Single-molecule fluorescence experiments determine protein folding transition path times

The transition path is the tiny fraction of an equilibrium molecular trajectory when a transition occurs as the free-energy barrier between two states is crossed. It is a single-molecule property that contains all the mechanistic information on how a process occurs. As a step toward observing transition paths in protein folding, we determined the average transition-path time for a fast- and a slow-folding protein from a photon-by-photon analysis of fluorescence trajectories in single-molecule F?rster resonance energy transfer experiments. Whereas the folding rate coefficients differ by a factor of 10,000, the transition-path times differ by a factor of less than 5, which shows that a fast- and a slow-folding protein take almost the same time to fold when folding actually happens. A very simple model based on energy landscape theory can explain this result.     

牛附红细胞体体外药物筛选试验

以RPMI-1640为基础培养液,加入30%犊牛血清,选用贝尼尔、四环素、敌百虫、泰妙菌素、咪唑苯脲、氟苯尼考、依沙吖啶、磷酸伯氨喹等药物原粉,在37℃,5%CO2气体条件下对牛附红细胞体进行了体外药物筛选试验。结果表明,依沙吖啶效果最好;敌百虫和磷酸伯氨喹次之,但敌百虫毒性较大;贝尼尔、咪唑苯脲、氟苯尼考有效。     

Single-molecule torsional pendulum

We have built a torsional pendulum based on an individual single-walled carbon nanotube, which is used as a torsional spring and mechanical support for the moving part. The moving part can be rotated by an electric field, resulting in large but fully elastic torsional deformations of the nanotube. As a result of the extremely small restoring force associated with the torsional deformation of a single molecule, unusually large oscillations are excited by the thermal energy of the pendulum. By diffraction analysis, we are able to determine the handedness of the molecule in our device. Mechanical devices with molecular-scale components are potential building blocks for nanoelectromechanical systems and may also serve as sensors or actuators.     

Single-molecule vibrational spectroscopy and microscopy

Vibrational spectra for a single molecule adsorbed on a solid surface have been obtained with a scanning tunneling microscope (STM). Inelastic electron tunneling spectra for an isolated acetylene (C2H2) molecule adsorbed on the copper (100) surface showed an increase in the tunneling conductance at 358 millivolts, resulting from excitation of the C-H stretch mode. An isotopic shift to 266 millivolts was observed for deuterated acetylene (C2D2). Vibrational microscopy from spatial imaging of the inelastic tunneling channels yielded additional data to further distinguish and characterize the two isotopes. Single-molecule vibrational analysis should lead to better understanding and control of surface chemistry at the atomic level.     

Single-molecule optomechanical cycle

Light-powered molecular machines are conjectured to be essential constituents of future nanoscale devices. As a model for such systems, we have synthesized a polymer of bistable photosensitive azobenzenes. Individual polymers were investigated by single-molecule force spectroscopy in combination with optical excitation in total internal reflection. We were able to optically lengthen and contract individual polymers by switching the azo groups between their trans and cis configurations. The polymer was found to contract against an external force acting along the polymer backbone, thus delivering mechanical work. As a proof of principle, the polymer was operated in a periodic mode, demonstrating for the first time optomechanical energy conversion in a single-molecule device.     

亨利兜兰茎尖诱导与离体快繁试验

以亨利兜兰的侧芽为试验材料,选择适宜的灭菌方式及筛选出较佳培养基,提高茎尖诱导率。研究基本培养基及激素浓度对继代增殖的影响。结果表明：适合兜兰侧芽的灭菌方式是将75%酒精倒入小烧杯浸泡1 min,无菌水冲洗3次进行消毒处理;倒入0.1%升汞浸泡约5 min,放入0.1%克拉霉素的溶液中5 min,再次放入0.1%升汞浸5 min,用无菌水冲洗5次,然后剥去外层,把5～7 mm茎尖接入培养基中。茎尖诱导培养基以Heller＋6-BA 2 mg/L＋NAA 0.2 mg/L＋C 0.5在继代培养中,试管苗在培养基1/2 MS＋6-BA 0.2 mg/L｜＋NAA 2 mg/L＋10%yz＋C 2中,丛生芽增殖为佳,增殖倍数为3.50。壮苗生根以1/2 MS＋NAA 1＋10%土豆汁＋C 2为好。     

Single-molecule cut-and-paste surface assembly

We introduce a method for the bottom-up assembly of biomolecular structures that combines the precision of the atomic force microscope (AFM) with the selectivity of DNA hybridization. Functional units coupled to DNA oligomers were picked up from a depot area by means of a complementary DNA strand bound to an AFM tip. These units were transferred to and deposited on a target area to create basic geometrical structures, assembled from units with different functions. Each of these cut-and-paste events was characterized by single-molecule force spectroscopy and single-molecule fluorescence microscopy. Transport and deposition of more than 5000 units were achieved, with less than 10% loss in transfer efficiency.     

