Single cell transcriptomics to explore the immune system in health and disease†

PubMed Central

Regev, Aviv; Teichmann, Sarah A.

2017-01-01

The immune system varies in cell types, states, and locations. The complex networks, interactions and responses of immune cells produce diverse cellular ecosystems composed of multiple cell types, accompanied by genetic diversity in antigen receptors. Within this ecosystem, innate and adaptive immune cells maintain and protect tissue function, integrity and homeostasis upon changes in functional demands and diverse insults. Characterizing this inherent complexity requires studies at single-cell resolution. Recent advances such as, massively-parallel single cell RNA-Seq and sophisticated computational methods are catalysing a revolution in our understanding of immunology. Here, we provide an overview of the state of single cell genomics methods and an outlook on the use of single-cell techniques to decipher the adaptive and innate components of immunity. PMID:28983043

In vivo and in situ monitoring of the nitric oxide stimulus response of single cancer cells by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Su, L.; Chen, Y.; Zhang, G. N.; Wang, L. H.; Shen, A. G.; Zhou, X. D.; Wang, X. H.; Hu, J. M.

2013-04-01

Raman spectroscopy is capable of studying time-resolved information of selected biomolecular distributions inside individual cells without labeling. In this study, Raman spectroscopy was for the first time utilized to in vivo and in situ monitor the cellular response to nitric oxide (NO) in single oral squamous cell carcinoma (OSCC) cells over a period of 24 h. Sodium nitroprusside (SNP) was chosen as a NO donor to be incubated with the OSCC cell line (TCA8113) for certain time intervals. In vivo and in situ Raman analysis revealed that the degradation and conformational changes of nucleic acids, lipids and proteins could be directly observed by changes in the characteristic Raman bands. In comparison with conventional flow cytometric analysis, Raman spectroscopy not only detected more subtle NO-induced chemical changes of cells, where the SNP concentration could be even less than 1 mM, but also provided a full view of the whole chemical components of single cells. Raman spectroscopy therefore is an important candidate for label-free, nondestructive and in situ monitoring of cellular changes in response to chemotherapeutic agents, which could potentially be used in rapid screening of novel drugs.

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).

Application of First Principles Model to Spacecraft Operations

NASA Technical Reports Server (NTRS)

Timmerman, Paul; Bugga, Ratnakumar; DiStefano, Salvidor

1996-01-01

Previous models use a single phase reaction; cycled cell predicts cannot be met with a single phase; interphase conversion provides means for film aging; aging cells predictions display typical behaviors: pressure changes in NiH² cells; voltage fading upon cycling; second plateau on discharge of cycled cells; negative limited behavior for Ni-Cds.

Integrated Droplet-Based Microextraction with ESI-MS for Removal of Matrix Interference in Single-Cell Analysis.

PubMed

Zhang, Xiao-Chao; Wei, Zhen-Wei; Gong, Xiao-Yun; Si, Xing-Yu; Zhao, Yao-Yao; Yang, Cheng-Dui; Zhang, Si-Chun; Zhang, Xin-Rong

2016-04-29

Integrating droplet-based microfluidics with mass spectrometry is essential to high-throughput and multiple analysis of single cells. Nevertheless, matrix effects such as the interference of culture medium and intracellular components influence the sensitivity and the accuracy of results in single-cell analysis. To resolve this problem, we developed a method that integrated droplet-based microextraction with single-cell mass spectrometry. Specific extraction solvent was used to selectively obtain intracellular components of interest and remove interference of other components. Using this method, UDP-Glc-NAc, GSH, GSSG, AMP, ADP and ATP were successfully detected in single MCF-7 cells. We also applied the method to study the change of unicellular metabolites in the biological process of dysfunctional oxidative phosphorylation. The method could not only realize matrix-free, selective and sensitive detection of metabolites in single cells, but also have the capability for reliable and high-throughput single-cell analysis.

Single-cell sequencing technologies: current and future.

