Detection of fluorescence dye-labeled proteins in 2-D gels using an Arthur 1442 Multiwavelength Fluoroimager

Labeling of proteins with SYPRO Orange, SYPRO Red, and SYPRO Ruby after 2-D polyacrylamide gel electrophoresis (PAGE) using plastic-backed immobilized pH gradient (IPG) strips and precast SDS polyacrylamide gels was tested. Protein spots were detected using an Arthur 1442 Multiwavelength Fluoroimager. The labeling methods described allow detection of proteins both after isoelectric focusing (IEF) and PAGE with a sensitivity higher than or comparable to standard silver staining methods. In addition to the post-labeling methods mentioned above, pre-labeling with the cysteine-specific fluorophore monobromobimane before 2-D PAGE is a sensitive, fast, and cost-effective alternative to existing staining protocols.     

无人机高光谱影像与冠层树种多样性监测

冠层树种多样性是自然森林生态系统功能和服务的重要基础。及时掌握冠层多样性的现状及变化趋势, 是探讨诸多重要生态学问题的前提, 更是制定合理生物多样性保护策略的基础。但受制于传统的多样性信息采集方法, 区域尺度的高精度冠层多样性监测发展较为缓慢; 许多在气候变化和人类干扰下的生物多样性分布信息得不到及时更新。近年来基于无人机的冠层高光谱影像收集与分析技术的发展, 使得冠层多样性监测迎来了新的发展契机。本文从森林冠层高光谱影像出发, 介绍了与多样性监测相关的无人机航拍和基于深度学习的图像处理技术, 并结合已有文献, 探讨了无人机高光谱应用于森林冠层树种多样性监测的研究现状、可行性、优势及缺陷等。我们认为冠层高光谱影像为多样性监测提供了不可或缺且丰富的原始信息; 而无人机与高光谱相机的结合, 使得区域化高频率(如每周)、高精度(如分米乃至厘米级)的冠层多样性信息自动化收集成为可能。然而高光谱影像数据量大、数据维度高与数据结构非线性的特点为影像处理带来了挑战, 而深度学习技术的飞跃, 使得从冠层高光谱影像中提取个体及物种信息达到了极高精度。恰当地使用这些技术将大大提升冠层树种多样性的自动化监测水平, 由此也将帮助我们在当前剧变环境下及时掌握森林冠层多样性的现状与变化, 为生物多样性研究与保护提供可靠的数据支撑。     

Detecting spatial regimes in ecosystems

Research on early warning indicators has generally focused on assessing temporal transitions with limited application of these methods to detecting spatial regimes. Traditional spatial boundary detection procedures that result in ecoregion maps are typically based on ecological potential (i.e. potential vegetation), and often fail to account for ongoing changes due to stressors such as land use change and climate change and their effects on plant and animal communities. We use Fisher information, an information theory‐based method, on both terrestrial and aquatic animal data (U.S. Breeding Bird Survey and marine zooplankton) to identify ecological boundaries, and compare our results to traditional early warning indicators, conventional ecoregion maps and multivariate analyses such as nMDS and cluster analysis. We successfully detected spatial regimes and transitions in both terrestrial and aquatic systems using Fisher information. Furthermore, Fisher information provided explicit spatial information about community change that is absent from other multivariate approaches. Our results suggest that defining spatial regimes based on animal communities may better reflect ecological reality than do traditional ecoregion maps, especially in our current era of rapid and unpredictable ecological change.     

Staining efficiency of specific proteins depends on the staining method: wheat gluten proteins

To analyze gluten proteins involved in celiac disease (CD) by proteomic analysis, prolamins extracted from hexaploid wheat varieties were analyzed by SDS-PAGE and 2-DE. Differences between staining methods (CBB, silver nitrate, SYPRO Ruby, and CyDye) were analyzed in comparison to immunoblotting. Staining efficiency varied per protein across methods, and complete staining of all gluten proteins could not be achieved by one of these methods. Care should be taken in the selection of staining method especially if one wants to relate the results to data obtained by immunoblotting.     

