Genome engineering of Agrobacterium tumefaciens using the lambda Red recombination system

Agrobacterium tumefaciens has been widely used as a tool for transgenesis in plants. The availability of its genome sequence should facilitate the directed engineering of improved properties; however, the current genome engineering options are laborious. Here, we investigated whether the lambda R ed operon can be applied for recombineering of the A. tumefaciens genome. First, we built an expression plasmid for A. tumefaciens employing a tetracycline-inducible promoter to regulate the Red operon. This multicopy plasmid was then itself modified in A. tumefaciens to verify that the Red operon was functional. Then, we modified the endogenous A. tumefaciens tumor-inducing plasmid and the linear chromosome. These results extend recombineering technology to a new host and indicate a fast and convenient way to engineer the A. tumefaciens genome for functional genomics and strain improvements.     

Mixed Polyethylene Glycol-Modified Breviscapine-Loaded Solid Lipid Nanoparticles for Improved Brain Bioavailability: Preparation,Characterization, and In Vivo Cerebral Microdialysis Evaluation in Adult Sprague Dawley Rats

Breviscapine is used in the treatment of ischemic cerebrovascular diseases, but it has a low bioavailability in the brain due to its poor physicochemical properties and the activity of P-glycoprotein efflux pumps located at the blood–brain barrier. In the present study, breviscapine-loaded solid lipid nanoparticles (SLN) coated with polyethylene glycol (PEG) derivatives were formulated and evaluated for their ability to enhance brain bioavailability. The SLNs were either coated with polyethylene glycol (40) (PEG-40) stearate alone (Bre-GBSLN-PS) or a mixture of PEG-40 stearate and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000 (DSPE-PEG₂₀₀₀) (Bre-GBSLN-PS-DSPE) and were characterized both in vitro and in vivo. The mean particle size, polydispersity index, and entrapment efficiency for Bre-GBSLN-PS and Bre-GBSLN-PS-DSPE were 21.60 ± 0.10 and 22.60 ± 0.70 nm, 0.27 ± 0.01 and 0.26 ± 0.04, and 46.89 ± 0.73% and 47.62 ± 1.86%, respectively. The brain pharmacokinetic parameters revealed that the brain bioavailability of breviscapine from the Bre-GBSLN-PS and Bre-GBSLN-PS-DSPE was significantly enhanced (p < 0.01) with the area under concentration–time curve (AUC) of 1.59 ± 0.39 and 1.42 ± 0.58 μg h/mL of breviscapine, respectively, in comparison to 0.11 ± 0.02 μg h/mL from the commercial breviscapine injection. The ratios of the brain AUC for scutellarin in comparison with the plasma scutellarin AUC for commercial breviscapine injection, Bre-GBSLN-PS, and Bre-GBSLN-PS-DSPE were 0.66%, 2.82%, and 4.51%, respectively. These results showed that though both SLN formulations increased brain uptake of breviscapine, Bre-GBSLN-PS-DSPE which was coated with a binary combination of PEG-40 stearate and DSPE-PEG₂₀₀₀ had a better brain bioavailability than Bre-GBSLN-PS. Thus, the coating of SLNs with the appropriate PEG derivative combination could improve brain bioavailability of breviscapine and can be a promising tool for brain drug delivery.KEY WORDS: breviscapine, microdialysis, mixed PEGylation, P-glycoprotein (P-gp), solid lipid nanoparticles     

铁锈色小孢子菌引起Majocchi肉芽肿1例临床分析及分子鉴定

目的报道1例铁锈色小孢子菌感染引起的结节性肉芽肿及病理改变,探讨诊断和治疗。方法反复取感染部位分泌物进行细菌和真菌培养,对皮损进行病理检查,将培养菌落进行rDNAITS测序鉴定。结果分泌物PDA培养基中可见灰色菌落,表面呈绒毛状,37℃生长良好,镜下可见分隔菌丝未见分生孢子。分子生物学ITS测序结果显示：病原菌与铁锈色小孢子菌相似率为100%。确诊后用伊曲康唑治疗取得良好效果。结论在新疆特别是南疆地区头癣是很常见的皮肤病。对皮肤出现结节性损害且长期抗炎治疗不愈的患者,要考虑真菌感染的可能,及时诊断并早期进行抗真菌治疗。     

Exploring the stereochemistry of CXCR4-peptide recognition and inhibiting HIV-1 entry with D-peptides derived from chemokines

Chemokine receptor CXCR4 plays an important role in the immune system and the cellular entry of human immunodeficiency virus type 1 (HIV-1). To probe the stereospecificity of the CXCR4-ligand interface, d-amino acid peptides derived from natural chemokines, viral macrophage inflammatory protein II (vMIP-II) and stromal cell-derived factor-1alpha (SDF-1alpha), were synthesized and found to compete with (125)I-SDF-1alpha and monoclonal antibody 12G5 binding to CXCR4 with potency and selectivity comparable with or higher than their l-peptide counterparts. This was surprising because of the profoundly different side chain topologies between d- and l-enantiomers, which circular dichroism spectroscopy showed adopt mirror image conformations. Further direct binding experiments using d-peptide labeled with fluorescein (designated as FAM-DV1) demonstrated that d- and l-peptides shared similar or at least overlapping binding site(s) on the CXCR4 receptor. Structure-activity analyses of related peptide analogs of mixed chiralities or containing alanine replacements revealed specific residues at the N-terminal half of the peptides as key binding determinants. Acting as CXCR4 antagonists and with much higher biological stability than l-counterparts, the d-peptides showed significant activity in inhibiting the replication of CXCR4-dependent HIV-1 strains. These results show the remarkable stereochemical flexibility of the CXCR4-peptide interface. Further studies to understand the mechanism of this unusual feature of the CXCR4 binding surface might aid the development of novel CXCR4-binding molecules like the d-peptides that have high affinity and stability.     

