Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

General Staining and Segmentation Procedures for High Content Imaging and Analysis.

PubMed

Chambers, Kevin M; Mandavilli, Bhaskar S; Dolman, Nick J; Janes, Michael S

2018-01-01

Automated quantitative fluorescence microscopy, also known as high content imaging (HCI), is a rapidly growing analytical approach in cell biology. Because automated image analysis relies heavily on robust demarcation of cells and subcellular regions, reliable methods for labeling cells is a critical component of the HCI workflow. Labeling of cells for image segmentation is typically performed with fluorescent probes that bind DNA for nuclear-based cell demarcation or with those which react with proteins for image analysis based on whole cell staining. These reagents, along with instrument and software settings, play an important role in the successful segmentation of cells in a population for automated and quantitative image analysis. In this chapter, we describe standard procedures for labeling and image segmentation in both live and fixed cell samples. The chapter will also provide troubleshooting guidelines for some of the common problems associated with these aspects of HCI.

Label free quantitative proteomics analysis on the cisplatin resistance in ovarian cancer cells.

PubMed

Wang, F; Zhu, Y; Fang, S; Li, S; Liu, S

2017-05-20

Quantitative proteomics has been made great progress in recent years. Label free quantitative proteomics analysis based on the mass spectrometry is widely used. Using this technique, we determined the differentially expressed proteins in the cisplatin-sensitive ovarian cancer cells COC1 and cisplatin-resistant cells COC1/DDP before and after the application of cisplatin. Using the GO analysis, we classified those proteins into different subgroups bases on their cellular component, biological process, and molecular function. We also used KEGG pathway analysis to determine the key signal pathways that those proteins were involved in. There are 710 differential proteins between COC1 and COC1/DDP cells, 783 between COC1 and COC1/DDP cells treated with cisplatin, 917 between the COC1/DDP cells and COC1/DDP cells treated with LaCl3, 775 between COC1/DDP cells treated with cisplatin and COC1/DDP cells treated with cisplatin and LaCl3. Among the same 411 differentially expressed proteins in cisplatin-sensitive COC1 cells and cisplain-resistant COC1/DDP cells before and after cisplatin treatment, 14% of them were localized on the cell membrane. According to the KEGG results, differentially expressed proteins were classified into 21 groups. The most abundant proteins were involved in spliceosome. This study lays a foundation for deciphering the mechanism for drug resistance in ovarian tumor.

A gradient method for the quantitative analysis of cell movement and tissue flow and its application to the analysis of multicellular Dictyostelium development.

PubMed

Siegert, F; Weijer, C J; Nomura, A; Miike, H

1994-01-01

We describe the application of a novel image processing method, which allows quantitative analysis of cell and tissue movement in a series of digitized video images. The result is a vector velocity field showing average direction and velocity of movement for every pixel in the frame. We apply this method to the analysis of cell movement during different stages of the Dictyostelium developmental cycle. We analysed time-lapse video recordings of cell movement in single cells, mounds and slugs. The program can correctly assess the speed and direction of movement of either unlabelled or labelled cells in a time series of video images depending on the illumination conditions. Our analysis of cell movement during multicellular development shows that the entire morphogenesis of Dictyostelium is characterized by rotational cell movement. The analysis of cell and tissue movement by the velocity field method should be applicable to the analysis of morphogenetic processes in other systems such as gastrulation and neurulation in vertebrate embryos.

Quantitative Analysis of Cancer Cell Migration in Gradients Of EGF, HGF, and SDF-alpha Using a Microfluidic Chemotaxis Device

DTIC Science & Technology

2005-01-01

Quantitative Analysis of Cancer Cell Migration in Gradients of EGF, HGF, and SDF-alpha Using a Microfluidic Chemotaxis Device The University of California...allowing for parallel analysis . Additionally, simple methods of localizing gels into microdevices are demonstrated. The device was characterized by...To overcome some of these drawbacks, several approaches have utilized free diffusion to produce gradients in static environ - ments.5-9 However

Visualization and quantitative analysis of extrachromosomal telomere-repeat DNA in individual human cells by Halo-FISH

PubMed Central

Komosa, Martin; Root, Heather; Meyn, M. Stephen

2015-01-01

Current methods for characterizing extrachromosomal nuclear DNA in mammalian cells do not permit single-cell analysis, are often semi-quantitative and frequently biased toward the detection of circular species. To overcome these limitations, we developed Halo-FISH to visualize and quantitatively analyze extrachromosomal DNA in single cells. We demonstrate Halo-FISH by using it to analyze extrachromosomal telomere-repeat (ECTR) in human cells that use the Alternative Lengthening of Telomeres (ALT) pathway(s) to maintain telomere lengths. We find that GM847 and VA13 ALT cells average ∼80 detectable G/C-strand ECTR DNA molecules/nucleus, while U2OS ALT cells average ∼18 molecules/nucleus. In comparison, human primary and telomerase-positive cells contain <5 ECTR DNA molecules/nucleus. ECTR DNA in ALT cells exhibit striking cell-to-cell variations in number (<20 to >300), range widely in length (<1 to >200 kb) and are composed of primarily G- or C-strand telomere-repeat DNA. Halo-FISH enables, for the first time, the simultaneous analysis of ECTR DNA and chromosomal telomeres in a single cell. We find that ECTR DNA comprises ∼15% of telomere-repeat DNA in GM847 and VA13 cells, but <4% in U2OS cells. In addition to its use in ALT cell analysis, Halo-FISH can facilitate the study of a wide variety of extrachromosomal DNA in mammalian cells. PMID:25662602

Proflavine Hemisulfate as a Fluorescent Contrast Agent for Point-of-Care Cytology

PubMed Central

Prieto, Sandra P.; Powless, Amy J.; Boice, Jackson W.; Sharma, Shree G.; Muldoon, Timothy J.

2015-01-01

Proflavine hemisulfate, an acridine-derived fluorescent dye, can be used as a rapid stain for cytologic examination of biological specimens. Proflavine fluorescently stains cell nuclei and cytoplasmic structures, owing to its small amphipathic structure and ability to intercalate DNA. In this manuscript, we demonstrated the use of proflavine as a rapid cytologic dye on a number of specimens, including normal exfoliated oral squamous cells, cultured human oral squamous carcinoma cells, and leukocytes derived from whole blood specimens using a custom-built, portable, LED-illuminated fluorescence microscope. No incubation time was needed after suspending cells in 0.01% (w/v) proflavine diluted in saline. Images of proflavine stained oral cells had clearly visible nuclei as well as granular cytoplasm, while stained leukocytes exhibited bright nuclei, and highlighted the multilobar nature of nuclei in neutrophils. We also demonstrated the utility of quantitative analysis of digital images of proflavine stained cells, which can be used to detect significant morphological differences between different cell types. Proflavine stained oral cells have well-defined nuclei and cell membranes which allowed for quantitative analysis of nuclear to cytoplasmic ratios, as well as image texture analysis to extract quantitative image features. PMID:25962131

Proflavine Hemisulfate as a Fluorescent Contrast Agent for Point-of-Care Cytology.

PubMed

Prieto, Sandra P; Powless, Amy J; Boice, Jackson W; Sharma, Shree G; Muldoon, Timothy J

2015-01-01

Proflavine hemisulfate, an acridine-derived fluorescent dye, can be used as a rapid stain for cytologic examination of biological specimens. Proflavine fluorescently stains cell nuclei and cytoplasmic structures, owing to its small amphipathic structure and ability to intercalate DNA. In this manuscript, we demonstrated the use of proflavine as a rapid cytologic dye on a number of specimens, including normal exfoliated oral squamous cells, cultured human oral squamous carcinoma cells, and leukocytes derived from whole blood specimens using a custom-built, portable, LED-illuminated fluorescence microscope. No incubation time was needed after suspending cells in 0.01% (w/v) proflavine diluted in saline. Images of proflavine stained oral cells had clearly visible nuclei as well as granular cytoplasm, while stained leukocytes exhibited bright nuclei, and highlighted the multilobar nature of nuclei in neutrophils. We also demonstrated the utility of quantitative analysis of digital images of proflavine stained cells, which can be used to detect significant morphological differences between different cell types. Proflavine stained oral cells have well-defined nuclei and cell membranes which allowed for quantitative analysis of nuclear to cytoplasmic ratios, as well as image texture analysis to extract quantitative image features.

Determination of the Absolute Number of Cytokine mRNA Molecules within Individual Activated Human T Cells

NASA Technical Reports Server (NTRS)

Karr, Laurel J.; Marshall, Gwen; Hockett, Richard D.; Bucy, R. Pat; Curreri, Peter A. (Technical Monitor)

2002-01-01

A primary function of activated T cells is the expression and subsequent secretion of cytokines, which orchestrate the differentiation of other lymphocytes, modulate antigen presenting cell activity, and alter vascular endothelium to mediate an immune response. Since many features of immune regulation probably result from modest alterations of endogenous rates of multiple interacting processes, quantitative analysis of the frequency and specific activity of individual T cells is critically important. Using a coordinated set of quantitative methods, the absolute number of molecules of several key cytokine mRNA species in individual T cells has been determined. The frequency of human blood T cells activated in vitro by mitogens and recall protein antigens was determined by intracellular cytokine protein staining, in situ hybridization for cytokine mRNA, and by limiting dilution analysis for cytokine mRNA+ cells. The absolute number of mRNA molecules was simultaneously determined in both homogenates of the entire population of cells and in individual cells obtained by limiting dilution, using a quantitative, competitive RT-PCR assay. The absolute numbers of mRNA molecules in a population of cells divided by the frequency of individual positive cells, yielded essentially the same number of mRNA molecules per cell as direct analysis of individual cells by limiting dilution analysis. Mean numbers of mRNA per positive cell from both mitogen and antigen activated T cells, using these stimulation conditions, were 6000 for IL-2, 6300 for IFN-gamma, and 1600 for IL-4.

Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice.

PubMed

Joshi, Molishree; Keith Pittman, H; Haisch, Carl; Verbanac, Kathryn

2008-09-01

Quantitative real-time PCR (qPCR) is a sensitive technique for the detection and quantitation of specific DNA sequences. Here we describe a Taqman qPCR assay for quantification of tissue-localized, adoptively transferred enhanced green fluorescent protein (EGFP)-transgenic cells. A standard curve constructed from serial dilutions of a plasmid containing the EGFP transgene was (i) highly reproducible, (ii) detected as few as two copies, and (iii) was included in each qPCR assay. qPCR analysis of genomic DNA was used to determine transgene copy number in several mouse strains. Fluorescent microscopy of tissue sections showed that adoptively transferred vascular endothelial cells (VEC) from EGFP-transgenic mice specifically localized to tissue with metastatic tumors in syngeneic recipients. VEC microscopic enumeration of liver metastases strongly correlated with qPCR analysis of identical sections (Pearson correlation 0.81). EGFP was undetectable in tissue from control mice by qPCR. In another study using intra-tumor EGFP-VEC delivery to subcutaneous tumors, manual cell count and qPCR analysis of alternating sections also strongly correlated (Pearson correlation 0.82). Confocal microscopy of the subcutaneous tumor sections determined that visual fluorescent signals were frequently tissue artifacts. This qPCR methodology offers specific, objective, and rapid quantitation, uncomplicated by tissue autofluorescence, and should be readily transferable to other in vivo models to quantitate the biolocalization of transplanted cells.

Subsurface imaging and cell refractometry using quantitative phase/ shear-force feedback microscopy

NASA Astrophysics Data System (ADS)

Edward, Kert; Farahi, Faramarz

2009-10-01

Over the last few years, several novel quantitative phase imaging techniques have been developed for the study of biological cells. However, many of these techniques are encumbered by inherent limitations including 2π phase ambiguities and diffraction limited spatial resolution. In addition, subsurface information in the phase data is not exploited. We hereby present a novel quantitative phase imaging system without 2 π ambiguities, which also allows for subsurface imaging and cell refractometry studies. This is accomplished by utilizing simultaneously obtained shear-force topography information. We will demonstrate how the quantitative phase and topography data can be used for subsurface and cell refractometry analysis and will present results for a fabricated structure and a malaria infected red blood cell.

IB-LBM simulation of the haemocyte dynamics in a stenotic capillary.

PubMed

Yuan-Qing, Xu; Xiao-Ying, Tang; Fang-Bao, Tian; Yu-Hua, Peng; Yong, Xu; Yan-Jun, Zeng

2014-01-01

To study the behaviour of a haemocyte when crossing a stenotic capillary, the immersed boundary-lattice Boltzmann method was used to establish a quantitative analysis model. The haemocyte was assumed to be spherical and to have an elastic cell membrane, which can be driven by blood flow to adopt a highly deformable character. In the stenotic capillary, the spherical blood cell was stressed both by the flow and the wall dimension, and the cell shape was forced to be stretched to cross the stenosis. Our simulation investigated the haemocyte crossing process in detail. The velocity and pressure were anatomised to obtain information on how blood flows through a capillary and to estimate the degree of cell damage caused by excessive pressure. Quantitative velocity analysis results demonstrated that a large haemocyte crossing a small stenosis would have a noticeable effect on blood flow, while quantitative pressure distribution analysis results indicated that the crossing process would produce a special pressure distribution in the cell interior and to some extent a sudden change between the cell interior and the surrounding plasma.

Development of CD3 cell quantitation algorithms for renal allograft biopsy rejection assessment utilizing open source image analysis software.

PubMed

Moon, Andres; Smith, Geoffrey H; Kong, Jun; Rogers, Thomas E; Ellis, Carla L; Farris, Alton B Brad

2018-02-01

Renal allograft rejection diagnosis depends on assessment of parameters such as interstitial inflammation; however, studies have shown interobserver variability regarding interstitial inflammation assessment. Since automated image analysis quantitation can be reproducible, we devised customized analysis methods for CD3+ T-cell staining density as a measure of rejection severity and compared them with established commercial methods along with visual assessment. Renal biopsy CD3 immunohistochemistry slides (n = 45), including renal allografts with various degrees of acute cellular rejection (ACR) were scanned for whole slide images (WSIs). Inflammation was quantitated in the WSIs using pathologist visual assessment, commercial algorithms (Aperio nuclear algorithm for CD3+ cells/mm 2 and Aperio positive pixel count algorithm), and customized open source algorithms developed in ImageJ with thresholding/positive pixel counting (custom CD3+%) and identification of pixels fulfilling "maxima" criteria for CD3 expression (custom CD3+ cells/mm 2 ). Based on visual inspections of "markup" images, CD3 quantitation algorithms produced adequate accuracy. Additionally, CD3 quantitation algorithms correlated between each other and also with visual assessment in a statistically significant manner (r = 0.44 to 0.94, p = 0.003 to < 0.0001). Methods for assessing inflammation suggested a progression through the tubulointerstitial ACR grades, with statistically different results in borderline versus other ACR types, in all but the custom methods. Assessment of CD3-stained slides using various open source image analysis algorithms presents salient correlations with established methods of CD3 quantitation. These analysis techniques are promising and highly customizable, providing a form of on-slide "flow cytometry" that can facilitate additional diagnostic accuracy in tissue-based assessments.

Analytical validation of quantitative immunohistochemical assays of tumor infiltrating lymphocyte biomarkers.

PubMed

Singh, U; Cui, Y; Dimaano, N; Mehta, S; Pruitt, S K; Yearley, J; Laterza, O F; Juco, J W; Dogdas, B

2018-06-04

Tumor infiltrating lymphocytes (TIL), especially T-cells, have both prognostic and therapeutic applications. The presence of CD8+ effector T-cells and the ratio of CD8+ cells to FOXP3+ regulatory T-cells have been used as biomarkers of disease prognosis to predict response to various immunotherapies. Blocking the interaction between inhibitory receptors on T-cells and their ligands with therapeutic antibodies including atezolizumab, nivolumab, pembrolizumab and tremelimumab increases the immune response against cancer cells and has shown significant improvement in clinical benefits and survival in several different tumor types. The improved clinical outcome is presumed to be associated with a higher tumor infiltration; therefore, it is thought that more accurate methods for measuring the amount of TIL could assist prognosis and predict treatment response. We have developed and validated quantitative immunohistochemistry (IHC) assays for CD3, CD8 and FOXP3 for immunophenotyping T-lymphocytes in tumor tissue. Various types of formalin fixed, paraffin embedded (FFPE) tumor tissues were immunolabeled with anti-CD3, anti-CD8 and anti-FOXP3 antibodies using an IHC autostainer. The tumor area of stained tissues, including the invasive margin of the tumor, was scored by a pathologist (visual scoring) and by computer-based quantitative image analysis. Two image analysis scores were obtained for the staining of each biomarker: the percent positive cells in the tumor area and positive cells/mm 2 tumor area. Comparison of visual vs. image analysis scoring methods using regression analysis showed high correlation and indicated that quantitative image analysis can be used to score the number of positive cells in IHC stained slides. To demonstrate that the IHC assays produce consistent results in normal daily testing, we evaluated the specificity, sensitivity and reproducibility of the IHC assays using both visual and image analysis scoring methods. We found that CD3, CD8 and FOXP3 IHC assays met the fit-for-purpose analytical acceptance validation criteria and that they can be used to support clinical studies.

Live-cell confocal microscopy and quantitative 4D image analysis of anchor cell invasion through the basement membrane in C. elegans

PubMed Central

Kelley, Laura C.; Wang, Zheng; Hagedorn, Elliott J.; Wang, Lin; Shen, Wanqing; Lei, Shijun; Johnson, Sam A.; Sherwood, David R.

2018-01-01

Cell invasion through basement membrane (BM) barriers is crucial during development, leukocyte trafficking, and for the spread of cancer. Despite its importance in normal and diseased states, the mechanisms that direct invasion are poorly understood, in large part because of the inability to visualize dynamic cell-basement membrane interactions in vivo. This protocol describes multi-channel time-lapse confocal imaging of anchor cell invasion in live C. elegans. Methods presented include outline slide preparation and worm growth synchronization (15 min), mounting (20 min), image acquisition (20-180 min), image processing (20 min), and quantitative analysis (variable timing). Images acquired enable direct measurement of invasive dynamics including invadopodia formation, cell membrane protrusions, and BM removal. This protocol can be combined with genetic analysis, molecular activity probes, and optogenetic approaches to uncover molecular mechanisms underlying cell invasion. These methods can also be readily adapted for real-time analysis of cell migration, basement membrane turnover, and cell membrane dynamics by any worm laboratory. PMID:28880279

Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA).

PubMed

Narayan, Malathi; Seeley, Kent W; Jinwal, Umesh K

2016-06-01

Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA) on a mouse microglial (N9) cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier PXD003032.

Screening hypochromism (sieve effect) in red blood cells: a quantitative analysis

PubMed Central

Razi Naqvi, K.

2014-01-01

Multiwavelength UV-visible spectroscopy, Kramers-Kronig analysis, and several other experimental and theoretical tools have been applied over the last several decades to fathom absorption and scattering of light by suspensions of micron-sized pigmented particles, including red blood cells, but a satisfactory quantitative analysis of the difference between the absorption spectra of suspension of intact and lysed red blood cells is still lacking. It is stressed that such a comparison is meaningful only if the pertinent spectra are free from, or have been corrected for, scattering losses, and it is shown that Duysens’ theory can, whereas that of Vekshin cannot, account satisfactorily for the observed hypochromism of suspensions of red blood cells. PMID:24761307

Screening hypochromism (sieve effect) in red blood cells: a quantitative analysis.

PubMed

Razi Naqvi, K

2014-04-01

Multiwavelength UV-visible spectroscopy, Kramers-Kronig analysis, and several other experimental and theoretical tools have been applied over the last several decades to fathom absorption and scattering of light by suspensions of micron-sized pigmented particles, including red blood cells, but a satisfactory quantitative analysis of the difference between the absorption spectra of suspension of intact and lysed red blood cells is still lacking. It is stressed that such a comparison is meaningful only if the pertinent spectra are free from, or have been corrected for, scattering losses, and it is shown that Duysens' theory can, whereas that of Vekshin cannot, account satisfactorily for the observed hypochromism of suspensions of red blood cells.

Automated analysis of individual sperm cells using stain-free interferometric phase microscopy and machine learning.

PubMed

Mirsky, Simcha K; Barnea, Itay; Levi, Mattan; Greenspan, Hayit; Shaked, Natan T

2017-09-01

Currently, the delicate process of selecting sperm cells to be used for in vitro fertilization (IVF) is still based on the subjective, qualitative analysis of experienced clinicians using non-quantitative optical microscopy techniques. In this work, a method was developed for the automated analysis of sperm cells based on the quantitative phase maps acquired through use of interferometric phase microscopy (IPM). Over 1,400 human sperm cells from 8 donors were imaged using IPM, and an algorithm was designed to digitally isolate sperm cell heads from the quantitative phase maps while taking into consideration both the cell 3D morphology and contents, as well as acquire features describing sperm head morphology. A subset of these features was used to train a support vector machine (SVM) classifier to automatically classify sperm of good and bad morphology. The SVM achieves an area under the receiver operating characteristic curve of 88.59% and an area under the precision-recall curve of 88.67%, as well as precisions of 90% or higher. We believe that our automatic analysis can become the basis for objective and automatic sperm cell selection in IVF. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Quantitative phase-digital holographic microscopy: a new imaging modality to identify original cellular biomarkers of diseases

NASA Astrophysics Data System (ADS)

Marquet, P.; Rothenfusser, K.; Rappaz, B.; Depeursinge, C.; Jourdain, P.; Magistretti, P. J.

2016-03-01

Quantitative phase microscopy (QPM) has recently emerged as a powerful label-free technique in the field of living cell imaging allowing to non-invasively measure with a nanometric axial sensitivity cell structure and dynamics. Since the phase retardation of a light wave when transmitted through the observed cells, namely the quantitative phase signal (QPS), is sensitive to both cellular thickness and intracellular refractive index related to the cellular content, its accurate analysis allows to derive various cell parameters and monitor specific cell processes, which are very likely to identify new cell biomarkers. Specifically, quantitative phase-digital holographic microscopy (QP-DHM), thanks to its numerical flexibility facilitating parallelization and automation processes, represents an appealing imaging modality to both identify original cellular biomarkers of diseases as well to explore the underlying pathophysiological processes.

Multiplex Quantitative Histologic Analysis of Human Breast Cancer Cell Signaling and Cell Fate

DTIC Science & Technology

2010-05-01

Breast cancer, cell signaling, cell proliferation, histology, image analysis 15. NUMBER OF PAGES - 51 16. PRICE CODE 17. SECURITY CLASSIFICATION...revealed by individual stains in multiplex combinations; and (3) software (FARSIGHT) for automated multispectral image analysis that (i) segments...Task 3. Develop computational algorithms for multispectral immunohistological image analysis FARSIGHT software was developed to quantify intrinsic

Noninvasive characterization of the fission yeast cell cycle by monitoring dry mass with digital holographic microscopy.

PubMed

Rappaz, Benjamin; Cano, Elena; Colomb, Tristan; Kühn, Jonas; Depeursinge, Christian; Simanis, Viesturs; Magistretti, Pierre J; Marquet, Pierre

2009-01-01

Digital holography microscopy (DHM) is an optical technique which provides phase images yielding quantitative information about cell structure and cellular dynamics. Furthermore, the quantitative phase images allow the derivation of other parameters, including dry mass production, density, and spatial distribution. We have applied DHM to study the dry mass production rate and the dry mass surface density in wild-type and mutant fission yeast cells. Our study demonstrates the applicability of DHM as a tool for label-free quantitative analysis of the cell cycle and opens the possibility for its use in high-throughput screening.

Use of MRI in Differentiation of Papillary Renal Cell Carcinoma Subtypes: Qualitative and Quantitative Analysis.

PubMed

Doshi, Ankur M; Ream, Justin M; Kierans, Andrea S; Bilbily, Matthew; Rusinek, Henry; Huang, William C; Chandarana, Hersh

2016-03-01

The purpose of this study was to determine whether qualitative and quantitative MRI feature analysis is useful for differentiating type 1 from type 2 papillary renal cell carcinoma (PRCC). This retrospective study included 21 type 1 and 17 type 2 PRCCs evaluated with preoperative MRI. Two radiologists independently evaluated various qualitative features, including signal intensity, heterogeneity, and margin. For the quantitative analysis, a radiology fellow and a medical student independently drew 3D volumes of interest over the entire tumor on T2-weighted HASTE images, apparent diffusion coefficient parametric maps, and nephrographic phase contrast-enhanced MR images to derive first-order texture metrics. Qualitative and quantitative features were compared between the groups. For both readers, qualitative features with greater frequency in type 2 PRCC included heterogeneous enhancement, indistinct margin, and T2 heterogeneity (all, p < 0.035). Indistinct margins and heterogeneous enhancement were independent predictors (AUC, 0.822). Quantitative analysis revealed that apparent diffusion coefficient, HASTE, and contrast-enhanced entropy were greater in type 2 PRCC (p < 0.05; AUC, 0.682-0.716). A combined quantitative and qualitative model had an AUC of 0.859. Qualitative features within the model had interreader concordance of 84-95%, and the quantitative data had intraclass coefficients of 0.873-0.961. Qualitative and quantitative features can help discriminate between type 1 and type 2 PRCC. Quantitative analysis may capture useful information that complements the qualitative appearance while benefiting from high interobserver agreement.

3D/4D multiscale imaging in acute lymphoblastic leukemia cells: visualizing dynamics of cell death

NASA Astrophysics Data System (ADS)

Sarangapani, Sreelatha; Mohan, Rosmin Elsa; Patil, Ajeetkumar; Lang, Matthew J.; Asundi, Anand

2017-06-01

Quantitative phase detection is a new methodology that provides quantitative information on cellular morphology to monitor the cell status, drug response and toxicity. In this paper the morphological changes in acute leukemia cells treated with chitosan were detected using d'Bioimager a robust imaging system. Quantitative phase image of the cells was obtained with numerical analysis. Results show that the average area and optical volume of the chitosan treated cells is significantly reduced when compared with the control cells, which reveals the effect of chitosan on the cancer cells. From the results it can be attributed that d'Bioimager can be used as a non-invasive imaging alternative to measure the morphological changes of the living cells in real time.

Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

PubMed

Wu, Yi-Hsuan; Hu, Chia-Wei; Chien, Chih-Wei; Chen, Yu-Ju; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

2013-01-01

ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

Quantitative Proteomic Analysis of Human Lung Tumor Xenografts Treated with the Ectopic ATP Synthase Inhibitor Citreoviridin

PubMed Central

Wu, Yi-Hsuan; Hu, Chia-Wei; Chien, Chih-Wei; Chen, Yu-Ju; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

2013-01-01

ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy. PMID:23990911

Mass-spectrometric analysis of hydroperoxy- and hydroxy-derivatives of cardiolipin and phosphatidylserine in cells and tissues induced by pro-apoptotic and pro-inflammatory stimuli

PubMed Central

Tyurin, Vladimir A.; Tyurina, Yulia Y.; Jung, Mi-Yeon; Tungekar, Muhammad A.; Wasserloos, Karla J.; Bayir, Hülya; Greenberger, Joel S.; Kochanek, Patrick M.; Shvedova, Anna A.; Pitt, Bruce; Kagan, Valerian E.

2009-01-01

Oxidation of two anionic phospholipids - cardiolipin (CL) in mitochondria and phosphatidylserine (PS) in extramitochondrial compartments - are important signaling events, particularly during the execution of programmed cell death and clearance of apoptotic cells. Quantitative analysis of CL and PS oxidation products is central to understanding their molecular mechanisms of action. We combined the identification of diverse phospholipid molecular species by ESI-MS with quantitative assessments of lipid hydroperoxides using a fluorescence HPLC-based protocol. We characterized CL and PS oxidation products formed in a model system (cyt c/H2O2), in apoptotic cells (neurons, pulmonary artery endothelial cells) and mouse lung under inflammatory/oxidative stress conditions (hyperoxia, inhalation of single walled carbon nanotubes). Our results demonstrate the usefulness of this approach for quantitative assessments, identification of individual molecular species and structural characterization of anionic phospholipids that are involved in oxidative modification in cells and tissues. PMID:19328050

SuperSegger: robust image segmentation, analysis and lineage tracking of bacterial cells.

PubMed

Stylianidou, Stella; Brennan, Connor; Nissen, Silas B; Kuwada, Nathan J; Wiggins, Paul A

2016-11-01

Many quantitative cell biology questions require fast yet reliable automated image segmentation to identify and link cells from frame-to-frame, and characterize the cell morphology and fluorescence. We present SuperSegger, an automated MATLAB-based image processing package well-suited to quantitative analysis of high-throughput live-cell fluorescence microscopy of bacterial cells. SuperSegger incorporates machine-learning algorithms to optimize cellular boundaries and automated error resolution to reliably link cells from frame-to-frame. Unlike existing packages, it can reliably segment microcolonies with many cells, facilitating the analysis of cell-cycle dynamics in bacteria as well as cell-contact mediated phenomena. This package has a range of built-in capabilities for characterizing bacterial cells, including the identification of cell division events, mother, daughter and neighbouring cells, and computing statistics on cellular fluorescence, the location and intensity of fluorescent foci. SuperSegger provides a variety of postprocessing data visualization tools for single cell and population level analysis, such as histograms, kymographs, frame mosaics, movies and consensus images. Finally, we demonstrate the power of the package by analyzing lag phase growth with single cell resolution. © 2016 John Wiley & Sons Ltd.

Defining High-Risk Precursor Signaling to Advance Breast Cancer Risk Assessment and Prevention

DTIC Science & Technology

2017-03-01

KEYWORDS: 3. ACCOMPLISHMENTS: Aim 1: Functional analysis of progenitor and stem cells in high-risk tissues. Major Task 1Functional...and stem cells in high-risk tissues. Major Task 1: Quantitation of LP (Luminal Progenitor) and basal stem cell (MASC) populations A. Quantitation of...LP and basal stem cell (MASC) populations We have continued to add patients to the cohorts between months 12 and 24. (This reporting period

Quantitative Analysis, Design, and Fabrication of Biosensing and Bioprocessing Devices in Living Cells

DTIC Science & Technology

2015-03-10

AFRL-OSR-VA-TR-2015-0080 Biosensing and Bioprocessing Devices in Living Cells Domitilla Del Vecchio MASSACHUSETTS INSTITUTE OF TECHNOLOGY Final...Of Biosensing And Bioprocessing Devices In Living Cells FA9550-12-1-0129 D. Del Vecchio Massachusetts Institute of Technology -- 77 Massachusetts...research is to develop quantitative techniques for the de novo design and fabrication of biosensing devices in living cells . Such devices will be entirely

Analyzing the texture changes in the quantitative phase maps of adipocytes

NASA Astrophysics Data System (ADS)

Roitshtain, Darina; Sharabani-Yosef, Orna; Gefen, Amit; Shaked, Natan T.

2016-03-01

We present a new analysis tool for studying texture changes in the quantitative phase maps of live cells acquired by wide-field interferometry. The sensitivity of wide-field interferometry systems to small changes in refractive index enables visualizing cells and inner cell organelles without the using fluorescent dyes or other cell-invasive approaches, which may affect the measurement and require external labeling. Our label-free texture-analysis tool is based directly on the optical path delay profile of the sample and does not necessitate decoupling refractive index and thickness in the cell quantitative phase profile; thus, relevant parameters can be calculated using a single-frame acquisition. Our experimental system includes low-coherence wide-field interferometer, combined with simultaneous florescence microscopy system for validation. We used this system and analysis tool for studying lipid droplets formation in adipocytes. The latter demonstration is relevant for various cellular functions such as lipid metabolism, protein storage and degradation to viral replication. These processes are functionally linked to several physiological and pathological conditions, including obesity and metabolic diseases. Quantification of these biological phenomena based on the texture changes in the cell phase map has a potential as a new cellular diagnosis tool.

Quantitative biology of single neurons

PubMed Central

Eberwine, James; Lovatt, Ditte; Buckley, Peter; Dueck, Hannah; Francis, Chantal; Kim, Tae Kyung; Lee, Jaehee; Lee, Miler; Miyashiro, Kevin; Morris, Jacqueline; Peritz, Tiina; Schochet, Terri; Spaethling, Jennifer; Sul, Jai-Yoon; Kim, Junhyong

2012-01-01

The building blocks of complex biological systems are single cells. Fundamental insights gained from single-cell analysis promise to provide the framework for understanding normal biological systems development as well as the limits on systems/cellular ability to respond to disease. The interplay of cells to create functional systems is not well understood. Until recently, the study of single cells has concentrated primarily on morphological and physiological characterization. With the application of new highly sensitive molecular and genomic technologies, the quantitative biochemistry of single cells is now accessible. PMID:22915636

Multiplex Quantitative Histologic Analysis of Human Breast Cancer Cell Signaling and Cell Fate

DTIC Science & Technology

2008-05-01

stains. 15. SUBJECT TERMS Breast cancer, cell signaling, cell proliferation, histology, image analysis 16. SECURITY CLASSIFICATION OF: 17...fluorescence, and these DAPI-stained nuclei are often not counted during subsequent image analysis ). To study two analytes in the same tumor section or...analytes (p-ERK, p-AKT, Ki67) and for epithelial cytokeratin (CK), so that tumor cells may be identified during subsequent automated image analysis (as

Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis

PubMed Central

Falter, Christian; Ellinger, Dorothea; von Hülsen, Behrend; Heim, René; Voigt, Christian A.

2015-01-01

The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape – liquid cover glass technique (ACT) for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection (LM) coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the ACT for simple leaf epidermis preparation and the compatibility to LM and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant–microbe interaction with their potential outreach into crop breeding. PMID:25870605

Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis.

PubMed

Falter, Christian; Ellinger, Dorothea; von Hülsen, Behrend; Heim, René; Voigt, Christian A

2015-01-01

The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape - liquid cover glass technique (ACT) for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection (LM) coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the ACT for simple leaf epidermis preparation and the compatibility to LM and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant-microbe interaction with their potential outreach into crop breeding.

Dynamically monitoring the gene expression of dual fluorophore in the cell cycle with quantitative spectrum analysis

NASA Astrophysics Data System (ADS)

Lee, Ja-Yun; Wu, Tzong-Yuan; Hsu, I.-Jen

2008-04-01

The cloning and transcription techniques on gene cloned fluorescent proteins have been widely used in many applications. They have been used as reporters of some conditions in a series of reactions. However, it is usually difficult to monitor the specific target with the exactly number of proteins during the process in turbid media, especially at micrometer scales. We successfully revealed an alternative way to monitor the cell cycle behavior and quantitatively analyzed the target cells with green and red fluorescent proteins (GFP and RFP) during different phases of the cell cycle by quantitatively analyzing its behavior and also monitoring its spatial distribution.

Hyperplasia of type 2 pneumocytes following 0. 34 ppm nitrogen dioxide exposure: quantitation by image analysis. [Mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sherwin, R.P.; Richters, V.

1982-09-01

Swiss Webster male mice were exposed to intermittent 0.34 ppm nitrogen dioxide for 6 wk. Quantitative image analysis showed increased Type 2 cell numbers in each of the three lobes measured, with and without adjustment to alveolar wall measurements for lung volume normalization (e.g., P < .037 for Type 2 cell number adjusted to alveolar wall perimeters, combined lobe analysis of variance). The exposed animals dominated the upper quartile ranking of the cell number/alveolar area ratio computations (P < .025), which implied the presence of an especially susceptible subpopulation of animals. The Type 2 cell increase is believed to resultmore » from damage and loss of Type 1 cells, the reversibility and progression of which are presently unknown. The data also suggest an increased size of the Type 2 cell, and possibly slight atelectasis and/or edema of the alveolar walls.« less

[Quantitative analysis of the structure of neuronal dendritic spines in the striatum using the Leitz-ASM system].

PubMed

Leontovich, T A; Zvegintseva, E G

1985-10-01

Two principal classes of striatum long axonal neurons (sparsely ramified reticular cells and densely ramified dendritic cells) were analyzed quantitatively in four animal species: hedgehog, rabbit, dog and monkey. The cross section area, total dendritic length and the area of dendritic field were measured using "LEITZ-ASM" system. Classes of neurons studied were significantly different in dogs and monkeys, while no differences were noted between hedgehog and rabbit. Reticular neurons of different species varied much more than dendritic ones. Quantitative analysis has revealed the progressive increase in the complexity of dendritic tree in mammals from rabbit to monkey.

Phenotype classification of single cells using SRS microscopy, RNA sequencing, and microfluidics (Conference Presentation)

NASA Astrophysics Data System (ADS)

Streets, Aaron M.; Cao, Chen; Zhang, Xiannian; Huang, Yanyi

2016-03-01

Phenotype classification of single cells reveals biological variation that is masked in ensemble measurement. This heterogeneity is found in gene and protein expression as well as in cell morphology. Many techniques are available to probe phenotypic heterogeneity at the single cell level, for example quantitative imaging and single-cell RNA sequencing, but it is difficult to perform multiple assays on the same single cell. In order to directly track correlation between morphology and gene expression at the single cell level, we developed a microfluidic platform for quantitative coherent Raman imaging and immediate RNA sequencing (RNA-Seq) of single cells. With this device we actively sort and trap cells for analysis with stimulated Raman scattering microscopy (SRS). The cells are then processed in parallel pipelines for lysis, and preparation of cDNA for high-throughput transcriptome sequencing. SRS microscopy offers three-dimensional imaging with chemical specificity for quantitative analysis of protein and lipid distribution in single cells. Meanwhile, the microfluidic platform facilitates single-cell manipulation, minimizes contamination, and furthermore, provides improved RNA-Seq detection sensitivity and measurement precision, which is necessary for differentiating biological variability from technical noise. By combining coherent Raman microscopy with RNA sequencing, we can better understand the relationship between cellular morphology and gene expression at the single-cell level.

A correlative and quantitative imaging approach enabling characterization of primary cell-cell communication: Case of human CD4+ T cell-macrophage immunological synapses.

PubMed

Kasprowicz, Richard; Rand, Emma; O'Toole, Peter J; Signoret, Nathalie

2018-05-22

Cell-to-cell communication engages signaling and spatiotemporal reorganization events driven by highly context-dependent and dynamic intercellular interactions, which are difficult to capture within heterogeneous primary cell cultures. Here, we present a straightforward correlative imaging approach utilizing commonly available instrumentation to sample large numbers of cell-cell interaction events, allowing qualitative and quantitative characterization of rare functioning cell-conjugates based on calcium signals. We applied this approach to examine a previously uncharacterized immunological synapse, investigating autologous human blood CD4 + T cells and monocyte-derived macrophages (MDMs) forming functional conjugates in vitro. Populations of signaling conjugates were visualized, tracked and analyzed by combining live imaging, calcium recording and multivariate statistical analysis. Correlative immunofluorescence was added to quantify endogenous molecular recruitments at the cell-cell junction. By analyzing a large number of rare conjugates, we were able to define calcium signatures associated with different states of CD4 + T cell-MDM interactions. Quantitative image analysis of immunostained conjugates detected the propensity of endogenous T cell surface markers and intracellular organelles to polarize towards cell-cell junctions with high and sustained calcium signaling profiles, hence defining immunological synapses. Overall, we developed a broadly applicable approach enabling detailed single cell- and population-based investigations of rare cell-cell communication events with primary cells.

Quantitative Cell Cycle Analysis Based on an Endogenous All-in-One Reporter for Cell Tracking and Classification.

PubMed

Zerjatke, Thomas; Gak, Igor A; Kirova, Dilyana; Fuhrmann, Markus; Daniel, Katrin; Gonciarz, Magdalena; Müller, Doris; Glauche, Ingmar; Mansfeld, Jörg

2017-05-30

Cell cycle kinetics are crucial to cell fate decisions. Although live imaging has provided extensive insights into this relationship at the single-cell level, the limited number of fluorescent markers that can be used in a single experiment has hindered efforts to link the dynamics of individual proteins responsible for decision making directly to cell cycle progression. Here, we present fluorescently tagged endogenous proliferating cell nuclear antigen (PCNA) as an all-in-one cell cycle reporter that allows simultaneous analysis of cell cycle progression, including the transition into quiescence, and the dynamics of individual fate determinants. We also provide an image analysis pipeline for automated segmentation, tracking, and classification of all cell cycle phases. Combining the all-in-one reporter with labeled endogenous cyclin D1 and p21 as prime examples of cell-cycle-regulated fate determinants, we show how cell cycle and quantitative protein dynamics can be simultaneously extracted to gain insights into G1 phase regulation and responses to perturbations. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Single cell elemental analysis using nuclear microscopy

NASA Astrophysics Data System (ADS)

Ren, M. Q.; Thong, P. S. P.; Kara, U.; Watt, F.

1999-04-01

The use of Particle Induced X-ray Emission (PIXE), Rutherford Backscattering Spectrometry (RBS) and Scanning Transmission Ion Microscopy (STIM) to provide quantitative elemental analysis of single cells is an area which has high potential, particularly when the trace elements such as Ca, Fe, Zn and Cu can be monitored. We describe the methodology of sample preparation for two cell types, the procedures of cell imaging using STIM, and the quantitative elemental analysis of single cells using RBS and PIXE. Recent work on single cells at the Nuclear Microscopy Research Centre,National University of Singapore has centred around two research areas: (a) Apoptosis (programmed cell death), which has been recently implicated in a wide range of pathological conditions such as cancer, Parkinson's disease etc, and (b) Malaria (infection of red blood cells by the malaria parasite). Firstly we present results on the elemental analysis of human Chang liver cells (ATTCC CCL 13) where vanadium ions were used to trigger apoptosis, and demonstrate that nuclear microscopy has the capability of monitoring vanadium loading within individual cells. Secondly we present the results of elemental changes taking place in individual mouse red blood cells which have been infected with the malaria parasite and treated with the anti-malaria drug Qinghaosu (QHS).

Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification.

PubMed

Fuchs-Telka, Sabine; Fister, Susanne; Mester, Patrick-Julian; Wagner, Martin; Rossmanith, Peter

2017-02-01

DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120-150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction because of limited quantitative PCR inhibition by the aqueous eluate as well as no detectable DNA uptake. The newly developed method was able to efficiently lyse Gram-negative bacterial cells, whereas Gram-positive cells were protected by their thick cell wall. The performance of the final protocol resulted in quantitative DNA extraction efficiencies for Gram-negative bacteria similar to those obtained with a commercial kit, whereas the number of handling steps, and especially the time required, was dramatically reduced. Graphical Abstract After careful evaluation of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMPyr + ][Ntf 2 - ]) was identified as the most suitable ionic liquid for quantitative cell lysis and DNA extraction. When used for Gram-negative bacteria, the protocol presented is simple and very fast and achieves DNA extraction efficiencies similar to those obtained with a commercial kit. ddH 2 O double-distilled water, qPCR quantitative PCR.

An Image Analysis Method for the Precise Selection and Quantitation of Fluorescently Labeled Cellular Constituents

PubMed Central

Agley, Chibeza C.; Velloso, Cristiana P.; Lazarus, Norman R.

2012-01-01

The accurate measurement of the morphological characteristics of cells with nonuniform conformations presents difficulties. We report here a straightforward method using immunofluorescent staining and the commercially available imaging program Adobe Photoshop, which allows objective and precise information to be gathered on irregularly shaped cells. We have applied this measurement technique to the analysis of human muscle cells and their immunologically marked intracellular constituents, as these cells are prone to adopting a highly branched phenotype in culture. Use of this method can be used to overcome many of the long-standing limitations of conventional approaches for quantifying muscle cell size in vitro. In addition, wider applications of Photoshop as a quantitative and semiquantitative tool in immunocytochemistry are explored. PMID:22511600

Quantitative image analysis for investigating cell-matrix interactions

NASA Astrophysics Data System (ADS)

Burkel, Brian; Notbohm, Jacob

2017-07-01

The extracellular matrix provides both chemical and physical cues that control cellular processes such as migration, division, differentiation, and cancer progression. Cells can mechanically alter the matrix by applying forces that result in matrix displacements, which in turn may localize to form dense bands along which cells may migrate. To quantify the displacements, we use confocal microscopy and fluorescent labeling to acquire high-contrast images of the fibrous material. Using a technique for quantitative image analysis called digital volume correlation, we then compute the matrix displacements. Our experimental technology offers a means to quantify matrix mechanics and cell-matrix interactions. We are now using these experimental tools to modulate mechanical properties of the matrix to study cell contraction and migration.

The absolute counting of red cell-derived microparticles with red cell bead by flow rate based assay.

PubMed

Nantakomol, Duangdao; Imwong, Malika; Soontarawirat, Ingfar; Kotjanya, Duangporn; Khakhai, Chulalak; Ohashi, Jun; Nuchnoi, Pornlada

2009-05-01

Activation of red blood cell is associated with the formation of red cell-derived microparticles (RMPs). Analysis of circulating RMPs is becoming more refined and clinically useful. A quantitative Trucount tube method is the conventional method uses for quantitating RMPs. In this study, we validated a quantitative method called "flow rate based assay using red cell bead (FCB)" to measure circulating RMPs in the peripheral blood of healthy subjects. Citrated blood samples collected from 30 cases of healthy subjects were determined the RMPs count by using double labeling of annexin V-FITC and anti-glycophorin A-PE. The absolute RMPs numbers were measured by FCB, and the results were compared with the Trucount or with flow rate based calibration (FR). Statistical correlation and agreement were analyzed using linear regression and Bland-Altman analysis. There was no significant difference in the absolute number of RMPs quantitated by FCB when compared with those two reference methods including the Trucount tube and FR method. The absolute RMPs count obtained from FCB method was highly correlated with those obtained from Trucount tube (r(2) = 0.98, mean bias 4 cell/microl, limit of agreement [LOA] -20.3 to 28.3 cell/microl), and FR method (r(2) = 1, mean bias 10.3 cell/microl, and LOA -5.5 to 26.2 cell/microl). This study demonstrates that FCB is suitable and more affordable for RMPs quantitation in the clinical samples. This method is a low cost and interchangeable to latex bead-based method for generating the absolute counts in the resource-limited areas. (c) 2008 Clinical Cytometry Society.

Analysis of defect structure in silicon. Characterization of samples from UCP ingot 5848-13C

NASA Technical Reports Server (NTRS)

Natesh, R.; Guyer, T.; Stringfellow, G. B.

1982-01-01

Statistically significant quantitative structural imperfection measurements were made on samples from ubiquitous crystalline process (UCP) Ingot 5848 - 13 C. Important trends were noticed between the measured data, cell efficiency, and diffusion length. Grain boundary substructure appears to have an important effect on the conversion efficiency of solar cells from Semix material. Quantitative microscopy measurements give statistically significant information compared to other microanalytical techniques. A surface preparation technique to obtain proper contrast of structural defects suitable for QTM analysis was perfected.

Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level.

PubMed

Zhu, Zhi; Zhang, Wenhua; Leng, Xuefei; Zhang, Mingxia; Guan, Zhichao; Lu, Jiangquan; Yang, Chaoyong James

2012-10-21

Genetic alternations can serve as highly specific biomarkers to distinguish fatal bacteria or cancer cells from their normal counterparts. However, these mutations normally exist in very rare amount in the presence of a large excess of non-mutated analogs. Taking the notorious pathogen E. coli O157:H7 as the target analyte, we have developed an agarose droplet-based microfluidic ePCR method for highly sensitive, specific and quantitative detection of rare pathogens in the high background of normal bacteria. Massively parallel singleplex and multiplex PCR at the single-cell level in agarose droplets have been successfully established. Moreover, we challenged the system with rare pathogen detection and realized the sensitive and quantitative analysis of a single E. coli O157:H7 cell in the high background of 100,000 excess normal K12 cells. For the first time, we demonstrated rare pathogen detection through agarose droplet microfluidic ePCR. Such a multiplex single-cell agarose droplet amplification method enables ultra-high throughput and multi-parameter genetic analysis of large population of cells at the single-cell level to uncover the stochastic variations in biological systems.

Combinatorial modification of human histone H4 quantitated by two-dimensional liquid chromatography coupled with top down mass spectrometry.

PubMed

Pesavento, James J; Bullock, Courtney R; LeDuc, Richard D; Mizzen, Craig A; Kelleher, Neil L

2008-05-30

Quantitative proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and absolute quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >10(4) using a strictly Top Down mass spectrometric approach coupled with two dimensions of liquid chromatography. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quantitative analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quantitative analysis. This work represents the most comprehensive analysis of histone H4 forms present in human cells reported to date.

EVALUATION OF RAPID DNA EXTRACTION PROCEDURES FOR THE QUANTITATIVE DETECTION OF FUNGAL CELLS USING REAL TIME PCR ANALYSIS

EPA Science Inventory

The ease and rapidity of quantitative DNA sequence detection by real-time PCR instruments promises to make their use increasingly common for the microbial analysis many different types of environmental samples. To fully exploit the capabilities of these instruments, correspondin...

Isolating specific cell and tissue compartments from 3D images for quantitative regional distribution analysis using novel computer algorithms.

PubMed

Fenrich, Keith K; Zhao, Ethan Y; Wei, Yuan; Garg, Anirudh; Rose, P Ken

2014-04-15

Isolating specific cellular and tissue compartments from 3D image stacks for quantitative distribution analysis is crucial for understanding cellular and tissue physiology under normal and pathological conditions. Current approaches are limited because they are designed to map the distributions of synapses onto the dendrites of stained neurons and/or require specific proprietary software packages for their implementation. To overcome these obstacles, we developed algorithms to Grow and Shrink Volumes of Interest (GSVI) to isolate specific cellular and tissue compartments from 3D image stacks for quantitative analysis and incorporated these algorithms into a user-friendly computer program that is open source and downloadable at no cost. The GSVI algorithm was used to isolate perivascular regions in the cortex of live animals and cell membrane regions of stained spinal motoneurons in histological sections. We tracked the real-time, intravital biodistribution of injected fluorophores with sub-cellular resolution from the vascular lumen to the perivascular and parenchymal space following a vascular microlesion, and mapped the precise distributions of membrane-associated KCC2 and gephyrin immunolabeling in dendritic and somatic regions of spinal motoneurons. Compared to existing approaches, the GSVI approach is specifically designed for isolating perivascular regions and membrane-associated regions for quantitative analysis, is user-friendly, and free. The GSVI algorithm is useful to quantify regional differences of stained biomarkers (e.g., cell membrane-associated channels) in relation to cell functions, and the effects of therapeutic strategies on the redistributions of biomolecules, drugs, and cells in diseased or injured tissues. Copyright © 2014 Elsevier B.V. All rights reserved.

Transcriptional profiling of CD31(+) cells isolated from murine embryonic stem cells.

PubMed

Mariappan, Devi; Winkler, Johannes; Chen, Shuhua; Schulz, Herbert; Hescheler, Jürgen; Sachinidis, Agapios

2009-02-01

Identification of genes involved in endothelial differentiation is of great interest for the understanding of the cellular and molecular mechanisms involved in the development of new blood vessels. Mouse embryonic stem (mES) cells serve as a potential source of endothelial cells for transcriptomic analysis. We isolated endothelial cells from 8-days old embryoid bodies by immuno-magnetic separation using platelet endothelial cell adhesion molecule-1 (also known as CD31) expressed on both early and mature endothelial cells. CD31(+) cells exhibit endothelial-like behavior by being able to incorporate DiI-labeled acetylated low-density lipoprotein as well as form tubular structures on matrigel. Quantitative and semi-quantitative PCR analysis further demonstrated the increased expression of endothelial transcripts. To ascertain the specific transcriptomic identity of the CD31(+) cells, large-scale microarray analysis was carried out. Comparative bioinformatic analysis reveals an enrichment of the gene ontology categories angiogenesis, blood vessel morphogenesis, vasculogenesis and blood coagulation in the CD31(+) cell population. Based on the transcriptomic signatures of the CD31(+) cells, we conclude that this ES cell-derived population contains endothelial-like cells expressing a mesodermal marker BMP2 and possess an angiogenic potential. The transcriptomic characterization of CD31(+) cells enables an in vitro functional genomic model to identify genes required for angiogenesis.

Automated classification of cell morphology by coherence-controlled holographic microscopy

NASA Astrophysics Data System (ADS)

Strbkova, Lenka; Zicha, Daniel; Vesely, Pavel; Chmelik, Radim

2017-08-01

In the last few years, classification of cells by machine learning has become frequently used in biology. However, most of the approaches are based on morphometric (MO) features, which are not quantitative in terms of cell mass. This may result in poor classification accuracy. Here, we study the potential contribution of coherence-controlled holographic microscopy enabling quantitative phase imaging for the classification of cell morphologies. We compare our approach with the commonly used method based on MO features. We tested both classification approaches in an experiment with nutritionally deprived cancer tissue cells, while employing several supervised machine learning algorithms. Most of the classifiers provided higher performance when quantitative phase features were employed. Based on the results, it can be concluded that the quantitative phase features played an important role in improving the performance of the classification. The methodology could be valuable help in refining the monitoring of live cells in an automated fashion. We believe that coherence-controlled holographic microscopy, as a tool for quantitative phase imaging, offers all preconditions for the accurate automated analysis of live cell behavior while enabling noninvasive label-free imaging with sufficient contrast and high-spatiotemporal phase sensitivity.

Automated classification of cell morphology by coherence-controlled holographic microscopy.

PubMed

Strbkova, Lenka; Zicha, Daniel; Vesely, Pavel; Chmelik, Radim

2017-08-01

In the last few years, classification of cells by machine learning has become frequently used in biology. However, most of the approaches are based on morphometric (MO) features, which are not quantitative in terms of cell mass. This may result in poor classification accuracy. Here, we study the potential contribution of coherence-controlled holographic microscopy enabling quantitative phase imaging for the classification of cell morphologies. We compare our approach with the commonly used method based on MO features. We tested both classification approaches in an experiment with nutritionally deprived cancer tissue cells, while employing several supervised machine learning algorithms. Most of the classifiers provided higher performance when quantitative phase features were employed. Based on the results, it can be concluded that the quantitative phase features played an important role in improving the performance of the classification. The methodology could be valuable help in refining the monitoring of live cells in an automated fashion. We believe that coherence-controlled holographic microscopy, as a tool for quantitative phase imaging, offers all preconditions for the accurate automated analysis of live cell behavior while enabling noninvasive label-free imaging with sufficient contrast and high-spatiotemporal phase sensitivity. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Quantitative investigation of red blood cell three-dimensional geometric and chemical changes in the storage lesion using digital holographic microscopy.

PubMed

Jaferzadeh, Keyvan; Moon, Inkyu

2015-11-01

Quantitative phase information obtained by digital holographic microscopy (DHM) can provide new insight into the functions and morphology of single red blood cells (RBCs). Since the functionality of a RBC is related to its three-dimensional (3-D) shape, quantitative 3-D geometric changes induced by storage time can help hematologists realize its optimal functionality period. We quantitatively investigate RBC 3-D geometric changes in the storage lesion using DHM. Our experimental results show that the substantial geometric transformation of the biconcave-shaped RBCs to the spherocyte occurs due to RBC storage lesion. This transformation leads to progressive loss of cell surface area, surface-to-volume ratio, and functionality of RBCs. Furthermore, our quantitative analysis shows that there are significant correlations between chemical and morphological properties of RBCs.

Multidimensional analysis of the frequencies and rates of cytokine secretion from single cells by quantitative microengraving.

PubMed

Han, Qing; Bradshaw, Elizabeth M; Nilsson, Björn; Hafler, David A; Love, J Christopher

2010-06-07

The large diversity of cells that comprise the human immune system requires methods that can resolve the individual contributions of specific subsets to an immunological response. Microengraving is process that uses a dense, elastomeric array of microwells to generate microarrays of proteins secreted from large numbers of individual live cells (approximately 10(4)-10(5) cells/assay). In this paper, we describe an approach based on this technology to quantify the rates of secretion from single immune cells. Numerical simulations of the microengraving process indicated an operating regime between 30 min-4 h that permits quantitative analysis of the rates of secretion. Through experimental validation, we demonstrate that microengraving can provide quantitative measurements of both the frequencies and the distribution in rates of secretion for up to four cytokines simultaneously released from individual viable primary immune cells. The experimental limits of detection ranged from 0.5 to 4 molecules/s for IL-6, IL-17, IFNgamma, IL-2, and TNFalpha. These multidimensional measures resolve the number and intensities of responses by cells exposed to stimuli with greater sensitivity than single-parameter assays for cytokine release. We show that cells from different donors exhibit distinct responses based on both the frequency and magnitude of cytokine secretion when stimulated under different activating conditions. Primary T cells with specific profiles of secretion can also be recovered after microengraving for subsequent expansion in vitro. These examples demonstrate the utility of quantitative, multidimensional profiles of single cells for analyzing the diversity and dynamics of immune responses in vitro and for identifying rare cells from clinical samples.

Flow cytometric determination of quantitative immunophenotypes

NASA Astrophysics Data System (ADS)

Redelman, Douglas; Ensign, Wayne; Roberts, Don

2001-05-01

Immunofluorescent flow cytometric analysis of peripheral blood leucocytes is most commonly used to identify and enumerate cells defined by one or more clusters of differentiation (CD) antigens. Although less widely employed, quantitative tests that measure the amounts of CD antigens expressed per cell are used in some situations such as the characterization of lymphomas and leukocytes or the measurement of CD38 on CD3plu8pluT cells in HIV infected individuals. The CD antigens used to identify leukocyte populations are functionally important molecules and it is known that under- or over-expression of some CD antigens can affect cellular responses. For example, high or low expression of CD19 on B cells is associated with autoimmune conditions or depressed antibody responses, respectively. In the current studies, the quantitative expression of CD antigens on T cells, B cells and monocytes was determined in a group of age and sex-matched Marines at several times before and after training exercises. There was substantial variation among these individuals in the quantitative expression of CD antigens and in the number of cells in various populations. However, there was relatively little variation within individuals during the two months they were examined. Thus, the number of cells in leukocyte sub-populations and the amount of CD antigens expressed per cell appear to comprise a characteristic quantitative immunophenotype.

Multichannel series piezoelectric quartz crystal cell sensor for real time and quantitative monitoring of the living cell and assessment of cytotoxicity.

PubMed

Tong, Feifei; Lian, Yan; Zhou, Huang; Shi, Xiaohong; He, Fengjiao

2014-10-21

A new multichannel series piezoelectric quartz crystal (MSPQC) cell sensor for real time monitoring of living cells in vitro was reported in this paper. The constructed sensor was used successfully to monitor adhesion, spreading, proliferation, and apoptosis of MG63 osteosarcoma cells and investigate the effects of different concentrations of cobalt chloride on MG63 cells. Quantitative real time and dynamic cell analyses data were conducted using the MSPQC cell sensor. Compared with methods such as fluorescence staining and morphology observation by microscopy, the MSPQC cell sensor is noninvasive, label free, simple, cheap, and capable of online monitoring. It can automatically record the growth status of cells and quantitatively evaluate cell proliferation and the apoptotic response to drugs. It will be a valuable detection and analysis tool for the acquisition of cellular level information and is anticipated to have application in the field of cell biology research or cytotoxicity testing in the future.

[The application of stereology in radiology imaging and cell biology fields].

PubMed

Hu, Na; Wang, Yan; Feng, Yuanming; Lin, Wang

2012-08-01

Stereology is an interdisciplinary method for 3D morphological study developed from mathematics and morphology. It is widely used in medical image analysis and cell biology studies. Because of its unbiased, simple, fast, reliable and non-invasive characteristics, stereology has been widely used in biomedical areas for quantitative analysis and statistics, such as histology, pathology and medical imaging. Because the stereological parameters show distinct differences in different pathology, many scholars use stereological methods to do quantitative analysis in their studies in recent years, for example, in the areas of the condition of cancer cells, tumor grade, disease development and the patient's prognosis, etc. This paper describes the stereological concept and estimation methods, also illustrates the applications of stereology in the fields of CT images, MRI images and cell biology, and finally reflects the universality, the superiority and reliability of stereology.

qSR: a quantitative super-resolution analysis tool reveals the cell-cycle dependent organization of RNA Polymerase I in live human cells.

PubMed

Andrews, J O; Conway, W; Cho, W -K; Narayanan, A; Spille, J -H; Jayanth, N; Inoue, T; Mullen, S; Thaler, J; Cissé, I I

2018-05-09

We present qSR, an analytical tool for the quantitative analysis of single molecule based super-resolution data. The software is created as an open-source platform integrating multiple algorithms for rigorous spatial and temporal characterizations of protein clusters in super-resolution data of living cells. First, we illustrate qSR using a sample live cell data of RNA Polymerase II (Pol II) as an example of highly dynamic sub-diffractive clusters. Then we utilize qSR to investigate the organization and dynamics of endogenous RNA Polymerase I (Pol I) in live human cells, throughout the cell cycle. Our analysis reveals a previously uncharacterized transient clustering of Pol I. Both stable and transient populations of Pol I clusters co-exist in individual living cells, and their relative fraction vary during cell cycle, in a manner correlating with global gene expression. Thus, qSR serves to facilitate the study of protein organization and dynamics with very high spatial and temporal resolutions directly in live cell.

Single shot white light interference microscopy with colour fringe analysis for quantitative phase imaging of biological cells

NASA Astrophysics Data System (ADS)

Srivastava, Vishal; Mehta, D. S.

2013-02-01

To quantitatively obtain the phase map of Onion and human red blood cell (RBC) from white light interferogram we used Hilbert transform color fringe analysis technique. The three Red, Blue and Green color components are decomposed from single white light interferogram and Refractive index profile for Red, Blue and Green colour were computed in a completely non-invasive manner for Onion and human RBC. The present technique might be useful for non-invasive determination of the refractive index variation within cells and tissues and morphological features of sample with ease of operation and low cost.

Quantitative proteomic analysis of intact plastids.

PubMed

Shiraya, Takeshi; Kaneko, Kentaro; Mitsui, Toshiaki

2014-01-01

Plastids are specialized cell organelles in plant cells that are differentiated into various forms including chloroplasts, chromoplasts, and amyloplasts, and fulfill important functions in maintaining the overall cell metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis possible while maintaining the plastid functions are provided; in addition, a quantitative shotgun method for analyzing the composition and changes in the content of proteins in plastids as a result of environmental impacts is described.

Segmentation and Quantitative Analysis of Apoptosis of Chinese Hamster Ovary Cells from Fluorescence Microscopy Images.

PubMed

Du, Yuncheng; Budman, Hector M; Duever, Thomas A

2017-06-01

Accurate and fast quantitative analysis of living cells from fluorescence microscopy images is useful for evaluating experimental outcomes and cell culture protocols. An algorithm is developed in this work to automatically segment and distinguish apoptotic cells from normal cells. The algorithm involves three steps consisting of two segmentation steps and a classification step. The segmentation steps are: (i) a coarse segmentation, combining a range filter with a marching square method, is used as a prefiltering step to provide the approximate positions of cells within a two-dimensional matrix used to store cells' images and the count of the number of cells for a given image; and (ii) a fine segmentation step using the Active Contours Without Edges method is applied to the boundaries of cells identified in the coarse segmentation step. Although this basic two-step approach provides accurate edges when the cells in a given image are sparsely distributed, the occurrence of clusters of cells in high cell density samples requires further processing. Hence, a novel algorithm for clusters is developed to identify the edges of cells within clusters and to approximate their morphological features. Based on the segmentation results, a support vector machine classifier that uses three morphological features: the mean value of pixel intensities in the cellular regions, the variance of pixel intensities in the vicinity of cell boundaries, and the lengths of the boundaries, is developed for distinguishing apoptotic cells from normal cells. The algorithm is shown to be efficient in terms of computational time, quantitative analysis, and differentiation accuracy, as compared with the use of the active contours method without the proposed preliminary coarse segmentation step.

Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells.

PubMed

Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

2016-01-29

Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics.

Note: An automated image analysis method for high-throughput classification of surface-bound bacterial cell motions.

PubMed

Shen, Simon; Syal, Karan; Tao, Nongjian; Wang, Shaopeng

2015-12-01

We present a Single-Cell Motion Characterization System (SiCMoCS) to automatically extract bacterial cell morphological features from microscope images and use those features to automatically classify cell motion for rod shaped motile bacterial cells. In some imaging based studies, bacteria cells need to be attached to the surface for time-lapse observation of cellular processes such as cell membrane-protein interactions and membrane elasticity. These studies often generate large volumes of images. Extracting accurate bacterial cell morphology features from these images is critical for quantitative assessment. Using SiCMoCS, we demonstrated simultaneous and automated motion tracking and classification of hundreds of individual cells in an image sequence of several hundred frames. This is a significant improvement from traditional manual and semi-automated approaches to segmenting bacterial cells based on empirical thresholds, and a first attempt to automatically classify bacterial motion types for motile rod shaped bacterial cells, which enables rapid and quantitative analysis of various types of bacterial motion.

Single-Cell Quantitative PCR: Advances and Potential in Cancer Diagnostics.

PubMed

Ok, Chi Young; Singh, Rajesh R; Salim, Alaa A

2016-01-01

Tissues are heterogeneous in their components. If cells of interest are a minor population of collected tissue, it would be difficult to obtain genetic or genomic information of the interested cell population with conventional genomic DNA extraction from the collected tissue. Single-cell DNA analysis is important in the analysis of genetics of cell clonality, genetic anticipation, and single-cell DNA polymorphisms. Single-cell PCR using Single Cell Ampligrid/GeXP platform is described in this chapter.

[Quantitative characterization of nuclear polymorphism of the epithelial cells in fibroadenomatosis and cancer of the breast].

PubMed

Ganina, K P; Petunin, Iu I; Timoshenko, Ia G

1989-01-01

A method for quantitative analysis of epithelial cell nuclear polymorphism was suggested, viz. identification of general statistical population using Petunin's criterion. This criterion was employed to assess heterogeneity of visible surface of interphase epithelial cell nuclei and to assay nuclear DNA level in fibroadenomatous hyperplasia and cancer of the breast. Heterogeneity index (h), alongside with other parameters, appeared useful for quantitative assessment of the disease: heterogeneity index values ranging 0.1-0.4 point to pronounced heterogeneity of epithelial cell nucleus surface and DNA level, and are suggestive of malignant transformation of tissue, whereas benign proliferation of the epithelium is usually characterized by 0.4 less than h less than or equal to 0.9.

Wavelet-based multiscale analysis of bioimpedance data measured by electric cell-substrate impedance sensing for classification of cancerous and normal cells.

PubMed

Das, Debanjan; Shiladitya, Kumar; Biswas, Karabi; Dutta, Pranab Kumar; Parekh, Aditya; Mandal, Mahitosh; Das, Soumen

2015-12-01

The paper presents a study to differentiate normal and cancerous cells using label-free bioimpedance signal measured by electric cell-substrate impedance sensing. The real-time-measured bioimpedance data of human breast cancer cells and human epithelial normal cells employs fluctuations of impedance value due to cellular micromotions resulting from dynamic structural rearrangement of membrane protrusions under nonagitated condition. Here, a wavelet-based multiscale quantitative analysis technique has been applied to analyze the fluctuations in bioimpedance. The study demonstrates a method to classify cancerous and normal cells from the signature of their impedance fluctuations. The fluctuations associated with cellular micromotion are quantified in terms of cellular energy, cellular power dissipation, and cellular moments. The cellular energy and power dissipation are found higher for cancerous cells associated with higher micromotions in cancer cells. The initial study suggests that proposed wavelet-based quantitative technique promises to be an effective method to analyze real-time bioimpedance signal for distinguishing cancer and normal cells.

Quantitative study of mammalian cells by scanning transmission soft X-ray microscopy

NASA Astrophysics Data System (ADS)

Shinohara, K.; Ohigashi, T.; Toné, S.; Kado, M.; Ito, A.

2017-06-01

Molecular distribution in mammalian cells was studied by soft X-ray scanning transmission microscopy with respect to the quantitative aspect of analysis. NEXAFS profiles at the C, N and O K-absorption edges were combined and used for the analysis. For the estimation of quantity for nucleic acids and proteins, NEXAFS profiles of DNA and bovine serum albumin (BSA) at the N K-absorption edge were applied assuming that those were their representatives. The method has a potential to explore the other molecular components than nucleic acids and proteins.