A sensitive method for determining the phosphorylation status of natriuretic peptide receptors: cGK-Ialpha does not regulate NPR-A

Natriuretic peptide receptor A (NPR-A) and natriuretic peptide receptor B (NPR-B) are transmembrane guanylyl cyclases that catalyze the synthesis of cGMP in response to natriuretic peptides. Phosphorylation and dephosphorylation regulate these receptors and have been traditionally studied by (32)PO(4) labeling of transfected cells. However, this approach cannot be used to determine the phosphorylation state of receptors isolated from unlabeled sources. Here, we use Pro-Q Diamond and SYPRO Ruby dyes to quantify the phosphorylation status and protein levels, respectively, of natriuretic peptide receptors from tissues and cells. Strong Pro-Q Diamond signals for NPR-A and NPR-B were obtained when receptors were isolated from lung tissue, liver tissue and overexpressing cells. The level of NPR-A Pro-Q staining was also high in kidney but was much lower in heart tissue. In contrast, the SYPRO Ruby protein signal was weaker and more variable. In a direct comparison, Pro-Q Diamond staining was as sensitive as but more specific than the (32)PO(4) labeling method. The two approaches were highly correlated (R(2) = 0.98). We exploited these techniques to measure the effect of cGMP-dependent protein kinase Ialpha on the phosphate content and guanylyl cyclase activity of NPR-A. Neither value was significantly affected in cells overexpressing cGK-Ialpha or in tissues from mice lacking cGK-I. We conclude that cGK-I does not regulate the cyclase activity or phosphorylation state of NPR-A. Furthermore, we find that Pro-Q Diamond staining is a sensitive method for measuring the phosphate levels of natriuretic peptide receptors, but protein levels are best detected by Western blot analysis, not SYPRO Ruby staining.     

Design and synthesis of ICT-based fluorescent probe for high-sensitivity protein detection and application to rapid protein staining for SDS-PAGE

A novel fluorescent molecular probe possessing styryl, sulfonyl, and cyanopyranyl moieties that was termed compound 1 was designed and synthesized to detect proteins through noncovalent bonding. Compound 1 did not produce fluorescence emission in the absence of proteins. However, its fluorescence spectrum showed a dramatic increase in the fluorescence intensity and strong orange emission after the addition of BSA. These changes were caused by intramolecular charge transfer (ICT). The fluorescence intensities of compound 1 were plotted as a function of the protein concentrations. A good linear relationship was observed up to a protein concentration of 325 mug/mL, and the detection limit was 70 ng/mL under the given assay conditions; this detection limit was higher than that of previously reported compounds. To demonstrate the application of compound 1, proteins in an SDS-PAGE gel were stained with compound 1 and were successfully imaged with a higher sensitivity and shorter staining operation time as compared to those of the silver staining method and SYPRO Ruby staining method. Thus, easy and high-sensitivity protein detection can be performed with the fluorescent probe, and this probe is ideally suited to proteomic applications.     

A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of about 470 nm. Fluorescence emission of the dye is approximately 618 nm. The stain can be visualized using a wide range of excitation sources utilized in image analysis systems including a UV-B transilluminator, 488-nm argon-ion laser, 532-nm yttrium-aluminum-garnet (YAG) laser, blue fluorescent light bulb, or blue light-emitting diode (LED). The detection sensitivity of SYPRO Ruby protein blot stain (0.25-1 ng protein/mm(2)) is superior to that of amido black, Coomassie blue, and india ink staining and nearly matches colloidal gold staining. SYPRO Ruby protein blot stain visualizes proteins more rapidly than colloidal gold stain and the linear dynamic range is more extensive. Unlike colloidal gold stain, SYPRO Ruby protein blot stain is fully compatible with subsequent biochemical applications including colorimetric and chemiluminescent immunoblotting, Edman-based sequencing and mass spectrometry.     