Nodule infection by bean yellow mosaic virus in Phaseolus vulgaris.

Infection of root nodules of beans, Phaseolus vulgaris L., by bean yellow mosaic virus (BYMV) and the effect of the disease on the specific activity of the nodule are reported. Infectivity and serological microprecipitin assays with two sources of BYMV antiserum demonstrated that nodules from bean plants whose leaves had been inoculated with BYMV contain BYMV antigen. The disease reduced the fresh weights of tops, roots, and root nodules and induced premature nodule decay and/or nodule drop. The disease also reduced leghemoglobin content, on a plant weight basis, and N2 fixation rate, on an individual plant basis, as measured by the acetylene reduction assay. The increased leghemoglobin content per gram-nodule in BYMV-infected nodules relative to healthy nodules might be associated with multiplication of the virus in the nodule and/or unknown cellular effects derived from the BYMV-Rhizobium interaction.     

柯萨奇B组病毒感染与胰岛素依赖型糖尿病关系的研究

探讨柯萨奇B组病毒(CBV)感染与胰岛素依赖型糖尿病之间的关系。用PT-PCR方法检测38例IDDM患者外周血中CBV-RNA,同时用ELISA法测定血清中CBV特异性IgM。结果显示,病例组38例患者外周血CBV-RNA阳性率为42.10％,血清中CBV-IgM阳性率为36.84％。对照组的阳性率分别为7.5％和5％。两组比较有显著性差别(P<0.05)。说明胰岛素依赖型糖尿病与柯萨奇B组病毒感染有关。     

A Multistage Gene Normalization System Integrating Multiple Effective Methods

Gene/protein recognition and normalization is an important preliminary step for many biological text mining tasks. In this paper, we present a multistage gene normalization system which consists of four major subtasks: pre-processing, dictionary matching, ambiguity resolution and filtering. For the first subtask, we apply the gene mention tagger developed in our earlier work, which achieves an F-score of 88.42% on the BioCreative II GM testing set. In the stage of dictionary matching, the exact matching and approximate matching between gene names and the EntrezGene lexicon have been combined. For the ambiguity resolution subtask, we propose a semantic similarity disambiguation method based on Munkres'' Assignment Algorithm. At the last step, a filter based on Wikipedia has been built to remove the false positives. Experimental results show that the presented system can achieve an F-score of 90.1%, outperforming most of the state-of-the-art systems.     

Development and Evaluation of a Pseudovirus-Luciferase Assay for Rapid and Quantitative Detection of Neutralizing Antibodies against Enterovirus 71

The level of neutralizing antibodies (NtAb) induced by vaccine inoculation is an important endpoint to evaluate the efficacy of EV71 vaccine. In order to evaluate the efficacy of EV71 vaccine, here, we reported the development of a novel pseudovirus system expression firefly luciferase (PVLA) for the quantitative measurement of NtAb. We first evaluated and validated the sensitivity and specificity of the PVLA method. A total of 326 serum samples from an epidemiological survey and 144 serum specimens from 3 clinical trials of EV71 vaccines were used, and the level of each specimen''s neutralizing antibodies (NtAb) was measured in parallel using both the conventional CPE-based and PVLA-based assay. Against the standard neutralization assay based on the inhibition of the cytopathic effect (CPE), the sensitivity and specificity of the PVLA method are 98% and 96%, respectively. Then, we tested the potential interference of NtAb against hepatitis A virus, Polio-I, Polio-II, and Polio-III standard antisera (WHO) and goat anti-G10/CA16 serum, the PVLA based assay showed no cross-reactivity with NtAb against other specific sera. Importantly, unlike CPE based method, no live replication-competent EV71 is used during the measurement. Taken together, PVLA is a rapid and specific assay with higher sensitivity and accuracy. It could serve as a valuable tool in assessing the efficacy of EV71 vaccines in clinical trials and disease surveillance in epidemiology studies.     

Characterization of saponins and phenolic compounds: antioxidant activity and inhibitory effects on α-glucosidase in different varieties of colored quinoa (Chenopodium quinoa Willd)

ABSTRACT

This study investigated the contents of saponins and phenolic compounds in relation to their antioxidant activity and α-glucosidase inhibition activity of 7 colored quinoa varieties. The total saponin content was significantly different among 7 varieties and ranged from 7.51 to 12.12 mg OAE/g DW. Darker quinoa had a higher content of phenolic compounds, as well as higher flavonoids and antioxidant activity than that of light varieties. Nine individual phenolic compounds were detected in free and bound form, with gallic acid and ferulic acid representing the major compounds. The free and bound phenolic compounds (gallic acid and ferulic acid in particular) exhibited high linear correlation with their corresponding antioxidant values. In addition, the free phenolic extracts from colored quinoa exhibited higher inhibitory activity against α-glucosidase than the bound phenolic extracts. These findings imply that colored quinoa with abundant bioactive phytochemicals could be an important natural source for preparing functional food.