Simultaneous collection of rapid chlorophyll fluorescence induction kinetics, fluorescence quenching parameters, and environmental data using an automated PAM-2000/CR10X data logging system

The PAM-2000 portable chlorophyll fluorometer represents one of the first commercially available instruments utilizing the Pulse Amplitude Modulation (PAM) measurement principle, and has become a widely used platform for measuring chlorophyll fluorescence in a wide range of study systems. In this paper, we describe a new method for externally driving and gathering data from the PAM-2000, a method that allows the user to execute a pre-defined user run (or runs) and capture (1) rapid induction kinetics (at 2 ms frequency) during all saturating pulses, (2) measures of F, F_o, F_o′, F_m, and F_m′ associated with those same pulses, and (3) changes in fluorescence F at user-defined intervals between pulses, for the entire user run, with all data compressed into a single, manageable data logger file. Practically, the method makes possible, for example, a post-hoc evaluation of the appropriateness of saturation pulse lengths and intensities during a user run. More importantly it captures, during entire user runs, the varied information contained in slow changes in fluorescence between saturating pulses, as well as rapid induction kinetics, quenching coefficients and quantum yields all gathered simultaneously from all saturating flashes.Electronic Supplementary Material Supplementary material is available for this article at     

N-glycoproteomics - an automated workflow approach

Glycan decorations dictate protein functions and thus have crucialimportance in life sciences. Previously glycoprotein analysiswas mainly focused on the analysis of the liberated glycansallowing detailed structural, but lacking positional information.Analysis of intact glycopeptides required purified glycoproteinsand manual interpretation of spectra. We developed an approachwhere mixtures of native glycopeptides were analyzed with tandemmass spectrometry and the spectra were analyzed with automatedin silico workflows. The latter included combination of theoriginal spectra, generation of a human N-glycopeptide library,matching the glycopeptide spectra to the theoretical peptidefragments, scoring the observations, predicting the glycan composition,which were then matched against the observed spectra, statisticalvalidation of the results with target–decoy filtering,and finally the calculation of glycan structures. We verifiedthis approach with the 150 serotransferrin glycopeptide spectra,where we automatically generated 10⁵ putative interpretationsfrom >10⁹ theoretical glycopeptides. After scoring 62 glycopeptidespectra obtained validated interpretation with concomitant aminoacid sequences, glycan compositions, and structures. When applyingthis method to an unknown mixture of human plasma glycoproteinswe identified 80 glycopeptides with their glycan compositionsor structures. Instead of weeks and months of interpretationwork of mass spectrometry files our automated workflow can beexecuted in few hours and provide information concomitantlyfrom both the amino acid and glycan moieties of intact glycopeptidesin mixtures. No advanced computational skills were needed touse these preformed and tested workflows. In case users wantto add complexity to the analysis they are allowed to alterall parameters and rebuild the workflows.     

昆虫图像分割方法及其应用

昆虫图像自动鉴定是一种快速鉴定昆虫的方法,图像分割则是其中关键步骤。通过搜集和整理国内外近年来针对昆虫图像的分割方法和研究,发现对昆虫图像分割的研究日趋增多。随着计算机图像技术的发展,昆虫图像分割方法吸收了许多图像分割领域中新兴的方法, 诸如采用水平集、边缘流以及结合形状、纹理、色彩等多种要素的智能分割(如JSEG方法)等。虽然大量的图像分割方法被引入到昆虫图像研究中,但是目前分割技术依然是阻碍昆虫图像广泛应用的关键。本文经过总结和分析,发现目前昆虫图像分割研究的往往在各自的测试集上有良好表现, 但是缺乏统一的评价标准, 因此很多方法在昆虫图像中应用难以推广。针对研究中的存在的这些问题,需建立良好的昆虫图像分割评价体系,本文建议通过建立统一的昆虫图像库以及对昆虫图像分割的评价方法深入研究,并且这些工作是当前昆虫图像分割研究亟待完善任务。     

蛋白质磁共振——从结构生物学到结构基因组学

在后基因组时代,随着大量物种全基因组序列的获得,结构生物学家面临着结构基因组学的新机遇和挑战。与传统的结构生物学不同的是,结构基因组学的研究主要集中在结构和功能未知并且与从前研究的蛋白质相似性很小的蛋白质。准确的来讲,结构基因组学通过高通量蛋白质表达、结构解析来完成所有蛋白质家族的结构表征,从而能够通过结构预测功能。加州结构基因组学联合实验室发展了高度自动化的蛋白质合成、结晶、结构解析生产线。然而由于一些蛋白质不能被结晶,要想覆盖所有蛋白质结构域还有很大困难。Wuthrich的研究小组通过一些高通量的目的蛋白质筛选和NMR结构解析的方法解决了这一难题。与X射线晶体学解析蛋白质结构相比,NMR技术由于能够解析更接近生理状态的溶液结构而具有互补性。通过获得溶液中的蛋白质稳定性、动力学特征和相互作用信息,正如在朊蛋白和SARS相关蛋白的研究中所表现的那样,NMR技术从扩大已知的蛋白质结构数据库、新的蛋白质功能到化学生物学研究中都扮演着激动人心的角色。     

Striatal dopamine signals are region specific and temporally stable across action-sequence habit formation

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Detection range of songbirds using a stopover site by automated radio‐telemetry

