Use of redundant exclusion PCR to identify a novel Bacillus thuringiensis Cry8 toxin gene from pooled genomic DNA

With the aim of optimizing the cloning of novel genes from a genomic pool containing many previously identified, homologous, genes we designed a redundant exclusion PCR technique. In RE-PCR a pair of generic amplification primers are combined with additional primers that are designed to specifically bind to redundant, unwanted genes that are a subset of those copied by the amplification primers. During RE-PCR the specific primer blocks amplification of the full length redundant gene. Using this method we managed to clone a number of cry8 or cry9 toxin genes from a pool of Bacillus thuringiensis genomic DNA while excluding amplicons for cry9Da, cry9Ea and cry9Eb. The method proved very efficient at increasing the number of rare genes in the resulting library. One such rare, and novel, cry8-like gene was expressed and the encoded toxin was shown to be toxic to Anomola corpulenta

TMFFA Two-Bead Multipole Force Field for Coarse-Grained Molecular Dynamics Simulation of Protein

Coarse-grained (CG) models are desirable for studying large and complex biological systems. In this paper, we propose a new two-bead multipole force field (TMFF) in which electric multipoles up to the quadrupole are included in the CG force field. The inclusion of electric multipoles in the proposed CG force field enables a more realistic description of the anisotropic electrostatic interactions in the protein system and, thus, provides an improvement over the standard isotropic two-bead CG models. In order to test the accuracy of the new CG force field model, extensive molecular dynamics simulations were carried out for a series of benchmark protein systems. These simulation studies showed that the TMFF model can realistically reproduce the structural and dynamical properties of proteins, as demonstrated by the close agreement of the CG results with those from the corresponding all-atom simulations in terms of root-mean-square deviations (RMSDs) and root-mean-square fluctuations (RMSFs) of the protein backbones. The current two-bead model is highly coarse-grained and is 50-fold more efficient than all-atom method in MD simulation of proteins in explicit water

Hepatitis E Virus Induces Hepatocyte Apoptosis via Mitochondrial Pathway in Mongolian Gerbils

Previous studies demonstrated that Mongolian gerbils can be infected by hepatitis E virus (HEV), which induces the hepatic injury. Here, the mitochondria in hepatocytes from HEV-infected gerbils were considerably swollen, thin cristae. After HEV infection, the activity of superoxide dismutase significantly decreased (p < 0.01), while malondialdehyde concentrations significantly increased, compared with those in the control group (p < 0.01). Adenosine triphosphatase levels decreased significantly in the hepatocyte of the inoculated groups, compared with those in control group (p < 0.05) at days 21, 28, 42 post-inoculation (dpi) as well. Furthermore, the levels of ATP synthetase ATP5A1 significantly decreased during HEV infection, compared with those in the control group (p < 0.05). According to the TdT mediated dUTP nick end labeling (TUNEL) detection, TUNEL positive hepatocytes increased in the inoculated group, compared with that in the control group (p < 0.05). Up-regulation of the mitochondrion-mediated apoptosis regulating proteins, Bax and Bcl-2, in the HEV-infected gerbils (p < 0.05) was observed. However, cytochrome c levels in mitochondria decreased, while this molecule was detected in the cytoplasm of the infected animals, in contrast to that in the control group. Apaf-1, and active caspase-9 and -3 levels were shown to be significantly higher in the inoculated group compared with those in the control group (p < 0.05). Taken together, our results demonstrated that HEV infection induces hepatocyte injuries and activity of the mitochondrial apoptotic pathway, which trigger the hepatocyte apoptosis in Mongolian gerbils

Physiological Evaluation of Alkali-Salt Tolerance of Thirty Switchgrass (Panicum virgatum) Lines.

Soil salt-alkalization is a major limiting factor for crop production in many regions. Switchgrass (Panicum virgatum L.) is a warm-season C4 perennial rhizomatous bunchgrass and a target lignocellulosic biofuel species. The objective of this study was to evaluate relative alkali-salt tolerance among 30 switchgrass lines. Tillers of each switchgrass line were transplanted into pots filled with fine sand. Two months after transplanting, plants at E5 developmental stage were grown in either half strength Hoagland's nutrient solution with 0 mM Na+ (control) or half strength Hoagland's nutrient solution with 150 mM Na+ and pH of 9.5 (alkali-salt stress treatment) for 20 d. Alkali-salt stress damaged cell membranes [higher electrolyte leakage (EL)], reduced leaf relative water content (RWC), net photosynthetic rate (Pn), stomatal conductance (gs), and transpiration rate (Tr). An alkali-salt stress tolerance trait index (ASTTI) for each parameter was calculated based on the ratio of the value under alkali-salt stress and the value under non-stress conditions for each parameter of each line. Relative alkali-salt tolerance was determined based on principal components analysis and cluster analysis of the physiological parameters and their ASTTI values. Significant differences in alkali-salt stress tolerance were found among the 30 lines. Lowland lines TEM-SEC, Alamo, TEM-SLC and Kanlow were classified as alkali-salt tolerant. In contrast, three lowland lines (AM-314/MS-155, BN-13645-64) and two upland lines (Caddo and Blackwell-1) were classified as alkali-salt sensitive. The results suggest wide variations exist in alkali-salt stress tolerance among the 30 switchgrass lines. The approach of using a combination of principal components and cluster analysis of the physiological parameters and related ASTTI is feasible for evaluating alkali-salt tolerance in switchgrass