PubMed

Liang, Jialong; Cai, Wanshi; Sun, Zhongsheng

2014-10-20

Intensively developed in the last few years, single-cell sequencing technologies now present numerous advantages over traditional sequencing methods for solving the problems of biological heterogeneity and low quantities of available biological materials. The application of single-cell sequencing technologies has profoundly changed our understanding of a series of biological phenomena, including gene transcription, embryo development, and carcinogenesis. However, before single-cell sequencing technologies can be used extensively, researchers face the serious challenge of overcoming inherent issues of high amplification bias, low accuracy and reproducibility. Here, we simply summarize the techniques used for single-cell isolation, and review the current technologies used in single-cell genomic, transcriptomic, and epigenomic sequencing. We discuss the merits, defects, and scope of application of single-cell sequencing technologies and then speculate on the direction of future developments. Copyright © 2014 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells

PubMed Central

Gole, Jeff; Gore, Athurva; Richards, Andrew; Chiu, Yu-Jui; Fung, Ho-Lim; Bushman, Diane; Chiang, Hsin-I; Chun, Jerold; Lo, Yu-Hwa; Zhang, Kun

2013-01-01

Genome sequencing of single cells has a variety of applications, including characterizing difficult-to-culture microorganisms and identifying somatic mutations in single cells from mammalian tissues. A major hurdle in this process is the bias in amplifying the genetic material from a single cell, a procedure known as polymerase cloning. Here we describe the microwell displacement amplification system (MIDAS), a massively parallel polymerase cloning method in which single cells are randomly distributed into hundreds to thousands of nanoliter wells and simultaneously amplified for shotgun sequencing. MIDAS reduces amplification bias because polymerase cloning occurs in physically separated nanoliter-scale reactors, facilitating the de novo assembly of near-complete microbial genomes from single E. coli cells. In addition, MIDAS allowed us to detect single-copy number changes in primary human adult neurons at 1–2 Mb resolution. MIDAS will further the characterization of genomic diversity in many heterogeneous cell populations. PMID:24213699

Mass sensors with mechanical traps for weighing single cells in different fluids.

PubMed

Weng, Yaochung; Delgado, Francisco Feijó; Son, Sungmin; Burg, Thomas P; Wasserman, Steven C; Manalis, Scott R

2011-12-21

We present two methods by which single cells can be mechanically trapped and continuously monitored within the suspended microchannel resonator (SMR) mass sensor. Since the fluid surrounding the trapped cell can be quickly and completely replaced on demand, our methods are well suited for measuring changes in cell size and growth in response to drugs or other chemical stimuli. We validate our methods by measuring the density of single polystyrene beads and Saccharomyces cerevisiae yeast cells with a precision of approximately 10(-3) g cm(-3), and by monitoring the growth of single mouse lymphoblast cells before and after drug treatment.

Sensitive Detection of Single-Cell Secreted H2O2 by Integrating a Microfluidic Droplet Sensor and Au Nanoclusters.

PubMed

Shen, Rui; Liu, Peipei; Zhang, Yiqiu; Yu, Zhao; Chen, Xuyue; Zhou, Lu; Nie, Baoqing; Żaczek, Anna; Chen, Jian; Liu, Jian

2018-04-03

As an important signaling molecule, hydrogen peroxide (H 2 O 2 ) secreted externally by the cells influences cell migration, immunity generation, and cellular communications. Herein, we have developed a microfluidic approach with droplets in combination with Au nanoclusters for the sensitive detection of H 2 O 2 secreted by a single cell. Isolated in the ultrasmall volume (4.2 nL) of a microdroplet, single-cell secreted H 2 O 2 can initiate dramatic fluorescence changes of horseradish peroxidase-Au nanoclusters. We have demonstrated an ultrahigh sensitivity (200-400 attomole H 2 O 2 directly measured from a single cell) with good specificity. It offers a useful research tool to study the cell-to-cell differences of H 2 O 2 secretion at the single-cell level.

Limiting Energy Dissipation Induces Glassy Kinetics in Single-Cell High-Precision Responses

PubMed Central

Das, Jayajit

2016-01-01

Single cells often generate precise responses by involving dissipative out-of-thermodynamic-equilibrium processes in signaling networks. The available free energy to fuel these processes could become limited depending on the metabolic state of an individual cell. How does limiting dissipation affect the kinetics of high-precision responses in single cells? I address this question in the context of a kinetic proofreading scheme used in a simple model of early-time T cell signaling. Using exact analytical calculations and numerical simulations, I show that limiting dissipation qualitatively changes the kinetics in single cells marked by emergence of slow kinetics, large cell-to-cell variations of copy numbers, temporally correlated stochastic events (dynamic facilitation), and ergodicity breaking. Thus, constraints in energy dissipation, in addition to negatively affecting ligand discrimination in T cells, can create a fundamental difficulty in determining single-cell kinetics from cell-population results. PMID:26958894

Advances in single-cell RNA sequencing and its applications in cancer research.