A major uncertainty in automated radio‐telemetry studies of small birds is the detection range of receiving antennas. We compared simultaneous daytime detections (± 30 s) by automated and manual radio‐telemetry to assess detection probability and the proportion of transmissions detected for birds on migratory stopover as a function of distance, foraging guild (Black‐throated Blue Warblers, Setophaga caerulescens, and Yellow‐rumped Warblers, Dendroica coronata coronata, represented mid‐canopy foliage gleaners and White‐throated Sparrows, Zonotrichia albicollis, represented a ground forager), habitat type, meteorological variables, tower antenna number (1–4), and the position of a bird relative to the receiving antenna's bearing (offset angle). Our study was conducted at a migratory stopover site in southern Ontario, Canada. Most detections were in dense to sparse forest, and all individuals were within 1.03 km of the automated receiving station. Daily detection probability was near 100% for both foraging guilds. However, within 30 s before and after a manual radio‐telemetry location was made, detection probability and the proportion of transmissions detected by automated radio‐telemetry declined with distance, was higher for warblers than sparrows, and was lowest for 90° offset angles. Our results suggest that when research goals do not require detections with high temporal frequency, e.g., estimation of departure date or daily departure probability, our study design had an effective detection range of at least 1 km. However, where temporal precision is required, e.g., to investigate movements and changes in activity levels during stopover, detection range was ~300 m for ground‐foraging sparrows and 600 m for mid‐canopy foraging warblers, which is much lower than the presumed detection range of antennas under optimal conditions (15 km). This corresponds to a spatial area of coverage for forest‐dwelling birds of ~0.3–1.1 km². Our results suggest that to optimally configure an automated radio‐telemetry array at the regional scale, investigators should carefully consider detection range and its underlying covariates, including species type, the habitat matrix, and the orientation of antennas relative to preferred habitat.     

Automated identification of live moths (Macrolepidoptera) using digital automated identification System (DAISY)

Two hundred and thirty‐seven species of Macrolepidoptera were light trapped at Treborth Botanical Garden, Gwynedd, UK. Live adults were digitally imaged using a simple, inexpensive method suitable for field use, then released. Inconsistent lighting, variation in resting posture and inclusion of worn individuals produced image sets high in intraspecific variation. Thirty‐five common species were selected to provide training images for the Digital Automated Identification SYstem (DAISY). Twenty individuals per species were pre‐processed to standardize size and posture and to enhance features. The right forewing of each was highlighted manually and the pattern rendered polar and greyscale for DAISY analysis. Despite poor quality of some images, 83% of unknown species were identified correctly. The best species had 100% correct identification and the worst 35%. The most poorly identified images were those of moths that had lost scales or been unevenly illuminated. The precision with which the forewing was highlighted affected performance. When highlighted carefully, Laothoe populi was identified correctly twice as successfully as when the same image was highlighted poorly. Size of the training set was also important. Sets of 5, 10, 15 and 20 training images, plotted against performance produced a curve of diminishing returns. Colourimages and inclusion of size should improve accuracy.     

Single-cell microliter-droplet screening system (MISS Cell): An integrated platform for automated high-throughput microbial monoclonal cultivation and picking

Solid plates have been used for microbial monoclonal isolation, cultivation, and colony picking since 1881. However, the process is labor- and resource-intensive for high-throughput requirements. Currently, several instruments have been integrated for automated and high-throughput picking, but complicated and expensive. To address these issues, we report a novel integrated platform, the single-cell microliter-droplet screening system (MISS Cell), for automated, high-throughput microbial monoclonal colony cultivation and picking. We verified the monoclonality of droplet cultures in the MISS Cell and characterized culture performance. Compared with solid plates, the MISS Cell generated a larger number of monoclonal colonies with higher initial growth rates using fewer resources. Finally, we established a workflow for automated high-throughput screening of Corynebacterium glutamicum using the MISS Cell and identified high glutamate-producing strains. The MISS Cell can serve as a universal platform to efficiently produce monoclonal colonies in high-throughput applications, overcoming the limitations of solid plates to promote rapid development in biotechnology.     

Automated analysis of alditols by anion-exchange chromatography with photometric and fluorimetric postcolumn derivatization

Eight alditols were separated in ca. 80 min as their borate complexes by stepwise elution with three borate buffers on a column packed with Hitachi 2633 resin. The alditols in the eluate were derivatized automatically to colored, fluorescent products by applying sequential reactions of periodate oxidation and Hantzsch condensation, and the products were detected either photometrically or fluorimetrically. This automated method allowed simultaneous determination of 20–500 and 20–200 nmol amounts of alditols by photometric and fluorimetric monitorings, respectively. The lower limits of detection were ca. 2 and 0.5 nmol, respectively. The interference by aldoses was slight. Aldoses may be also determined as alditols by direct injection of aqueous solutions to which excess amounts of sodium borohydride have been added. This method was applied with success to urinary alditol assay and to molecular weight determination by end group analysis.     

Automated analysis of uronic acids by high-performance liquid chromatography with photometric and fluorimetric postcolumn labelling using 2-cyanoacetamide

A convenient method for the rapid and sensitive automated analysis of uronic acids, based on high-performance anion-exchange chromatography on a Hitachi 2633 column and photometric as well as fluorimetric postcolumn labeling with 2-cyanoacetamide, has been developed. This method allows the simultaneous determination of 1-1000 nmol of D-mannuronic and D-galacturonic acids and 5-1000 nmol of L-iduronic and D-glucuronic acids in approx 70 min with high precision by photometric monitoring. In fluorimetric monitoring the linearity range was 1-1000 nmol for all these uronic acids, but reproducibility was rather low at the lowest limit of linearity. Application of this method to the analysis of component uronic acids in some polyuronides has suggested the inadequacy of generally accepted conditions for hydrolysis.