番茄褪绿病毒TaqMan荧光定量PCR快速检测方法的建立与应用

【目的】本研究旨在建立TaqMan实时荧光定量PCR(TaqMan RT-qPCR)技术,快速检测单头烟粉虱Bemisia tabaci体内的番茄褪绿病毒(tomato chlorosis virus,ToCV)。【方法】根据ToCV外壳蛋白保守序列设计了1对特异性引物和1条TaqMan探针,建立了TaqMan RT-qPCR方法;与常规PCR检测进行比较,检测该方法的灵敏度与特异性;并应用该方法对单头烟粉虱成虫体内ToCV进行了快速检测。【结果】本研究构建的TaqMan RT-qPCR检测ToCV的标准曲线,其循环阈值(Ct值)与模板浓度具有良好的线性关系,扩增效率为98%。该方法对ToCV的最低检测浓度为8.3×10 copies/μL,灵敏度是常规RT-PCR的1000倍。该方法与田间番茄两种重要病毒番茄黄化曲叶病毒(tomato yellow leaf curl virus,TYLCV)和番茄斑萎病毒(tomato spotted wilt virus,TSWV)检测无交叉反应。单头烟粉虱成虫ToCV检测结果表明,温室内ToCV侵染植株上烟粉虱携毒率为100%,田间烟粉虱的携毒率为30%。【结论】本研究建立的TaqMan RT-qPCR检测方法,可快速有效检测单头烟粉虱体内ToCV携毒情况,为该病毒病的防控提供了技术支撑。     

Dielectric Properties of Normal and Metastatic Lymph Nodes Ex Vivo From Lung Cancer Surgeries

The dielectric properties of normal and tumor human tissues have been widely reported in recent years. However, the dielectric properties of intrathoracic lymph nodes (LNs) have not been reported. In this communication, we measured the dielectric properties (i.e., permittivity and conductivity) of ex vivo intrathoracic LNs obtained from lung cancer surgeries. Results show that the permittivity and conductivity of metastatic LNs are higher than those of normal LNs over the frequency range of 1 MHz–4 GHz. Statistically significant differences are observed at single specific frequencies (64, 128, 298, 433, and 915 MHz and 2.45 GHz). Our study provides the basic data to support future-related research and fills the research gap on the dielectric properties of LNs in the lungs. Bioelectromagnetics. 2020;41:148–155. © 2020 Bioelectromagnetics Society.     

Texture perception through direct and indirect touch: An analysis of perceptual space for tactile textures in two modes of exploration

Considerable information about the texture of objects can be perceived remotely through a probe. It is not clear, however, how texture perception with a probe compares with texture perception with the bare finger. Here we investigate the perception of a variety of textured surfaces encountered daily (e.g., corduroy, paper, and rubber) using the two scanning modes—direct touch through the finger and indirect touch through a probe held in the hand—in two tasks. In the first task, subjects rated the overall pair-wise dissimilarity of the textures. In the second task, subjects rated each texture along three continua, namely, perceived roughness, hardness, and stickiness of the surfaces, shown previously as the primary dimensions of texture perception in direct touch. From the dissimilarity judgment experiment, we found that the texture percept is similar though not identical in the two scanning modes. From the adjective rating experiments, we found that while roughness ratings are similar, hardness and stickiness ratings tend to differ between scanning conditions. These differences between the two modes of scanning are apparent in perceptual space for tactile textures based on multidimensional scaling (MDS) analysis. Finally, we demonstrate that three physical quantities, vibratory power, compliance, and friction carry roughness, hardness, and stickiness information, predicting perceived dissimilarity of texture pairs with indirect touch. Given that different types of texture information are processed by separate groups of neurons across direct and indirect touch, we propose that the neural mechanisms underlying texture perception differ between scanning modes.     

An estuarine species of the alga Vaucheria (Xanthophyceae) displays an increased capacity for turgor regulation when compared to a freshwater species

Turgor regulation is the process by which walled organisms alter their internal osmotic potential to adapt to osmotic changes in the environment. Apart from a few studies on freshwater oomycetes, the ability of stramenopiles to turgor regulate has not been investigated. In this study, turgor regulation and growth were compared in two species of the stramenopile alga Vaucheria, Vaucheria erythrospora isolated from an estuarine habitat, and Vaucheria repens isolated from a freshwater habitat. Species were identified using their rbcL sequences and respective morphologies. Using a single cell pressure probe to directly measure turgor in Vaucheria after hyperosmotic shock, V. erythrospora was found to recover turgor after a larger shock than V. repens. Threshold shock values for this ability were >0.5 MPa for V. erythrospora and <0.5 MPa for V. repens. Recovery was more rapid in V. erythrospora than V. repens after comparable shocks. Turgor recovery in V. erythrospora was inhibited by Gd³⁺ and TEA, suggesting a role for mechanosensitive channels, nonselective cation channels, and K⁺ channels in the process. Growth studies showed that V. erythrospora was able to grow over a wider range of NaCl concentrations. These responses may underlie the ability of V. erythrospora to survive in an estuarine habitat and restrict V. repens to freshwater. The fact that both species can turgor regulate may indicate a fundamental difference between members of the Stramenopila, as research to date on oomycetes suggests they are unable to turgor regulate.     