PubMed

Zhu, Sibo; Qing, Tao; Zheng, Yuanting; Jin, Li; Shi, Leming

2017-08-08

Unlike population-level approaches, single-cell RNA sequencing enables transcriptomic analysis of an individual cell. Through the combination of high-throughput sequencing and bioinformatic tools, single-cell RNA-seq can detect more than 10,000 transcripts in one cell to distinguish cell subsets and dynamic cellular changes. After several years' development, single-cell RNA-seq can now achieve massively parallel, full-length mRNA sequencing as well as in situ sequencing and even has potential for multi-omic detection. One appealing area of single-cell RNA-seq is cancer research, and it is regarded as a promising way to enhance prognosis and provide more precise target therapy by identifying druggable subclones. Indeed, progresses have been made regarding solid tumor analysis to reveal intratumoral heterogeneity, correlations between signaling pathways, stemness, drug resistance, and tumor architecture shaping the microenvironment. Furthermore, through investigation into circulating tumor cells, many genes have been shown to promote a propensity toward stemness and the epithelial-mesenchymal transition, to enhance anchoring and adhesion, and to be involved in mechanisms of anoikis resistance and drug resistance. This review focuses on advances and progresses of single-cell RNA-seq with regard to the following aspects: 1. Methodologies of single-cell RNA-seq 2. Single-cell isolation techniques 3. Single-cell RNA-seq in solid tumor research 4. Single-cell RNA-seq in circulating tumor cell research 5.

Advances in single-cell RNA sequencing and its applications in cancer research

PubMed Central

Zhu, Sibo; Qing, Tao; Zheng, Yuanting; Jin, Li; Shi, Leming

2017-01-01

Unlike population-level approaches, single-cell RNA sequencing enables transcriptomic analysis of an individual cell. Through the combination of high-throughput sequencing and bioinformatic tools, single-cell RNA-seq can detect more than 10,000 transcripts in one cell to distinguish cell subsets and dynamic cellular changes. After several years’ development, single-cell RNA-seq can now achieve massively parallel, full-length mRNA sequencing as well as in situ sequencing and even has potential for multi-omic detection. One appealing area of single-cell RNA-seq is cancer research, and it is regarded as a promising way to enhance prognosis and provide more precise target therapy by identifying druggable subclones. Indeed, progresses have been made regarding solid tumor analysis to reveal intratumoral heterogeneity, correlations between signaling pathways, stemness, drug resistance, and tumor architecture shaping the microenvironment. Furthermore, through investigation into circulating tumor cells, many genes have been shown to promote a propensity toward stemness and the epithelial-mesenchymal transition, to enhance anchoring and adhesion, and to be involved in mechanisms of anoikis resistance and drug resistance. This review focuses on advances and progresses of single-cell RNA-seq with regard to the following aspects: 1. Methodologies of single-cell RNA-seq 2. Single-cell isolation techniques 3. Single-cell RNA-seq in solid tumor research 4. Single-cell RNA-seq in circulating tumor cell research 5. Perspectives PMID:28881849

Single-Cell Analysis of Experience-Dependent Transcriptomic States in Mouse Visual Cortex

PubMed Central

Hrvatin, Sinisa; Hochbaum, Daniel R.; Nagy, M. Aurel; Cicconet, Marcelo; Robertson, Keiramarie; Cheadle, Lucas; Zilionis, Rapolas; Ratner, Alex; Borges-Monroy, Rebeca; Klein, Allon M.; Sabatini, Bernardo L.; Greenberg, Michael E.

2017-01-01

Activity-dependent transcriptional responses shape cortical function. However, we lack a comprehensive understanding of the diversity of these responses across the full range of cortical cell types, and how these changes contribute to neuronal plasticity and disease. Here we applied high-throughput single-cell RNA-sequencing to investigate the breadth of transcriptional changes that occur across cell types in mouse visual cortex following exposure to light. We identified significant and divergent transcriptional responses to stimulation in each of the 30 cell types characterized, revealing 611 stimulus-responsive genes. Excitatory pyramidal neurons exhibit inter- and intra-laminar heterogeneity in the induction of stimulus responsive genes. Non-neuronal cells demonstrated clear transcriptional responses that may regulate experience-dependent changes in neurovascular coupling and myelination. Together, these results reveal the dynamic landscape of stimulus-dependent transcriptional changes that occur across cell types in visual cortex, which are likely critical for cortical function and may be sites of de-regulation in developmental brain disorders. PMID:29230054