Hyperspectral darkfield microscopy of PEGylated gold nanoparticles targeting CD44‐expressing cancer cells

We present a new hyperspectral darkfield imaging system with a scanned broadband supercontinuum light source. We observed the specific attachment of the functionalized gold plasmonic nanoparticles (AuNPs) targeting CD44⁺ human breast cancer cells by conventional and by proposed hyperspectral darkfield microscopy. This wide‐field and low phototoxic hyperspectral imaging system has been successful for performing spectral three‐dimensional (3D) localization and spectroscopic identification of CD44‐targeted PEGylated AuNPs in fixed cell preparations. Such spatial and spectral information is essential for the improvement of nanoplasmonic‐based imaging, disease detection and treatment in complex biological environment. Presented system capability for 3D NP tracking will also enable investigation of specific sub‐cellular activity with the use of NPs as spectral sensors. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Use of a fluorescent internal protein standard to achieve quantitative two-dimensional gel electrophoresis

2-DE is a powerful separation method for complex protein mixtures. However, large intergel variations in spot intensity limit its use for quantitative proteomics studies. To address this issue, we developed a fluorescent internal protein standard for use in 2-DE analysis. Protein samples are spiked with an Alexa-labeled internal standard (ALIS) prior to separation with 2-DE. Due to the high extinction coefficient of the Alexa-fluor, incorporation of 0.1% of total protein is sufficient to allow visualization of the internal standard yet low enough to avoid interference in subsequent quantification and identification steps. Following 2-DE, total proteins are visualized with fluorescent postelectrophoretic stains spectrally separated from ALIS. Four protein stains, Deep Purple, Sulforhodamine G, ruthenium II-tris(bathophenanthroline disulfonate) (RuTBS), and SYPRO Ruby, including improved purification and staining protocols for RuTBS and ten-fold dilutions of SYPRO Ruby were evaluated. All staining protocols were compatible with the ALIS method and had similar LODs (1-4 ng) and dynamic ranges (10(3)). ALIS is a powerful normalization method for quantitative 2-DE which avoids potential problems associated with dual spot migration patterns observed in the DIGE method. Furthermore, ALIS provides significantly improved normality in the distribution of spot abundance-variance compared to normalization through division by the total spot volume.     

Benefits of hyperspectral remote sensing for tracking plant invasions

Aim We aim to report what hyperspectral remote sensing can offer for invasion ecologists and review recent progress made in plant invasion research using hyperspectral remote sensing. Location United States. Methods We review the utility of hyperspectral remote sensing for detecting, mapping and predicting the spatial spread of invasive species. We cover a range of topics including the trade‐off between spatial and spectral resolutions and classification accuracy, the benefits of using time series to incorporate phenology in mapping species distribution, the potential of biochemical and physiological properties in hyperspectral spectral reflectance for tracking ecosystem changes caused by invasions, and the capacity of hyperspectral data as a valuable input for quantitative models developed for assessing the future spread of invasive species. Results Hyperspectral remote sensing holds great promise for invasion research. Spectral information provided by hyperspectral sensors can detect invaders at the species level across a range of community and ecosystem types. Furthermore, hyperspectral data can be used to assess habitat suitability and model the future spread of invasive species, thus providing timely information for invasion risk analysis. Main conclusions Our review suggests that hyperspectral remote sensing can effectively provide a baseline of invasive species distributions for future monitoring and control efforts. Furthermore, information on the spatial distribution of invasive species can help land managers to make long‐term constructive conservation plans for protecting and maintaining natural ecosystems.     

