QCM detection of DNA targets with single-base mutation based on DNA ligase reaction and biocatalyzed deposition amplification

A novel biosensing technique for highly specific identification of gene with single-base mutation is proposed based on the implementation of the DNA ligase reaction and the biocatalyzed deposition of an insoluble product. The target gene mediated deposition of an insoluble precipitate is then transduced by quartz crystal microbalance (QCM) measurements. In this method, the DNA target hybridizes with a capture DNA probe tethered onto the gold electrode and then with a biotinylated allele-specific detection DNA. A ligase reaction is performed to generate the ligation between the capture and the detection probes, provided there is perfect match between the DNA target and the detection probe. Otherwise even when there is an allele mismatch between them, no ligation would take place. After thermal treatment at an elevated temperature, the formed duplex melts apart that merely allows the detection probe perfectly matched with the target to remain on the electrode surface. The presence of the biotinylated allele-matched probe is then detected by the QCM via the binding to streptavidin-peroxide horseradish (SA-HRP), which catalyzes the oxidative precipitation of 3,3-diaminobenzidine (DAB) by H2O2 on the electrode and provides an amplified frequency response. The proposed approach has been successfully implemented for the identification of single-base mutation in -28 site of the beta-thalassemia gene with a detection limit of 0.1 nM, demonstrating that this method provides a highly specific and cost-efficient approach for point mutation detection.     

Elevated O3 reduces the fitness of Bemisia tabaci via enhancement of the SA-dependent defense of the tomato plant

The effect of elevated O₃ on tomato plants of three different genotypes (wild-type, a jasmonic acid (JA) defense-enhanced genotype (35S) and a JA-deficient genotype (spr2)) grown in association with the whitefly Bemisia tabaci Gennadius biotype B was examined in the field in open-top chambers. We experimentally tested the hypothesis that elevated O₃ tends to reduce the nutrition of tomato plants, and to increase the SA-dependent pathway defenses and the secondary metabolites, and therefore decrease the population fitness of the whitefly. The results show that for all three tomato genotypes, elevated O₃ reduced the soluble sugars and free amino acids, increased the phenylalanine ammonia-lyase enzyme activity and the accumulated salicylic acid (SA), and up-regulated the pathogenesis-related protein (PR1), which is commonly considered to be the whitefly-resistance gene product involved in SA-dependent defense. Elevated O₃ did not affect the JA level in any of the three plant genotypes, but it increased the levels of some secondary metabolites, including total phenolics and condensed tannins. Elevated O₃ prolonged the developmental time of whiteflies fed on the three plant genotypes, and it also reduced the fecundity and the intrinsic rate of increase of whiteflies fed on either the 35S or the wild-type plants. These results suggest that elevated O₃ reduces the nutrition of tomato plants and enhances their SA content, relative PR mRNA expression and secondary metabolism, resulting in decreased fitness of whiteflies on these tomato plants.     

GC-GAP,a Rho family GTPase-activating protein that interacts with signaling adapters Gab1 and Gab2

Gab1 and Gab2 are scaffolding proteins acting downstream of cell surface receptors and interact with a variety of cytoplasmic signaling proteins such as Grb2, Shp-2, phosphatidylinositol 3-kinase, Shc, and Crk. To identify new binding partners for GAB proteins and better understand their functions, we performed a yeast two-hybrid screening with hGab2-(120-587) as bait. This work led to identification of a novel GTPase-activating protein (GAP) for Rho family GTPases. The GAP domain shows high similarity to the recently cloned CdGAP and displays activity toward RhoA, Rac1, and Cdc42 in vitro. The protein was named GC-GAP for its ability to interact with GAB proteins and its activity toward Rac and Cdc42. GC-GAP is predominantly expressed in the brain with low levels detected in other tissues. Antibodies directed against GC-GAP recognized a protein of approximately 200 kDa. Expression of GC-GAP in 293T cells led to a reduction in active Rac1 and Cdc42 levels but not RhoA. Suppression of GC-GAP expression by siRNA inhibited proliferation of C6 astroglioma cells. In addition, GC-GAP contains several classic proline-rich motifs, and it interacts with the first SH3 domain of Crk and full-length Nck in vitro. We propose that Gab1 and Gab2 in cooperation with other adapter molecules might regulate the cellular localization of GC-GAP under specific stimuli, acting to regulate precisely Rac and Cdc42 activities. Given that GC-GAP is specifically expressed in the nervous system and that it is localized to the dendritic processes of cultured neurons, GC-GAP may play a role in dendritic morphogenesis and also possibly in neural/glial cell proliferation.     