Magnetic levitation of single cells

PubMed Central

Durmus, Naside Gozde; Tekin, H. Cumhur; Guven, Sinan; Sridhar, Kaushik; Arslan Yildiz, Ahu; Calibasi, Gizem; Davis, Ronald W.; Steinmetz, Lars M.; Demirci, Utkan

2015-01-01

Several cellular events cause permanent or transient changes in inherent magnetic and density properties of cells. Characterizing these changes in cell populations is crucial to understand cellular heterogeneity in cancer, immune response, infectious diseases, drug resistance, and evolution. Although magnetic levitation has previously been used for macroscale objects, its use in life sciences has been hindered by the inability to levitate microscale objects and by the toxicity of metal salts previously applied for levitation. Here, we use magnetic levitation principles for biological characterization and monitoring of cells and cellular events. We demonstrate that each cell type (i.e., cancer, blood, bacteria, and yeast) has a characteristic levitation profile, which we distinguish at an unprecedented resolution of 1 × 10−4 g⋅mL−1. We have identified unique differences in levitation and density blueprints between breast, esophageal, colorectal, and nonsmall cell lung cancer cell lines, as well as heterogeneity within these seemingly homogenous cell populations. Furthermore, we demonstrate that changes in cellular density and levitation profiles can be monitored in real time at single-cell resolution, allowing quantification of heterogeneous temporal responses of each cell to environmental stressors. These data establish density as a powerful biomarker for investigating living systems and their responses. Thereby, our method enables rapid, density-based imaging and profiling of single cells with intriguing applications, such as label-free identification and monitoring of heterogeneous biological changes under various physiological conditions, including antibiotic or cancer treatment in personalized medicine. PMID:26124131

The role of the cytoskeleton in sensing changes in gravity by nonspecialized cells.

PubMed

Vorselen, Daan; Roos, Wouter H; MacKintosh, Fred C; Wuite, Gijs J L; van Loon, Jack J W A

2014-02-01

A large body of evidence indicates that single cells in vitro respond to changes in gravity, and that this response might play an important role for physiological changes at the organism level during spaceflight. Gravity can lead to changes in cell proliferation, differentiation, signaling, and gene expression. At first glance, gravitational forces seem too small to affect bodies with the size of a cell. Thus, the initial response to gravity is both puzzling and important for understanding physiological changes in space. This also offers a unique environment to study the mechanical response of cells. In the past 2 decades, important steps have been made in the field of mechanobiology, and we use these advances to reevaluate the response of single cells to changes in gravity. Recent studies have focused on the cytoskeleton as initial gravity sensor. Thus, we review the observed changes in the cytoskeleton in a microgravity environment, both during spaceflight and in ground-based simulation techniques. We also evaluate to what degree the current experimental evidence supports the cytoskeleton as primary gravity sensor. Finally, we consider how the cytoskeleton itself could be affected by changed gravity. To make the next step toward understanding the response of cells to altered gravity, the challenge will be to track changes quantitatively and on short timescales.

Single-neuron identification of chemical constituents, physiological changes, and metabolism using mass spectrometry.

PubMed

Zhu, Hongying; Zou, Guichang; Wang, Ning; Zhuang, Meihui; Xiong, Wei; Huang, Guangming

2017-03-07

The use of single-cell assays has emerged as a cutting-edge technique during the past decade. Although single-cell mass spectrometry (MS) has recently achieved remarkable results, deep biological insights have not yet been obtained, probably because of various technical issues, including the unavoidable use of matrices, the inability to maintain cell viability, low throughput because of sample pretreatment, and the lack of recordings of cell physiological activities from the same cell. In this study, we describe a patch clamp/MS-based platform that enables the sensitive, rapid, and in situ chemical profiling of single living neurons. This approach integrates modified patch clamp technique and modified MS measurements to directly collect and detect nanoliter-scale samples from the cytoplasm of single neurons in mice brain slices. Abundant possible cytoplasmic constituents were detected in a single neuron at a relatively fast rate, and over 50 metabolites were identified in this study. The advantages of direct, rapid, and in situ sampling and analysis enabled us to measure the biological activities of the cytoplasmic constituents in a single neuron, including comparing neuron types by cytoplasmic chemical constituents; observing changes in constituent concentrations as the physiological conditions, such as age, vary; and identifying the metabolic pathways of small molecules.