航空航天遥感在物种多样性研究与保护中的应用

人类活动导致全球范围内生物多样性丧失日趋严重。物种多样性是研究最为深入以及最贴近生物多样性管理的层次。物种多样性的研究往往受到多时空尺度生态过程的影响, 传统物种多样性调查方法受到人力物力影响, 局限性大, 物种多样性的研究与管理亟需整合不同来源的数据。遥感技术从传统的光学遥感阶段发展到不同平台、不同维度相结合的多源遥感阶段, 并逐渐进入以高空间分辨率和高光谱为特征、以激光雷达为前沿发展方向的综合遥感阶段。遥感技术因为其监测范围广、能监测人迹罕至地区以及长期可重复等特性, 为研究不同时空尺度的生态学科学问题提供了更新更优的研究手段。本文围绕种群动态、种间关系与群落多样性、功能属性及功能多样性以及生物多样性保护管理等生物多样性研究热点问题, 系统地论述了航空航天遥感技术在物种多样性研究与保护领域的应用, 总结了航空航天遥感技术在研究与物种多样性有关的主要生态学问题中的机遇与挑战。我们认为航空航天遥感技术利用多光谱甚至高光谱与激光技术从空中监测物种多样性, 从不同视角、基于不同光源提供了物种多样性不同侧面的信息, 能够减小地面调查强度, 在大范围和边远地区的物种多样性调查研究中有着至关重要的作用。依据光谱特性的物种判别以及依据激光雷达的三维结构量测将促进生物多样性的研究与管理, 加强遥感学家和生物多样性研究者的沟通交流将有助于促进不同时空尺度的生物多样性与遥感技术的结合。     

Imaging technologies for the detection of multiple stains in proteomics

Laser-based scanners and charge-coupled device (CCD) camera systems are evolving to have greater functional capabilities for capturing images from a range of staining technologies used in gel electrophoresis and electroblotting. Digitizing Coomassie Brilliant Blue (CBB) stained gels and silver stained gels has now become possible using a laser-based gel scanner, the FLA-5000 fluorescent image analyzer system. Also, a simultaneous dual fluorescent imaging function has been incorporated into the FLA-5000 system, utilizing dichroic mirrors with both the optical system and the emission filter. In the workflow of routine proteomics research, the relationship between SYPRO dye staining and fluorescent detection using the FLA-5000 system have become symbiotic. Additionally in many cases, subsequent staining of the gel with CBB is useful for future research, and thus imaging instruments should be able to handle both staining formats. Digitizing the CBB stained gel can now be easily performed by the FLA-5000 fluorescent image analyzer system using a fluorescent board as an epi-illumination background. A cooled CCD camera system has the potential of imaging not only chemiluminescent membranes but also digitizing molecular weight markers and fluorescent detection of SYPRO dye-stained gels. With Multi Gauge software version 2.0 it is now a simple task to combine two images into one, as commonly required in dual detection experiments. The LAS-3000 system was designed to capture chemiluminescent images and to digitize the images automatically. Thus, new capabilities added to gel imaging systems make them capable of detecting and displaying multiple signals more conveniently.     

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.Download video file.^{(121M, mp4)}     

Comparison of the Mantel test and alternative approaches for detecting complex multivariate relationships in the spatial analysis of genetic data

The Mantel test is widely used to test the linear or monotonic independence of the elements in two distance matrices. It is one of the few appropriate tests when the hypothesis under study can only be formulated in terms of distances; this is often the case with genetic data. In particular, the Mantel test has been widely used to test for spatial relationship between genetic data and spatial layout of the sampling locations. We describe the domain of application of the Mantel test and derived forms. Formula development demonstrates that the sum-of-squares (SS) partitioned in Mantel tests and regression on distance matrices differs from the SS partitioned in linear correlation, regression and canonical analysis. Numerical simulations show that in tests of significance of the relationship between simple variables and multivariate data tables, the power of linear correlation, regression and canonical analysis is far greater than that of the Mantel test and derived forms, meaning that the former methods are much more likely than the latter to detect a relationship when one is present in the data. Examples of difference in power are given for the detection of spatial gradients. Furthermore, the Mantel test does not correctly estimate the proportion of the original data variation explained by spatial structures. The Mantel test should not be used as a general method for the investigation of linear relationships or spatial structures in univariate or multivariate data. Its use should be restricted to tests of hypotheses that can only be formulated in terms of distances.     