A versatile pipeline for the multi-scale digital reconstruction and quantitative analysis of 3D tissue architecture

PubMed Central

Morales-Navarrete, Hernán; Segovia-Miranda, Fabián; Klukowski, Piotr; Meyer, Kirstin; Nonaka, Hidenori; Marsico, Giovanni; Chernykh, Mikhail; Kalaidzidis, Alexander; Zerial, Marino; Kalaidzidis, Yannis

2015-01-01

A prerequisite for the systems biology analysis of tissues is an accurate digital three-dimensional reconstruction of tissue structure based on images of markers covering multiple scales. Here, we designed a flexible pipeline for the multi-scale reconstruction and quantitative morphological analysis of tissue architecture from microscopy images. Our pipeline includes newly developed algorithms that address specific challenges of thick dense tissue reconstruction. Our implementation allows for a flexible workflow, scalable to high-throughput analysis and applicable to various mammalian tissues. We applied it to the analysis of liver tissue and extracted quantitative parameters of sinusoids, bile canaliculi and cell shapes, recognizing different liver cell types with high accuracy. Using our platform, we uncovered an unexpected zonation pattern of hepatocytes with different size, nuclei and DNA content, thus revealing new features of liver tissue organization. The pipeline also proved effective to analyse lung and kidney tissue, demonstrating its generality and robustness. DOI: http://dx.doi.org/10.7554/eLife.11214.001 PMID:26673893

Integrative Analysis of Subcellular Quantitative Proteomics Studies Reveals Functional Cytoskeleton Membrane-Lipid Raft Interactions in Cancer.

PubMed

Shah, Anup D; Inder, Kerry L; Shah, Alok K; Cristino, Alexandre S; McKie, Arthur B; Gabra, Hani; Davis, Melissa J; Hill, Michelle M

2016-10-07

Lipid rafts are dynamic membrane microdomains that orchestrate molecular interactions and are implicated in cancer development. To understand the functions of lipid rafts in cancer, we performed an integrated analysis of quantitative lipid raft proteomics data sets modeling progression in breast cancer, melanoma, and renal cell carcinoma. This analysis revealed that cancer development is associated with increased membrane raft-cytoskeleton interactions, with ∼40% of elevated lipid raft proteins being cytoskeletal components. Previous studies suggest a potential functional role for the raft-cytoskeleton in the action of the putative tumor suppressors PTRF/Cavin-1 and Merlin. To extend the observation, we examined lipid raft proteome modulation by an unrelated tumor suppressor opioid binding protein cell-adhesion molecule (OPCML) in ovarian cancer SKOV3 cells. In agreement with the other model systems, quantitative proteomics revealed that 39% of OPCML-depleted lipid raft proteins are cytoskeletal components, with microfilaments and intermediate filaments specifically down-regulated. Furthermore, protein-protein interaction network and simulation analysis showed significantly higher interactions among cancer raft proteins compared with general human raft proteins. Collectively, these results suggest increased cytoskeleton-mediated stabilization of lipid raft domains with greater molecular interactions as a common, functional, and reversible feature of cancer cells.

Quantitative protein expression and cell surface characteristics of Escherichia coli MG1655 biofilms.

PubMed

Mukherjee, Joy; Ow, Saw Yen; Noirel, Josselin; Biggs, Catherine A

2011-02-01

Cell surface physicochemical characterization techniques were combined with quantitative changes in protein expression, to investigate the biological and biophysical changes of Escherichia coli MG1655 cells when grown as a biofilm (BIO). The overall surface charge of BIO cells was found to be less negative, highlighting the need for a lower electrophoretic mobility for attachment to occur. Comparison of the chemical functional groups on the cell surface showed similar profiles, with the absorbance intensity higher for proteins and carbohydrates in the BIO cells. Quantitative proteomic analysis demonstrated that 3 proteins were significantly increased, and 9 proteins significantly decreased in abundance, in cells grown as a BIO compared to their planktonic counterparts, with 7 of these total 12 proteins unique to this study. Proteins showing significant increased or decreased abundance include proteins involved in acid resistance, DNA protection and binding and ABC transporters. Further predictive analysis of the metabolic pathways showed an increased abundance of the amino acid metabolism and tricarboxylic acid (TCA) cycle, with a decrease in expression within the pentose phosphate and glycolysis pathways. It is therefore hypothesized that cells grown as a BIO are still energetically viable potentially using amino acids as an indirect carbon backbone source into the TCA cycle. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-Content Screening for Quantitative Cell Biology.

PubMed

Mattiazzi Usaj, Mojca; Styles, Erin B; Verster, Adrian J; Friesen, Helena; Boone, Charles; Andrews, Brenda J

2016-08-01

High-content screening (HCS), which combines automated fluorescence microscopy with quantitative image analysis, allows the acquisition of unbiased multiparametric data at the single cell level. This approach has been used to address diverse biological questions and identify a plethora of quantitative phenotypes of varying complexity in numerous different model systems. Here, we describe some recent applications of HCS, ranging from the identification of genes required for specific biological processes to the characterization of genetic interactions. We review the steps involved in the design of useful biological assays and automated image analysis, and describe major challenges associated with each. Additionally, we highlight emerging technologies and future challenges, and discuss how the field of HCS might be enhanced in the future. Copyright © 2016 Elsevier Ltd. All rights reserved.

A strategy to apply quantitative epistasis analysis on developmental traits.

PubMed

Labocha, Marta K; Yuan, Wang; Aleman-Meza, Boanerges; Zhong, Weiwei

2017-05-15

Genetic interactions are keys to understand complex traits and evolution. Epistasis analysis is an effective method to map genetic interactions. Large-scale quantitative epistasis analysis has been well established for single cells. However, there is a substantial lack of such studies in multicellular organisms and their complex phenotypes such as development. Here we present a method to extend quantitative epistasis analysis to developmental traits. In the nematode Caenorhabditis elegans, we applied RNA interference on mutants to inactivate two genes, used an imaging system to quantitatively measure phenotypes, and developed a set of statistical methods to extract genetic interactions from phenotypic measurement. Using two different C. elegans developmental phenotypes, body length and sex ratio, as examples, we showed that this method could accommodate various metazoan phenotypes with performances comparable to those methods in single cell growth studies. Comparing with qualitative observations, this method of quantitative epistasis enabled detection of new interactions involving subtle phenotypes. For example, several sex-ratio genes were found to interact with brc-1 and brd-1, the orthologs of the human breast cancer genes BRCA1 and BARD1, respectively. We confirmed the brc-1 interactions with the following genes in DNA damage response: C34F6.1, him-3 (ortholog of HORMAD1, HORMAD2), sdc-1, and set-2 (ortholog of SETD1A, SETD1B, KMT2C, KMT2D), validating the effectiveness of our method in detecting genetic interactions. We developed a reliable, high-throughput method for quantitative epistasis analysis of developmental phenotypes.

FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles.

PubMed

Kuipers, Jeroen; van Ham, Tjakko J; Kalicharan, Ruby D; Veenstra-Algra, Anneke; Sjollema, Klaas A; Dijk, Freark; Schnell, Ulrike; Giepmans, Ben N G

2015-04-01

Ultrastructural examination of cells and tissues by electron microscopy (EM) yields detailed information on subcellular structures. However, EM is typically restricted to small fields of view at high magnification; this makes quantifying events in multiple large-area sample sections extremely difficult. Even when combining light microscopy (LM) with EM (correlated LM and EM: CLEM) to find areas of interest, the labeling of molecules is still a challenge. We present a new genetically encoded probe for CLEM, named "FLIPPER", which facilitates quantitative analysis of ultrastructural features in cells. FLIPPER consists of a fluorescent protein (cyan, green, orange, or red) for LM visualization, fused to a peroxidase allowing visualization of targets at the EM level. The use of FLIPPER is straightforward and because the module is completely genetically encoded, cells can be optimally prepared for EM examination. We use FLIPPER to quantify cellular morphology at the EM level in cells expressing a normal and disease-causing point-mutant cell-surface protein called EpCAM (epithelial cell adhesion molecule). The mutant protein is retained in the endoplasmic reticulum (ER) and could therefore alter ER function and morphology. To reveal possible ER alterations, cells were co-transfected with color-coded full-length or mutant EpCAM and a FLIPPER targeted to the ER. CLEM examination of the mixed cell population allowed color-based cell identification, followed by an unbiased quantitative analysis of the ER ultrastructure by EM. Thus, FLIPPER combines bright fluorescent proteins optimized for live imaging with high sensitivity for EM labeling, thereby representing a promising tool for CLEM.

Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis.

PubMed

Fox, Bridget C; Devonshire, Alison S; Baradez, Marc-Olivier; Marshall, Damian; Foy, Carole A

2012-08-15

Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT-qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis. Copyright © 2012 Elsevier Inc. All rights reserved.

Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (Phospho)Proteomic Profiling.

PubMed

Singec, Ilyas; Crain, Andrew M; Hou, Junjie; Tobe, Brian T D; Talantova, Maria; Winquist, Alicia A; Doctor, Kutbuddin S; Choy, Jennifer; Huang, Xiayu; La Monaca, Esther; Horn, David M; Wolf, Dieter A; Lipton, Stuart A; Gutierrez, Gustavo J; Brill, Laurence M; Snyder, Evan Y

2016-09-13

Controlled differentiation of human embryonic stem cells (hESCs) can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs). This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites) provides unique molecular insights into pluripotency and neural lineage entry. Systems-level comparative analysis of proteins (e.g., transcription factors, epigenetic regulators, kinase families), phosphorylation sites, and numerous biological pathways allowed the identification of distinct signatures in pluripotent and multipotent cells. Furthermore, as predicted by the dataset, we functionally validated an autocrine/paracrine mechanism by demonstrating that the secreted protein midkine is a regulator of neural specification. This resource is freely available to the scientific community, including a searchable website, PluriProt. Published by Elsevier Inc.

Analysis of defect structure in silicon. Characterization of SEMIX material. Silicon sheet growth development for the large area silicon sheet task of the low-cost solar array project

NASA Technical Reports Server (NTRS)

Natesh, R.; Stringfellow, G. B.; Virkar, A. V.; Dunn, J.; Guyer, T.

1983-01-01

Statistically significant quantitative structural imperfection measurements were made on samples from ubiquitous crystalline process (UCP) Ingot 5848 - 13C. Important correlation was obtained between defect densities, cell efficiency, and diffusion length. Grain boundary substructure displayed a strong influence on the conversion efficiency of solar cells from Semix material. Quantitative microscopy measurements gave statistically significant information compared to other microanalytical techniques. A surface preparation technique to obtain proper contrast of structural defects suitable for quantimet quantitative image analyzer (QTM) analysis was perfected and is used routinely. The relationships between hole mobility and grain boundary density was determined. Mobility was measured using the van der Pauw technique, and grain boundary density was measured using quantitative microscopy technique. Mobility was found to decrease with increasing grain boundary density.

[Quantitative data analysis for live imaging of bone.

PubMed

Seno, Shigeto

Bone tissue is a hard tissue, it was difficult to observe the interior of the bone tissue alive. With the progress of microscopic technology and fluorescent probe technology in recent years, it becomes possible to observe various activities of various cells forming bone society. On the other hand, the quantitative increase in data and the diversification and complexity of the images makes it difficult to perform quantitative analysis by visual inspection. It has been expected to develop a methodology for processing microscopic images and data analysis. In this article, we introduce the research field of bioimage informatics which is the boundary area of biology and information science, and then outline the basic image processing technology for quantitative analysis of live imaging data of bone.

COMPARISON OF ENTEROCOCCUS MEASUREMENTS IN FRESHWATER AT TWO RECREATIONAL BEACHES BY QUANTITATIVE POLYMERASE CHAIN REACTION AND MEMBRANE FILER CULTURE ANALYSIS

EPA Science Inventory

Cell densities of the fecal pollution indicator genus, Enterococcus, were determined by a rapid (2-3 hr) quantitative PCR (QPCR) analysis based method in 100 ml water samples collected from recreational beaches on Lake Michigan and Lake Erie during the summer of 2003. Enumeration...

The new numerology of immunity mediated by virus-specific CD8(+) T cells.

PubMed

Doherty, P C

1998-08-01

Our understanding of virus-specific CD8(+) T cell responses is currently being revolutionized by peptide-based assay systems that allow flow cytometric analysis of effector and memory cytotoxic T lymphocyte populations. These techniques are, for the first time, putting the analysis of T-cell-mediated immunity on a quantitative basis.

Novel Quantitative Autophagy Analysis by Organelle Flow Cytometry after Cell Sonication

PubMed Central

Degtyarev, Michael; Reichelt, Mike; Lin, Kui

2014-01-01

Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication), takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis. PMID:24489953

Quantitative phase imaging of human red blood cells using phase-shifting white light interference microscopy with colour fringe analysis

NASA Astrophysics Data System (ADS)

Singh Mehta, Dalip; Srivastava, Vishal

2012-11-01

We report quantitative phase imaging of human red blood cells (RBCs) using phase-shifting interference microscopy. Five phase-shifted white light interferograms are recorded using colour charge coupled device camera. White light interferograms were decomposed into red, green, and blue colour components. The phase-shifted interferograms of each colour were then processed by phase-shifting analysis and phase maps for red, green, and blue colours were reconstructed. Wavelength dependent refractive index profiles of RBCs were computed from the single set of white light interferogram. The present technique has great potential for non-invasive determination of refractive index variation and morphological features of cells and tissues.

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis.

PubMed

Ong, Edmund; Cahill, Catherine

2015-07-03

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Quantitative Analysis of Rat Dorsal Root Ganglion Neurons Cultured on Microelectrode Arrays Based on Fluorescence Microscopy Image Processing.

PubMed

Mari, João Fernando; Saito, José Hiroki; Neves, Amanda Ferreira; Lotufo, Celina Monteiro da Cruz; Destro-Filho, João-Batista; Nicoletti, Maria do Carmo

2015-12-01

Microelectrode Arrays (MEA) are devices for long term electrophysiological recording of extracellular spontaneous or evocated activities on in vitro neuron culture. This work proposes and develops a framework for quantitative and morphological analysis of neuron cultures on MEAs, by processing their corresponding images, acquired by fluorescence microscopy. The neurons are segmented from the fluorescence channel images using a combination of segmentation by thresholding, watershed transform, and object classification. The positioning of microelectrodes is obtained from the transmitted light channel images using the circular Hough transform. The proposed method was applied to images of dissociated culture of rat dorsal root ganglion (DRG) neuronal cells. The morphological and topological quantitative analysis carried out produced information regarding the state of culture, such as population count, neuron-to-neuron and neuron-to-microelectrode distances, soma morphologies, neuron sizes, neuron and microelectrode spatial distributions. Most of the analysis of microscopy images taken from neuronal cultures on MEA only consider simple qualitative analysis. Also, the proposed framework aims to standardize the image processing and to compute quantitative useful measures for integrated image-signal studies and further computational simulations. As results show, the implemented microelectrode identification method is robust and so are the implemented neuron segmentation and classification one (with a correct segmentation rate up to 84%). The quantitative information retrieved by the method is highly relevant to assist the integrated signal-image study of recorded electrophysiological signals as well as the physical aspects of the neuron culture on MEA. Although the experiments deal with DRG cell images, cortical and hippocampal cell images could also be processed with small adjustments in the image processing parameter estimation.

[Study of Cervical Exfoliated Cell's DNA Quantitative Analysis Based on Multi-Spectral Imaging Technology].

PubMed

Wu, Zheng; Zeng, Li-bo; Wu, Qiong-shui

2016-02-01

The conventional cervical cancer screening methods mainly include TBS (the bethesda system) classification method and cellular DNA quantitative analysis, however, by using multiple staining method in one cell slide, which is staining the cytoplasm with Papanicolaou reagent and the nucleus with Feulgen reagent, the study of achieving both two methods in the cervical cancer screening at the same time is still blank. Because the difficulty of this multiple staining method is that the absorbance of the non-DNA material may interfere with the absorbance of DNA, so that this paper has set up a multi-spectral imaging system, and established an absorbance unmixing model by using multiple linear regression method based on absorbance's linear superposition character, and successfully stripped out the absorbance of DNA to run the DNA quantitative analysis, and achieved the perfect combination of those two kinds of conventional screening method. Through a series of experiment we have proved that between the absorbance of DNA which is calculated by the absorbance unmixxing model and the absorbance of DNA which is measured there is no significant difference in statistics when the test level is 1%, also the result of actual application has shown that there is no intersection between the confidence interval of the DNA index of the tetraploid cells which are screened by using this paper's analysis method when the confidence level is 99% and the DNA index's judging interval of cancer cells, so that the accuracy and feasibility of the quantitative DNA analysis with multiple staining method expounded by this paper have been verified, therefore this analytical method has a broad application prospect and considerable market potential in early diagnosis of cervical cancer and other cancers.

Application of adenosine triphosphate affinity probe and scheduled multiple-reaction monitoring analysis for profiling global kinome in human cells in response to arsenite treatment.

PubMed

Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

2014-11-04

Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli.

Application of Adenosine Triphosphate Affinity Probe and Scheduled Multiple-Reaction Monitoring Analysis for Profiling Global Kinome in Human Cells in Response to Arsenite Treatment

PubMed Central

2015-01-01

Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli. PMID:25301106

Quantitative Schlieren analysis applied to holograms of crystals grown on Spacelab 3

NASA Technical Reports Server (NTRS)

Brooks, Howard L.

1986-01-01

In order to extract additional information about crystals grown in the microgravity environment of Spacelab, a quantitative schlieren analysis technique was developed for use in a Holography Ground System of the Fluid Experiment System. Utilizing the Unidex position controller, it was possible to measure deviation angles produced by refractive index gradients of 0.5 milliradians. Additionally, refractive index gradient maps for any recorded time during the crystal growth were drawn and used to create solute concentration maps for the environment around the crystal. The technique was applied to flight holograms of Cell 204 of the Fluid Experiment System that were recorded during the Spacelab 3 mission on STS 51B. A triglycine sulfate crystal was grown under isothermal conditions in the cell and the data gathered with the quantitative schlieren analysis technique is consistent with a diffusion limited growth process.

Optimizing morphology through blood cell image analysis.

PubMed

Merino, A; Puigví, L; Boldú, L; Alférez, S; Rodellar, J

2018-05-01

Morphological review of the peripheral blood smear is still a crucial diagnostic aid as it provides relevant information related to the diagnosis and is important for selection of additional techniques. Nevertheless, the distinctive cytological characteristics of the blood cells are subjective and influenced by the reviewer's interpretation and, because of that, translating subjective morphological examination into objective parameters is a challenge. The use of digital microscopy systems has been extended in the clinical laboratories. As automatic analyzers have some limitations for abnormal or neoplastic cell detection, it is interesting to identify quantitative features through digital image analysis for morphological characteristics of different cells. Three main classes of features are used as follows: geometric, color, and texture. Geometric parameters (nucleus/cytoplasmic ratio, cellular area, nucleus perimeter, cytoplasmic profile, RBC proximity, and others) are familiar to pathologists, as they are related to the visual cell patterns. Different color spaces can be used to investigate the rich amount of information that color may offer to describe abnormal lymphoid or blast cells. Texture is related to spatial patterns of color or intensities, which can be visually detected and quantitatively represented using statistical tools. This study reviews current and new quantitative features, which can contribute to optimize morphology through blood cell digital image processing techniques. © 2018 John Wiley & Sons Ltd.

Associative image analysis: a method for automated quantification of 3D multi-parameter images of brain tissue

PubMed Central

Bjornsson, Christopher S; Lin, Gang; Al-Kofahi, Yousef; Narayanaswamy, Arunachalam; Smith, Karen L; Shain, William; Roysam, Badrinath

2009-01-01

Brain structural complexity has confounded prior efforts to extract quantitative image-based measurements. We present a systematic ‘divide and conquer’ methodology for analyzing three-dimensional (3D) multi-parameter images of brain tissue to delineate and classify key structures, and compute quantitative associations among them. To demonstrate the method, thick (~100 μm) slices of rat brain tissue were labeled using 3 – 5 fluorescent signals, and imaged using spectral confocal microscopy and unmixing algorithms. Automated 3D segmentation and tracing algorithms were used to delineate cell nuclei, vasculature, and cell processes. From these segmentations, a set of 23 intrinsic and 8 associative image-based measurements was computed for each cell. These features were used to classify astrocytes, microglia, neurons, and endothelial cells. Associations among cells and between cells and vasculature were computed and represented as graphical networks to enable further analysis. The automated results were validated using a graphical interface that permits investigator inspection and corrective editing of each cell in 3D. Nuclear counting accuracy was >89%, and cell classification accuracy ranged from 81–92% depending on cell type. We present a software system named FARSIGHT implementing our methodology. Its output is a detailed XML file containing measurements that may be used for diverse quantitative hypothesis-driven and exploratory studies of the central nervous system. PMID:18294697

An easy and inexpensive method for quantitative analysis of endothelial damage by using vital dye staining and Adobe Photoshop software.

PubMed

Saad, Hisham A; Terry, Mark A; Shamie, Neda; Chen, Edwin S; Friend, Daniel F; Holiman, Jeffrey D; Stoeger, Christopher

2008-08-01

We developed a simple, practical, and inexpensive technique to analyze areas of endothelial cell loss and/or damage over the entire corneal area after vital dye staining by using a readily available, off-the-shelf, consumer software program, Adobe Photoshop. The purpose of this article is to convey a method of quantifying areas of cell loss and/or damage. Descemet-stripping automated endothelial keratoplasty corneal transplant surgery was performed by using 5 precut corneas on a human cadaver eye. Corneas were removed and stained with trypan blue and alizarin red S and subsequently photographed. Quantitative assessment of endothelial damage was performed by using Adobe Photoshop 7.0 software. The average difference for cell area damage for analyses performed by 1 observer twice was 1.41%. For analyses performed by 2 observers, the average difference was 1.71%. Three masked observers were 100% successful in matching the randomized stained corneas to their randomized processed Adobe images. Vital dye staining of corneal endothelial cells can be combined with Adobe Photoshop software to yield a quantitative assessment of areas of acute endothelial cell loss and/or damage. This described technique holds promise for a more consistent and accurate method to evaluate the surgical trauma to the endothelial cell layer in laboratory models. This method of quantitative analysis can probably be generalized to any area of research that involves areas that are differentiated by color or contrast.

Quantitative method of measuring cancer cell urokinase and metastatic potential

NASA Technical Reports Server (NTRS)

Morrison, Dennis R. (Inventor)

1993-01-01

The metastatic potential of tumors can be evaluated by the quantitative detection of urokinase and DNA. The cell sample selected for examination is analyzed for the presence of high levels of urokinase and abnormal DNA using analytical flow cytometry and digital image analysis. Other factors such as membrane associated urokinase, increased DNA synthesis rates and certain receptors can be used in the method for detection of potentially invasive tumors.

Quantitative Microbial Community Analysis of Three Different Sulfidic Mine Tailing Dumps Generating Acid Mine Drainage▿

PubMed Central

Kock, Dagmar; Schippers, Axel

2008-01-01

The microbial communities of three different sulfidic and acidic mine waste tailing dumps located in Botswana, Germany, and Sweden were quantitatively analyzed using quantitative real-time PCR (Q-PCR), fluorescence in situ hybridization (FISH), catalyzed reporter deposition-FISH (CARD-FISH), Sybr green II direct counting, and the most probable number (MPN) cultivation technique. Depth profiles of cell numbers showed that the compositions of the microbial communities are greatly different at the three sites and also strongly varied between zones of oxidized and unoxidized tailings. Maximum cell numbers of up to 109 cells g−1 dry weight were determined in the pyrite or pyrrhotite oxidation zones, whereas cell numbers in unoxidized tailings were significantly lower. Bacteria dominated over Archaea and Eukarya at all tailing sites. The acidophilic Fe(II)- and/or sulfur-oxidizing Acidithiobacillus spp. dominated over the acidophilic Fe(II)-oxidizing Leptospirillum spp. among the Bacteria at two sites. The two genera were equally abundant at the third site. The acidophilic Fe(II)- and sulfur-oxidizing Sulfobacillus spp. were generally less abundant. The acidophilic Fe(III)-reducing Acidiphilium spp. could be found at only one site. The neutrophilic Fe(III)-reducing Geobacteraceae as well as the dsrA gene of sulfate reducers were quantifiable at all three sites. FISH analysis provided reliable data only for tailing zones with high microbial activity, whereas CARD-FISH, Q-PCR, Sybr green II staining, and MPN were suitable methods for a quantitative microbial community analysis of tailings in general. PMID:18586975

Quantitative microbial community analysis of three different sulfidic mine tailing dumps generating acid mine drainage.

PubMed

Kock, Dagmar; Schippers, Axel

2008-08-01

The microbial communities of three different sulfidic and acidic mine waste tailing dumps located in Botswana, Germany, and Sweden were quantitatively analyzed using quantitative real-time PCR (Q-PCR), fluorescence in situ hybridization (FISH), catalyzed reporter deposition-FISH (CARD-FISH), Sybr green II direct counting, and the most probable number (MPN) cultivation technique. Depth profiles of cell numbers showed that the compositions of the microbial communities are greatly different at the three sites and also strongly varied between zones of oxidized and unoxidized tailings. Maximum cell numbers of up to 10(9) cells g(-1) dry weight were determined in the pyrite or pyrrhotite oxidation zones, whereas cell numbers in unoxidized tailings were significantly lower. Bacteria dominated over Archaea and Eukarya at all tailing sites. The acidophilic Fe(II)- and/or sulfur-oxidizing Acidithiobacillus spp. dominated over the acidophilic Fe(II)-oxidizing Leptospirillum spp. among the Bacteria at two sites. The two genera were equally abundant at the third site. The acidophilic Fe(II)- and sulfur-oxidizing Sulfobacillus spp. were generally less abundant. The acidophilic Fe(III)-reducing Acidiphilium spp. could be found at only one site. The neutrophilic Fe(III)-reducing Geobacteraceae as well as the dsrA gene of sulfate reducers were quantifiable at all three sites. FISH analysis provided reliable data only for tailing zones with high microbial activity, whereas CARD-FISH, Q-PCR, Sybr green II staining, and MPN were suitable methods for a quantitative microbial community analysis of tailings in general.

Quantitative molecular analysis in mantle cell lymphoma.

PubMed

Brízová, H; Hilská, I; Mrhalová, M; Kodet, R

2011-07-01

A molecular analysis has three major roles in modern oncopathology--as an aid in the differential diagnosis, in molecular monitoring of diseases, and in estimation of the potential prognosis. In this report we review the application of the molecular analysis in a group of patients with mantle cell lymphoma (MCL). We demonstrate that detection of the cyclin D1 mRNA level is a molecular marker in 98% of patients with MCL. Cyclin D1 quantitative monitoring is specific and sensitive for the differential diagnosis and for the molecular monitoring of the disease in the bone marrow. Moreover, the dynamics of cyclin D1 in bone marrow reflects the disease development and it predicts the clinical course. We employed the molecular analysis for a precise quantitative detection of proliferation markers, Ki-67, topoisomerase IIalpha, and TPX2, that are described as effective prognostic factors. Using the molecular approach it is possible to measure the proliferation rate in a reproducible, standard way which is an essential prerequisite for using the proliferation activity as a routine clinical tool. Comparing with immunophenotyping we may conclude that the quantitative PCR-based analysis is a useful, reliable, rapid, reproducible, sensitive and specific method broadening our diagnostic tools in hematopathology. In comparison to interphase FISH in paraffin sections quantitative PCR is less technically demanding and less time-consuming and furthermore it is more sensitive in detecting small changes in the mRNA level. Moreover, quantitative PCR is the only technology which provides precise and reproducible quantitative information about the expression level. Therefore it may be used to demonstrate the decrease or increase of a tumor-specific marker in bone marrow in comparison with a previously aspirated specimen. Thus, it has a powerful potential to monitor the course of the disease in correlation with clinical data.

A Semi-Automatic Method for Image Analysis of Edge Dynamics in Living Cells

PubMed Central

Huang, Lawrence; Helmke, Brian P.

2011-01-01

Spatial asymmetry of actin edge ruffling contributes to the process of cell polarization and directional migration, but mechanisms by which external cues control actin polymerization near cell edges remain unclear. We designed a quantitative image analysis strategy to measure the spatiotemporal distribution of actin edge ruffling. Time-lapse images of endothelial cells (ECs) expressing mRFP-actin were segmented using an active contour method. In intensity line profiles oriented normal to the cell edge, peak detection identified the angular distribution of polymerized actin within 1 µm of the cell edge, which was localized to lamellipodia and edge ruffles. Edge features associated with filopodia and peripheral stress fibers were removed. Circular statistical analysis enabled detection of cell polarity, indicated by a unimodal distribution of edge ruffles. To demonstrate the approach, we detected a rapid, nondirectional increase in edge ruffling in serum-stimulated ECs and a change in constitutive ruffling orientation in quiescent, nonpolarized ECs. Error analysis using simulated test images demonstrate robustness of the method to variations in image noise levels, edge ruffle arc length, and edge intensity gradient. These quantitative measurements of edge ruffling dynamics enable investigation at the cellular length scale of the underlying molecular mechanisms regulating actin assembly and cell polarization. PMID:21643526

Photothermal technique in cell microscopy studies

NASA Astrophysics Data System (ADS)

Lapotko, Dmitry; Chebot'ko, Igor; Kutchinsky, Georgy; Cherenkevitch, Sergey

1995-01-01

Photothermal (PT) method is applied for a cell imaging and quantitative studies. The techniques for cell monitoring, imaging and cell viability test are developed. The method and experimental set up for optical and PT-image acquisition and analysis is described. Dual- pulsed laser set up combined with phase contrast illumination of a sample provides visualization of temperature field or absorption structure of a sample with spatial resolution 0.5 micrometers . The experimental optics, hardware and software are designed using the modular principle, so the whole set up can be adjusted for various experiments: PT-response monitoring or photothermal spectroscopy studies. Sensitivity of PT-method provides the imaging of the structural elements of live (non-stained) white blood cells. The results of experiments with normal and subnormal blood cells (red blood cells, lymphocytes, neutrophyles and lymphoblasts) are reported. Obtained PT-images are different from optical analogs and deliver additional information about cell structure. The quantitative analysis of images was used for cell population comparative diagnostic. The viability test for red blood cell differentiation is described. During the study of neutrophyles in norma and sarcoidosis disease the differences in PT-images of cells were found.

Sequential processing of quantitative phase images for the study of cell behaviour in real-time digital holographic microscopy.

PubMed

Zikmund, T; Kvasnica, L; Týč, M; Křížová, A; Colláková, J; Chmelík, R

2014-11-01

Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The study of the cell is based on extraction of the dynamic data on cell behaviour from the time-lapse sequence of the phase images. However, the phase images are affected by the phase aberrations that make the analysis particularly difficult. This is because the phase deformation is prone to change during long-term experiments. Here, we present a novel algorithm for sequential processing of living cells phase images in a time-lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least-squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. All these procedures are performed automatically and applied immediately after obtaining every single phase image. This property of the algorithm is important for real-time cell quantitative phase imaging and instantaneous control of the course of the experiment by playback of the recorded sequence up to actual time. Such operator's intervention is a forerunner of process automation derived from image analysis. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off-axis holographic microscope. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

Nanoscale nuclear architecture for cancer diagnosis beyond pathology via spatial-domain low-coherence quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Wang, Pin; Bista, Rajan K.; Khalbuss, Walid E.; Qiu, Wei; Uttam, Shikhar; Staton, Kevin; Zhang, Lin; Brentnall, Teresa A.; Brand, Randall E.; Liu, Yang

2010-11-01

Definitive diagnosis of malignancy is often challenging due to limited availability of human cell or tissue samples and morphological similarity with certain benign conditions. Our recently developed novel technology-spatial-domain low-coherence quantitative phase microscopy (SL-QPM)-overcomes the technical difficulties and enables us to obtain quantitative information about cell nuclear architectural characteristics with nanoscale sensitivity. We explore its ability to improve the identification of malignancy, especially in cytopathologically non-cancerous-appearing cells. We perform proof-of-concept experiments with an animal model of colorectal carcinogenesis-APCMin mouse model and human cytology specimens of colorectal cancer. We show the ability of in situ nanoscale nuclear architectural characteristics in identifying cancerous cells, especially in those labeled as ``indeterminate or normal'' by expert cytopathologists. Our approach is based on the quantitative analysis of the cell nucleus on the original cytology slides without additional processing, which can be readily applied in a conventional clinical setting. Our simple and practical optical microscopy technique may lead to the development of novel methods for early detection of cancer.

Proteomic analysis of mitochondria in respiratory epithelial cells infected with human respiratory syncytial virus and functional implications for virus and cell biology.

PubMed

Munday, Diane C; Howell, Gareth; Barr, John N; Hiscox, Julian A

2015-03-01

The aim of this study was to quantitatively characterise the mitochondrial proteome of airway epithelial cells infected with human respiratory syncytial virus (HRSV), a major cause of paediatric illness. Quantitative proteomics, underpinned by stable isotope labelling with amino acids in cell culture, coupled to LC-MS/MS, was applied to mitochondrial fractions prepared from HRSV-infected and mock-infected cells 12 and 24 h post-infection. Datasets were analysed using ingenuity pathway analysis, and the results were validated and characterised using bioimaging, targeted inhibition and gene depletion. The data quantitatively indicated that antiviral signalling proteins converged on mitochondria during HRSV infection. The mitochondrial receptor protein Tom70 was found to act in an antiviral manner, while its chaperone, Hsp90, was confirmed to be a positive viral factor. Proteins associated with different organelles were also co-enriched in the mitochondrial fractions from HRSV-infected cells, suggesting that alterations in organelle dynamics and membrane associations occur during virus infection. Protein and pathway-specific alterations occur to the mitochondrial proteome in a spatial and temporal manner during HRSV infection, suggesting that this organelle may have altered functions. These could be targeted as part of potential therapeutic strategies to disrupt virus biology. © 2014 Royal Pharmaceutical Society.

Diversity in ATP concentrations in a single bacterial cell population revealed by quantitative single-cell imaging

PubMed Central

Yaginuma, Hideyuki; Kawai, Shinnosuke; Tabata, Kazuhito V.; Tomiyama, Keisuke; Kakizuka, Akira; Komatsuzaki, Tamiki; Noji, Hiroyuki; Imamura, Hiromi

2014-01-01

Recent advances in quantitative single-cell analysis revealed large diversity in gene expression levels between individual cells, which could affect the physiology and/or fate of each cell. In contrast, for most metabolites, the concentrations were only measureable as ensemble averages of many cells. In living cells, adenosine triphosphate (ATP) is a critically important metabolite that powers many intracellular reactions. Quantitative measurement of the absolute ATP concentration in individual cells has not been achieved because of the lack of reliable methods. In this study, we developed a new genetically-encoded ratiometric fluorescent ATP indicator “QUEEN”, which is composed of a single circularly-permuted fluorescent protein and a bacterial ATP binding protein. Unlike previous FRET-based indicators, QUEEN was apparently insensitive to bacteria growth rate changes. Importantly, intracellular ATP concentrations of numbers of bacterial cells calculated from QUEEN fluorescence were almost equal to those from firefly luciferase assay. Thus, QUEEN is suitable for quantifying the absolute ATP concentration inside bacteria cells. Finally, we found that, even for a genetically-identical Escherichia coli cell population, absolute concentrations of intracellular ATP were significantly diverse between individual cells from the same culture, by imaging QUEEN signals from single cells. PMID:25283467

Multispectral Imaging Broadens Cellular Analysis

NASA Technical Reports Server (NTRS)

2007-01-01

Amnis Corporation, a Seattle-based biotechnology company, developed ImageStream to produce sensitive fluorescence images of cells in flow. The company responded to an SBIR solicitation from Ames Research Center, and proposed to evaluate several methods of extending the depth of field for its ImageStream system and implement the best as an upgrade to its commercial products. This would allow users to view whole cells at the same time, rather than just one section of each cell. Through Phase I and II SBIR contracts, Ames provided Amnis the funding the company needed to develop this extended functionality. For NASA, the resulting high-speed image flow cytometry process made its way into Medusa, a life-detection instrument built to collect, store, and analyze sample organisms from erupting hydrothermal vents, and has the potential to benefit space flight health monitoring. On the commercial end, Amnis has implemented the process in ImageStream, combining high-resolution microscopy and flow cytometry in a single instrument, giving researchers the power to conduct quantitative analyses of individual cells and cell populations at the same time, in the same experiment. ImageStream is also built for many other applications, including cell signaling and pathway analysis; classification and characterization of peripheral blood mononuclear cell populations; quantitative morphology; apoptosis (cell death) assays; gene expression analysis; analysis of cell conjugates; molecular distribution; and receptor mapping and distribution.

EasyFRAP-web: a web-based tool for the analysis of fluorescence recovery after photobleaching data.

PubMed

Koulouras, Grigorios; Panagopoulos, Andreas; Rapsomaniki, Maria A; Giakoumakis, Nickolaos N; Taraviras, Stavros; Lygerou, Zoi

2018-06-13

Understanding protein dynamics is crucial in order to elucidate protein function and interactions. Advances in modern microscopy facilitate the exploration of the mobility of fluorescently tagged proteins within living cells. Fluorescence recovery after photobleaching (FRAP) is an increasingly popular functional live-cell imaging technique which enables the study of the dynamic properties of proteins at a single-cell level. As an increasing number of labs generate FRAP datasets, there is a need for fast, interactive and user-friendly applications that analyze the resulting data. Here we present easyFRAP-web, a web application that simplifies the qualitative and quantitative analysis of FRAP datasets. EasyFRAP-web permits quick analysis of FRAP datasets through an intuitive web interface with interconnected analysis steps (experimental data assessment, different types of normalization and estimation of curve-derived quantitative parameters). In addition, easyFRAP-web provides dynamic and interactive data visualization and data and figure export for further analysis after every step. We test easyFRAP-web by analyzing FRAP datasets capturing the mobility of the cell cycle regulator Cdt2 in the presence and absence of DNA damage in cultured cells. We show that easyFRAP-web yields results consistent with previous studies and highlights cell-to-cell heterogeneity in the estimated kinetic parameters. EasyFRAP-web is platform-independent and is freely accessible at: https://easyfrap.vmnet.upatras.gr/.

Quantitative two-dimensional gel electrophoresis analysis of human fibroblasts transformed by ras oncogenes.

PubMed

Miller, M J; Maher, V M; McCormick, J J

1992-11-01

Quantitative two-dimensional gel electrophoresis was used to compare the cellular protein patterns of a normal foreskin-derived human fibroblasts cell line (LG1) and three immortal derivatives of LG1. One derivative, designated MSU-1.1 VO, was selected for its ability to grow in the absence of serum and is non-tumorigenic in athymic mice. The other two strains were selected for focus-formation following transfection with either Ha-ras or N-ras oncogenes and form high grade malignant tumors. Correspondence and cluster analysis provided a nonbiased estimate of the relative similarity of the different two-dimensional patterns. These techniques separated the gel patterns into three distinct classes: LG1, MSU-1.1 VO, and the ras transformed cell strains. The MSU-1.1 VO cells were more closely related to the parental LG1 than to the ras-transformed cells. The differences between the three classes were primarily quantitative in nature: 16% of the spots demonstrated statistically significant changes (P < 0.01, T test, mean ratio of intensity > 2) in the rate of incorporation of radioactive amino acids. The patterns from the two ras-transformed cell strains were similar, and variations in the expression of proteins that occurred between the separate experiments obscured consistent differences between the Ha-ras and N-ras transformed cells. However, while only 9 out of 758 spots were classified as different (1%), correspondence analysis could consistently separate the two ras transformants. One of these spots was five times more intense in the Ha-ras transformed cells than the N-ras.(ABSTRACT TRUNCATED AT 250 WORDS)

An analysis toolbox to explore mesenchymal migration heterogeneity reveals adaptive switching between distinct modes

PubMed Central

Shafqat-Abbasi, Hamdah; Kowalewski, Jacob M; Kiss, Alexa; Gong, Xiaowei; Hernandez-Varas, Pablo; Berge, Ulrich; Jafari-Mamaghani, Mehrdad; Lock, John G; Strömblad, Staffan

2016-01-01

Mesenchymal (lamellipodial) migration is heterogeneous, although whether this reflects progressive variability or discrete, 'switchable' migration modalities, remains unclear. We present an analytical toolbox, based on quantitative single-cell imaging data, to interrogate this heterogeneity. Integrating supervised behavioral classification with multivariate analyses of cell motion, membrane dynamics, cell-matrix adhesion status and F-actin organization, this toolbox here enables the detection and characterization of two quantitatively distinct mesenchymal migration modes, termed 'Continuous' and 'Discontinuous'. Quantitative mode comparisons reveal differences in cell motion, spatiotemporal coordination of membrane protrusion/retraction, and how cells within each mode reorganize with changed cell speed. These modes thus represent distinctive migratory strategies. Additional analyses illuminate the macromolecular- and cellular-scale effects of molecular targeting (fibronectin, talin, ROCK), including 'adaptive switching' between Continuous (favored at high adhesion/full contraction) and Discontinuous (low adhesion/inhibited contraction) modes. Overall, this analytical toolbox now facilitates the exploration of both spontaneous and adaptive heterogeneity in mesenchymal migration. DOI: http://dx.doi.org/10.7554/eLife.11384.001 PMID:26821527

Low-frequency quantitative ultrasound imaging of cell death in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sadeghi-Naini, Ali; Falou, Omar; Czarnota, Gregory J.

Purpose: Currently, no clinical imaging modality is used routinely to assess tumor response to cancer therapies within hours to days of the delivery of treatment. Here, the authors demonstrate the efficacy of ultrasound at a clinically relevant frequency to quantitatively detect changes in tumors in response to cancer therapies using preclinical mouse models.Methods: Conventional low-frequency and corresponding high-frequency ultrasound (ranging from 4 to 28 MHz) were used along with quantitative spectroscopic and signal envelope statistical analyses on data obtained from xenograft tumors treated with chemotherapy, x-ray radiation, as well as a novel vascular targeting microbubble therapy.Results: Ultrasound-based spectroscopic biomarkers indicatedmore » significant changes in cell-death associated parameters in responsive tumors. Specifically changes in the midband fit, spectral slope, and 0-MHz intercept biomarkers were investigated for different types of treatment and demonstrated cell-death related changes. The midband fit and 0-MHz intercept biomarker derived from low-frequency data demonstrated increases ranging approximately from 0 to 6 dBr and 0 to 8 dBr, respectively, depending on treatments administrated. These data paralleled results observed for high-frequency ultrasound data. Statistical analysis of ultrasound signal envelope was performed as an alternative method to obtain histogram-based biomarkers and provided confirmatory results. Histological analysis of tumor specimens indicated up to 61% cell death present in the tumors depending on treatments administered, consistent with quantitative ultrasound findings indicating cell death. Ultrasound-based spectroscopic biomarkers demonstrated a good correlation with histological morphological findings indicative of cell death (r{sup 2}= 0.71, 0.82; p < 0.001).Conclusions: In summary, the results provide preclinical evidence, for the first time, that quantitative ultrasound used at a clinically relevant frequency, in addition to high-frequency ultrasound, can detect tissue changes associated with cell death in vivo in response to cancer treatments.« less

Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy

PubMed Central

Young, Jonathan W; Locke, James C W; Altinok, Alphan; Rosenfeld, Nitzan; Bacarian, Tigran; Swain, Peter S; Mjolsness, Eric; Elowitz, Michael B

2014-01-01

Quantitative single-cell time-lapse microscopy is a powerful method for analyzing gene circuit dynamics and heterogeneous cell behavior. We describe the application of this method to imaging bacteria by using an automated microscopy system. This protocol has been used to analyze sporulation and competence differentiation in Bacillus subtilis, and to quantify gene regulation and its fluctuations in individual Escherichia coli cells. The protocol involves seeding and growing bacteria on small agarose pads and imaging the resulting microcolonies. Images are then reviewed and analyzed using our laboratory's custom MATLAB analysis code, which segments and tracks cells in a frame-to-frame method. This process yields quantitative expression data on cell lineages, which can illustrate dynamic expression profiles and facilitate mathematical models of gene circuits. With fast-growing bacteria, such as E. coli or B. subtilis, image acquisition can be completed in 1 d, with an additional 1–2 d for progressing through the analysis procedure. PMID:22179594

EDITORIAL: SPECTROSCOPIC IMAGING

EPA Science Inventory

A foremost goal in biology is understanding the molecular basis of single cell behavior, as well as cell interactions that result in functioning tissues. Accomplishing this goal requires quantitative analysis of multiple, specific macromolecules (e.g. proteins, ligands and enzyme...

Semi-quantitative proteomics of mammalian cells upon short-term exposure to non-ionizing electromagnetic fields.

PubMed

Kuzniar, Arnold; Laffeber, Charlie; Eppink, Berina; Bezstarosti, Karel; Dekkers, Dick; Woelders, Henri; Zwamborn, A Peter M; Demmers, Jeroen; Lebbink, Joyce H G; Kanaar, Roland

2017-01-01

The potential effects of non-ionizing electromagnetic fields (EMFs), such as those emitted by power-lines (in extremely low frequency range), mobile cellular systems and wireless networking devices (in radio frequency range) on human health have been intensively researched and debated. However, how exposure to these EMFs may lead to biological changes underlying possible health effects is still unclear. To reveal EMF-induced molecular changes, unbiased experiments (without a priori focusing on specific biological processes) with sensitive readouts are required. We present the first proteome-wide semi-quantitative mass spectrometry analysis of human fibroblasts, osteosarcomas and mouse embryonic stem cells exposed to three types of non-ionizing EMFs (ELF 50 Hz, UMTS 2.1 GHz and WiFi 5.8 GHz). We performed controlled in vitro EMF exposures of metabolically labeled mammalian cells followed by reliable statistical analyses of differential protein- and pathway-level regulations using an array of established bioinformatics methods. Our results indicate that less than 1% of the quantitated human or mouse proteome responds to the EMFs by small changes in protein abundance. Further network-based analysis of the differentially regulated proteins did not detect significantly perturbed cellular processes or pathways in human and mouse cells in response to ELF, UMTS or WiFi exposure. In conclusion, our extensive bioinformatics analyses of semi-quantitative mass spectrometry data do not support the notion that the short-time exposures to non-ionizing EMFs have a consistent biologically significant bearing on mammalian cells in culture.

Semi-quantitative proteomics of mammalian cells upon short-term exposure to non-ionizing electromagnetic fields

PubMed Central

Laffeber, Charlie; Eppink, Berina; Bezstarosti, Karel; Dekkers, Dick; Woelders, Henri; Zwamborn, A. Peter M.; Demmers, Jeroen; Lebbink, Joyce H. G.; Kanaar, Roland

2017-01-01

The potential effects of non-ionizing electromagnetic fields (EMFs), such as those emitted by power-lines (in extremely low frequency range), mobile cellular systems and wireless networking devices (in radio frequency range) on human health have been intensively researched and debated. However, how exposure to these EMFs may lead to biological changes underlying possible health effects is still unclear. To reveal EMF-induced molecular changes, unbiased experiments (without a priori focusing on specific biological processes) with sensitive readouts are required. We present the first proteome-wide semi-quantitative mass spectrometry analysis of human fibroblasts, osteosarcomas and mouse embryonic stem cells exposed to three types of non-ionizing EMFs (ELF 50 Hz, UMTS 2.1 GHz and WiFi 5.8 GHz). We performed controlled in vitro EMF exposures of metabolically labeled mammalian cells followed by reliable statistical analyses of differential protein- and pathway-level regulations using an array of established bioinformatics methods. Our results indicate that less than 1% of the quantitated human or mouse proteome responds to the EMFs by small changes in protein abundance. Further network-based analysis of the differentially regulated proteins did not detect significantly perturbed cellular processes or pathways in human and mouse cells in response to ELF, UMTS or WiFi exposure. In conclusion, our extensive bioinformatics analyses of semi-quantitative mass spectrometry data do not support the notion that the short-time exposures to non-ionizing EMFs have a consistent biologically significant bearing on mammalian cells in culture. PMID:28234898

Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells.

PubMed

Tan, Hwee Tong; Tan, Sandra; Lin, Qingsong; Lim, Teck Kwang; Hew, Choy Leong; Chung, Maxey C M

2008-06-01

Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a short-chain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer.

CMEIAS color segmentation: an improved computing technology to process color images for quantitative microbial ecology studies at single-cell resolution.

PubMed

Gross, Colin A; Reddy, Chandan K; Dazzo, Frank B

2010-02-01

Quantitative microscopy and digital image analysis are underutilized in microbial ecology largely because of the laborious task to segment foreground object pixels from background, especially in complex color micrographs of environmental samples. In this paper, we describe an improved computing technology developed to alleviate this limitation. The system's uniqueness is its ability to edit digital images accurately when presented with the difficult yet commonplace challenge of removing background pixels whose three-dimensional color space overlaps the range that defines foreground objects. Image segmentation is accomplished by utilizing algorithms that address color and spatial relationships of user-selected foreground object pixels. Performance of the color segmentation algorithm evaluated on 26 complex micrographs at single pixel resolution had an overall pixel classification accuracy of 99+%. Several applications illustrate how this improved computing technology can successfully resolve numerous challenges of complex color segmentation in order to produce images from which quantitative information can be accurately extracted, thereby gain new perspectives on the in situ ecology of microorganisms. Examples include improvements in the quantitative analysis of (1) microbial abundance and phylotype diversity of single cells classified by their discriminating color within heterogeneous communities, (2) cell viability, (3) spatial relationships and intensity of bacterial gene expression involved in cellular communication between individual cells within rhizoplane biofilms, and (4) biofilm ecophysiology based on ribotype-differentiated radioactive substrate utilization. The stand-alone executable file plus user manual and tutorial images for this color segmentation computing application are freely available at http://cme.msu.edu/cmeias/ . This improved computing technology opens new opportunities of imaging applications where discriminating colors really matter most, thereby strengthening quantitative microscopy-based approaches to advance microbial ecology in situ at individual single-cell resolution.

SPECHT - single-stage phosphopeptide enrichment and stable-isotope chemical tagging: quantitative phosphoproteomics of insulin action in muscle.

PubMed

Kettenbach, Arminja N; Sano, Hiroyuki; Keller, Susanna R; Lienhard, Gustav E; Gerber, Scott A

2015-01-30

The study of cellular signaling remains a significant challenge for translational and clinical research. In particular, robust and accurate methods for quantitative phosphoproteomics in tissues and tumors represent significant hurdles for such efforts. In the present work, we design, implement and validate a method for single-stage phosphopeptide enrichment and stable isotope chemical tagging, or SPECHT, that enables the use of iTRAQ, TMT and/or reductive dimethyl-labeling strategies to be applied to phosphoproteomics experiments performed on primary tissue. We develop and validate our approach using reductive dimethyl-labeling and HeLa cells in culture, and find these results indistinguishable from data generated from more traditional SILAC-labeled HeLa cells mixed at the cell level. We apply the SPECHT approach to the quantitative analysis of insulin signaling in a murine myotube cell line and muscle tissue, identify known as well as new phosphorylation events, and validate these phosphorylation sites using phospho-specific antibodies. Taken together, our work validates chemical tagging post-single-stage phosphoenrichment as a general strategy for studying cellular signaling in primary tissues. Through the use of a quantitatively reproducible, proteome-wide phosphopeptide enrichment strategy, we demonstrated the feasibility of post-phosphopeptide purification chemical labeling and tagging as an enabling approach for quantitative phosphoproteomics of primary tissues. Using reductive dimethyl labeling as a generalized chemical tagging strategy, we compared the performance of post-phosphopeptide purification chemical tagging to the well established community standard, SILAC, in insulin-stimulated tissue culture cells. We then extended our method to the analysis of low-dose insulin signaling in murine muscle tissue, and report on the analytical and biological significance of our results. Copyright © 2014 Elsevier B.V. All rights reserved.

Analysis of ribosomal RNA stability in dead cells of wine yeast by quantitative PCR.

PubMed

Sunyer-Figueres, Merce; Wang, Chunxiao; Mas, Albert

2018-04-02

During wine production, some yeasts enter a Viable But Not Culturable (VBNC) state, which may influence the quality and stability of the final wine through remnant metabolic activity or by resuscitation. Culture-independent techniques are used for obtaining an accurate estimation of the number of live cells, and quantitative PCR could be the most accurate technique. As a marker of cell viability, rRNA was evaluated by analyzing its stability in dead cells. The species-specific stability of rRNA was tested in Saccharomyces cerevisiae, as well as in three species of non-Saccharomyces yeast (Hanseniaspora uvarum, Torulaspora delbrueckii and Starmerella bacillaris). High temperature and antimicrobial dimethyl dicarbonate (DMDC) treatments were efficient in lysing the yeast cells. rRNA gene and rRNA (as cDNA) were analyzed over 48 h after cell lysis by quantitative PCR. The results confirmed the stability of rRNA for 48 h after the cell lysis treatments. To sum up, rRNA may not be a good marker of cell viability in the wine yeasts that were tested. Copyright © 2018 Elsevier B.V. All rights reserved.

Determination of cell metabolite VEGF₁₆₅ and dynamic analysis of protein-DNA interactions by combination of microfluidic technique and luminescent switch-on probe.

PubMed

Lin, Xuexia; Leung, Ka-Ho; Lin, Ling; Lin, Luyao; Lin, Sheng; Leung, Chung-Hang; Ma, Dik-Lung; Lin, Jin-Ming

2016-05-15

In this paper, we rationally design a novel G-quadruplex-selective luminescent iridium (III) complex for rapid detection of oligonucleotide and VEGF165 in microfluidics. This new probe is applied as a convenient biosensor for label-free quantitative analysis of VEGF165 protein from cell metabolism, as well as for studying the kinetics of the aptamer-protein interaction combination with a microfluidic platform. As a result, we have successfully established a quantitative analysis of VEGF165 from cell metabolism. Furthermore, based on the principles of hydrodynamic focusing and diffusive mixing, different transient states during kinetics process were monitored and recorded. Thus, the combination of microfluidic technique and G-quadruplex luminescent probe will be potentially applied in the studies of intramolecular interactions and molecule recognition in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

Gene expression analysis of immunostained endothelial cells isolated from formaldehyde-fixated paraffin embedded tumors using laser capture microdissection--a technical report.

PubMed

Kaneko, Tomoatsu; Okiji, Takashi; Kaneko, Reika; Suda, Hideaki; Nör, Jacques E

2009-12-01

Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by reverse transcription-PCR (RT-PCR) and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4 degrees C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. Glyceraldehyde-3-phosphate dehydrogenase and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM.

TSCAN: Pseudo-time reconstruction and evaluation in single-cell RNA-seq analysis

PubMed Central

Ji, Zhicheng; Ji, Hongkai

2016-01-01

When analyzing single-cell RNA-seq data, constructing a pseudo-temporal path to order cells based on the gradual transition of their transcriptomes is a useful way to study gene expression dynamics in a heterogeneous cell population. Currently, a limited number of computational tools are available for this task, and quantitative methods for comparing different tools are lacking. Tools for Single Cell Analysis (TSCAN) is a software tool developed to better support in silico pseudo-Time reconstruction in Single-Cell RNA-seq ANalysis. TSCAN uses a cluster-based minimum spanning tree (MST) approach to order cells. Cells are first grouped into clusters and an MST is then constructed to connect cluster centers. Pseudo-time is obtained by projecting each cell onto the tree, and the ordered sequence of cells can be used to study dynamic changes of gene expression along the pseudo-time. Clustering cells before MST construction reduces the complexity of the tree space. This often leads to improved cell ordering. It also allows users to conveniently adjust the ordering based on prior knowledge. TSCAN has a graphical user interface (GUI) to support data visualization and user interaction. Furthermore, quantitative measures are developed to objectively evaluate and compare different pseudo-time reconstruction methods. TSCAN is available at https://github.com/zji90/TSCAN and as a Bioconductor package. PMID:27179027

Redox environment in stem and differentiated cells: A quantitative approach.

PubMed

Lyublinskaya, O G; Ivanova, Ju S; Pugovkina, N A; Kozhukharova, I V; Kovaleva, Z V; Shatrova, A N; Aksenov, N D; Zenin, V V; Kaulin, Yu A; Gamaley, I A; Nikolsky, N N

2017-08-01

Stem cells are believed to maintain a specific intracellular redox status through a combination of enhanced removal capacity and limited production of ROS. In the present study, we challenge this assumption by developing a quantitative approach for the analysis of the pro- and antioxidant ability of human embryonic stem cells in comparison with their differentiated descendants, as well as adult stem and non-stem cells. Our measurements showed that embryonic stem cells are characterized by low ROS level, low rate of extracellular hydrogen peroxide removal and low threshold for peroxide-induced cytotoxicity. However, biochemical normalization of these parameters to cell volume/protein leads to matching of normalized values in stem and differentiated cells and shows that tested in the present study cells (human embryonic stem cells and their fibroblast-like progenies, adult mesenchymal stem cells, lymphocytes, HeLa) maintain similar intracellular redox status. Based on these observations, we propose to use ROS concentration averaged over the cell volume instead of ROS level as a measure of intracellular redox balance. We show that attempts to use ROS level for comparative analysis of redox status of morphologically different cells could lead to false conclusions. Methods for the assessment of ROS concentration based on flow cytometry analysis with the use of H 2 DCFDA dye and HyPer, genetically encoded probe for hydrogen peroxide, are discussed. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

TSCAN: Pseudo-time reconstruction and evaluation in single-cell RNA-seq analysis.

PubMed

Ji, Zhicheng; Ji, Hongkai

2016-07-27

When analyzing single-cell RNA-seq data, constructing a pseudo-temporal path to order cells based on the gradual transition of their transcriptomes is a useful way to study gene expression dynamics in a heterogeneous cell population. Currently, a limited number of computational tools are available for this task, and quantitative methods for comparing different tools are lacking. Tools for Single Cell Analysis (TSCAN) is a software tool developed to better support in silico pseudo-Time reconstruction in Single-Cell RNA-seq ANalysis. TSCAN uses a cluster-based minimum spanning tree (MST) approach to order cells. Cells are first grouped into clusters and an MST is then constructed to connect cluster centers. Pseudo-time is obtained by projecting each cell onto the tree, and the ordered sequence of cells can be used to study dynamic changes of gene expression along the pseudo-time. Clustering cells before MST construction reduces the complexity of the tree space. This often leads to improved cell ordering. It also allows users to conveniently adjust the ordering based on prior knowledge. TSCAN has a graphical user interface (GUI) to support data visualization and user interaction. Furthermore, quantitative measures are developed to objectively evaluate and compare different pseudo-time reconstruction methods. TSCAN is available at https://github.com/zji90/TSCAN and as a Bioconductor package. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Localisation and semi-quantitative measurement of lipocortin 1 in rat anterior pituitary cells by fluorescence-activated cell analysis/sorting and electron microscopy.

PubMed

Christian, H C; Flower, R J; Morris, J F; Buckingham, J C

1999-09-01

Lipocortin 1 (LC1, also called annexin 1), a Ca2(+)- and phospholipid-binding protein, is an important mediator of glucocorticoid action in the anterior pituitary gland. Previous studies based on immunoprecipitation and Western blot analysis suggest that LC1 is found intracellularly both in the cytoplasm and in association with membranes and also on the cell surface where it attaches to the membrane by a Ca2(+)-dependent mechanism. However, as yet it is unclear which anterior pituitary cell types express the protein. Accordingly, we have developed a method based on a combination of fluorescence activated cell (FAC) analysis/sorting and electron microscopy to detect and quantify intracellular LC1 in rat anterior pituitary cells and to identify the cell types in which it is expressed. In addition, we have measured cell surface LC1 and examined the influence of glucocorticoids on the cellular disposition of the protein. Anterior pituitary cells were dispersed with collagenase. For experiments measuring intracellular LC1, three cell fixation/permeabilisation methods were examined initially, i.e. (1) Zamboni's fluid (30 min) and Triton-X-100 (0.12%, 1 or 12 h); (2) paraformaldehyde (2%, 1 h) and Triton-X-100 (0.2%, 10 min); and (3) paraformaldehyde (0.2%, 15 min) and saponin (0.1%, 5 min). The protocol using paraformaldehyde/Triton-X-100 provided optimal preservation of cell ultrastructure and of LC1 immunoreactivity (ir-LC1) while also effectively permeabilising the cells; it was therefore used in subsequent studies. Using an anti-LC1 monoclonal antibody as a probe, 82+/-5% of the secretory cells in the heterogeneous anterior pituitary cell preparation were shown by FAC analysis to display specific fluorescence for intracellular ir-LC1. Morphological analysis and immunogold-histochemistry of cells separated by FAC sorting identified corticotrophs, lactotrophs, somatotrophs and gonadotrophs in the population displaying LC1 immunofluorescence. LC1 was also detected on the surface of anterior pituitary cells by FACS analysis. Incubation of anterior pituitary cells with dexamethasone or corticosterone (0.1 and 1.0 microM) prior to fixation and analysis produced a significant, concentration-dependent decrease in intracellular ir-LC1 and a concomitant increase in the amount of ir-LC1 detected on the surface of the cells; the effects of the two steroids were indistinguishable quantitatively. In conclusion, we report a novel method which permits (1) the detection and semi-quantitative measurement of intracellular and surface LC1 in anterior pituitary cells; and (2) the identification of the cell types in which the protein is found.

40 CFR 799.9780 - TSCA immunotoxicity.

Code of Federal Regulations, 2012 CFR

2012-07-01

... quantitative analysis of the effects of a chemical on the numbers of cells in major lymphocyte populations and..., and the research sample shall be stored under conditions that maintain its purity and stability. Prior... type of effect. (ii) All observed results, quantitative and incidental, shall be evaluated by an...

40 CFR 799.9780 - TSCA immunotoxicity.

Code of Federal Regulations, 2013 CFR

2013-07-01

... quantitative analysis of the effects of a chemical on the numbers of cells in major lymphocyte populations and..., and the research sample shall be stored under conditions that maintain its purity and stability. Prior... type of effect. (ii) All observed results, quantitative and incidental, shall be evaluated by an...

40 CFR 799.9780 - TSCA immunotoxicity.

Code of Federal Regulations, 2010 CFR

2010-07-01

... quantitative analysis of the effects of a chemical on the numbers of cells in major lymphocyte populations and..., and the research sample shall be stored under conditions that maintain its purity and stability. Prior... type of effect. (ii) All observed results, quantitative and incidental, shall be evaluated by an...

40 CFR 799.9780 - TSCA immunotoxicity.

Code of Federal Regulations, 2014 CFR

2014-07-01

... quantitative analysis of the effects of a chemical on the numbers of cells in major lymphocyte populations and..., and the research sample shall be stored under conditions that maintain its purity and stability. Prior... type of effect. (ii) All observed results, quantitative and incidental, shall be evaluated by an...

40 CFR 799.9780 - TSCA immunotoxicity.

Code of Federal Regulations, 2011 CFR

2011-07-01

... quantitative analysis of the effects of a chemical on the numbers of cells in major lymphocyte populations and..., and the research sample shall be stored under conditions that maintain its purity and stability. Prior... type of effect. (ii) All observed results, quantitative and incidental, shall be evaluated by an...

Quantitative Structure-Cytotoxicity Relationship of Cinnamic Acid Phenetyl Esters.

PubMed

Uesawa, Yoshihiro; Sakagami, Hiroshi; Okudaira, Noriyuki; Toda, Kazuhiro; Takao, Koichi; Kagaya, Hajime; Sugita, Yoshiaki

2018-02-01

Many phenolic acid phenethyl esters possess diverse biological effects including antioxidant, cytoprotective, anti-inflammation and anti-tumor activities. However, most previous antitumor studies have not considered the cytotoxicity against normal cells. Ten cinnamic acid phenetyl esters were subjected to quantitative structure-activity relationship (QSAR) analysis, based on their cytotoxicity and tumor-specificity, in order to find their new biological activities. Cytotoxicity against four human oral squamous cell carcinoma cell lines and three oral normal mesenchymal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Tumor specificity (TS) was evaluated by the ratio of the mean 50% cytotoxic concentration (CC 50 ) against normal oral cells to that against human oral squamous cell carcinoma cell lines. Potency-selectivity expression (PSE) value was calculated by dividing the TS value by CC 50 against tumor cells. Apoptosis markers were detected by western blot analysis. Physicochemical, structural and quantum-chemical parameters were calculated based on the conformations optimized by force-field minimization. Western blot analysis demonstrated that [ 9 ] stimulated the cleavage of caspase-3, suggesting the induction of apoptosis. QSAR analysis demonstrated that TS values were correlated with shape, size and ionization potential. Chemical modification of the lead compound may be a potential choice for designing a new type of anticancer drugs. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Quantitative imaging assay for NF-κB nuclear translocation in primary human macrophages

PubMed Central

Noursadeghi, Mahdad; Tsang, Jhen; Haustein, Thomas; Miller, Robert F.; Chain, Benjamin M.; Katz, David R.

2008-01-01

Quantitative measurement of NF-κB nuclear translocation is an important research tool in cellular immunology. Established methodologies have a number of limitations, such as poor sensitivity, high cost or dependence on cell lines. Novel imaging methods to measure nuclear translocation of transcriptionally active components of NF-κB are being used but are also partly limited by the need for specialist imaging equipment or image analysis software. Herein we present a method for quantitative detection of NF-κB rel A nuclear translocation, using immunofluorescence microscopy and the public domain image analysis software ImageJ that can be easily adopted for cellular immunology research without the need for specialist image analysis expertise and at low cost. The method presented here is validated by demonstrating the time course and dose response of NF-κB nuclear translocation in primary human macrophages stimulated with LPS, and by comparison with a commercial NF-κB activation reporter cell line. PMID:18036607

Global, quantitative and dynamic mapping of protein subcellular localization.

PubMed

Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg Hh

2016-06-09

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology.

Quantitative evaluation of bone resorption activity of osteoclast-like cells by measuring calcium phosphate resorbing area using incubator-facilitated and video-enhanced microscopy.

PubMed

Morimoto, Yoshitaka; Hoshino, Hironobu; Sakurai, Takashi; Terakawa, Susumu; Nagano, Akira

2009-04-01

Quantitative evaluation of the ability of bone resorption activity in live osteoclast-like cells (OCLs) has not yet been reported on. In this study, we observed the sequential morphological change of OCLs and measured the resorbing calcium phosphate (CP) area made by OCLs alone and with the addition of elcatonin utilizing incubator facilitated video-enhanced microscopy. OCLs, which were obtained from a coculture of ddy-mouse osteoblastic cells and bone marrow cells, were cultured on CP-coated quartz cover slips. The CP-free area increased constantly in the OCLs alone, whereas it did not increase after the addition of elcatonin. This study showed that analysis of the resorbed areas under the OCL body using this method enables the sequential quantitative evaluation of the bone resorption activity and the effect of several therapeutic agents on bone resorption in vitro.

Membrane-based electrochemical nanobiosensor for Escherichia coli detection and analysis of cells viability.

PubMed

Cheng, Ming Soon; Lau, Suk Hiang; Chow, Vincent T; Toh, Chee-Seng

2011-08-01

A sensitive and selective membrane-based electrochemical nanobiosensor is developed for specific quantitative label-free detection of Escherichia coli (E. coli) cells and analysis of viable but nonculturable (VBNC) E. coli cells which remain mostly undetected using current methods. The sensing mechanism relies on the blocking of nanochannels of a nanoporous alumina-membrane modified electrode, upon the formation of immune complexes at the nanoporous membrane. The resulting obstacle to diffusive mass transfer of a redox probe in the analysis solution to the underlying platinum electrode reduces the Faradaic signal response of the biosensor, measured using cyclic voltammetry. Antibody loading under conditions of varying antibody concentrations and pHs are optimized. The biosensor gives a low detection limit of 22 cfu mL(-1) (R(2) = 0.999) over a wide linear working range of 10 to 10(6) cfu mL(-1). It is specific toward E. coli with minimal cross-reactivity to two other pathogenic bacteria (commonly found in waters). Relative standard deviation (RSD) for triplicate measurements of 2.5% indicates reasonably useful level of reproducibility. Differentiation of live, VBNC, and dead cells are carried out after the cell capture and quantitation step, by simple monitoring of the cells' enzyme activity using the same redox probe in the analysis solution, in the presence of glucose.

Three-dimensional modeling and quantitative analysis of gap junction distributions in cardiac tissue.