TaqMan-MGB探针检测文森巴尔通体博格霍夫亚种实时荧光定量PCR方法的建立及应用

【目的】应用TaqMan-MGB探针技术,建立具有种水平特异性、高敏感性的荧光定量PCR方法,用于快速检测文森巴尔通体博格霍夫亚种。【方法】在序列特异性扩增区标记(Sequence characterized amplifiedregion,SCAR)技术基础上,依据文森巴尔通体博格霍夫亚种一段特有的基因序列设计探针和引物,分别优化扩增反应的退火温度、探针和引物的反应浓度;分析此方法的特异性、敏感性及重复性;绘制标准曲线,评估PCR反应的扩增效率和稳定性。【结果】本研究设计的TaqMan-MGB探针具有种水平特异性;最低检出限为每个PCR反应11个拷贝;组内和组间的变异系数CV值分别为0.12%-0.70%和0.14%-0.55%,在允许范围内;标准曲线线性关系良好(R2=1),扩增效率高(E=104.7%)。【结论】本研究建立的基于TaqMan-MGB探针技术的荧光定量PCR方法能够在种水平特异性、高灵敏度检出文森巴尔通体博格霍夫亚种,为这种巴尔通体所引起的一系列疾病的早期快速诊断、监测和流行病学调查等研究提供有效手段。     

Imaging of jasmonic acid binding sites in tissue

Hormones regulate the mechanism of plant growth and development, senescence, and plants’ adaptation to the environment; studies of the molecular mechanisms of plant hormone action are necessary for the understanding of these complex phenomena. However, there is no measurable signal for the hormone signal transduction process. We synthesized and applied a quantum dot-based fluorescent probe for the labeling of jasmonic acid (JA) binding sites in plants. This labeling probe was obtained by coupling mercaptoethylamine-modified CdTe quantum dots with JA using N-hydroxysuccinimide (NHS) as a coupling agent. The probe, CdTe–JA, was characterized by transmission electron microscopy, dynamic light scattering, and fluorescent spectrum and applied in labeling JA binding sites in tissue sections of mung bean seedlings and Arabidopsis thaliana root tips. Laser scanning confocal microscopy (LSCM) revealed that the probe selectively labeled JA receptor. The competition assays demonstrated that the CdTe–JA probe retained the original bioactivity of JA. An LSCM three-dimensional reconstruction experiment demonstrated excellent photostability of the probe.     

Phenanthroimidazole-based thiobenzamide as an effective sensor for highly selective detection of mercury(II)

Based on highly selective and irreversible Hg²⁺-promoted desulfurization reaction, a new and simple phenanthroimidazole-type sensor was prepared and exhibited high selectivity towards Hg²⁺ ion over other metal ions, accompanied by transformation of a weakly fluorescent thioamide moiety (colorless) to a highly fluorescent amide one (blue), with a 136-fold increase in fluorescent intensity in aqueous solution with a pH span 2.57–9.12.     

8-Aminoquinoline-based ratiometric zinc probe: Unexpected binding mode and its application in living cells

PMQA, an 8-aminoquinoline-based ratiometric fluorescent sensor, demonstrates the Zn²⁺-induced red-shift of emission (85 nm), and was successfully applied to image zinc in living cells. Compared to 2:1 stoichiometry in PMQA–Zn²⁺, PMQA–Cu²⁺ shows 1:1 composition. Both nitrogen atoms from the aminoquinoline are missing in binding of zinc, while they are critically involved in Cu²⁺ chelation. The structure difference between PMQA–Zn²⁺ and PMQA–Cu²⁺ might shed light in designing novel zinc probes without suffering from copper interference.     

Synchronous plasma membrane electrochemical potential oscillations during yeast colony development and aging

Microorganisms that survive in natural environments form organized multicellular communities, biofilms and colonies with specific properties. During stress and nutrient limitation, slow growing and senescent cells in such communities retain vital processes by maintaining plasma membrane integrity and retaining the ability to generate transmembrane electrochemical gradients. We report the use of a Saccharomyces cerevisiae colonial model to show that population growth in a multicellular community depends on nutrient diffusion and that resting cells start to accumulate from the beginning of the second acidic phase of colony development. Despite differentiation of colony members, synchronous transmembrane potential oscillation was detected in the organized colony. The electrochemical membrane potential periodically oscillated at frequencies between those for circadian to infradian rhythms during colony aging and transiently decreased at time points previously linked with rebuilding of yeast metabolism. Despite extensive decreases in the intracellular ATP concentration and in the amount and activity of the plasma membrane proton pump during nutrient limited growth and colony aging, the transmembrane electrochemical potential appeared to be maintained above a level critical for population survival.     

Multiplex ligation-dependant probe amplification study of children with idiopathic mental retardation in South India

BACKGROUND:

Mental retardation (MR) is a heterogeneous dysfunction of the central nervous system exhibiting complex phenotypes and has an estimated prevalence of 1-3% in the general population. However, in about 50% of the children diagnosed with any form of intellectual disability or developmental delay the cause goes undetected contributing to idiopathic intellectual disability.

MATERIALS AND METHODS:

A total of 122 children with developmental delay/MR were studied to identify the microscopic and submicroscopic chromosome rearrangements by using the conventional cytogenetics and multiplex ligation dependent probe amplification (MLPA) analysis using SALSA MLPA kits from Microbiology Research Centre Holland [MRC] Holland.

RESULTS:

All the recruited children were selected for this study, after thorough clinical assessment and metaphases prepared were analyzed by using automated karyotyping system. None was found to have chromosomal abnormality; MLPA analysis was carried out in all subjects and identified in 11 (9%) patients.

CONCLUSION:

Karyotype analysis in combination with MLPA assays for submicroscopic micro-deletions may be recommended for children with idiopathic MR.