimensional (2D) streptavidin crystals as an affinity surface for biotinylated DNA (Crucifix et al., 2004), we propose to use the crystals to tether liposomes doped with biotinylated lipids. The 2D crystal image also serves as a calibration of the image formation process, providing an absolute conversion from electrostatic potentials in the specimen to the EM image intensity, and serving as a quality control of acquired cryo-EM images. We were able to grow streptavidin crystals covering more than 90% of the holes in an EM grid, and which remained stable even under negative stain. The liposome density in the resulting cryo-EM sample was uniform and high due to the high-affinity binding of biotin to streptavidin. Using computational methods, the 2D crystal background can be removed from images without noticeable effect on image properties.     

Oleic acid Enhances Dengue Virus But Not Dengue Virus-Like Particle Production from Mammalian Cells

Despite the recent introduction of a commercial vaccine, the mosquito-transmitted dengue virus is still a worldwide public health problem. Based on the live attenuated vaccine strategy, the commercial vaccine has a less than optimal protective profile. Virus-like particles (VLPs) offer an attractive alternate vaccination strategy due to the effectively native presentation of epitopes in the absence of any infectious genetic material. However, the production of amounts of VLP in a platform that can support commercial development remains a major obstacle. This study generated two DENV 2 VLPs [codon-optimized and chimeric DENV/Japanese encephalitis virus (JEV)] and directly compared yields of these constructs by western blotting and dot blot hybridization. The effect of oleic acid supplementation, a process known to increase DENV production in natural infection, was also investigated. Results showed that the chimeric construct gave a two- to threefold higher yield than the codon-optimized construct and that while oleic acid increased DENV virion production in natural infection, it inhibited VLP production. These results suggest that further optimization of DENV VLP expression is possible, but it will require more understanding of how native DENV infection remodels the host cell machinery.     

Bacillus thuringiensis Cry4A and Cry4B mosquito-larvicidal proteins: homology-based 3D model and implications for toxin activity

Three-dimensional (3D) models for the 65-kDa activated Cry4A and Cry4B delta-endotoxins from Bacillus thuringiensis subsp. israelensis that are specifically toxic to mosquito-larvae were constructed by homology modeling, based on atomic coordinates of the Cry1Aa and Cry3Aa crystal structures. They were structurally similar to the known structures, both derived 3D models displayed a three-domain organization: the N-terminal domain (I) is a seven-helix bundle, while the middle and C-terminal domains are primarily comprise of anti-parallel beta-sheets. Circular dichroism spectroscopy confirmed the secondary structural contents of the two homology-based Cry4 structures. A structural analysis of both Cry4 models revealed the following: (a) Residues Arg-235 and Arg-203 are located in the interhelical 5/6 loop within the domain I of Cry4A and Cry4B, respectively. Both are solvent exposed. This suggests that they are susceptible to tryptic cleavage. (b) The unique disulphide bond, together with a proline-rich region within the long loop connecting alpha4 and alpha5 of Cry4A, were identified. This implies their functional significance for membrane insertion. (c) Significant structural differences between both models were found within domain II that may reflect their different activity spectra. Structural insights from this molecular modeling study would therefore increase our understanding of the mechanic aspects of these two closely related mosquito-larvicidal proteins.     

Refining DNA Barcoding Coupled High Resolution Melting for Discrimination of 12 Closely Related Croton Species

DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Croton (Euphorbiaceae), one of the largest genera of plants with over 1,200 species. Seven primer pairs were evaluated (matK, rbcL1, rbcL2, rbcL3, rpoC, trnL and ITS1) from four plastid regions, matK, rbcL, rpoC, and trnL, and the nuclear ribosomal marker ITS1. The primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL1 primer pair gave the lowest resolution. It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Our Bar-HRM results here also provide further support for the hypothesis that both sequence and base composition affect DNA duplex stability.     

A gaze bias with coarse spatial indexing during a gambling task

Researchers have used eye-tracking methods to infer cognitive processes during decision making in choice tasks involving visual materials. Gaze likelihood analysis has shown a cascading effect, suggestive of a causal role for the gaze in preference formation during evaluative decision making. According to the gaze bias hypothesis, the gaze serves to build commitment gradually towards a choice. Here, we applied gaze likelihood analysis in a two-choice version of the well-known Iowa Gambling Task. This task requires active learning of the value of different choice options. As such, it does not involve visual preference formation, but choice optimization through learning. In Experiment 1 we asked subjects to choose between two decks with different payoff structures, and to give their responses using mouse clicks. Two groups of subjects were exposed to stable versus varying outcome contingencies. The analysis revealed a pronounced gaze bias towards the chosen stimuli in both groups of subjects, plateauing at more than 400 ms before the choice. The early plateauing suggested that the gaze effect partially reflected eye-hand coordination. In Experiment 2 we asked subjects to give responses using a key press. The results again showed a clear gaze bias towards the chosen deck, this time without any influence from eye-hand coordination. In both experiments, there was a clear gaze bias towards the choice even though the gaze fixations did not narrowly focus on the spatial positions of choice options. Taken together, the data suggested a role for gaze in coarse spatial indexing during non-perceptual decision making.