PubMed

Lackey, Daniel P; Carruth, Eric D; Lasher, Richard A; Boenisch, Jan; Sachse, Frank B; Hitchcock, Robert W

2011-11-01

Gap junctions play a fundamental role in intercellular communication in cardiac tissue. Various types of heart disease including hypertrophy and ischemia are associated with alterations of the spatial arrangement of gap junctions. Previous studies applied two-dimensional optical and electron-microscopy to visualize gap junction arrangements. In normal cardiomyocytes, gap junctions were primarily found at cell ends, but can be found also in more central regions. In this study, we extended these approaches toward three-dimensional reconstruction of gap junction distributions based on high-resolution scanning confocal microscopy and image processing. We developed methods for quantitative characterization of gap junction distributions based on analysis of intensity profiles along the principal axes of myocytes. The analyses characterized gap junction polarization at cell ends and higher-order statistical image moments of intensity profiles. The methodology was tested in rat ventricular myocardium. Our analysis yielded novel quantitative data on gap junction distributions. In particular, the analysis demonstrated that the distributions exhibit significant variability with respect to polarization, skewness, and kurtosis. We suggest that this methodology provides a quantitative alternative to current approaches based on visual inspection, with applications in particular in characterization of engineered and diseased myocardium. Furthermore, we propose that these data provide improved input for computational modeling of cardiac conduction.

The Focinator v2-0 - Graphical Interface, Four Channels, Colocalization Analysis and Cell Phase Identification.

PubMed

Oeck, Sebastian; Malewicz, Nathalie M; Hurst, Sebastian; Al-Refae, Klaudia; Krysztofiak, Adam; Jendrossek, Verena

2017-07-01

The quantitative analysis of foci plays an important role in various cell biological methods. In the fields of radiation biology and experimental oncology, the effect of ionizing radiation, chemotherapy or molecularly targeted drugs on DNA damage induction and repair is frequently performed by the analysis of protein clusters or phosphorylated proteins recruited to so called repair foci at DNA damage sites, involving for example γ-H2A.X, 53BP1 or RAD51. We recently developed "The Focinator" as a reliable and fast tool for automated quantitative and qualitative analysis of nuclei and DNA damage foci. The refined software is now even more user-friendly due to a graphical interface and further features. Thus, we included an R-script-based mode for automated image opening, file naming, progress monitoring and an error report. Consequently, the evaluation no longer required the attendance of the operator after initial parameter definition. Moreover, the Focinator v2-0 is now able to perform multi-channel analysis of four channels and evaluation of protein-protein colocalization by comparison of up to three foci channels. This enables for example the quantification of foci in cells of a specific cell cycle phase.

Proteomic analysis of acquired tamoxifen resistance in MCF-7 cells reveals expression signatures associated with enhanced migration

PubMed Central

2012-01-01

Introduction Acquired tamoxifen resistance involves complex signaling events that are not yet fully understood. Successful therapeutic intervention to delay the onset of hormone resistance depends critically on mechanistic elucidation of viable molecular targets associated with hormone resistance. This study was undertaken to investigate the global proteomic alterations in a tamoxifen resistant MCF-7 breast cancer cell line obtained by long term treatment of the wild type MCF-7 cell line with 4-hydroxytamoxifen (4-OH Tam). Methods We cultured MCF-7 cells with 4-OH Tam over a period of 12 months to obtain the resistant cell line. A gel-free, quantitative proteomic method was used to identify and quantify the proteome of the resistant cell line. Nano-flow high-performance liquid chromatography coupled to high resolution Fourier transform mass spectrometry was used to analyze fractionated peptide mixtures that were isobarically labeled from the resistant and control cell lysates. Real time quantitative PCR and Western blots were used to verify selected proteomic changes. Lentiviral vector transduction was used to generate MCF-7 cells stably expressing S100P. Online pathway analysis was performed to assess proteomic signatures in tamoxifen resistance. Survival analysis was done to evaluate clinical relevance of altered proteomic expressions. Results Quantitative proteomic analysis revealed a wide breadth of signaling events during transition to acquired tamoxifen resistance. A total of 629 proteins were found significantly changed with 364 up-regulated and 265 down-regulated. Collectively, these changes demonstrated the suppressed state of estrogen receptor (ER) and ER-regulated genes, activated survival signaling and increased migratory capacity of the resistant cell line. The protein S100P was found to play a critical role in conferring tamoxifen resistance and enhanced cell motility. Conclusions Our data demonstrate that the adaptive changes in the proteome of tamoxifen resistant breast cancer cells are characterized by down-regulated ER signaling, activation of alternative survival pathways, and enhanced cell motility through regulation of the actin cytoskeleton dynamics. Evidence also emerged that S100P mediates acquired tamoxifen resistance and migration capacity. PMID:22417809

ACVP-06: Cell-Associated SIV/SHIV RNA and DNA Analysis on Cell/Tissue Specimens  | Frederick National Laboratory for Cancer Research

Cancer.gov

This testing is available only after mandatory prior consultation with Dr. Jeff Lifson (lifsonj@mail.nih.gov) of the Quantitative Molecular Diagnostics Core (QMDC). This assay is performed by the QMDC for measuring levels of cell- or t

Quantitative assessment of neurite outgrowth in human embryonic stem cell derived hN2 cells using automated high-content image analysis

EPA Science Inventory

Throughout development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxic chemicals that interfere with this process may result in permanent deficits in nervous system function. Traditionally, rodent primary ne...

qFlow Cytometry-Based Receptoromic Screening: A High-Throughput Quantification Approach Informing Biomarker Selection and Nanosensor Development.

PubMed

Chen, Si; Weddell, Jared; Gupta, Pavan; Conard, Grace; Parkin, James; Imoukhuede, Princess I

2017-01-01

Nanosensor-based detection of biomarkers can improve medical diagnosis; however, a critical factor in nanosensor development is deciding which biomarker to target, as most diseases present several biomarkers. Biomarker-targeting decisions can be informed via an understanding of biomarker expression. Currently, immunohistochemistry (IHC) is the accepted standard for profiling biomarker expression. While IHC provides a relative mapping of biomarker expression, it does not provide cell-by-cell readouts of biomarker expression or absolute biomarker quantification. Flow cytometry overcomes both these IHC challenges by offering biomarker expression on a cell-by-cell basis, and when combined with calibration standards, providing quantitation of biomarker concentrations: this is known as qFlow cytometry. Here, we outline the key components for applying qFlow cytometry to detect biomarkers within the angiogenic vascular endothelial growth factor receptor family. The key aspects of the qFlow cytometry methodology include: antibody specificity testing, immunofluorescent cell labeling, saturation analysis, fluorescent microsphere calibration, and quantitative analysis of both ensemble and cell-by-cell data. Together, these methods enable high-throughput quantification of biomarker expression.

Quantitative assessment of cancer cell morphology and movement using telecentric digital holographic microscopy

NASA Astrophysics Data System (ADS)

Nguyen, Thanh C.; Nehmetallah, George; Lam, Van; Chung, Byung Min; Raub, Christopher

2017-02-01

Digital holographic microscopy (DHM) provides label-free and real-time quantitative phase information relevant to the analysis of dynamic biological systems. A DHM based on telecentric configuration optically mitigates phase aberrations due to the microscope objective and linear high frequency fringes due to the reference beam thus minimizing digital aberration correction needed for distortion free 3D reconstruction. The purpose of this work is to quantitatively assess growth and migratory behavior of invasive cancer cells using a telecentric DHM system. Together, the height and lateral shape features of individual cells, determined from time-lapse series of phase reconstructions, should reveal aspects of cell migration, cell-matrix adhesion, and cell cycle phase transitions. To test this, MDA-MB-231 breast cancer cells were cultured on collagen-coated or un-coated glass, and 3D holograms were reconstructed over 2 hours. Cells on collagencoated glass had an average 14% larger spread area than cells on uncoated glass (n=18-22 cells/group). The spread area of cells on uncoated glass were 15-21% larger than cells seeded on collagen hydrogels (n=18-22 cells/group). Premitotic cell rounding was observed with average phase height increasing 57% over 10 minutes. Following cell division phase height decreased linearly (R2=0.94) to 58% of the original height pre-division. Phase objects consistent with lamellipodia were apparent from the reconstructions at the leading edge of migrating cells. These data demonstrate the ability to track quantitative phase parameters and relate them to cell morphology during cell migration and division on adherent substrates, using telecentric DHM. The technique enables future studies of cell-matrix interactions relevant to cancer.

A multiplexed quantitative proteomics approach for investigating protein expression in the developing central nervous system.

PubMed

Orme, Rowan P; Gates, Monte A; Fricker-Gates, Rosemary A

2010-08-15

Cell transplantation using stem cell-derived neurons is commonly viewed as a candidate therapy for neurodegenerative diseases. However, methods for differentiating stem cells into homogenous populations of neurons suitable for transplant remain elusive. This suggests that there are as yet unknown signalling factors working in vivo to specify neuronal cell fate during development. These factors could be manipulated to better differentiate stem cells into neural populations useful for therapeutic transplantation. Here a quantitative proteomics approach is described for investigating cell signalling in the developing central nervous system (CNS), using the embryonic ventral mesencephalon as a model. Briefly, total protein was extracted from embryonic ventral midbrain tissue before, during and after the birth of dopaminergic neurons, and digested using trypsin. Two-dimensional liquid chromatography, coupled with tandem mass spectrometry, was then used to identify proteins from the tryptic peptides. Isobaric tagging for relative and absolute quantification (iTRAQ) reagents were used to label the tryptic peptides and facilitate relative quantitative analysis. The success of the experiment was confirmed by the identification of proteins known to be expressed in the developing ventral midbrain, as well as by Western blotting, and immunolabelling of embryonic tissue sections. This method of protein discovery improves upon previous attempts to identify novel signalling factors through microarray analysis. Importantly, the methods described here could be applied to virtually any aspect of development. (c) 2010 Elsevier B.V. All rights reserved.

Application of Organosilane Monolayer Template to Quantitative Evaluation of Cancer Cell Adhesive Ability

NASA Astrophysics Data System (ADS)

Tanii, Takashi; Sasaki, Kosuke; Ichisawa, Kota; Demura, Takanori; Beppu, Yuichi; Vu, Hoan Anh; Thanh Chi, Hoan; Yamamoto, Hideaki; Sato, Yuko

2011-06-01

The adhesive ability of two human pancreatic cancer cell lines was evaluated using organosilane monolayer templates (OMTs). Using the OMT, the spreading area of adhered cells can be limited, and this enables us to focus on the initial attachment process of adhesion. Moreover, it becomes possible to arrange the cells in an array and to quantitatively evaluate the number of attached cells. The adhesive ability of the cancer cells cultured on the OMT was controlled by adding (-)-epigallocatechin-3-gallate (EGCG), which blocks a receptor that mediates cell adhesion and is overexpressed in cancer cells. Measurement of the relative ability of the cancer cells to attach to the OMT revealed that the ability for attachment decreased with increasing EGCG concentration. The results agreed well with the western blot analysis, indicating that the OMT can potentially be employed to evaluate the adhesive ability of various cancer cells.

HISTORY OF GERM CELL MUTAGENESIS

EPA Science Inventory

Much of the early work on germ cell mutation analysis was conducted with nonmammalian species, but this historical overview will begin with the rodent studies that provided quantitative data on induced mutations. The initial studies of mutation induction utilized the newly develo...

Quantitative proteomics in biological research.

PubMed

Wilm, Matthias

2009-10-01

Proteomics has enabled the direct investigation of biological material, at first through the analysis of individual proteins, then of lysates from cell cultures, and finally of extracts from tissues and biopsies from entire organisms. Its latest manifestation - quantitative proteomics - allows deeper insight into biological systems. This article reviews the different methods used to extract quantitative information from mass spectra. It follows the technical developments aimed toward global proteomics, the attempt to characterize every expressed protein in a cell by at least one peptide. When applications of the technology are discussed, the focus is placed on yeast biology. In particular, differential quantitative proteomics, the comparison between an experiment and its control, is very discriminating for proteins involved in the process being studied. When trying to understand biological processes on a molecular level, differential quantitative proteomics tends to give a clearer picture than global transcription analyses. As a result, MS has become an even more indispensable tool for biochemically motivated biological research.

Predicting human blood viscosity in silico

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fedosov, Dmitry A.; Pan, Wenxiao; Caswell, Bruce

2011-07-05

Cellular suspensions such as blood are a part of living organisms and their rheological and flow characteristics determine and affect majority of vital functions. The rheological and flow properties of cell suspensions are determined by collective dynamics of cells, their structure or arrangement, cell properties and interactions. We study these relations for blood in silico using a mesoscopic particle-based method and two different models (multi-scale/low-dimensional) of red blood cells. The models yield accurate quantitative predictions of the dependence of blood viscosity on shear rate and hematocrit. We explicitly model cell aggregation interactions and demonstrate the formation of reversible rouleaux structuresmore » resulting in a tremendous increase of blood viscosity at low shear rates and yield stress, in agreement with experiments. The non-Newtonian behavior of such cell suspensions (e.g., shear thinning, yield stress) is analyzed and related to the suspension’s microstructure, deformation and dynamics of single cells. We provide the flrst quantitative estimates of normal stress differences and magnitude of aggregation forces in blood. Finally, the flexibility of the cell models allows them to be employed for quantitative analysis of a much wider class of complex fluids including cell, capsule, and vesicle suspensions.« less

Method and apparatus for chromatographic quantitative analysis

DOEpatents

Fritz, James S.; Gjerde, Douglas T.; Schmuckler, Gabriella

1981-06-09

An improved apparatus and method for the quantitative analysis of a solution containing a plurality of anion species by ion exchange chromatography which utilizes a single eluent and a single ion exchange bed which does not require periodic regeneration. The solution containing the anions is added to an anion exchange resin bed which is a low capacity macroreticular polystyrene-divinylbenzene resin containing quarternary ammonium functional groups, and is eluted therefrom with a dilute solution of a low electrical conductance organic acid salt. As each anion species is eluted from the bed, it is quantitatively sensed by conventional detection means such as a conductivity cell.

Quantitative impedimetric monitoring of cell migration under the stimulation of cytokine or anti-cancer drug in a microfluidic chip

PubMed Central

Xiao, Xia; Lei, Kin Fong; Huang, Chia-Hao

2015-01-01

Cell migration is a cellular response and results in various biological processes such as cancer metastasis, that is, the primary cause of death for cancer patients. Quantitative investigation of the correlation between cell migration and extracellular stimulation is essential for developing effective therapeutic strategies for controlling invasive cancer cells. The conventional method to determine cell migration rate based on comparison of successive images may not be an objective approach. In this work, a microfluidic chip embedded with measurement electrodes has been developed to quantitatively monitor the cell migration activity based on the impedimetric measurement technique. A no-damage wound was constructed by microfluidic phenomenon and cell migration activity under the stimulation of cytokine and an anti-cancer drug, i.e., interleukin-6 and doxorubicin, were, respectively, investigated. Impedance measurement was concurrently performed during the cell migration process. The impedance change was directly correlated to the cell migration activity; therefore, the migration rate could be calculated. In addition, a good match was found between impedance measurement and conventional imaging analysis. But the impedimetric measurement technique provides an objective and quantitative measurement. Based on our technique, cell migration rates were calculated to be 8.5, 19.1, and 34.9 μm/h under the stimulation of cytokine at concentrations of 0 (control), 5, and 10 ng/ml. This technique has high potential to be developed into a powerful analytical platform for cancer research. PMID:26180566

Nonlinear optical microscopy: use of second harmonic generation and two-photon microscopy for automated quantitative liver fibrosis studies.

PubMed

Sun, Wanxin; Chang, Shi; Tai, Dean C S; Tan, Nancy; Xiao, Guangfa; Tang, Huihuan; Yu, Hanry

2008-01-01

Liver fibrosis is associated with an abnormal increase in an extracellular matrix in chronic liver diseases. Quantitative characterization of fibrillar collagen in intact tissue is essential for both fibrosis studies and clinical applications. Commonly used methods, histological staining followed by either semiquantitative or computerized image analysis, have limited sensitivity, accuracy, and operator-dependent variations. The fibrillar collagen in sinusoids of normal livers could be observed through second-harmonic generation (SHG) microscopy. The two-photon excited fluorescence (TPEF) images, recorded simultaneously with SHG, clearly revealed the hepatocyte morphology. We have systematically optimized the parameters for the quantitative SHG/TPEF imaging of liver tissue and developed fully automated image analysis algorithms to extract the information of collagen changes and cell necrosis. Subtle changes in the distribution and amount of collagen and cell morphology are quantitatively characterized in SHG/TPEF images. By comparing to traditional staining, such as Masson's trichrome and Sirius red, SHG/TPEF is a sensitive quantitative tool for automated collagen characterization in liver tissue. Our system allows for enhanced detection and quantification of sinusoidal collagen fibers in fibrosis research and clinical diagnostics.

Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts.

PubMed

Milovancev, Milan; Hilgart-Martiszus, Ian; McNamara, Michael J; Goodall, Cheri P; Seguin, Bernard; Bracha, Shay; Wickramasekara, Samanthi I

2013-06-13

Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis.

Quantifying the pattern of microbial cell dispersion, density and clustering on surfaces of differing chemistries and topographies using multifractal analysis.

PubMed

Wickens, David; Lynch, Stephen; West, Glen; Kelly, Peter; Verran, Joanna; Whitehead, Kathryn A

2014-09-01

The effects of surface topography on bacterial distribution across a surface are of extreme importance when designing novel, hygienic or antimicrobial surface coatings. The majority of methods that are deployed to describe the pattern of cell dispersion, density and clustering across surfaces are currently qualitative. This paper presents a novel application of multifractal analysis to quantitatively measure these factors using medically relevant microorganisms (Staphylococcus aureus or Staphylococcus epidermidis). Surfaces (medical grade 316 stainless steel) and coatings (Ti-ZrN, Ti-ZrN/6.0%Ag, Ti-ZrN/15.6%Ag, TiZrN/24.7%Ag) were used in microbiological retention assays. Results demonstrated that S. aureus displayed a more heterogeneous cell dispersion (∆αAS<1) whilst the dispersion of S. epidermidis was more symmetric and homogeneous (∆αAS≥1). Further, although the surface topography and chemistry had an effect on cell dispersion, density and clustering, the type of bonding that occurred at the surface interface was also important. Both types of cells were influenced by both surface topographical and chemical effects; however, S. aureus was influenced marginally more by surface chemistry whilst S. epidermidis cells was influenced marginally more by surface topography. Thus, this effect was bacterially species specific. The results demonstrate that multifractal analysis is a method that can be used to quantitatively analyse the cell dispersion, density and clustering of retained microorganisms on surfaces. Using quantitative descriptors has the potential to aid the understanding the effect of surface properties on the production of hygienic and antimicrobial coatings. Copyright © 2014 Elsevier B.V. All rights reserved.

Microscopic optical path length difference and polarization measurement system for cell analysis

NASA Astrophysics Data System (ADS)

Satake, H.; Ikeda, K.; Kowa, H.; Hoshiba, T.; Watanabe, E.

2018-03-01

In recent years, noninvasive, nonstaining, and nondestructive quantitative cell measurement techniques have become increasingly important in the medical field. These cell measurement techniques enable the quantitative analysis of living cells, and are therefore applied to various cell identification processes, such as those determining the passage number limit during cell culturing in regenerative medicine. To enable cell measurement, we developed a quantitative microscopic phase imaging system based on a Mach-Zehnder interferometer that measures the optical path length difference distribution without phase unwrapping using optical phase locking. The applicability of our phase imaging system was demonstrated by successful identification of breast cancer cells amongst normal cells. However, the cell identification method using this phase imaging system exhibited a false identification rate of approximately 7%. In this study, we implemented a polarimetric imaging system by introducing a polarimetric module to one arm of the Mach-Zehnder interferometer of our conventional phase imaging system. This module was comprised of a quarter wave plate and a rotational polarizer on the illumination side of the sample, and a linear polarizer on the optical detector side. In addition, we developed correction methods for the measurement errors of the optical path length and birefringence phase differences that arose through the influence of elements other than cells, such as the Petri dish. As the Petri dish holding the fluid specimens was transparent, it did not affect the amplitude information; however, the optical path length and birefringence phase differences were affected. Therefore, we proposed correction of the optical path length and birefringence phase for the influence of elements other than cells, as a prerequisite for obtaining highly precise phase and polarimetric images.

Quantitative Analysis of Dendritic Cell Haptotaxis.

PubMed

Schwarz, Jan; Sixt, Michael

2016-01-01

Chemokines are the main guidance cues directing leukocyte migration. Opposed to early assumptions, chemokines do not necessarily act as soluble cues but are often immobilized within tissues, e.g., dendritic cell migration toward lymphatic vessels is guided by a haptotactic gradient of the chemokine CCL21. Controlled assay systems to quantitatively study haptotaxis in vitro are still missing. In this chapter, we describe an in vitro haptotaxis assay optimized for the unique properties of dendritic cells. The chemokine CCL21 is immobilized in a bioactive state, using laser-assisted protein adsorption by photobleaching. The cells follow this immobilized CCL21 gradient in a haptotaxis chamber, which provides three dimensionally confined migration conditions. © 2016 Elsevier Inc. All rights reserved.

Quantitative Confocal Microscopy Analysis as a Basis for Search and Study of Potassium Kv1.x Channel Blockers

NASA Astrophysics Data System (ADS)

Feofanov, Alexey V.; Kudryashova, Kseniya S.; Nekrasova, Oksana V.; Vassilevski, Alexander A.; Kuzmenkov, Alexey I.; Korolkova, Yuliya V.; Grishin, Eugene V.; Kirpichnikov, Mikhail P.

Artificial KcsA-Kv1.x (x = 1, 3) receptors were recently designed by transferring the ligand-binding site from human Kv1.x voltage-gated potassium channels into corresponding domain of the bacterial KscA channel. We found that KcsA-Kv1.x receptors expressed in E. coli cells are embedded into cell membrane and bind ligands when the cells are transformed to spheroplasts. We supposed that E. coli spheroplasts with membrane-embedded KcsA-Kv1.x and fluorescently labeled ligand agitoxin-2 (R-AgTx2) can be used as elements of an advanced analytical system for search and study of Kv1-channel blockers. To realize this idea, special procedures were developed for measurement and quantitative treatment of fluorescence signals obtained from spheroplast membrane using confocal laser scanning microscopy (CLSM). The worked out analytical "mix and read" systems supported by quantitative CLSM analysis were demonstrated to be reliable alternative to radioligand and electrophysiology techniques in the search and study of selective Kv1.x channel blockers of high scientific and medical importance.

Development of cell-based quantitative evaluation method for cell cycle-arrest type cancer drugs for apoptosis by high precision surface plasmon resonance sensor

NASA Astrophysics Data System (ADS)

Ona, Toshihiro; Nishijima, Hiroshi; Kosaihira, Atsushi; Shibata, Junko

2008-04-01

In vitro rapid and quantitative cell-based assay is demanded to verify the efficacy prediction of cancer drugs since a cancer patient may have unconventional aspects of tumor development. Here, we show the rapid and non-label quantitative verifying method and instrumentation of apoptosis for cell cycle-arrest type cancer drugs (Roscovitine and D-allose) by reaction analysis of living liver cancer cells cultured on a sensor chip with a newly developed high precision (50 ndeg s -1 average fluctuation) surface plasmon resonance (SPR) sensor. The time-course cell reaction as the SPR angle change rate for 10 min from 30 min cell culture with a drug was significantly related to cell viability. By the simultaneous detection of differential SPR angle change and fluorescence by specific probes using the new instrument, the SPR angle was related to the nano-order potential decrease in inner mitochondrial membrane potential. The results obtained are universally valid for the cell cycle-arrest type cancer drugs, which mediate apoptosis through different cell-signaling pathways, by a liver cancer cell line of Hep G2 (P<0.001). This system towards the application to evaluate personal therapeutic potentials of drugs using cancer cells from patients in clinical use.

Automated Detection of P. falciparum Using Machine Learning Algorithms with Quantitative Phase Images of Unstained Cells.

PubMed

Park, Han Sang; Rinehart, Matthew T; Walzer, Katelyn A; Chi, Jen-Tsan Ashley; Wax, Adam

2016-01-01

Malaria detection through microscopic examination of stained blood smears is a diagnostic challenge that heavily relies on the expertise of trained microscopists. This paper presents an automated analysis method for detection and staging of red blood cells infected by the malaria parasite Plasmodium falciparum at trophozoite or schizont stage. Unlike previous efforts in this area, this study uses quantitative phase images of unstained cells. Erythrocytes are automatically segmented using thresholds of optical phase and refocused to enable quantitative comparison of phase images. Refocused images are analyzed to extract 23 morphological descriptors based on the phase information. While all individual descriptors are highly statistically different between infected and uninfected cells, each descriptor does not enable separation of populations at a level satisfactory for clinical utility. To improve the diagnostic capacity, we applied various machine learning techniques, including linear discriminant classification (LDC), logistic regression (LR), and k-nearest neighbor classification (NNC), to formulate algorithms that combine all of the calculated physical parameters to distinguish cells more effectively. Results show that LDC provides the highest accuracy of up to 99.7% in detecting schizont stage infected cells compared to uninfected RBCs. NNC showed slightly better accuracy (99.5%) than either LDC (99.0%) or LR (99.1%) for discriminating late trophozoites from uninfected RBCs. However, for early trophozoites, LDC produced the best accuracy of 98%. Discrimination of infection stage was less accurate, producing high specificity (99.8%) but only 45.0%-66.8% sensitivity with early trophozoites most often mistaken for late trophozoite or schizont stage and late trophozoite and schizont stage most often confused for each other. Overall, this methodology points to a significant clinical potential of using quantitative phase imaging to detect and stage malaria infection without staining or expert analysis.

Automated Detection of P. falciparum Using Machine Learning Algorithms with Quantitative Phase Images of Unstained Cells

PubMed Central

Park, Han Sang; Rinehart, Matthew T.; Walzer, Katelyn A.; Chi, Jen-Tsan Ashley; Wax, Adam

2016-01-01

Malaria detection through microscopic examination of stained blood smears is a diagnostic challenge that heavily relies on the expertise of trained microscopists. This paper presents an automated analysis method for detection and staging of red blood cells infected by the malaria parasite Plasmodium falciparum at trophozoite or schizont stage. Unlike previous efforts in this area, this study uses quantitative phase images of unstained cells. Erythrocytes are automatically segmented using thresholds of optical phase and refocused to enable quantitative comparison of phase images. Refocused images are analyzed to extract 23 morphological descriptors based on the phase information. While all individual descriptors are highly statistically different between infected and uninfected cells, each descriptor does not enable separation of populations at a level satisfactory for clinical utility. To improve the diagnostic capacity, we applied various machine learning techniques, including linear discriminant classification (LDC), logistic regression (LR), and k-nearest neighbor classification (NNC), to formulate algorithms that combine all of the calculated physical parameters to distinguish cells more effectively. Results show that LDC provides the highest accuracy of up to 99.7% in detecting schizont stage infected cells compared to uninfected RBCs. NNC showed slightly better accuracy (99.5%) than either LDC (99.0%) or LR (99.1%) for discriminating late trophozoites from uninfected RBCs. However, for early trophozoites, LDC produced the best accuracy of 98%. Discrimination of infection stage was less accurate, producing high specificity (99.8%) but only 45.0%-66.8% sensitivity with early trophozoites most often mistaken for late trophozoite or schizont stage and late trophozoite and schizont stage most often confused for each other. Overall, this methodology points to a significant clinical potential of using quantitative phase imaging to detect and stage malaria infection without staining or expert analysis. PMID:27636719

Assessment of stem cell differentiation based on genome-wide expression profiles.

PubMed

Godoy, Patricio; Schmidt-Heck, Wolfgang; Hellwig, Birte; Nell, Patrick; Feuerborn, David; Rahnenführer, Jörg; Kattler, Kathrin; Walter, Jörn; Blüthgen, Nils; Hengstler, Jan G

2018-07-05

In recent years, protocols have been established to differentiate stem and precursor cells into more mature cell types. However, progress in this field has been hampered by difficulties to assess the differentiation status of stem cell-derived cells in an unbiased manner. Here, we present an analysis pipeline based on published data and methods to quantify the degree of differentiation and to identify transcriptional control factors explaining differences from the intended target cells or tissues. The pipeline requires RNA-Seq or gene array data of the stem cell starting population, derived 'mature' cells and primary target cells or tissue. It consists of a principal component analysis to represent global expression changes and to identify possible problems of the dataset that require special attention, such as: batch effects; clustering techniques to identify gene groups with similar features; over-representation analysis to characterize biological motifs and transcriptional control factors of the identified gene clusters; and metagenes as well as gene regulatory networks for quantitative cell-type assessment and identification of influential transcription factors. Possibilities and limitations of the analysis pipeline are illustrated using the example of human embryonic stem cell and human induced pluripotent cells to generate 'hepatocyte-like cells'. The pipeline quantifies the degree of incomplete differentiation as well as remaining stemness and identifies unwanted features, such as colon- and fibroblast-associated gene clusters that are absent in real hepatocytes but typically induced by currently available differentiation protocols. Finally, transcription factors responsible for incomplete and unwanted differentiation are identified. The proposed method is widely applicable and allows an unbiased and quantitative assessment of stem cell-derived cells.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'. © 2018 The Author(s).

Quantitative Analysis of Intracellular Motility Based on Optical Flow Model

PubMed Central

Li, Heng

2017-01-01

Analysis of cell mobility is a key issue for abnormality identification and classification in cell biology research. However, since cell deformation induced by various biological processes is random and cell protrusion is irregular, it is difficult to measure cell morphology and motility in microscopic images. To address this dilemma, we propose an improved variation optical flow model for quantitative analysis of intracellular motility, which not only extracts intracellular motion fields effectively but also deals with optical flow computation problem at the border by taking advantages of the formulation based on L1 and L2 norm, respectively. In the energy functional of our proposed optical flow model, the data term is in the form of L2 norm; the smoothness of the data changes with regional features through an adaptive parameter, using L1 norm near the edge of the cell and L2 norm away from the edge. We further extract histograms of oriented optical flow (HOOF) after optical flow field of intracellular motion is computed. Then distances of different HOOFs are calculated as the intracellular motion features to grade the intracellular motion. Experimental results show that the features extracted from HOOFs provide new insights into the relationship between the cell motility and the special pathological conditions. PMID:29065574

Quantitative analysis of changes in cell shape of Amoeba proteus during locomotion and upon responses to salt stimuli.

PubMed

Ueda, T; Kobatake, Y

1983-09-01

A new parameter expressing the complexity of cell shape defined as (periphery)2/(area) in 2D projection was found useful for a quantitative analysis of changes in the cell shape of Amoeba proteus and potentially of any amoeboid cells. During locomotion the complexity and the motive force of the protoplasmic streaming in amoeba varied periodically, and the Fourier analysis of the two showed a similar pattern in the power spectrum, giving a rather broad peak at about 2.5 X 10(-3) Hz. The complexity increased mainly due to elongation of the cell as external Ca2+ increased. This effect was blocked by La3+, half the inhibition being attained at 1/200 amount of the coexisting Ca2+. On the other hand, the complexity decreased due to rounding up of the cell as the concentration of other cations, such as Sr2+, Mg2+, Co2+, Ni2+, Na+, K+ etc., increased. Irrespective of the opposite effects of Ca2+ and other cations on the cell shape, the ATP concentration in amoeba decreased in both cases with increase of all these cations. The irregularity in amoeboid motility is discussed in terms of a dynamic system theory.

Cell classification using big data analytics plus time stretch imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Jalali, Bahram; Chen, Claire L.; Mahjoubfar, Ata

2016-09-01

We show that blood cells can be classified with high accuracy and high throughput by combining machine learning with time stretch quantitative phase imaging. Our diagnostic system captures quantitative phase images in a flow microscope at millions of frames per second and extracts multiple biophysical features from individual cells including morphological characteristics, light absorption and scattering parameters, and protein concentration. These parameters form a hyperdimensional feature space in which supervised learning and cell classification is performed. We show binary classification of T-cells against colon cancer cells, as well classification of algae cell strains with high and low lipid content. The label-free screening averts the negative impact of staining reagents on cellular viability or cell signaling. The combination of time stretch machine vision and learning offers unprecedented cell analysis capabilities for cancer diagnostics, drug development and liquid biopsy for personalized genomics.

Quantitative fluorescence correlation spectroscopy on DNA in living cells

NASA Astrophysics Data System (ADS)

Hodges, Cameron; Kafle, Rudra P.; Meiners, Jens-Christian

2017-02-01

FCS is a fluorescence technique conventionally used to study the kinetics of fluorescent molecules in a dilute solution. Being a non-invasive technique, it is now drawing increasing interest for the study of more complex systems like the dynamics of DNA or proteins in living cells. Unlike an ordinary dye solution, the dynamics of macromolecules like proteins or entangled DNA in crowded environments is often slow and subdiffusive in nature. This in turn leads to longer residence times of the attached fluorophores in the excitation volume of the microscope and artifacts from photobleaching abound that can easily obscure the signature of the molecular dynamics of interest and make quantitative analysis challenging.We discuss methods and procedures to make FCS applicable to quantitative studies of the dynamics of DNA in live prokaryotic and eukaryotic cells. The intensity autocorrelation is computed function from weighted arrival times of the photons on the detector that maximizes the information content while simultaneously correcting for the effect of photobleaching to yield an autocorrelation function that reflects only the underlying dynamics of the sample. This autocorrelation function in turn is used to calculate the mean square displacement of the fluorophores attached to DNA. The displacement data is more amenable to further quantitative analysis than the raw correlation functions. By using a suitable integral transform of the mean square displacement, we can then determine the viscoelastic moduli of the DNA in its cellular environment. The entire analysis procedure is extensively calibrated and validated using model systems and computational simulations.

In silico designing of power conversion efficient organic lead dyes for solar cells using todays innovative approaches to assure renewable energy for future

NASA Astrophysics Data System (ADS)

Kar, Supratik; Roy, Juganta K.; Leszczynski, Jerzy

2017-06-01

Advances in solar cell technology require designing of new organic dye sensitizers for dye-sensitized solar cells with high power conversion efficiency to circumvent the disadvantages of silicon-based solar cells. In silico studies including quantitative structure-property relationship analysis combined with quantum chemical analysis were employed to understand the primary electron transfer mechanism and photo-physical properties of 273 arylamine organic dyes from 11 diverse chemical families explicit to iodine electrolyte. The direct quantitative structure-property relationship models enable identification of the essential electronic and structural attributes necessary for quantifying the molecular prerequisites of 11 classes of arylamine organic dyes, responsible for high power conversion efficiency of dye-sensitized solar cells. Tetrahydroquinoline, N,N'-dialkylaniline and indoline have been least explored classes under arylamine organic dyes for dye-sensitized solar cells. Therefore, the identified properties from the corresponding quantitative structure-property relationship models of the mentioned classes were employed in designing of "lead dyes". Followed by, a series of electrochemical and photo-physical parameters were computed for designed dyes to check the required variables for electron flow of dye-sensitized solar cells. The combined computational techniques yielded seven promising lead dyes each for all three chemical classes considered. Significant (130, 183, and 46%) increment in predicted %power conversion efficiency was observed comparing with the existing dye with highest experimental %power conversion efficiency value for tetrahydroquinoline, N,N'-dialkylaniline and indoline, respectively maintaining required electrochemical parameters.

Putative regulatory sites unraveled by network-embedded thermodynamic analysis of metabolome data

PubMed Central

Kümmel, Anne; Panke, Sven; Heinemann, Matthias

2006-01-01

As one of the most recent members of the omics family, large-scale quantitative metabolomics data are currently complementing our systems biology data pool and offer the chance to integrate the metabolite level into the functional analysis of cellular networks. Network-embedded thermodynamic analysis (NET analysis) is presented as a framework for mechanistic and model-based analysis of these data. By coupling the data to an operating metabolic network via the second law of thermodynamics and the metabolites' Gibbs energies of formation, NET analysis allows inferring functional principles from quantitative metabolite data; for example it identifies reactions that are subject to active allosteric or genetic regulation as exemplified with quantitative metabolite data from Escherichia coli and Saccharomyces cerevisiae. Moreover, the optimization framework of NET analysis was demonstrated to be a valuable tool to systematically investigate data sets for consistency, for the extension of sub-omic metabolome data sets and for resolving intracompartmental concentrations from cell-averaged metabolome data. Without requiring any kind of kinetic modeling, NET analysis represents a perfectly scalable and unbiased approach to uncover insights from quantitative metabolome data. PMID:16788595

Muscadine Grape Skin Extract Induces an Unfolded Protein Response-Mediated Autophagy in Prostate Cancer Cells: A TMT-Based Quantitative Proteomic Analysis

PubMed Central

Burton, Liza J.; Rivera, Mariela; Hawsawi, Ohuod; Zou, Jin; Hudson, Tamaro; Wang, Guangdi; Zhang, Qiang; Cubano, Luis; Boukli, Nawal; Odero-Marah, Valerie

2016-01-01

Muscadine grape skin extract (MSKE) is derived from muscadine grape (Vitis rotundifolia), a common red grape used to produce red wine. Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR) that serves as a survival mechanism to relieve ER stress and restore ER homeostasis. However, when persistent, ER stress can alter the cytoprotective functions of the UPR to promote autophagy and cell death. Although MSKE has been documented to induce apoptosis, it has not been linked to ER stress/UPR/autophagy. We hypothesized that MSKE may induce a severe ER stress response-mediated autophagy leading to apoptosis. As a model, we treated C4-2 prostate cancer cells with MSKE and performed a quantitative Tandem Mass Tag Isobaric Labeling proteomic analysis. ER stress response, autophagy and apoptosis were analyzed by western blot, acridine orange and TUNEL/Annexin V staining, respectively. Quantitative proteomics analysis indicated that ER stress response proteins, such as GRP78 were greatly elevated following treatment with MSKE. The up-regulation of pro-apoptotic markers PARP, caspase-12, cleaved caspase-3, -7, BAX and down-regulation of anti-apoptotic marker BCL2 was confirmed by Western blot analysis and apoptosis was visualized by increased TUNEL/Annexin V staining upon MSKE treatment. Moreover, increased acridine orange, and LC3B staining was detected in MSKE-treated cells, suggesting an ER stress/autophagy response. Finally, MSKE-mediated autophagy and apoptosis was antagonized by co-treatment with chloroquine, an autophagy inhibitor. Our results indicate that MSKE can elicit an UPR that can eventually lead to apoptosis in prostate cancer cells. PMID:27755556

Muscadine Grape Skin Extract Induces an Unfolded Protein Response-Mediated Autophagy in Prostate Cancer Cells: A TMT-Based Quantitative Proteomic Analysis.

PubMed

Burton, Liza J; Rivera, Mariela; Hawsawi, Ohuod; Zou, Jin; Hudson, Tamaro; Wang, Guangdi; Zhang, Qiang; Cubano, Luis; Boukli, Nawal; Odero-Marah, Valerie

2016-01-01

Muscadine grape skin extract (MSKE) is derived from muscadine grape (Vitis rotundifolia), a common red grape used to produce red wine. Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR) that serves as a survival mechanism to relieve ER stress and restore ER homeostasis. However, when persistent, ER stress can alter the cytoprotective functions of the UPR to promote autophagy and cell death. Although MSKE has been documented to induce apoptosis, it has not been linked to ER stress/UPR/autophagy. We hypothesized that MSKE may induce a severe ER stress response-mediated autophagy leading to apoptosis. As a model, we treated C4-2 prostate cancer cells with MSKE and performed a quantitative Tandem Mass Tag Isobaric Labeling proteomic analysis. ER stress response, autophagy and apoptosis were analyzed by western blot, acridine orange and TUNEL/Annexin V staining, respectively. Quantitative proteomics analysis indicated that ER stress response proteins, such as GRP78 were greatly elevated following treatment with MSKE. The up-regulation of pro-apoptotic markers PARP, caspase-12, cleaved caspase-3, -7, BAX and down-regulation of anti-apoptotic marker BCL2 was confirmed by Western blot analysis and apoptosis was visualized by increased TUNEL/Annexin V staining upon MSKE treatment. Moreover, increased acridine orange, and LC3B staining was detected in MSKE-treated cells, suggesting an ER stress/autophagy response. Finally, MSKE-mediated autophagy and apoptosis was antagonized by co-treatment with chloroquine, an autophagy inhibitor. Our results indicate that MSKE can elicit an UPR that can eventually lead to apoptosis in prostate cancer cells.

Simple and fast spectral domain algorithm for quantitative phase imaging of living cells with digital holographic microscopy

NASA Astrophysics Data System (ADS)

Min, Junwei; Yao, Baoli; Ketelhut, Steffi; Kemper, Björn

2017-02-01

The modular combination of optical microscopes with digital holographic microscopy (DHM) has been proven to be a powerful tool for quantitative live cell imaging. The introduction of condenser and different microscope objectives (MO) simplifies the usage of the technique and makes it easier to measure different kinds of specimens with different magnifications. However, the high flexibility of illumination and imaging also causes variable phase aberrations that need to be eliminated for high resolution quantitative phase imaging. The existent phase aberrations compensation methods either require add additional elements into the reference arm or need specimen free reference areas or separate reference holograms to build up suitable digital phase masks. These inherent requirements make them unpractical for usage with highly variable illumination and imaging systems and prevent on-line monitoring of living cells. In this paper, we present a simple numerical method for phase aberration compensation based on the analysis of holograms in spatial frequency domain with capabilities for on-line quantitative phase imaging. From a single shot off-axis hologram, the whole phase aberration can be eliminated automatically without numerical fitting or pre-knowledge of the setup. The capabilities and robustness for quantitative phase imaging of living cancer cells are demonstrated.

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.

PubMed

Rusin, Scott F; Schlosser, Kate A; Adamo, Mark E; Kettenbach, Arminja N

2015-10-13

Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. Copyright © 2015, American Association for the Advancement of Science.

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells

PubMed Central

Rusin, Scott F.; Schlosser, Kate A.; Adamo, Mark E.; Kettenbach, Arminja N.

2017-01-01

Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry–based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c–dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2–dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. PMID:26462736

Dancing Styles of Collective Cell Migration: Image-Based Computational Analysis of JRAB/MICAL-L2.

PubMed

Sakane, Ayuko; Yoshizawa, Shin; Yokota, Hideo; Sasaki, Takuya

2018-01-01

Collective cell migration is observed during morphogenesis, angiogenesis, and wound healing, and this type of cell migration also contributes to efficient metastasis in some kinds of cancers. Because collectively migrating cells are much better organized than a random assemblage of individual cells, there seems to be a kind of order in migrating clusters. Extensive research has identified a large number of molecules involved in collective cell migration, and these factors have been analyzed using dramatic advances in imaging technology. To date, however, it remains unclear how myriad cells are integrated as a single unit. Recently, we observed unbalanced collective cell migrations that can be likened to either precision dancing or awa-odori , Japanese traditional dancing similar to the style at Rio Carnival, caused by the impairment of the conformational change of JRAB/MICAL-L2. This review begins with a brief history of image-based computational analyses on cell migration, explains why quantitative analysis of the stylization of collective cell behavior is difficult, and finally introduces our recent work on JRAB/MICAL-L2 as a successful example of the multidisciplinary approach combining cell biology, live imaging, and computational biology. In combination, these methods have enabled quantitative evaluations of the "dancing style" of collective cell migration.

Evaluation of p16 hypermethylation in oral submucous fibrosis: A quantitative and comparative analysis in buccal cells and saliva using real-time methylation-specific polymerase chain reaction.

PubMed

Kaliyaperumal, Subadra; Sankarapandian, Sathasivasubramanian

2016-01-01

The aim of this study was to quantitatively investigate the hypermethylation of p16 gene in buccal cells and saliva of oral submucous fibrosis (OSMF) patients using real-time quantitative methylation-specific polymerase chain reaction (PCR) and to compare the values of two methods. A total of 120 samples were taken from 60 subjects selected for this study, of which 30 were controls and 30 patients were clinically and histopathologically diagnosed with OSMF. In both groups, two sets of samples were collected, one directly from the buccal cells through cytobrush technique and the other through salivary rinse. We analyzed the samples for the presence of p16 hypermethylation using quantitative real-time PCR. In OSMF, the hypermethylation status of p16 in buccal cells was very high (93.3%) and in salivary samples, it was partially methylated (50%). However, no hypermethylation was found in controls suggesting that significant quantity of p16 hypermethylation was present in buccal cells and saliva in OSMF. This study indicates that buccal cell sampling may be a better method for evaluation than the salivary samples. It signifies that hypermethylation of p16 is an important factor to be considered in epigenetic alterations of normal cells to oral precancer, i.e. OSMF.

Establishment of a sensitive system for analysis of human vaginal microbiota on the basis of rRNA-targeted reverse transcription-quantitative PCR.

PubMed

Kurakawa, Takashi; Ogata, Kiyohito; Tsuji, Hirokazu; Kado, Yukiko; Takahashi, Takuya; Kida, Yumi; Ito, Masahiro; Okada, Nobuhiko; Nomoto, Koji

2015-04-01

Ten specific primer sets, for Lactobacillus gasseri, Lactobacillus crispatus, Atopobium vaginae, Gardnerella vaginalis, Mobiluncus curtisii, Chlamydia trachomatis/muridarum, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, and Bifidobacterium angulatum, were developed for quantitative analysis of vaginal microbiota. rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) analysis of the vaginal samples from 12 healthy Japanese volunteers using the new primer sets together with 25 existing primer sets revealed the diversity of their vaginal microbiota: Lactobacilli such as L. crispatus, L. gasseri, Lactobacillus jensenii, Lactobacillus iners, and Lactobacillus vaginalis, as the major populations at 10(7) cells/ml vaginal fluid, were followed by facultative anaerobes such as Streptococcus and strict anaerobes at lower population levels of 10(4) cells/ml or less. Certain bacterial vaginosis (BV)-related bacteria, such as G. vaginalis, A. vaginae, M. curtisii, and Prevotella, were also detected in some subjects. Especially in one subject, both G. vaginalis and A. vaginae were detected at high population levels of 10(8.8) and 10(8.9) cells/ml vaginal fluid, suggesting that she is an asymptomatic BV patient. These results suggest that the RT-qPCR system is effective for accurate analysis of major vaginal commensals and diagnosis of several vaginal infections. Copyright © 2015 Elsevier B.V. All rights reserved.

Glycoproteomic Analysis of Glioblastoma Stem Cell Differentiation

PubMed Central

He, Jintang; Liu, Yashu; Zhu, Thant S.; Xie, Xiaolei; Costello, Mark A.; Talsma, Caroline E.; Flack, Callie G.; Crowley, Jessica G.; DiMeco, Francesco; Vescovi, Angelo L.; Fan, Xing; Lubman, David M.

2010-01-01

Cancer stem cells are responsible for tumor formation through self-renewal and differentiation into multiple cell types, and thus represent a new therapeutic target for tumors. Glycoproteins play a critical role in determining the fates of stem cells such as self-renewal, proliferation and differentiation. Here we applied a multi-lectin affinity chromatography and quantitative glycoproteomics approach to analyze alterations of glycoproteins relevant to the differentiation of a glioblastoma-derived stem cell line HSR-GBM1. Three lectins including concanavalin A (Con A), wheat germ agglutinin (WGA) and peanut agglutinin (PNA) were used to capture glycoproteins, followed by LC-MS/MS analysis. A total of 73 and 79 high-confidence (FDR < 0.01) glycoproteins were identified from the undifferentiated and differentiated cells, respectively. Label-free quantitation resulted in the discovery of 18 differentially expressed glycoproteins, wherein 9 proteins are localized in the lysosome. All of these lysosomal glycoproteins were up-regulated after differentiation, where their principal function was hydrolysis of glycosyl residues. Protein-protein interaction and functional analyses revealed the active involvement of lysosomes during the process of glioblastoma stem cell differentiation. This work provides glycoprotein markers to characterize differentiation status of glioblastoma stem cells which may be useful in stemcell therapy of glioblastoma. PMID:21110520

The protein expression landscape of mitosis and meiosis in diploid budding yeast.

PubMed

Becker, Emmanuelle; Com, Emmanuelle; Lavigne, Régis; Guilleux, Marie-Hélène; Evrard, Bertrand; Pineau, Charles; Primig, Michael

2017-03-06

Saccharomyces cerevisiae is an established model organism for the molecular analysis of fundamental biological processes. The genomes of numerous strains have been sequenced, and the transcriptome and proteome ofmajor phases during the haploid and diploid yeast life cycle have been determined. However, much less is known about dynamic changes of the proteome when cells switch from mitotic growth to meiotic development. We report a quantitative protein profiling analysis of yeast cell division and differentiation based on mass spectrometry. Information about protein levels was integrated with strand-specific tiling array expression data. We identified a total of 2366 proteins in at least one condition, including 175 proteins showing a statistically significant>5-fold change across the sample set, and 136 proteins detectable in sporulating but not respiring cells. We correlate protein expression patterns with biological processes and molecular function by Gene Ontology term enrichment, chemoprofiling, transcription interference and the formation of double stranded RNAs by overlapping sense/antisense transcripts. Our work provides initial quantitative insight into protein expression in diploid respiring and differentiating yeast cells. Critically, it associates developmentally regulated induction of antisense long noncoding RNAs and double stranded RNAs with fluctuating protein concentrations during growth and development. This integrated genomics analysis helps better understand how the transcriptome and the proteome correlate in diploid yeast cells undergoing mitotic growth in the presence of acetate (respiration) versus meiotic differentiation (Meiosis I and II). The study (i) provides quantitative expression data for 2366 proteins and their cognate mRNAs in at least one sample, (ii) shows strongly fluctuating protein levels during growth and differentiation for 175 cases, and (iii) identifies 136 proteins absent in mitotic but present in meiotic yeast cells. We have integrated protein profiling data using mass spectrometry with tiling array RNA profiling data and information on double-stranded RNAs (dsRNAs) by overlapping sense/antisense transcripts from an RNA-Sequencing experiment. This work therefore provides quantitative insight into protein expression during cell division and development and associates changing protein levels with developmental stage specific induction of antisense transcripts and the formation of dsRNAs. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative phosphoproteomic analysis of host responses in human lung epithelial (A549) cells during influenza virus infection.

PubMed

Dapat, Clyde; Saito, Reiko; Suzuki, Hiroshi; Horigome, Tsuneyoshi

2014-01-22

The emergence of antiviral drug-resistant influenza viruses highlights the need for alternative therapeutic strategies. Elucidation of host factors required during virus infection provides information not only on the signaling pathways involved but also on the identification of novel drug targets. RNA interference screening method had been utilized by several studies to determine these host factors; however, proteomics data on influenza host factors are currently limited. In this study, quantitative phosphoproteomic analysis of human lung cell line (A549) infected with 2009 pandemic influenza virus A (H1N1) virus was performed. Phosphopeptides were enriched from tryptic digests of total protein of infected and mock-infected cells using a titania column on an automated purification system followed by iTRAQ labeling. Identification and quantitative analysis of iTRAQ-labeled phosphopeptides were performed using LC-MS/MS. We identified 366 phosphorylation sites on 283 proteins. Of these, we detected 43 upregulated and 35 downregulated proteins during influenza virus infection. Gene ontology enrichment analysis showed that majority of the identified proteins are phosphoproteins involved in RNA processing, immune system process and response to infection. Host-virus interaction network analysis had identified 23 densely connected subnetworks. Of which, 13 subnetworks contained proteins with altered phosphorylation levels during by influenza virus infection. Our results will help to identify potential drug targets that can be pursued for influenza antiviral drug development. Copyright © 2013 Elsevier B.V. All rights reserved.

Groping for quantitative digital 3-D image analysis: an approach to quantitative fluorescence in situ hybridization in thick tissue sections of prostate carcinoma.

PubMed

Rodenacker, K; Aubele, M; Hutzler, P; Adiga, P S

1997-01-01

In molecular pathology numerical chromosome aberrations have been found to be decisive for the prognosis of malignancy in tumours. The existence of such aberrations can be detected by interphase fluorescence in situ hybridization (FISH). The gain or loss of certain base sequences in the desoxyribonucleic acid (DNA) can be estimated by counting the number of FISH signals per cell nucleus. The quantitative evaluation of such events is a necessary condition for a prospective use in diagnostic pathology. To avoid occlusions of signals, the cell nucleus has to be analyzed in three dimensions. Confocal laser scanning microscopy is the means to obtain series of optical thin sections from fluorescence stained or marked material to fulfill the conditions mentioned above. A graphical user interface (GUI) to a software package for display, inspection, count and (semi-)automatic analysis of 3-D images for pathologists is outlined including the underlying methods of 3-D image interaction and segmentation developed. The preparative methods are briefly described. Main emphasis is given to the methodical questions of computer-aided analysis of large 3-D image data sets for pathologists. Several automated analysis steps can be performed for segmentation and succeeding quantification. However tumour material is in contrast to isolated or cultured cells even for visual inspection, a difficult material. For the present a fully automated digital image analysis of 3-D data is not in sight. A semi-automatic segmentation method is thus presented here.

Wires in the soup: quantitative models of cell signaling

PubMed Central

Cheong, Raymond; Levchenko, Andre

2014-01-01

Living cells are capable of extracting information from their environments and mounting appropriate responses to a variety of associated challenges. The underlying signal transduction networks enabling this can be quite complex, necessitating for their unraveling by sophisticated computational modeling coupled with precise experimentation. Although we are still at the beginning of this process, some recent examples of integrative analysis of cell signaling are very encouraging. This review highlights the case of the NF-κB pathway in order to illustrate how a quantitative model of a signaling pathway can be gradually constructed through continuous experimental validation, and what lessons one might learn from such exercises. PMID:18291655

Quantitative analysis of eosinophil chemotaxis tracked using a novel optical device -- TAXIScan.

PubMed

Nitta, Nao; Tsuchiya, Tomoko; Yamauchi, Akira; Tamatani, Takuya; Kanegasaki, Shiro

2007-03-30

We have reported previously the development of an optically accessible, horizontal chemotaxis apparatus, in which migration of cells in the channel from a start line can be traced with time-lapse intervals using a CCD camera (JIM 282, 1-11, 2003). To obtain statistical data of migrating cells, we have developed quantitative methods to calculate various parameters in the process of chemotaxis, employing human eosinophil and CXCL12 as a model cell and a model chemoattractant, respectively. Median values of velocity and directionality of each cell within an experimental period could be calculated from the migratory pathway data obtained from time-lapse images and the data were expressed as Velocity-Directionality (VD) plot. This plot is useful for quantitatively analyzing multiple migrating cells exposed to a certain chemoattractant, and can distinguish chemotaxis from random migration. Moreover precise observation of cell migration revealed that each cell had a different lag period before starting chemotaxis, indicating variation in cell sensitivity to the chemoattractant. Thus lag time of each cell before migration, and time course of increment of the migrating cell ratio at the early stages could be calculated. We also graphed decrement of still moving cell ratio at the later stages by calculating the duration time of cell migration of each cell. These graphs could distinguish different motion patterns of chemotaxis of eosinophils, in response to a range of chemoattractants; PGD(2), fMLP, CCL3, CCL5 and CXCL12. Finally, we compared parameters of eosinophils from normal volunteers, allergy patients and asthma patients and found significant difference in response to PGD(2). The quantitative methods described here could be applicable to image data obtained with any combination of cells and chemoattractants and useful not only for basic studies of chemotaxis but also for diagnosis and for drug screening.

Evolutionary Game Theory Analysis of Tumor Progression

NASA Astrophysics Data System (ADS)

Wu, Amy; Liao, David; Sturm, James; Austin, Robert

2014-03-01

Evolutionary game theory applied to two interacting cell populations can yield quantitative prediction of the future densities of the two cell populations based on the initial interaction terms. We will discuss how in a complex ecology that evolutionary game theory successfully predicts the future densities of strains of stromal and cancer cells (multiple myeloma), and discuss the possible clinical use of such analysis for predicting cancer progression. Supported by the National Science Foundation and the National Cancer Institute.

Quantitative analysis of three-dimensional biological cells using interferometric microscopy

NASA Astrophysics Data System (ADS)

Shaked, Natan T.; Wax, Adam

2011-06-01

Live biological cells are three-dimensional microscopic objects that constantly adjust their sizes, shapes and other biophysical features. Wide-field digital interferometry (WFDI) is a holographic technique that is able to record the complex wavefront of the light which has interacted with in-vitro cells in a single camera exposure, where no exogenous contrast agents are required. However, simple quasi-three-dimensional holographic visualization of the cell phase profiles need not be the end of the process. Quantitative analysis should permit extraction of numerical parameters which are useful for cytology or medical diagnosis. Using a transmission-mode setup, the phase profile represents the multiplication between the integral refractive index and the thickness of the sample. These coupled variables may not be distinct when acquiring the phase profiles of dynamic cells. Many morphological parameters which are useful for cell biologists are based on the cell thickness profile rather than on its phase profile. We first overview methods to decouple the cell thickness and its refractive index using the WFDI-based phase profile. Then, we present a whole-cell-imaging approach which is able to extract useful numerical parameters on the cells even in cases where decoupling of cell thickness and refractive index is not possible or desired.

Quantitative Raman spectral changes of the differentiation of mesenchymal stem cells into islet-like cells by biochemical component analysis and multiple peak fitting

NASA Astrophysics Data System (ADS)

Su, Xin; Fang, Shaoyin; Zhang, Daosen; Zhang, Qinnan; He, Yingtian; Lu, Xiaoxu; Liu, Shengde; Zhong, Liyun

2015-12-01

Mesenchymal stem cells (MSCs) differentiate into islet-like cells, providing a possible solution for type I diabetes treatment. To search for the precise molecular mechanism of the directional differentiation of MSC-derived islet-like cells, biomolecular composition, and structural conformation information during MSC differentiation, is required. Because islet-like cells lack specific surface markers, the commonly employed immunostaining technique is not suitable for their identification, physical separation, and enrichment. Combining Raman spectroscopic data, a fitting accuracy-improved biochemical component analysis, and multiple peaks fitting approach, we identified the quantitative biochemical and intensity change of Raman peaks that show the differentiation of MSCs into islet-like cells. Along with increases in protein and glycogen content, and decreases in deoxyribonucleic acid and ribonucleic acid content, in islet-like cells relative to MSCs, it was found that a characteristic peak of insulin (665 cm-1) has twice the intensity in islet-like cells relative to MSCs, indicating differentiation of MSCs into islet-like cells was successful. Importantly, these Raman signatures provide useful information on the structural and pathological states during MSC differentiation and help to develop noninvasive and label-free Raman sorting methods for stem cells and their lineages.

Methodological Variables in the Analysis of Cell-Free DNA.

PubMed

Bronkhorst, Abel Jacobus; Aucamp, Janine; Pretorius, Piet J

2016-01-01

In recent years, cell-free DNA (cfDNA) analysis has received increasing amounts of attention as a potential non-invasive screening tool for the early detection of genetic aberrations and a wide variety of diseases, especially cancer. However, except for some prenatal tests and BEAMing, a technique used to detect mutations in various genes of cancer patients, cfDNA analysis is not yet routinely applied in clinical practice. Although some confusing biological factors inherent to the in vivo setting play a key part, it is becoming increasingly clear that this struggle is mainly due to the lack of an analytical consensus, especially as regards quantitative analyses of cfDNA. In order to use quantitative analysis of cfDNA with confidence, process optimization and standardization are crucial. In this work we aim to elucidate the most confounding variables of each preanalytical step that must be considered for process optimization and equivalence of procedures.

In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling*

PubMed Central

Boersema, Paul J.; Foong, Leong Yan; Ding, Vanessa M. Y.; Lemeer, Simone; van Breukelen, Bas; Philp, Robin; Boekhorst, Jos; Snel, Berend; den Hertog, Jeroen; Choo, Andre B. H.; Heck, Albert J. R.

2010-01-01

Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated. PMID:19770167

Automated image analysis for quantitative fluorescence in situ hybridization with environmental samples.

PubMed

Zhou, Zhi; Pons, Marie Noëlle; Raskin, Lutgarde; Zilles, Julie L

2007-05-01

When fluorescence in situ hybridization (FISH) analyses are performed with complex environmental samples, difficulties related to the presence of microbial cell aggregates and nonuniform background fluorescence are often encountered. The objective of this study was to develop a robust and automated quantitative FISH method for complex environmental samples, such as manure and soil. The method and duration of sample dispersion were optimized to reduce the interference of cell aggregates. An automated image analysis program that detects cells from 4',6'-diamidino-2-phenylindole (DAPI) micrographs and extracts the maximum and mean fluorescence intensities for each cell from corresponding FISH images was developed with the software Visilog. Intensity thresholds were not consistent even for duplicate analyses, so alternative ways of classifying signals were investigated. In the resulting method, the intensity data were divided into clusters using fuzzy c-means clustering, and the resulting clusters were classified as target (positive) or nontarget (negative). A manual quality control confirmed this classification. With this method, 50.4, 72.1, and 64.9% of the cells in two swine manure samples and one soil sample, respectively, were positive as determined with a 16S rRNA-targeted bacterial probe (S-D-Bact-0338-a-A-18). Manual counting resulted in corresponding values of 52.3, 70.6, and 61.5%, respectively. In two swine manure samples and one soil sample 21.6, 12.3, and 2.5% of the cells were positive with an archaeal probe (S-D-Arch-0915-a-A-20), respectively. Manual counting resulted in corresponding values of 22.4, 14.0, and 2.9%, respectively. This automated method should facilitate quantitative analysis of FISH images for a variety of complex environmental samples.

Endogenous protein "barcode" for data validation and normalization in quantitative MS analysis.

PubMed

Lee, Wooram; Lazar, Iulia M

2014-07-01

Quantitative proteomic experiments with mass spectrometry detection are typically conducted by using stable isotope labeling and label-free quantitation approaches. Proteins with housekeeping functions and stable expression level such actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase are frequently used as endogenous controls. Recent studies have shown that the expression level of such common housekeeping proteins is, in fact, dependent on various factors such as cell type, cell cycle, or disease status and can change in response to a biochemical stimulation. The interference of such phenomena can, therefore, substantially compromise their use for data validation, alter the interpretation of results, and lead to erroneous conclusions. In this work, we advance the concept of a protein "barcode" for data normalization and validation in quantitative proteomic experiments. The barcode comprises a novel set of proteins that was generated from cell cycle experiments performed with MCF7, an estrogen receptor positive breast cancer cell line, and MCF10A, a nontumorigenic immortalized breast cell line. The protein set was selected from a list of ~3700 proteins identified in different cellular subfractions and cell cycle stages of MCF7/MCF10A cells, based on the stability of spectral count data generated with an LTQ ion trap mass spectrometer. A total of 11 proteins qualified as endogenous standards for the nuclear and 62 for the cytoplasmic barcode, respectively. The validation of the protein sets was performed with a complementary SKBR3/Her2+ cell line.

Quantitation of TGF-beta1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: comparison with ribonuclease protection assay and in situ hybridization.

PubMed

Ceol, M; Forino, M; Gambaro, G; Sauer, U; Schleicher, E D; D'Angelo, A; Anglani, F

2001-01-01

Gene expression can be examined with different techniques including ribonuclease protection assay (RPA), in situ hybridisation (ISH), and quantitative reverse transcription-polymerase chain reaction (RT/PCR). These methods differ considerably in their sensitivity and precision in detecting and quantifying low abundance mRNA. Although there is evidence that RT/PCR can be performed in a quantitative manner, the quantitative capacity of this method is generally underestimated. To demonstrate that the comparative kinetic RT/PCR strategy-which uses a housekeeping gene as internal standard-is a quantitative method to detect significant differences in mRNA levels between different samples, the inhibitory effect of heparin on phorbol 12-myristate 13-acetate (PMA)-induced-TGF-beta1 mRNA expression was evaluated by RT/PCR and RPA, the standard method of mRNA quantification, and the results were compared. The reproducibility of RT/PCR amplification was calculated by comparing the quantity of G3PDH and TGF-beta1 PCR products, generated during the exponential phases, estimated from two different RT/PCR (G3PDH, r = 0.968, P = 0.0000; TGF-beta1, r = 0.966, P = 0.0000). The quantitative capacity of comparative kinetic RT/PCR was demonstrated by comparing the results obtained from RPA and RT/PCR using linear regression analysis. Starting from the same RNA extraction, but using only 1% of the RNA for the RT/PCR compared to RPA, significant correlation was observed (r = 0.984, P = 0.0004). Moreover the morphometric analysis of ISH signal was applied for the semi-quantitative evaluation of the expression and localisation of TGF-beta1 mRNA in the entire cell population. Our results demonstrate the close similarity of the RT/PCR and RPA methods in giving quantitative information on mRNA expression and indicate the possibility to adopt the comparative kinetic RT/PCR as reliable quantitative method of mRNA analysis. Copyright 2001 Wiley-Liss, Inc.

A novel method for morphological pleomorphism and heterogeneity quantitative measurement: Named cell feature level co-occurrence matrix.

PubMed

Saito, Akira; Numata, Yasushi; Hamada, Takuya; Horisawa, Tomoyoshi; Cosatto, Eric; Graf, Hans-Peter; Kuroda, Masahiko; Yamamoto, Yoichiro

2016-01-01

Recent developments in molecular pathology and genetic/epigenetic analysis of cancer tissue have resulted in a marked increase in objective and measurable data. In comparison, the traditional morphological analysis approach to pathology diagnosis, which can connect these molecular data and clinical diagnosis, is still mostly subjective. Even though the advent and popularization of digital pathology has provided a boost to computer-aided diagnosis, some important pathological concepts still remain largely non-quantitative and their associated data measurements depend on the pathologist's sense and experience. Such features include pleomorphism and heterogeneity. In this paper, we propose a method for the objective measurement of pleomorphism and heterogeneity, using the cell-level co-occurrence matrix. Our method is based on the widely used Gray-level co-occurrence matrix (GLCM), where relations between neighboring pixel intensity levels are captured into a co-occurrence matrix, followed by the application of analysis functions such as Haralick features. In the pathological tissue image, through image processing techniques, each nucleus can be measured and each nucleus has its own measureable features like nucleus size, roundness, contour length, intra-nucleus texture data (GLCM is one of the methods). In GLCM each nucleus in the tissue image corresponds to one pixel. In this approach the most important point is how to define the neighborhood of each nucleus. We define three types of neighborhoods of a nucleus, then create the co-occurrence matrix and apply Haralick feature functions. In each image pleomorphism and heterogeneity are then determined quantitatively. For our method, one pixel corresponds to one nucleus feature, and we therefore named our method Cell Feature Level Co-occurrence Matrix (CFLCM). We tested this method for several nucleus features. CFLCM is showed as a useful quantitative method for pleomorphism and heterogeneity on histopathological image analysis.

Adaptation of a Simple Microfluidic Platform for High-Dimensional Quantitative Morphological Analysis of Human Mesenchymal Stromal Cells on Polystyrene-Based Substrates.

PubMed

Lam, Johnny; Marklein, Ross A; Jimenez-Torres, Jose A; Beebe, David J; Bauer, Steven R; Sung, Kyung E

2017-12-01

Multipotent stromal cells (MSCs, often called mesenchymal stem cells) have garnered significant attention within the field of regenerative medicine because of their purported ability to differentiate down musculoskeletal lineages. Given the inherent heterogeneity of MSC populations, recent studies have suggested that cell morphology may be indicative of MSC differentiation potential. Toward improving current methods and developing simple yet effective approaches for the morphological evaluation of MSCs, we combined passive pumping microfluidic technology with high-dimensional morphological characterization to produce robust tools for standardized high-throughput analysis. Using ultraviolet (UV) light as a modality for reproducible polystyrene substrate modification, we show that MSCs seeded on microfluidic straight channel devices incorporating UV-exposed substrates exhibited morphological changes that responded accordingly to the degree of substrate modification. Substrate modification also effected greater morphological changes in MSCs seeded at a lower rather than higher density within microfluidic channels. Despite largely comparable trends in morphology, MSCs seeded in microscale as opposed to traditional macroscale platforms displayed much higher sensitivity to changes in substrate properties. In summary, we adapted and qualified microfluidic cell culture platforms comprising simple straight channel arrays as a viable and robust tool for high-throughput quantitative morphological analysis to study cell-material interactions.