多层螺旋CT在诊断小儿急性阑尾炎中的应用

目的:探讨多层螺旋CT对小儿急性阑尾炎的诊断价值。方法:回顾分析经临床手术病理证实的53例小儿急性阑尾炎的CT表现特点。所有患儿均行多排CT横断面扫描及MPVR重建。结果:53例中,单纯性阑尾炎2例;急性化脓性阑尾炎伴周围炎4例,其中1例合并紫癜;急性化脓性阑尾炎伴穿孔33例,其中1例合并回肠末端美克尔憩室;坏疽性阑尾炎伴穿孔6例;阑尾脓肿8例。CT平扫以及MPVR重建显示阑尾肿大、增粗(直径大于6mm)30例,阑尾内粪石27例,阑尾周围蜂窝织炎23例,阑尾周围脓肿8例,大量腹水6例。结论:多层螺旋CT扫描及重建技术能为小儿阑尾炎的诊断提供有力依据,提高临床术前诊断能力。     

Capsaicin inhibits intestinal Cl- secretion and promotes Na+ absorption by blocking TRPV4 channels in healthy and colitic mice

Although capsaicin has been studied extensively as an activator of the transient receptor potential vanilloid cation channel subtype 1 (TRPV1) channels in sensory neurons, little is known about its TRPV1-independent actions in gastrointestinal health and disease. Here, we aimed to investigate the pharmacological actions of capsaicin as a food additive and medication on intestinal ion transporters in mouse models of ulcerative colitis (UC). The short-circuit current (I_sc) of the intestine from WT, TRPV1-, and TRPV4-KO mice were measured in Ussing chambers, and Ca²⁺ imaging was performed on small intestinal epithelial cells. We also performed Western blots, immunohistochemistry, and immunofluorescence on intestinal epithelial cells and on intestinal tissues following UC induction with dextran sodium sulfate. We found that capsaicin did not affect basal intestinal I_sc but significantly inhibited carbachol- and caffeine-induced intestinal I_sc in WT mice. Capsaicin similarly inhibited the intestinal I_sc in TRPV1 KO mice, but this inhibition was absent in TRPV4 KO mice. We also determined that Ca²⁺ influx via TRPV4 was required for cholinergic signaling–mediated intestinal anion secretion, which was inhibited by capsaicin. Moreover, the glucose-induced jejunal I_scvia Na⁺/glucose cotransporter was suppressed by TRPV4 activation, which could be relieved by capsaicin. Capsaicin also stimulated ouabain- and amiloride-sensitive colonic I_sc. Finally, we found that dietary capsaicin ameliorated the UC phenotype, suppressed hyperaction of TRPV4 channels, and rescued the reduced ouabain- and amiloride-sensitive I_sc. We therefore conclude that capsaicin inhibits intestinal Cl^- secretion and promotes Na⁺ absorption predominantly by blocking TRPV4 channels to exert its beneficial anti-colitic action.     

Glutamate Decarboxylase Genes as a Prescreening Marker for Detection of Pathogenic Escherichia coli Groups

The enzyme glutamate decarboxylase (GAD) is prevalent in Escherichia coli but few strains in the various pathogenic E. coli groups have been tested for GAD. Using PCR primers that amplify a 670-bp segment from the gadA and gadB genes encoding GAD, we examined the distribution of the gadAB genes among enteric bacteria. Analysis of 173 pathogenic E. coli strains, including 125 enterohemorrhagic E. coli isolates of the O157:H7 serotype and its phenotypic variants and 48 isolates of enteropathogenic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and other Shiga toxin-producing E. coli (STEC) serotypes, showed that gadAB genes were present in all these strains. Among the 22 non-E. coli isolates tested, only the 6 Shigella spp. carried gadAB. Analysis of naturally contaminated water and food samples using a gadAB-specific DNA probe that was labeled with digoxigenin showed that a gadAB-based assay is as reliable as standard methods that enumerate E. coli organisms on the basis of lactose fermentation. The presence of few E. coli cells initially seeded into produce rinsates could be detected by PCR to gadA/B genes after overnight enrichment. A multiplex PCR assay using the gadAB primers in combination with primers to Shiga toxin (Stx) genes stx₁ and stx₂ was effective in detecting STEC from the enrichment medium after seeding produce rinsate samples with as few as 2 CFU. The gadAB primers may be multiplexed with primers to other trait virulence markers to specifically identify other pathogenic E. coli groups.     

广西石山人工林灌草多样性与环境因子的关系

以广西石山人工林群落调查数据为材料,采用双向指示种分类(TWINSPAN)、冗余分析(RDA)和典范对应分析(CCA)研究了9个生境变量、1林分类型因子与石山人工林多样性、草本和灌木植物组成的关系。结果表明,坡位是影响石山人工林物种组成多样性的最主要因子,露石率和坡度对石山人工林林下有害物种分布影响最大。石山人工林林下植被组成的主要决定因素是生境因子(解释率23%~55%),而树种选择是次要因素(解释率11%~17%)。石山梯地人工林的有害草本种类多于坡地,梯地的有害灌木种类少于坡地。石山人工林的有害植物种类少于封山育林地。