Application of high-resolution melting for variant scanning in chloroplast gene atpB and atpB-rbcL intergenic spacer region of Crucifer species

High-resolution melting (HRM) analysis is a rapid and sensitive method for single nucleotide polymorphism (SNP) analysis. In this study, a novel HRM assay was carried out to detect SNPs in the chloroplast gene atpB which encodes the beta subunit of the ATP synthase and atpB upstream intergenic region. The polymorphisms of the two fragments in intertribal samples from the Cruciferae family and within the species of Brassica napus were detected. Based on this results, we found that HRM were able to determine over 90% of the variants which included single or multiple variants and insertion-deletion polymorphisms (INDELs) and rendered possible genotyping of more closely spaced polymorphisms, although there were several false positives (FPs) and misclassification. Six haplotypes were identified in the intertribal materials. The analysis of 90 B. napus found five variation types and the variations were all located in the intergenic region. In conclusion, HRM analysis is a closed tube assay that is easy to perform and is a more effective approach to identify variant of chloroplast genes. This study will facilitate further functional investigations into the role of chloroplast genes in photosynthesis, phylogeny and molecular evolution.Key words: atpB gene, chloroplast genome, crucifer, high-resolution melt curve analysis, SNP, INDEL

Cloning and characterization of peptidylprolyl isomerase B in the silkworm, Bombyx mori

Peptidylprolyl isomerases (PPIases) play essential roles in protein folding and are implicated in immune response and cell cycle control. Our previous proteomic analysis indicated that Bombyx mori PPIases may be involved in anti- Bombyx mori nucleopolyhedrovirus (BmNPV) response. To help investigate this mechanism, we cloned a B. mori PPIase gene PPIB and characterized it by bioinformatic and experimental analysis. We found that the B. mori PPIB gene contains 4 exons and its cDNA is about of618 bp, encoding a protein of 205 amino acid residues (21474.41 Da) with an isoelectric point of 8.05. PPIB contains conserved and unique cyclophilin domain and belongs to cyclophilin superfamily. Its transcription could be detected by PCR in all the B. mori tissue samples, which is consistent withnormal PPIase expression pattern and their essential roles. It is localized in cytoplasm revealed by fluorescence microscopy. We also successfully expressed this protein in E. coli and characterized it by SDS-PAGE and Mass Spectrometry. The cloned DNA sequence was submitted to GenBank (EU583493)

Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo.

Spores of some species of the strictly anaerobic bacteria Clostridium naturally target and partially lyse the hypoxic cores of tumors, which tend to be refractory to conventional therapies. The anti-tumor effect can be augmented by engineering strains to convert a non-toxic prodrug into a cytotoxic drug specifically at the tumor site by expressing a prodrug-converting enzyme (PCE). Safe doses of the favored prodrug CB1954 lead to peak concentrations of 6.3 μM in patient sera, but at these concentration(s) known nitroreductase (NTR) PCEs for this prodrug show low activity. Furthermore, efficacious and safe Clostridium strains that stably express a PCE have not been reported. Here we identify a novel nitroreductase from Neisseria meningitidis, NmeNTR, which is able to activate CB1954 at clinically-achievable serum concentrations. An NmeNTR expression cassette, which does not contain an antibiotic resistance marker, was stably localized to the chromosome of Clostridium sporogenes using a new integration method, and the strain was disabled for safety and containment by making it a uracil auxotroph. The efficacy of Clostridium-Directed Enzyme Prodrug Therapy (CDEPT) using this system was demonstrated in a mouse xenograft model of human colon carcinoma. Substantial tumor suppression was achieved, and several animals were cured. These encouraging data suggest that the novel enzyme and strain engineering approach represent a promising platform for the clinical development of CDEPT

Antiferromagnetic fluctuations in the normal state of LiFeAs

We present a detailed study of 75As NMR Knight shift and spin-lattice relaxation rate in the normal state of stoichiometric polycrystalline LiFeAs. Our analysis of the Korringa relation suggests that LiFeAs exhibits strong antiferromagnetic fluctuations, if transferred hyperfine coupling is a dominant interaction between 75As nuclei and Fe electronic spins, whereas for an on-site hyperfine coupling scenario, these are weaker, but still present to account for our experimental observations. Density-functional calculations of electric field gradient correctly reproduce the experimental values for both 75As and 7Li sites.Comment: 5 pages, 3 figures, thoroughly revised version with refined experimental data, accepted for publication as a Rapid Communication in Physical Review B