SSBD: a database of quantitative data of spatiotemporal dynamics of biological phenomena

PubMed Central

Tohsato, Yukako; Ho, Kenneth H. L.; Kyoda, Koji; Onami, Shuichi

2016-01-01

Motivation: Rapid advances in live-cell imaging analysis and mathematical modeling have produced a large amount of quantitative data on spatiotemporal dynamics of biological objects ranging from molecules to organisms. There is now a crucial need to bring these large amounts of quantitative biological dynamics data together centrally in a coherent and systematic manner. This will facilitate the reuse of this data for further analysis. Results: We have developed the Systems Science of Biological Dynamics database (SSBD) to store and share quantitative biological dynamics data. SSBD currently provides 311 sets of quantitative data for single molecules, nuclei and whole organisms in a wide variety of model organisms from Escherichia coli to Mus musculus. The data are provided in Biological Dynamics Markup Language format and also through a REST API. In addition, SSBD provides 188 sets of time-lapse microscopy images from which the quantitative data were obtained and software tools for data visualization and analysis. Availability and Implementation: SSBD is accessible at http://ssbd.qbic.riken.jp. Contact: sonami@riken.jp PMID:27412095

SSBD: a database of quantitative data of spatiotemporal dynamics of biological phenomena.

PubMed

Tohsato, Yukako; Ho, Kenneth H L; Kyoda, Koji; Onami, Shuichi

2016-11-15

Rapid advances in live-cell imaging analysis and mathematical modeling have produced a large amount of quantitative data on spatiotemporal dynamics of biological objects ranging from molecules to organisms. There is now a crucial need to bring these large amounts of quantitative biological dynamics data together centrally in a coherent and systematic manner. This will facilitate the reuse of this data for further analysis. We have developed the Systems Science of Biological Dynamics database (SSBD) to store and share quantitative biological dynamics data. SSBD currently provides 311 sets of quantitative data for single molecules, nuclei and whole organisms in a wide variety of model organisms from Escherichia coli to Mus musculus The data are provided in Biological Dynamics Markup Language format and also through a REST API. In addition, SSBD provides 188 sets of time-lapse microscopy images from which the quantitative data were obtained and software tools for data visualization and analysis. SSBD is accessible at http://ssbd.qbic.riken.jp CONTACT: sonami@riken.jp. © The Author 2016. Published by Oxford University Press.

Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

PubMed

Mas, Abraham; Amenós, Montse; Lois, L Maria

2016-01-01

Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected.

Electron paramagnetic resonance oximetry as a quantitative method to measure cellular respiration: a consideration of oxygen diffusion interference.

PubMed

Presley, Tennille; Kuppusamy, Periannan; Zweier, Jay L; Ilangovan, Govindasamy

2006-12-15

Electron paramagnetic resonance (EPR) oximetry is being widely used to measure the oxygen consumption of cells, mitochondria, and submitochondrial particles. However, further improvement of this technique, in terms of data analysis, is required to use it as a quantitative tool. Here, we present a new approach for quantitative analysis of cellular respiration using EPR oximetry. The course of oxygen consumption by cells in suspension has been observed to have three distinct zones: pO(2)-independent respiration at higher pO(2) ranges, pO(2)-dependent respiration at low pO(2) ranges, and a static equilibrium with no change in pO(2) at very low pO(2) values. The approach here enables one to comprehensively analyze all of the three zones together-where the progression of O(2) diffusion zones around each cell, their overlap within time, and their potential impact on the measured pO(2) data are considered. The obtained results agree with previously established methods such as high-resolution respirometry measurements. Additionally, it is also demonstrated how the diffusion limitations can depend on cell density and consumption rate. In conclusion, the new approach establishes a more accurate and meaningful model to evaluate the EPR oximetry data on cellular respiration to quantify related parameters using EPR oximetry.

Proteomic and transcriptional analysis of Lactobacillus johnsonii PF01 during bile salt exposure by iTRAQ shotgun proteomics and quantitative RT-PCR.

PubMed

Lee, Ji Yoon; Pajarillo, Edward Alain B; Kim, Min Jeong; Chae, Jong Pyo; Kang, Dae-Kyung

2013-01-04

Lactobacillus johnsonii PF01 has been reported to be highly resistant to bile, a key property of probiotic microorganisms. Here, we examine the nature of the bile-salt tolerance of L. johnsonii PF01. Growth inhibition and surface morphology and physiology aberrations were observed after overnight exposure to bile stress. Quantitative proteomic profiles using iTRAQ-LC-MS/MS technology identified 8307 peptides from both untreated PF01 cells and those exposed to 0.1%, 0.2%, and 0.3% bile salts. Some 215 proteins exhibited changed levels in response to bile stress; of these, levels of 94 peptides increased while those of 121 decreased. These were classified into the following categories: stress responses, cell division, transcription, translation, nucleotide metabolism, carbohydrate transport and metabolism, cell wall biosynthesis, and amino acid biosynthesis, and 16 of unidentified function. Analysis of the mRNA expression of selected genes by quantitative reverse transcriptase-PCR verified the proteomic data. Both proteomic and mRNA data provided evidence for increased phosphotransferase activity and cell wall biosynthesis. In addition, three bile salt hydrolases were significantly upregulated by bile exposure. These findings provide a basis for future evaluations of the tolerance of potential probiotic strains toward the various gastrointestinal challenges, including bile stress.

Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumors.

PubMed

Shackney, Stanley; Emlet, David R; Pollice, Agnese; Smith, Charles; Brown, Kathryn; Kociban, Deborah

2006-01-01

Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell-based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell-based immunofluorescence measurements. We describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell-based analysis. Improvements in the quality of cell-based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided. Advances in methodology can improve cell-based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications. Copyright (c) 2005 Wiley-Liss, Inc.

[Methods of quantitative proteomics].

PubMed

Kopylov, A T; Zgoda, V G

2007-01-01

In modern science proteomic analysis is inseparable from other fields of systemic biology. Possessing huge resources quantitative proteomics operates colossal information on molecular mechanisms of life. Advances in proteomics help researchers to solve complex problems of cell signaling, posttranslational modification, structure and functional homology of proteins, molecular diagnostics etc. More than 40 various methods have been developed in proteomics for quantitative analysis of proteins. Although each method is unique and has certain advantages and disadvantages all these use various isotope labels (tags). In this review we will consider the most popular and effective methods employing both chemical modifications of proteins and also metabolic and enzymatic methods of isotope labeling.

Improved method and apparatus for chromatographic quantitative analysis

DOEpatents

Fritz, J.S.; Gjerde, D.T.; Schmuckler, G.

An improved apparatus and method are described for the quantitative analysis of a solution containing a plurality of anion species by ion exchange chromatography which utilizes a single element and a single ion exchange bed which does not require periodic regeneration. The solution containing the anions is added to an anion exchange resin bed which is a low capacity macroreticular polystyrene-divinylbenzene resin containing quarternary ammonium functional groups, and is eluted therefrom with a dilute solution of a low electrical conductance organic acid salt. As each anion species is eluted from the bed, it is quantitatively sensed by conventional detection means such as a conductivity cell.

Impact of carotid stent cell design on vessel scaffolding: a case study comparing experimental investigation and numerical simulations.

PubMed

Conti, Michele; Van Loo, Denis; Auricchio, Ferdinando; De Beule, Matthieu; De Santis, Gianluca; Verhegghe, Benedict; Pirrelli, Stefano; Odero, Attilio

2011-06-01

To quantitatively evaluate the impact of carotid stent cell design on vessel scaffolding by using patient-specific finite element analysis of carotid artery stenting (CAS). The study was organized in 2 parts: (1) validation of a patient-specific finite element analysis of CAS and (2) evaluation of vessel scaffolding. Micro-computed tomography (CT) images of an open-cell stent deployed in a patient-specific silicone mock artery were compared with the corresponding finite element analysis results. This simulation was repeated for the closed-cell counterpart. In the second part, the stent strut distribution, as reflected by the inter-strut angles, was evaluated for both cell types in different vessel cross sections as a measure of scaffolding. The results of the patient-specific finite element analysis of CAS matched well with experimental stent deployment both qualitatively and quantitatively, demonstrating the reliability of the numerical approach. The measured inter-strut angles suggested that the closed-cell design provided superior vessel scaffolding compared to the open-cell counterpart. However, the full strut interconnection of the closed-cell design reduced the stent's ability to accommodate to the irregular eccentric profile of the vessel cross section, leading to a gap between the stent surface and the vessel wall. Even though this study was limited to a single stent design and one vascular anatomy, the study confirmed the capability of dedicated computer simulations to predict differences in scaffolding by open- and closed-cell carotid artery stents. These simulations have the potential to be used in the design of novel carotid stents or for procedure planning.

Global, quantitative and dynamic mapping of protein subcellular localization

PubMed Central

Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg HH

2016-01-01

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775

Quantitative analysis of multi-component gas mixture based on AOTF-NIR spectroscopy

NASA Astrophysics Data System (ADS)

Hao, Huimin; Zhang, Yong; Liu, Junhua

2007-12-01

Near Infrared (NIR) spectroscopy analysis technology has attracted many eyes and has wide application in many domains in recent years because of its remarkable advantages. But the NIR spectrometer can only be used for liquid and solid analysis by now. In this paper, a new quantitative analysis method of gas mixture by using new generation NIR spectrometer is explored. To collect the NIR spectra of gas mixtures, a vacuumable gas cell was designed and assembled to Luminar 5030-731 Acousto-Optic Tunable Filter (AOTF)-NIR spectrometer. Standard gas samples of methane (CH 4), ethane (C IIH 6) and propane (C 3H 8) are diluted with super pure nitrogen via precision volumetric gas flow controllers to obtain gas mixture samples of different concentrations dynamically. The gas mixtures were injected into the gas cell and the spectra of wavelength between 1100nm-2300nm were collected. The feature components extracted from gas mixture spectra by using Partial Least Squares (PLS) were used as the inputs of the Support Vector Regress Machine (SVR) to establish the quantitative analysis model. The effectiveness of the model is tested by the samples of predicting set. The prediction Root Mean Square Error (RMSE) of CH 4, C IIH 6 and C 3H 8 is respectively 1.27%, 0.89%, and 1.20% when the concentrations of component gas are over 0.5%. It shows that the AOTF-NIR spectrometer with gas cell can be used for gas mixture analysis. PLS combining with SVR has a good performance in NIR spectroscopy analysis. This paper provides the bases for extending the application of NIR spectroscopy analysis to gas detection.

Quantitative prediction of phase transformations in silicon during nanoindentation

NASA Astrophysics Data System (ADS)

Zhang, Liangchi; Basak, Animesh

2013-08-01

This paper establishes the first quantitative relationship between the phases transformed in silicon and the shape characteristics of nanoindentation curves. Based on an integrated analysis using TEM and unit cell properties of phases, the volumes of the phases emerged in a nanoindentation are formulated as a function of pop-out size and depth of nanoindentation impression. This simple formula enables a fast, accurate and quantitative prediction of the phases in a nanoindentation cycle, which has been impossible before.

Label-free distinguishing between neurons and glial cells based on two-photon excited fluorescence signal of neuron perinuclear granules

NASA Astrophysics Data System (ADS)

Du, Huiping; Jiang, Liwei; Wang, Xingfu; Liu, Gaoqiang; Wang, Shu; Zheng, Liqin; Li, Lianhuang; Zhuo, Shuangmu; Zhu, Xiaoqin; Chen, Jianxin

2016-08-01

Neurons and glial cells are two critical cell types of brain tissue. Their accurate identification is important for the diagnosis of psychiatric disorders such as depression and schizophrenia. In this paper, distinguishing between neurons and glial cells by using the two-photon excited fluorescence (TPEF) signals of intracellular intrinsic sources was performed. TPEF microscopy combined with TUJ-1 and GFAP immunostaining and quantitative image analysis demonstrated that the perinuclear granules of neurons in the TPEF images of brain tissue and the primary cultured cortical cells were a unique characteristic of neurons compared to glial cells which can become a quantitative feature to distinguish neurons from glial cells. With the development of miniaturized TPEF microscope (‘two-photon fiberscopes’) imaging devices, TPEF microscopy can be developed into an effective diagnostic and monitoring tool for psychiatric disorders such as depression and schizophrenia.

Quantitation of influenza virus using field flow fractionation and multi-angle light scattering for quantifying influenza A particles

PubMed Central

Bousse, Tatiana; Shore, David A.; Goldsmith, Cynthia S.; Hossain, M. Jaber; Jang, Yunho; Davis, Charles T.; Donis, Ruben O.; Stevens, James

2017-01-01

Summary Recent advances in instrumentation and data analysis in field flow fractionation and multi-angle light scattering (FFF-MALS) have enabled greater use of this technique to characterize and quantitate viruses. In this study, the FFF-MALS technique was applied to the characterization and quantitation of type A influenza virus particles to assess its usefulness for vaccine preparation. The use of FFF-MALS for quantitation and measurement of control particles provided data accurate to within 5% of known values, reproducible with a coefficient of variation of 1.9 %. The methods, sensitivity and limit of detection were established by analyzing different volumes of purified virus, which produced a linear regression with fitting value R2 of 0.99. FFF-MALS was further applied to detect and quantitate influenza virus in the supernatant of infected MDCK cells and allantoic fluids of infected eggs. FFF fractograms of the virus present in these different fluids revealed similar distribution of monomeric and oligomeric virions. However, the monomer fraction of cell grown virus has greater size variety. Notably, β-propialactone (BPL) inactivation of influenza viruses did not influence any of the FFF-MALS measurements. Quantitation analysis by FFF-MALS was compared to infectivity assays and real-time RT-PCR (qRT-PCR) and the limitations of each assay were discussed. PMID:23916678

High content screening of ToxCast compounds using Vala Sciences’ complex cell culturing systems (SOT)

EPA Science Inventory

US EPA’s ToxCast research program evaluates bioactivity for thousands of chemicals utilizing high-throughput screening assays to inform chemical testing decisions. Vala Sciences provides high content, multiplexed assays that utilize quantitative cell-based digital image analysis....

Infrared Multiphoton Dissociation for Quantitative Shotgun Proteomics

PubMed Central

Ledvina, Aaron R.; Lee, M. Violet; McAlister, Graeme C.; Westphall, Michael S.; Coon, Joshua J.

2012-01-01

We modified a dual-cell linear ion trap mass spectrometer to perform infrared multiphoton dissociation (IRMPD) in the low pressure trap of a dual-cell quadrupole linear ion trap (dual cell QLT) and perform large-scale IRMPD analyses of complex peptide mixtures. Upon optimization of activation parameters (precursor q-value, irradiation time, and photon flux), IRMPD subtly, but significantly outperforms resonant excitation CAD for peptides identified at a 1% false-discovery rate (FDR) from a yeast tryptic digest (95% confidence, p = 0.019). We further demonstrate that IRMPD is compatible with the analysis of isobaric-tagged peptides. Using fixed QLT RF amplitude allows for the consistent retention of reporter ions, but necessitates the use of variable IRMPD irradiation times, dependent upon precursor mass-to-charge (m/z). We show that IRMPD activation parameters can be tuned to allow for effective peptide identification and quantitation simultaneously. We thus conclude that IRMPD performed in a dual-cell ion trap is an effective option for the large-scale analysis of both unmodified and isobaric-tagged peptides. PMID:22480380

Quantitative phase imaging characterization of tumor-associated blood vessel formation on a chip

NASA Astrophysics Data System (ADS)

Guo, Peng; Huang, Jing; Moses, Marsha A.

2018-02-01

Angiogenesis, the formation of new blood vessels from existing ones, is a biological process that has an essential role in solid tumor growth, development, and progression. Recent advances in Lab-on-a-Chip technology has created an opportunity for scientists to observe endothelial cell (EC) behaviors during the dynamic process of angiogenesis using a simple and economical in vitro platform that recapitulates in vivo blood vessel formation. Here, we use quantitative phase imaging (QPI) microscopy to continuously and non-invasively characterize the dynamic process of tumor cell-induced angiogenic sprout formation on a microfluidic chip. The live tumor cell-induced angiogenic sprouts are generated by multicellular endothelial sprouting into 3 dimensional (3D) Matrigel using human umbilical vein endothelial cells (HUVECs). By using QPI, we quantitatively measure a panel of cellular morphological and behavioral parameters of each individual EC participating in this sprouting. In this proof-of-principle study, we demonstrate that QPI is a powerful tool that can provide real-time quantitative analysis of biological processes in in vitro 3D biomimetic devices, which, in turn, can improve our understanding of the biology underlying functional tissue engineering.

Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation

PubMed Central

2012-01-01

Background Fluorescence loss in photobleaching (FLIP) is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. In FLIP, a small cellular region is repeatedly illuminated by an intense laser pulse, while images are taken with reduced laser power with a time lag between the bleaches. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. Typically, the user defines regions of interest (ROIs) for further analysis which is subjective and does not allow for comparing different cells and experimental settings. Results We present two complementary methods to detect and quantify protein transport and aggregation in living cells from FLIP image series. In the first approach, a stretched exponential (StrExp) function is fitted to fluorescence loss (FL) inside and outside the bleached region. We show by reaction–diffusion simulations, that the StrExp function can describe both, binding/barrier–limited and diffusion-limited FL kinetics. By pixel-wise regression of that function to FL kinetics of enhanced green fluorescent protein (eGFP), we determined in a user-unbiased manner from which cellular regions eGFP can be replenished in the bleached area. Spatial variation in the parameters calculated from the StrExp function allow for detecting diffusion barriers for eGFP in the nucleus and cytoplasm of living cells. Polyglutamine (polyQ) disease proteins like mutant huntingtin (mtHtt) can form large aggregates called inclusion bodies (IB’s). The second method combines single particle tracking with multi-compartment modelling of FL kinetics in moving IB’s to determine exchange rates of eGFP-tagged mtHtt protein (eGFP-mtHtt) between aggregates and the cytoplasm. This method is self-calibrating since it relates the FL inside and outside the bleached regions. It makes it therefore possible to compare release kinetics of eGFP-mtHtt between different cells and experiments. Conclusions We present two complementary methods for quantitative analysis of FLIP experiments in living cells. They provide spatial maps of exchange dynamics and absolute binding parameters of fluorescent molecules to moving intracellular entities, respectively. Our methods should be of great value for quantitative studies of intracellular transport. PMID:23148417

Spectral analysis of fundus autofluorescence pattern as a tool to detect early stages of degeneration in the retina and retinal pigment epithelium.

PubMed

Feldman, Tatiana B; Yakovleva, Marina A; Larichev, Andrey V; Arbukhanova, Patimat M; Radchenko, Alexandra Sh; Borzenok, Sergey A; Kuzmin, Vladimir A; Ostrovsky, Mikhail A

2018-05-22

The aim of this work is the determination of quantitative diagnostic criteria based on the spectral characteristics of fundus autofluorescence to detect early stages of degeneration in the retina and retinal pigment epithelium (RPE). RPE cell suspension samples were obtained from the cadaver eyes with and without signs of age-related macular degeneration (AMD). Fluorescence analysis at an excitation wavelength of 488 nm was performed. The fluorescence lifetimes of lipofuscin-granule fluorophores were measured by counting time-correlated photon method. Comparative analysis of fluorescence spectra of RPE cell suspensions from the cadaver eyes with and without signs of AMD showed a significant difference in fluorescence intensity at 530-580 nm in response to fluorescence excitation at 488 nm. It was notably higher in eyes with visual pathology than in normal eyes regardless of the age of the eye donor. Measurements of fluorescence lifetimes of lipofuscin fluorophores showed that the contribution of photooxidation and photodegradation products of bisretinoids to the total fluorescence at 530-580 nm of RPE cell suspensions was greater in eyes with visual pathology than in normal eyes. Because photooxidation and photodegradation products of bisretinoids are markers of photodestructive processes, which can cause RPE cell death and initiate degenerative processes in the retina, quantitative determination of increases in these bisretinoid products in lipofuscin granules may be used to establish quantitative diagnostic criteria for degenerative processes in the retina and RPE.

Quantitative Chemical Imaging and Unsupervised Analysis Using Hyperspectral Coherent Anti-Stokes Raman Scattering Microscopy

PubMed Central

2013-01-01

In this work, we report a method to acquire and analyze hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy images of organic materials and biological samples resulting in an unbiased quantitative chemical analysis. The method employs singular value decomposition on the square root of the CARS intensity, providing an automatic determination of the components above noise, which are retained. Complex CARS susceptibility spectra, which are linear in the chemical composition, are retrieved from the CARS intensity spectra using the causality of the susceptibility by two methods, and their performance is evaluated by comparison with Raman spectra. We use non-negative matrix factorization applied to the imaginary part and the nonresonant real part of the susceptibility with an additional concentration constraint to obtain absolute susceptibility spectra of independently varying chemical components and their absolute concentration. We demonstrate the ability of the method to provide quantitative chemical analysis on known lipid mixtures. We then show the relevance of the method by imaging lipid-rich stem-cell-derived mouse adipocytes as well as differentiated embryonic stem cells with a low density of lipids. We retrieve and visualize the most significant chemical components with spectra given by water, lipid, and proteins segmenting the image into the cell surrounding, lipid droplets, cytosol, and the nucleus, and we reveal the chemical structure of the cells, with details visualized by the projection of the chemical contrast into a few relevant channels. PMID:24099603

A quantitative image cytometry technique for time series or population analyses of signaling networks.

PubMed

Ozaki, Yu-ichi; Uda, Shinsuke; Saito, Takeshi H; Chung, Jaehoon; Kubota, Hiroyuki; Kuroda, Shinya

2010-04-01

Modeling of cellular functions on the basis of experimental observation is increasingly common in the field of cellular signaling. However, such modeling requires a large amount of quantitative data of signaling events with high spatio-temporal resolution. A novel technique which allows us to obtain such data is needed for systems biology of cellular signaling. We developed a fully automatable assay technique, termed quantitative image cytometry (QIC), which integrates a quantitative immunostaining technique and a high precision image-processing algorithm for cell identification. With the aid of an automated sample preparation system, this device can quantify protein expression, phosphorylation and localization with subcellular resolution at one-minute intervals. The signaling activities quantified by the assay system showed good correlation with, as well as comparable reproducibility to, western blot analysis. Taking advantage of the high spatio-temporal resolution, we investigated the signaling dynamics of the ERK pathway in PC12 cells. The QIC technique appears as a highly quantitative and versatile technique, which can be a convenient replacement for the most conventional techniques including western blot, flow cytometry and live cell imaging. Thus, the QIC technique can be a powerful tool for investigating the systems biology of cellular signaling.

A microdosimetric study of {sup 10}B(n,{alpha}){sup 7}Li and {sup 157}Gd(n,{gamma}) reactions for neutron capture therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, C.K.C.; Sutton, M.; Evans, T.M.

1999-01-01

This paper presents the microdosimetric analysis for the most interesting cell survival experiment recently performed at the Brookhaven National Laboratory (BNL). In this experiment, the cells were first treated with a gadolinium (Gd) labeled tumor-seeking boronated porphyrin (Gd-BOPP) or with BOPP alone, and then irradiated with thermal neutrons. The resulting cell-survival curves indicate that the {sup 157}Gd(n,{gamma}) reactions are very effective in cell killing. The death of a cell treated with Gd-BOPP was attributed to either the {sup 10}B(n,{alpha}){sup 7}Li reactions or the {sup 157}Gd(n,{gamma}) reactions (or both). However, the quantitative relationship between the two types of reaction and themore » cell-survival fraction was not clear. This paper presents the microdosimetric analysis for the BNL experiment based on the measured experimental parameters, and the results clearly suggest a quantitative relationship between the two types of reaction and the cell survival fraction. The results also suggest new research in gadolinium neutron capture therapy (GdNCT) which may lead to a more practical modality than the boron neutron capture therapy (BNCT) for treating cancers.« less

Effects of Amphotericin B and Three Azole Derivatives on the Lipids of Yeast Cells of Paracoccidioides brasiliensis

PubMed Central

Hahn, Rosane Christine; Hamdan, Júnia Soares

2000-01-01

Yeast cells of five different strains of Paracoccidioides brasiliensis were obtained for partial analysis of lipid composition, and sterol content was determined quantitatively and qualitatively. The determinations were conducted with cells cultured in the presence and absence of amphotericin B and azole derivatives at levels below the MIC. PMID:10858371

Quantitative Image Restoration in Bright Field Optical Microscopy.

PubMed

Gutiérrez-Medina, Braulio; Sánchez Miranda, Manuel de Jesús

2017-11-07

Bright field (BF) optical microscopy is regarded as a poor method to observe unstained biological samples due to intrinsic low image contrast. We introduce quantitative image restoration in bright field (QRBF), a digital image processing method that restores out-of-focus BF images of unstained cells. Our procedure is based on deconvolution, using a point spread function modeled from theory. By comparing with reference images of bacteria observed in fluorescence, we show that QRBF faithfully recovers shape and enables quantify size of individual cells, even from a single input image. We applied QRBF in a high-throughput image cytometer to assess shape changes in Escherichia coli during hyperosmotic shock, finding size heterogeneity. We demonstrate that QRBF is also applicable to eukaryotic cells (yeast). Altogether, digital restoration emerges as a straightforward alternative to methods designed to generate contrast in BF imaging for quantitative analysis. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Single-cell-type quantitative proteomic and ionomic analysis of epidermal bladder cells from the halophyte model plant Mesembryanthemum crystallinum to identify salt-responsive proteins.

PubMed

Barkla, Bronwyn J; Vera-Estrella, Rosario; Raymond, Carolyn

2016-05-10

Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum. Recent development of a simple but high throughput technique to extract the contents from these cells has provided an opportunity to conduct detailed single-cell-type analyses of their molecular characteristics at high resolution to gain insight into the role of these cells in the salt tolerance of the plant. In this study, we carry out large-scale complementary quantitative proteomic studies using both a label (DIGE) and label-free (GeLC-MS) approach to identify salt-responsive proteins in the EBC extract. Additionally we perform an ionomics analysis (ICP-MS) to follow changes in the amounts of 27 different elements. Using these methods, we were able to identify 54 proteins and nine elements that showed statistically significant changes in the EBC from salt-treated plants. GO enrichment analysis identified a large number of transport proteins but also proteins involved in photosynthesis, primary metabolism and Crassulacean acid metabolism (CAM). Validation of results by western blot, confocal microscopy and enzyme analysis helped to strengthen findings and further our understanding into the role of these specialized cells. As expected EBC accumulated large quantities of sodium, however, the most abundant element was chloride suggesting the sequestration of this ion into the EBC vacuole is just as important for salt tolerance. This single-cell type omics approach shows that epidermal bladder cells of M. crystallinum are metabolically active modified trichomes, with primary metabolism supporting cell growth, ion accumulation, compatible solute synthesis and CAM. Data are available via ProteomeXchange with identifier PXD004045.

Analysis of live cell images: Methods, tools and opportunities.

PubMed

Nketia, Thomas A; Sailem, Heba; Rohde, Gustavo; Machiraju, Raghu; Rittscher, Jens

2017-02-15

Advances in optical microscopy, biosensors and cell culturing technologies have transformed live cell imaging. Thanks to these advances live cell imaging plays an increasingly important role in basic biology research as well as at all stages of drug development. Image analysis methods are needed to extract quantitative information from these vast and complex data sets. The aim of this review is to provide an overview of available image analysis methods for live cell imaging, in particular required preprocessing image segmentation, cell tracking and data visualisation methods. The potential opportunities recent advances in machine learning, especially deep learning, and computer vision provide are being discussed. This review includes overview of the different available software packages and toolkits. Copyright © 2017. Published by Elsevier Inc.

Quantitative analysis of serotonin secreted by human embryonic stem cells-derived serotonergic neurons via pH-mediated online stacking-CE-ESI-MRM.

PubMed

Zhong, Xuefei; Hao, Ling; Lu, Jianfeng; Ye, Hui; Zhang, Su-Chun; Li, Lingjun

2016-04-01

A CE-ESI-MRM-based assay was developed for targeted analysis of serotonin released by human embryonic stem cells-derived serotonergic neurons in a chemically defined environment. A discontinuous electrolyte system was optimized for pH-mediated online stacking of serotonin. Combining with a liquid-liquid extraction procedure, LOD of serotonin in the Krebs'-Ringer's solution by CE-ESI-MS/MS on a 3D ion trap MS was0.15 ng/mL. The quantitative results confirmed the serotonergic identity of the in vitro developed neurons and the capacity of these neurons to release serotonin in response to stimulus. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detecting ordered small molecule drug aggregates in live macrophages: a multi-parameter microscope image data acquisition and analysis strategy

PubMed Central

Rzeczycki, Phillip; Yoon, Gi Sang; Keswani, Rahul K.; Sud, Sudha; Stringer, Kathleen A.; Rosania, Gus R.

2017-01-01

Following prolonged administration, certain orally bioavailable but poorly soluble small molecule drugs are prone to precipitate out and form crystal-like drug inclusions (CLDIs) within the cells of living organisms. In this research, we present a quantitative multi-parameter imaging platform for measuring the fluorescence and polarization diattenuation signals of cells harboring intracellular CLDIs. To validate the imaging system, the FDA-approved drug clofazimine (CFZ) was used as a model compound. Our results demonstrated that a quantitative multi-parameter microscopy image analysis platform can be used to study drug sequestering macrophages, and to detect the formation of ordered molecular aggregates formed by poorly soluble small molecule drugs in animals. PMID:28270989

Detecting ordered small molecule drug aggregates in live macrophages: a multi-parameter microscope image data acquisition and analysis strategy.

PubMed

Rzeczycki, Phillip; Yoon, Gi Sang; Keswani, Rahul K; Sud, Sudha; Stringer, Kathleen A; Rosania, Gus R

2017-02-01

Following prolonged administration, certain orally bioavailable but poorly soluble small molecule drugs are prone to precipitate out and form crystal-like drug inclusions (CLDIs) within the cells of living organisms. In this research, we present a quantitative multi-parameter imaging platform for measuring the fluorescence and polarization diattenuation signals of cells harboring intracellular CLDIs. To validate the imaging system, the FDA-approved drug clofazimine (CFZ) was used as a model compound. Our results demonstrated that a quantitative multi-parameter microscopy image analysis platform can be used to study drug sequestering macrophages, and to detect the formation of ordered molecular aggregates formed by poorly soluble small molecule drugs in animals.

Segmentation and Quantitative Analysis of Epithelial Tissues.

PubMed

Aigouy, Benoit; Umetsu, Daiki; Eaton, Suzanne

2016-01-01

Epithelia are tissues that regulate exchanges with the environment. They are very dynamic and can acquire virtually any shape; at the cellular level, they are composed of cells tightly connected by junctions. Most often epithelia are amenable to live imaging; however, the large number of cells composing an epithelium and the absence of informatics tools dedicated to epithelial analysis largely prevented tissue scale studies. Here we present Tissue Analyzer, a free tool that can be used to segment and analyze epithelial cells and monitor tissue dynamics.

Two-Photon Flow Cytometry

NASA Technical Reports Server (NTRS)

Zhog, Cheng Frank; Ye, Jing Yong; Norris, Theodore B.; Myc, Andrzej; Cao, Zhengyl; Bielinska, Anna; Thomas, Thommey; Baker, James R., Jr.

2004-01-01

Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantitation is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. In this work, we demonstrate a two-beam, two- channel detection and two-photon excitation flow cytometry (T(sup 3)FC) system that enables multi-dye analysis to be performed very simply, with greatly relaxed requirements on the fluid flow. Two-photon excitation using a femtosecond near-infrared (NIR) laser has the advantages that it enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling of cells by targeted nanoparticles with multiple fluorophores enables normalization of the fluorescence signal and thus ratiometric measurements under nonuniform excitation. Quantitative size measurements can also be done even under conditions of nonuniform flow via a two-beam layout. This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or in vivo. Real-time detection of fluorescent microbeads in the vasculature of mouse ear demonstrates the ability to do flow cytometry in vivo. The conditions required to perform quantitative in vivo cytometry on labeled cells will be presented.

Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis.

PubMed

Varga, Viktor Sebestyén; Bocsi, József; Sipos, Ferenc; Csendes, Gábor; Tulassay, Zsolt; Molnár, Béla

2004-07-01

Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. We applied a commercial motorized fluorescent microscope system. The scanning was done at 20 x (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno-Brite beads were used. With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after +/-5 microm error. SFM is a valuable method for the evaluation of fluorescently labeled cells. Copyright 2004 Wiley-Liss, Inc.

Isolation of RNA for dot hybridization by heparin-DNase I treatment of whole cell lysate.

PubMed

Krawczyk, Z; Wu, C

1987-08-15

We have developed a new procedure for the rapid preparation of undegraded total RNA from cultured cells for specific quantitation by dot blotting analysis. Pelleted cells are resuspended in hypotonic solution containing a ribonuclease inhibitor and heparin and disrupted by freeze-thaw. Heparin is employed as an agent for nuclear lysis, dissociation of chromosomal protein, and release of mRNA from rough endoplasmic reticulum. We eliminate chromosomal DNA by digestion with DNase I and denature the RNA in the lysate with formaldehyde. After centrifugation to remove debris, the supernatant is used directly for dot blotting. All manipulations are performed in the same microfuge tube and recovery of RNA is quantitative. The procedure is especially useful for processing large numbers of samples. We illustrate its versatility by analysis of specific RNAs in Drosophila, rat, and human cell lines. In reconstruction experiments, less than 80 molecules per cell of a small RNA (beta-globin) can be detected under highly stringent hybridization conditions, using only moderately labeled double-stranded plasmid DNA probes and short film exposures.

Quantitative assessment of image motion blur in diffraction images of moving biological cells

NASA Astrophysics Data System (ADS)

Wang, He; Jin, Changrong; Feng, Yuanming; Qi, Dandan; Sa, Yu; Hu, Xin-Hua

2016-02-01

Motion blur (MB) presents a significant challenge for obtaining high-contrast image data from biological cells with a polarization diffraction imaging flow cytometry (p-DIFC) method. A new p-DIFC experimental system has been developed to evaluate the MB and its effect on image analysis using a time-delay-integration (TDI) CCD camera. Diffraction images of MCF-7 and K562 cells have been acquired with different speed-mismatch ratios and compared to characterize MB quantitatively. Frequency analysis of the diffraction images shows that the degree of MB can be quantified by bandwidth variations of the diffraction images along the motion direction. The analytical results were confirmed by the p-DIFC image data acquired at different speed-mismatch ratios and used to validate a method of numerical simulation of MB on blur-free diffraction images, which provides a useful tool to examine the blurring effect on diffraction images acquired from the same cell. These results provide insights on the dependence of diffraction image on MB and allow significant improvement on rapid biological cell assay with the p-DIFC method.

Quantitative determination of optical and recombination losses in thin-film photovoltaic devices based on external quantum efficiency analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakane, Akihiro; Tamakoshi, Masato; Fujimoto, Shohei

2016-08-14

In developing photovoltaic devices with high efficiencies, quantitative determination of the carrier loss is crucial. In conventional solar-cell characterization techniques, however, photocurrent reduction originating from parasitic light absorption and carrier recombination within the light absorber cannot be assessed easily. Here, we develop a general analysis scheme in which the optical and recombination losses in submicron-textured solar cells are evaluated systematically from external quantum efficiency (EQE) spectra. In this method, the optical absorption in solar cells is first deduced by imposing the anti-reflection condition in the calculation of the absorptance spectrum, and the carrier extraction from the light absorber layer ismore » then modeled by considering a carrier collection length from the absorber interface. Our analysis method is appropriate for a wide variety of photovoltaic devices, including kesterite solar cells [Cu{sub 2}ZnSnSe{sub 4}, Cu{sub 2}ZnSnS{sub 4}, and Cu{sub 2}ZnSn(S,Se){sub 4}], zincblende CdTe solar cells, and hybrid perovskite (CH{sub 3}NH{sub 3}PbI{sub 3}) solar cells, and provides excellent fitting to numerous EQE spectra reported earlier. Based on the results obtained from our EQE analyses, we discuss the effects of parasitic absorption and carrier recombination in different types of solar cells.« less

Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity

PubMed Central

Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A.; Bradford, William D.; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S.; Li, Rong

2015-01-01

Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein−based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. PMID:25823586

Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity.

PubMed

Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A; Bradford, William D; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S; Li, Rong

2015-03-30

Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein-based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. Copyright © 2015 Zhu et al.

Wide-scale quantitative phosphoproteomic analysis reveals that cold treatment of T cells closely mimics soluble antibody stimulation

PubMed Central

Ji, Qinqin; Salomon, Arthur R.

2015-01-01

The activation of T-lymphocytes through antigen-mediated T-cell receptor (TCR) clustering is vital in regulating the adaptive-immune response. Although T cell receptor signaling has been extensively studied, the fundamental mechanisms for signal initiation are not fully understood. Reduced temperature initiated some of the hallmarks of TCR signaling such as increased phosphorylation and activation on ERK and calcium release from the endoplasmic reticulum as well as coalesce T-cell membrane microdomains. The precise mechanism of TCR signaling initiation due to temperature change remains obscure. One critical question is whether signaling initiated by cold treatment of T cells differs from signaling initiated by crosslinking of the T cell receptor. To address this uncertainty, a wide-scale, quantitative mass spectrometry-based phosphoproteomic analysis was performed on T cells stimulated either by temperature shift or through crosslinking of the TCR. Careful statistical comparison between the two stimulations revealed a striking level of identity between the subset of 339 sites that changed significantly with both stimulations. This study demonstrates for the first time, at unprecedented detail, that T cell cold treatment was sufficient to initiate signaling patterns nearly identical to soluble antibody stimulation, shedding new light on the mechanism of activation of these critically important immune cells. PMID:25839225

Quantitative analysis of the human AKR family members in cancer cell lines using the mTRAQ/MRM approach.

PubMed

Zhang, Shenyan; Wen, Bo; Zhou, Baojin; Yang, Lei; Cha, Chao; Xu, Shaoxing; Qiu, Xuemei; Wang, Quanhui; Sun, Haidan; Lou, Xiaomin; Zi, Jin; Zhang, Yong; Lin, Liang; Liu, Siqi

2013-05-03

Members of human aldo-keto reductase (AKR) superfamily have been reported to be involved in cancer progression, whereas the final conclusion is not generally accepted. Herein, we propose a quantitative method to measure human AKR proteins in cells using mTRAQ-based multiple reaction monitoring (MRM). AKR peptides with multiple transitions were carefully selected upon tryptic digestion of the recombinant AKR proteins, while AKR proteins were identified by SDS-PAGE fractionation coupled with LC-MS/MS. Utilizing mTRAQ triplex labeling to produce the derivative peptides, calibration curves were generated using the mixed lysate as background, and no significantly different quantification of AKRs was elicited from the two sets of calibration curves under the mixed and single lysate as background. We employed this approach to quantitatively determine the 6 AKR proteins, AKR1A1, AKR1B1, AKR1B10, AKR1C1/C2, AKR1C3, and AKR1C4, in 7 different cancer cell lines and for the first time to obtain the absolute quantities of all the AKR proteins in each cell. The cluster plot revealed that AKR1A and AKR1B were widely distributed in most cancer cells with relatively stable abundances, whereas AKR1Cs were unevenly detected among these cells with diverse dynamic abundances. The AKR quantitative distribution in different cancer cells, therefore, may assist further exploration toward how the AKR proteins are involved in tumorigenesis.

Peptidoglycan and Teichoic Acid Levels and Alterations in Staphylococcus aureus by Cell-Wall and Whole-Cell Nuclear Magnetic Resonance.

PubMed

Romaniuk, Joseph A H; Cegelski, Lynette

2018-06-11

Gram-positive bacteria surround themselves with a multilayered macromolecular cell wall that is essential to cell survival and serves as a major target for antibiotics. The cell wall of Staphylococcus aureus is composed of two major structural components, peptidoglycan (PG) and wall teichoic acid (WTA), together creating a heterogeneous and insoluble matrix that poses a challenge to quantitative compositional analysis. Here, we present 13 C cross polarization magic angle spinning solid-state nuclear magnetic resonance (NMR) spectra of intact cell walls, purified PG, and purified WTA. The spectra reveal the clear molecular differences in the two polymers and enable quantification of PG and WTA in isolated cell walls, an attractive alternative to estimating teichoic acid content from a phosphate analysis of completely pyrolyzed cell walls. Furthermore, we discovered that unique PG and WTA spectral signatures could be identified in whole-cell NMR spectra and used to compare PG and WTA levels among intact bacterial cell samples. The distinguishing whole-cell 13 C NMR contributions associated with PG include the GlcNAc-MurNAc sugar carbons and glycyl α-carbons. WTA contributes carbons from the phosphoribitol backbone. Distinguishing 15 N spectral signatures include glycyl amide nitrogens in PG and the esterified d-alanyl amine nitrogens in WTA. 13 C NMR analysis was performed with samples at natural abundance and included 10 whole-cell sample comparisons. Changes consistent with altered PG and WTA content were detected in whole-cell spectra of bacteria harvested at different growth times and in cells treated with tunicamycin. This use of whole-cell NMR provides quantitative parameters of composition in the context of whole-cell activity.

Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription.

PubMed

Lee, Jinwoo; Foong, Yee Hoon; Musaitif, Ibrahim; Tong, Tiegang; Jefcoate, Colin

2016-07-05

The steroidogenic acute regulatory protein (StAR) has been proposed to serve as the switch that can turn on/off steroidogenesis. We investigated the events that facilitate dynamic StAR transcription in response to cAMP stimulation in MA-10 Leydig cells, focusing on splicing anomalies at StAR gene loci. We used 3' reverse primers in a single reaction to respectively quantify StAR primary (p-RNA), spliced (sp-RNA/mRNA), and extended 3' untranslated region (UTR) transcripts, which were quantitatively imaged by high-resolution fluorescence in situ hybridization (FISH). This approach delivers spatio-temporal resolution of initiation and splicing at single StAR loci, and transfers individual mRNA molecules to cytoplasmic sites. Gene expression was biphasic, initially showing slow splicing, transitioning to concerted splicing. The alternative 3.5-kb mRNAs were distinguished through the use of extended 3'UTR probes, which exhibited distinctive mitochondrial distribution. Combining quantitative PCR and FISH enables imaging of localization of RNA expression and analysis of RNA processing rates. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

MIiSR: Molecular Interactions in Super-Resolution Imaging Enables the Analysis of Protein Interactions, Dynamics and Formation of Multi-protein Structures.

PubMed

Caetano, Fabiana A; Dirk, Brennan S; Tam, Joshua H K; Cavanagh, P Craig; Goiko, Maria; Ferguson, Stephen S G; Pasternak, Stephen H; Dikeakos, Jimmy D; de Bruyn, John R; Heit, Bryan

2015-12-01

Our current understanding of the molecular mechanisms which regulate cellular processes such as vesicular trafficking has been enabled by conventional biochemical and microscopy techniques. However, these methods often obscure the heterogeneity of the cellular environment, thus precluding a quantitative assessment of the molecular interactions regulating these processes. Herein, we present Molecular Interactions in Super Resolution (MIiSR) software which provides quantitative analysis tools for use with super-resolution images. MIiSR combines multiple tools for analyzing intermolecular interactions, molecular clustering and image segmentation. These tools enable quantification, in the native environment of the cell, of molecular interactions and the formation of higher-order molecular complexes. The capabilities and limitations of these analytical tools are demonstrated using both modeled data and examples derived from the vesicular trafficking system, thereby providing an established and validated experimental workflow capable of quantitatively assessing molecular interactions and molecular complex formation within the heterogeneous environment of the cell.

Quantitative Magnetic Separation of Particles and Cells using Gradient Magnetic Ratcheting

PubMed Central

Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

2016-01-01

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting (MACS), are robust but perform coarse, qualitative separations based on surface antigen expression. We report a quantitative magnetic separation technology using high-force magnetic ratcheting over arrays of magnetically soft micro-pillars with gradient spacing, and use the system to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micro-pillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic-field. Particles with higher IOC separate and equilibrate along the miro-pillar array at larger pitches. We develop a semi-analytical model that predicts behavior for particles and cells. Using the system, LNCaP cells were separated based on the bound quantity of 1μm anti-EpCAM particles as a metric for expression. The ratcheting cytometry system was able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof of concept, EpCAM-labeled cells from patient blood were isolated with 74% purity, demonstrating potential towards a quantitative magnetic separation instrument. PMID:26890496

Quantitative analysis of mechanical force required for cell extrusion in zebrafish embryonic epithelia.

PubMed

Yamada, Sohei; Iino, Takanori; Bessho, Yasumasa; Hosokawa, Yoichiroh; Matsui, Takaaki

2017-10-15

When cells in epithelial sheets are damaged by intrinsic or extrinsic causes, they are eliminated by extrusion from the sheet. Cell extrusion, which is required for maintenance of tissue integrity, is the consequence of contraction of actomyosin rings, as demonstrated by both molecular/cellular biological experimentation and numerical simulation. However, quantitative evaluation of actomyosin contraction has not been performed because of the lack of a suitable direct measurement system. In this study, we developed a new method using a femtosecond laser to quantify the contraction force of the actomyosin ring during cell extrusion in zebrafish embryonic epithelia. In this system, an epithelial cell in zebrafish embryo is first damaged by direct femtosecond laser irradiation. Next, a femtosecond laser-induced impulsive force is loaded onto the actomyosin ring, and the contraction force is quantified to be on the order of kPa as a unit of pressure. We found that cell extrusion was delayed when the contraction force was slightly attenuated, suggesting that a relatively small force is sufficient to drive cell extrusion. Thus, our method is suitable for the relative quantitative evaluation of mechanical dynamics in the process of cell extrusion, and in principle the method is applicable to similar phenomena in different tissues and organs of various species. © 2017. Published by The Company of Biologists Ltd.

Proteomic analysis of single mammalian cells enabled by microfluidic nanodroplet sample preparation and ultrasensitive nanoLC-MS.

PubMed

Zhu, Ying; Clair, Geremy; Chrisler, William; Shen, Yufeng; Zhao, Rui; Shukla, Anil; Moore, Ronald; Misra, Ravi; Pryhuber, Gloria; Smith, Richard; Ansong, Charles; Kelly, Ryan T

2018-05-24

We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analysed by ultrasensitive nanoLC-MS. An average of ~670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single cell proteomics platform can be used to differentiate cell types from enzyme-dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cell bioprocessing in space - Applications of analytical cytology

NASA Technical Reports Server (NTRS)

Todd, P.; Hymer, W. C.; Goolsby, C. L.; Hatfield, J. M.; Morrison, D. R.

1988-01-01

Cell bioprocessing experiments in space are reviewed and the development of on-board cell analytical cytology techniques that can serve such experiments is discussed. Methods and results of experiments involving the cultivation and separation of eukaryotic cells in space are presented. It is suggested that an advanced cytometer should be developed for the quantitative analysis of large numbers of specimens of suspended eukaryotic cells and bioparticles in experiments on the Space Station.

Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts

PubMed Central

2013-01-01

Background Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. Results Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. Conclusions The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis. PMID:23758893

Tension and Elasticity Contribute to Fibroblast Cell Shape in Three Dimensions.

PubMed

Brand, Christoph A; Linke, Marco; Weißenbruch, Kai; Richter, Benjamin; Bastmeyer, Martin; Schwarz, Ulrich S

2017-08-22

The shape of animal cells is an important regulator for many essential processes such as cell migration or division. It is strongly determined by the organization of the actin cytoskeleton, which is also the main regulator of cell forces. Quantitative analysis of cell shape helps to reveal the physical processes underlying cell shape and forces, but it is notoriously difficult to conduct it in three dimensions. Here we use direct laser writing to create 3D open scaffolds for adhesion of connective tissue cells through well-defined adhesion platforms. Due to actomyosin contractility in the cell contour, characteristic invaginations lined by actin bundles form between adjacent adhesion sites. Using quantitative image processing and mathematical modeling, we demonstrate that the resulting shapes are determined not only by contractility, but also by elastic stress in the peripheral actin bundles. In this way, cells can generate higher forces than through contractility alone. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Dual reporter transgene driven by 2.3Col1a1 promoter is active in differentiated osteoblasts

NASA Technical Reports Server (NTRS)

Marijanovic, Inga; Jiang, Xi; Kronenberg, Mark S.; Stover, Mary Louise; Erceg, Ivana; Lichtler, Alexander C.; Rowe, David W.

2003-01-01

AIM: As quantitative and spatial analyses of promoter reporter constructs are not easily performed in intact bone, we designed a reporter gene specific to bone, which could be analyzed both visually and quantitatively by using chloramphenicol acetyltransferase (CAT) and a cyan version of green fluorescent protein (GFPcyan), driven by a 2.3-kb fragment of the rat collagen promoter (Col2.3). METHODS: The construct Col2.3CATiresGFPcyan was used for generating transgenic mice. Quantitative measurement of promoter activity was performed by CAT analysis of different tissues derived from transgenic animals; localization was performed by visualized GFP in frozen bone sections. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed for CAT and GFP activity. RESULTS: In mice, CAT activity was detected in the calvaria, long bone, teeth, and tendon, whereas histology showed that GFP expression was limited to osteoblasts and osteocytes. In cell culture, increased activity of CAT correlated with increased differentiation, and GFP activity was restricted to mineralized nodules. CONCLUSION: The concept of a dual reporter allows a simultaneous visual and quantitative analysis of transgene activity in bone.

Scanning transmission ion microscopy mass measurements for quantitative trace element analysis within biological samples and validation using atomic force microscopy thickness measurements

NASA Astrophysics Data System (ADS)

Devès, Guillaume; Cohen-Bouhacina, Touria; Ortega, Richard

2004-10-01

We used the nuclear microprobe techniques, micro-PIXE (particle-induced X-ray emission), micro-RBS (Rutherford backscattering spectrometry) and scanning transmission ion microscopy (STIM) in order to perform the characterization of trace element content and spatial distribution within biological samples (dehydrated cultured cells, tissues). The normalization of PIXE results was usually expressed in terms of sample dry mass as determined by micro-RBS recorded simultaneously to micro-PIXE. However, the main limit of RBS mass measurement is the sample mass loss occurring during irradiation and which could be up to 30% of the initial sample mass. We present here a new methodology for PIXE normalization and quantitative analysis of trace element within biological samples based on dry mass measurement performed by mean of STIM. The validation of STIM cell mass measurements was obtained in comparison with AFM sample thickness measurements. Results indicated the reliability of STIM mass measurement performed on biological samples and suggested that STIM should be performed for PIXE normalization. Further information deriving from direct confrontation of AFM and STIM analysis could as well be obtained, like in situ measurements of cell specific gravity within cells compartment (nucleolus and cytoplasm).

Quantitative assessment of neurite outgrowth in human embryonic stem-cell derived neurons using automated high-content image analysis

EPA Science Inventory

During development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxicants that interfere with this process may cause in permanent deficits in nervous system function. While many studies have used rodent primary...

Analysis of high accuracy, quantitative proteomics data in the MaxQB database.

PubMed

Schaab, Christoph; Geiger, Tamar; Stoehr, Gabriele; Cox, Juergen; Mann, Matthias

2012-03-01

MS-based proteomics generates rapidly increasing amounts of precise and quantitative information. Analysis of individual proteomic experiments has made great strides, but the crucial ability to compare and store information across different proteome measurements still presents many challenges. For example, it has been difficult to avoid contamination of databases with low quality peptide identifications, to control for the inflation in false positive identifications when combining data sets, and to integrate quantitative data. Although, for example, the contamination with low quality identifications has been addressed by joint analysis of deposited raw data in some public repositories, we reasoned that there should be a role for a database specifically designed for high resolution and quantitative data. Here we describe a novel database termed MaxQB that stores and displays collections of large proteomics projects and allows joint analysis and comparison. We demonstrate the analysis tools of MaxQB using proteome data of 11 different human cell lines and 28 mouse tissues. The database-wide false discovery rate is controlled by adjusting the project specific cutoff scores for the combined data sets. The 11 cell line proteomes together identify proteins expressed from more than half of all human genes. For each protein of interest, expression levels estimated by label-free quantification can be visualized across the cell lines. Similarly, the expression rank order and estimated amount of each protein within each proteome are plotted. We used MaxQB to calculate the signal reproducibility of the detected peptides for the same proteins across different proteomes. Spearman rank correlation between peptide intensity and detection probability of identified proteins was greater than 0.8 for 64% of the proteome, whereas a minority of proteins have negative correlation. This information can be used to pinpoint false protein identifications, independently of peptide database scores. The information contained in MaxQB, including high resolution fragment spectra, is accessible to the community via a user-friendly web interface at http://www.biochem.mpg.de/maxqb.

Dual-color dual-focus line-scanning FCS for quantitative analysis of receptor-ligand interactions in living specimens.

PubMed

Dörlich, René M; Chen, Qing; Niklas Hedde, Per; Schuster, Vittoria; Hippler, Marc; Wesslowski, Janine; Davidson, Gary; Nienhaus, G Ulrich

2015-05-07

Cellular communication in multi-cellular organisms is mediated to a large extent by a multitude of cell-surface receptors that bind specific ligands. An in-depth understanding of cell signaling networks requires quantitative information on ligand-receptor interactions within living systems. In principle, fluorescence correlation spectroscopy (FCS) based methods can provide such data, but live-cell applications have proven extremely challenging. Here, we have developed an integrated dual-color dual-focus line-scanning fluorescence correlation spectroscopy (2c2f lsFCS) technique that greatly facilitates live-cell and tissue experiments. Absolute ligand and receptor concentrations and their diffusion coefficients within the cell membrane can be quantified without the need to perform additional calibration experiments. We also determine the concentration of ligands diffusing in the medium outside the cell within the same experiment by using a raster image correlation spectroscopy (RICS) based analysis. We have applied this robust technique to study the interactions of two Wnt antagonists, Dickkopf1 and Dickkopf2 (Dkk1/2), to their cognate receptor, low-density-lipoprotein-receptor related protein 6 (LRP6), in the plasma membrane of living HEK293T cells. We obtained significantly lower affinities than previously reported using in vitro studies, underscoring the need to measure such data on living cells or tissues.

A temperature-controlled photoelectrochemical cell for quantitative product analysis.

PubMed

Corson, Elizabeth R; Creel, Erin B; Kim, Youngsang; Urban, Jeffrey J; Kostecki, Robert; McCloskey, Bryan D

2018-05-01

In this study, we describe the design and operation of a temperature-controlled photoelectrochemical cell for analysis of gaseous and liquid products formed at an illuminated working electrode. This cell is specifically designed to quantitatively analyze photoelectrochemical processes that yield multiple gas and liquid products at low current densities and exhibit limiting reactant concentrations that prevent these processes from being studied in traditional single chamber electrolytic cells. The geometry of the cell presented in this paper enables front-illumination of the photoelectrode and maximizes the electrode surface area to electrolyte volume ratio to increase liquid product concentration and hence enhances ex situ spectroscopic sensitivity toward them. Gas is bubbled through the electrolyte in the working electrode chamber during operation to maintain a saturated reactant concentration and to continuously mix the electrolyte. Gaseous products are detected by an in-line gas chromatograph, and liquid products are analyzed ex situ by nuclear magnetic resonance. Cell performance was validated by examining carbon dioxide reduction on a silver foil electrode, showing comparable results both to those reported in the literature and identical experiments performed in a standard parallel-electrode electrochemical cell. To demonstrate a photoelectrochemical application of the cell, CO 2 reduction experiments were carried out on a plasmonic nanostructured silver photocathode and showed different product distributions under dark and illuminated conditions.

A temperature-controlled photoelectrochemical cell for quantitative product analysis

NASA Astrophysics Data System (ADS)

Corson, Elizabeth R.; Creel, Erin B.; Kim, Youngsang; Urban, Jeffrey J.; Kostecki, Robert; McCloskey, Bryan D.

2018-05-01

In this study, we describe the design and operation of a temperature-controlled photoelectrochemical cell for analysis of gaseous and liquid products formed at an illuminated working electrode. This cell is specifically designed to quantitatively analyze photoelectrochemical processes that yield multiple gas and liquid products at low current densities and exhibit limiting reactant concentrations that prevent these processes from being studied in traditional single chamber electrolytic cells. The geometry of the cell presented in this paper enables front-illumination of the photoelectrode and maximizes the electrode surface area to electrolyte volume ratio to increase liquid product concentration and hence enhances ex situ spectroscopic sensitivity toward them. Gas is bubbled through the electrolyte in the working electrode chamber during operation to maintain a saturated reactant concentration and to continuously mix the electrolyte. Gaseous products are detected by an in-line gas chromatograph, and liquid products are analyzed ex situ by nuclear magnetic resonance. Cell performance was validated by examining carbon dioxide reduction on a silver foil electrode, showing comparable results both to those reported in the literature and identical experiments performed in a standard parallel-electrode electrochemical cell. To demonstrate a photoelectrochemical application of the cell, CO2 reduction experiments were carried out on a plasmonic nanostructured silver photocathode and showed different product distributions under dark and illuminated conditions.

Quantitative Evaluation of Cisplatin Uptake in Sensitive and Resistant Individual Cells by Single-Cell ICP-MS (SC-ICP-MS).

PubMed

Corte Rodríguez, M; Álvarez-Fernández García, R; Blanco, E; Bettmer, J; Montes-Bayón, M

2017-11-07

One of the main limitations to the Pt-therapy in cancer is the development of associated drug resistance that can be associated with a significant reduction of the intracellular platinum concentration. Thus, intracellular Pt concentration could be considered as a biomarker of cisplatin resistance. In this work, an alternative method to address intracellular Pt concentration in individual cells is explored to permit the evaluation of different cell models and alternative therapies in a relatively fast way. For this aim, total Pt analysis in single cells has been implemented using a total consumption nebulizer coupled to inductively coupled plasma mass spectrometric detection (ICP-MS). The efficiency of the proposed device has been evaluated in combination with flow cytometry and turned out to be around 25% (cells entering the ICP-MS from the cells in suspension). Quantitative uptake studies of a nontoxic Tb-containing compound by individual cells were conducted and the results compared to those obtained by bulk analysis of the same cells. Both sets of data were statistically comparable. Thus, final application of the developed methodology to the comparative uptake of Pt-species in cisplatin resistant and sensitive cell lines (A2780cis and A2780) was conducted. The results obtained revealed the potential of this analytical strategy to differentiate between different cell lines of different sensitivity to the drug which might be of high medical interest.

Characterization of T cell repertoire changes in acute Kawasaki disease

PubMed Central

1993-01-01

Kawasaki disease (KD) is an acute multisystem vasculitis of unknown etiology that is associated with marked activation of T cells and monocyte/macrophages. Using a quantitative polymerase chain reaction (PCR) technique, we recently found that the acute phase of KD is associated with the expansion of T cells expressing the V beta 2 and V beta 8.1 gene segments. In the present work, we used a newly developed anti-V beta 2 monoclonal antibody (mAb) and studied a new group of KD patients to extend our previous PCR results. Immunofluorescence analysis confirmed that V beta 2-bearing T cells are selectively increased in patients with acute KD. The increase occurred primarily in the CD4 T cell subset. The percentages of V beta 2+ T cells as determined by mAb reactivity and flow cytometry correlated linearly with V beta expression as quantitated by PCR. However, T cells from acute KD patients appeared to express proportionately higher levels of V beta 2 transcripts per cell as compared with healthy controls or convalescent KD patients. Sequence analysis of T cell receptor beta chain genes of V beta 2 and V beta 8.1 expressing T cells from acute KD patients showed extensive junctional region diversity. These data showing polyclonal expansion of V beta 2+ and V beta 8+ T cells in acute KD provide additional insight into the immunopathogenesis of this disease. PMID:8094737

Quantitative analysis of ChIP-seq data uncovers dynamic and sustained H3K4me3 and H3K27me3 modulation in cancer cells under hypoxia.

PubMed

Adriaens, Michiel E; Prickaerts, Peggy; Chan-Seng-Yue, Michelle; van den Beucken, Twan; Dahlmans, Vivian E H; Eijssen, Lars M; Beck, Timothy; Wouters, Bradly G; Voncken, Jan Willem; Evelo, Chris T A

2016-01-01

A comprehensive assessment of the epigenetic dynamics in cancer cells is the key to understanding the molecular mechanisms underlying cancer and to improving cancer diagnostics, prognostics and treatment. By combining genome-wide ChIP-seq epigenomics and microarray transcriptomics, we studied the effects of oxygen deprivation and subsequent reoxygenation on histone 3 trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3) in a breast cancer cell line, serving as a model for abnormal oxygenation in solid tumors. A priori, epigenetic markings and gene expression levels not only are expected to vary greatly between hypoxic and normoxic conditions, but also display a large degree of heterogeneity across the cell population. Where traditionally ChIP-seq data are often treated as dichotomous data, the model and experiment here necessitate a quantitative, data-driven analysis of both datasets. We first identified genomic regions with sustained epigenetic markings, which provided a sample-specific reference enabling quantitative ChIP-seq data analysis. Sustained H3K27me3 marking was located around centromeres and intergenic regions, while sustained H3K4me3 marking is associated with genes involved in RNA binding, translation and protein transport and localization. Dynamic marking with both H3K4me3 and H3K27me3 (hypoxia-induced bivalency) was found in CpG-rich regions at loci encoding factors that control developmental processes, congruent with observations in embryonic stem cells. In silico -identified epigenetically sustained and dynamic genomic regions were confirmed through ChIP-PCR in vitro, and obtained results are corroborated by published data and current insights regarding epigenetic regulation.

New Tools for Comparing Microscopy Images: Quantitative Analysis of Cell Types in Bacillus subtilis

PubMed Central

van Gestel, Jordi; Vlamakis, Hera

2014-01-01

Fluorescence microscopy is a method commonly used to examine individual differences between bacterial cells, yet many studies still lack a quantitative analysis of fluorescence microscopy data. Here we introduce some simple tools that microbiologists can use to analyze and compare their microscopy images. We show how image data can be converted to distribution data. These data can be subjected to a cluster analysis that makes it possible to objectively compare microscopy images. The distribution data can further be analyzed using distribution fitting. We illustrate our methods by scrutinizing two independently acquired data sets, each containing microscopy images of a doubly labeled Bacillus subtilis strain. For the first data set, we examined the expression of srfA and tapA, two genes which are expressed in surfactin-producing and matrix-producing cells, respectively. For the second data set, we examined the expression of eps and tapA; these genes are expressed in matrix-producing cells. We show that srfA is expressed by all cells in the population, a finding which contrasts with a previously reported bimodal distribution of srfA expression. In addition, we show that eps and tapA do not always have the same expression profiles, despite being expressed in the same cell type: both operons are expressed in cell chains, while single cells mainly express eps. These findings exemplify that the quantification and comparison of microscopy data can yield insights that otherwise would go unnoticed. PMID:25448819

New tools for comparing microscopy images: quantitative analysis of cell types in Bacillus subtilis.

PubMed

van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

2015-02-15

Fluorescence microscopy is a method commonly used to examine individual differences between bacterial cells, yet many studies still lack a quantitative analysis of fluorescence microscopy data. Here we introduce some simple tools that microbiologists can use to analyze and compare their microscopy images. We show how image data can be converted to distribution data. These data can be subjected to a cluster analysis that makes it possible to objectively compare microscopy images. The distribution data can further be analyzed using distribution fitting. We illustrate our methods by scrutinizing two independently acquired data sets, each containing microscopy images of a doubly labeled Bacillus subtilis strain. For the first data set, we examined the expression of srfA and tapA, two genes which are expressed in surfactin-producing and matrix-producing cells, respectively. For the second data set, we examined the expression of eps and tapA; these genes are expressed in matrix-producing cells. We show that srfA is expressed by all cells in the population, a finding which contrasts with a previously reported bimodal distribution of srfA expression. In addition, we show that eps and tapA do not always have the same expression profiles, despite being expressed in the same cell type: both operons are expressed in cell chains, while single cells mainly express eps. These findings exemplify that the quantification and comparison of microscopy data can yield insights that otherwise would go unnoticed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis.

PubMed

Özlü, Nurhan; Qureshi, Mohammad H; Toyoda, Yusuke; Renard, Bernhard Y; Mollaoglu, Gürkan; Özkan, Nazlı E; Bulbul, Selda; Poser, Ina; Timm, Wiebke; Hyman, Anthony A; Mitchison, Timothy J; Steen, Judith A

2015-01-13

The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy. © 2014 The Authors.

Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis

PubMed Central

Özlü, Nurhan; Qureshi, Mohammad H; Toyoda, Yusuke; Renard, Bernhard Y; Mollaoglu, Gürkan; Özkan, Nazlı E; Bulbul, Selda; Poser, Ina; Timm, Wiebke; Hyman, Anthony A; Mitchison, Timothy J; Steen, Judith A

2015-01-01

The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy. PMID:25476450

Integrated quantitative fractal polarimetric analysis of monolayer lung cancer cells

NASA Astrophysics Data System (ADS)

Shrestha, Suman; Zhang, Lin; Quang, Tri; Farrahi, Tannaz; Narayan, Chaya; Deshpande, Aditi; Na, Ying; Blinzler, Adam; Ma, Junyu; Liu, Bo; Giakos, George C.

2014-05-01

Digital diagnostic pathology has become one of the most valuable and convenient advancements in technology over the past years. It allows us to acquire, store and analyze pathological information from the images of histological and immunohistochemical glass slides which are scanned to create digital slides. In this study, efficient fractal, wavelet-based polarimetric techniques for histological analysis of monolayer lung cancer cells will be introduced and different monolayer cancer lines will be studied. The outcome of this study indicates that application of fractal, wavelet polarimetric principles towards the analysis of squamous carcinoma and adenocarcinoma cancer cell lines may be proved extremely useful in discriminating among healthy and lung cancer cells as well as differentiating among different lung cancer cells.

Influence of sample preparation and reliability of automated numerical refocusing in stain-free analysis of dissected tissues with quantitative phase digital holographic microscopy

NASA Astrophysics Data System (ADS)

Kemper, Björn; Lenz, Philipp; Bettenworth, Dominik; Krausewitz, Philipp; Domagk, Dirk; Ketelhut, Steffi

2015-05-01

Digital holographic microscopy (DHM) has been demonstrated to be a versatile tool for high resolution non-destructive quantitative phase imaging of surfaces and multi-modal minimally-invasive monitoring of living cell cultures in-vitro. DHM provides quantitative monitoring of physiological processes through functional imaging and structural analysis which, for example, gives new insight into signalling of cellular water permeability and cell morphology changes due to toxins and infections. Also the analysis of dissected tissues quantitative DHM phase contrast prospects application fields by stain-free imaging and the quantification of tissue density changes. We show that DHM allows imaging of different tissue layers with high contrast in unstained tissue sections. As the investigation of fixed samples represents a very important application field in pathology, we also analyzed the influence of the sample preparation. The retrieved data demonstrate that the quality of quantitative DHM phase images of dissected tissues depends strongly on the fixing method and common staining agents. As in DHM the reconstruction is performed numerically, multi-focus imaging is achieved from a single digital hologram. Thus, we evaluated the automated refocussing feature of DHM for application on different types of dissected tissues and revealed that on moderately stained samples highly reproducible holographic autofocussing can be achieved. Finally, it is demonstrated that alterations of the spatial refractive index distribution in murine and human tissue samples represent a reliable absolute parameter that is related of different degrees of inflammation in experimental colitis and Crohn's disease. This paves the way towards the usage of DHM in digital pathology for automated histological examinations and further studies to elucidate the translational potential of quantitative phase microscopy for the clinical management of patients, e.g., with inflammatory bowel disease.

Accurate quantitation of D+ fetomaternal hemorrhage by flow cytometry using a novel reagent to eliminate granulocytes from analysis.

