Upgrading of high-boiling fraction of bio-oil in supercritical methanol

In this work, the upgrading reactions of high-boiling fraction (HBF) of bio-oil were carried out over a series of supported mono- and bi-metallic catalysts under the supercritical methanol condition. During these reactions, esterification and cracking (alcoholysis and hydrocracking) were the two dominant processes. PtNi/MgO exhibited good performance, and gave a high yield (72.4 wt.%) of refined oil. The acid-base properties of the supports have an important effect on the coke deposition on the catalyst surface. The acidic catalysts gave the somewhat lower product yields, but tended to inhibit coking reaction. This would improve the life of the catalysts in the practical applications. The refined oil is believed to be a potential substitute or partial substitute for the fossil transportation fuel.     

Single amino acid changes in the virus capsid permit coxsackievirus B3 to bind decay-accelerating factor

Many coxsackievirus B isolates bind to human decay-accelerating factor (DAF) as well as to the coxsackievirus and adenovirus receptor (CAR). The first-described DAF-binding isolate, coxsackievirus B3 (CB3)-RD, was obtained during passage of the prototype strain CB3-Nancy on RD cells, which express DAF but very little CAR. CB3-RD binds to human DAF, whereas CB3-Nancy does not. To determine the molecular basis for the specific interaction of CB3-RD with DAF, we produced cDNA clones encoding both CB3-RD and CB3-Nancy and mutated each of the sites at which the RD and Nancy sequences diverged. We found that a single amino acid change, the replacement of a glutamate within VP3 (VP3-234E) with a glutamine residue (Q), conferred upon CB3-Nancy the capacity to bind DAF and to infect RD cells. Readaptation of molecularly cloned CB3-Nancy to RD cells selected for a new virus with the same VP3-234Q residue. In experiments with CB3-H3, another virus isolate that does not bind measurably to DAF, adaptation to RD cells resulted in a DAF-binding isolate with a single amino acid change within VP2 (VP2-138 N to D). Both VP3-234Q and VP2-138D were required for binding of CB3-RD to DAF. In the structure of the CB3-RD-DAF complex determined by cryo-electron microscopy, both VP3-234Q and VP2-138D are located at the contact site between the virus and DAF.     

Metabolic engineering of isoflavone genistein in Brassica napus with soybean isoflavone synthase

Genistein, 4′,5,7-trihydroxyisoflavone, is an isoflavonoid compound predominantly restricted to legumes and known to possess phyto-oestrogenic and antioxidative activities. The key enzyme that redirects phenylpropanoid pathway intermediates from flavonoids to isoflavonoids is the isoflavone synthase (IFS). Brassica napus is a non-legume oilseed crop with vegetative tissues producing phenylpropanoids and flavonoids, but does not naturally accumulate isoflavones due to the absence of IFS. To demonstrate whether exogenous IFS is able to use endogenous substrate to produce isoflavone genistein in oilseed crop, the soybean IFS gene (GmIFS2) was incorporated into B. napus plants. The presence of GmIFS2 in B. napus was shown to direct the synthesis and accumulation of genistein derivatives in leaves up to 0.72 mg g⁻¹ DW. In addition, expression levels for most B. napus genes in the phenylpropanoid pathway were altered. These results suggest that the heterologous GmIFS2 enzyme is functionally active at using the B. napus naringenin as a substrate to produce genistein in oilseed rape.     

Taccasubosides A-D, four new steroidal glycosides from Tacca subflabellata

By analyzing the steroidal content of fresh whole plants of Tacca subflabellata (Taccaceae), we isolated one sapogenin and eight glycosides with four kinds of steroidal skeletons including four new glycosides, named taccasubosides A-D (1-4), together with five known compounds. Among them, compound 1 is the first pentacyclic sterol glycoside with 6-6-6-5-6 fused rings. The structures of 1-4 were elucidated on the basis of extensive spectroscopic analysis, including that of 2D NMR data, and the results of acidic hydrolysis. The cytotoxicity of the selected steroidal glycosides (1-4, 8, and 9) was evaluated in vitro against five human cancer cell lines. Compound 9 showed significant inhibitory activity against all five cell lines.     

Modulation of synaptic plasticity by stress hormone associates with plastic alteration of synaptic NMDA receptor in the adult hippocampus

Stress exerts a profound impact on learning and memory, in part, through the actions of adrenal corticosterone (CORT) on synaptic plasticity, a cellular model of learning and memory. Increasing findings suggest that CORT exerts its impact on synaptic plasticity by altering the functional properties of glutamate receptors, which include changes in the motility and function of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype of glutamate receptor (AMPAR) that are responsible for the expression of synaptic plasticity. Here we provide evidence that CORT could also regulate synaptic plasticity by modulating the function of synaptic N-methyl-D-aspartate receptors (NMDARs), which mediate the induction of synaptic plasticity. We found that stress level CORT applied to adult rat hippocampal slices potentiated evoked NMDAR-mediated synaptic responses within 30 min. Surprisingly, following this fast-onset change, we observed a slow-onset (>1 hour after termination of CORT exposure) increase in synaptic expression of GluN2A-containing NMDARs. To investigate the consequences of the distinct fast- and slow-onset modulation of NMDARs for synaptic plasticity, we examined the formation of long-term potentiation (LTP) and long-term depression (LTD) within relevant time windows. Paralleling the increased NMDAR function, both LTP and LTD were facilitated during CORT treatment. However, 1-2 hours after CORT treatment when synaptic expression of GluN2A-containing NMDARs is increased, bidirectional plasticity was no longer facilitated. Our findings reveal the remarkable plasticity of NMDARs in the adult hippocampus in response to CORT. CORT-mediated slow-onset increase in GluN2A in hippocampal synapses could be a homeostatic mechanism to normalize synaptic plasticity following fast-onset stress-induced facilitation.     

Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.     

Site-specific solvent exposure analysis of a membrane protein using unnatural amino acids and 19F nuclear magnetic resonance

Membrane proteins play an essential role in cellular metabolism, transportation and signal transduction across cell membranes. The scarcity of membrane protein structures has thus far prevented a full understanding of their molecular mechanisms. Preliminary topology studies and residue solvent exposure analysis have the potential to provide valuable information on membrane proteins of unknown structure. Here, a (19)F-containing unnatural amino acid (trimethylfluoro-phenylalanine, tfmF) was applied to accomplish site-specific (19)F spin incorporation at different sites in diacylglycerol kinase (DAGK, an Escherichia coli membrane protein) for site-specific solvent exposure analysis. Due to isotope effect on (19)F spins, a standard curve for (19)F-tfmF chemical shifts was drawn for varying solvent H(2)O/D(2)O ratios. Further site-specific (19)F solvent isotope shift analysis was conducted for DAGK to distinguish residues in water-soluble loops, interfacial areas or hydrophobic membrane regions. This site-specific solvent exposure analysis method could be applied for further topological analysis of other membrane proteins.