Improved Visualization of Cell Nuclei in Unstained Cultured Cells

Nuclear morphology is useful in tissue culture studies in determining the presence and grade of transformed cells as well as in determining the heterogeneity of the cell population (Grogan el al. 1981, Hustin 1976, Siracky et al. 1978, Siracky 1979). The ratio of long and short nuclear axes provides a useful numerical expression of nuclear shape (Hustin 1976). Clear visualization of nuclei is critical for making the necessary measurements.     

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death ¹. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection ^2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature ⁴.Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin ^5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology ^7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin ⁹, allowed breakthroughs in the field.In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma ^10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.     

Visualization of Negative Signaling in B Cells by Quantitative Confocal Microscopy

Numerous biochemical experiments have invoked a model in which B-cell antigen receptor (BCR)-Fc receptor for immunoglobulin (Ig) G (FcgammaRII) coclustering provides a dominant negative signal that blocks B-cell activation. Here, we tested this model using quantitative confocal microscopic techniques applied to ex vivo splenic B cells. We found that FcgammaRII and BCR colocalized with intact anti-Ig and that the SH2 domain-containing inositol 5'-phosphatase (SHIP) was recruited to the same site. Colocalization of BCR and SHIP was inefficient in FcgammaRII-/- but not gamma chain-/- splenic B cells. We also examined the subcellular location of a variety of enzymes and adapter proteins involved in signal transduction. Several proteins (CD19, CD22, SHP-1, and Dok) and a lipid raft marker were co-recruited to the BCR, regardless of the presence or absence of FcgammaRII and SHIP. Other proteins (Btk, Vav, Rac, and F-actin) displayed reduced colocalization with BCR in the presence of FcgammaRII and SHIP. Colocalization of BCR and F-actin required phosphatidylinositol (PtdIns) 3-kinase and was inhibited by SHIP, because the block in BCR/F-actin colocalization was not seen in B cells of SHIP-/- animals. Furthermore, BCR internalization was inhibited with intact anti-Ig stimulation or by expression of a dominant-negative mutant form of Rac. From these results, we propose that SHIP recruitment to BCR/FcgammaRII and the resulting hydrolysis of PtdIns-3,4,5-trisphosphate prevents the appropriate spatial redistribution and activation of enzymes distal to PtdIns 3-kinase, including those that promote Rac activation, actin polymerization, and receptor internalization.     

An Improved UPLC-MS/MS Platform for Quantitative Analysis of Glycerophosphoinositol in Mammalian Cells

The glycerophosphoinositols constitute a class of biologically active lipid-derived mediators whose intracellular levels are modulated during physiological and pathological cell processes. Comprehensive assessment of the role of these compounds expands beyond the cellular biology of lipids and includes rapid and unambiguous measurement in cells and tissues. Here we describe a sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative analysis of the most abundant among these phosphoinositide derivatives in mammalian cells, the glycerophosphoinositol (GroPIns). The method has been developed in mouse Raw 264.7 macrophages with limits of quantitation at 3 ng/ml. Validation on the same cell line showed excellent response in terms of linear dynamic range (from 3 to 3,000 ng/ml), intra-day and inter-day precision (coefficient of variation ≤ 7.10%) and accuracy (between 98.1 and 109.0%) in the range 10-320 ng/ml. As proof of concept, a simplified analytical platform based on this method and external calibration was also tested on four stimulated and unstimulated cell lines, including Raw 264.7 macrophages, Jurkat T-cells, A375MM melanoma cells and rat basophilic leukemia RBL-2H3 cells. The results indicate a wide variation in GroPIns levels among different cell lines and stimulation conditions, although the measurements were always in line with the literature. No significant matrix effects were observed thus indicating that the here proposed method can be of general use for similar determinations in cells of different origin.     

Visualization and Molecular Analysis of Actin Assembly in Living Cells

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.     