PubMed

Kumpel, Belinda; Hazell, Matthew; Guest, Alan; Dixey, Jonathan; Mushens, Rosey; Bishop, Debbie; Wreford-Bush, Tim; Lee, Edmond

2014-05-01

Quantitation of fetomaternal hemorrhage (FMH) is performed to determine the dose of prophylactic anti-D (RhIG) required to prevent D immunization of D- women. Flow cytometry (FC) is the most accurate method. However, maternal white blood cells (WBCs) can give high background by binding anti-D nonspecifically, compromising accuracy. Maternal blood samples (69) were sent for FC quantitation of FMH after positive Kleihauer-Betke test (KBT) analysis and RhIG administration. Reagents used were BRAD-3-fluorescein isothiocyanate (FITC; anti-D), AEVZ5.3-FITC (anti-varicella zoster [anti-VZ], negative control), anti-fetal hemoglobin (HbF)-FITC, blended two-color reagents, BRAD-3-FITC/anti-CD45-phycoerythrin (PE; anti-D/L), and BRAD-3-FITC/anti-CD66b-PE (anti-D/G). PE-positive WBCs were eliminated from analysis by gating. Full blood counts were performed on maternal samples and female donors. Elevated numbers of neutrophils were present in 80% of patients. Red blood cell (RBC) indices varied widely in maternal blood. D+ FMH values obtained with anti-D/L, anti-D/G, and anti-HbF-FITC were very similar (r = 0.99, p < 0.001). Correlation between KBT and anti-HbF-FITC FMH results was low (r = 0.716). Inaccurate FMH quantitation using the current method (anti-D minus anti-VZ) occurred with 71% samples having less than 15 mL of D+ FMH (RBCs) and insufficient RhIG calculated for 9%. Using two-color reagents and anti-HbF-FITC, approximately 30% patients had elevated F cells, 26% had no fetal cells, 6% had D- FMH, 26% had 4 to 15 mL of D+ FMH, and 12% patients had more than 15 mL of D+ FMH (RBCs) requiring more than 300 μg of RhIG. Without accurate quantitation of D+ FMH by FC, some women would receive inappropriate or inadequate anti-D prophylaxis. The latter may be at risk of immunization leading to hemolytic disease of the newborn. © 2013 American Association of Blood Banks.

Quantitative tracking of tumor cells in phase-contrast microscopy exploiting halo artifact pattern

NASA Astrophysics Data System (ADS)

Kang, Mi-Sun; Song, Soo-Min; Lee, Hana; Kim, Myoung-Hee

2012-03-01

Tumor cell morphology is closely related to its invasiveness characteristics and migratory behaviors. An invasive tumor cell has a highly irregular shape, whereas a spherical cell is non-metastatic. Thus, quantitative analysis of cell features is crucial to determine tumor malignancy or to test the efficacy of anticancer treatment. We use phase-contrast microscopy to analyze single cell morphology and to monitor its change because it enables observation of long-term activity of living cells without photobleaching and phototoxicity, which is common in other fluorescence-labeled microscopy. Despite this advantage, there are image-level drawbacks to phase-contrast microscopy, such as local light effect and contrast interference ring, among others. Thus, we first applied a local filter to compensate for non-uniform illumination. Then, we used intensity distribution information to detect the cell boundary. In phase-contrast microscopy images, the cell normally appears as a dark region surrounded by a bright halo. As the halo artifact around the cell body is minimal and has an asymmetric diffusion pattern, we calculated the cross-sectional plane that intersected the center of each cell and was orthogonal to the first principal axis. Then, we extracted the dark cell region by level set. However, a dense population of cultured cells still rendered single-cell analysis difficult. Finally, we measured roundness and size to classify tumor cells into malignant and benign groups. We validated segmentation accuracy by comparing our findings with manually obtained results.

Evaluation of a rapid, quantitative real-time PCR method for enumeration of pathogenic Candida cells in water

USGS Publications Warehouse

Brinkman, Nichole E.; Haugland, Richard A.; Wymer, Larry J.; Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Vesper, Stephen J.

2003-01-01

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5′ nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

Comparison of different approaches to quantitative adenovirus detection in stool specimens of hematopoietic stem cell transplant recipients.

PubMed

Kosulin, K; Dworzak, S; Lawitschka, A; Matthes-Leodolter, S; Lion, T

2016-12-01

Adenoviruses almost invariably proliferate in the gastrointestinal tract prior to dissemination, and critical threshold concentrations in stool correlate with the risk of viremia. Monitoring of adenovirus loads in stool may therefore be important for timely initiation of treatment in order to prevent invasive infection. Comparison of a manual DNA extraction kit in combination with a validated in-house PCR assay with automated extraction on the NucliSENS-EasyMAG device coupled with the Adenovirus R-gene kit (bioMérieux) for quantitative adenovirus analysis in stool samples. Stool specimens spiked with adenovirus concentrations in a range from 10E2-10E11 copies/g and 32 adenovirus-positive clinical stool specimens from pediatric stem cell transplant recipients were tested along with appropriate negative controls. Quantitative analysis of viral load in adenovirus-positive stool specimens revealed a median difference of 0.5 logs (range 0.1-2.2) between the detection systems tested and a difference of 0.3 logs (range 0.0-1.7) when the comparison was restricted to the PCR assays only. Spiking experiments showed a detection limit of 10 2 -10 3 adenovirus copies/g stool revealing a somewhat higher sensitivity offered by the automated extraction. The dynamic range of accurate quantitative analysis by both systems investigated was between 10 3 and 10 8 virus copies/g. The differences in quantitative analysis of adenovirus copy numbers between the systems tested were primarily attributable to the DNA extraction method used, while the qPCR assays revealed a high level of concordance. Both systems showed adequate performance for detection and monitoring of adenoviral load in stool specimens. Copyright © 2016 Elsevier B.V. All rights reserved.

Semi-automated discrimination of retinal pigmented epithelial cells in two-photon fluorescence images of mouse retinas.

PubMed

Alexander, Nathan S; Palczewska, Grazyna; Palczewski, Krzysztof

2015-08-01

Automated image segmentation is a critical step toward achieving a quantitative evaluation of disease states with imaging techniques. Two-photon fluorescence microscopy (TPM) has been employed to visualize the retinal pigmented epithelium (RPE) and provide images indicating the health of the retina. However, segmentation of RPE cells within TPM images is difficult due to small differences in fluorescence intensity between cell borders and cell bodies. Here we present a semi-automated method for segmenting RPE cells that relies upon multiple weak features that differentiate cell borders from the remaining image. These features were scored by a search optimization procedure that built up the cell border in segments around a nucleus of interest. With six images used as a test, our method correctly identified cell borders for 69% of nuclei on average. Performance was strongly dependent upon increasing retinosome content in the RPE. TPM image analysis has the potential of providing improved early quantitative assessments of diseases affecting the RPE.

Elemental analysis of occupational and environmental lung diseases by electron probe microanalyzer with wavelength dispersive spectrometer.

PubMed

Takada, Toshinori; Moriyama, Hiroshi; Suzuki, Eiichi

2014-01-01

Occupational and environmental lung diseases are a group of pulmonary disorders caused by inhalation of harmful particles, mists, vapors or gases. Mineralogical analysis is not generally required in the diagnosis of most cases of these diseases. Apart from minerals that are encountered rarely or only in specific occupations, small quantities of mineral dusts are present in the healthy lung. As such when mineralogical analysis is required, quantitative or semi-quantitative methods must be employed. An electron probe microanalyzer with wavelength dispersive spectrometer (EPMA-WDS) enables analysis of human lung tissue for deposits of elements by both qualitative and semi-quantitative methods. Since 1993, we have analyzed 162 cases of suspected occupational and environmental lung diseases using an EPMA-WDS. Our institute has been accepting online requests for elemental analysis of lung tissue samples by EPMA-WDS since January 2011. Hard metal lung disease is an occupational interstitial lung disease that primarily affects workers exposed to the dust of tungsten carbide. The characteristic pathological findings of the disease are giant cell interstitial pneumonia (GIP) with centrilobular fibrosis, surrounded by mild alveolitis with giant cells within the alveolar space. EPMA-WDS analysis of biopsied lung tissue from patients with GIP has demonstrated that tungsten and/or cobalt is distributed in the giant cells and centrilobular fibrosing lesion in GIP. Pneumoconiosis, caused by amorphous silica, and acute interstitial pneumonia, associated with the giant tsunami, were also elementally analyzed by EPMA-WDS. The results suggest that commonly found elements, such as silicon, aluminum, and iron, may cause occupational and environmental lung diseases. Copyright © 2013 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

Simultaneous quantitation of oxidized and reduced glutathione via LC-MS/MS: An insight into the redox state of hematopoietic stem cells.

PubMed

Carroll, Dustin; Howard, Diana; Zhu, Haining; Paumi, Christian M; Vore, Mary; Bondada, Subbarao; Liang, Ying; Wang, Chi; St Clair, Daret K

2016-08-01

Cellular redox balance plays a significant role in the regulation of hematopoietic stem-progenitor cell (HSC/MPP) self-renewal and differentiation. Unregulated changes in cellular redox homeostasis are associated with the onset of most hematological disorders. However, accurate measurement of the redox state in stem cells is difficult because of the scarcity of HSC/MPPs. Glutathione (GSH) constitutes the most abundant pool of cellular antioxidants. Thus, GSH metabolism may play a critical role in hematological disease onset and progression. A major limitation to studying GSH metabolism in HSC/MPPs has been the inability to measure quantitatively GSH concentrations in small numbers of HSC/MPPs. Current methods used to measure GSH levels not only rely on large numbers of cells, but also rely on the chemical/structural modification or enzymatic recycling of GSH and therefore are likely to measure only total glutathione content accurately. Here, we describe the validation of a sensitive method used for the direct and simultaneous quantitation of both oxidized and reduced GSH via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) in HSC/MPPs isolated from bone marrow. The lower limit of quantitation (LLOQ) was determined to be 5.0ng/mL for GSH and 1.0ng/mL for GSSG with lower limits of detection at 0.5ng/mL for both glutathione species. Standard addition analysis utilizing mouse bone marrow shows that this method is both sensitive and accurate with reproducible analyte recovery. This method combines a simple extraction with a platform for the high-throughput analysis, allows for efficient determination of GSH/GSSG concentrations within the HSC/MPP populations in mouse, chemotherapeutic treatment conditions within cell culture, and human normal/leukemia patient samples. The data implicate the importance of the modulation of GSH/GSSG redox couple in stem cells related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Visualisation and quantitative analysis of the rodent malaria liver stage by real time imaging.

PubMed

Ploemen, Ivo H J; Prudêncio, Miguel; Douradinha, Bruno G; Ramesar, Jai; Fonager, Jannik; van Gemert, Geert-Jan; Luty, Adrian J F; Hermsen, Cornelus C; Sauerwein, Robert W; Baptista, Fernanda G; Mota, Maria M; Waters, Andrew P; Que, Ivo; Lowik, Clemens W G M; Khan, Shahid M; Janse, Chris J; Franke-Fayard, Blandine M D

2009-11-18

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc(con), expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1-5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.

Visualisation and Quantitative Analysis of the Rodent Malaria Liver Stage by Real Time Imaging

PubMed Central

Douradinha, Bruno G.; Ramesar, Jai; Fonager, Jannik; van Gemert, Geert-Jan; Luty, Adrian J. F.; Hermsen, Cornelus C.; Sauerwein, Robert W.; Baptista, Fernanda G.; Mota, Maria M.; Waters, Andrew P.; Que, Ivo; Lowik, Clemens W. G. M.; Khan, Shahid M.; Janse, Chris J.; Franke-Fayard, Blandine M. D.

2009-01-01

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luccon, expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1–5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium. PMID:19924309

A new 3D tracking method for cell mechanics investigation exploiting the capabilities of digital holography in microscopy

NASA Astrophysics Data System (ADS)

Miccio, L.; Memmolo, P.; Merola, F.; Fusco, S.; Netti, P. A.; Ferraro, P.

2014-03-01

A method for 3D tracking has been developed exploiting Digital Holography features in Microscopy (DHM). In the framework of self-consistent platform for manipulation and measurement of biological specimen we use DHM for quantitative and completely label free analysis of samples with low amplitude contrast. Tracking capability extend the potentiality of DHM allowing to monitor the motion of appropriate probes and correlate it with sample properties. Complete 3D tracking has been obtained for the probes avoiding the amplitude refocusing in traditional tracking processes. Moreover, in biology and biomedical research fields one of the main topic is the understanding of morphology and mechanics of cells and microorganisms. Biological samples present low amplitude contrast that limits the information that can be retrieved through optical bright-field microscope measurements. The main effect on light propagating in such objects is in phase. This is known as phase-retardation or phase-shift. DHM is an innovative and alternative approach in microscopy, it's a good candidate for no-invasive and complete specimen analysis because its main characteristic is the possibility to discern between intensity and phase information performing quantitative mapping of the Optical Path Length. In this paper, the flexibility of DH is employed to analyze cell mechanics of unstained cells subjected to appropriate stimuli. DHM is used to measure all the parameters useful to understand the deformations induced by external and controlled stresses on in-vitro cells. Our configuration allows 3D tracking of micro-particles and, simultaneously, furnish quantitative phase-contrast maps. Experimental results are presented and discussed for in vitro cells.

cgCorrect: a method to correct for confounding cell-cell variation due to cell growth in single-cell transcriptomics

NASA Astrophysics Data System (ADS)

Blasi, Thomas; Buettner, Florian; Strasser, Michael K.; Marr, Carsten; Theis, Fabian J.

2017-06-01

Accessing gene expression at a single-cell level has unraveled often large heterogeneity among seemingly homogeneous cells, which remains obscured when using traditional population-based approaches. The computational analysis of single-cell transcriptomics data, however, still imposes unresolved challenges with respect to normalization, visualization and modeling the data. One such issue is differences in cell size, which introduce additional variability into the data and for which appropriate normalization techniques are needed. Otherwise, these differences in cell size may obscure genuine heterogeneities among cell populations and lead to overdispersed steady-state distributions of mRNA transcript numbers. We present cgCorrect, a statistical framework to correct for differences in cell size that are due to cell growth in single-cell transcriptomics data. We derive the probability for the cell-growth-corrected mRNA transcript number given the measured, cell size-dependent mRNA transcript number, based on the assumption that the average number of transcripts in a cell increases proportionally to the cell’s volume during the cell cycle. cgCorrect can be used for both data normalization and to analyze the steady-state distributions used to infer the gene expression mechanism. We demonstrate its applicability on both simulated data and single-cell quantitative real-time polymerase chain reaction (PCR) data from mouse blood stem and progenitor cells (and to quantitative single-cell RNA-sequencing data obtained from mouse embryonic stem cells). We show that correcting for differences in cell size affects the interpretation of the data obtained by typically performed computational analysis.

Computerized image analysis of cell-cell interactions in human renal tissue by using multi-channel immunoflourescent confocal microscopy

NASA Astrophysics Data System (ADS)

Peng, Yahui; Jiang, Yulei; Liarski, Vladimir M.; Kaverina, Natalya; Clark, Marcus R.; Giger, Maryellen L.

2012-03-01

Analysis of interactions between B and T cells in tubulointerstitial inflammation is important for understanding human lupus nephritis. We developed a computer technique to perform this analysis, and compared it with manual analysis. Multi-channel immunoflourescent-microscopy images were acquired from 207 regions of interest in 40 renal tissue sections of 19 patients diagnosed with lupus nephritis. Fresh-frozen renal tissue sections were stained with combinations of immunoflourescent antibodies to membrane proteins and counter-stained with a cell nuclear marker. Manual delineation of the antibodies was considered as the reference standard. We first segmented cell nuclei and cell membrane markers, and then determined corresponding cell types based on the distances between cell nuclei and specific cell-membrane marker combinations. Subsequently, the distribution of the shortest distance from T cell nuclei to B cell nuclei was obtained and used as a surrogate indicator of cell-cell interactions. The computer and manual analyses results were concordant. The average absolute difference was 1.1+/-1.2% between the computer and manual analysis results in the number of cell-cell distances of 3 μm or less as a percentage of the total number of cell-cell distances. Our computerized analysis of cell-cell distances could be used as a surrogate for quantifying cell-cell interactions as either an automated and quantitative analysis or for independent confirmation of manual analysis.

Silicon solar cell process development, fabrication and analysis

NASA Technical Reports Server (NTRS)

Leung, D. C.; Iles, P. A.

1983-01-01

Measurements of minority carrier diffusion lengths were made on the small mesa diodes from HEM Si and SILSO Si. The results were consistent with previous Voc and Isc measurements. Only the medium grain SILSO had a distinct advantage for the non grain boundary diodes. Substantial variations were observed for the HEM ingot 4141C. Also a quantitatively scaled light spot scan was being developed for localized diffusion length measurements in polycrystalline silicon solar cells. A change to a more monochromatic input for the light spot scan results in greater sensitivity and in principle, quantitative measurement of local material qualities is now possible.

The Use of Mouse Models of Breast Cancer and Quantitative Image Analysis to Evaluate Hormone Receptor Antigenicity after Microwave-assisted Formalin Fixation

PubMed Central

Engelberg, Jesse A.; Giberson, Richard T.; Young, Lawrence J.T.; Hubbard, Neil E.

2014-01-01

Microwave methods of fixation can dramatically shorten fixation times while preserving tissue structure; however, it remains unclear if adequate tissue antigenicity is preserved. To assess and validate antigenicity, robust quantitative methods and animal disease models are needed. We used two mouse mammary models of human breast cancer to evaluate microwave-assisted and standard 24-hr formalin fixation. The mouse models expressed four antigens prognostic for breast cancer outcome: estrogen receptor, progesterone receptor, Ki67, and human epidermal growth factor receptor 2. Using pathologist evaluation and novel methods of quantitative image analysis, we measured and compared the quality of antigen preservation, percentage of positive cells, and line plots of cell intensity. Visual evaluations by pathologists established that the amounts and patterns of staining were similar in tissues fixed by the different methods. The results of the quantitative image analysis provided a fine-grained evaluation, demonstrating that tissue antigenicity is preserved in tissues fixed using microwave methods. Evaluation of the results demonstrated that a 1-hr, 150-W fixation is better than a 45-min, 150-W fixation followed by a 15-min, 650-W fixation. The results demonstrated that microwave-assisted formalin fixation can standardize fixation times to 1 hr and produce immunohistochemistry that is in every way commensurate with longer conventional fixation methods. PMID:24682322

Quantitative Proteomics Analysis of the cAMP/Protein Kinase A Signaling Pathway

PubMed Central

2012-01-01

To define the proteins whose expression is regulated by cAMP and protein kinase A (PKA), we used a quantitative proteomics approach in studies of wild-type (WT) and kin- (PKA-null) S49 murine T lymphoma cells. We also compared the impact of endogenous increases in the level of cAMP [by forskolin (Fsk) and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX)] or by a cAMP analogue (8-CPT-cAMP). We identified 1056 proteins in WT and kin- S49 cells and found that 8-CPT-cAMP and Fsk with IBMX produced differences in protein expression. WT S49 cells had a correlation coefficient of 0.41 between DNA microarray data and the proteomics analysis in cells incubated with 8-CPT-cAMP for 24 h and a correlation coefficient of 0.42 between the DNA microarray data obtained at 6 h and the changes in protein expression after incubation with 8-CPT-cAMP for 24 h. Glutathione reductase (Gsr) had a higher level of basal expression in kin- S49 cells than in WT cells. Consistent with this finding, kin- cells are less sensitive to cell killing and generation of malondialdehyde than are WT cells incubated with H2O2. Cyclic AMP acting via PKA thus has a broad impact on protein expression in mammalian cells, including in the regulation of Gsr and oxidative stress. PMID:23110364

[The risk of neoplastic processes transformation in cervix uteri].

PubMed

Kiseleva, V I; Krikunova, L I; Mkrtchian, L S; Liubina, L V; Beziaeva, G P; Panarina, L V; Zamuliaeva, I A

2014-01-01

There was performed a comparative analysis of quantitative load and physical status of human papillomavirus (HPV) type 16 in groups of patients with cervical intraepithelial neoplasia (CIN)--25 people and cervical cancer (CC)--85 people. According to the analysis there were selected criteria appropriate to a combination of adverse factors that characterized HPV- infection and at the same time estimated both quantitative load and physical status of the virus: high viral load (> 6,5 lg copies of HPV DNA per 100000 cells) in episomal form or low load (< 6,5 lg copies of HPV DNA per 100000 cells) in integrated form of the virus. According to calculations a relative chance of appearing of CC in CIN patients with unfavorable combination of factors was 7,5 times higher than in other patients.

Electron Paramagnetic Resonance Oximetry as a Quantitative Method to Measure Cellular Respiration: A Consideration of Oxygen Diffusion Interference

PubMed Central

Presley, Tennille; Kuppusamy, Periannan; Zweier, Jay L.; Ilangovan, Govindasamy

2006-01-01

Electron paramagnetic resonance (EPR) oximetry is being widely used to measure the oxygen consumption of cells, mitochondria, and submitochondrial particles. However, further improvement of this technique, in terms of data analysis, is required to use it as a quantitative tool. Here, we present a new approach for quantitative analysis of cellular respiration using EPR oximetry. The course of oxygen consumption by cells in suspension has been observed to have three distinct zones: pO2-independent respiration at higher pO2 ranges, pO2-dependent respiration at low pO2 ranges, and a static equilibrium with no change in pO2 at very low pO2 values. The approach here enables one to comprehensively analyze all of the three zones together—where the progression of O2 diffusion zones around each cell, their overlap within time, and their potential impact on the measured pO2 data are considered. The obtained results agree with previously established methods such as high-resolution respirometry measurements. Additionally, it is also demonstrated how the diffusion limitations can depend on cell density and consumption rate. In conclusion, the new approach establishes a more accurate and meaningful model to evaluate the EPR oximetry data on cellular respiration to quantify related parameters using EPR oximetry. PMID:17012319

Precise Quantitation of MicroRNA in a Single Cell with Droplet Digital PCR Based on Ligation Reaction.

PubMed

Tian, Hui; Sun, Yuanyuan; Liu, Chenghui; Duan, Xinrui; Tang, Wei; Li, Zhengping

2016-12-06

MicroRNA (miRNA) analysis in a single cell is extremely important because it allows deep understanding of the exact correlation between the miRNAs and cell functions. Herein, we wish to report a highly sensitive and precisely quantitative assay for miRNA detection based on ligation-based droplet digital polymerase chain reaction (ddPCR), which permits the quantitation of miRNA in a single cell. In this ligation-based ddPCR assay, two target-specific oligonucleotide probes can be simply designed to be complementary to the half-sequence of the target miRNA, respectively, which avoids the sophisticated design of reverse transcription and provides high specificity to discriminate a single-base difference among miRNAs with simple operations. After the miRNA-templated ligation, the ddPCR partitions individual ligated products into a water-in-oil droplet and digitally counts the fluorescence-positive and negative droplets after PCR amplification for quantification of the target molecules, which possesses the power of precise quantitation and robustness to variation in PCR efficiency. By integrating the advantages of the precise quantification of ddPCR and the simplicity of the ligation-based PCR, the proposed method can sensitively measure let-7a miRNA with a detection limit of 20 aM (12 copies per microliter), and even a single-base difference can be discriminated in let-7 family members. More importantly, due to its high selectivity and sensitivity, the proposed method can achieve precise quantitation of miRNAs in single-cell lysate. Therefore, the ligation-based ddPCR assay may serve as a useful tool to exactly reveal the miRNAs' actions in a single cell, which is of great importance for the study of miRNAs' biofunction as well as for the related biomedical studies.

Single-cell quantitative HER2 measurement identifies heterogeneity and distinct subgroups within traditionally defined HER2-positive patients.

PubMed

Onsum, Matthew D; Geretti, Elena; Paragas, Violette; Kudla, Arthur J; Moulis, Sharon P; Luus, Lia; Wickham, Thomas J; McDonagh, Charlotte F; MacBeath, Gavin; Hendriks, Bart S

2013-11-01

Human epidermal growth factor receptor 2 (HER2) is an important biomarker for breast and gastric cancer prognosis and patient treatment decisions. HER2 positivity, as defined by IHC or fluorescent in situ hybridization testing, remains an imprecise predictor of patient response to HER2-targeted therapies. Challenges to correct HER2 assessment and patient stratification include intratumoral heterogeneity, lack of quantitative and/or objective assays, and differences between measuring HER2 amplification at the protein versus gene level. We developed a novel immunofluorescence method for quantitation of HER2 protein expression at the single-cell level on FFPE patient samples. Our assay uses automated image analysis to identify and classify tumor versus non-tumor cells, as well as quantitate the HER2 staining for each tumor cell. The HER2 staining level is converted to HER2 protein expression using a standard cell pellet array stained in parallel with the tissue sample. This approach allows assessment of HER2 expression and heterogeneity within a tissue section at the single-cell level. By using this assay, we identified distinct subgroups of HER2 heterogeneity within traditional definitions of HER2 positivity in both breast and gastric cancers. Quantitative assessment of intratumoral HER2 heterogeneity may offer an opportunity to improve the identification of patients likely to respond to HER2-targeted therapies. The broad applicability of the assay was demonstrated by measuring HER2 expression profiles on multiple tumor types, and on normal and diseased heart tissues. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

An integrated enhancement and reconstruction strategy for the quantitative extraction of actin stress fibers from fluorescence micrographs.

PubMed

Zhang, Zhen; Xia, Shumin; Kanchanawong, Pakorn

2017-05-22

The stress fibers are prominent organization of actin filaments that perform important functions in cellular processes such as migration, polarization, and traction force generation, and whose collective organization reflects the physiological and mechanical activities of the cells. Easily visualized by fluorescence microscopy, the stress fibers are widely used as qualitative descriptors of cell phenotypes. However, due to the complexity of the stress fibers and the presence of other actin-containing cellular features, images of stress fibers are relatively challenging to quantitatively analyze using previously developed approaches, requiring significant user intervention. This poses a challenge for the automation of their detection, segmentation, and quantitative analysis. Here we describe an open-source software package, SFEX (Stress Fiber Extractor), which is geared for efficient enhancement, segmentation, and analysis of actin stress fibers in adherent tissue culture cells. Our method made use of a carefully chosen image filtering technique to enhance filamentous structures, effectively facilitating the detection and segmentation of stress fibers by binary thresholding. We subdivided the skeletons of stress fiber traces into piecewise-linear fragments, and used a set of geometric criteria to reconstruct the stress fiber networks by pairing appropriate fiber fragments. Our strategy enables the trajectory of a majority of stress fibers within the cells to be comprehensively extracted. We also present a method for quantifying the dimensions of the stress fibers using an image gradient-based approach. We determine the optimal parameter space using sensitivity analysis, and demonstrate the utility of our approach by analyzing actin stress fibers in cells cultured on various micropattern substrates. We present an open-source graphically-interfaced computational tool for the extraction and quantification of stress fibers in adherent cells with minimal user input. This facilitates the automated extraction of actin stress fibers from fluorescence images. We highlight their potential uses by analyzing images of cells with shapes constrained by fibronectin micropatterns. The method we reported here could serve as the first step in the detection and characterization of the spatial properties of actin stress fibers to enable further detailed morphological analysis.

An atlas of histone deacetylase expression in breast cancer: fluorescence methodology for comparative semi-quantitative analysis

PubMed Central

Ververis, Katherine; Karagiannis, Tom C

2012-01-01

The histone deacetylase inhibitors, suberoylanilide hydroxamic acid (Vorinostat, Zolinza™) and depsipeptide (Romidepsin, Istodax™) have been approved by the US Food and Drug Administration for the treatment of refractory cutaneous T-cell lymphoma. Numerous histone deacetylase inhibitors are currently undergoing clinical trials, predominantly in combination with other cancer modalities, for the treatment of various haematological and solid malignancies. Most of the traditional compounds are known as broad-spectrum or pan-histone deacetylase inhibitors, possessing activity against a number of the 11 metal-dependent enzymes. One of the main questions in the field is whether class- or isoform-specific compounds would offer a therapeutic benefit compared to broad-spectrum inhibitors. Therefore, analysis of the relative expression of the different histone deacetylase enzymes in cancer cells and tissues is important to determine whether there are specific targets. We used a panel of antibodies directed against the 11 known mammalian histone deacetylases to determine expression levels in MCF7 breast cancer cells and in tissue representative of invasive ductal cell carcinoma and ductal carcinoma in situ. Firstly, we utilized a semi-quantitative method based on immunofluorescence staining to examine expression of the different histone deacetylases in MCF7 cells. Our findings indicate high expression levels of HDAC1, 3 and 6 in accordance with findings from others using RT-PCR and immunoblotting. Following validation of our approach we examined the expression of the different isoforms in representative control and breast cancer tissue. In general, our findings indicate higher expression of class I histone deacetylases compared to class II enzymes in breast cancer tissue. Analysis of individual cancer cells in the same tissue indicated marked heterogeneity in the expression of most class I enzymes indicating potential complications with the use of class- or isoform-specific compounds. Overall, our approach can be utilized to rapidly compare, in an unbiased semi-quantitative manner, the differential levels of expression of histone deacetylase enzymes in cells and tissues using widely available imaging software. It is anticipated that such analysis will become increasingly important as class- or isoform-specific histone deacetylase inhibitors become more readily available. PMID:22347520

An atlas of histone deacetylase expression in breast cancer: fluorescence methodology for comparative semi-quantitative analysis.

PubMed

Ververis, Katherine; Karagiannis, Tom C

2012-01-01

The histone deacetylase inhibitors, suberoylanilide hydroxamic acid (Vorinostat, Zolinza™) and depsipeptide (Romidepsin, Istodax™) have been approved by the US Food and Drug Administration for the treatment of refractory cutaneous T-cell lymphoma. Numerous histone deacetylase inhibitors are currently undergoing clinical trials, predominantly in combination with other cancer modalities, for the treatment of various haematological and solid malignancies. Most of the traditional compounds are known as broad-spectrum or pan-histone deacetylase inhibitors, possessing activity against a number of the 11 metal-dependent enzymes. One of the main questions in the field is whether class- or isoform-specific compounds would offer a therapeutic benefit compared to broad-spectrum inhibitors. Therefore, analysis of the relative expression of the different histone deacetylase enzymes in cancer cells and tissues is important to determine whether there are specific targets. We used a panel of antibodies directed against the 11 known mammalian histone deacetylases to determine expression levels in MCF7 breast cancer cells and in tissue representative of invasive ductal cell carcinoma and ductal carcinoma in situ. Firstly, we utilized a semi-quantitative method based on immunofluorescence staining to examine expression of the different histone deacetylases in MCF7 cells. Our findings indicate high expression levels of HDAC1, 3 and 6 in accordance with findings from others using RT-PCR and immunoblotting. Following validation of our approach we examined the expression of the different isoforms in representative control and breast cancer tissue. In general, our findings indicate higher expression of class I histone deacetylases compared to class II enzymes in breast cancer tissue. Analysis of individual cancer cells in the same tissue indicated marked heterogeneity in the expression of most class I enzymes indicating potential complications with the use of class- or isoform-specific compounds. Overall, our approach can be utilized to rapidly compare, in an unbiased semi-quantitative manner, the differential levels of expression of histone deacetylase enzymes in cells and tissues using widely available imaging software. It is anticipated that such analysis will become increasingly important as class- or isoform-specific histone deacetylase inhibitors become more readily available.

Quantitative Magnetic Separation of Particles and Cells Using Gradient Magnetic Ratcheting.

PubMed

Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

2016-04-13

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting, are robust but perform coarse, qualitative separations based on surface antigen expression. A quantitative magnetic separation technology is reported using high-force magnetic ratcheting over arrays of magnetically soft micropillars with gradient spacing, and the system is used to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micropillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic field. Particles with higher IOC separate and equilibrate along the miropillar array at larger pitches. A semi-analytical model is developed that predicts behavior for particles and cells. Using the system, LNCaP cells are separated based on the bound quantity of 1 μm anti-epithelial cell adhesion molecule (EpCAM) particles as a metric for expression. The ratcheting cytometry system is able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof-of-concept, EpCAM-labeled cells from patient blood are isolated with 74% purity, demonstrating potential toward a quantitative magnetic separation instrument. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Elucidating dynamic metabolic physiology through network integration of quantitative time-course metabolomics

DOE PAGES

Bordbar, Aarash; Yurkovich, James T.; Paglia, Giuseppe; ...

2017-04-07

In this study, the increasing availability of metabolomics data necessitates novel methods for deeper data analysis and interpretation. We present a flux balance analysis method that allows for the computation of dynamic intracellular metabolic changes at the cellular scale through integration of time-course absolute quantitative metabolomics. This approach, termed “unsteady-state flux balance analysis” (uFBA), is applied to four cellular systems: three dynamic and one steady-state as a negative control. uFBA and FBA predictions are contrasted, and uFBA is found to be more accurate in predicting dynamic metabolic flux states for red blood cells, platelets, and Saccharomyces cerevisiae. Notably, only uFBAmore » predicts that stored red blood cells metabolize TCA intermediates to regenerate important cofactors, such as ATP, NADH, and NADPH. These pathway usage predictions were subsequently validated through 13C isotopic labeling and metabolic flux analysis in stored red blood cells. Utilizing time-course metabolomics data, uFBA provides an accurate method to predict metabolic physiology at the cellular scale for dynamic systems.« less

Elucidating dynamic metabolic physiology through network integration of quantitative time-course metabolomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bordbar, Aarash; Yurkovich, James T.; Paglia, Giuseppe

In this study, the increasing availability of metabolomics data necessitates novel methods for deeper data analysis and interpretation. We present a flux balance analysis method that allows for the computation of dynamic intracellular metabolic changes at the cellular scale through integration of time-course absolute quantitative metabolomics. This approach, termed “unsteady-state flux balance analysis” (uFBA), is applied to four cellular systems: three dynamic and one steady-state as a negative control. uFBA and FBA predictions are contrasted, and uFBA is found to be more accurate in predicting dynamic metabolic flux states for red blood cells, platelets, and Saccharomyces cerevisiae. Notably, only uFBAmore » predicts that stored red blood cells metabolize TCA intermediates to regenerate important cofactors, such as ATP, NADH, and NADPH. These pathway usage predictions were subsequently validated through 13C isotopic labeling and metabolic flux analysis in stored red blood cells. Utilizing time-course metabolomics data, uFBA provides an accurate method to predict metabolic physiology at the cellular scale for dynamic systems.« less

Combining FT-IR spectroscopy and multivariate analysis for qualitative and quantitative analysis of the cell wall composition changes during apples development.

PubMed

Szymanska-Chargot, M; Chylinska, M; Kruk, B; Zdunek, A

2015-01-22

The aim of this work was to quantitatively and qualitatively determine the composition of the cell wall material from apples during development by means of Fourier transform infrared (FT-IR) spectroscopy. The FT-IR region of 1500-800 cm(-1), containing characteristic bands for galacturonic acid, hemicellulose and cellulose, was examined using principal component analysis (PCA), k-means clustering and partial least squares (PLS). The samples were differentiated by development stage and cultivar using PCA and k-means clustering. PLS calibration models for galacturonic acid, hemicellulose and cellulose content from FT-IR spectra were developed and validated with the reference data. PLS models were tested using the root-mean-square errors of cross-validation for contents of galacturonic acid, hemicellulose and cellulose which was 8.30 mg/g, 4.08% and 1.74%, respectively. It was proven that FT-IR spectroscopy combined with chemometric methods has potential for fast and reliable determination of the main constituents of fruit cell walls. Copyright © 2014 Elsevier Ltd. All rights reserved.

Recent advances in mass spectrometry-based proteomics of gastric cancer.

PubMed

Kang, Changwon; Lee, Yejin; Lee, J Eugene

2016-10-07

The last decade has witnessed remarkable technological advances in mass spectrometry-based proteomics. The development of proteomics techniques has enabled the reliable analysis of complex proteomes, leading to the identification and quantification of thousands of proteins in gastric cancer cells, tissues, and sera. This quantitative information has been used to profile the anomalies in gastric cancer and provide insights into the pathogenic mechanism of the disease. In this review, we mainly focus on the advances in mass spectrometry and quantitative proteomics that were achieved in the last five years and how these up-and-coming technologies are employed to track biochemical changes in gastric cancer cells. We conclude by presenting a perspective on quantitative proteomics and its future applications in the clinic and translational gastric cancer research.

A quantitative spatiotemporal analysis of microglia morphology during ischemic stroke and reperfusion

PubMed Central

2013-01-01

Background Microglia cells continuously survey the healthy brain in a ramified morphology and, in response to injury, undergo progressive morphological and functional changes that encompass microglia activation. Although ideally positioned for immediate response to ischemic stroke (IS) and reperfusion, their progressive morphological transformation into activated cells has not been quantified. In addition, it is not well understood if diverse microglia morphologies correlate to diverse microglia functions. As such, the dichotomous nature of these cells continues to confound our understanding of microglia-mediated injury after IS and reperfusion. The purpose of this study was to quantitatively characterize the spatiotemporal pattern of microglia morphology during the evolution of cerebral injury after IS and reperfusion. Methods Male C57Bl/6 mice were subjected to focal cerebral ischemia and periods of reperfusion (0, 8 and 24 h). The microglia process length/cell and number of endpoints/cell was quantified from immunofluorescent confocal images of brain regions using a skeleton analysis method developed for this study. Live cell morphology and process activity were measured from movies acquired in acute brain slices from GFP-CX3CR1 transgenic mice after IS and 24-h reperfusion. Regional CD11b and iNOS expressions were measured from confocal images and Western blot, respectively, to assess microglia proinflammatory function. Results Quantitative analysis reveals a significant spatiotemporal relationship between microglia morphology and evolving cerebral injury in the ipsilateral hemisphere after IS and reperfusion. Microglia were both hyper- and de-ramified in striatal and cortical brain regions (respectively) after 60 min of focal cerebral ischemia. However, a de-ramified morphology was prominent when ischemia was coupled to reperfusion. Live microglia were de-ramified, and, in addition, process activity was severely blunted proximal to the necrotic core after IS and 24 h of reperfusion. CD11b expression, but not iNOS expression, was increased in regions of hyper- and de-ramified microglia during the course of ischemic stroke and 24 h of reperfusion. Conclusions Our findings illustrate that microglia activation after stroke includes both increased and decreased cell ramification. Importantly, quantitative analyses of microglial morphology and activity are feasible and, in future studies, would assist in the comprehensive identification and stratification of their dichotomous contribution toward cerebral injury and recovery during IS and reperfusion. PMID:23311642

CellStress - open source image analysis program for single-cell analysis

NASA Astrophysics Data System (ADS)

Smedh, Maria; Beck, Caroline; Sott, Kristin; Goksör, Mattias

2010-08-01

This work describes our image-analysis software, CellStress, which has been developed in Matlab and is issued under a GPL license. CellStress was developed in order to analyze migration of fluorescent proteins inside single cells during changing environmental conditions. CellStress can also be used to score information regarding protein aggregation in single cells over time, which is especially useful when monitoring cell signaling pathways involved in e.g. Alzheimer's or Huntington's disease. Parallel single-cell analysis of large numbers of cells is an important part of the research conducted in systems biology and quantitative biology in order to mathematically describe cellular processes. To quantify properties for single cells, large amounts of data acquired during extended time periods are needed. Manual analyses of such data involve huge efforts and could also include a bias, which complicates the use and comparison of data for further simulations or modeling. Therefore, it is necessary to have an automated and unbiased image analysis procedure, which is the aim of CellStress. CellStress utilizes cell contours detected by CellStat (developed at Fraunhofer-Chalmers Centre), which identifies cell boundaries using bright field images, and thus reduces the fluorescent labeling needed.

Inhibition of glycogen phosphorylation induces changes in cellular proteome and signaling pathways in MIA pancreatic cancer cells

PubMed Central

Ma, Danjun; Wang, Jiarui; Zhao, Yingchun; Lee, Wai-Nang Paul; Xiao, Jing; Go, Vay Liang W.; Wang, Qi; Recker, Robert; Xiao, Gary Guishan

2011-01-01

Objectives Novel quantitative proteomic approaches were used to study the effects of inhibition of glycogen phosphorylase on proteome and signaling pathways in MIA PaCa-2 pancreatic cancer cells. Methods We performed quantitative proteomic analysis in MIA PaCa-2 cancer cells treated with a stratified dose of CP-320626 (25 μM, 50 μM and 100 μM). The effect of metabolic inhibition on cellular protein turnover dynamics was also studied using the modified SILAC method (mSILAC). Results A total of twenty-two protein spots and four phosphoprotein spots were quantitatively analyzed. We found that dynamic expression of total proteins and phosphoproteins was significantly changed in MIA PaCa-2 cells treated with an incremental dose of CP-320626. Functional analyses suggested that most of the proteins differentially expressed were in the pathways of MAPK/ERK and TNF-α/NF-κB. Conclusions Signaling pathways and metabolic pathways share many common cofactors and substrates forming an extended metabolic network. The restriction of substrate through one pathway such as inhibition of glycogen phosphorylation induces pervasive metabolomic and proteomic changes manifested in protein synthesis, breakdown and post-translational modification of signaling molecules. Our results suggest that quantitative proteomic is an important approach to understand the interaction between metabolism and signaling pathways. PMID:22158071

Quantitative Proteomics Analysis of Streptomyces coelicolor Development Demonstrates That Onset of Secondary Metabolism Coincides with Hypha Differentiation*

PubMed Central

Manteca, Angel; Sanchez, Jesus; Jung, Hye R.; Schwämmle, Veit; Jensen, Ole N.

2010-01-01

Streptomyces species produce many clinically important secondary metabolites, including antibiotics and antitumorals. They have a complex developmental cycle, including programmed cell death phenomena, that makes this bacterium a multicellular prokaryotic model. There are two differentiated mycelial stages: an early compartmentalized vegetative mycelium (first mycelium) and a multinucleated reproductive mycelium (second mycelium) arising after programmed cell death processes. In the present study, we made a detailed proteomics analysis of the distinct developmental stages of solid confluent Streptomyces coelicolor cultures using iTRAQ (isobaric tags for relative and absolute quantitation) labeling and LC-MS/MS. A new experimental approach was developed to obtain homogeneous samples at each developmental stage (temporal protein analysis) and also to obtain membrane and cytosolic protein fractions (spatial protein analysis). A total of 345 proteins were quantified in two biological replicates. Comparative bioinformatics analyses revealed the switch from primary to secondary metabolism between the initial compartmentalized mycelium and the multinucleated hyphae. PMID:20224110

Parsing Stem Cell Lineage Development Using High Content Image Analysis of Epigenetic Spatial Markers.

PubMed

Kim, Joseph J; Moghe, Prabhas V

2018-06-14

This unit describes a protocol for acquiring and analyzing high-content super-resolution images of human stem cell nuclei for the characterization and classification of the cell differentiation paths based on distinct patterns of epigenetic mark organization. Here, we describe the cell culture, immunocytochemical labeling, super-resolution imaging parameters, and MATLAB-based quantitative image analysis approaches for monitoring human mesenchymal stem cells (hMSCs) and human induced pluripotent stem cells (hiPSCs) as the cells differentiate towards various lineages. Although this protocol uses specific cell types as examples, this approach could be easily extended to a variety of cell types and nuclear epigenetic and mechanosensitive biomarkers that are relevant to specific cell developmental scenarios. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

Engineering cell-compatible paper chips for cell culturing, drug screening, and mass spectrometric sensing.

PubMed

Chen, Qiushui; He, Ziyi; Liu, Wu; Lin, Xuexia; Wu, Jing; Li, Haifang; Lin, Jin-Ming

2015-10-28

Paper-supported cell culture is an unprecedented development for advanced bioassays. This study reports a strategy for in vitro engineering of cell-compatible paper chips that allow for adherent cell culture, quantitative assessment of drug efficiency, and label-free sensing of intracellular molecules via paper spray mass spectrometry. The polycarbonate paper is employed as an excellent alternative bioscaffold for cell distribution, adhesion, and growth, as well as allowing for fluorescence imaging without light scattering. The cell-cultured paper chips are thus amenable to fabricate 3D tissue construction and cocultures by flexible deformation, stacks and assembly by layers of cells. As a result, the successful development of cell-compatible paper chips subsequently offers a uniquely flexible approach for in situ sensing of live cell components by paper spray mass spectrometry, allowing profiling the cellular lipids and quantitative measurement of drug metabolism with minimum sample pretreatment. Consequently, the developed paper chips for adherent cell culture are inexpensive for one-time use, compatible with high throughputs, and amenable to label-free and rapid analysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mechanical behavior in living cells consistent with the tensegrity model

NASA Technical Reports Server (NTRS)

Wang, N.; Naruse, K.; Stamenovic, D.; Fredberg, J. J.; Mijailovich, S. M.; Tolic-Norrelykke, I. M.; Polte, T.; Mannix, R.; Ingber, D. E.

2001-01-01

Alternative models of cell mechanics depict the living cell as a simple mechanical continuum, porous filament gel, tensed cortical membrane, or tensegrity network that maintains a stabilizing prestress through incorporation of discrete structural elements that bear compression. Real-time microscopic analysis of cells containing GFP-labeled microtubules and associated mitochondria revealed that living cells behave like discrete structures composed of an interconnected network of actin microfilaments and microtubules when mechanical stresses are applied to cell surface integrin receptors. Quantitation of cell tractional forces and cellular prestress by using traction force microscopy confirmed that microtubules bear compression and are responsible for a significant portion of the cytoskeletal prestress that determines cell shape stability under conditions in which myosin light chain phosphorylation and intracellular calcium remained unchanged. Quantitative measurements of both static and dynamic mechanical behaviors in cells also were consistent with specific a priori predictions of the tensegrity model. These findings suggest that tensegrity represents a unified model of cell mechanics that may help to explain how mechanical behaviors emerge through collective interactions among different cytoskeletal filaments and extracellular adhesions in living cells.

Quantitative 4D analyses of epithelial folding during Drosophila gastrulation.

PubMed

Khan, Zia; Wang, Yu-Chiun; Wieschaus, Eric F; Kaschube, Matthias

2014-07-01

Understanding the cellular and mechanical processes that underlie the shape changes of individual cells and their collective behaviors in a tissue during dynamic and complex morphogenetic events is currently one of the major frontiers in developmental biology. The advent of high-speed time-lapse microscopy and its use in monitoring the cellular events in fluorescently labeled developing organisms demonstrate tremendous promise in establishing detailed descriptions of these events and could potentially provide a foundation for subsequent hypothesis-driven research strategies. However, obtaining quantitative measurements of dynamic shapes and behaviors of cells and tissues in a rapidly developing metazoan embryo using time-lapse 3D microscopy remains technically challenging, with the main hurdle being the shortage of robust imaging processing and analysis tools. We have developed EDGE4D, a software tool for segmenting and tracking membrane-labeled cells using multi-photon microscopy data. Our results demonstrate that EDGE4D enables quantification of the dynamics of cell shape changes, cell interfaces and neighbor relations at single-cell resolution during a complex epithelial folding event in the early Drosophila embryo. We expect this tool to be broadly useful for the analysis of epithelial cell geometries and movements in a wide variety of developmental contexts. © 2014. Published by The Company of Biologists Ltd.

Differential diagnosis of lung carcinoma with three-dimensional quantitative molecular vibrational imaging

NASA Astrophysics Data System (ADS)

Gao, Liang; Hammoudi, Ahmad A.; Li, Fuhai; Thrall, Michael J.; Cagle, Philip T.; Chen, Yuanxin; Yang, Jian; Xia, Xiaofeng; Fan, Yubo; Massoud, Yehia; Wang, Zhiyong; Wong, Stephen T. C.

2012-06-01

The advent of molecularly targeted therapies requires effective identification of the various cell types of non-small cell lung carcinomas (NSCLC). Currently, cell type diagnosis is performed using small biopsies or cytology specimens that are often insufficient for molecular testing after morphologic analysis. Thus, the ability to rapidly recognize different cancer cell types, with minimal tissue consumption, would accelerate diagnosis and preserve tissue samples for subsequent molecular testing in targeted therapy. We report a label-free molecular vibrational imaging framework enabling three-dimensional (3-D) image acquisition and quantitative analysis of cellular structures for identification of NSCLC cell types. This diagnostic imaging system employs superpixel-based 3-D nuclear segmentation for extracting such disease-related features as nuclear shape, volume, and cell-cell distance. These features are used to characterize cancer cell types using machine learning. Using fresh unstained tissue samples derived from cell lines grown in a mouse model, the platform showed greater than 97% accuracy for diagnosis of NSCLC cell types within a few minutes. As an adjunct to subsequent histology tests, our novel system would allow fast delineation of cancer cell types with minimum tissue consumption, potentially facilitating on-the-spot diagnosis, while preserving specimens for additional tests. Furthermore, 3-D measurements of cellular structure permit evaluation closer to the native state of cells, creating an alternative to traditional 2-D histology specimen evaluation, potentially increasing accuracy in diagnosing cell type of lung carcinomas.

Serum Squamous Cell Carcinoma Antigen in Psoriasis: A Potential Quantitative Biomarker for Disease Severity.

PubMed

Sun, Ziwen; Shi, Xiaomin; Wang, Yun; Zhao, Yi

2018-06-05

An objective and quantitative method to evaluate psoriasis severity is important for practice and research in the precision care of psoriasis. We aimed to explore serum biomarkers quantitatively in association with disease severity and treatment response in psoriasis patients, with serum squamous cell carcinoma antigen (SCCA) evaluated in this pilot study. 15 psoriasis patients were treated with adalimumab. At different visits before and after treatment, quantitative body surface area (qBSA) was obtained from standardized digital body images of the patients, and the psoriasis area severity index (PASI) was also monitored. SCCA were detected by using microparticle enzyme immunoassay. The serum biomarkers were also tested in healthy volunteers as normal controls. Receiver-operating characteristic (ROC) curve analysis was used to explore the optimal cutoff point of SCCA to differentiate mild and moderate-to-severe psoriasis. The serum SCCA level in the psoriasis group was significantly higher (p < 0.05) than in the normal control group. After treatment, the serum SCCA levels were significantly decreased (p < 0.05). The SCCA level was well correlated with PASI and qBSA. In ROC analysis, when taking PASI = 10 or qBSA = 10% as the threshold, an optimal cutoff point of SCCA was found at 2.0 ng/mL with the highest Youden index. Serum SCCA might be a useful quantitative biomarker for psoriasis disease severity. © 2018 S. Karger AG, Basel.

Quantitative Method of Measuring Metastatic Activity

NASA Technical Reports Server (NTRS)

Morrison, Dennis R. (Inventor)

1999-01-01

The metastatic potential of tumors can be evaluated by the quantitative detection of urokinase and DNA. The cell sample selected for examination is analyzed for the presence of high levels of urokinase and abnormal DNA using analytical flow cytometry and digital image analysis. Other factors such as membrane associated uroldnase, increased DNA synthesis rates and certain receptors can be used in the method for detection of potentially invasive tumors.

Quantitative and semi-quantitative immunoassay of growth factors and cytokines in the conditioned medium of STO and CF-1 mouse feeder cells

USDA-ARS?s Scientific Manuscript database

Feeder-cells of irradiated mouse fibroblasts are commonly used for, and are generally necessary for, the in vitro maintenance and growth of many fastidious cell types, particularly embryonic stem cells or induced pluripotent stem cells. Quantitative and semi-quantitative immunoassays were performed...

Mass cytometry: a highly multiplexed single-cell technology for advancing drug development.

PubMed

Atkuri, Kondala R; Stevens, Jeffrey C; Neubert, Hendrik

2015-02-01

Advanced single-cell analysis technologies (e.g., mass cytometry) that help in multiplexing cellular measurements in limited-volume primary samples are critical in bridging discovery efforts to successful drug approval. Mass cytometry is the state-of-the-art technology in multiparametric single-cell analysis. Mass cytometers (also known as cytometry by time-of-flight or CyTOF) combine the cellular analysis principles of traditional fluorescence-based flow cytometry with the selectivity and quantitative power of inductively coupled plasma-mass spectrometry. Standard flow cytometry is limited in the number of parameters that can be measured owing to the overlap in signal when detecting fluorescently labeled antibodies. Mass cytometry uses antibodies tagged to stable isotopes of rare earth metals, which requires minimal signal compensation between the different metal tags. This unique feature enables researchers to seamlessly multiplex up to 40 independent measurements on single cells. In this overview we first present an overview of mass cytometry and compare it with traditional flow cytometry. We then discuss the emerging and potential applications of CyTOF technology in the pharmaceutical industry, including quantitative and qualitative deep profiling of immune cells and their applications in assessing drug immunogenicity, extensive mapping of signaling networks in single cells, cell surface receptor quantification and multiplexed internalization kinetics, multiplexing sample analysis by barcoding, and establishing cell ontologies on the basis of phenotype and/or function. We end with a discussion of the anticipated impact of this technology on drug development lifecycle with special emphasis on the utility of mass cytometry in deciphering a drug's pharmacokinetics and pharmacodynamics relationship. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

High-throughput microfluidics to control and measure signaling dynamics in single yeast cells

PubMed Central

Hansen, Anders S.; Hao, Nan; O'Shea, Erin K.

2015-01-01

Microfluidics coupled to quantitative time-lapse fluorescence microscopy is transforming our ability to control, measure, and understand signaling dynamics in single living cells. Here we describe a pipeline that incorporates multiplexed microfluidic cell culture, automated programmable fluid handling for cell perturbation, quantitative time-lapse microscopy, and computational analysis of time-lapse movies. We illustrate how this setup can be used to control the nuclear localization of the budding yeast transcription factor Msn2. Using this protocol, we generate oscillations of Msn2 localization and measure the dynamic gene expression response of individual genes in single cells. The protocol allows a single researcher to perform up to 20 different experiments in a single day, whilst collecting data for thousands of single cells. Compared to other protocols, the present protocol is relatively easy to adopt and higher-throughput. The protocol can be widely used to control and monitor single-cell signaling dynamics in other signal transduction systems in microorganisms. PMID:26158443

Single cell model for simultaneous drug delivery and efflux.

PubMed

Yi, C; Saidel, G M; Gratzl, M

1999-01-01

Multidrug resistance (MDR) of some cancer cells is a major challenge for chemotherapy of systemic cancers to overcome. To experimentally uncover the cellular mechanisms leading to MDR, it is necessary to quantitatively assess both drug influx into, and efflux from, the cells exposed to drug treatment. By using a novel molecular microdelivery system to enforce continuous and adjustable drug influx into single cells by controlled diffusion through a gel plug in a micropipet tip, drug resistance studies can now be performed on the single cell level. Our dynamic model of this scheme incorporates drug delivery, diffusive mixing, and accumulation inside the cytoplasm, and efflux by both passive and active membrane transport. Model simulations using available experimental information on these processes can assist in the design of MDR related experiments on single cancer cells which are expected to lead to a quantitative evaluation of mechanisms. Simulations indicate that drug resistance of a cancer cell can be quantified better by its dynamic response than by steady-state analysis.

The quantitative proteomes of human-induced pluripotent stem cells and embryonic stem cells

PubMed Central

Munoz, Javier; Low, Teck Y; Kok, Yee J; Chin, Angela; Frese, Christian K; Ding, Vanessa; Choo, Andre; Heck, Albert J R

2011-01-01

Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. PMID:22108792

Harnessing cell-to-cell variations to probe bacterial structure and biophysics

NASA Astrophysics Data System (ADS)

Cass, Julie A.

Advances in microscopy and biotechnology have given us novel insights into cellular biology and physics. While bacteria were long considered to be relatively unstructured, the development of fluorescence microscopy techniques, and spatially and temporally resolved high-throughput quantitative studies, have uncovered that the bacterial cell is highly organized, and its structure rigorously maintained. In this thesis I will describe our gateTool software, designed to harness cell-to-cell variations to probe bacterial structure, and discuss two exciting aspects of structure that we have employed gateTool to investigate: (i) chromosome organization and the cellular mechanisms for controlling DNA dynamics, and (ii) the study of cell wall synthesis, and how the genes in the synthesis pathway impact cellular shape. In the first project, we develop a spatial and temporal mapping of cell-cycle-dependent chromosomal organization, and use this quantitative map to discover that chromosomal loci segregate from midcell with universal dynamics. In the second project, I describe preliminary time- lapse and snapshot imaging analysis suggesting phentoypical coherence across peptidoglycan synthesis pathways.

NHS-based Tandem Mass Tagging of Proteins at the Level of Whole Cells: A Critical Evaluation in Comparison to Conventional TMT-Labeling Approaches for Quantitative Proteome Analysis.

PubMed

Megger, Dominik A; Pott, Leona L; Rosowski, Kristin; Zülch, Birgit; Tautges, Stephanie; Bracht, Thilo; Sitek, Barbara

2017-01-01

Tandem mass tags (TMT) are usually introduced at the levels of isolated proteins or peptides. Here, for the first time, we report the labeling of whole cells and a critical evaluation of its performance in comparison to conventional labeling approaches. The obtained results indicated that TMT protein labeling using intact cells is generally possible, if it is coupled to a subsequent enrichment using anti-TMT antibody. The quantitative results were similar to those obtained after labeling of isolated proteins and both were found to be slightly complementary to peptide labeling. Furthermore, when using NHS-based TMT, no specificity towards cell surface proteins was observed in the case of cell labeling. In summary, the conducted study revealed first evidence for the general possibility of TMT cell labeling and highlighted limitations of NHS-based labeling reagents. Future studies should therefore focus on the synthesis and investigation of membrane impermeable TMTs to increase specificity towards cell surface proteins.

Dissecting Embryonic Stem Cell Self-Renewal and Differentiation Commitment from Quantitative Models.

PubMed

Hu, Rong; Dai, Xianhua; Dai, Zhiming; Xiang, Qian; Cai, Yanning

2016-10-01

To model quantitatively embryonic stem cell (ESC) self-renewal and differentiation by computational approaches, we developed a unified mathematical model for gene expression involved in cell fate choices. Our quantitative model comprised ESC master regulators and lineage-specific pivotal genes. It took the factors of multiple pathways as input and computed expression as a function of intrinsic transcription factors, extrinsic cues, epigenetic modifications, and antagonism between ESC master regulators and lineage-specific pivotal genes. In the model, the differential equations of expression of genes involved in cell fate choices from regulation relationship were established according to the transcription and degradation rates. We applied this model to the Murine ESC self-renewal and differentiation commitment and found that it modeled the expression patterns with good accuracy. Our model analysis revealed that Murine ESC was an attractor state in culture and differentiation was predominantly caused by antagonism between ESC master regulators and lineage-specific pivotal genes. Moreover, antagonism among lineages played a critical role in lineage reprogramming. Our results also uncovered that the ordered expression alteration of ESC master regulators over time had a central role in ESC differentiation fates. Our computational framework was generally applicable to most cell-type maintenance and lineage reprogramming.

Cells and Stripes: A novel quantitative photo-manipulation technique

PubMed Central

Mistrik, Martin; Vesela, Eva; Furst, Tomas; Hanzlikova, Hana; Frydrych, Ivo; Gursky, Jan; Majera, Dusana; Bartek, Jiri

2016-01-01

Laser micro-irradiation is a technology widely used in the DNA damage response, checkpoint signaling, chromatin remodeling and related research fields, to assess chromatin modifications and recruitment of diverse DNA damage sensors, mediators and repair proteins to sites of DNA lesions. While this approach has aided numerous discoveries related to cell biology, maintenance of genome integrity, aging and cancer, it has so far been limited by a tedious manual definition of laser-irradiated subcellular regions, with the ensuing restriction to only a small number of cells treated and analyzed in a single experiment. Here, we present an improved and versatile alternative to the micro-irradiation approach: Quantitative analysis of photo-manipulated samples using innovative settings of standard laser-scanning microscopes. Up to 200 cells are simultaneously exposed to a laser beam in a defined pattern of collinear rays. The induced striation pattern is then automatically evaluated by a simple algorithm, which provides a quantitative assessment of various laser-induced phenotypes in live or fixed cells. Overall, this new approach represents a more robust alternative to existing techniques, and provides a versatile tool for a wide range of applications in biomedicine. PMID:26777522

Detecting Autophagy and Autophagy Flux in Chronic Myeloid Leukemia Cells Using a Cyto-ID Fluorescence Spectrophotometric Assay.

PubMed

Guo, Sujuan; Pridham, Kevin J; Sheng, Zhi

2016-01-01

Autophagy is a catabolic process whereby cellular components are degraded to fuel cells for longer survival during stress. Hence, autophagy plays a vital role in determining cell fate and is central for homeostasis and pathogenesis of many human diseases including chronic myeloid leukemia (CML). It has been well established that autophagy is important for the leukemogenesis as well as drug resistance in CML. Thus, autophagy is an intriguing therapeutic target. However, current approaches that detect autophagy lack reliability and often fail to provide quantitative measurements. To overcome this hurdle and facilitate the development of autophagy-related therapies, we have recently developed an autophagy assay termed as the Cyto-ID fluorescence spectrophotometric assay. This method uses a cationic fluorescence dye, Cyto-ID, which specifically labels autophagic compartments and is detected by a spectrophotometer to permit a large-scale and quantitative analysis. As such, it allows rapid, reliable, and quantitative detection of autophagy and estimation of autophagy flux. In this chapter, we further provide technical details of this method and step-by-step protocols for measuring autophagy or autophagy flux in CML cell lines as well as primary hematopoietic cells.

Supramolecular assembly affording a ratiometric two-photon fluorescent nanoprobe for quantitative detection and bioimaging.

PubMed

Wang, Peng; Zhang, Cheng; Liu, Hong-Wen; Xiong, Mengyi; Yin, Sheng-Yan; Yang, Yue; Hu, Xiao-Xiao; Yin, Xia; Zhang, Xiao-Bing; Tan, Weihong

2017-12-01

Fluorescence quantitative analyses for vital biomolecules are in great demand in biomedical science owing to their unique detection advantages with rapid, sensitive, non-damaging and specific identification. However, available fluorescence strategies for quantitative detection are usually hard to design and achieve. Inspired by supramolecular chemistry, a two-photon-excited fluorescent supramolecular nanoplatform ( TPSNP ) was designed for quantitative analysis with three parts: host molecules (β-CD polymers), a guest fluorophore of sensing probes (Np-Ad) and a guest internal reference (NpRh-Ad). In this strategy, the TPSNP possesses the merits of (i) improved water-solubility and biocompatibility; (ii) increased tissue penetration depth for bioimaging by two-photon excitation; (iii) quantitative and tunable assembly of functional guest molecules to obtain optimized detection conditions; (iv) a common approach to avoid the limitation of complicated design by adjustment of sensing probes; and (v) accurate quantitative analysis by virtue of reference molecules. As a proof-of-concept, we utilized the two-photon fluorescent probe NHS-Ad-based TPSNP-1 to realize accurate quantitative analysis of hydrogen sulfide (H 2 S), with high sensitivity and good selectivity in live cells, deep tissues and ex vivo -dissected organs, suggesting that the TPSNP is an ideal quantitative indicator for clinical samples. What's more, TPSNP will pave the way for designing and preparing advanced supramolecular sensors for biosensing and biomedicine.

CellShape: A user-friendly image analysis tool for quantitative visualization of bacterial cell factories inside.

PubMed

Goñi-Moreno, Ángel; Kim, Juhyun; de Lorenzo, Víctor

2017-02-01

Visualization of the intracellular constituents of individual bacteria while performing as live biocatalysts is in principle doable through more or less sophisticated fluorescence microscopy. Unfortunately, rigorous quantitation of the wealth of data embodied in the resulting images requires bioinformatic tools that are not widely extended within the community-let alone that they are often subject to licensing that impedes software reuse. In this context we have developed CellShape, a user-friendly platform for image analysis with subpixel precision and double-threshold segmentation system for quantification of fluorescent signals stemming from single-cells. CellShape is entirely coded in Python, a free, open-source programming language with widespread community support. For a developer, CellShape enhances extensibility (ease of software improvements) by acting as an interface to access and use existing Python modules; for an end-user, CellShape presents standalone executable files ready to open without installation. We have adopted this platform to analyse with an unprecedented detail the tridimensional distribution of the constituents of the gene expression flow (DNA, RNA polymerase, mRNA and ribosomal proteins) in individual cells of the industrial platform strain Pseudomonas putida KT2440. While the CellShape first release version (v0.8) is readily operational, users and/or developers are enabled to expand the platform further. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Resistin modulates glucose uptake and glucose transporter-1 (GLUT-1) expression in trophoblast cells.

PubMed

Di Simone, Nicoletta; Di Nicuolo, Fiorella; Marzioni, Daniela; Castellucci, Mario; Sanguinetti, Maurizio; D'lppolito, Silvia; Caruso, Alessandro

2009-02-01

The adipocytokine resistin impairs glucose tolerance and insulin sensitivity. Here, we examine the effect of resistin on glucose uptake in human trophoblast cells and we demonstrate that transplacental glucose transport is mediated by glucose transporter (GLUT)-1. Furthermore, we evaluate the type of signal transduction induced by resistin in GLUT-1 regulation. BeWo choriocarcinoma cells and primary cytotrophoblast cells were cultured with increasing resistin concentrations for 24 hrs. The main outcome measures include glucose transport assay using [(3)H]-2-deoxy glucose, GLUT-1 protein expression by Western blot analysis and GLUT-1 mRNA detection by quantitative real-time RT-PCR. Quantitative determination of phospho(p)-ERK1/2 in cell lysates was performed by an Enzyme Immunometric Assay and Western blot analysis. Our data demonstrate a direct effect of resistin on normal cytotrophoblastic and on BeWo cells: resistin modulates glucose uptake, GLUT-1 messenger ribonucleic acid (mRNA) and protein expression in placental cells. We suggest that ERK1/2 phosphorylation is involved in the GLUT-1 regulation induced by resistin. In conclusion, resistin causes activation of both the ERK1 and 2 pathway in trophoblast cells. ERK1 and 2 activation stimulated GLUT-1 synthesis and resulted in increase of placental glucose uptake. High resistin levels (50-100 ng/ml) seem able to affect glucose-uptake, presumably by decreasing the cell surface glucose transporter.

The design analysis of a rechargeable lithium cell for space applications

NASA Technical Reports Server (NTRS)

Subba Rao, S.; Shen, D. H.; Yen, S. P. S.; Somoano, R. B.

1986-01-01

Ambient temperature rechargeable lithium batteries are needed by NASA for advanced space power applications for future missions. Specific energies of not less than 100 Wh/kg and long cycle life are critical performance goals. A design analysis of a 35 Ah Li-TiS2 cell was carried out using literature and experimental data to identify key design parameters governing specific energy. It is found that high specific energies are achievable in prismatic cells, especially with the use of advanced hardware materials. There is a serious need for a greatly expanded engineering database in order to enable more quantitative design analysis.

[Morphometric analysis of lymphocyte nuclei in chronic lymphocytic leukemia].

PubMed

Ostapenko, V A; Kruchinskiĭ, N G; Smirnova, L A; Cherednik, A B; Nesterov, V N; Tepliakov, A I

1994-01-01

This work is dedicated to the study of use of quantitative analysis of cell nucleus structure for the analysis of peripheral blood lymphocytes in patients with chronic lymphocytic leukaemia. The structure of lymphocytic nuclei of healthy donors was evaluated by means of staining by toluidine blue purified cell suspensions smears. The preparations were analysed on the television measuring system "omnicon" with measurements of the following parameters: square of the nucleus, euchromatin, heterochromatin, and the ratio of heterochromatin and euchromatin squares. Actuarial analysis and nuclei classification of the previously mentioned parameters showed, that in peripheral blood of patients with chronic lymphocytic leukemia a large amount of atypical lymphocytes is present with reduced nucleus sizes. Atypical cells retain the ratio of structural components of chromatine, characteristic to normal cells, which show their low proliferative activity.

iTRAQ-based proteomic analysis of LI-F type peptides produced by Paenibacillus polymyxa JSa-9 mode of action against Bacillus cereus.