An improved formulation of SYPRO Ruby protein gel stain: comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation

SYPRO Ruby protein gel stain is compatible with a variety of imaging platforms since it absorbs maximally in the ultraviolet (280 nm) and visible (470 nm) regions of the spectrum. Dye localization is achieved by noncovalent, electrostatic and hydrophobic binding to proteins, with signal being detected at 610 nm. Since proteins are not covalently modified by the dye, compatibility with downstream proteomics techniques such as matrix-assisted laser desorption/ionisation-time of flight mass spectrometry is assured. The principal limitation of the original formulation of SYPRO Ruby protein gel stain, is that it was only compatible with a limited number of gel fixation procedures. Too aggressive a fixation protocol led to diminished signal intensity and poor detection sensitivity. This is particularly apparent when post-staining gels subjected to labeling with other fluorophores such as Schiff's base staining of glycoproteins with fluorescent hydrazides. Consequently, we have developed an improved formulation of SYPRO Ruby protein gel stain that is fully compatible with commonly implemented protein fixation procedures and is suitable for post-staining gels after detection of glycoproteins using the green fluorescent Pro-Q Emerald 300 glycoprotein stain or detection of beta-glucuronidase using the green fluorescent ELF 97 beta-D-glucuronide. The new stain formulation is brighter, making it easier to manually excise spots for peptide mass profiling. An additional benefit of the improved formulation is that it permits staining of proteins in isoelectric focusing gels, without the requirement for caustic acids.     

具有左截断、右删失寿命数据类型的生命表编制方法

左截断数据是一类特殊的寿命数据类型,其定义为一些动物个体并非在初始时间（出生或孵化）而是在某个时间（年龄）延滞之后才进入调查取样范围而收集到的一类寿命数据。传统的乘积限估计法只能处理寿终数据和右删失数据,对左截断数据则无能为力。本文提出一种对乘积限估计法改进方法,此种方法能同时处理寿终数据、右删失数据和左截断数据,从而有效地利用了左截断数据所含有的物种存活信息。     

Mass spectral compatibility of four proteomics stains

With the recent introduction of new fluorescence stains to the proteomics market, there is now more choice available. SYPRO Ruby, LavaPurple, Flamingo, and Krypton total protein stains were compared for ease of use, image quality, and compatibility with protein identification by peptide mass fingerprinting (PMF) (MALDI-TOF). All four stains produced good images but with slightly different staining patterns. SYPRO was found to inhibit identification of cysteine and tryptophan containing peptides, which reduced protein identification.     

In vivo cytometry: a spectrum of possibilities.

BACKGROUND: We investigate whether optical imaging can reliably detect abnormalities in tissue, in a range of specimens (live cells in vitro; fixed, fresh ex-vivo and in vivo tissue), without the use of added contrast agents, and review our promising spectral methods for achieving quantitative, real-time, high resolution intrasurgical optical diagnostics. METHODS: We use reflectance, fluorescence, two-photon, and Mie scattering imaging, performed with instrumentation we developed or modified, to detect intrinsic tissue signatures. Emphasis is on spectral/hyperspectral imaging approaches allowing the equivalent of in vivo pathology. RESULTS: With experimental focus on unstained specimens, we demonstrate the ability to segment tissue images for cancer detection. Spectral reflectance imaging, coupled with advanced analysis, typically yields 90% specificity and sensitivity. Autofluorescence is also shown to be diagnostically useful, with lymph nodes results highlighted here. Elastic scattering hyperspectral imaging endoscopy, using a new instrument we designed and built, shows promise in bronchoscopic detection of dysplasia and early cancer in patients. CONCLUSIONS: The results demonstrate that advanced optical imaging can detect and localize cellular signatures of cancer in real-time, in vivo, without the use of contrast agents, in animals and humans. This is an important step towards tight spatio-temporal coupling between such detection and clinical intervention.