彩色马蹄莲的离体繁殖试验

以彩色马蹄莲初代培养中产生的丛芽块为供试材料,以改良的MS作为基本培养基,外加不同浓度的激素6-BA和NAA的组合,研究其丛芽增殖、丛芽块单芽分化生长、不定芽生根及根芽同步生长分化情况.筛选出了根芽同步分化的激素浓度组合6-BA 1 mg/L NAA0.1 mg/L,从而在保持一定增殖系数的同时,缩短了试管苗出瓶的时间,大大加快了种苗的繁育进程,降低了种苗的生产成本.     

Single-molecule measurement of protein folding kinetics

In order to investigate the behavior of single molecules under conditions far from equilibrium, we have coupled a microfabricated laminar-flow mixer to a confocal optical system. This combination enables time-resolved measurement of F?rster resonance energy transfer after an abrupt change in solution conditions. Observations of a small protein show the evolution of the intramolecular distance distribution as folding progresses. This technique can expose subpopulations, such as unfolded protein under conditions favoring the native structure, that would be obscured in equilibrium experiments.     

Single-molecule speckle analysis of actin filament turnover in lamellipodia

Lamellipodia are thin, veil-like extensions at the edge of cells that contain a dynamic array of actin filaments. We describe an approach for analyzing spatial regulation of actin polymerization and depolymerization in vivo in which we tracked single molecules of actin fused to the green fluorescent protein. Polymerization and the lifetime of actin filaments in lamellipodia were measured with high spatial precision. Basal polymerization and depolymerization occurred throughout lamellipodia with largely constant kinetics, and polymerization was promoted within one micron of the lamellipodium tip. Most of the actin filaments in the lamellipodium were generated by polymerization away from the tip.     

Single-molecule DNA sequencing of a viral genome

The full promise of human genomics will be realized only when the genomes of thousands of individuals can be sequenced for comparative analysis. A reference sequence enables the use of short read length. We report an amplification-free method for determining the nucleotide sequence of more than 280,000 individual DNA molecules simultaneously. A DNA polymerase adds labeled nucleotides to surface-immobilized primer-template duplexes in stepwise fashion, and the asynchronous growth of individual DNA molecules was monitored by fluorescence imaging. Read lengths of >25 bases and equivalent phred software program quality scores approaching 30 were achieved. We used this method to sequence the M13 virus to an average depth of >150x and with 100% coverage; thus, we resequenced the M13 genome with high-sensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.     

Single-molecule lysozyme dynamics monitored by an electronic circuit

Tethering a single lysozyme molecule to a carbon nanotube field-effect transistor produced a stable, high-bandwidth transducer for protein motion. Electronic monitoring during 10-minute periods extended well beyond the limitations of fluorescence techniques to uncover dynamic disorder within a single molecule and establish lysozyme as a processive enzyme. On average, 100 chemical bonds are processively hydrolyzed, at 15-hertz rates, before lysozyme returns to its nonproductive, 330-hertz hinge motion. Statistical analysis differentiated single-step hinge closure from enzyme opening, which requires two steps. Seven independent time scales governing lysozyme's activity were observed. The pH dependence of lysozyme activity arises not from changes to its processive kinetics but rather from increasing time spent in either nonproductive rapid motions or an inactive, closed conformation.     

In vitro and in silico studies of salicylic acid on systemic induced resistance against bacterial leaf blight disease and enhancement of crop yield

Salicylic acid (SA) is an effective elicitor to promote plant defenses and growth.This study aimed to investigate rice(Oryza sativa L.) cv.Khao Dawk Mali 105 treated with salicylic acid (SA)-Ricemate as an enhanced plant protection mechanism against bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv.oryzae (Xoo).Results indicated that the use of SA-Ricemate as a foliar spray at concentrations of more than 100 mg L^–1 can reduce the severity of BLB disease by 71%.SA-Ric...     

Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder

We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.     

基于生物信息学方法挖掘奶山羊miRNAs研究

microRNAs(miRNAs)是长约22nt的内源非编码小分子RNA,在转录后基因调控中发挥重要作用。奶山羊是具有重要经济价值的产乳动物,有关奶山羊miRNAs研究相对匮乏,识别和鉴定新的奶山羊miRNA至关重要。文章以与奶山羊高度同源的绵羊基因组为参考数据库,应用生物信息学方法得到101条新的奶山羊miRNAs序列,并对其进行序列特性分析,为今后基因组信息不全物种的miRNAs挖掘与鉴定提供参考。     

Single-molecule studies of heterogeneous dynamics in polymer melts near the glass transition

Single-molecule spectroscopy was used to follow the orientation of a single probe molecule in a polymer film in real time. Broad spatially heterogeneous dynamics were observed on long time scales, which result from simple diffusive rotational motions on short time scales. This diffusive behavior persists for many rotations before the molecule's local environment changes to one characterized by a new time scale. This environmental exchange occurs instantaneously on the time scale of the experiment and may arise from large-scale collective motions. The distribution of exchange times for these environments was measured for several temperatures near the glass transition.