Single-neuron identification of chemical constituents, physiological changes, and metabolism using mass spectrometry

PubMed Central

Zhu, Hongying; Zou, Guichang; Wang, Ning; Zhuang, Meihui; Xiong, Wei; Huang, Guangming

2017-01-01

The use of single-cell assays has emerged as a cutting-edge technique during the past decade. Although single-cell mass spectrometry (MS) has recently achieved remarkable results, deep biological insights have not yet been obtained, probably because of various technical issues, including the unavoidable use of matrices, the inability to maintain cell viability, low throughput because of sample pretreatment, and the lack of recordings of cell physiological activities from the same cell. In this study, we describe a patch clamp/MS-based platform that enables the sensitive, rapid, and in situ chemical profiling of single living neurons. This approach integrates modified patch clamp technique and modified MS measurements to directly collect and detect nanoliter-scale samples from the cytoplasm of single neurons in mice brain slices. Abundant possible cytoplasmic constituents were detected in a single neuron at a relatively fast rate, and over 50 metabolites were identified in this study. The advantages of direct, rapid, and in situ sampling and analysis enabled us to measure the biological activities of the cytoplasmic constituents in a single neuron, including comparing neuron types by cytoplasmic chemical constituents; observing changes in constituent concentrations as the physiological conditions, such as age, vary; and identifying the metabolic pathways of small molecules. PMID:28223513

Fibronectin Modulates Cell Adhesion and Signaling to Promote Single Cell Migration of Highly Invasive Oral Squamous Cell Carcinoma

PubMed Central

Ramos, Grasieli de Oliveira; Bernardi, Lisiane; Lauxen, Isabel; Sant’Ana Filho, Manoel; Horwitz, Alan Rick; Lamers, Marcelo Lazzaron

2016-01-01

Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad) or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad), plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization. PMID:26978651

Self-identity reprogrammed by a single residue switch in a cell surface receptor of a social bacterium.

PubMed

Cao, Pengbo; Wall, Daniel

2017-04-04

The ability to recognize close kin confers survival benefits on single-celled microbes that live in complex and changing environments. Microbial kinship detection relies on perceptible cues that reflect relatedness between individuals, although the mechanisms underlying recognition in natural populations remain poorly understood. In myxobacteria, cells identify related individuals through a polymorphic cell surface receptor, TraA. Recognition of compatible receptors leads to outer membrane exchange among clonemates and fitness consequences. Here, we investigated how a single receptor creates a diversity in recognition across myxobacterial populations. We first show that TraA requires its partner protein TraB to function in cell-cell adhesion. Recognition is shown to be traA allele-specific, where polymorphisms within TraA dictate binding selectivity. We reveal the malleability of TraA recognition, and seemingly minor changes to its variable region reprogram recognition outcomes. Strikingly, we identify a single residue (A/P205) as a molecular switch for TraA recognition. Substitutions at this position change the specificity of a diverse panel of environmental TraA receptors. In addition, we engineered a receptor with unique specificity by simply creating an A205P substitution, suggesting that modest changes in TraA can lead to diversification of new recognition groups in nature. We hypothesize that the malleable property of TraA has allowed it to evolve and create social barriers between myxobacterial populations and in turn avoid adverse interactions with relatives.

Integrated time-lapse and single-cell transcription studies highlight the variable and dynamic nature of human hematopoietic cell fate commitment

PubMed Central

Moussy, Alice; Cosette, Jérémie; Parmentier, Romuald; da Silva, Cindy; Corre, Guillaume; Richard, Angélique; Gandrillon, Olivier; Stockholm, Daniel

2017-01-01

Individual cells take lineage commitment decisions in a way that is not necessarily uniform. We address this issue by characterising transcriptional changes in cord blood-derived CD34+ cells at the single-cell level and integrating data with cell division history and morphological changes determined by time-lapse microscopy. We show that major transcriptional changes leading to a multilineage-primed gene expression state occur very rapidly during the first cell cycle. One of the 2 stable lineage-primed patterns emerges gradually in each cell with variable timing. Some cells reach a stable morphology and molecular phenotype by the end of the first cell cycle and transmit it clonally. Others fluctuate between the 2 phenotypes over several cell cycles. Our analysis highlights the dynamic nature and variable timing of cell fate commitment in hematopoietic cells, links the gene expression pattern to cell morphology, and identifies a new category of cells with fluctuating phenotypic characteristics, demonstrating the complexity of the fate decision process (which is different from a simple binary switch between 2 options, as it is usually envisioned). PMID:28749943

Single-cell sequencing reveals karyotype heterogeneity in murine and human malignancies.

