Fluorescence sensing of intermolecular interactions and development of direct molecular biosensors

Molecular biosensors are devices of molecular size that are designed for sensing different analytes on the basis of biospecific recognition. They should provide two coupled functions - the recognition (specific binding) of the target and the transduction of information about the recognition event into a measurable signal. The present review highlights the achievements and prospects in design and operation of molecular biosensors for which the transduction mechanism is based on fluorescence. We focus on the general strategy of fluorescent molecular sensing, construction of sensor elements, based on natural and designed biopolymers (proteins and nucleic acids). Particular attention is given to the coupling of sensing elements with fluorescent reporter dyes and to the methods for producing efficient fluorescence responses.     

食品中沙门氏菌分子检测靶点的筛选与评价

[目的]发掘新的沙门氏菌分子检测靶点,筛选检测性能优秀的引物.[方法]利用BLAST程序比较沙门氏菌属内基因组DNA序列的同源性以及沙门氏菌与非沙门氏菌基因组DNA序列之间的特异性,发掘出100多个检测沙门氏菌属的特异性片段,并从中随机挑选出15个片段作为候选靶点,一共设计了27对引物(FS1～FS27),对它们的特异性、灵敏度加以评价,从中筛选检测性能最好的引物.[结果]在27对引物中,检测性能最优的引物为FS23,采用该引物对供试菌株的相应检测靶点进行PCR扩增,44株沙门氏菌都能扩增到一条492 bp特异性片段,而22株非沙门氏菌则不能扩增出这一特异性片段.以FS23为引物建立PCR方法检测猪霍乱沙门氏菌基因组DNA的灵敏度为11.9 fg/μL,细菌纯培养物灵敏度为4.9×102cfu/mL;用猪霍乱沙门氏菌人工污染牛奶样品,如果接种起始菌量为100 cfu/25 mL时,只需要增菌5 h,采用上述方法即能检测出沙门氏菌.[结论]引物FS23对应的基因序列是一个性能优良的新分子检测靶点,具备很高的特异性和灵敏性,能够广泛应用于食品中沙门氏菌的快速检测.     

Classifying compound mechanism of action for linking whole cell phenotypes to molecular targets

Drug development programs have proven successful when performed at a whole cell level, thus incorporating solubility and permeability into the primary screen. However, linking those results to the target within the cell has been a major setback. The Phenotype Microarray system, marketed and sold by Biolog, seeks to address this need by assessing the phenotype in combination with a variety of chemicals with known mechanism of action (MOA). We have evaluated this system for usefulness in deducing the MOA for three test compounds. To achieve this, we constructed a database with 21 known antimicrobials, which served as a comparison for grouping our unknown MOA compounds. Pearson correlation and Ward linkage calculations were used to generate a dendrogram that produced clustering largely by known MOA, although there were exceptions. Of the three unknown compounds, one was definitively placed as an antifolate. The second and third compounds' MOA were not clearly identified, likely because the unique MOA was not represented within the database. The availability of the database generated in this report for Staphylococcus aureus ATCC 29213 will increase the accessibility of this technique to other investigators. From our analysis, the Phenotype Microarray system can group compounds with clear MOA, but the distinction of unique or broadly acting MOA at this time is less clear.     

环境因素诱导昆虫滞育发生的分子机制研究进展

滞育是生物体为了渡过恶劣环境而进行的一种停滞生长发育的生理状态,主要受遗传和环境因素的影响。研究发现光周期、温度、食物营养和体内激素水平等都能诱导昆虫滞育发生,然而滞育诱导是一个复杂的调控过程,对其分子机制的研究仍不清楚。本文对近年来在滞育诱导相关分子和分子信号通路方面取得的研究进展进行梳理,重点介绍了光周期和温度诱导昆虫滞育发生的分子级联系统,包括生物钟Per/Tim和Clk反馈回路、温度敏感型瞬时受体电位通道、γ-氨基丁酸GABA信号通路、珊瑚青素Corazonin信号通路和胰岛素信号途径等,该综述内容将为昆虫滞育研究提供帮助,同时为利用昆虫滞育进行农林害虫的防治及天敌保护提供理论参考。     

The value of applying molecular biology tools in environmental engineering: Academic and industry perspective in the USA

The integration of molecular biology tools in environmental engineering is a challenge. We discuss our views on the following four critical issues: (i) faculty career development, (ii) tool standardization, (iii) teaching, and (iv) the application of molecular biology tools in practice. For (i), we suggest that administrators and faculty need to understand the special challenges inherent to research and teaching within this highly interdisciplinary area. Furthermore, we suggest preparing two white papers aimed at educating administrators in universities and agencies. For (ii), we conclude that, because molecular biology tools are still in a state of rapid development, proposing standards at this time is premature. In the future, standards for widely applied tools should be in an on-line, peer-reviewed format. Concerning (iii), we believe that molecular biology should be taught only to the degree needed to achieve program goals. For example, environmental engineering practitioners only need to know the vocabulary and basic concepts of molecular biology tools, not be experts at doing them hands on. To help engineering students gain the right level and type of information, learning modules should be developed for them. Finally, although engineering successes applying molecular biology tools are available (iv), the biggest value will come when the tools are fully integrated with practice. Therefore, we encourage the creation of a demonstration project to document the value of applying molecular biology tools in environmental engineering. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role of sulfate in microbial transformations of environmental contaminants: Chlorinated aromatic compounds