PubMed

Han, Jinzhi; Gao, Peng; Zhao, Shengming; Bie, Xiaomei; Lu, Zhaoxin; Zhang, Chong; Lv, Fengxia

2017-01-06

LI-F type peptides (AMP-jsa9) produced by Paenibacillus polymyxa JSa-9 are a group of cyclic lipodepsipeptide antibiotics that exhibit a broad antimicrobial spectrum against Gram-positive bacteria and filamentous fungi, especially Bacillus cereus and Fusarium moniliforme. In this study, to better understand the antibacterial mechanism of AMP-jsa9 against B. cereus, the ultrastructure of AMP-jsa9-treated B. cereus cells was observed by both atomic force microscopy and transmission electron microscopy, and quantitative proteomic analysis was performed on proteins extracted from treated and untreated bacterial cells by using isobaric tag for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to access differentially expressed proteins. Furthermore, multiple experiments were conducted to validate the results of the proteomic analysis, including determinations of ATP, NAD (+) H, NADP (+) H, reactive oxygen species (ROS), the activities of catalase (CAT) and superoxide dismutase (SOD), and the relative expression of target genes by quantitative real-time PCR. Bacterial cells exposed to AMP-jsa9 showed irregular surfaces with bleb projections and concaves; we hypothesize that AMP-jsa9 penetrated the cell wall and was anchored on the cytoplasmic membrane and that ROS accumulated in the cell membrane after treatment with AMP-jsa9, modulating the bacterial membrane properties and increasing membrane permeability. Consequently, the blebs were formed on the cell wall by the impulsive force of the leakage of intercellular contents. iTRAQ-based proteomic analysis detected a total of 1317 proteins, including 176 differentially expressed proteins (75 upregulated (fold >2) and 101 downregulated (fold <0.5)). Based on proteome analysis, the putative pathways of AMP-jsa9 action against B. cereus can be summarized as: (i) inhibition of bacterial sporulation, thiamine biosynthesis, energy metabolism, DNA transcription and translation, and cell wall biosynthesis, through direct regulation of protein levels; and (ii) indirect effects on the same pathways through the accumulation of ROS and the consequent impairment of cellular functions, resulting from downregulation of antioxidant proteins, especially CAT and SOD. The mode of action of LI-F type antimicrobial peptides (AMP-jsa9) against B. cereus was elucidated at the proteomic level. Two pathways of AMP-jsa9 action upon B. cereus cells were identified and the mechanism of bleb formation on the surfaces of bacterial cells was predicted based on the results of ultrastructural observation and proteomic analysis. These results are helpful in understanding the mechanism of LI-F type peptides and in providing the theoretical base for applying AMP-jsa9 or its analogs to combat Gram-positive pathogenic bacteria in the food and feed industries. Copyright © 2016 Elsevier B.V. All rights reserved.

Droplet microfluidics--a tool for single-cell analysis.

PubMed

Joensson, Haakan N; Andersson Svahn, Helene

2012-12-03

Droplet microfluidics allows the isolation of single cells and reagents in monodisperse picoliter liquid capsules and manipulations at a throughput of thousands of droplets per second. These qualities allow many of the challenges in single-cell analysis to be overcome. Monodispersity enables quantitative control of solute concentrations, while encapsulation in droplets provides an isolated compartment for the single cell and its immediate environment. The high throughput allows the processing and analysis of the tens of thousands to millions of cells that must be analyzed to accurately describe a heterogeneous cell population so as to find rare cell types or access sufficient biological space to find hits in a directed evolution experiment. The low volumes of the droplets make very large screens economically viable. This Review gives an overview of the current state of single-cell analysis involving droplet microfluidics and offers examples where droplet microfluidics can further biological understanding. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A review of state-of-the-art stereology for better quantitative 3D morphology in cardiac research.

PubMed

Mühlfeld, Christian; Nyengaard, Jens Randel; Mayhew, Terry M

2010-01-01

The aim of stereological methods in biomedical research is to obtain quantitative information about three-dimensional (3D) features of tissues, cells, or organelles from two-dimensional physical or optical sections. With immunogold labeling, stereology can even be used for the quantitative analysis of the distribution of molecules within tissues and cells. Nowadays, a large number of design-based stereological methods offer an efficient quantitative approach to intriguing questions in cardiac research, such as "Is there a significant loss of cardiomyocytes during progression from ventricular hypertrophy to heart failure?" or "Does a specific treatment reduce the degree of fibrosis in the heart?" Nevertheless, the use of stereological methods in cardiac research is rare. The present review article demonstrates how some of the potential pitfalls in quantitative microscopy may be avoided. To this end, we outline the concepts of design-based stereology and illustrate their practical applications to a wide range of biological questions in cardiac research. We hope that the present article will stimulate researchers in cardiac research to incorporate design-based stereology into their study designs, thus promoting an unbiased quantitative 3D microscopy.

Screening circular RNA related to chemotherapeutic resistance in breast cancer.

PubMed

Gao, Danfeng; Zhang, Xiufen; Liu, Beibei; Meng, Dong; Fang, Kai; Guo, Zijian; Li, Lihua

2017-09-01

We aimed to identify circular RNAs (circRNAs) associated with breast cancer chemoresistance. CircRNA microarray expression profiles were obtained from Adriamycin (ADM) resistant MCF-7 breast cancer cells (MCF-7/ADM) and parental MCF-7 cells and were validated using quantitative real-time reverse transcription PCR. The expression data were analyzed bioinformatically. We detected 3093 circRNAs and identified 18 circRNAs that are differentially expressed between MCF-7/ADM and MCF-7 cells; after validating by quantitative real-time reverse transcription PCR, we predicted the possible miRNAs and potential target genes of the seven upregulated circRNAs using TargetScan and miRanda. The bioinformatics analysis revealed several target genes related to cancer-related signaling pathways. Additionally, we discovered a regulatory role of the circ_0006528-miR-7-5p-Raf1 axis in ADM-resistant breast cancer. These results revealed that circRNAs may play a role in breast cancer chemoresistance and that hsa_circ_0006528 might be a promising candidate for further functional analysis.

Identification of Salmonella Typhimurium deubiquitinase SseL substrates by immunoaffinity enrichment and quantitative proteomic analysis

DOE PAGES

Nakayasu, Ernesto S.; Sydor, Michael A.; Brown, Roslyn N.; ...

2015-07-06

Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification and global quantitative proteomic analysis. As model system to identify substrates, we used a virulence-related deubiquitinase secreted by Salmonella enterica serovar Typhimurium into the host cells, SseL. By using this approach two SseL substrates were identified in RAW 264.7 murine macrophage-like cell line, S100A6 and het-erogeneous nuclear ribonuclear protein K, inmore » addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. Finally, this method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.« less

Identification of Salmonella Typhimurium deubiquitinase SseL substrates by immunoaffinity enrichment and quantitative proteomic analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakayasu, Ernesto S.; Sydor, Michael A.; Brown, Roslyn N.

Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification and global quantitative proteomic analysis. As model system to identify substrates, we used a virulence-related deubiquitinase secreted by Salmonella enterica serovar Typhimurium into the host cells, SseL. By using this approach two SseL substrates were identified in RAW 264.7 murine macrophage-like cell line, S100A6 and het-erogeneous nuclear ribonuclear protein K, inmore » addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. Finally, this method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.« less

Large-scale label-free quantitative proteomics of the pea aphid-Buchnera symbiosis.

PubMed

Poliakov, Anton; Russell, Calum W; Ponnala, Lalit; Hoops, Harold J; Sun, Qi; Douglas, Angela E; van Wijk, Klaas J

2011-06-01

Many insects are nutritionally dependent on symbiotic microorganisms that have tiny genomes and are housed in specialized host cells called bacteriocytes. The obligate symbiosis between the pea aphid Acyrthosiphon pisum and the γ-proteobacterium Buchnera aphidicola (only 584 predicted proteins) is particularly amenable for molecular analysis because the genomes of both partners have been sequenced. To better define the symbiotic relationship between this aphid and Buchnera, we used large-scale, high accuracy tandem mass spectrometry (nanoLC-LTQ-Orbtrap) to identify aphid and Buchnera proteins in the whole aphid body, purified bacteriocytes, isolated Buchnera cells and the residual bacteriocyte fraction. More than 1900 aphid and 400 Buchnera proteins were identified. All enzymes in amino acid metabolism annotated in the Buchnera genome were detected, reflecting the high (68%) coverage of the proteome and supporting the core function of Buchnera in the aphid symbiosis. Transporters mediating the transport of predicted metabolites were present in the bacteriocyte. Label-free spectral counting combined with hierarchical clustering, allowed to define the quantitative distribution of a subset of these proteins across both symbiotic partners, yielding no evidence for the selective transfer of protein among the partners in either direction. This is the first quantitative proteome analysis of bacteriocyte symbiosis, providing a wealth of information about molecular function of both the host cell and bacterial symbiont.

Quantifying Low Energy Proton Damage in Multijunction Solar Cells

NASA Technical Reports Server (NTRS)

Messenger, Scott R.; Burke, Edward A.; Walters, Robert J.; Warner, Jeffrey H.; Summers, Geoffrey P.; Lorentzen, Justin R.; Morton, Thomas L.; Taylor, Steven J.

2007-01-01

An analysis of the effects of low energy proton irradiation on the electrical performance of triple junction (3J) InGaP2/GaAs/Ge solar cells is presented. The Monte Carlo ion transport code (SRIM) is used to simulate the damage profile induced in a 3J solar cell under the conditions of typical ground testing and that of the space environment. The results are used to present a quantitative analysis of the defect, and hence damage, distribution induced in the cell active region by the different radiation conditions. The modelling results show that, in the space environment, the solar cell will experience a uniform damage distribution through the active region of the cell. Through an application of the displacement damage dose analysis methodology, the implications of this result on mission performance predictions are investigated.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions.

PubMed

Wolters, Manuel; Zobiak, Bernd; Nauth, Theresa; Aepfelbacher, Martin

2015-10-13

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Functional Module Search in Protein Networks based on Semantic Similarity Improves the Analysis of Proteomics Data*

PubMed Central

Boyanova, Desislava; Nilla, Santosh; Klau, Gunnar W.; Dandekar, Thomas; Müller, Tobias; Dittrich, Marcus

2014-01-01

The continuously evolving field of proteomics produces increasing amounts of data while improving the quality of protein identifications. Albeit quantitative measurements are becoming more popular, many proteomic studies are still based on non-quantitative methods for protein identification. These studies result in potentially large sets of identified proteins, where the biological interpretation of proteins can be challenging. Systems biology develops innovative network-based methods, which allow an integrated analysis of these data. Here we present a novel approach, which combines prior knowledge of protein-protein interactions (PPI) with proteomics data using functional similarity measurements of interacting proteins. This integrated network analysis exactly identifies network modules with a maximal consistent functional similarity reflecting biological processes of the investigated cells. We validated our approach on small (H9N2 virus-infected gastric cells) and large (blood constituents) proteomic data sets. Using this novel algorithm, we identified characteristic functional modules in virus-infected cells, comprising key signaling proteins (e.g. the stress-related kinase RAF1) and demonstrate that this method allows a module-based functional characterization of cell types. Analysis of a large proteome data set of blood constituents resulted in clear separation of blood cells according to their developmental origin. A detailed investigation of the T-cell proteome further illustrates how the algorithm partitions large networks into functional subnetworks each representing specific cellular functions. These results demonstrate that the integrated network approach not only allows a detailed analysis of proteome networks but also yields a functional decomposition of complex proteomic data sets and thereby provides deeper insights into the underlying cellular processes of the investigated system. PMID:24807868

AI-augmented time stretch microscopy

NASA Astrophysics Data System (ADS)

Mahjoubfar, Ata; Chen, Claire L.; Lin, Jiahao; Jalali, Bahram

2017-02-01

Cell reagents used in biomedical analysis often change behavior of the cells that they are attached to, inhibiting their native signaling. On the other hand, label-free cell analysis techniques have long been viewed as challenging either due to insufficient accuracy by limited features, or because of low throughput as a sacrifice of improved precision. We present a recently developed artificial-intelligence augmented microscope, which builds upon high-throughput time stretch quantitative phase imaging (TS-QPI) and deep learning to perform label-free cell classification with record high-accuracy. Our system captures quantitative optical phase and intensity images simultaneously by frequency multiplexing, extracts multiple biophysical features of the individual cells from these images fused, and feeds these features into a supervised machine learning model for classification. The enhanced performance of our system compared to other label-free assays is demonstrated by classification of white blood T-cells versus colon cancer cells and lipid accumulating algal strains for biofuel production, which is as much as five-fold reduction in inaccuracy. This system obtains the accuracy required in practical applications such as personalized drug development, while the cells remain intact and the throughput is not sacrificed. Here, we introduce a data acquisition scheme based on quadrature phase demodulation that enables interruptionless storage of TS-QPI cell images. Our proof of principle demonstration is capable of saving 40 TB of cell images in about four hours, i.e. pictures of every single cell in 10 mL of a sample.

Optical computed tomography for spatially isotropic four-dimensional imaging of live single cells

PubMed Central

Kelbauskas, Laimonas; Shetty, Rishabh; Cao, Bin; Wang, Kuo-Chen; Smith, Dean; Wang, Hong; Chao, Shi-Hui; Gangaraju, Sandhya; Ashcroft, Brian; Kritzer, Margaret; Glenn, Honor; Johnson, Roger H.; Meldrum, Deirdre R.

2017-01-01

Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field. PMID:29226240

Quantitative analysis of osteoblast behavior on microgrooved hydroxyapatite and titanium substrata.

PubMed

Lu, Xiong; Leng, Yang

2003-09-01

The effects of implant surface topography and chemistry on osteoblast behavior have been a research focus because of their potential importance in orthopedic and dental applications. This work focused on the topographic effects of hydroxyapatite (HA) and titanium (Ti) surface that had identical micropatterns to determine whether there was synergistic interaction between surface chemistry and surface topography. Surface microgrooves with six different groove widths (4, 8, 16, 24, 30, and 38 microm) and three different groove depths (2, 4, and 10 microm) were made on single crystalline silicon wafers using microfabrication techniques. Ti and HA thin films were coated on the microgrooves by radio-frequency magnetron sputtering. After that, human osteoblast-like cells were seeded and cultured on the microgrooved surfaces for up to 7 days. The cells' behavior was examined using scanning electron microscopy after cells were fixed and dehydrated. Statistical analysis was based on quantitative data of orientation angle, evaluating the contact guidance, and form index, describing cell shape or cell morphology changes. The contact guidance and cell shape changes were observed on the HA and Ti microgrooves. No difference in orientation angle between HA and Ti microgrooves was found. This might suggest that surface chemistry was not a significant influence on cell guidance. However, the form index analysis indicated an interaction between topographic effects and surface chemistry. Thus, conclusions about surface topographic effects on cell behavior drawn from one type of material cannot simply be applied to another type of material. Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 677-687, 2003

Antitumor effect of CXCR4 antagonist AMD3100 on the tumorigenic cell line of BHP10-3 papillary thyroid cancer cells.

PubMed

Jung, Young Ho; Lee, Doh Young; Cha, Wonjae; Kim, Bo Hae; Sung, Myung-Whun; Kim, Kwang Hyun; Ahn, Soon-Hyun

2016-10-01

A tumorigenic cell line (BHP10-3M) derived from nontumorigenic papillary thyroid carcinoma (PTC) cells (BHP10-3) having rearranged during transfection (RET)/PTC1 gene rearrangement might have a higher expression of CXCR4, either quantitatively or functionally. The authors also postulated that CXCR4-mediated invasion or tumorigenesis could be blocked by CXCR4 antagonists, including AMD3100. The expression of CXCR4 in BHP10-3 and BHP10-3M cells was assessed using immunoblot analysis, flow cytometry, and quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The effect of AMD3100 on BHP10-3 and BHP10-3M cell lines was evaluated using cell proliferation assay, invasion assay, and tumor growth experiment in nude mice. Immunoblotting, flow cytometry, and quantitative RT-PCR proved that BHP10-3M cells expressed a higher level of CXCR4 than BHP10-3 cells. Although blocking CXCR4 with AMD3100 did not suppress cell proliferation in both cell lines from 1 ng/mL to 100 ng/mL concentration, AMD3100 suppressed invasion of BHP10-3M cells in vitro in a dose-dependent manner. At higher concentrations from 10(3) ng/mL to 10(5) ng/mL, the proliferation of BHP10-3M cells was inhibited more strongly by AMD3100 than that of BHP10-3 cells. Intraperitoneal injection of AMD3100 inhibited tumor formation by BHP10-3M cells in the thyroid of nude mice. A tumorigenic cell line (BHP10-3M) of PTC showed higher expression of CXCR4 quantitatively and functionally than a nontumorigenic cell line (BHP10-3). The CXCR4 antagonist (AMD3100) showed a significant antitumor effect on the tumorigenic cell line of PTC BHP10-3 cells both in vitro and in vivo. CXCR4 antagonist can be expected to have an adjuvant role in the management of PTC. © 2016 Wiley Periodicals, Inc. Head Neck, 2016 © 2016 Wiley Periodicals, Inc. Head Neck 38: First-1486, 2016. © 2016 Wiley Periodicals, Inc.

Quantitative analysis of phosphoinositide 3-kinase (PI3K) signaling using live-cell total internal reflection fluorescence (TIRF) microscopy.

PubMed

Johnson, Heath E; Haugh, Jason M

2013-12-02

This unit focuses on the use of total internal reflection fluorescence (TIRF) microscopy and image analysis methods to study the dynamics of signal transduction mediated by class I phosphoinositide 3-kinases (PI3Ks) in mammalian cells. The first four protocols cover live-cell imaging experiments, image acquisition parameters, and basic image processing and segmentation. These methods are generally applicable to live-cell TIRF experiments. The remaining protocols outline more advanced image analysis methods, which were developed in our laboratory for the purpose of characterizing the spatiotemporal dynamics of PI3K signaling. These methods may be extended to analyze other cellular processes monitored using fluorescent biosensors. Copyright © 2013 John Wiley & Sons, Inc.

Systems cell biology

PubMed Central

Mast, Fred D.; Ratushny, Alexander V.

2014-01-01

Systems cell biology melds high-throughput experimentation with quantitative analysis and modeling to understand many critical processes that contribute to cellular organization and dynamics. Recently, there have been several advances in technology and in the application of modeling approaches that enable the exploration of the dynamic properties of cells. Merging technology and computation offers an opportunity to objectively address unsolved cellular mechanisms, and has revealed emergent properties and helped to gain a more comprehensive and fundamental understanding of cell biology. PMID:25225336

Systems Imaging of the Immune Synapse.

PubMed

Ambler, Rachel; Ruan, Xiangtao; Murphy, Robert F; Wülfing, Christoph

2017-01-01

Three-dimensional live cell imaging of the interaction of T cells with antigen-presenting cells (APCs) visualizes the subcellular distributions of signaling intermediates during T cell activation at thousands of resolved positions within a cell. These information-rich maps of local protein concentrations are a valuable resource in understanding T cell signaling. Here, we describe a protocol for the efficient acquisition of such imaging data and their computational processing to create four-dimensional maps of local concentrations. This protocol allows quantitative analysis of T cell signaling as it occurs inside live cells with resolution in time and space across thousands of cells.

Seasonal development of cambial activity in relation to xylem formation in Chinese fir.

PubMed

Wu, Hongyang; Xu, Huimin; Li, Hanyin; Wei, Dongmei; Lin, Jinxing; Li, Xiaojuan

2016-05-20

The vascular cambium is a lateral meristem which can differentiate into secondary phloem and xylem. The secondary growth of woody plants resulting from vascular cambium activity has been a focus of considerable attention, but the quantitative relationships between cambial activity and secondary xylem formation have been little studied. Our analysis of cytological changes in the cambium of Chinese fir (Cunninghamia lanceolata), revealed a significant positive correlation between vascular cambium cell numbers and cambium zone width through the seasonal cycle. Cambium cell numbers and the cambium cell radial diameter were closely related to xylem formation. Immuno-labeling showed that de-esterified homogalacturonan and (1-4)-β-d-galactan epitopes were highly abundant in cell walls of dormant-stage cambium, whereas high methylesterified homogalacturonan was strongly labeled in the active stage. Raman spectroscopy detected significant changes in the chemical composition of cell walls during the active-dormant stage transition. More pectin and less monolignols occurred in radial cell walls than in tangential walls during the dormant stage, but no significant changes were found in other stages, indicating that pectin accumulation facilitates cell wall expansion, with cambium activity transition. Our quantitative analysis of the relationship between cambial activity and xylem formation, as well as the cell wall modification during the active stage provides useful information about cambial characteristics and xylogenesis. Copyright © 2016. Published by Elsevier GmbH.

Substance P Promotes the Progression of Endometrial Adenocarcinoma.

PubMed

Ma, Jing; Yuan, Shifa; Cheng, Jianxin; Kang, Shan; Zhao, Wenhong; Zhang, Jie

2016-06-01

It has been demonstrated that substance P (SP) promotes while neurokinin-1 receptor (NK-1R) antagonist inhibits the proliferation of several human cancer cells. Currently, it is still unknown whether such actions exist in human endometrial carcinoma. This study aimed to explore the role of SP/NK-1R signaling in the progression of endometrial adenocarcinoma. The expression levels of SP and NK-1R in endometrial adenocarcinoma tissues and Ishikawa cell line were detected by real-time quantitative PCR and Western blot analysis. The effects of SP on Ishikawa cells proliferation and invasion were analyzed using MTT assay and transwell matrigel invasion assay, respectively. The expression levels of matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor C (VEGF-C) in Ishikawa cells after administration of SP were detected by real-time quantitative RCR and Western blot analysis. The expression levels of SP and NK-1R were significantly higher in endometrial adenocarcinoma tissues and Ishikawa cells than in normal endometrium. Substance P significantly enhanced the proliferation and invasion of Ishikawa cells. In addition, SP induced the expression of MMP-9 and VEGF-C in Ishikawa cells, whereas NK-1R antagonist inhibited these effects. Substance P plays an important role in the development of endometrial carcinoma by inducing the expression of MMP-9 and VEGF-C and promoting cancer cell proliferation and metastasis, which can be blocked by NK-1R antagonist.

Quantitative image analysis of cellular heterogeneity in breast tumors complements genomic profiling.

PubMed

Yuan, Yinyin; Failmezger, Henrik; Rueda, Oscar M; Ali, H Raza; Gräf, Stefan; Chin, Suet-Feung; Schwarz, Roland F; Curtis, Christina; Dunning, Mark J; Bardwell, Helen; Johnson, Nicola; Doyle, Sarah; Turashvili, Gulisa; Provenzano, Elena; Aparicio, Sam; Caldas, Carlos; Markowetz, Florian

2012-10-24

Solid tumors are heterogeneous tissues composed of a mixture of cancer and normal cells, which complicates the interpretation of their molecular profiles. Furthermore, tissue architecture is generally not reflected in molecular assays, rendering this rich information underused. To address these challenges, we developed a computational approach based on standard hematoxylin and eosin-stained tissue sections and demonstrated its power in a discovery and validation cohort of 323 and 241 breast tumors, respectively. To deconvolute cellular heterogeneity and detect subtle genomic aberrations, we introduced an algorithm based on tumor cellularity to increase the comparability of copy number profiles between samples. We next devised a predictor for survival in estrogen receptor-negative breast cancer that integrated both image-based and gene expression analyses and significantly outperformed classifiers that use single data types, such as microarray expression signatures. Image processing also allowed us to describe and validate an independent prognostic factor based on quantitative analysis of spatial patterns between stromal cells, which are not detectable by molecular assays. Our quantitative, image-based method could benefit any large-scale cancer study by refining and complementing molecular assays of tumor samples.

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues.

PubMed

Molenaar, Remco J; Khurshed, Mohammed; Hira, Vashendriya V V; Van Noorden, Cornelis J F

2018-05-26

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation. We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P) + and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase's activity. The formazan's absorbance is therefore a direct measure of the dehydrogenase's activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays. Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Building quantitative, three-dimensional atlases of gene expression and morphology at cellular resolution.

PubMed

Knowles, David W; Biggin, Mark D

2013-01-01

Animals comprise dynamic three-dimensional arrays of cells that express gene products in intricate spatial and temporal patterns that determine cellular differentiation and morphogenesis. A rigorous understanding of these developmental processes requires automated methods that quantitatively record and analyze complex morphologies and their associated patterns of gene expression at cellular resolution. Here we summarize light microscopy-based approaches to establish permanent, quantitative datasets-atlases-that record this information. We focus on experiments that capture data for whole embryos or large areas of tissue in three dimensions, often at multiple time points. We compare and contrast the advantages and limitations of different methods and highlight some of the discoveries made. We emphasize the need for interdisciplinary collaborations and integrated experimental pipelines that link sample preparation, image acquisition, image analysis, database design, visualization, and quantitative analysis. Copyright © 2013 Wiley Periodicals, Inc.

Phenotypic analysis of prostate-infiltrating lymphocytes reveals TH17 and Treg skewing.

PubMed

Sfanos, Karen Sandell; Bruno, Tullia C; Maris, Charles H; Xu, Lauren; Thoburn, Christopher J; DeMarzo, Angelo M; Meeker, Alan K; Isaacs, William B; Drake, Charles G

2008-06-01

Pathologic examination of prostate glands removed from patients with prostate cancer commonly reveals infiltrating CD4+ and CD8+ T cells. Little is known about the phenotype of these cells, despite accumulating evidence suggesting a potential role for chronic inflammation in the etiology of prostate cancer. We developed a technique that samples the majority of the peripheral prostate through serial needle aspirates. CD4+ prostate-infiltrating lymphocytes (PIL) were isolated using magnetic beads and analyzed for subset skewing using both flow cytometry and quantitative reverse transcription-PCR. The transcriptional profile of fluorescence-activated cell sorted prostate-infiltrating regulatory T cells (CD4+, CD25+, GITR+) was compared with naïve, peripheral blood T cells using microarray analysis. CD4+ PIL showed a paucity of TH2 (interleukin-4-secreting) cells, a surprising finding given the generally accepted association of these cells with chronic, smoldering inflammation. Instead, CD4+ PIL seemed to be skewed towards a regulatory Treg phenotype (FoxP3+) as well as towards the TH17 phenotype (interleukin-17+). We also found that a preponderance of TH17-mediated inflammation was associated with a lower pathologic Gleason score. These protein level data were reflected at the message level, as analyzed by quantitative reverse transcription-PCR. Microarray analysis of pooled prostate-infiltrating T(reg) revealed expected Treg-associated transcripts (FoxP3, CTLA-4, GITR, LAG-3) as well as a number of unique cell surface markers that may serve as additional Treg markers. Taken together, these data suggest that TH17 and/or Treg CD4+ T cells (rather than TH2 T cells) may be involved in the development or progression of prostate cancer.

Phenotypic Analysis of Prostate-Infiltrating Lymphocytes Reveals TH17 and Treg Skewing

PubMed Central

Sfanos, Karen Sandell; Bruno, Tullia C.; Maris, Charles H.; Xu, Lauren; Thoburn, Christopher J.; DeMarzo, Angelo M.; Meeker, Alan K.; Isaacs, William B.; Drake, Charles G.

2011-01-01

Purpose Pathologic examination of prostate glands removed from patients with prostate cancer commonly reveals infiltrating CD4+ and CD8+ T cells. Little is known about the phenotype of these cells, despite accumulating evidence suggesting a potential role for chronic inflammation in the etiology of prostate cancer. Experimental Design We developed a technique that samples the majority of the peripheral prostate through serial needle aspirates. CD4+ prostate-infiltrating lymphocytes (PIL) were isolated using magnetic beads and analyzed for subset skewing using both flow cytometry and quantitative reverse transcription-PCR. The transcriptional profile of fluorescence-activated cell sorted prostate-infiltrating regulatory T cells (CD4+, CD25+, GITR+) was compared with naïve, peripheral blood T cells using microarray analysis. Results CD4+ PIL showed a paucity of TH2 (interleukin-4– secreting) cells, a surprising finding given the generally accepted association of these cells with chronic, smoldering inflammation. Instead, CD4+ PIL seemed to be skewed towards a regulatory Treg phenotype (FoxP3+) as well as towards the TH17 phenotype (interleukin-17+). We also found that a preponderance of TH17-mediated inflammation was associated with a lower pathologic Gleason score. These protein level data were reflected at the message level, as analyzed by quantitative reverse transcription-PCR. Microarray analysis of pooled prostate-infiltrating Treg revealed expected Treg-associated transcripts (FoxP3, CTLA-4, GITR, LAG-3) as well as a number of unique cell surface markers that may serve as additional Treg markers. Conclusion Taken together, these data suggest that TH17 and/or Treg CD4+ T cells (rather than TH2 T cells) may be involved in the development or progression of prostate cancer. PMID:18519750

MCM - 2 and Ki - 67 as proliferation markers in renal cell carcinoma: A quantitative and semi - quantitative analysis

PubMed Central

Mehdi, Muhammad Zain; Nagi, Abdul Hanan; Naseem, Nadia

2016-01-01

ABSTRACT Introduction/Background: Fuhrman nuclear grade is the most important histological parameter to predict prognosis in a patient of renal cell carcinoma (RCC). However, it suffers from inter-observer and intra-observer variation giving rise to need of a parameter that not only correlates with nuclear grade but is also objective and reproducible. Proliferation is the measure of aggressiveness of a tumour and it is strongly correlated with Fuhrman nuclear grade, clinical survival and recurrence in RCC. Ki-67 is conventionally used to assess proliferation. Mini-chromosome maintenance 2 (MCM-2) is a lesser known marker of proliferation and identifies a greater proliferation faction. This study was designed to assess the prognostic significance of MCM-2 by comparing it with Fuhrman nuclear grade and Ki-67. Material and Methods: n=50 cases of various ages, stages, histological subtypes and grades of RCC were selected for this study. Immunohistochemical staining using Ki-67(MIB-1, Mouse monoclonal antibody, Dako) and MCM-2 (Mouse monoclonal antibody, Thermo) was performed on the paraffin embedded blocks in the department of Morbid anatomy and Histopathology, University of Health Sciences, Lahore. Labeling indices (LI) were determined by two pathologists independently using quantitative and semi-quantitative analysis. Statistical analysis was carried out using SPSS 20.0. Kruskall-Wallis test was used to determine a correlation of proliferation markers with grade, and Pearson's correlate was used to determine correlation between the two proliferation markers. Results: Labeling index of MCM-2 (median=24.29%) was found to be much higher than Ki-67(median=13.05%). Both markers were significantly related with grade (p=0.00; Kruskall-Wallis test). LI of MCM-2 was found to correlate significantly with LI of Ki-67(r=0.0934;p=0.01 with Pearson's correlate). Results of semi-quantitative analysis correlated well with quantitative analysis. Conclusion: Both Ki-67 and MCM-2 are markers of proliferation which are closely linked to grade. Therefore, they can act as surrogate markers for grade in a manner that is more objective and reproducible. PMID:27532114

MCM - 2 and Ki - 67 as proliferation markers in renal cell carcinoma: A quantitative and semi - quantitative analysis.

PubMed

Mehdi, Muhammad Zain; Nagi, Abdul Hanan; Naseem, Nadia

2016-01-01

Fuhrman nuclear grade is the most important histological parameter to predict prognosis in a patient of renal cell carcinoma (RCC). However, it suffers from inter-observer and intra-observer variation giving rise to need of a parameter that not only correlates with nuclear grade but is also objective and reproducible. Proliferation is the measure of aggressiveness of a tumour and it is strongly correlated with Fuhrman nuclear grade, clinical survival and recurrence in RCC. Ki-67 is conventionally used to assess proliferation. Mini-chromosome maintenance 2 (MCM-2) is a lesser known marker of proliferation and identifies a greater proliferation faction. This study was designed to assess the prognostic significance of MCM-2 by comparing it with Fuhrman nuclear grade and Ki-67. n=50 cases of various ages, stages, histological subtypes and grades of RCC were selected for this study. Immunohistochemical staining using Ki-67(MIB-1, Mouse monoclonal antibody, Dako) and MCM-2 (Mouse monoclonal antibody, Thermo) was performed on the paraffin embedded blocks in the department of Morbid anatomy and Histopathology, University of Health Sciences, Lahore. Labeling indices (LI) were determined by two pathologists independently using quantitative and semi-quantitative analysis. Statistical analysis was carried out using SPSS 20.0. Kruskall-Wallis test was used to determine a correlation of proliferation markers with grade, and Pearson's correlate was used to determine correlation between the two proliferation markers. Labeling index of MCM-2 (median=24.29%) was found to be much higher than Ki-67(median=13.05%). Both markers were significantly related with grade (p=0.00; Kruskall-Wallis test). LI of MCM-2 was found to correlate significantly with LI of Ki-67(r=0.0934;p=0.01 with Pearson's correlate). Results of semi-quantitative analysis correlated well with quantitative analysis. Both Ki-67 and MCM-2 are markers of proliferation which are closely linked to grade. Therefore, they can act as surrogate markers for grade in a manner that is more objective and reproducible. Copyright® by the International Brazilian Journal of Urology.

The Human Respiratory Syncytial Virus Nonstructural Protein 1 Regulates Type I and Type II Interferon Pathways*

PubMed Central

Hastie, Marcus L.; Headlam, Madeleine J.; Patel, Nirav B.; Bukreyev, Alexander A.; Buchholz, Ursula J.; Dave, Keyur A.; Norris, Emma L.; Wright, Cassandra L.; Spann, Kirsten M.; Collins, Peter L.; Gorman, Jeffrey J.

2012-01-01

Respiratory syncytial viruses encode a nonstructural protein (NS1) that interferes with type I and III interferon and other antiviral responses. Proteomic studies were conducted on human A549 type II alveolar epithelial cells and type I interferon-deficient Vero cells (African green monkey kidney cells) infected with wild-type and NS1-deficient clones of human respiratory syncytial virus to identify other potential pathway and molecular targets of NS1 interference. These analyses included two-dimensional differential gel electrophoresis and quantitative Western blotting. Surprisingly, NS1 was found to suppress the induction of manganese superoxide dismutase (SOD2) expression in A549 cells and to a much lesser degree Vero cells in response to infection. Because SOD2 is not directly inducible by type I interferons, it served as a marker to probe the impact of NS1 on signaling of other cytokines known to induce SOD2 expression and/or indirect effects of type I interferon signaling. Deductive analysis of results obtained from cell infection and cytokine stimulation studies indicated that interferon-γ signaling was a potential target of NS1, possibly as a result of modulation of STAT1 levels. However, this was not sufficient to explain the magnitude of the impact of NS1 on SOD2 induction in A549 cells. Vero cell infection experiments indicated that NS1 targeted a component of the type I interferon response that does not directly induce SOD2 expression but is required to induce another initiator of SOD2 expression. STAT2 was ruled out as a target of NS1 interference using quantitative Western blot analysis of infected A549 cells, but data were obtained to indicate that STAT1 was one of a number of potential targets of NS1. A label-free mass spectrometry-based quantitative approach is proposed as a means of more definitive identification of NS1 targets. PMID:22322095

Protein content and functional characteristics of serum-purified exosomes from patients with colorectal cancer revealed by quantitative proteomics.

PubMed

Chen, Yanyu; Xie, Yong; Xu, Lai; Zhan, Shaohua; Xiao, Yi; Gao, Yanpan; Wu, Bin; Ge, Wei

2017-02-15

Tumor cells of colorectal cancer (CRC) release exosomes into the circulation. These exosomes can mediate communication between cells and affect various tumor-related processes in their target cells. We present a quantitative proteomics analysis of the exosomes purified from serum of patients with CRC and normal volunteers; data are available via ProteomeXchange with identifier PXD003875. We identified 918 proteins with an overlap of 725 Gene IDs in the Exocarta proteins list. Compared with the serum-purified exosomes (SPEs) of normal volunteers, we found 36 proteins upregulated and 22 proteins downregulated in the SPEs of CRC patients. Bioinformatics analysis revealed that upregulated proteins are involved in processes that modulate the pretumorigenic microenvironment for metastasis. In contrast, differentially expressed proteins (DEPs) that play critical roles in tumor growth and cell survival were principally downregulated. Our study demonstrates that SPEs of CRC patients play a pivotal role in promoting the tumor invasiveness, but have minimal influence on putative alterations in tumor survival or proliferation. According to bioinformatics analysis, we speculate that the protein contents of exosomes might be associated with whether they are involved in premetastatic niche establishment or growth and survival of metastatic tumor cells. This information will be helpful in elucidating the pathophysiological functions of tumor-derived exosomes, and aid in the development of CRC diagnostics and therapeutics. © 2016 UICC.

Automated quantitative cytological analysis using portable microfluidic microscopy.

PubMed

Jagannadh, Veerendra Kalyan; Murthy, Rashmi Sreeramachandra; Srinivasan, Rajesh; Gorthi, Sai Siva

2016-06-01

In this article, a portable microfluidic microscopy based approach for automated cytological investigations is presented. Inexpensive optical and electronic components have been used to construct a simple microfluidic microscopy system. In contrast to the conventional slide-based methods, the presented method employs microfluidics to enable automated sample handling and image acquisition. The approach involves the use of simple in-suspension staining and automated image acquisition to enable quantitative cytological analysis of samples. The applicability of the presented approach to research in cellular biology is shown by performing an automated cell viability assessment on a given population of yeast cells. Further, the relevance of the presented approach to clinical diagnosis and prognosis has been demonstrated by performing detection and differential assessment of malaria infection in a given sample. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of molecular assemblies by flow cytometry: determinants of Gi1 and by binding

NASA Astrophysics Data System (ADS)

Sarvazyan, Noune A.; Neubig, Richard R.

1998-05-01

We report here a novel application of flow cytometry for the quantitative analysis of the high affinity interaction between membrane proteins both in detergent solutions and when reconstituted into lipid vesicles. The approach is further advanced to permit the analysis of binding to expressed protein complexes in native cell membranes. The G protein heterotrimer signal transduction function links the extracellularly activated transmembrane receptors and intracellular effectors. Upon activation, (alpha) and (beta) (gamma) subunits of G protein undergo a dissociation/association cycle on the cell membrane interface. The binding parameters of solubilized G protein (alpha) and (beta) (gamma) subunits have been defined but little is known quantitatively about their interactions in the membrane. Using a novel flow cytometry approach, the binding of low nanomolar concentrations of fluorescein-labeled G(alpha) i1 (F- (alpha) ) to (beta) (gamma) both in detergent solution and in a lipid environment was quantitatively compared. Unlabeled (beta) $gama reconstituted in biotinylated phospholipid vesicles bound F-(alpha) tightly (Kd 6 - 12 nM) while the affinity for biotinylated-(beta) (gamma) in Lubrol was even higher (Kd of 2.9 nM). The application of this approach to proteins expressed in native cell membranes will advance our understanding of G protein function in context of receptor and effector interaction. More generally, this approach can be applied to study the interaction of any fluorescently labeled protein with a membrane protein which can be expressed in Sf9 plasma membranes.

Connecting Neuronal Cell Protective Pathways and Drug Combinations in a Huntington's Disease Model through the Application of Quantitative Systems Pharmacology.

PubMed

Pei, Fen; Li, Hongchun; Henderson, Mark J; Titus, Steven A; Jadhav, Ajit; Simeonov, Anton; Cobanoglu, Murat Can; Mousavi, Seyed H; Shun, Tongying; McDermott, Lee; Iyer, Prema; Fioravanti, Michael; Carlisle, Diane; Friedlander, Robert M; Bahar, Ivet; Taylor, D Lansing; Lezon, Timothy R; Stern, Andrew M; Schurdak, Mark E

2017-12-19

Quantitative Systems Pharmacology (QSP) is a drug discovery approach that integrates computational and experimental methods in an iterative way to gain a comprehensive, unbiased understanding of disease processes to inform effective therapeutic strategies. We report the implementation of QSP to Huntington's Disease, with the application of a chemogenomics platform to identify strategies to protect neuronal cells from mutant huntingtin induced death. Using the STHdh Q111 cell model, we investigated the protective effects of small molecule probes having diverse canonical modes-of-action to infer pathways of neuronal cell protection connected to drug mechanism. Several mechanistically diverse protective probes were identified, most of which showed less than 50% efficacy. Specific combinations of these probes were synergistic in enhancing efficacy. Computational analysis of these probes revealed a convergence of pathways indicating activation of PKA. Analysis of phospho-PKA levels showed lower cytoplasmic levels in STHdh Q111 cells compared to wild type STHdh Q7 cells, and these levels were increased by several of the protective compounds. Pharmacological inhibition of PKA activity reduced protection supporting the hypothesis that protection may be working, in part, through activation of the PKA network. The systems-level studies described here can be broadly applied to any discovery strategy involving small molecule modulation of disease phenotype.

Quantitative proteomic analysis of the chemolithoautotrophic bacterium Nitrosomonas europaea: comparison of growing- and energy-starved cells.

PubMed

Pellitteri-Hahn, Molly C; Halligan, Brian D; Scalf, Mark; Smith, Lloyd; Hickey, William J

2011-04-01

Obligately aerobic ammonia-oxidizing bacteria (AOB) like Nitrosomonas europaea play a pivotal role in the global nitrogen cycle. Although starvation tolerance is a key environmental adaptation, little is known about this response in AOB. The goal of these studies was to compare the composition of the N. europaea proteome in growing- and energy-starved cells using ¹⁵N labeling and HPLC-ESI-MS/MS. More than 6500 peptides were sequenced with high confidence, and matched to 876 proteins (34% of the protein coding genes). Of these, 126 proteins had two or more peptide forms identified by 10 or more scans, and were used in quantitative analysis and 27 were found to be significantly different in abundance between growing and starved cells. Proteins showing greater abundance in growing cells are geared toward biosynthesis, particularly DNA replication. Energy-starved cells were shifted away from biosynthesis and toward survival functions that included: cell envelope modification, protein protection/degradation, detoxification, and implementation of alternative energy generation mechanisms. Most of these activities have not previously been reported as associated with energy-starvation stress in N. europaea. This study provides insights into the potential effects of fluctuating environmental conditions on the regulation of physiological networks in N. europaea. Copyright © 2010 Elsevier B.V. All rights reserved.

A Quantitative Three-Dimensional Image Analysis Tool for Maximal Acquisition of Spatial Heterogeneity Data.

PubMed

Allenby, Mark C; Misener, Ruth; Panoskaltsis, Nicki; Mantalaris, Athanasios

2017-02-01

Three-dimensional (3D) imaging techniques provide spatial insight into environmental and cellular interactions and are implemented in various fields, including tissue engineering, but have been restricted by limited quantification tools that misrepresent or underutilize the cellular phenomena captured. This study develops image postprocessing algorithms pairing complex Euclidean metrics with Monte Carlo simulations to quantitatively assess cell and microenvironment spatial distributions while utilizing, for the first time, the entire 3D image captured. Although current methods only analyze a central fraction of presented confocal microscopy images, the proposed algorithms can utilize 210% more cells to calculate 3D spatial distributions that can span a 23-fold longer distance. These algorithms seek to leverage the high sample cost of 3D tissue imaging techniques by extracting maximal quantitative data throughout the captured image.

COMPARISON OF GENETIC METHODS TO OPTICAL METHODS IN THE IDENTIFICATION AND ASSESSMENT OF MOLD IN THE BUILT ENVIRONMENT -- COMPARISON OF TAQMAN AND MICROSCOPIC ANALYSIS OF CLADOSPORIUM SPORES RETRIEVED FROM ZEFON AIR-O-CELL TRACES

EPA Science Inventory

Recent advances in the sequencing of relevant water intrusion fungi by the EPA, combined with the development of probes and primers have allowed for the unequivocal quantitative and qualitative identification of fungi in selected matrices.

In this pilot study, quantitative...

Cell Specific eQTL Analysis without Sorting Cells

PubMed Central

Esko, Tõnu; Peters, Marjolein J.; Schurmann, Claudia; Schramm, Katharina; Kettunen, Johannes; Yaghootkar, Hanieh; Fairfax, Benjamin P.; Andiappan, Anand Kumar; Li, Yang; Fu, Jingyuan; Karjalainen, Juha; Platteel, Mathieu; Visschedijk, Marijn; Weersma, Rinse K.; Kasela, Silva; Milani, Lili; Tserel, Liina; Peterson, Pärt; Reinmaa, Eva; Hofman, Albert; Uitterlinden, André G.; Rivadeneira, Fernando; Homuth, Georg; Petersmann, Astrid; Lorbeer, Roberto; Prokisch, Holger; Meitinger, Thomas; Herder, Christian; Roden, Michael; Grallert, Harald; Ripatti, Samuli; Perola, Markus; Wood, Andrew R.; Melzer, David; Ferrucci, Luigi; Singleton, Andrew B.; Hernandez, Dena G.; Knight, Julian C.; Melchiotti, Rossella; Lee, Bernett; Poidinger, Michael; Zolezzi, Francesca; Larbi, Anis; Wang, De Yun; van den Berg, Leonard H.; Veldink, Jan H.; Rotzschke, Olaf; Makino, Seiko; Salomaa, Veikko; Strauch, Konstantin; Völker, Uwe; van Meurs, Joyce B. J.; Metspalu, Andres; Wijmenga, Cisca; Jansen, Ritsert C.; Franke, Lude

2015-01-01

The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE) meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn’s disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus. PMID:25955312

Quantitative imaging by pixel-based contrast-enhanced ultrasound reveals a linear relationship between synovial vascular perfusion and the recruitment of pathogenic IL-17A-F+IL-23+ CD161+ CD4+ T helper cells in psoriatic arthritis joints.

PubMed

Fiocco, Ugo; Stramare, Roberto; Martini, Veronica; Coran, Alessandro; Caso, Francesco; Costa, Luisa; Felicetti, Mara; Rizzo, Gaia; Tonietto, Matteo; Scanu, Anna; Oliviero, Francesca; Raffeiner, Bernd; Vezzù, Maristella; Lunardi, Francesca; Scarpa, Raffaele; Sacerdoti, David; Rubaltelli, Leopoldo; Punzi, Leonardo; Doria, Andrea; Grisan, Enrico

2017-02-01

To develop quantitative imaging biomarkers of synovial tissue perfusion by pixel-based contrast-enhanced ultrasound (CEUS), we studied the relationship between CEUS synovial vascular perfusion and the frequencies of pathogenic T helper (Th)-17 cells in psoriatic arthritis (PsA) joints. Eight consecutive patients with PsA were enrolled in this study. Gray scale CEUS evaluation was performed on the same joint immediately after joint aspiration, by automatic assessment perfusion data, using a new quantification approach of pixel-based analysis and the gamma-variate model. The set of perfusional parameters considered by the time intensity curve includes the maximum value (peak) of the signal intensity curve, the blood volume index or area under the curve, (BVI, AUC) and the contrast mean transit time (MTT). The direct ex vivo analysis of the frequencies of SF IL17A-F + CD161 + IL23 + CD4 + T cells subsets were quantified by fluorescence-activated cell sorter (FACS). In cross-sectional analyses, when tested for multiple comparison setting, a false discovery rate at 10%, a common pattern of correlations between CEUS Peak, AUC (BVI) and MTT parameters with the IL17A-F + IL23 + - IL17A-F + CD161 + - and IL17A-F + CD161 + IL23 + CD4 + T cells subsets, as well as lack of correlation between both peak and AUC values and both CD4 + T and CD4 + IL23 + T cells, was observed. The pixel-based CEUS assessment is a truly measure synovial inflammation, as a useful tool to develop quantitative imaging biomarker for monitoring target therapeutics in PsA.

Quantitative Analysis of Defects in Silicon. [to predict energy conversion efficiency of silicon samples for solar cells

NASA Technical Reports Server (NTRS)

Natesh, R.; Smith, J. M.; Qidwai, H. A.; Bruce, T.

1979-01-01

The evaluation and prediction of the conversion efficiency for a variety of silicon samples with differences in structural defects, such as grain boundaries, twin boundaries, precipitate particles, dislocations, etc. are discussed. Quantitative characterization of these structural defects, which were revealed by etching the surface of silicon samples, is performed by using an image analyzer. Due to different crystal growth and fabrication techniques the various types of silicon contain a variety of trace impurity elements and structural defects. The two most important criteria in evaluating the various silicon types for solar cell applications are cost and conversion efficiency.

Rapid analysis and exploration of fluorescence microscopy images.

PubMed

Pavie, Benjamin; Rajaram, Satwik; Ouyang, Austin; Altschuler, Jason M; Steininger, Robert J; Wu, Lani F; Altschuler, Steven J

2014-03-19

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard. Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

High-Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion Quantification.

PubMed

Kroll, Torsten; Schmidt, David; Schwanitz, Georg; Ahmad, Mubashir; Hamann, Jana; Schlosser, Corinne; Lin, Yu-Chieh; Böhm, Konrad J; Tuckermann, Jan; Ploubidou, Aspasia

2016-07-01

High-content analysis (HCA) converts raw light microscopy images to quantitative data through the automated extraction, multiparametric analysis, and classification of the relevant information content. Combined with automated high-throughput image acquisition, HCA applied to the screening of chemicals or RNAi-reagents is termed high-content screening (HCS). Its power in quantifying cell phenotypes makes HCA applicable also to routine microscopy. However, developing effective HCA and bioinformatic analysis pipelines for acquisition of biologically meaningful data in HCS is challenging. Here, the step-by-step development of an HCA assay protocol and an HCS bioinformatics analysis pipeline are described. The protocol's power is demonstrated by application to focal adhesion (FA) detection, quantitative analysis of multiple FA features, and functional annotation of signaling pathways regulating FA size, using primary data of a published RNAi screen. The assay and the underlying strategy are aimed at researchers performing microscopy-based quantitative analysis of subcellular features, on a small scale or in large HCS experiments. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Quantifying Golgi structure using EM: combining volume-SEM and stereology for higher throughput.

PubMed

Ferguson, Sophie; Steyer, Anna M; Mayhew, Terry M; Schwab, Yannick; Lucocq, John Milton

2017-06-01

Investigating organelles such as the Golgi complex depends increasingly on high-throughput quantitative morphological analyses from multiple experimental or genetic conditions. Light microscopy (LM) has been an effective tool for screening but fails to reveal fine details of Golgi structures such as vesicles, tubules and cisternae. Electron microscopy (EM) has sufficient resolution but traditional transmission EM (TEM) methods are slow and inefficient. Newer volume scanning EM (volume-SEM) methods now have the potential to speed up 3D analysis by automated sectioning and imaging. However, they produce large arrays of sections and/or images, which require labour-intensive 3D reconstruction for quantitation on limited cell numbers. Here, we show that the information storage, digital waste and workload involved in using volume-SEM can be reduced substantially using sampling-based stereology. Using the Golgi as an example, we describe how Golgi populations can be sensed quantitatively using single random slices and how accurate quantitative structural data on Golgi organelles of individual cells can be obtained using only 5-10 sections/images taken from a volume-SEM series (thereby sensing population parameters and cell-cell variability). The approach will be useful in techniques such as correlative LM and EM (CLEM) where small samples of cells are treated and where there may be variable responses. For Golgi study, we outline a series of stereological estimators that are suited to these analyses and suggest workflows, which have the potential to enhance the speed and relevance of data acquisition in volume-SEM.

Comparative and quantitative proteomic analysis of normal and degenerated human annulus fibrosus cells.

PubMed

Ye, Dongping; Liang, Weiguo; Dai, Libing; Zhou, Longqiang; Yao, Yicun; Zhong, Xin; Chen, Honghui; Xu, Jiake

2015-05-01

Degeneration of the intervertebral disc (IVD) is a major chronic medical condition associated with back pain. To better understand the pathogenesis of IVD degeneration, we performed comparative and quantitative proteomic analyses of normal and degenerated human annulus fibrosus (AF) cells and identified proteins that are differentially expressed between them. Annulus fibrosus cells were isolated and cultured from patients with lumbar disc herniation (the experimental group, degenerated AF cells) and scoliosis patients who underwent orthopaedic surgery (the control group, normal AF cells). Comparative proteomic analyses of normal and degenerated cultured AF cells were carried out using 2-D electrophoresis, mass spectrometric analyses, and database searching. Quantitative analyses of silver-stained 2-D electrophoresis gels of normal and degenerated cultured AF cells identified 10 protein spots that showed the most altered differential expression levels between the two groups. Among these, three proteins were decreased, including heat shock cognate 71-kDa protein, glucose-6-phosphate 1-dehydrogenase, and protocadherin-23, whereas seven proteins were increased, including guanine nucleotide-binding protein G(i) subunit α-2, superoxide dismutase, transmembrane protein 51, adenosine receptor A3, 26S protease regulatory subunit 8, lipid phosphate phosphatase-related protein, and fatty acyl-crotonic acid reductase 1. These differentially expressed proteins might be involved in the pathophysiological process of IVD degeneration and have potential values as biomarkers of the degeneration of IVD. © 2015 Wiley Publishing Asia Pty Ltd.

Quantitative phosphoproteomic analysis of RIP3-dependent protein phosphorylation in the course of TNF-induced necroptosis.

PubMed

Zhong, Chuan-Qi; Li, Yuanyue; Yang, Daowei; Zhang, Na; Xu, Xiaozheng; Wu, Yaying; Chen, Jinan; Han, Jiahuai

2014-03-01

Tumor necrosis factor (TNF) induced cell death in murine fibrosarcoma L929 cells is a model system in studying programed necrosis (also known as necroptosis). Receptor interacting protein 3 (RIP3), a serine-threonine kinase, is known to play an essential role in TNF-induced necroptosis; however, the phosphorylation events initiated by RIP3 activation in necroptotic process is still largely unknown. Here, we performed a quantitative MS based analysis to compare TNF-induced changes in the global phosphoproteome of wild-type (RIP3(+/+) ) and RIP3-knockdown L929 cells at different time points after TNF treatment. A total of 8058 phosphopeptides spanning 6892 phosphorylation sites in 2762 proteins were identified in the three experiments, in which cells were treated with TNF for 0.5, 2, and 4 h. By comparing the phosphorylation sites in wild-type and RIP3-knockdown L929 cells, 174, 167, and 177 distinct phosphorylation sites were revealed to be dependent on RIP3 at the 0.5, 2, and 4 h time points after TNF treatment, respectively. Notably, most of them were not detected in a previous phosphoproteomic analysis of RIP3-dependent phosphorylation in lipopolysaccharide-stimulated peritoneal macrophages and TNF-treated murine embryonic fibroblasts (MEFs), suggesting that the data presented in this report are highly relevant to the study of TNF-induced necroptosis of L929 cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes.

PubMed

Clark, David J; Fondrie, William E; Yang, Austin; Mao, Li

2016-02-05

Exosomes are 30-100 nm sized membrane vesicles released by cells into the extracellular space that mediate intercellular communication via transfer of proteins and other biological molecules. To better understand the role of these microvesicles in lung carcinogenesis, we employed a Triple SILAC quantitative proteomic strategy to examine the differential protein abundance between exosomes derived from an immortalized normal bronchial epithelial cell line and two non-small cell lung cancer (NSCLC) cell lines harboring distinct activating mutations in the cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) or epidermal growth factor receptor (EGFR). In total, we were able to quantify 721 exosomal proteins derived from the three cell lines. Proteins associated with signal transduction, including EGFR, GRB2 and SRC, were enriched in NSCLC exosomes, and could actively regulate cell proliferation in recipient cells. This study's investigation of the NSCLC exosomal proteome has identified enriched protein cargo that can contribute to lung cancer progression, which may have potential clinical implications in biomarker development for patients with NSCLC. The high mortality associated with lung cancer is a result of late-stage diagnosis of the disease. Current screening techniques used for early detection of lung cancer lack the specificity for accurate diagnosis. Exosomes are nano-sized extracellular vesicles, and the increased abundance of select protein cargo in exosomes derived from cancer cells may be used for diagnostic purposes. In this paper, we applied quantitative proteomic analysis to elucidate abundance differences in exosomal protein cargo between two NSCLC cell lines with distinctive oncogene mutations and an immortalized normal bronchial epithelial cell line. This study revealed proteins associated with cell adhesion, the extracellular matrix, and a variety of signaling molecules were enriched in NSCLC exosomes. The present data reveals a protein profile associated with NSCLC exosomes that suggests a role these vesicles have in the progression of lung carcinogenesis, as well as identifies several promising candidates that could be utilized as a multi-marker protein panel in a diagnostic platform for NSCLC. Copyright © 2015 Elsevier B.V. All rights reserved.

Proteome-wide detection and quantitative analysis of irreversible cysteine oxidation using long column UPLC-pSRM.

PubMed

Lee, Chia-Fang; Paull, Tanya T; Person, Maria D

2013-10-04

Reactive oxygen species (ROS) play an important role in normal biological functions and pathological processes. ROS is one of the driving forces for oxidizing proteins, especially on cysteine thiols. The labile, transient, and dynamic nature of oxidative modifications poses enormous technical challenges for both accurate modification site determination and quantitation of cysteine thiols. The present study describes a mass spectrometry-based approach that allows effective discovery and quantification of irreversible cysteine modifications. The utilization of a long reverse phase column provides high-resolution chromatography to separate different forms of modified cysteine thiols from protein complexes or cell lysates. This Fourier transform mass spectrometry (FT-MS) approach enabled detection and quantitation of ataxia telangiectasia mutated (ATM) complex cysteine sulfoxidation states using Skyline MS1 filtering. When we applied the long column ultra high pressure liquid chromatography (UPLC)-MS/MS analysis, 61 and 44 peptides from cell lysates and cells were identified with cysteine modifications in response to in vitro and in vivo H2O2 oxidation, respectively. Long column ultra high pressure liquid chromatography pseudo selected reaction monitoring (UPLC-pSRM) was then developed to monitor the oxidative level of cysteine thiols in cell lysate under varying concentrations of H2O2 treatment. From UPLC-pSRM analysis, the dynamic conversion of sulfinic (S-O2H) and sulfonic acid (S-O3H) was observed within nucleoside diphosphate kinase (Nm23-H1) and heat shock 70 kDa protein 8 (Hsc70). These methods are suitable for proteome-wide studies, providing a highly sensitive, straightforward approach to identify proteins containing redox-sensitive cysteine thiols in biological systems.

Classification-based quantitative analysis of stable isotope labeling by amino acids in cell culture (SILAC) data.

PubMed

Kim, Seongho; Carruthers, Nicholas; Lee, Joohyoung; Chinni, Sreenivasa; Stemmer, Paul

2016-12-01

Stable isotope labeling by amino acids in cell culture (SILAC) is a practical and powerful approach for quantitative proteomic analysis. A key advantage of SILAC is the ability to simultaneously detect the isotopically labeled peptides in a single instrument run and so guarantee relative quantitation for a large number of peptides without introducing any variation caused by separate experiment. However, there are a few approaches available to assessing protein ratios and none of the existing algorithms pays considerable attention to the proteins having only one peptide hit. We introduce new quantitative approaches to dealing with SILAC protein-level summary using classification-based methodologies, such as Gaussian mixture models with EM algorithms and its Bayesian approach as well as K-means clustering. In addition, a new approach is developed using Gaussian mixture model and a stochastic, metaheuristic global optimization algorithm, particle swarm optimization (PSO), to avoid either a premature convergence or being stuck in a local optimum. Our simulation studies show that the newly developed PSO-based method performs the best among others in terms of F1 score and the proposed methods further demonstrate the ability of detecting potential markers through real SILAC experimental data. No matter how many peptide hits the protein has, the developed approach can be applicable, rescuing many proteins doomed to removal. Furthermore, no additional correction for multiple comparisons is necessary for the developed methods, enabling direct interpretation of the analysis outcomes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Multidimensional electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) for quantitative analysis of the proteome and phosphoproteome in clinical and biomedical research.

PubMed

Loroch, Stefan; Schommartz, Tim; Brune, Wolfram; Zahedi, René Peiman; Sickmann, Albert

2015-05-01

Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150 μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ~75% more proteins and even ~134% more peptides compared to the 2D strategy. Additionally, we could quantify ~50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research. Copyright © 2015. Published by Elsevier B.V.

Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

PubMed

Xin, Qi-Lin; Deng, Cheng-Lin; Chen, Xi; Wang, Jun; Wang, Shao-Bo; Wang, Wei; Deng, Fei; Zhang, Bo; Xiao, Gengfu; Zhang, Lei-Ke

2017-06-15

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate antiviral responses. Identification of host proteins involved in the ZIKV replication process may lead to the discovery of antiviral targets. In this study, the first quantitative proteomic analysis of ZIKV-infected cells was performed to investigate host proteins involved in the ZIKV replication process. Bioinformatics analysis highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the UPS, bortezomib, can inhibit ZIKV infection in vivo Our study not only illustrated how host cells respond to ZIKV infection but also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. Copyright © 2017 American Society for Microbiology.

Ultrastructural analysis of cell component distribution in the apical cell of Ceratodon protonemata

NASA Technical Reports Server (NTRS)

Walker, L. M.; Sack, F. D.

1995-01-01

A distinctive feature of tip-growing plant cells is that cell components are distributed differentially along the length of the cell, although most ultrastructural analyses have been qualitative. The longtitudinal distribution of cell components was studied both qualitatively and quantitatively in the apical cell of dark-grown protonemata of the moss Ceratodon. The first 35 micrometers of the apical cell was analyzed stereologically using transmission electron microscopy. There were four types of distributions along the cell's axis, three of them differential: (1) tubular endoplasmic reticulum was evenly distributed, (2) cisternal endoplasmic reticulum and Golgi vesicles were distributed in a tip-to-base gradient, (3) plastids, vacuoles, and Golgi stacks were enriched in specific areas, although the locations of the enrichments varied, and (4) mitochondria were excluded in the tip-most 5 micrometers and evenly distributed throughout the remaining 30 micrometers. This study provides one of the most comprehensive quantitative, ultrastructural analyses of the distribution of cell components in the apex of any tip-growing plant cell. The finding that almost every component had its own spatial arrangement demonstrates the complexity of the organization and regulation of the distribution of components in tip-growing cells.

In-vitro analysis of early calcification in aortic valvular interstitial cells using Laser-Induced Breakdown Spectroscopy (LIBS).

PubMed

Davari, Seyyed Ali; Masjedi, Shirin; Ferdous, Zannatul; Mukherjee, Dibyendu

2018-01-01

Calcific aortic valve disease (CAVD) is a major cardiovascular disorder caused by osteogenic differentiation of valvular interstitial cells (VICs) within aortic valves. Conventional methods like colorimetric assays and histology fail to detect small calcium depositions during in-vitro VIC cultures. Laser-induced breakdown spectroscopy (LIBS) is a robust analytical tool used for inorganic materials characterizations, but relatively new to biomedical applications. We employ LIBS, for the first time, for quantitative in-vitro detection of calcium depositions in VICs at various osteogenic differentiation stages. VICs isolated from porcine aortic valves were cultured in osteogenic media over various days. Colorimetric calcium assays based on arsenazo dye and Von Kossa staining measured the calcium depositions within VICs. Simultaneously, LIBS signatures for Ca I (422.67 nm) atomic emission lines were collected for estimating calcium depositions in lyophilized VIC samples. Our results indicate excellent linear correlation between the calcium assay and our LIBS measurements. Furthermore, unlike the assay results, the LIBS results could resolve calcium signals from cell samples with as early as 2 days of osteogenic culture. Quantitatively, the LIBS measurements establish the limit of detection for calcium content in VICs to be ∼0.17±0.04 μg which indicates a 5-fold improvement over calcium assay. Picture: Quantitative LIBS enables in-vitro analysis for early stage detection of calcium deposition within aortic valvular interstitial cells (VICs). © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

On-Chip Quantitative Measurement of Mechanical Stresses During Cell Migration with Emulsion Droplets

NASA Astrophysics Data System (ADS)

Molino, D.; Quignard, S.; Gruget, C.; Pincet, F.; Chen, Y.; Piel, M.; Fattaccioli, J.

2016-07-01

The ability of immune cells to migrate within narrow and crowded spaces is a critical feature involved in various physiological processes from immune response to metastasis. Several in-vitro techniques have been developed so far to study the behaviour of migrating cells, the most recent being based on the fabrication of microchannels within which cells move. To address the question of the mechanical stress a cell is able to produce during the encounter of an obstacle while migrating, we developed a hybrid microchip made of parallel PDMS channels in which oil droplets are sparsely distributed and serve as deformable obstacles. We thus show that cells strongly deform droplets while passing them. Then, we show that the microdevice can be used to study the influence of drugs on migration at the population level. Finally, we describe a quantitative analysis method of the droplet deformation that allows measuring in real-time the mechanical stress exerted by a single cell. The method presented herein thus constitutes a powerful analytical tool for cell migration studies under confinement.

Changes in cell morphology due to plasma membrane wounding by acoustic cavitation

PubMed Central

Schlicher, Robyn K.; Hutcheson, Joshua D.; Radhakrishna, Harish; Apkarian, Robert P.; Prausnitz, Mark R.

2010-01-01

Acoustic cavitation-mediated wounding (i.e., sonoporation) has great potential to improve medical and laboratory applications requiring intracellular uptake of exogenous molecules; however, the field lacks detailed understanding of cavitation-induced morphological changes in cells and their relative importance. Here, we present an in-depth study of the effects of acoustic cavitation on cells using electron and confocal microscopy coupled with quantitative flow cytometry. High resolution images of treated cells show that morphologically different types of blebs can occur after wounding conditions caused by ultrasound exposure as well as by mechanical shear and strong laser ablation. In addition, these treatments caused wound-induced non-lytic necrotic death resulting in cell bodies we call wound-derived perikarya (WD-P). However, only cells exposed to acoustic cavitation experienced ejection of intact nuclei and nearly instant lytic necrosis. Quantitative analysis by flow cytometry indicates that wound-derived perikarya are the dominant morphology of nonviable cells, except at the strongest wounding conditions, where nuclear ejection accounts for a significant portion of cell death after ultrasound exposure. PMID:20350691

Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vesper, Stephen; McKinstry, Craig A.; Hartmann, Chris

2007-11-28

A method is described to discriminate between live and dead cells of the infectious fungi Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Mucor racemosus, Rhizopus stolonifer and Paecilomyces variotii. To test the method, conidial suspensions were heat inactivated at 85 °C or held at 5 °C (controls) for 1 h. Polycarbonate filters (25 mm diameter, 0.8 μm pore size) were placed on "welled" slides (14 mm diameter) and the filters treated with either PBS or PMA. Propidium monoazide (PMA), which enters dead cells but not live cells, was incubated with cell suspensions, exposed to blue wavelength light-emitting diodes (LED) to inactivatemore » remaining PMA and secure intercalation of PMAwith DNA of dead cells. Treated cells were extracted and the live and dead cells evaluated with quantitative PCR (QPCR). After heat treatment and DNA modification with PMA, all fungal species tested showed an approximate 100- to 1000-fold difference in cell viability estimated by QPCR analysis which was consistent with estimates of viability based on culturing.« less

Ultrastructural and mechanical changes in tubular epithelial cells by angiotensin II and aldosterone as observed with atomic force microscopy.

PubMed

Quan, Fu-Shi; Jeong, Kyung Hwan; Lee, Gi-Ja

2018-07-01

Tubular epithelial cells (TECs) play an important pathophysiological role in the promotion of renal fibrosis. Quantitative analysis of the mechanical changes in TECs may be helpful in evaluating novel pharmacological strategies. Atomic force microscopy (AFM) is a common nanotechnology tool used for imaging and measuring interaction forces in biological systems. In this study, we used AFM to study ultrastructural and mechanical changes in TECs mediated by the renin-angiotensin-aldosterone system. We quantitatively analyzed changes in the mechanical properties of TECs using three extrinsic factors, namely, chemical fixation, angiotensin II (AT II), and aldosterone (AD). Fixed TECs were 11 times stiffer at the cell body and 3 times stiffer at the cell-cell junction compared to live TECs. After stimulation with AT II, live TECs were four times stiffer at the junctional area than at the cell body, while fixed TECs after AT II stimulation were approximately two times stiffer at the both cell body and cell-cell junction compared to fixed unstimulated TECs. Fixed TECs also reflected changes in the mechanical properties of TECs at the cell body region after AD stimulation. Together, our results suggest that cell stiffness at the cell body region may serve as an effective index for evaluating drugs and stimulation, regardless of whether the cells are live or fixed at the time of analysis. In addition, studying the changes to the intrinsic mechanical property of TECs after application of external stimuli may be useful for investigating pathophysiologic mechanisms and effective therapeutic strategies for renal injury. Copyright © 2018 Elsevier Ltd. All rights reserved.