定量细胞化学分析大蒜油抗癌的作用

本文应用细胞化学和显微分光光度计对癌细胞的 DNA,ACP,ANAE、SDH 和G6PDH 进行定量测定,又用流式细胞计分析癌细胞 RNA 含量的变化.实验证明大蒜油能明显抑制癌细胞 DNA 与 RNA 合成,并减低五种酶的活性。表明大蒜油能影响癌细胞的核酸代谢、能量代谢及功能活动、抑制癌细胞的分裂增殖,从而起到抗癌效应.     

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.     

Multiple Quantitative Trait Analysis Using Bayesian Networks

Models for genome-wide prediction and association studies usually target a single phenotypic trait. However, in animal and plant genetics it is common to record information on multiple phenotypes for each individual that will be genotyped. Modeling traits individually disregards the fact that they are most likely associated due to pleiotropy and shared biological basis, thus providing only a partial, confounded view of genetic effects and phenotypic interactions. In this article we use data from a Multiparent Advanced Generation Inter-Cross (MAGIC) winter wheat population to explore Bayesian networks as a convenient and interpretable framework for the simultaneous modeling of multiple quantitative traits. We show that they are equivalent to multivariate genetic best linear unbiased prediction (GBLUP) and that they are competitive with single-trait elastic net and single-trait GBLUP in predictive performance. Finally, we discuss their relationship with other additive-effects models and their advantages in inference and interpretation. MAGIC populations provide an ideal setting for this kind of investigation because the very low population structure and large sample size result in predictive models with good power and limited confounding due to relatedness.     

Quantitative Analysis of Cell Migration Using Optical Flow

Neural crest cells exhibit dramatic migration behaviors as they populate their distant targets. Using a line of zebrafish expressing green fluorescent protein (sox10:EGFP) in neural crest cells we developed an assay to analyze and quantify cell migration as a population, and use it here to characterize in detail the subtle defects in cell migration caused by ethanol exposure during early development. The challenge was to quantify changes in the in vivo migration of all Sox10:EGFP expressing cells in the visual field of time-lapse movies. To perform this analysis we used an Optical Flow algorithm for motion detection and combined the analysis with a fit to an affine transformation. Through this analysis we detected and quantified significant differences in the cell migrations of Sox10:EGFP positive cranial neural crest populations in ethanol treated versus untreated embryos. Specifically, treatment affected migration by increasing the left-right asymmetry of the migrating cells and by altering the direction of cell movements. Thus, by applying this novel computational analysis, we were able to quantify the movements of populations of cells, allowing us to detect subtle changes in cell behaviors. Because cranial neural crest cells contribute to the formation of the frontal mass these subtle differences may underlie commonly observed facial asymmetries in normal human populations.     

Visualization and Analysis of Microtubule Dynamics Using Dual Color-Coded Display of Plus-End Labels

Investigating spatial and temporal control of microtubule dynamics in live cells is critical to understanding cell morphogenesis in development and disease. Tracking fluorescently labeled plus-end-tracking proteins over time has become a widely used method to study microtubule assembly. Here, we report a complementary approach that uses only two images of these labels to visualize and analyze microtubule dynamics at any given time. Using a simple color-coding scheme, labeled plus-ends from two sequential images are pseudocolored with different colors and then merged to display color-coded ends. Based on object recognition algorithms, these colored ends can be identified and segregated into dynamic groups corresponding to four events, including growth, rescue, catastrophe, and pause. Further analysis yields not only their spatial distribution throughout the cell but also provides measurements such as growth rate and direction for each labeled end. We have validated the method by comparing our results with ground-truth data derived from manual analysis as well as with data obtained using the tracking method. In addition, we have confirmed color-coded representation of different dynamic events by analyzing their history and fate. Finally, we have demonstrated the use of the method to investigate microtubule assembly in cells and provided guidance in selecting optimal image acquisition conditions. Thus, this simple computer vision method offers a unique and quantitative approach to study spatial regulation of microtubule dynamics in cells.     