Despite recent progress made in describing microbial transformations that occur under anaerobic conditions, our understanding of the role sulfate‐reducing bacteria may play in the remediation of environmental contaminants is still very limited. The objective of this mini‐review is to summarize what is currently known of the metabolism of chlorinated aromatic compounds in the presence of sulfate. Sulfidogenic processes are discussed with respect to the thermodynamics of haloaromatic oxidation and to their potential use in the in situ bioremediation of hazardous organic wastes. A comprehensive listing is made of anaerobic transformations that involve both halogenated and nonhalogenated monoaromatic substrates by denitrifiers, dissimilatory iron‐reducing bacteria, and methanogenic consortia. In contrast to other anaerobic processes, studies involving sulfate‐mediated metabolism of hazardous organic compounds have been neglected; however, the recent success in defining methanogenic transformations, in particular, has enhanced expectations of defining an analogous role for sulfate‐reducing microbial communities in low redox environments that have become contaminated with hazardous substances.     

Structure-based screening and molecular dynamics simulations offer novel natural compounds as potential inhibitors of Mycobacterium tuberculosis isocitrate lyase

Mycobacterium tuberculosis is the etiological agent of tuberculosis in humans and is responsible for more than two million deaths annually. M. tuberculosis isocitrate lyase (MtbICL) catalyzes the first step in the glyoxylate cycle, plays a pivotal role in the persistence of M. tuberculosis, which acts as a potential target for an anti-tubercular drug. To identify the potential anti-tuberculosis compound, we conducted a structure-based virtual screening of natural compounds from the ZINC database (n = 1,67,748) against the MtbICL structure. The ligands were docked against MtbICL in three sequential docking modes that resulted in 340 ligands having better docking score. These compounds were evaluated for Lipinski and ADMET prediction, and 27 compounds were found to fit well with re-docking studies. After refinement by molecular docking and drug-likeness analyses, three potential inhibitors (ZINC1306071, ZINC2111081, and ZINC2134917) were identified. These three ligands and the reference compounds were further subjected to molecular dynamics simulation and binding energy analyses to compare the dynamic structure of protein after ligand binding and the stability of the MtbICL and bound complexes. The binding free energy analyses were calculated to validate and capture the intermolecular interactions. The results suggested that the three compounds had a negative binding energy with ?96.462, ?143.549, and ?122.526 kJ mol^?1 for compounds with IDs ZINC1306071, ZINC2111081, and ZINC2134917, respectively. These lead compounds displayed substantial pharmacological and structural properties to be drug candidates. We concluded that ZINC2111081 has a great potential to inhibit MtbICL and would add to the drug discovery process against tuberculosis.     

Rates of molecular evolution in tree ferns are associated with body size,environmental temperature,and biological productivity

Variation in rates of molecular evolution (heterotachy) is a common phenomenon among plants. Although multiple theoretical models have been proposed, fundamental questions remain regarding the combined effects of ecological and morphological traits on rate heterogeneity. Here, we used tree ferns to explore the correlation between rates of molecular evolution in chloroplast DNA sequences and several morphological and environmental factors within a Bayesian framework. We revealed direct and indirect effects of body size, biological productivity, and temperature on substitution rates, where smaller tree ferns living in warmer and less productive environments tend to have faster rates of molecular evolution. In addition, we found that variation in the ratio of nonsynonymous to synonymous substitution rates (dN/dS) in the chloroplast rbcL gene was significantly correlated with ecological and morphological variables. Heterotachy in tree ferns may be influenced by effective population size associated with variation in body size and productivity. Macroevolutionary hypotheses should go beyond explaining heterotachy in terms of mutation rates and instead, should integrate population‐level factors to better understand the processes affecting the tempo of evolution at the molecular level.     

Identification of low molecular weight aromatic compounds by gas chromatography–mass spectrometry (GC–MS) from kraft lignin degradation by three Bacillus sp.

Three bacterial strains identified as Paenibacillus sp., Aneurinibacillus aneurinilyticus and Bacillus sp. have been shown to decolourise kraft lignin in 6 days of incubation. The release of low molecular weight aromatic compounds by these bacterial strains during degradation of kraft lignin was analysed by GC–MS analysis. The total ion chromatograph (TIC) of ethyl acetate extract from kraft lignin sample inoculated by Paenibacillus sp. was similar to control except some minor changes in the chromatographic profile indicating incapability of this bacterium to modify kraft lignin. On the other hand the TIC of ethyl acetate extract from kraft lignin inoculated by A. aneurinilyticus and Bacillus sp. caused formation of several aromatic lignin-related compound that were not present in the extract of control. The compounds identified in extract of the sample degraded by A. aneurinilyticus were guaiacol, acetoguiacone, gallic acid and ferulic acid while t-cinnamic acid, 3,4,5 trimethoxy benzaldehyde, and ferulic acid by Bacillus sp. indicating oxidization of coniferylic (G units) and sinapylic (S units) alcohol of lignin polymer. Differences between the identified compounds from different bacterial treatment were strain-specific. Among the identified aromatic compounds, ferulic acid and 3,4,5-trimethoxy benzaldehyde could be useful to the industry of preservatives, aromas and perfumes.