Probing the stochastic property of endoreduplication in cell size determination of Arabidopsis thaliana leaf epidermal tissue

PubMed Central

2017-01-01

Cell size distribution is highly reproducible, whereas the size of individual cells often varies greatly within a tissue. This is obvious in a population of Arabidopsis thaliana leaf epidermal cells, which ranged from 1,000 to 10,000 μm2 in size. Endoreduplication is a specialized cell cycle in which nuclear genome size (ploidy) is doubled in the absence of cell division. Although epidermal cells require endoreduplication to enhance cellular expansion, the issue of whether this mechanism is sufficient for explaining cell size distribution remains unclear due to a lack of quantitative understanding linking the occurrence of endoreduplication with cell size diversity. Here, we addressed this question by quantitatively summarizing ploidy profile and cell size distribution using a simple theoretical framework. We first found that endoreduplication dynamics is a Poisson process through cellular maturation. This finding allowed us to construct a mathematical model to predict the time evolution of a ploidy profile with a single rate constant for endoreduplication occurrence in a given time. We reproduced experimentally measured ploidy profile in both wild-type leaf tissue and endoreduplication-related mutants with this analytical solution, further demonstrating the probabilistic property of endoreduplication. We next extended the mathematical model by incorporating the element that cell size is determined according to ploidy level to examine cell size distribution. This analysis revealed that cell size is exponentially enlarged 1.5 times every endoreduplication round. Because this theoretical simulation successfully recapitulated experimentally observed cell size distributions, we concluded that Poissonian endoreduplication dynamics and exponential size-boosting are the sources of the broad cell size distribution in epidermal tissue. More generally, this study contributes to a quantitative understanding whereby stochastic dynamics generate steady-state biological heterogeneity. PMID:28926847

A general method for bead-enhanced quantitation by flow cytometry

PubMed Central

Montes, Martin; Jaensson, Elin A.; Orozco, Aaron F.; Lewis, Dorothy E.; Corry, David B.

2009-01-01

Flow cytometry provides accurate relative cellular quantitation (percent abundance) of cells from diverse samples, but technical limitations of most flow cytometers preclude accurate absolute quantitation. Several quantitation standards are now commercially available which, when added to samples, permit absolute quantitation of CD4+ T cells. However, these reagents are limited by their cost, technical complexity, requirement for additional software and/or limited applicability. Moreover, few studies have validated the use of such reagents in complex biological samples, especially for quantitation of non-T cells. Here we show that addition to samples of known quantities of polystyrene fluorescence standardization beads permits accurate quantitation of CD4+ T cells from complex cell samples. This procedure, here termed single bead-enhanced cytofluorimetry (SBEC), was equally capable of enumerating eosinophils as well as subcellular fragments of apoptotic cells, moieties with very different optical and fluorescent characteristics. Relative to other proprietary products, SBEC is simple, inexpensive and requires no special software, suggesting that the method is suitable for the routine quantitation of most cells and other particles by flow cytometry. PMID:17067632

A quantitative approach to the study of cell shapes and interactions during early chordate embryogenesis.

PubMed

Tassy, Olivier; Daian, Fabrice; Hudson, Clare; Bertrand, Vincent; Lemaire, Patrick

2006-02-21

The prospects of deciphering the genetic program underlying embryonic development were recently boosted by the generation of large sets of precisely organized quantitative molecular data. In contrast, although the precise arrangement, interactions, and shapes of cells are crucial for the fulfilment of this program, their description remains coarse and qualitative. To bridge this gap, we developed a generic software, 3D Virtual Embryo, to quantify the geometry and interactions of cells in interactive three-dimensional embryo models. We applied this approach to early ascidian embryos, chosen because of their simplicity and their phylogenetic proximity to vertebrates. We generated a collection of 19 interactive ascidian embryos between the 2- and 44-cell stages. We characterized the evolution with time, and in different cell lineages, of the volume of cells and of eight mathematical descriptors of their geometry, and we measured the surface of contact between neighboring blastomeres. These analyses first revealed that early embryonic blastomeres adopt a surprising variety of shapes, which appeared to be under strict and dynamic developmental control. Second, we found novel asymmetric cell divisions in the posterior vegetal lineages, which gave birth to sister cells with different fates. Third, during neural induction, differences in the area of contact between individual competent animal cells and inducing vegetal blastomeres appeared important to select the induced cells. In addition to novel insight into both cell-autonomous and inductive processes controlling early ascidian development, we establish a generic conceptual framework for the quantitative analysis of embryo geometry that can be applied to other model organisms.

Quantitative Microscopic Analysis of Plasma Membrane Receptor Dynamics in Living Plant Cells.

PubMed

Luo, Yu; Russinova, Eugenia

2017-01-01

Plasma membrane-localized receptors are essential for cellular communication and signal transduction. In Arabidopsis thaliana, BRASSINOSTEROID INSENSITIVE1 (BRI1) is one of the receptors that is activated by binding to its ligand, the brassinosteroid (BR) hormone, at the cell surface to regulate diverse plant developmental processes. The availability of BRI1 in the plasma membrane is related to its signaling output and is known to be controlled by the dynamic endomembrane trafficking. Advances in fluorescence labeling and confocal microscopy techniques enabled us to gain a better understanding of plasma membrane receptor dynamics in living cells. Here we describe different quantitative microscopy methods to monitor the relative steady-state levels of the BRI1 protein in the plasma membrane of root epidermal cells and its relative exocytosis and recycling rates. The methods can be applied also to analyze similar dynamics of other plasma membrane-localized receptors.

Extracting microtubule networks from superresolution single-molecule localization microscopy data

PubMed Central

Zhang, Zhen; Nishimura, Yukako; Kanchanawong, Pakorn

2017-01-01

Microtubule filaments form ubiquitous networks that specify spatial organization in cells. However, quantitative analysis of microtubule networks is hampered by their complex architecture, limiting insights into the interplay between their organization and cellular functions. Although superresolution microscopy has greatly facilitated high-resolution imaging of microtubule filaments, extraction of complete filament networks from such data sets is challenging. Here we describe a computational tool for automated retrieval of microtubule filaments from single-molecule-localization–based superresolution microscopy images. We present a user-friendly, graphically interfaced implementation and a quantitative analysis of microtubule network architecture phenotypes in fibroblasts. PMID:27852898

Highly sensitive transient absorption imaging of graphene and graphene oxide in living cells and circulating blood.

PubMed

Li, Junjie; Zhang, Weixia; Chung, Ting-Fung; Slipchenko, Mikhail N; Chen, Yong P; Cheng, Ji-Xin; Yang, Chen

2015-07-23

We report a transient absorption (TA) imaging method for fast visualization and quantitative layer analysis of graphene and GO. Forward and backward imaging of graphene on various substrates under ambient condition was imaged with a speed of 2 μs per pixel. The TA intensity linearly increased with the layer number of graphene. Real-time TA imaging of GO in vitro with capability of quantitative analysis of intracellular concentration and ex vivo in circulating blood were demonstrated. These results suggest that TA microscopy is a valid tool for the study of graphene based materials.

Novel image cytometric method for detection of physiological and metabolic changes in Saccharomyces cerevisiae.

PubMed

Chan, Leo L; Kury, Alexandria; Wilkinson, Alisha; Berkes, Charlotte; Pirani, Alnoor

2012-11-01

The studying and monitoring of physiological and metabolic changes in Saccharomyces cerevisiae (S. cerevisiae) has been a key research area for the brewing, baking, and biofuels industries, which rely on these economically important yeasts to produce their products. Specifically for breweries, physiological and metabolic parameters such as viability, vitality, glycogen, neutral lipid, and trehalose content can be measured to better understand the status of S. cerevisiae during fermentation. Traditionally, these physiological and metabolic changes can be qualitatively observed using fluorescence microscopy or flow cytometry for quantitative fluorescence analysis of fluorescently labeled cellular components associated with each parameter. However, both methods pose known challenges to the end-users. Specifically, conventional fluorescent microscopes lack automation and fluorescence analysis capabilities to quantitatively analyze large numbers of cells. Although flow cytometry is suitable for quantitative analysis of tens of thousands of fluorescently labeled cells, the instruments require a considerable amount of maintenance, highly trained technicians, and the system is relatively expensive to both purchase and maintain. In this work, we demonstrate the first use of Cellometer Vision for the kinetic detection and analysis of vitality, glycogen, neutral lipid, and trehalose content of S. cerevisiae. This method provides an important research tool for large and small breweries to study and monitor these physiological behaviors during production, which can improve fermentation conditions to produce consistent and higher-quality products.

Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells

PubMed Central

Howes, Mark T.; Kirkham, Matthew; Riches, James; Cortese, Katia; Walser, Piers J.; Simpson, Fiona; Hill, Michelle M.; Jones, Alun; Lundmark, Richard; Lindsay, Margaret R.; Hernandez-Deviez, Delia J.; Hadzic, Gordana; McCluskey, Adam; Bashir, Rumasia; Liu, Libin; Pilch, Paul; McMahon, Harvey; Robinson, Phillip J.; Hancock, John F.; Mayor, Satyajit

2010-01-01

Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells. PMID:20713605

Mapping the Extracellular and Membrane Proteome Associated with the Vasculature and the Stroma in the Embryo*

PubMed Central

Soulet, Fabienne; Kilarski, Witold W.; Roux-Dalvai, Florence; Herbert, John M. J.; Sacewicz, Izabela; Mouton-Barbosa, Emmanuelle; Bicknell, Roy; Lalor, Patricia; Monsarrat, Bernard; Bikfalvi, Andreas

2013-01-01

In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments. PMID:23674615

Systems cell biology.

PubMed

Mast, Fred D; Ratushny, Alexander V; Aitchison, John D

2014-09-15

Systems cell biology melds high-throughput experimentation with quantitative analysis and modeling to understand many critical processes that contribute to cellular organization and dynamics. Recently, there have been several advances in technology and in the application of modeling approaches that enable the exploration of the dynamic properties of cells. Merging technology and computation offers an opportunity to objectively address unsolved cellular mechanisms, and has revealed emergent properties and helped to gain a more comprehensive and fundamental understanding of cell biology. © 2014 Mast et al.

PAd-shRNA-PTN reduces pleiotrophin of pancreatic cancer cells and inhibits neurite outgrowth of DRG

PubMed Central

Yao, Jun; Zhang, Min; Ma, Qing-Yong; Wang, Zheng; Wang, Lian-Cai; Zhang, Dong

2011-01-01

AIM: To investigate the silencing effects of pAd-shRNA-pleiotrophin (PTN) on PTN in pancreatic cancer cells, and to observe the inhibition of pAd-shRNA-PTN on neurite outgrowth from dorsal root ganglion (DRG) neurons in vitro. METHODS: PAd-shRNA-PTN was used to infect pancreatic cancer BxPC-3 cells; assays were conducted for knockdown of the PTN gene on the 0th, 1st, 3rd, 5th, 7th and 9th d after infection using immunocytochemistry, real-time quantitative polymerase chain reaction (PCR), and Western blotting analysis. The morphologic changes of cultured DRG neurons were observed by mono-culture of DRG neurons and co-culture with BXPC-3 cells in vitro. RESULTS: The real-time quantitative PCR showed that the inhibition rates of PTN mRNA expression in the BxPC-3 cells were 20%, 80%, 50% and 25% on the 1st, 3rd, 5th and 7th d after infection. Immunocytochemistry and Western blotting analysis also revealed the same tendency. In contrast to the control, the DRG neurons co-cultured with the infected BxPC-3 cells shrunk; the number and length of neurites were significantly decreased. CONCLUSION: Efficient and specific knockdown of PTN in pancreatic cancer cells and the reduction in PTN expression resulted in the inhibition of neurite outgrowth from DRG neurons. PMID:21677838

Image processing and machine learning in the morphological analysis of blood cells.

PubMed

Rodellar, J; Alférez, S; Acevedo, A; Molina, A; Merino, A

2018-05-01

This review focuses on how image processing and machine learning can be useful for the morphological characterization and automatic recognition of cell images captured from peripheral blood smears. The basics of the 3 core elements (segmentation, quantitative features, and classification) are outlined, and recent literature is discussed. Although red blood cells are a significant part of this context, this study focuses on malignant lymphoid cells and blast cells. There is no doubt that these technologies may help the cytologist to perform efficient, objective, and fast morphological analysis of blood cells. They may also help in the interpretation of some morphological features and may serve as learning and survey tools. Although research is still needed, it is important to define screening strategies to exploit the potential of image-based automatic recognition systems integrated in the daily routine of laboratories along with other analysis methodologies. © 2018 John Wiley & Sons Ltd.

Selection of stable reference genes for quantitative rt-PCR comparisons of mouse embryonic and extra-embryonic stem cells.

PubMed

Veazey, Kylee J; Golding, Michael C

2011-01-01

Isolation and culture of both embryonic and tissue specific stem cells provide an enormous opportunity to study the molecular processes driving development. To gain insight into the initial events underpinning mammalian embryogenesis, pluripotent stem cells from each of the three distinct lineages present within the preimplantation blastocyst have been derived. Embryonic (ES), trophectoderm (TS) and extraembryonic endoderm (XEN) stem cells possess the developmental potential of their founding lineages and seemingly utilize distinct epigenetic modalities to program gene expression. However, the basis for these differing cellular identities and epigenetic properties remain poorly defined.Quantitative reverse transcription-polymerase chain reaction (qPCR) is a powerful and efficient means of rapidly comparing patterns of gene expression between different developmental stages and experimental conditions. However, careful, empirical selection of appropriate reference genes is essential to accurately measuring transcriptional differences. Here we report the quantitation and evaluation of fourteen commonly used references genes between ES, TS and XEN stem cells. These included: Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sk, Sdha, Tbp and Ywhaz. Utilizing three independent statistical analysis, we identify Pgk1, Sdha and Tbp as the most stable reference genes between each of these stem cell types. Furthermore, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of embryonic and trophectoderm stem cells respectively.Understanding the transcriptional and epigenetic regulatory mechanisms controlling cellular identity within these distinct stem cell types provides essential insight into cellular processes controlling both embryogenesis and stem cell biology. Normalizing quantitative RT-PCR measurements using the geometric mean CT values obtained for the identified mRNAs, offers a reliable method to assess differing patterns of gene expression between the three founding stem cell lineages present within the mammalian preimplantation embryo.

Detection of early changes in lung cell cytology by flow-systems analysis techniques, July 1--December 31, 1975

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinkamp, J.A.; Ingram, M.; Hansen, K.M.

1976-03-01

This report summarizes results of preliminary experiments to demonstrate the feasibility of using automated flow-systems analysis in detecting early changes of respiratory epithelium exposed to physical and chemical agents associated with the by-products of nonnuclear energy production. The Syrian hamster was selected as the experimental test animal to begin investigation of the effects of toxic agents to cells of the respiratory tract. Since initiation of the program approximately six months ago, the goals have been acquisition of adequate numbers of exfoliated cells from the lung; adaptation of cytological techniques developed on human exfoliated gynecological samples to hamster lung epithelium formore » obtaining single-cell suspensions; utilization of existing cell staining methods to measure DNA content in lung cells; and analysis of DNA content and cell size. As the flow-system cell analysis technology is adapted to the measurement of exfoliated lung cells, rapid and quantitative determination of early changes in the physical and biochemical cellular properties will be attempted as a function of exposure to the toxic agents. (auth)« less

Automatic detection and quantitative analysis of cells in the mouse primary motor cortex

NASA Astrophysics Data System (ADS)

Meng, Yunlong; He, Yong; Wu, Jingpeng; Chen, Shangbin; Li, Anan; Gong, Hui

2014-09-01

Neuronal cells play very important role on metabolism regulation and mechanism control, so cell number is a fundamental determinant of brain function. Combined suitable cell-labeling approaches with recently proposed three-dimensional optical imaging techniques, whole mouse brain coronal sections can be acquired with 1-μm voxel resolution. We have developed a completely automatic pipeline to perform cell centroids detection, and provided three-dimensional quantitative information of cells in the primary motor cortex of C57BL/6 mouse. It involves four principal steps: i) preprocessing; ii) image binarization; iii) cell centroids extraction and contour segmentation; iv) laminar density estimation. Investigations on the presented method reveal promising detection accuracy in terms of recall and precision, with average recall rate 92.1% and average precision rate 86.2%. We also analyze laminar density distribution of cells from pial surface to corpus callosum from the output vectorizations of detected cell centroids in mouse primary motor cortex, and find significant cellular density distribution variations in different layers. This automatic cell centroids detection approach will be beneficial for fast cell-counting and accurate density estimation, as time-consuming and error-prone manual identification is avoided.

Tunable Single-Cell Extraction for Molecular Analyses.

PubMed

Guillaume-Gentil, Orane; Grindberg, Rashel V; Kooger, Romain; Dorwling-Carter, Livie; Martinez, Vincent; Ossola, Dario; Pilhofer, Martin; Zambelli, Tomaso; Vorholt, Julia A

2016-07-14

Because of cellular heterogeneity, the analysis of endogenous molecules from single cells is of significant interest and has major implications. While micromanipulation or cell sorting followed by cell lysis is already used for subsequent molecular examinations, approaches to directly extract the content of living cells remain a challenging but promising alternative to achieving non-destructive sampling and cell-context preservation. Here, we demonstrate the quantitative extraction from single cells with spatiotemporal control using fluidic force microscopy. We further present a comprehensive analysis of the soluble molecules withdrawn from the cytoplasm or the nucleus, including the detection of enzyme activities and transcript abundances. This approach has uncovered the ability of cells to withstand extraction of up to several picoliters and opens opportunities to study cellular dynamics and cell-cell communication under physiological conditions at the single-cell level. Copyright © 2016 Elsevier Inc. All rights reserved.

Integrated stoichiometric, thermodynamic and kinetic modelling of steady state metabolism

PubMed Central

Fleming, R.M.T.; Thiele, I.; Provan, G.; Nasheuer, H.P.

2010-01-01

The quantitative analysis of biochemical reactions and metabolites is at frontier of biological sciences. The recent availability of high-throughput technology data sets in biology has paved the way for new modelling approaches at various levels of complexity including the metabolome of a cell or an organism. Understanding the metabolism of a single cell and multi-cell organism will provide the knowledge for the rational design of growth conditions to produce commercially valuable reagents in biotechnology. Here, we demonstrate how equations representing steady state mass conservation, energy conservation, the second law of thermodynamics, and reversible enzyme kinetics can be formulated as a single system of linear equalities and inequalities, in addition to linear equalities on exponential variables. Even though the feasible set is non-convex, the reformulation is exact and amenable to large-scale numerical analysis, a prerequisite for computationally feasible genome scale modelling. Integrating flux, concentration and kinetic variables in a unified constraint-based formulation is aimed at increasing the quantitative predictive capacity of flux balance analysis. Incorporation of experimental and theoretical bounds on thermodynamic and kinetic variables ensures that the predicted steady state fluxes are both thermodynamically and biochemically feasible. The resulting in silico predictions are tested against fluxomic data for central metabolism in E. coli and compare favourably with in silico prediction by flux balance analysis. PMID:20230840

A method for rapid quantitative assessment of biofilms with biomolecular staining and image analysis.

PubMed

Larimer, Curtis; Winder, Eric; Jeters, Robert; Prowant, Matthew; Nettleship, Ian; Addleman, Raymond Shane; Bonheyo, George T

2016-01-01

The accumulation of bacteria in surface-attached biofilms can be detrimental to human health, dental hygiene, and many industrial processes. Natural biofilms are soft and often transparent, and they have heterogeneous biological composition and structure over micro- and macroscales. As a result, it is challenging to quantify the spatial distribution and overall intensity of biofilms. In this work, a new method was developed to enhance the visibility and quantification of bacterial biofilms. First, broad-spectrum biomolecular staining was used to enhance the visibility of the cells, nucleic acids, and proteins that make up biofilms. Then, an image analysis algorithm was developed to objectively and quantitatively measure biofilm accumulation from digital photographs and results were compared to independent measurements of cell density. This new method was used to quantify the growth intensity of Pseudomonas putida biofilms as they grew over time. This method is simple and fast, and can quantify biofilm growth over a large area with approximately the same precision as the more laborious cell counting method. Stained and processed images facilitate assessment of spatial heterogeneity of a biofilm across a surface. This new approach to biofilm analysis could be applied in studies of natural, industrial, and environmental biofilms.

Quantitative Analysis of Signaling Networks across Differentially Embedded Tumors Highlights Interpatient Heterogeneity in Human Glioblastoma

PubMed Central

2015-01-01

Glioblastoma multiforme (GBM) is the most aggressive malignant primary brain tumor, with a dismal mean survival even with the current standard of care. Although in vitro cell systems can provide mechanistic insight into the regulatory networks governing GBM cell proliferation and migration, clinical samples provide a more physiologically relevant view of oncogenic signaling networks. However, clinical samples are not widely available and may be embedded for histopathologic analysis. With the goal of accurately identifying activated signaling networks in GBM tumor samples, we investigated the impact of embedding in optimal cutting temperature (OCT) compound followed by flash freezing in LN2 vs immediate flash freezing (iFF) in LN2 on protein expression and phosphorylation-mediated signaling networks. Quantitative proteomic and phosphoproteomic analysis of 8 pairs of tumor specimens revealed minimal impact of the different sample processing strategies and highlighted the large interpatient heterogeneity present in these tumors. Correlation analyses of the differentially processed tumor sections identified activated signaling networks present in selected tumors and revealed the differential expression of transcription, translation, and degradation associated proteins. This study demonstrates the capability of quantitative mass spectrometry for identification of in vivo oncogenic signaling networks from human tumor specimens that were either OCT-embedded or immediately flash-frozen. PMID:24927040

Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy

DOE PAGES

Jin, Qiaoling; Paunesku, Tatjana; Lai, Barry; ...

2016-08-31

Trace metals play important roles in biological function, and x-ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side-by-side comparison shows that plunge-freezing-based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with freshmore » media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. Lastly, when chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x-ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.« less

Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Qiaoling; Paunesku, Tatjana; Lai, Barry

Trace metals play important roles in biological function, and x-ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side-by-side comparison shows that plunge-freezing-based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with freshmore » media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. Lastly, when chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x-ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.« less

Quantitative proteomic analysis of cultured skin fibroblast cells derived from patients with triglyceride deposit cardiomyovasculopathy

PubMed Central

2013-01-01

Background Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare disease, characterized by the massive accumulation of triglyceride (TG) in multiple tissues, especially skeletal muscle, heart muscle and the coronary artery. TGCV is caused by mutation of adipose triglyceride lipase, which is an essential molecule for the hydrolysis of TG. TGCV is at high risk for skeletal myopathy and heart dysfunction, and therefore premature death. Development of therapeutic methods for TGCV is highly desirable. This study aims to discover specific molecules responsible for TGCV pathogenesis. Methods To identify differentially expressed proteins in TGCV patient cells, the stable isotope labeling with amino acids in cell culture (SILAC) method coupled with LC-MS/MS was performed using skin fibroblast cells derived from two TGCV patients and three healthy volunteers. Altered protein expression in TGCV cells was confirmed using the selected reaction monitoring (SRM) method. Microarray-based transcriptome analysis was simultaneously performed to identify changes in gene expression in TGCV cells. Results Using SILAC proteomics, 4033 proteins were quantified, 53 of which showed significantly altered expression in both TGCV patient cells. Twenty altered proteins were chosen and confirmed using SRM. SRM analysis successfully quantified 14 proteins, 13 of which showed the same trend as SILAC proteomics. The altered protein expression data set was used in Ingenuity Pathway Analysis (IPA), and significant networks were identified. Several of these proteins have been previously implicated in lipid metabolism, while others represent new therapeutic targets or markers for TGCV. Microarray analysis quantified 20743 transcripts, and 252 genes showed significantly altered expression in both TGCV patient cells. Ten altered genes were chosen, 9 of which were successfully confirmed using quantitative RT-PCR. Biological networks of altered genes were analyzed using an IPA search. Conclusions We performed the SILAC- and SRM-based identification-through-confirmation study using skin fibroblast cells derived from TGCV patients, and first identified altered proteins specific for TGCV. Microarray analysis also identified changes in gene expression. The functional networks of the altered proteins and genes are discussed. Our findings will be exploited to elucidate the pathogenesis of TGCV and discover clinically relevant molecules for TGCV in the near future. PMID:24360150

Quantitative Proteomics Analysis Identifies Mitochondria as Therapeutic Targets of Multidrug-Resistance in Ovarian Cancer

PubMed Central

Chen, Xiulan; Wei, Shasha; Ma, Ying; Lu, Jie; Niu, Gang; Xue, Yanhong; Chen, Xiaoyuan; Yang, Fuquan

2014-01-01

Doxorubicin is a widely used chemotherapeutic agent for the treatment of a variety of solid tumors. However, resistance to this anticancer drug is a major obstacle to the effective treatment of tumors. As mitochondria play important roles in cell life and death, we anticipate that mitochondria may be related to drug resistance. Here, stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic strategy was applied to compare mitochondrial protein expression in doxorubicin sensitive OVCAR8 cells and its doxorubicin-resistant variant NCI_ADR/RES cells. A total of 2085 proteins were quantified, of which 122 proteins displayed significant changes in the NCI_ADR/RES cells. These proteins participated in a variety of cell processes including cell apoptosis, substance metabolism, transport, detoxification and drug metabolism. Then qRT-PCR and western blot were applied to validate the differentially expressed proteins quantified by SILAC. Further functional studies with RNAi demonstrated TOP1MT, a mitochondrial protein participated in DNA repair, was involved in doxorubicin resistance in NCI_ADR/RES cells. Besides the proteomic study, electron microscopy and fluorescence analysis also observed that mitochondrial morphology and localization were greatly altered in NCI_ADR/RES cells. Mitochondrial membrane potential was also decreased in NCI_ADR/RES cells. All these results indicate that mitochondrial function is impaired in doxorubicin-resistant cells and mitochondria play an important role in doxorubicin resistance. This research provides some new information about doxorubicin resistance, indicating that mitochondria could be therapeutic targets of doxorubicin resistance in ovarian cancer cells. PMID:25285166

Automated tumor analysis for molecular profiling in lung cancer

PubMed Central

Boyd, Clinton; James, Jacqueline A.; Loughrey, Maurice B.; Hougton, Joseph P.; Boyle, David P.; Kelly, Paul; Maxwell, Perry; McCleary, David; Diamond, James; McArt, Darragh G.; Tunstall, Jonathon; Bankhead, Peter; Salto-Tellez, Manuel

2015-01-01

The discovery and clinical application of molecular biomarkers in solid tumors, increasingly relies on nucleic acid extraction from FFPE tissue sections and subsequent molecular profiling. This in turn requires the pathological review of haematoxylin & eosin (H&E) stained slides, to ensure sample quality, tumor DNA sufficiency by visually estimating the percentage tumor nuclei and tumor annotation for manual macrodissection. In this study on NSCLC, we demonstrate considerable variation in tumor nuclei percentage between pathologists, potentially undermining the precision of NSCLC molecular evaluation and emphasising the need for quantitative tumor evaluation. We subsequently describe the development and validation of a system called TissueMark for automated tumor annotation and percentage tumor nuclei measurement in NSCLC using computerized image analysis. Evaluation of 245 NSCLC slides showed precise automated tumor annotation of cases using Tissuemark, strong concordance with manually drawn boundaries and identical EGFR mutational status, following manual macrodissection from the image analysis generated tumor boundaries. Automated analysis of cell counts for % tumor measurements by Tissuemark showed reduced variability and significant correlation (p < 0.001) with benchmark tumor cell counts. This study demonstrates a robust image analysis technology that can facilitate the automated quantitative analysis of tissue samples for molecular profiling in discovery and diagnostics. PMID:26317646

Glycosyltransferases as marker genes for the quantitative polymerase chain reaction-based detection of circulating tumour cells from blood samples of patients with breast cancer undergoing adjuvant therapy.

PubMed

Kölbl, Alexandra C; Hiller, Roman A; Ilmer, Mathias; Liesche, Friederike; Heublein, Sabine; Schröder, Lennard; Hutter, Stefan; Friese, Klaus; Jeschke, Udo; Andergassen, Ulrich

2015-08-01

Altered glycosylation is a predominant feature of tumour cells; it serves for cell adhesion and detachment, respectively, and facilitates the immune escape of these cells. Therefore changes in the expression of glycosyltransferase genes could help to identify circulating tumour cells (CTCs) in the blood samples of cancer patients using a quantitative polymerase chain reaction (PCR) approach. Blood samples of healthy donors were inoculated with certain numbers of established breast cancer cell line cells, thus creating a model system. These samples were analysed by quantitative PCR for the expression of six different glycosyltransferase genes. The three genes with the best results in the model system were consecutively applied to samples from adjuvant breast cancer patients and of healthy donors. FUT3 and GALNT6 showed the highest increase in relative expression, while GALNT6 and ST3GAL3 were the first to reach statistically significant different ∆CT-values comparing the sample with and without addition of tumour cells. These three genes were applied to patient samples, but did not show any significant results that may suggest the presence of CTCs in the blood. Although the relative expression of some of the glycosyltransferase genes exhibited reasonable results in the model system, their application to breast cancer patient samples will have to be further improved, e.g. by co-analysis of patient blood samples by gold-standard methods.

An optimized method for measuring fatty acids and cholesterol in stable isotope-labeled cells

PubMed Central

Argus, Joseph P.; Yu, Amy K.; Wang, Eric S.; Williams, Kevin J.; Bensinger, Steven J.

2017-01-01

Stable isotope labeling has become an important methodology for determining lipid metabolic parameters of normal and neoplastic cells. Conventional methods for fatty acid and cholesterol analysis have one or more issues that limit their utility for in vitro stable isotope-labeling studies. To address this, we developed a method optimized for measuring both fatty acids and cholesterol from small numbers of stable isotope-labeled cultured cells. We demonstrate quantitative derivatization and extraction of fatty acids from a wide range of lipid classes using this approach. Importantly, cholesterol is also recovered, albeit at a modestly lower yield, affording the opportunity to quantitate both cholesterol and fatty acids from the same sample. Although we find that background contamination can interfere with quantitation of certain fatty acids in low amounts of starting material, our data indicate that this optimized method can be used to accurately measure mass isotopomer distributions for cholesterol and many fatty acids isolated from small numbers of cultured cells. Application of this method will facilitate acquisition of lipid parameters required for quantifying flux and provide a better understanding of how lipid metabolism influences cellular function. PMID:27974366

Analysis of interphase node proteins in fission yeast by quantitative and superresolution fluorescence microscopy

PubMed Central

Akamatsu, Matthew; Lin, Yu; Bewersdorf, Joerg; Pollard, Thomas D.

2017-01-01

We used quantitative confocal microscopy and FPALM superresolution microscopy of live fission yeast to investigate the structures and assembly of two types of interphase nodes—multiprotein complexes associated with the plasma membrane that merge together and mature into the precursors of the cytokinetic contractile ring. During the long G2 phase of the cell cycle, seven different interphase node proteins maintain constant concentrations as they accumulate in proportion to cell volume. During mitosis, the total numbers of type 1 node proteins (cell cycle kinases Cdr1p, Cdr2p, Wee1p, and anillin Mid1p) are constant even when the nodes disassemble. Quantitative measurements provide strong evidence that both types of nodes have defined sizes and numbers of constituent proteins, as observed for cytokinesis nodes. Type 1 nodes assemble in two phases—a burst at the end of mitosis, followed by steady increase during interphase to double the initial number. Type 2 nodes containing Blt1p, Rho-GEF Gef2p, and kinesin Klp8p remain intact throughout the cell cycle and are constituents of the contractile ring. They are released from the contractile ring as it disassembles and then associate with type 1 nodes around the equator of the cell during interphase. PMID:28539404

Comparison of four glycosyl residue composition methods for effectiveness in detecting sugars from cell walls of dicot and grass tissues.

PubMed

Biswal, Ajaya K; Tan, Li; Atmodjo, Melani A; DeMartini, Jaclyn; Gelineo-Albersheim, Ivana; Hunt, Kimberly; Black, Ian M; Mohanty, Sushree S; Ryno, David; Wyman, Charles E; Mohnen, Debra

2017-01-01

The effective use of plant biomass for biofuel and bioproduct production requires a comprehensive glycosyl residue composition analysis to understand the different cell wall polysaccharides present in the different biomass sources. Here we compared four methods side-by-side for their ability to measure the neutral and acidic sugar composition of cell walls from herbaceous, grass, and woody model plants and bioenergy feedstocks. Arabidopsis, Populus , rice, and switchgrass leaf cell walls, as well as cell walls from Populus wood, rice stems, and switchgrass tillers, were analyzed by (1) gas chromatography-mass spectrometry (GC-MS) of alditol acetates combined with a total uronic acid assay; (2) carbodiimide reduction of uronic acids followed by GC-MS of alditol acetates; (3) GC-MS of trimethylsilyl (TMS) derivatives; and (4) high-pressure, anion-exchange chromatography (HPAEC). All four methods gave comparable abundance ranking of the seven neutral sugars, and three of the methods were able to quantify unique acidic sugars. The TMS, HPAEC, and carbodiimide methods provided comparable quantitative results for the specific neutral and acidic sugar content of the biomass, with the TMS method providing slightly greater yield of specific acidic sugars and high total sugar yields. The alditol acetate method, while providing comparable information on the major neutral sugars, did not provide the requisite quantitative information on the specific acidic sugars in plant biomass. Thus, the alditol acetate method is the least informative of the four methods. This work provides a side-by-side comparison of the efficacy of four different established glycosyl residue composition analysis methods in the analysis of the glycosyl residue composition of cell walls from both dicot (Arabidopsis and Populus ) and grass (rice and switchgrass) species. Both primary wall-enriched leaf tissues and secondary wall-enriched wood/stem tissues were analyzed for mol% and mass yield of the non-cellulosic sugars. The TMS, HPAEC, and carbodiimide methods were shown to provide comparable quantitative data on the nine neutral and acidic sugars present in all plant cell walls.

Tissue matrix arrays for high throughput screening and systems analysis of cell function

PubMed Central

Beachley, Vince Z.; Wolf, Matthew T.; Sadtler, Kaitlyn; Manda, Srikanth S.; Jacobs, Heather; Blatchley, Michael; Bader, Joel S.; Pandey, Akhilesh; Pardoll, Drew; Elisseeff, Jennifer H.

2015-01-01

Cell and protein arrays have demonstrated remarkable utility in the high-throughput evaluation of biological responses; however, they lack the complexity of native tissue and organs. Here, we describe tissue extracellular matrix (ECM) arrays for screening biological outputs and systems analysis. We spotted processed tissue ECM particles as two-dimensional arrays or incorporated them with cells to generate three-dimensional cell-matrix microtissue arrays. We then investigated the response of human stem, cancer, and immune cells to tissue ECM arrays originating from 11 different tissues, and validated the 2D and 3D arrays as representative of the in vivo microenvironment through quantitative analysis of tissue-specific cellular responses, including matrix production, adhesion and proliferation, and morphological changes following culture. The biological outputs correlated with tissue proteomics, and network analysis identified several proteins linked to cell function. Our methodology enables broad screening of ECMs to connect tissue-specific composition with biological activity, providing a new resource for biomaterials research and translation. PMID:26480475

Image segmentation and dynamic lineage analysis in single-cell fluorescence microscopy.

PubMed

Wang, Quanli; Niemi, Jarad; Tan, Chee-Meng; You, Lingchong; West, Mike

2010-01-01

An increasingly common component of studies in synthetic and systems biology is analysis of dynamics of gene expression at the single-cell level, a context that is heavily dependent on the use of time-lapse movies. Extracting quantitative data on the single-cell temporal dynamics from such movies remains a major challenge. Here, we describe novel methods for automating key steps in the analysis of single-cell, fluorescent images-segmentation and lineage reconstruction-to recognize and track individual cells over time. The automated analysis iteratively combines a set of extended morphological methods for segmentation, and uses a neighborhood-based scoring method for frame-to-frame lineage linking. Our studies with bacteria, budding yeast and human cells, demonstrate the portability and usability of these methods, whether using phase, bright field or fluorescent images. These examples also demonstrate the utility of our integrated approach in facilitating analyses of engineered and natural cellular networks in diverse settings. The automated methods are implemented in freely available, open-source software.

A method for rapid quantitative assessment of biofilms with biomolecular staining and image analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larimer, Curtis J.; Winder, Eric M.; Jeters, Robert T.

Here, the accumulation of bacteria in surface attached biofilms, or biofouling, can be detrimental to human health, dental hygiene, and many industrial processes. A critical need in identifying and preventing the deleterious effects of biofilms is the ability to observe and quantify their development. Analytical methods capable of assessing early stage fouling are cumbersome or lab-confined, subjective, and qualitative. Herein, a novel photographic method is described that uses biomolecular staining and image analysis to enhance contrast of early stage biofouling. A robust algorithm was developed to objectively and quantitatively measure surface accumulation of Pseudomonas putida from photographs and results weremore » compared to independent measurements of cell density. Results from image analysis quantified biofilm growth intensity accurately and with approximately the same precision of the more laborious cell counting method. This simple method for early stage biofilm detection enables quantifiable measurement of surface fouling and is flexible enough to be applied from the laboratory to the field. Broad spectrum staining highlights fouling biomass, photography quickly captures a large area of interest, and image analysis rapidly quantifies fouling in the image.« less

A method for rapid quantitative assessment of biofilms with biomolecular staining and image analysis

DOE PAGES

Larimer, Curtis J.; Winder, Eric M.; Jeters, Robert T.; ...

2015-12-07

Here, the accumulation of bacteria in surface attached biofilms, or biofouling, can be detrimental to human health, dental hygiene, and many industrial processes. A critical need in identifying and preventing the deleterious effects of biofilms is the ability to observe and quantify their development. Analytical methods capable of assessing early stage fouling are cumbersome or lab-confined, subjective, and qualitative. Herein, a novel photographic method is described that uses biomolecular staining and image analysis to enhance contrast of early stage biofouling. A robust algorithm was developed to objectively and quantitatively measure surface accumulation of Pseudomonas putida from photographs and results weremore » compared to independent measurements of cell density. Results from image analysis quantified biofilm growth intensity accurately and with approximately the same precision of the more laborious cell counting method. This simple method for early stage biofilm detection enables quantifiable measurement of surface fouling and is flexible enough to be applied from the laboratory to the field. Broad spectrum staining highlights fouling biomass, photography quickly captures a large area of interest, and image analysis rapidly quantifies fouling in the image.« less

Envisioning migration: Mathematics in both experimental analysis and modeling of cell behavior

PubMed Central

Zhang, Elizabeth R.; Wu, Lani F.; Altschuler, Steven J.

2013-01-01

The complex nature of cell migration highlights the power and challenges of applying mathematics to biological studies. Mathematics may be used to create model equations that recapitulate migration, which can predict phenomena not easily uncovered by experiments or intuition alone. Alternatively, mathematics may be applied to interpreting complex data sets with better resolution—potentially empowering scientists to discern subtle patterns amid the noise and heterogeneity typical of migrating cells. Iteration between these two methods is necessary in order to reveal connections within the cell migration signaling network, as well as to understand the behavior that arises from those connections. Here, we review recent quantitative analysis and mathematical modeling approaches to the cell migration problem. PMID:23660413

Envisioning migration: mathematics in both experimental analysis and modeling of cell behavior.

PubMed

Zhang, Elizabeth R; Wu, Lani F; Altschuler, Steven J

2013-10-01

The complex nature of cell migration highlights the power and challenges of applying mathematics to biological studies. Mathematics may be used to create model equations that recapitulate migration, which can predict phenomena not easily uncovered by experiments or intuition alone. Alternatively, mathematics may be applied to interpreting complex data sets with better resolution--potentially empowering scientists to discern subtle patterns amid the noise and heterogeneity typical of migrating cells. Iteration between these two methods is necessary in order to reveal connections within the cell migration signaling network, as well as to understand the behavior that arises from those connections. Here, we review recent quantitative analysis and mathematical modeling approaches to the cell migration problem. Copyright © 2013 Elsevier Ltd. All rights reserved.

Quantitative proteomic analysis of human breast epithelial cells with differential telomere length

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Li-Rong; Chan, King C.; Tahara, Hidetoshi

Telomeres play important functional roles in cell proliferation, cell cycle regulation, and genetic stability, in which telomere length is critical. In this study, quantitative proteome comparisons for the human breast epithelial cells with short and long telomeres (184-hTERT{sub L} vs. 184-hTERT{sub S} and 90P-hTERT{sub L} vs. 90P-hTERT{sub S}), resulting from transfection of the human telomerase reverse transcriptase (hTERT) gene, were performed using cleavable isotope-coded affinity tags. More than 2000 proteins were quantified in each comparative experiment, with approximately 77% of the proteins identified in both analyses. In the cells with long telomeres, significant and consistent alterations were observed in metabolismmore » (amino acid, nucleotide, and lipid metabolism), genetic information transmission (transcription and translation regulation, spliceosome and ribosome complexes), and cell signaling. Interestingly, the DNA excision repair pathway is enhanced, while integrin and its ligands are downregulated in the cells with long telomeres. These results may provide valuable information related to telomere functions.« less

Deep Learning in Label-free Cell Classification

PubMed Central

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

2016-01-01

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells. PMID:26975219

Deep Learning in Label-free Cell Classification

NASA Astrophysics Data System (ADS)

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

2016-03-01

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.

Analysis of cell division patterns in the Arabidopsis shoot apical meristem

DOE PAGES

Shapiro, Bruce E.; Tobin, Cory; Mjolsness, Eric; ...

2015-03-30

The stereotypic pattern of cell shapes in the Arabidopsis shoot apical meristem (SAM) suggests that strict rules govern the placement of new walls during cell division. When a cell in the SAM divides, a new wall is built that connects existing walls and divides the cytoplasm of the daughter cells. Because features that are determined by the placement of new walls such as cell size, shape, and number of neighbors are highly regular, rules must exist for maintaining such order. Here in this paper we present a quantitative model of these rules that incorporates different observed features of cell division.more » Each feature is incorporated into a "potential function" that contributes a single term to a total analog of potential energy. New cell walls are predicted to occur at locations where the potential function is minimized. Quantitative terms that represent the well-known historical rules of plant cell division, such as those given by Hofmeister, Errera, and Sachs are developed and evaluated against observed cell divisions in the epidermal layer (L1) of Arabidopsis thaliana SAM. The method is general enough to allow additional terms for nongeometric properties such as internal concentration gradients and mechanical tensile forces.« less

Development of a facile droplet-based single-cell isolation platform for cultivation and genomic analysis in microorganisms.

PubMed

Zhang, Qiang; Wang, Tingting; Zhou, Qian; Zhang, Peng; Gong, Yanhai; Gou, Honglei; Xu, Jian; Ma, Bo

2017-01-23

Wider application of single-cell analysis has been limited by the lack of an easy-to-use and low-cost strategy for single-cell isolation that can be directly coupled to single-cell sequencing and single-cell cultivation, especially for small-size microbes. Herein, a facile droplet microfluidic platform was developed to dispense individual microbial cells into conventional standard containers for downstream analysis. Functional parts for cell encapsulation, droplet inspection and sorting, as well as a chip-to-tube capillary interface were integrated on one single chip with simple architecture, and control of the droplet sorting was achieved by a low-cost solenoid microvalve. Using microalgal and yeast cells as models, single-cell isolation success rate of over 90% and single-cell cultivation success rate of 80% were demonstrated. We further showed that the individual cells isolated can be used in high-quality DNA and RNA analyses at both gene-specific and whole-genome levels (i.e. real-time quantitative PCR and genome sequencing). The simplicity and reliability of the method should improve accessibility of single-cell analysis and facilitate its wider application in microbiology researches.

Development of a facile droplet-based single-cell isolation platform for cultivation and genomic analysis in microorganisms

PubMed Central

Zhang, Qiang; Wang, Tingting; Zhou, Qian; Zhang, Peng; Gong, Yanhai; Gou, Honglei; Xu, Jian; Ma, Bo

2017-01-01

Wider application of single-cell analysis has been limited by the lack of an easy-to-use and low-cost strategy for single-cell isolation that can be directly coupled to single-cell sequencing and single-cell cultivation, especially for small-size microbes. Herein, a facile droplet microfluidic platform was developed to dispense individual microbial cells into conventional standard containers for downstream analysis. Functional parts for cell encapsulation, droplet inspection and sorting, as well as a chip-to-tube capillary interface were integrated on one single chip with simple architecture, and control of the droplet sorting was achieved by a low-cost solenoid microvalve. Using microalgal and yeast cells as models, single-cell isolation success rate of over 90% and single-cell cultivation success rate of 80% were demonstrated. We further showed that the individual cells isolated can be used in high-quality DNA and RNA analyses at both gene-specific and whole-genome levels (i.e. real-time quantitative PCR and genome sequencing). The simplicity and reliability of the method should improve accessibility of single-cell analysis and facilitate its wider application in microbiology researches. PMID:28112223

Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo.

PubMed

Rice, G C; Bump, E A; Shrieve, D C; Lee, W; Kovacs, M

1986-12-01

An assay using a bimane derivative has been developed to detect free glutathione (GSH) in individual viable cells by flow cytometry. Monochlorobimane [syn-(ClCH2CH3)-1,5-diazabicycla[3.30]acta-3,6-diene-2,8-dio ne], itself nonfluorescent, reacts with GSH to form a highly fluorescent derivative. High pressure liquid chromatography analysis showed that, using specific staining conditions, the only low molecular weight fluorescent derivative formed in Chinese hamster ovary cells was that formed with GSH. Very little reaction with protein sulfhydryls was observed. Rates of GSH depletion in Chinese hamster ovary cells exposed to diethylmaleate were essentially the same, whether measured by relative fluorescence intensity, by flow cytometry or by enzymatic assay on cellular extracts. This method was shown to be useful for measurement of GSH resynthesis, uptake, and depletion by prolonged hypoxia and misonidazole treatment. Since measurements are made on individual cells, cell-to-cell variation and populational heterogeneity in GSH content are revealed by flow cytometry. Although under most conditions in vitro GSH content is relatively homogeneous, under certain circumstances, such as release from hypoxia, heterogeneity in populational GSH levels was observed. The significance of this heterogeneity is discussed in regard to the induction of gene amplification and drug resistance by transient hypoxia. Numerous subclones of Chinese hamster ovary cells selected by growth in Adriamycin or methotrexate-containing medium express elevated levels of GSH per cell. The method was extended to quantitate the GSH content of cells excised from EMT-6/SF mouse tumors that had been treated in vivo with L-buthionine-S-R-sulfoximine, an inhibitor of GSH synthesis. The bivariate analysis (forward angle light scatter versus monochlorobimane fluorescence) of cells derived from these tumors gave excellent resolution of normal and tumor cells and demonstrated extensive heterogeneity in the tumor cell population with respect to GSH content per cell.

Identification of Immunogenic Targets for Lung Cancer Vaccines

DTIC Science & Technology

2017-09-01

quantitative proteomic analysis to identify proteins overexpressed in non-small cell lung cancer cell lines compared with normal lung epithelial...Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for Public Release; Distribution...Department of the Army position, policy or decision unless so designated by other documentation. REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704-0188

Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.

2006-07-01

Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewermore » stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1 > LPA4 > LPA2 >> LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G protein-coupled receptor LPA1.« less

Non-invasive quality evaluation of confluent cells by image-based orientation heterogeneity analysis.

PubMed

Sasaki, Kei; Sasaki, Hiroto; Takahashi, Atsuki; Kang, Siu; Yuasa, Tetsuya; Kato, Ryuji

2016-02-01

In recent years, cell and tissue therapy in regenerative medicine have advanced rapidly towards commercialization. However, conventional invasive cell quality assessment is incompatible with direct evaluation of the cells produced for such therapies, especially in the case of regenerative medicine products. Our group has demonstrated the potential of quantitative assessment of cell quality, using information obtained from cell images, for non-invasive real-time evaluation of regenerative medicine products. However, image of cells in the confluent state are often difficult to evaluate, because accurate recognition of cells is technically difficult and the morphological features of confluent cells are non-characteristic. To overcome these challenges, we developed a new image-processing algorithm, heterogeneity of orientation (H-Orient) processing, to describe the heterogeneous density of cells in the confluent state. In this algorithm, we introduced a Hessian calculation that converts pixel intensity data to orientation data and a statistical profiling calculation that evaluates the heterogeneity of orientations within an image, generating novel parameters that yield a quantitative profile of an image. Using such parameters, we tested the algorithm's performance in discriminating different qualities of cellular images with three types of clinically important cell quality check (QC) models: remaining lifespan check (QC1), manipulation error check (QC2), and differentiation potential check (QC3). Our results show that our orientation analysis algorithm could predict with high accuracy the outcomes of all types of cellular quality checks (>84% average accuracy with cross-validation). Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Micrometer-sized iron oxide particle labeling of mesenchymal stem cells for magnetic resonance imaging-based monitoring of cartilage tissue engineering.

PubMed

Saldanha, Karl J; Doan, Ryan P; Ainslie, Kristy M; Desai, Tejal A; Majumdar, Sharmila

2011-01-01

To examine mesenchymal stem cell (MSC) labeling with micrometer-sized iron oxide particles (MPIOs) for magnetic resonance imaging (MRI)-based tracking and its application to monitoring articular cartilage regeneration. Rabbit MSCs were labeled using commercial MPIOs. In vitro MRI was performed with gradient echo (GRE) and spin echo (SE) sequences at 3T and quantitatively characterized using line profile and region of interest analysis. Ex vivo MRI of hydrogel-encapsulated labeled MSCs implanted within a bovine knee was performed with spoiled GRE (SPGR) and T(1ρ) sequences. Fluorescence microscopy, labeling efficiency, and chondrogenesis of MPIO-labeled cells were also examined. MPIO labeling results in efficient contrast uptake and signal loss that can be visualized and quantitatively characterized via MRI. SPGR imaging of implanted cells results in ex vivo detection within native tissue, and T(1ρ) imaging is unaffected by the presence of labeled cells immediately following implantation. MPIO labeling does not affect quantitative glycosaminoglycan production during chondrogenesis, but iron aggregation hinders extracellular matrix visualization. This aggregation may result from excess unincorporated particles following labeling and is an issue that necessitates further investigation. This study demonstrates the promise of MPIO labeling for monitoring cartilage regeneration and highlights its potential in the development of cell-based tissue engineering strategies. Published by Elsevier Inc.

Nucleolar molecular signature of pluripotent stem cells.

PubMed

Pliss, Artem; Kuzmin, Andrey N; Kachynski, Aliaksandr V; Jiang, Houbo; Hu, Zhixing; Ren, Yong; Feng, Jian; Prasad, Paras N

2013-04-02

Induced pluripotent stem cells (iPSC) are generated by reprogramming somatic cells to the pluripotent state. Identification and quantitative characterization of changes in the molecular organization of the cell during the process of cellular reprogramming is valuable for stem cell research and advancement of its therapeutic applications. Here we employ quantitative Raman microspectroscopy and biomolecular component analysis (BCA) for a comparative analysis of the molecular composition of nucleoli in skin fibroblasts and iPSC derived from them. We report that the cultured fibroblasts obtained from different human subjects, share comparable concentrations of proteins, RNA, DNA, and lipids in the molecular composition of nucleoli. The nucleolar molecular environment is drastically changed in the corresponding iPSC. We measured that the transition from skin fibroblasts to iPSC is accompanied by a statistically significant increase in protein concentrations ~1.3-fold, RNA concentrations ~1.3-fold, and DNA concentrations ~1.4-fold, while no statistically significant difference was found for the lipid concentrations. The analysis of molecular vibrations associated with diverse aminoacids and protein conformations indicates that nucleoli of skin fibroblasts contain similar subsets of proteins, with prevalence of tyrosine. In iPSC, we observed a higher signal from tryptophan with an increase in the random coil and α helix protein conformations, indicating changes in the subset of nucleolar proteins during cell reprogramming. At the same time, the concentrations of major types of macromolecules and protein conformations in the nucleoli of iPSC and human embryonic stem cells (hESC) were found to be similar. We discuss these results in the context of nucleolar function and conclude that the nucleolar molecular content is correlated with the cellular differentiation status. The approach described here shows the potential for spectroscopically monitoring changes in macromolecular organization of the cell at different stages of reprogramming.

Endorsing cellular competitiveness in aberrant epithelium of oral submucous fibrosis progression: neighbourhood analysis of immunohistochemical attributes.

PubMed

Anura, Anji; Kazi, Anees; Pal, Mousumi; Paul, Ranjan Rashmi; Sengupta, Sanghamitra; Chatterjee, Jyotirmoy

2018-04-23

Epithelial abnormality during the transformation of oral submucous fibrosis (OSF) into oral squamous cell carcinoma has been well studied and documented. However, the differential contribution of atrophy and hyperplasia for malignant potentiality of OSF is yet to be resolved. Existing diagnostic conjectures lack precise diagnostic attributes which may be effectively resolved by substantiation of specific molecular pathology signatures. Present study elucidates existence of cellular competitiveness in OSF conditions using computer-assisted neighbourhood analysis in quantitative immunohistochemistry (IHC) framework. The concept of field cancerization was contributory in finding correspondence among neighbouring cells of epithelial layers with reference to differential expression of cardinal cancer-related genes [c-Myc (oncogene), p53 (tumour suppressor), and HIF-1α (hypoxia regulator)] which are known to be important sensors in recognizing cellular competitive interface. Our analyses indicate that different states of OSF condition may be associated with different forms of competitiveness within epithelial neighbouring cells which might be responsible to shape the present and future of the pre-malignant condition. Analytical findings indicated association of atrophic epithelium with stress-driven competitive environment having low c-Myc, high-p53, and stable HIF-1α (the looser cells) which undergo apoptosis. Whereas, the cells with high c-Myc + (winner cells) give rise to hyperplastic epithelium via possible mutation in p53. The epithelial dysplasia plausibly occurs due to clonal expansion of c-Myc and p53 positive supercompetitor cells. Present study proposes quantitative IHC along with neighbourhood analysis which might help us to dig deeper on to the interaction among epithelial cell population to provide a better understanding of field cancerization and malignant transformation of pre-malignancy.

Correlation of quantitative histopathological morphology and quantitative radiological analysis during aseptic loosening of hip endoprostheses.

PubMed

Bertz, S; Kriegsmann, J; Eckardt, A; Delank, K-S; Drees, P; Hansen, T; Otto, M

2006-01-01

Aseptic hip prosthesis loosening is the most important long-term complication in total hip arthroplasty. Polyethylene (PE) wear is the dominant etiologic factor in aseptic loosening, which together with other factors induces mechanisms resulting in bone loss, and finally in implant loosening. The single-shot radiograph analysis (EBRA, abbreviation for the German term "Einzel-Bild-Röntgenanalyse") is a computerized method for early radiological prediction of aseptic loosening. In this study, EBRA parameters were correlated with histomorphological parameters of the periprosthetic membrane. Periprosthetic membranes obtained from 19 patients during revision surgery of loosened ABG I-type total hip pros-theses were analyzed histologically and morphometrically. The pre-existing EBRA parameters, the thickness of the PE debris lay-er and the dimension of inclination and anteversion, were compared with the density of macrophages and giant cells. Addi-tionally, the semiquantitatively determined density of lymphocytes, plasma cells, giant cells and the size of the necrotic areas were correlated with the EBRA results. All periprosthetic membranes were classified as debris-induced type membranes. We found a positive correlation between the number of giant cells and the thickness of the PE debris layer. There was no significant correlation between the number of macrophages or all semiquantitative parameters and EBRA parameters. The number of giant cells decreased with implant duration. The morphometrically measured number of foreign body giant cells more closely reflects the results of the EBRA. The semiquantitative estimation of giant cell density could not substitute for the morphometrical analysis. The density of macrophages, lymphocytes, plasma cells and the size of necrotic areas did not correlate with the EBRA parameters, indicating that there is no correlation with aseptic loosening.

Quantitative proteomic profiling of paired cancerous and normal colon epithelial cells isolated freshly from colorectal cancer patients.

PubMed

Tu, Chengjian; Mojica, Wilfrido; Straubinger, Robert M; Li, Jun; Shen, Shichen; Qu, Miao; Nie, Lei; Roberts, Rick; An, Bo; Qu, Jun

2017-05-01

The heterogeneous structure in tumor tissues from colorectal cancer (CRC) patients excludes an informative comparison between tumors and adjacent normal tissues. Here, we develop and apply a strategy to compare paired cancerous (CEC) versus normal (NEC) epithelial cells enriched from patients and discover potential biomarkers and therapeutic targets for CRC. CEC and NEC cells are respectively isolated from five different tumor and normal locations in the resected colon tissue from each patient (N = 12 patients) using an optimized epithelial cell adhesion molecule (EpCAM)-based enrichment approach. An ion current-based quantitative method is employed to perform comparative proteomic analysis for each patient. A total of 458 altered proteins that are common among >75% of patients are observed and selected for further investigation. Besides known findings such as deregulation of mitochondrial function, tricarboxylic acid cycle, and RNA post-transcriptional modification, functional analysis further revealed RAN signaling pathway, small nucleolar ribonucleoproteins (snoRNPs), and infection by RNA viruses are altered in CEC cells. A selection of the altered proteins of interest is validated by immunohistochemistry analyses. The informative comparison between matched CEC and NEC enhances our understanding of molecular mechanisms of CRC development and provides biomarker candidates and new pathways for therapeutic intervention. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative Analysis of Cell Migration Using Optical Flow

PubMed Central

Boric, Katica; Orio, Patricio; Viéville, Thierry; Whitlock, Kathleen

2013-01-01

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. PMID:23936049

StWRKY8 transcription factor regulates benzylisoquinoline alkaloid pathway in potato conferring resistance to late blight.

PubMed

Yogendra, Kalenahalli N; Dhokane, Dhananjay; Kushalappa, Ajjamada C; Sarmiento, Felipe; Rodriguez, Ernesto; Mosquera, Teresa

2017-03-01

The resistance to late blight is either qualitative or quantitative in nature. Quantitative resistance is durable, but challenging due to polygenic inheritance. In the present study, the diploid potato genotypes resistant and susceptible to late blight, were profiled for metabolites. Tissue specific metabolite analysis of benzylisoquinoline alkaloids (BIAs) in response to pathogen infection revealed increased accumulation of morphinone, codeine-6-glucuronide and morphine-3-glucuronides. These BIAs are antimicrobial compounds and possibly involved in cell wall reinforcement, especially through cross-linking cell wall pectins. Quantitative reverse transcription-PCR studies revealed higher expressions of TyDC, NCS, COR-2 and StWRKY8 transcription factor genes, in resistant genotypes than in susceptible genotype, following pathogen inoculation. A luciferase transient expression assay confirmed the binding of the StWRKY8 TF to promoters of downstream genes, elucidating a direct regulatory role on BIAs biosynthetic genes. Sequence analysis of StWRKY8 in potato genotypes revealed polymorphism in the WRKY DNA binding domain in the susceptible genotype, which is important for the regulatory function of this gene. A complementation assay of StWRKY8 in Arabidopsis wrky33 mutant background was associated with decreased fungal biomass. In conclusion, StWRKY8 regulates the biosynthesis of BIAs that are both antimicrobial and reinforce cell walls to contain the pathogen to initial infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Response of Human Osteoblast to n-HA/PEEK—Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite

NASA Astrophysics Data System (ADS)

Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng

2016-03-01

Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn’t increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca2+ concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery.

Response of Human Osteoblast to n-HA/PEEK--Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite.

PubMed

Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng

2016-03-09

Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn't increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca(2+) concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery.

Expression and immunoaffinity purification of recombinant soluble human GPR56 protein for the analysis of GPR56 receptor shedding by ELISA.

PubMed

Yang, Tai-Yun; Chiang, Nien-Yi; Tseng, Wen-Yi; Pan, Hsiao-Lin; Peng, Yen-Ming; Shen, Jiann-Jong; Wu, Kuo-An; Kuo, Ming-Ling; Chang, Gin-Wen; Lin, Hsi-Hsien

2015-05-01

GPR56 is a multi-functional adhesion-class G protein-coupled receptor involved in biological systems as diverse as brain development, male gonad development, myoblast fusion, hematopoietic stem cell maintenance, tumor growth and metastasis, and immune-regulation. Ectodomain shedding of human GPR56 receptor has been demonstrated previously, however the quantitative detection of GPR56 receptor shedding has not been investigated fully due to the lack of appropriate assays. Herein, an efficient system of expression and immune-affinity purification of the recombinant soluble extracellular domain of human GPR56 (sGPR56) protein from a stably transduced human melanoma cell line was established. The identity and functionality of the recombinant human sGPR56 protein were verified by Western blotting and mass spectrometry, and ligand-binding assays, respectively. Combined with the use of two recently generated anti-GPR56 monoclonal antibodies, a sensitive sandwich ELISA assay was successfully developed for the quantitative detection of human sGPR56 molecule. We found that GPR56 receptor shedding occurred constitutively and was further increased in activated human melanoma cells expressing endogenous GPR56. In conclusion, we report herein an efficient system for the production and purification of human sGPR56 protein for the establishment of a quantitative ELISA analysis of GPR56 receptor shedding. Copyright © 2014 Elsevier Inc. All rights reserved.

Quantitative Analysis of Electrochemical and Electrode Stability with Low Self-Discharge Lithium-Sulfur Batteries

DOE PAGES

Chung, Sheng-Heng; Han, Pauline; Manthiram, Arumugam

2017-06-07

The viability of employing high-capacity sulfur cathodes in building high-energy-density lithium-sulfur batteries is limited by rapid self-discharge, short shelf life, and severe structural degradation during cell resting (static instability). Unfortunately, the static instability has largely been ignored in the literature. We present in this letter a longterm self-discharge study by quantitatively analyzing the control lithium-sulfur batteries with a conventional cathode configuration, which provides meaningful insights into the cathode failure mechanisms during resting. Lastly, utilizing the understanding obtained with the control cells, we design and present low self-discharge (LSD) lithium-sulfur batteries for investigating the long-term self-discharge effect and electrode stability.

Phase noise optimization in temporal phase-shifting digital holography with partial coherence light sources and its application in quantitative cell imaging.

PubMed

Remmersmann, Christian; Stürwald, Stephan; Kemper, Björn; Langehanenberg, Patrik; von Bally, Gert

2009-03-10

In temporal phase-shifting-based digital holographic microscopy, high-resolution phase contrast imaging requires optimized conditions for hologram recording and phase retrieval. To optimize the phase resolution, for the example of a variable three-step algorithm, a theoretical analysis on statistical errors, digitalization errors, uncorrelated errors, and errors due to a misaligned temporal phase shift is carried out. In a second step the theoretically predicted results are compared to the measured phase noise obtained from comparative experimental investigations with several coherent and partially coherent light sources. Finally, the applicability for noise reduction is demonstrated by quantitative phase contrast imaging of pancreas tumor cells.

Quantitative Analysis of Electrochemical and Electrode Stability with Low Self-Discharge Lithium-Sulfur Batteries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chung, Sheng-Heng; Han, Pauline; Manthiram, Arumugam

The viability of employing high-capacity sulfur cathodes in building high-energy-density lithium-sulfur batteries is limited by rapid self-discharge, short shelf life, and severe structural degradation during cell resting (static instability). Unfortunately, the static instability has largely been ignored in the literature. We present in this letter a longterm self-discharge study by quantitatively analyzing the control lithium-sulfur batteries with a conventional cathode configuration, which provides meaningful insights into the cathode failure mechanisms during resting. Lastly, utilizing the understanding obtained with the control cells, we design and present low self-discharge (LSD) lithium-sulfur batteries for investigating the long-term self-discharge effect and electrode stability.

Comparative analysis of quantitative methodologies for Vibrionaceae biofilms.

PubMed

Chavez-Dozal, Alba A; Nourabadi, Neda; Erken, Martina; McDougald, Diane; Nishiguchi, Michele K

2016-11-01

Multiple symbiotic and free-living Vibrio spp. grow as a form of microbial community known as a biofilm. In the laboratory, methods to quantify Vibrio biofilm mass include crystal violet staining, direct colony-forming unit (CFU) counting, dry biofilm cell mass measurement, and observation of development of wrinkled colonies. Another approach for bacterial biofilms also involves the use of tetrazolium (XTT) assays (used widely in studies of fungi) that are an appropriate measure of metabolic activity and vitality of cells within the biofilm matrix. This study systematically tested five techniques, among which the XTT assay and wrinkled colony measurement provided the most reproducible, accurate, and efficient methods for the quantitative estimation of Vibrionaceae biofilms.

Label-Free Relative Quantitation of Isobaric and Isomeric Human Histone H2A and H2B Variants by Fourier Transform Ion Cyclotron Resonance Top-Down MS/MS.

PubMed

Dang, Xibei; Singh, Amar; Spetman, Brian D; Nolan, Krystal D; Isaacs, Jennifer S; Dennis, Jonathan H; Dalton, Stephen; Marshall, Alan G; Young, Nicolas L

2016-09-02

Histone variants are known to play a central role in genome regulation and maintenance. However, many variants are inaccessible by antibody-based methods or bottom-up tandem mass spectrometry due to their highly similar sequences. For many, the only tractable approach is with intact protein top-down tandem mass spectrometry. Here, ultra-high-resolution FT-ICR MS and MS/MS yield quantitative relative abundances of all detected HeLa H2A and H2B isobaric and isomeric variants with a label-free approach. We extend the analysis to identify and relatively quantitate 16 proteoforms from 12 sequence variants of histone H2A and 10 proteoforms of histone H2B from three other cell lines: human embryonic stem cells (WA09), U937, and a prostate cancer cell line LaZ. The top-down MS/MS approach provides a path forward for more extensive elucidation of the biological role of many previously unstudied histone variants and post-translational modifications.

Biological Dynamics Markup Language (BDML): an open format for representing quantitative biological dynamics data

PubMed Central

Kyoda, Koji; Tohsato, Yukako; Ho, Kenneth H. L.; Onami, Shuichi

2015-01-01

Motivation: Recent progress in live-cell imaging and modeling techniques has resulted in generation of a large amount of quantitative data (from experimental measurements and computer simulations) on spatiotemporal dynamics of biological objects such as molecules, cells and organisms. Although many research groups have independently dedicated their efforts to developing software tools for visualizing and analyzing these data, these tools are often not compatible with each other because of different data formats. Results: We developed an open unified format, Biological Dynamics Markup Language (BDML; current version: 0.2), which provides a basic framework for representing quantitative biological dynamics data for objects ranging from molecules to cells to organisms. BDML is based on Extensible Markup Language (XML). Its advantages are machine and human readability and extensibility. BDML will improve the efficiency of development and evaluation of software tools for data visualization and analysis. Availability and implementation: A specification and a schema file for BDML are freely available online at http://ssbd.qbic.riken.jp/bdml/. Contact: sonami@riken.jp Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:25414366

Biological Dynamics Markup Language (BDML): an open format for representing quantitative biological dynamics data.

PubMed

Kyoda, Koji; Tohsato, Yukako; Ho, Kenneth H L; Onami, Shuichi

2015-04-01

Recent progress in live-cell imaging and modeling techniques has resulted in generation of a large amount of quantitative data (from experimental measurements and computer simulations) on spatiotemporal dynamics of biological objects such as molecules, cells and organisms. Although many research groups have independently dedicated their efforts to developing software tools for visualizing and analyzing these data, these tools are often not compatible with each other because of different data formats. We developed an open unified format, Biological Dynamics Markup Language (BDML; current version: 0.2), which provides a basic framework for representing quantitative biological dynamics data for objects ranging from molecules to cells to organisms. BDML is based on Extensible Markup Language (XML). Its advantages are machine and human readability and extensibility. BDML will improve the efficiency of development and evaluation of software tools for data visualization and analysis. A specification and a schema file for BDML are freely available online at http://ssbd.qbic.riken.jp/bdml/. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis.

PubMed

Zhang, Xue; Zhang, Chong; Zhou, Qian-Qian; Zhang, Xiao-Fei; Wang, Li-Yan; Chang, Hai-Bo; Li, He-Ping; Oda, Yoshimitsu; Xing, Xin-Hui

2015-07-01

DNA damage is the dominant source of mutation, which is the driving force of evolution. Therefore, it is important to quantitatively analyze the DNA damage caused by different mutagenesis methods, the subsequent mutation rates, and their relationship. Atmospheric and room temperature plasma (ARTP) mutagenesis has been used for the mutation breeding of more than 40 microorganisms. However, ARTP mutagenesis has not been quantitatively compared with conventional mutation methods. In this study, the umu test using a flow-cytometric analysis was developed to quantify the DNA damage in individual viable cells using Salmonella typhimurium NM2009 as the model strain and to determine the mutation rate. The newly developed method was used to evaluate four different mutagenesis systems: a new ARTP tool, ultraviolet radiation, 4-nitroquinoline-1-oxide (4-NQO), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The mutation rate was proportional to the corresponding SOS response induced by DNA damage. ARTP caused greater DNA damage to individual living cells than the other conventional mutagenesis methods, and the mutation rate was also higher. By quantitatively comparing the DNA damage and consequent mutation rate after different types of mutagenesis, we have shown that ARTP is a potentially powerful mutagenesis tool with which to improve the characteristics of microbial cell factories.