Qualitative and Quantitative Analysis of DNA Fragmentation Using Digital Imaging

Apoptosis is an important and common pathway of cellular death. Differentiation from cellular necrosis and quantitation of apoptosis within the milieu of necrosis are analytical challenges. We describe the use of the RIT120 digital imaging software package for quantitative and qualitative analysis of apoptotic DNA ladders induced by a variety of agents, such as serum, tumor necrosis factor-α, transforming growth factor-β1, and nitric oxide. Autoradiographs of DNA ladders are densitometrically scanned to yield a set of curves with peaks corresponding to specific DNA fragments, thereby allowing quantitative subtraction of concurrent DNA degradation from necrotic death. Integration of the areas specifically under the peaks yields a quantitative measure of apoptosis. We provide a useful, rapid, and objective means to quantitate apoptosis, using relatively inexpensive hardware and software.     

Development of a Quantitative Polyacrylamide Gel Electrophoresis Analysis Using a Multichannel Radioactivity Counter for the Evaluation of Oligonucleotide-Bound Drug Carrier

A quantitative polyacrylamide gel electrophoresis (PAGE) analysis using a multichannel radioactivity counter was designed for the evaluation of³³P-labeled antisense oligonucleotide associated with polymeric drug carrier (nanoparticles). The proposed analytical method was first validated. The criteria of specificity, linearity, reliability, detection and quantification limits, and resolution power were determined. Results were compared to those obtained using liquid scintillation counting of crude samples or after solubilization of gel slices. The proposed method gave a better linearity and reliability than liquid scintillation counting of solubilized gel slices. In comparison with the liquid scintillation counting of crude samples, the method presented the advantage of being able to directly separate oligonucleotides differing by only one nucleotide in length. This method was applied for the separation of free oligonucleotides and oligonucleotides bound onto nanoparticles, allowing quantification of the amount of free and bound oligonucleotides without any further separation steps. Thus, because it is easy and rapid, the quantitative PAGE analysis using a multichannel radioactivity counter offers interesting possibilities for the characterization of oligonucleotide nanoparticles.     

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues¹. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.Download video file.^{(77M, mp4)}     

Label-Free Quantitative Proteomics and N-terminal Analysis of Human Metastatic Lung Cancer Cells

Proteomic analysis is helpful in identifying cancer-associated proteins that are differentially expressed and fragmented that can be annotated as dysregulated networks and pathways during metastasis. To examine meta-static process in lung cancer, we performed a proteomics study by label-free quantitative analysis and N-terminal analysis in 2 human non-small-cell lung cancer cell lines with disparate metastatic potentials—NCI-H1703 (primary cell, stage I) and NCI-H1755 (metastatic cell, stage IV). We identified 2130 proteins, 1355 of which were common to both cell lines. In the label-free quantitative analysis, we used the NSAF normalization method, resulting in 242 differential expressed proteins. For the N-terminal proteome analysis, 325 N-terminal peptides, including 45 novel fragments, were identified in the 2 cell lines. Based on two proteomic analysis, 11 quantitatively expressed proteins and 8 N-terminal peptides were enriched for the focal adhesion pathway. Most proteins from the quantitative analysis were upregulated in metastatic cancer cells, whereas novel fragment of CRKL was detected only in primary cancer cells. This study increases our understanding of the NSCLC metastasis proteome.     

Simulating Serial-Target Antibacterial Drug Synergies Using Flux Balance Analysis

Flux balance analysis (FBA) is an increasingly useful approach for modeling the behavior of metabolic systems. However, standard FBA modeling of genetic knockouts cannot predict drug combination synergies observed between serial metabolic targets, even though such synergies give rise to some of the most widely used antibiotic treatments. Here we extend FBA modeling to simulate responses to chemical inhibitors at varying concentrations, by diverting enzymatic flux to a waste reaction. This flux diversion yields very similar qualitative predictions to prior methods for single target activity. However, we find very different predictions for combinations, where flux diversion, which mimics the kinetics of competitive metabolic inhibitors, can explain serial target synergies between metabolic enzyme inhibitors that we confirmed in Escherichia coli cultures. FBA flux diversion opens the possibility for more accurate genome-scale predictions of drug synergies, which can be used to suggest treatments for infections and other diseases.     