Modulation of cellular redox homeostasis by the endocannabinoid system

The endocannabinoid system (ECS) and reactive oxygen species (ROS) constitute two key cellular signalling systems that participate in the modulation of diverse cellular functions. Importantly, growing evidence suggests that cross-talk between these two prominent signalling systems acts to modulate functionality of the ECS as well as redox homeostasis in different cell types. Herein, we review and discuss evidence pertaining to ECS-induced regulation of ROS generating and scavenging mechanisms, as well as highlighting emerging work that supports redox modulation of ECS function. Functionally, the studies outlined reveal that interactions between the ECS and ROS signalling systems can be both stimulatory and inhibitory in nature, depending on cell stimulus, the source of ROS species and cell context. Importantly, such cross-talk may act to maintain cell function, whereas abnormalities in either system may propagate and undermine the stability of both systems, thereby contributing to various pathologies associated with their dysregulation

Regulation and Regulatory Role of WNT Signaling in Potentiating FSH Action during Bovine Dominant Follicle Selection

Follicular development occurs in wave like patterns in monotocous species such as cattle and humans and is regulated by a complex interaction of gonadotropins with local intrafollicular regulatory molecules. To further elucidate potential mechanisms controlling dominant follicle selection, granulosa cell RNA harvested from F1 (largest) and F2 (second largest) follicles isolated at predeviation (PD) and onset of diameter deviation (OD) stages of the first follicular wave was subjected to preliminary RNA transcriptome analysis. Expression of numerous WNT system components was observed. Hence experiments were performed to test the hypothesis that WNT signaling modulates FSH action on granulosa cells during follicular waves. Abundance of mRNA for WNT pathway members was evaluated in granulosa cells harvested from follicles at emergence (EM), PD, OD and early dominance (ED) stages of the first follicular wave. In F1 follicles, abundance of CTNNB1 and DVL1 mRNAs was higher and AXIN2 mRNA was lower at ED versus EM stages and DVL1 and FZD6 mRNAs were higher and AXIN2mRNA was lower in F1 versus F2 follicle at the ED stage. Bovine granulosa cells were treated in vitro with increasing doses of the WNT inhibitor IWR-1+/− maximal stimulatory dose of FSH. IWR-1 treatment blocked the FSH-induced increase in granulosa cell numbers and reduced the FSH-induced increase in estradiol. Granulosa cells were also cultured in the presence or absence of FSH +/− IWR-1 and hormonal regulation of mRNA for WNT pathway members and known FSH targets determined. FSH treatment increased CYP19A1, CCND2, CTNNB1, AXIN2and FZD6 mRNAs and the stimulatory effect on CYP19A1 mRNA was reduced by IWR-1. In contrast, FSH reduced CARTPT mRNA and IWR-1 partially reversed the inhibitory effect of FSH. Results support temporal and hormonal regulation and a potential role for WNT signaling in potentiating FSH action during dominant follicle selection

A study on experiment and numerical simulation of heat exchanger in heating furnace

In this paper, air preheater is used the research object and its heat transfer law is studied by experiment and numerical simulation. The experimental data showed that with the increases of inlet air velocity, the comprehensive heat transfer coefficient and heat transfer efficiency increase, but the temperature efficiency decreases and the resistance loss on the air side increases. The numerical simulation results showed that the larger the diameter of the tube, the better the heat transfer effect. When horizontal spacing in the range of 290 - 305 mm and longitudinal spacing is 70 - 90 mm, the heat transfer effect is best. The optimized heat exchanger structure is that diameter is 60 mm, horizontal spacing is 300 mm, longitudinal spacing is 90 mm. As the inlet air flow rate increases, the heat transfer efficiency increases, but the temperature efficiency decreases and the resistance loss on the air side increases