GAPDH, β-actin and β2-microglobulin, as three common reference genes, are not reliable for gene expression studies in equine adipose- and marrow-derived mesenchymal stem cells.

PubMed

Nazari, Fatemeh; Parham, Abbas; Maleki, Adham Fani

2015-01-01

Quantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on expression level of three common reference genes (GAPDH, β-actin and β2-microglobulin) in equine marrow- and adipose- derived undifferentiated MSCs and consequently their reliability for comparative qRT-PCR. Adipose tissue (AT) and bone marrow (BM) samples were harvested from 3 mares. MSCs were isolated and cultured until passage 3 (P3). Total RNA of P3 cells was extracted for cDNA synthesis. The generated cDNAs were analyzed by quantitative real-time PCR. The PCR reactions were ended with a melting curve analysis to verify the specificity of amplicon. The expression levels of GAPDH were significantly different between AT- and BM- derived MSCs (p < 0.05). Differences in expression level of β-actin (P < 0.001) and B2M (P < 0.006.) between MSCs derived from AT and BM were substantially higher than GAPDH. In addition, the fold change in expression levels of GAPDH, β-actin and B2M in AT-derived MSCs compared to BM-derived MSCs were 2.38, 6.76 and 7.76, respectively. This study demonstrated that GAPDH and especially β-actin and B2M express in different levels in equine AT- and BM- derived MSCs. Thus they cannot be considered as reliable reference genes for comparative quantitative gene expression analysis in MSCs derived from equine bone marrow and adipose tissue.

Quantification of focal adhesion dynamics of cell movement based on cell-induced collagen matrix deformation using second-harmonic generation microscopy.

PubMed

Kang, Yong Guk; Jang, Hwanseok; Yang, Taeseok Daniel; Notbohm, Jacob; Choi, Youngwoon; Park, Yongdoo; Kim, Beop-Min

2018-06-01

Mechanical interactions of living cells with the surrounding environment via focal adhesion (FA) in three dimensions (3-D) play a key role in dynamic biological events, such as tissue regeneration, wound healing, and cancer invasion. Recently, several methods for observing 3-D cell-extracellular matrix (ECM) interactions have been reported, lacking solid and quantitative analysis on the dynamics of the physical interaction between the cell and the ECM. We measured the submicron displacements of ECM deformation in 3-D due to protrusion-retraction dynamics during cell migration, using second-harmonic generation without labeling the matrix structures. We then quantitatively analyzed the mechanical deformation between the ECM and the cells based on spatiotemporal volumetric correlations. The greatest deformations within the collagen matrix were found to occur at sites of colocalization of the FA site-related proteins vinculin and actin, which confirms that FA sites play a critical role in living cells within the ECM as a point for adhesion, traction, and migration. We believe that this modality can be used in studies of cell-ECM interaction during angiogenesis, wound healing, and metastasis. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Quantitative proteomic characterization of the lung extracellular matrix in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.

PubMed

Åhrman, Emma; Hallgren, Oskar; Malmström, Lars; Hedström, Ulf; Malmström, Anders; Bjermer, Leif; Zhou, Xiao-Hong; Westergren-Thorsson, Gunilla; Malmström, Johan

2018-03-01

Remodeling of the extracellular matrix (ECM) is a common feature in lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Here, we applied a sequential tissue extraction strategy to describe disease-specific remodeling of human lung tissue in disease, using end-stages of COPD and IPF. Our strategy was based on quantitative comparison of the disease proteomes, with specific focus on the matrisome, using data-independent acquisition and targeted data analysis (SWATH-MS). Our work provides an in-depth proteomic characterization of human lung tissue during impaired tissue remodeling. In addition, we show important quantitative and qualitative effects of the solubility of matrisome proteins. COPD was characterized by a disease-specific increase in ECM regulators, metalloproteinase inhibitor 3 (TIMP3) and matrix metalloproteinase 28 (MMP-28), whereas for IPF, impairment in cell adhesion proteins, such as collagen VI and laminins, was most prominent. For both diseases, we identified increased levels of proteins involved in the regulation of endopeptidase activity, with several proteins belonging to the serpin family. The established human lung quantitative proteome inventory and the construction of a tissue-specific protein assay library provides a resource for future quantitative proteomic analyses of human lung tissues. We present a sequential tissue extraction strategy to determine changes in extractability of matrisome proteins in end-stage COPD and IPF compared to healthy control tissue. Extensive quantitative analysis of the proteome changes of the disease states revealed altered solubility of matrisome proteins involved in ECM regulators and cell-ECM communication. The results highlight disease-specific remodeling mechanisms associated with COPD and IPF. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantitative identification of senescent cells in aging and disease.

PubMed

Biran, Anat; Zada, Lior; Abou Karam, Paula; Vadai, Ezra; Roitman, Lior; Ovadya, Yossi; Porat, Ziv; Krizhanovsky, Valery

2017-08-01

Senescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single-cell basis. The method combines a senescence-associated beta-galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high-content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Combinatorial peptide libraries and biometric score matrices permit the quantitative analysis of specific and degenerate interactions between clonotypic TCR and MHC peptide ligands.

PubMed

Zhao, Y; Gran, B; Pinilla, C; Markovic-Plese, S; Hemmer, B; Tzou, A; Whitney, L W; Biddison, W E; Martin, R; Simon, R

2001-08-15

The interaction of TCRs with MHC peptide ligands can be highly flexible, so that many different peptides are recognized by the same TCR in the context of a single restriction element. We provide a quantitative description of such interactions, which allows the identification of T cell epitopes and molecular mimics. The response of T cell clones to positional scanning synthetic combinatorial libraries is analyzed with a mathematical approach that is based on a model of independent contribution of individual amino acids to peptide Ag recognition. This biometric analysis compares the information derived from these libraries composed of trillions of decapeptides with all the millions of decapeptides contained in a protein database to rank and predict the most stimulatory peptides for a given T cell clone. We demonstrate the predictive power of the novel strategy and show that, together with gene expression profiling by cDNA microarrays, it leads to the identification of novel candidate autoantigens in the inflammatory autoimmune disease, multiple sclerosis.

Fluorescence-based detection and quantification of features of cellular senescence.

PubMed

Cho, Sohee; Hwang, Eun Seong

2011-01-01

Cellular senescence is a spontaneous organismal defense mechanism against tumor progression which is raised upon the activation of oncoproteins or other cellular environmental stresses that must be circumvented for tumorigenesis to occur. It involves growth-arrest state of normal cells after a number of active divisions. There are multiple experimental routes that can drive cells into a state of senescence. Normal somatic cells and cancer cells enter a state of senescence upon overexpression of oncogenic Ras or Raf protein or by imposing certain kinds of stress such as cellular tumor suppressor function. Both flow cytometry and confocal imaging analysis techniques are very useful in quantitative analysis of cellular senescence phenomenon. They allow quantitative estimates of multiple different phenotypes expressed in multiple cell populations simultaneously. Here we review the various types of fluorescence methodologies including confocal imaging and flow cytometry that are frequently utilized to study a variety of senescence. First, we discuss key cell biological changes occurring during senescence and review the current understanding on the mechanisms of these changes with the goal of improving existing protocols and further developing new ones. Next, we list specific senescence phenotypes associated with each cellular trait along with the principles of their assay methods and the significance of the assay outcomes. We conclude by selecting appropriate references that demonstrate a typical example of each method. Copyright © 2011 Elsevier Inc. All rights reserved.

Methods for Characterization of Alternative RNA Splicing.

PubMed

Harvey, Samuel E; Cheng, Chonghui

2016-01-01

Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semi-quantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We also describe analysis of alternative splicing using a splicing "minigene" in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest.

Identification of internal control genes for quantitative expression analysis by real-time PCR in bovine peripheral lymphocytes.

PubMed

Spalenza, Veronica; Girolami, Flavia; Bevilacqua, Claudia; Riondato, Fulvio; Rasero, Roberto; Nebbia, Carlo; Sacchi, Paola; Martin, Patrice

2011-09-01

Gene expression studies in blood cells, particularly lymphocytes, are useful for monitoring potential exposure to toxicants or environmental pollutants in humans and livestock species. Quantitative PCR is the method of choice for obtaining accurate quantification of mRNA transcripts although variations in the amount of starting material, enzymatic efficiency, and the presence of inhibitors can lead to evaluation errors. As a result, normalization of data is of crucial importance. The most common approach is the use of endogenous reference genes as an internal control, whose expression should ideally not vary among individuals and under different experimental conditions. The accurate selection of reference genes is therefore an important step in interpreting quantitative PCR studies. Since no systematic investigation in bovine lymphocytes has been performed, the aim of the present study was to assess the expression stability of seven candidate reference genes in circulating lymphocytes collected from 15 dairy cows. Following the characterization by flow cytometric analysis of the cell populations obtained from blood through a density gradient procedure, three popular softwares were used to evaluate the gene expression data. The results showed that two genes are sufficient for normalization of quantitative PCR studies in cattle lymphocytes and that YWAHZ, S24 and PPIA are the most stable genes. Copyright © 2010 Elsevier Ltd. All rights reserved.

Targeting Neuronal-like Metabolism of Metastatic Tumor Cells as a Novel Therapy for Breast Cancer Brain Metastasis

DTIC Science & Technology

2017-03-01

Contribution to Project: Ian primarily focuses on developing tissue imaging pipeline and perform imaging data analysis . Funding Support: Partially...3D ReconsTruction), a multi-faceted image analysis pipeline , permitting quantitative interrogation of functional implications of heterogeneous... analysis pipeline , to observe and quantify phenotypic metastatic landscape heterogeneity in situ with spatial and molecular resolution. Our implementation

Spectrally And Temporally Resolved Low-Light Level Video Microscopy

NASA Astrophysics Data System (ADS)

Wampler, John E.; Furukawa, Ruth; Fechheimer, Marcus

1989-12-01

The IDG law-light video microscope system was designed to aid studies of localization of subcellular luminescence sources and stimulus/response coupling in single living cells using luminescent probes. Much of the motivation for design of this instrument system came from the pioneering efforts of Dr. Reynolds (Reynolds, Q. Rev. Biophys. 5, 295-347; Reynolds and Taylor, Bioscience 30, 586-592) who showed the value of intensified video camera systems for detection and localizion of fluorescence and bioluminescence signals from biological tissues. Our instrument system has essentially two roles, 1) localization and quantitation of very weak bioluminescence signals and 2) quantitation of intracellular environmental characteristics such as pH and calcium ion concentrations using fluorescent and bioluminescent probes. The instrument system exhibits over one million fold operating range allowing visualization and enhancement of quantum limited images with quantum limited response, spectral analysis of fluorescence signals, and transmitted light imaging. The computer control of the system implements rapid switching between light regimes, spatially resolved spectral scanning, and digital data processing for spectral shape analysis and for detailed analysis of the statistical distribution of single cell measurements. The system design and software algorithms used by the system are summarized. These design criteria are illustrated with examples taken from studies of bioluminescence, applications of bioluminescence to study developmental processes and gene expression in single living cells, and applications of fluorescent probes to study stimulus/response coupling in living cells.

Automated segmentation of retinal pigment epithelium cells in fluorescence adaptive optics images.

PubMed

Rangel-Fonseca, Piero; Gómez-Vieyra, Armando; Malacara-Hernández, Daniel; Wilson, Mario C; Williams, David R; Rossi, Ethan A

2013-12-01

Adaptive optics (AO) imaging methods allow the histological characteristics of retinal cell mosaics, such as photoreceptors and retinal pigment epithelium (RPE) cells, to be studied in vivo. The high-resolution images obtained with ophthalmic AO imaging devices are rich with information that is difficult and/or tedious to quantify using manual methods. Thus, robust, automated analysis tools that can provide reproducible quantitative information about the cellular mosaics under examination are required. Automated algorithms have been developed to detect the position of individual photoreceptor cells; however, most of these methods are not well suited for characterizing the RPE mosaic. We have developed an algorithm for RPE cell segmentation and show its performance here on simulated and real fluorescence AO images of the RPE mosaic. Algorithm performance was compared to manual cell identification and yielded better than 91% correspondence. This method can be used to segment RPE cells for morphometric analysis of the RPE mosaic and speed the analysis of both healthy and diseased RPE mosaics.

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

PubMed

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

Smooth muscle cells of penis in the rat: noninvasive quantification with shear wave elastography.

PubMed

Zhang, Jia-Jie; Qiao, Xiao-Hui; Gao, Feng; Bai, Ming; Li, Fan; Du, Lian-Fang; Xing, Jin-Fang

2015-01-01

Smooth muscle cells (SMCs) of cavernosum play an important role in erection. It is of great significance to quantitatively analyze the level of SMCs in penis. In this study, we investigated the feasibility of shear wave elastography (SWE) on evaluating the level of SMCs in penis quantitatively. Twenty healthy male rats were selected. The SWE imaging of penis was carried out and then immunohistochemistry analysis of penis was performed to analyze the expression of alpha smooth muscle actin in penis. The measurement index of SWE examination was tissue stiffness (TS). The measurement index of immunohistochemistry analysis was positive area percentage of alpha smooth muscle actin (AP). Sixty sets of data of TS and AP were obtained. The results showed that TS was significantly correlated with AP and the correlation coefficient was -0.618 (p < 0.001). The result of TS had been plotted against the AP measurements. The relation between the two results has been fitted with quadric curve; the goodness-of-fit index was 0.364 (p < 0.001). The level of SMCs in penis was successfully quantified in vivo with SWE. SWE can be used clinically for evaluating the level of SMCs in penis quantitatively.

Simple and rapid method for isolation and quantitation of polyhydroxyalkanoate by SDS-sonication treatment.

PubMed

Arikawa, Hisashi; Sato, Shunsuke; Fujiki, Tetsuya; Matsumoto, Keiji

2017-08-01

We developed a new method for isolation and quantitation of polyhydroxyalkanoate (PHA) from culture broth. In this method, the cells were sonicated in sodium dodecyl sulfate (SDS) solution and centrifuged to recover PHA. The recovered PHA was rinsed with deionized water and ethanol, and then weighed after drying. Hazardous chemicals such as chloroform, methanol, and sulfuric acid were not used, and no expensive analytical instruments were needed. We applied this method to Cupriavidus necator culture broths that included various amounts of poly(3-hydroxybutyrate) (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) from flasks and jar fermentors. The quantitation by this method was practical for use with a wide range of production amounts and PHA monomer compositions compared to the conventional whole-cell methanolysis method with gas chromatographic analysis, and besides, the recovered PHAs were adequately pure (≥96% purity). Therefore, this new method would be valuable not only for quantitation of PHA but also for preparation of samples to characterize their mechanical properties. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

NASA Astrophysics Data System (ADS)

Smith, Elizabeth Myhra

The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

Single Cell Mass Cytometry for Analysis of Immune System Functional States

PubMed Central

Bjornson, Zach B.; Nolan, Garry P.; Fantl, Wendy J.

2013-01-01

Single cell mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with antibody panels (upwards of 40) in which each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the Lanthanide series of the periodic table. Isotope labelled antibodies recognize surface markers to delineate cell types and intracellular signaling molecules to provide a measure of the network state—and thereby demarcating multiple cell state functions such as apoptosis, DNA damage and cell cycle. By measuring all these parameters simultaneously, the signaling state of an individual cell can be measured at its network state. This review will cover the basics of mass cytometry as well as outline steps already taken to allow it to stand aside traditional fluorescence based cytometry in the immunologist’s analytical arsenal in their study of immune states during infection. PMID:23999316

Centered reduced moments and associate density functions applied to alkaline comet assay.

PubMed

Castaneda, Roman; Pelaez, Alejandro; Marquez, Maria-Elena; Abad, Pablo

2005-01-01

The single cell gel electrophoresis assay is a sensitive, rapid, and visual technique for deoxyribonucleic acid (DNA) strand-break detection in individual mammalian cells, whose application has significantly increased in the past few years. The cells are embedded in agarose on glass slides followed by lyses of the cell membrane. Thereafter, damaged DNA strands are electrophoresed away from the nucleus towards the anode giving the appearance of a comet tail. Nowadays, charge coupled device cameras are attached at optical microscopes for recording the images of the cells, and digital image processing is applied for obtaining quantitative descriptors. However, the conventional software is usually expensive, inflexible and, in many cases, can only provide low-order descriptors based in image segmentation, determination of centers of mass, and Euclidean distances. Associated density functions and centered reduced moments offer an effective and flexible alternative for quantitative analysis of the comet cells. We will show how the position of the center of mass, the lengths and orientation of the main semiaxes, and the eccentricity of such images can be accurately determined by this method.

SILAC-Based Comparative Proteomic Analysis of Lysosomes from Mammalian Cells Using LC-MS/MS.

PubMed

Thelen, Melanie; Winter, Dominic; Braulke, Thomas; Gieselmann, Volkmar

2017-01-01

Mass spectrometry-based proteomics of lysosomal proteins has led to significant advances in understanding lysosomal function and pathology. The ever-increasing sensitivity and resolution of mass spectrometry in combination with labeling procedures which allow comparative quantitative proteomics can be applied to shed more light on the steadily increasing range of lysosomal functions. In addition, investigation of alterations in lysosomal protein composition in the many lysosomal storage diseases may yield further insights into the molecular pathology of these disorders. Here, we describe a protocol which allows to determine quantitative differences in the lysosomal proteome of cells which are genetically and/or biochemically different or have been exposed to certain stimuli. The method is based on stable isotope labeling of amino acids in cell culture (SILAC). Cells are exposed to superparamagnetic iron oxide particles which are endocytosed and delivered to lysosomes. After homogenization of cells, intact lysosomes are rapidly enriched by passing the cell homogenates over a magnetic column. Lysosomes are eluted after withdrawal of the magnetic field and subjected to mass spectrometry.

Quantitative analysis of RNA-protein interactions on a massively parallel array for mapping biophysical and evolutionary landscapes

PubMed Central

Buenrostro, Jason D.; Chircus, Lauren M.; Araya, Carlos L.; Layton, Curtis J.; Chang, Howard Y.; Snyder, Michael P.; Greenleaf, William J.

2015-01-01

RNA-protein interactions drive fundamental biological processes and are targets for molecular engineering, yet quantitative and comprehensive understanding of the sequence determinants of affinity remains limited. Here we repurpose a high-throughput sequencing instrument to quantitatively measure binding and dissociation of MS2 coat protein to >107 RNA targets generated on a flow-cell surface by in situ transcription and inter-molecular tethering of RNA to DNA. We decompose the binding energy contributions from primary and secondary RNA structure, finding that differences in affinity are often driven by sequence-specific changes in association rates. By analyzing the biophysical constraints and modeling mutational paths describing the molecular evolution of MS2 from low- to high-affinity hairpins, we quantify widespread molecular epistasis, and a long-hypothesized structure-dependent preference for G:U base pairs over C:A intermediates in evolutionary trajectories. Our results suggest that quantitative analysis of RNA on a massively parallel array (RNAMaP) relationships across molecular variants. PMID:24727714

Improved method for HPLC analysis of polyamines, agmatine and aromatic monoamines in plant tissue

NASA Technical Reports Server (NTRS)

Slocum, R. D.; Flores, H. E.; Galston, A. W.; Weinstein, L. H.

1989-01-01

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucas carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.

Highly sensitive transient absorption imaging of graphene and graphene oxide in living cells and circulating blood

PubMed Central

Li, Junjie; Zhang, Weixia; Chung, Ting-Fung; Slipchenko, Mikhail N.; Chen, Yong P.; Cheng, Ji-Xin; Yang, Chen

2015-01-01

We report a transient absorption (TA) imaging method for fast visualization and quantitative layer analysis of graphene and GO. Forward and backward imaging of graphene on various substrates under ambient condition was imaged with a speed of 2 μs per pixel. The TA intensity linearly increased with the layer number of graphene. Real-time TA imaging of GO in vitro with capability of quantitative analysis of intracellular concentration and ex vivo in circulating blood were demonstrated. These results suggest that TA microscopy is a valid tool for the study of graphene based materials. PMID:26202216

Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring

PubMed Central

Cao, Yuhong; Chen, Haodong; Birey, Fikri; Leal-Ortiz, Sergio A.; Han, Crystal M.; Santiago, Juan G.; Paşca, Sergiu P.; Wu, Joseph C.; Melosh, Nicholas A.

2017-01-01

Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation. PMID:28223521

Recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma on behalf of the International Neuroblastoma Response Criteria Bone Marrow Working Group.

PubMed

Burchill, Susan A; Beiske, Klaus; Shimada, Hiroyuki; Ambros, Peter F; Seeger, Robert; Tytgat, Godelieve A M; Brock, Penelope R; Haber, Michelle; Park, Julie R; Berthold, Frank

2017-04-01

The current study was conducted to expedite international standardized reporting of bone marrow disease in children with neuroblastoma and to improve equivalence of care. A multidisciplinary International Neuroblastoma Response Criteria Bone Marrow Working Group was convened by the US National Cancer Institute in January 2012 with representation from Europe, North America, and Australia. Practical transferable recommendations to standardize the reporting of bone marrow disease were developed. To the authors' knowledge, the current study is the first to comprehensively present consensus criteria for the collection, analysis, and reporting of the percentage area of bone marrow parenchyma occupied by tumor cells in trephine-biopsies. The quantitative analysis of neuroblastoma content in bone marrow aspirates by immunocytology and reverse transcriptase-quantitative polymerase chain reaction are revised. The inclusion of paired-like homeobox 2b (PHOX2B) for immunohistochemistry and reverse transcriptase-quantitative polymerase chain reaction is recommended. Recommendations for recording bone marrow response are provided. The authors endorse the quantitative assessment of neuroblastoma cell content in bilateral core needle biopsies-trephines and aspirates in all children with neuroblastoma, with the exception of infants, in whom the evaluation of aspirates alone is advised. It is interesting to note that 5% disease is accepted as an internationally achievable level for disease assessment. The quantitative assessment of neuroblastoma cells is recommended to provide data from which evidence-based numerical criteria for the reporting of bone marrow response can be realized. This is particularly important in the minimal disease setting and when neuroblastoma detection in bone marrow is intermittent, where clinical impact has yet to be validated. The wide adoption of these harmonized criteria will enhance the ability to compare outcomes from different trials and facilitate collaborative trial design. Cancer 2017;123:1095-1105. © 2016 American Cancer Society. © 2016 American Cancer Society.

[Development of non-invasive clinical examination methods for the anterior segment of the eye and their clinical significance].

PubMed

Sawa, Mitsuru

2011-03-01

1. Slit-lamp microscopy is a principal ophthalmic clinical method, because it provides microscopic findings of the anterior segment of the eye noninvasively. Its findings, however, are qualitative and there are large inter-observer variations in their evaluation. Furthermore, slit-lamp microscopy provides morphological findings, but a functional evaluation is difficult. We developed two novel methods that establish a qualitative methodology of the slit-lamp microscope and the pathophysiology of the anterior segment of the eye. One is the flare-cell photometer to evaluate flare and cells in the aqueous humor of the eye and the other is an immunohistochemical examination method using tear fluid to evaluate ocular surface disorders. The comprehensive evaluation of these studies is herein overviewed. 2. INNOVATION OF THE FLARE-CELL PHOTOMETER AND ITS CLINICAL SIGNIFICANCE: The breakdown of the blood-aqueous barrier (BAB) causes an increase in protein (flare) and leakage of blood cells (cell) into the aqueous humor of the eye and the severity of BAB breakdown has a positive correlation with the intensity of flare and cells. The flare and cells in the aqueous can be observed qualitatively by slit-lamp microscopy. These findings are primarily distinguished in optics by light scattering. Therefore, detection of the intensity of light scattering due to flare and cells can evaluate the BAB function. The flare-cell photometer comprises 3 novel components: a laser beam system as an incident light, a photomultiplier to detect scattered light intensity and a computer-assisted system to operate the whole system and analyze detected scattered light signals due to flare and cells. The instrument enables us to quantitatively analyze the flare and cells non-invasively and accurately with a wide dynamic measurement range, resulting in a repeated examination of each individual case. It also enables the evaluation of inflammation in the aqueous not only postoperatively but also in endogenous uveitis, evaluation of the effects of anti-inflammatory drugs on BAB and evaluation of aqueous humor dynamics. Furthermore, repeating the examination can minimize inter-individual variations and reduce the number of animals in animal experiments. 3. Sampling of tears can be performed noninvasively, but the obtainable volume is limited. Therefore, a determination of targeting biomarkers and a development of their micro-volume analysis methods play a crucial role in pathophysiological studies of the ocular surface. Targeting biomarkers should be determined according to the various specified bioactive substances such as eosinophil cationic protein (ECP), cytokines and others. A number of microvolume analysis methods, such as chemiluminescent enzyme immunoassay, immunochromatography, micro-array system and polymerase chain reaction method are used. Objective disorders in the studies include allergic conjunctivitis and infectious diseases such as herpetic keratitis. Quantitative evaluation methods for ECP concentration, antigen-specific secretory IgA in allergic diseases and herpetic keratitis, herpes simplex virus-DNA and cytokine and chemokine profile in tear fluid sampled by filter paper method were investigated. We developed a clinically applicable quantitative immunochemical method for ECP concentration in tear fluid. The results revealed that tear fluid analysis using the above mentioned methods is a clinically useful to investigate the pathophysiology of the ocular surface. 4. Laser flare-cell photometer and tear fluid analysis are potent clinical quantitative methods to investigate the pathophysiology of the anterior segment of the eye.

Biomarkers for Prostate and Bladder Cancer — EDRN Public Portal

Cancer.gov

CD26+ cancer cells and CD90+ tumor-associated stromal cells were sorted from tumor tissue. Dataset analysis with transcriptomes of CD26+ luminal, CD104+ basal, CD49a+ stromal and CD31+ endothelial was carried out to identify candidates: CD90, AGR2, BCMP11, CEACAM5, CRISP3. Quantitative protomics was applied to measure these proteins in urine samples.

Three-Dimensional Computer Graphics Brain-Mapping Project.

DTIC Science & Technology

1987-03-15

NEUROQUANT . This package was directed towards quantitative microneuroanatomic data acquisition and analysis. Using this interface, image frames captured...populations of brains. This would have been aprohibitive task if done manually with a densitometer and film, due to user error and bias. NEUROQUANT functioned...of cells were of interest. NEUROQUANT is presently being implemented with a more fully automatic method of localizing the cell bodies directly

Quantitative probe of the transition metal redox in battery electrodes through soft x-ray absorption spectroscopy

NASA Astrophysics Data System (ADS)

Li, Qinghao; Qiao, Ruimin; Wray, L. Andrew; Chen, Jun; Zhuo, Zengqing; Chen, Yanxue; Yan, Shishen; Pan, Feng; Hussain, Zahid; Yang, Wanli

2016-10-01

Most battery positive electrodes operate with a 3d transition-metal (TM) reaction centre. A direct and quantitative probe of the TM states upon electrochemical cycling is valuable for understanding the detailed cycling mechanism and charge diffusion in the electrodes, which is related with many practical parameters of a battery. This review includes a comprehensive summary of our recent demonstrations of five different types of quantitative analysis of the TM states in battery electrodes based on soft x-ray absorption spectroscopy and multiplet calculations. In LiFePO4, a system of a well-known two-phase transformation type, the TM redox could be strictly determined through a simple linear combination of the two end-members. In Mn-based compounds, the Mn states could also be quantitatively evaluated, but a set of reference spectra with all the three possible Mn valences needs to be deliberately selected and considered in the fitting. Although the fluorescence signals suffer the self-absorption distortion, the multiplet calculations could consider the distortion effect, which allows a quantitative determination of the overall Ni oxidation state in the bulk. With the aid of multiplet calculations, one could also achieve a quasi-quantitative analysis of the Co redox evolution in LiCoO2 based on the energy position of the spectroscopic peak. The benefit of multiplet calculations is more important for studying electrode materials with TMs of mixed spin states, as exemplified by the quantitative analysis of the mixed spin Na2-x Fe2(CN)6 system. At the end, we showcase that such quantitative analysis could provide valuable information for optimizing the electrochemical performance of Na0.44MnO2 electrodes for Na-ion batteries. The methodology summarized in this review could be extended to other energy application systems with TM redox centre for detailed analysis, for example, fuel cell and catalytic materials.

Histopathological image analysis of chemical-induced hepatocellular hypertrophy in mice.

PubMed

Asaoka, Yoshiji; Togashi, Yuko; Mutsuga, Mayu; Imura, Naoko; Miyoshi, Tomoya; Miyamoto, Yohei

2016-04-01

Chemical-induced hepatocellular hypertrophy is frequently observed in rodents, and is mostly caused by the induction of phase I and phase II drug metabolic enzymes and peroxisomal lipid metabolic enzymes. Liver weight is a sensitive and commonly used marker for detecting hepatocellular hypertrophy, but is also increased by a number of other factors. Histopathological observations subjectively detect changes such as hepatocellular hypertrophy based on the size of a hepatocyte. Therefore, quantitative microscopic observations are required to evaluate histopathological alterations objectively. In the present study, we developed a novel quantitative method for an image analysis of hepatocellular hypertrophy using liver sections stained with hematoxylin and eosin, and demonstrated its usefulness for evaluating hepatocellular hypertrophy induced by phenobarbital (a phase I and phase II enzyme inducer) and clofibrate (a peroxisomal enzyme inducer) in mice. The algorithm of this imaging analysis was designed to recognize an individual hepatocyte through a combination of pixel-based and object-based analyses. Hepatocellular nuclei and the surrounding non-hepatocellular cells were recognized by the pixel-based analysis, while the areas of the recognized hepatocellular nuclei were then expanded until they ran against their expanding neighboring hepatocytes and surrounding non-hepatocellular cells by the object-based analysis. The expanded area of each hepatocellular nucleus was regarded as the size of an individual hepatocyte. The results of this imaging analysis showed that changes in the sizes of hepatocytes corresponded with histopathological observations in phenobarbital and clofibrate-treated mice, and revealed a correlation between hepatocyte size and liver weight. In conclusion, our novel image analysis method is very useful for quantitative evaluations of chemical-induced hepatocellular hypertrophy. Copyright © 2015 Elsevier GmbH. All rights reserved.

Statistical Model to Analyze Quantitative Proteomics Data Obtained by 18O/16O Labeling and Linear Ion Trap Mass Spectrometry

PubMed Central

Jorge, Inmaculada; Navarro, Pedro; Martínez-Acedo, Pablo; Núñez, Estefanía; Serrano, Horacio; Alfranca, Arántzazu; Redondo, Juan Miguel; Vázquez, Jesús

2009-01-01

Statistical models for the analysis of protein expression changes by stable isotope labeling are still poorly developed, particularly for data obtained by 16O/18O labeling. Besides large scale test experiments to validate the null hypothesis are lacking. Although the study of mechanisms underlying biological actions promoted by vascular endothelial growth factor (VEGF) on endothelial cells is of considerable interest, quantitative proteomics studies on this subject are scarce and have been performed after exposing cells to the factor for long periods of time. In this work we present the largest quantitative proteomics study to date on the short term effects of VEGF on human umbilical vein endothelial cells by 18O/16O labeling. Current statistical models based on normality and variance homogeneity were found unsuitable to describe the null hypothesis in a large scale test experiment performed on these cells, producing false expression changes. A random effects model was developed including four different sources of variance at the spectrum-fitting, scan, peptide, and protein levels. With the new model the number of outliers at scan and peptide levels was negligible in three large scale experiments, and only one false protein expression change was observed in the test experiment among more than 1000 proteins. The new model allowed the detection of significant protein expression changes upon VEGF stimulation for 4 and 8 h. The consistency of the changes observed at 4 h was confirmed by a replica at a smaller scale and further validated by Western blot analysis of some proteins. Most of the observed changes have not been described previously and are consistent with a pattern of protein expression that dynamically changes over time following the evolution of the angiogenic response. With this statistical model the 18O labeling approach emerges as a very promising and robust alternative to perform quantitative proteomics studies at a depth of several thousand proteins. PMID:19181660

Quantitative Imaging in Cancer Evolution and Ecology

PubMed Central

Grove, Olya; Gillies, Robert J.

2013-01-01

Cancer therapy, even when highly targeted, typically fails because of the remarkable capacity of malignant cells to evolve effective adaptations. These evolutionary dynamics are both a cause and a consequence of cancer system heterogeneity at many scales, ranging from genetic properties of individual cells to large-scale imaging features. Tumors of the same organ and cell type can have remarkably diverse appearances in different patients. Furthermore, even within a single tumor, marked variations in imaging features, such as necrosis or contrast enhancement, are common. Similar spatial variations recently have been reported in genetic profiles. Radiologic heterogeneity within tumors is usually governed by variations in blood flow, whereas genetic heterogeneity is typically ascribed to random mutations. However, evolution within tumors, as in all living systems, is subject to Darwinian principles; thus, it is governed by predictable and reproducible interactions between environmental selection forces and cell phenotype (not genotype). This link between regional variations in environmental properties and cellular adaptive strategies may permit clinical imaging to be used to assess and monitor intratumoral evolution in individual patients. This approach is enabled by new methods that extract, report, and analyze quantitative, reproducible, and mineable clinical imaging data. However, most current quantitative metrics lack spatialness, expressing quantitative radiologic features as a single value for a region of interest encompassing the whole tumor. In contrast, spatially explicit image analysis recognizes that tumors are heterogeneous but not well mixed and defines regionally distinct habitats, some of which appear to harbor tumor populations that are more aggressive and less treatable than others. By identifying regional variations in key environmental selection forces and evidence of cellular adaptation, clinical imaging can enable us to define intratumoral Darwinian dynamics before and during therapy. Advances in image analysis will place clinical imaging in an increasingly central role in the development of evolution-based patient-specific cancer therapy. © RSNA, 2013 PMID:24062559

Long Noncoding RNA PANDA Positively Regulates Proliferation of Osteosarcoma Cells.

PubMed

Kotake, Yojiro; Goto, Taiki; Naemura, Madoka; Inoue, Yasutoshi; Okamoto, Haruna; Tahara, Keiichiro

2017-01-01

A long noncoding RNA, p21-associated ncRNA DNA damage-activated (PANDA), associates with nuclear transcription factor Y subunit alpha (NF-YA) and inhibits its binding to promoters of apoptosis-related genes, thereby repressing apoptosis in normal human fibroblasts. Here, we show that PANDA is involved in regulating proliferation in the U2OS human osteosarcoma cell line. U2OS cells were transfected with siRNAs against PANDA 72 h later and they were subjected to reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR and cell-cycle analysis. PANDA was highly expressed in U2OS cells, and its expression was induced by DNA damage. Silencing PANDA caused arrest at the G 1 phase of the cell cycle, leading to inhibition of cell proliferation. Quantitative RT-PCR showed that silencing PANDA increased mRNA levels of the cyclin-dependent kinase inhibitor p18, which caused G 1 phase arrest. These results suggest that PANDA promotes G 1 -S transition by repressing p18 transcription, and thus promotes U2OS cell proliferation. Copyright© 2017 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

The Use of Multidimensional Image-Based Analysis to Accurately Monitor Cell Growth in 3D Bioreactor Culture

PubMed Central

Baradez, Marc-Olivier; Marshall, Damian

2011-01-01

The transition from traditional culture methods towards bioreactor based bioprocessing to produce cells in commercially viable quantities for cell therapy applications requires the development of robust methods to ensure the quality of the cells produced. Standard methods for measuring cell quality parameters such as viability provide only limited information making process monitoring and optimisation difficult. Here we describe a 3D image-based approach to develop cell distribution maps which can be used to simultaneously measure the number, confluency and morphology of cells attached to microcarriers in a stirred tank bioreactor. The accuracy of the cell distribution measurements is validated using in silico modelling of synthetic image datasets and is shown to have an accuracy >90%. Using the cell distribution mapping process and principal component analysis we show how cell growth can be quantitatively monitored over a 13 day bioreactor culture period and how changes to manufacture processes such as initial cell seeding density can significantly influence cell morphology and the rate at which cells are produced. Taken together, these results demonstrate how image-based analysis can be incorporated in cell quality control processes facilitating the transition towards bioreactor based manufacture for clinical grade cells. PMID:22028809

The use of multidimensional image-based analysis to accurately monitor cell growth in 3D bioreactor culture.

PubMed

Baradez, Marc-Olivier; Marshall, Damian

2011-01-01

The transition from traditional culture methods towards bioreactor based bioprocessing to produce cells in commercially viable quantities for cell therapy applications requires the development of robust methods to ensure the quality of the cells produced. Standard methods for measuring cell quality parameters such as viability provide only limited information making process monitoring and optimisation difficult. Here we describe a 3D image-based approach to develop cell distribution maps which can be used to simultaneously measure the number, confluency and morphology of cells attached to microcarriers in a stirred tank bioreactor. The accuracy of the cell distribution measurements is validated using in silico modelling of synthetic image datasets and is shown to have an accuracy >90%. Using the cell distribution mapping process and principal component analysis we show how cell growth can be quantitatively monitored over a 13 day bioreactor culture period and how changes to manufacture processes such as initial cell seeding density can significantly influence cell morphology and the rate at which cells are produced. Taken together, these results demonstrate how image-based analysis can be incorporated in cell quality control processes facilitating the transition towards bioreactor based manufacture for clinical grade cells.

HeLa cells response to photodynamic treatment with Radachlorin at various irradiation parameters

NASA Astrophysics Data System (ADS)

Belashov, A. V.; Zhikhoreva, A. A.; Belyaeva, T. N.; Kornilova, E. S.; Petrov, N. V.; Salova, A. V.; Semenova, I. V.; Vasyutinskii, O. S.

2017-07-01

Measurements of average phase shifts introduced by living HeLa cells to probe wave front were carried out. Variations of this value were monitored in the course of morphological changes caused by photodynamic treatment at various irradiation doses. Observations of changes in living cells were also performed by means of far field optical microscopy and confocal fluorescent microscopy. Quantitative analysis of the data obtained shows that average phase shift introduced by the cells may either increase or decrease depending upon major parameters of the treatment.

Upsets in Erased Floating Gate Cells With High-Energy Protons

DOE PAGES

Gerardin, S.; Bagatin, M.; Paccagnella, A.; ...

2017-01-01

We discuss upsets in erased floating gate cells, due to large threshold voltage shifts, using statistical distributions collected on a large number of memory cells. The spread in the neutral threshold voltage appears to be too low to quantitatively explain the experimental observations in terms of simple charge loss, at least in SLC devices. The possibility that memories exposed to high energy protons and heavy ions exhibit negative charge transfer between programmed and erased cells is investigated, although the analysis does not provide conclusive support to this hypothesis.

An integrated microfluidic sensor for real-time detection of RNA in seawater using preserved reagents

NASA Astrophysics Data System (ADS)

Tsaloglou, M.-N.; Loukas, C. M.; Ruano-López, J. M.; Morgan, H.; Mowlem, M. C.

2012-04-01

Quantitation of RNA sequences coding either for key metabolic proteins or highly conserved ribosomal subunits can provide insight on cell abundance, speciation and viability. Nucleic sequence-based amplification (NASBA) is an isothermal alternative to traditional nucleic acid amplification methods, such as quantitative PCR. We present here an integrated microfluidic sensor for cell concentration and lysis, RNA extraction/purification and quantitative RNA detection for environmental applications. The portable system uses pre-loaded reagents, stored as a gel on a disposable microfluidic cartridge, which is manufactured using low-cost injection moulding. The NASBA reaction is monitored real-time using a bespoke control unit which includes: an external fluorescence detector, three peristaltic micro-pumps, two heaters and temperature sensors, a battery, seven pin actuated micro-motors (or valve actuators), and an automatic cartridge insertion mechanism. The system has USB connectivity and none of the expensive components require replacing between reactions. Long-term storage of reagents is critically important for any diagnostic tool that will be used in the field, whether for medical or environmental analysis and has not been previously demonstrated for NASBA reagents on-chip. We have shown effective amplification, for as little as 500 cells of the toxic microalga Karenia brevis using reagents which had been preserved as a gel for 45 days. This is the first reported real-time isothermal RNA amplification using with on-chip preservation. Annealing of primers, amplification at 41 °C and real-time fluorescence detection using, also for the first time, an internal control and sequence-specific molecular beacons was all performed on our microfluidic sensor. Our results show excellent promise as a future quantitative tool of in situ phytoplankton analysis and other environmental applications, where long-term reagent storage and low power consumption is essential.

Image-based quantification and mathematical modeling of spatial heterogeneity in ESC colonies.

PubMed

Herberg, Maria; Zerjatke, Thomas; de Back, Walter; Glauche, Ingmar; Roeder, Ingo

2015-06-01

Pluripotent embryonic stem cells (ESCs) have the potential to differentiate into cells of all three germ layers. This unique property has been extensively studied on the intracellular, transcriptional level. However, ESCs typically form clusters of cells with distinct size and shape, and establish spatial structures that are vital for the maintenance of pluripotency. Even though it is recognized that the cells' arrangement and local interactions play a role in fate decision processes, the relations between transcriptional and spatial patterns have not yet been studied. We present a systems biology approach which combines live-cell imaging, quantitative image analysis, and multiscale, mathematical modeling of ESC growth. In particular, we develop quantitative measures of the morphology and of the spatial clustering of ESCs with different expression levels and apply them to images of both in vitro and in silico cultures. Using the same measures, we are able to compare model scenarios with different assumptions on cell-cell adhesions and intercellular feedback mechanisms directly with experimental data. Applying our methodology to microscopy images of cultured ESCs, we demonstrate that the emerging colonies are highly variable regarding both morphological and spatial fluorescence patterns. Moreover, we can show that most ESC colonies contain only one cluster of cells with high self-renewing capacity. These cells are preferentially located in the interior of a colony structure. The integrated approach combining image analysis with mathematical modeling allows us to reveal potential transcription factor related cellular and intercellular mechanisms behind the emergence of observed patterns that cannot be derived from images directly. © 2015 International Society for Advancement of Cytometry.

Characterization of human myoblast differentiation for tissue-engineering purposes by quantitative gene expression analysis.

PubMed

Stern-Straeter, Jens; Bonaterra, Gabriel Alejandro; Kassner, Stefan S; Zügel, Stefanie; Hörmann, Karl; Kinscherf, Ralf; Goessler, Ulrich Reinhart

2011-08-01

Tissue engineering of skeletal muscle is an encouraging possibility for the treatment of muscle loss through the creation of functional muscle tissue in vitro from human stem cells. Currently, the preferred stem cells are primary, non-immunogenic satellite cells ( = myoblasts). The objective of this study was to determine the expression patterns of myogenic markers within the human satellite cell population during their differentiation into multinucleated myotubes for an accurate characterization of stem cell behaviour. Satellite cells were incubated (for 1, 4, 8, 12 or 16 days) with a culture medium containing either a low [ = differentiation medium (DM)] or high [ = growth medium (GM)] concentration of growth factors. Furthermore, we performed a quantitative gene expression analysis of well-defined differentiation makers: myogenic factor 5 (MYF5), myogenin (MYOG), skeletal muscle αactin1 (ACTA1), embryonic (MYH3), perinatal (MYH8) and adult skeletal muscle myosin heavy chain (MYH1). Additionally, the fusion indices of forming myotubes of MYH1, MYH8 and ACTA1 were calculated. We show that satellite cells incubated with DM expressed multiple characteriztic features of mature skeletal muscles, verified by time-dependent upregulation of MYOG, MYH1, MYH3, MYH8 and ACTA1. However, satellite cells incubated with GM did not reveal all morphological aspects of muscle differentiation. Immunocytochemical investigations with antibodies directed against the differentiation markers showed correlations between the gene expression and differentiation. Our data provide information about time-dependent gene expression of differentiation markers in human satellite cells, which can be used for maturation analyses in skeletal muscle tissue-engineering applications. Copyright © 2011 John Wiley & Sons, Ltd.

Recent Advances in Nanobiotechnology and High-Throughput Molecular Techniques for Systems Biomedicine

PubMed Central

Kim, Eung-Sam; Ahn, Eun Hyun; Chung, Euiheon; Kim, Deok-Ho

2013-01-01

Nanotechnology-based tools are beginning to emerge as promising platforms for quantitative high-throughput analysis of live cells and tissues. Despite unprecedented progress made over the last decade, a challenge still lies in integrating emerging nanotechnology-based tools into macroscopic biomedical apparatuses for practical purposes in biomedical sciences. In this review, we discuss the recent advances and limitations in the analysis and control of mechanical, biochemical, fluidic, and optical interactions in the interface areas of nanotechnology-based materials and living cells in both in vitro and in vivo settings. PMID:24258011

Recent advances in nanobiotechnology and high-throughput molecular techniques for systems biomedicine.

PubMed

Kim, Eung-Sam; Ahn, Eun Hyun; Chung, Euiheon; Kim, Deok-Ho

2013-12-01

Nanotechnology-based tools are beginning to emerge as promising platforms for quantitative high-throughput analysis of live cells and tissues. Despite unprecedented progress made over the last decade, a challenge still lies in integrating emerging nanotechnology-based tools into macroscopic biomedical apparatuses for practical purposes in biomedical sciences. In this review, we discuss the recent advances and limitations in the analysis and control of mechanical, biochemical, fluidic, and optical interactions in the interface areas of nanotechnologybased materials and living cells in both in vitro and in vivo settings.

iTRAQ-Based Quantitative Proteomic Analysis of the Initiation of Head Regeneration in Planarians.

PubMed

Geng, Xiaofang; Wang, Gaiping; Qin, Yanli; Zang, Xiayan; Li, Pengfei; Geng, Zhi; Xue, Deming; Dong, Zimei; Ma, Kexue; Chen, Guangwen; Xu, Cunshuan

2015-01-01

The planarian Dugesia japonica has amazing ability to regenerate a head from the anterior ends of the amputated stump with maintenance of the original anterior-posterior polarity. Although planarians present an attractive system for molecular investigation of regeneration and research has focused on clarifying the molecular mechanism of regeneration initiation in planarians at transcriptional level, but the initiation mechanism of planarian head regeneration (PHR) remains unclear at the protein level. Here, a global analysis of proteome dynamics during the early stage of PHR was performed using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy, and our data are available via ProteomeXchange with identifier PXD002100. The results showed that 162 proteins were differentially expressed at 2 h and 6 h following amputation. Furthermore, the analysis of expression patterns and functional enrichment of the differentially expressed proteins showed that proteins involved in muscle contraction, oxidation reduction and protein synthesis were up-regulated in the initiation of PHR. Moreover, ingenuity pathway analysis showed that predominant signaling pathways such as ILK, calcium, EIF2 and mTOR signaling which were associated with cell migration, cell proliferation and protein synthesis were likely to be involved in the initiation of PHR. The results for the first time demonstrated that muscle contraction and ILK signaling might played important roles in the initiation of PHR at the global protein level. The findings of this research provide a molecular basis for further unraveling the mechanism of head regeneration initiation in planarians.

Histone deacetylase inhibitors suppress ABO transcription in vitro, leading to reduced expression of the antigens.

PubMed

Takahashi, Yoichiro; Kubo, Rieko; Sano, Rie; Nakajima, Tamiko; Takahashi, Keiko; Kobayashi, Momoko; Handa, Hiroshi; Tsukada, Junichi; Kominato, Yoshihiko

2017-03-01

The ABO system is of fundamental importance in the fields of transfusion and transplantation and has apparent associations with certain diseases, including cardiovascular disorders. ABO expression is reduced in the late phase of erythroid differentiation in vitro, whereas histone deacetylase inhibitors (HDACIs) are known to promote cell differentiation. Therefore, whether or not HDACIs could reduce the amount of ABO transcripts and A or B antigens is an intriguing issue. Quantitative polymerase chain reactions were carried out for the ABO transcripts in erythroid-lineage K562 and epithelial-lineage KATOIII cells after incubation with HDACIs, such as sodium butyrate, panobinostat, vorinostat, and sodium valproate. Flow cytometric analysis was conducted to evaluate the amounts of antigen in KATOIII cells treated with panobinostat. Quantitative chromatin immunoprecipitation (ChIP) assays and luciferase assays were performed on both cell types to examine the mechanisms of ABO suppression. HDACIs reduced the ABO transcripts in both K562 and KATOIII cells, with panobinostat exerting the most significant effect. Flow cytometric analysis demonstrated a decrease in B-antigen expression on panobinostat-treated KATOIII cells. ChIP assays indicated that panobinostat altered the modification of histones in the transcriptional regulatory regions of ABO, and luciferase assays demonstrated reduced activity of these elements. ABO transcription seems to be regulated by an epigenetic mechanism. Panobinostat appears to suppress ABO transcription, reducing the amount of antigens on the surface of cultured cells. © 2016 AABB.

Fusion-Related Host Proteins Are Actively Regulated by NA during Influenza Infection as Revealed by Quantitative Proteomics Analysis

PubMed Central

Sui, Zhiwei; Wen, Bo; Gao, Zhimin; Chen, Quanjiao

2014-01-01

Three recombinant influenza A viruses with different neuraminidases (NAs) in the background of A/PR/8/34 (PR8), named rPR8-H5N1NA, rPR8-H9N2NA, and rPR8-H1N1NA, derived from H5N1, H9N2, H1N1 (swine) viruses, respectively, were constructed. We performed a quantitative proteomics analysis to investigate differential protein expression in Madin-Darby canine kidney (MDCK) cells infected with recombinant and wild-type influenza viruses to determine whether NA replacement would alter host cell gene expression. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-TOF MS) and two-dimensional gel electrophoresis (2-DE), we identified 12 up-regulated and 49 down-regulated protein spots, including cytoskeletal proteins, molecular biosynthesis proteins, ubiquitin-proteasome pathway proteins, and heat shock proteins. The most significant changes in infected cells were observed for molecular biosynthesis proteins. We found more differentially expressed protein spots in cells infected with rPR8-H5N1NA or rPR8-H9N2NA viruses than cells infected with wild-type virus. Many of those proteins are postulated to be involved in cell-cell fusion, but the full mechanism remains to be explored. Meanwhile, our data demonstrate that the wild-type virus has evolutionary advantages over recombinant viruses. PMID:25153908

Quantitative Analysis of Energy Metabolic Pathways in MCF-7 Breast Cancer Cells by Selected Reaction Monitoring Assay*

PubMed Central

Drabovich, Andrei P.; Pavlou, Maria P.; Dimitromanolakis, Apostolos; Diamandis, Eleftherios P.

2012-01-01

To investigate the quantitative response of energy metabolic pathways in human MCF-7 breast cancer cells to hypoxia, glucose deprivation, and estradiol stimulation, we developed a targeted proteomics assay for accurate quantification of protein expression in glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate pathways. Cell growth conditions were selected to roughly mimic the exposure of cells in the cancer tissue to the intermittent hypoxia, glucose deprivation, and hormonal stimulation. Targeted proteomics assay allowed for reproducible quantification of 76 proteins in four different growth conditions after 24 and 48 h of perturbation. Differential expression of a number of control and metabolic pathway proteins in response to the change of growth conditions was found. Elevated expression of the majority of glycolytic enzymes was observed in hypoxia. Cancer cells, as opposed to near-normal MCF-10A cells, exhibited significantly increased expression of key energy metabolic pathway enzymes (FBP1, IDH2, and G6PD) that are known to redirect cellular metabolism and increase carbon flux through the pentose phosphate pathway. Our quantitative proteomic protocol is based on a mass spectrometry-compatible acid-labile detergent and is described in detail. Optimized parameters of a multiplex selected reaction monitoring (SRM) assay for 76 proteins, 134 proteotypic peptides, and 401 transitions are included and can be downloaded and used with any SRM-compatible mass spectrometer. The presented workflow is an integrated tool for hypothesis-driven studies of mammalian cells as well as functional studies of proteins, and can greatly complement experimental methods in systems biology, metabolic engineering, and metabolic transformation of cancer cells. PMID:22535206

Miniaturization of Fresnel lenses for solar concentration: a quantitative investigation.

PubMed

Duerr, Fabian; Meuret, Youri; Thienpont, Hugo

2010-04-20

Sizing down the dimensions of solar concentrators for photovoltaic applications offers a number of promising advantages. It provides thinner modules and smaller solar cells, which reduces thermal issues. In this work a plane Fresnel lens design is introduced that is first analyzed with geometrical optics. Because of miniaturization, pure ray tracing may no longer be valid to determine the concentration performance. Therefore, a quantitative wave optical analysis of the miniaturization's influence on the obtained concentration performance is presented. This better quantitative understanding of the impact of diffraction in microstructured Fresnel lenses might help to optimize the design of several applications in nonimaging optics.

LobeFinder: A Convex Hull-Based Method for Quantitative Boundary Analyses of Lobed Plant Cells1[OPEN

PubMed Central

Wu, Tzu-Ching; Belteton, Samuel A.; Szymanski, Daniel B.; Umulis, David M.

2016-01-01

Dicot leaves are composed of a heterogeneous mosaic of jigsaw puzzle piece-shaped pavement cells that vary greatly in size and the complexity of their shape. Given the importance of the epidermis and this particular cell type for leaf expansion, there is a strong need to understand how pavement cells morph from a simple polyhedral shape into highly lobed and interdigitated cells. At present, it is still unclear how and when the patterns of lobing are initiated in pavement cells, and one major technological bottleneck to addressing the problem is the lack of a robust and objective methodology to identify and track lobing events during the transition from simple cell geometry to lobed cells. We developed a convex hull-based algorithm termed LobeFinder to identify lobes, quantify geometric properties, and create a useful graphical output of cell coordinates for further analysis. The algorithm was validated against manually curated images of pavement cells of widely varying sizes and shapes. The ability to objectively count and detect new lobe initiation events provides an improved quantitative framework to analyze mutant phenotypes, detect symmetry-breaking events in time-lapse image data, and quantify the time-dependent correlation between cell shape change and intracellular factors that may play a role in the morphogenesis process. PMID:27288363

SET mediates TCE-induced liver cell apoptosis through dephosphorylation and upregulation of nucleolin

PubMed Central

Ren, Xiaohu; Huang, Xinfeng; Yang, Xifei; Liu, Yungang; Liu, Wei; Huang, Haiyan; Wu, Desheng; Zou, Fei; Liu, Jianjun

2017-01-01

Trichloroethylene (TCE) is an occupational and environmental chemical that can cause severe hepatotoxicity. While our previous studies showed that the phosphatase inhibitor SET is a key mediator of TCE-induced liver cell apoptosis, the molecular mechanisms remain elusive. Using quantitative phosphoproteomic analysis, we report here that nucleolin is a SET-regulated phosphoprotein in human liver HL-7702 cells. Functional analysis suggested that SET promoted dephosphorylation of nucleolin, decreased its binding to its transcriptional activator, c-myc, and upregulated nucleolin expression in TCE-treated cells. Importantly, TCE-induced hepatocyte apoptosis was significantly attenuated when nucleolin was downregulated with specific siRNAs. These findings indicate that TCE may induce hepatocyte apoptosis via SET-mediated dephosphorylation and overexpression of nucleolin. PMID:28402964

SET mediates TCE-induced liver cell apoptosis through dephosphorylation and upregulation of nucleolin.

PubMed

Ren, Xiaohu; Huang, Xinfeng; Yang, Xifei; Liu, Yungang; Liu, Wei; Huang, Haiyan; Wu, Desheng; Zou, Fei; Liu, Jianjun

2017-06-20

Trichloroethylene (TCE) is an occupational and environmental chemical that can cause severe hepatotoxicity. While our previous studies showed that the phosphatase inhibitor SET is a key mediator of TCE-induced liver cell apoptosis, the molecular mechanisms remain elusive. Using quantitative phosphoproteomic analysis, we report here that nucleolin is a SET-regulated phosphoprotein in human liver HL-7702 cells. Functional analysis suggested that SET promoted dephosphorylation of nucleolin, decreased its binding to its transcriptional activator, c-myc, and upregulated nucleolin expression in TCE-treated cells. Importantly, TCE-induced hepatocyte apoptosis was significantly attenuated when nucleolin was downregulated with specific siRNAs. These findings indicate that TCE may induce hepatocyte apoptosis via SET-mediated dephosphorylation and overexpression of nucleolin.

Prospects and challenges of quantitative phase imaging in tumor cell biology

NASA Astrophysics Data System (ADS)

Kemper, Björn; Götte, Martin; Greve, Burkhard; Ketelhut, Steffi

2016-03-01

Quantitative phase imaging (QPI) techniques provide high resolution label-free quantitative live cell imaging. Here, prospects and challenges of QPI in tumor cell biology are presented, using the example of digital holographic microscopy (DHM). It is shown that the evaluation of quantitative DHM phase images allows the retrieval of different parameter sets for quantification of cellular motion changes in migration and motility assays that are caused by genetic modifications. Furthermore, we demonstrate simultaneously label-free imaging of cell growth and morphology properties.

Quantitative diagnosis of bladder cancer by morphometric analysis of HE images

NASA Astrophysics Data System (ADS)

Wu, Binlin; Nebylitsa, Samantha V.; Mukherjee, Sushmita; Jain, Manu

2015-02-01

In clinical practice, histopathological analysis of biopsied tissue is the main method for bladder cancer diagnosis and prognosis. The diagnosis is performed by a pathologist based on the morphological features in the image of a hematoxylin and eosin (HE) stained tissue sample. This manuscript proposes algorithms to perform morphometric analysis on the HE images, quantify the features in the images, and discriminate bladder cancers with different grades, i.e. high grade and low grade. The nuclei are separated from the background and other types of cells such as red blood cells (RBCs) and immune cells using manual outlining, color deconvolution and image segmentation. A mask of nuclei is generated for each image for quantitative morphometric analysis. The features of the nuclei in the mask image including size, shape, orientation, and their spatial distributions are measured. To quantify local clustering and alignment of nuclei, we propose a 1-nearest-neighbor (1-NN) algorithm which measures nearest neighbor distance and nearest neighbor parallelism. The global distributions of the features are measured using statistics of the proposed parameters. A linear support vector machine (SVM) algorithm is used to classify the high grade and low grade bladder cancers. The results show using a particular group of nuclei such as large ones, and combining multiple parameters can achieve better discrimination. This study shows the proposed approach can potentially help expedite pathological diagnosis by triaging potentially suspicious biopsies.

Introduction to Modern Methods in Light Microscopy.

PubMed

Ryan, Joel; Gerhold, Abby R; Boudreau, Vincent; Smith, Lydia; Maddox, Paul S

2017-01-01

For centuries, light microscopy has been a key method in biological research, from the early work of Robert Hooke describing biological organisms as cells, to the latest in live-cell and single-molecule systems. Here, we introduce some of the key concepts related to the development and implementation of modern microscopy techniques. We briefly discuss the basics of optics in the microscope, super-resolution imaging, quantitative image analysis, live-cell imaging, and provide an outlook on active research areas pertaining to light microscopy.

Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq

PubMed Central

Shepard, Peter J.; Choi, Eun-A; Lu, Jente; Flanagan, Lisa A.; Hertel, Klemens J.; Shi, Yongsheng

2011-01-01

Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq analyses of human and mouse transcriptomes showed that 40%–50% of all expressed genes produce alternatively polyadenylated mRNAs. Furthermore, our study detected evolutionarily conserved polyadenylation of histone mRNAs and revealed novel features of mitochondrial RNA polyadenylation. Finally, PAS-Seq analyses of mouse embryonic stem (ES) cells, neural stem/progenitor (NSP) cells, and neurons not only identified more poly(A) sites than what was found in the entire mouse EST database, but also detected significant changes in the global APA profile that lead to lengthening of 3′ untranslated regions (UTR) in many mRNAs during stem cell differentiation. Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation. PMID:21343387

Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cui, Yi; Hu, Dehong; Markillie, Lye Meng

Quantitative gene expression analysis in intact single cells can be achieved using single molecule- based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple FISH sub-probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific binding of sub-probes and tissue autofluorescence, limiting its accuracy. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on blinking frequencies of photoswitchable dyes. This fluctuation localization imaging-based FISH (fliFISH) uses blinking frequency patterns, emitted frommore » a transcript bound to multiple sub-probes, which are distinct from blinking patterns emitted from partial or nonspecifically bound sub-probes and autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2, and their ratio (Nkx2-2/Ins2). These radial patterns, showing higher values in β cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct blinking frequency patterns, laying the foundation for reliable single-cell transcriptomics.« less

A new holistic 3D non-invasive analysis of cellular distribution and motility on fibroin-alginate microcarriers using light sheet fluorescent microscopy

PubMed Central

Pierini, Michela; Bevilacqua, Alessandro; Torre, Maria Luisa; Lucarelli, Enrico

2017-01-01

Cell interaction with biomaterials is one of the keystones to developing medical devices for tissue engineering applications. Biomaterials are the scaffolds that give three-dimensional support to the cells, and are vectors that deliver the cells to the injured tissue requiring repair. Features of biomaterials can influence the behaviour of the cells and consequently the efficacy of the tissue-engineered product. The adhesion, distribution and motility of the seeded cells onto the scaffold represent key aspects, and must be evaluated in vitro during the product development, especially when the efficacy of a specific tissue-engineered product depends on viable and functional cell loading. In this work, we propose a non-invasive and non-destructive imaging analysis for investigating motility, viability and distribution of Mesenchymal Stem Cells (MSCs) on silk fibroin-based alginate microcarriers, to test the adhesion capacity of the fibroin coating onto alginate which is known to be unsuitable for cell adhesion. However, in depth characterization of the biomaterial is beyond the scope of this paper. Scaffold-loaded MSCs were stained with Calcein-AM and Ethidium homodimer-1 to detect live and dead cells, respectively, and counterstained with Hoechst to label cell nuclei. Time-lapse Light Sheet Fluorescent Microscopy (LSFM) was then used to produce three-dimensional images of the entire cells-loaded fibroin/alginate microcarriers. In order to quantitatively track the cell motility over time, we also developed an open source user friendly software tool called Fluorescent Cell Tracker in Three-Dimensions (F-Tracker3D). Combining LSFM with F-Tracker3D we were able for the first time to assess the distribution and motility of stem cells in a non-invasive, non-destructive, quantitative, and three-dimensional analysis of the entire surface of the cell-loaded scaffold. We therefore propose this imaging technique as an innovative holistic tool for monitoring cell-biomaterial interactions, and as a tool for the design, fabrication and functionalization of a scaffold as a medical device. PMID:28817694

Quantitative Analysis of Cell Nucleus Organisation

PubMed Central

Shiels, Carol; Adams, Niall M; Islam, Suhail A; Stephens, David A; Freemont, Paul S

2007-01-01

There are almost 1,300 entries for higher eukaryotes in the Nuclear Protein Database. The proteins' subcellular distribution patterns within interphase nuclei can be complex, ranging from diffuse to punctate or microspeckled, yet they all work together in a coordinated and controlled manner within the three-dimensional confines of the nuclear volume. In this review we describe recent advances in the use of quantitative methods to understand nuclear spatial organisation and discuss some of the practical applications resulting from this work. PMID:17676980

Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells

NASA Astrophysics Data System (ADS)

Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

2016-04-01

Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue.

Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells

PubMed Central

Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

2016-01-01

Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue. PMID:27063397

Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome.

PubMed

Panizza, Elena; Branca, Rui M M; Oliviusson, Peter; Orre, Lukas M; Lehtiö, Janne

2017-07-03

Protein phosphorylation is involved in the regulation of most eukaryotic cells functions and mass spectrometry-based analysis has made major contributions to our understanding of this regulation. However, low abundance of phosphorylated species presents a major challenge in achieving comprehensive phosphoproteome coverage and robust quantification. In this study, we developed a workflow employing titanium dioxide phospho-enrichment coupled with isobaric labeling by Tandem Mass Tags (TMT) and high-resolution isoelectric focusing (HiRIEF) fractionation to perform in-depth quantitative phosphoproteomics starting with a low sample quantity. To benchmark the workflow, we analyzed HeLa cells upon pervanadate treatment or cell cycle arrest in mitosis. Analyzing 300 µg of peptides per sample, we identified 22,712 phosphorylation sites, of which 19,075 were localized with high confidence and 1,203 are phosphorylated tyrosine residues, representing 6.3% of all detected phospho-sites. HiRIEF fractions with the most acidic isoelectric points are enriched in multiply phosphorylated peptides, which represent 18% of all the phospho-peptides detected in the pH range 2.5-3.7. Cross-referencing with the PhosphoSitePlus database reveals 1,264 phosphorylation sites that have not been previously reported and kinase association analysis suggests that a subset of these may be functional during the mitotic phase.

Dynamic Contrast-enhanced MR Imaging in Renal Cell Carcinoma: Reproducibility of Histogram Analysis on Pharmacokinetic Parameters

PubMed Central

Wang, Hai-yi; Su, Zi-hua; Xu, Xiao; Sun, Zhi-peng; Duan, Fei-xue; Song, Yuan-yuan; Li, Lu; Wang, Ying-wei; Ma, Xin; Guo, Ai-tao; Ma, Lin; Ye, Hui-yi

2016-01-01

Pharmacokinetic parameters derived from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) have been increasingly used to evaluate the permeability of tumor vessel. Histogram metrics are a recognized promising method of quantitative MR imaging that has been recently introduced in analysis of DCE-MRI pharmacokinetic parameters in oncology due to tumor heterogeneity. In this study, 21 patients with renal cell carcinoma (RCC) underwent paired DCE-MRI studies on a 3.0 T MR system. Extended Tofts model and population-based arterial input function were used to calculate kinetic parameters of RCC tumors. Mean value and histogram metrics (Mode, Skewness and Kurtosis) of each pharmacokinetic parameter were generated automatically using ImageJ software. Intra- and inter-observer reproducibility and scan–rescan reproducibility were evaluated using intra-class correlation coefficients (ICCs) and coefficient of variation (CoV). Our results demonstrated that the histogram method (Mode, Skewness and Kurtosis) was not superior to the conventional Mean value method in reproducibility evaluation on DCE-MRI pharmacokinetic parameters (K trans & Ve) in renal cell carcinoma, especially for Skewness and Kurtosis which showed lower intra-, inter-observer and scan-rescan reproducibility than Mean value. Our findings suggest that additional studies are necessary before wide incorporation of histogram metrics in quantitative analysis of DCE-MRI pharmacokinetic parameters. PMID:27380733

Proteomic characterization of EL4 lymphoma-derived tumors upon chemotherapy treatment reveals potential roles for lysosomes and caspase-6 during tumor cell death in vivo.

PubMed

Kramer, David A; Eldeeb, Mohamed A; Wuest, Melinda; Mercer, John; Fahlman, Richard P

2017-06-01

The murine mouse lymphoblastic lymphoma cell line (EL4) tumor model is an established in vivo apoptosis model for the investigation of novel cancer imaging agents and immunological treatments due to the rapid and significant response of the EL4 tumors to cyclophosphamide and etoposide combination chemotherapy. Despite the utility of this model system in cancer research, little is known regarding the molecular details of in vivo tumor cell death. Here, we report the first in-depth quantitative proteomic analysis of the changes that occur in these tumors upon cyclophosphamide and etoposide treatment in vivo. Using a label-free quantitative proteomic approach a total of 5838 proteins were identified in the treated and untreated tumors, of which 875 were determined to change in abundance with statistical significance. Initial analysis of the data reveals changes that may have been predicted, such as the downregulation of ribosomes, but demonstrates the robustness of the dataset. Analysis of the dataset also reveals the unexpected downregulation of caspase-3 and an upregulation of caspase-6 in addition to a global upregulation of lysosomal proteins in the bulk of the tumor. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells*

PubMed Central

An, Hyun Joo; Gip, Phung; Kim, Jaehan; Wu, Shuai; Park, Kun Wook; McVaugh, Cheryl T.; Schaffer, David V.; Bertozzi, Carolyn R.; Lebrilla, Carlito B.

2012-01-01

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation. PMID:22147732

Highly Reproducible Label Free Quantitative Proteomic Analysis of RNA Polymerase Complexes*

PubMed Central

Mosley, Amber L.; Sardiu, Mihaela E.; Pattenden, Samantha G.; Workman, Jerry L.; Florens, Laurence; Washburn, Michael P.

2011-01-01

The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports. PMID:21048197

Deep Learning in Label-free Cell Classification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individualmore » cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. In conclusion, this system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.« less