吸入麻醉诱导阶段药物动力学模型的定量分析

考虑了给药浓度、氧流量对吸入麻醉诱导阶段的影响,建立了诱导期药物动力学模型.利用非线性拟合的方法给出了诱导阶段的麻醉药血药浓度的拟合曲线;讨论了给药浓度相同的情况下,氧流量对诱导阶段所需时间的影响,得到了诱导阶段的氧流量分别与吸入肺部的药物浓度和体内消除系数的关系式,利用这两个关系式和诱导阶段的一般解可确定在此阶段体内血药浓度和完成诱导的时间.     

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.     

Visualization of Ovarian Cancer Cells with Peptide VEGEGEEGEEY

The binding of peptide VEGEGEEGEEY to the surface of ovarian cancer cells was studied by confocal microscopy. It has been demonstrated that the peptide competes with hyaluronan for the binding to RHAMM (receptor for hyaluronan-mediated motility). It was found that peptide VEGEGEEGEEY specifically bound to RHAMM on ovarian cancer cell surface, and this interaction was blocked by anti-RHAMM antibodies. The peptide did not bind to RHAMM-knockout fibroblasts RHAMM^(–/–) but interacted with transfected fibroblasts RHAMM^(+/+), which further confirms the binding of the peptide to RHAMM. Evaluation of the peptide binding selectivity showed that the peptide reacts with cancer cells and does not bind to the surface of fibroblasts and normal cells. Thus, the specificity of binding of peptide FITC-VEGEGEEGEEY to RHAMM suggests a possibility of its application as a molecular probe for imaging and diagnosis of ovarian cancer at early stages.     

Intercellular Bridge Mediates Ca2+ Signals between Micropatterned Cells via IP3 and Ca2+ Diffusion

Intercellular bridges are plasma continuities formed at the end of the cytokinesis process that facilitate intercellular mass transport between the two daughter cells. However, it remains largely unknown how the intercellular bridge mediates Ca²⁺ communication between postmitotic cells. In this work, we utilize BV-2 microglial cells planted on dumbbell-shaped micropatterned assemblies to resolve spatiotemporal characteristics of Ca²⁺ signal transfer over the intercellular bridges. With the use of such micropatterns, considerably longer and more regular intercellular bridges can be obtained than in conventional cell cultures. The initial Ca²⁺ signal is evoked by mechanical stimulation of one of the daughter cells. A considerable time delay is observed between the arrivals of passive Ca²⁺ diffusion and endogenous Ca²⁺ response in the intercellular-bridge-connected cell, indicating two different pathways of the Ca²⁺ communication. Extracellular Ca²⁺ and the paracrine pathway have practically no effect on the endogenous Ca²⁺ response, demonstrated by application of Ca²⁺-free medium, exogenous ATP, and P2Y₁₃ receptor antagonist. In contrast, the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin and inositol trisphosphate (IP₃) receptor blocker 2-aminoethyl diphenylborate significantly inhibit the endogenous Ca²⁺ increase, which signifies involvement of IP₃-sensitive calcium store release. Notably, passive Ca²⁺ diffusion into the connected cell can clearly be detected when IP₃-sensitive calcium store release is abolished by 2-aminoethyl diphenylborate. Those observations prove that both passive Ca²⁺ diffusion and IP₃-mediated endogenous Ca²⁺ response contribute to the Ca²⁺ increase in intercellular-bridge-connected cells. Moreover, a simulation model agreed well with the experimental observations.