PubMed

Bakker, Bjorn; Taudt, Aaron; Belderbos, Mirjam E; Porubsky, David; Spierings, Diana C J; de Jong, Tristan V; Halsema, Nancy; Kazemier, Hinke G; Hoekstra-Wakker, Karina; Bradley, Allan; de Bont, Eveline S J M; van den Berg, Anke; Guryev, Victor; Lansdorp, Peter M; Colomé-Tatché, Maria; Foijer, Floris

2016-05-31

Chromosome instability leads to aneuploidy, a state in which cells have abnormal numbers of chromosomes, and is found in two out of three cancers. In a chromosomal instable p53 deficient mouse model with accelerated lymphomagenesis, we previously observed whole chromosome copy number changes affecting all lymphoma cells. This suggests that chromosome instability is somehow suppressed in the aneuploid lymphomas or that selection for frequently lost/gained chromosomes out-competes the CIN-imposed mis-segregation. To distinguish between these explanations and to examine karyotype dynamics in chromosome instable lymphoma, we use a newly developed single-cell whole genome sequencing (scWGS) platform that provides a complete and unbiased overview of copy number variations (CNV) in individual cells. To analyse these scWGS data, we develop AneuFinder, which allows annotation of copy number changes in a fully automated fashion and quantification of CNV heterogeneity between cells. Single-cell sequencing and AneuFinder analysis reveals high levels of copy number heterogeneity in chromosome instability-driven murine T-cell lymphoma samples, indicating ongoing chromosome instability. Application of this technology to human B cell leukaemias reveals different levels of karyotype heterogeneity in these cancers. Our data show that even though aneuploid tumours select for particular and recurring chromosome combinations, single-cell analysis using AneuFinder reveals copy number heterogeneity. This suggests ongoing chromosome instability that other platforms fail to detect. As chromosome instability might drive tumour evolution, karyotype analysis using single-cell sequencing technology could become an essential tool for cancer treatment stratification.

Single-cell-precision microplasma-induced cancer cell apoptosis.

PubMed

Tan, Xiao; Zhao, Shasha; Lei, Qian; Lu, Xinpei; He, Guangyuan; Ostrikov, Kostya

2014-01-01

The issue of single-cell control has recently attracted enormous interest. However, in spite of the presently achievable intracellular-level physiological probing through bio-photonics, nano-probe-based, and some other techniques, the issue of inducing selective, single-cell-precision apoptosis, without affecting neighbouring cells remains essentially open. Here we resolve this issue and report on the effective single-cell-precision cancer cell treatment using the reactive chemistry of the localized corona-type plasma discharge around a needle-like electrode with the spot size ∼1 µm. When the electrode is positioned with the micrometer precision against a selected cell, a focused and highly-localized micro-plasma discharge induces apoptosis in the selected individual HepG2 and HeLa cancer cells only, without affecting any surrounding cells, even in small cell clusters. This is confirmed by the real-time monitoring of the morphological and structural changes at the cellular and cell nucleus levels after the plasma exposure.

Single Cell Oncogenesis

NASA Astrophysics Data System (ADS)

Lu, Xin

It is believed that cancer originates from a single cell that has gone through generations of evolution of genetic and epigenetic changes that associate with the hallmarks of cancer. In some cancers such as various types of leukemia, cancer is clonal. Yet in other cancers like glioblastoma (GBM), there is tremendous tumor heterogeneity that is likely to be caused by simultaneous evolution of multiple subclones within the same tissue. It is obvious that understanding how a single cell develops into a clonal tumor upon genetic alterations, at molecular and cellular levels, holds the key to the real appreciation of tumor etiology and ultimate solution for therapeutics. Surprisingly very little is known about the process of spontaneous tumorigenesis from single cells in human or vertebrate animal models. The main reason is the lack of technology to track the natural process of single cell changes from a homeostatic state to a progressively cancerous state. Recently, we developed a patented compound, photoactivatable (''caged'') tamoxifen analogue 4-OHC and associated technique called optochemogenetic switch (OCG switch), which we believe opens the opportunity to address this urgent biological as well as clinical question about cancer. We propose to combine OCG switch with genetically engineered mouse models of head and neck squamous cell carcinoma and high grade astrocytoma (including GBM) to study how single cells, when transformed through acute loss of tumor suppressor genes PTEN and TP53 and gain of oncogenic KRAS, can develop into tumor colonies with cellular and molecular heterogeneity in these tissues. The abstract is for my invited talk in session ``Beyond Darwin: Evolution in Single Cells'' 3/18/2016 11:15 AM.

Limiting Energy Dissipation Induces Glassy Kinetics in Single-Cell High-Precision Responses.

PubMed

Das, Jayajit

2016-03-08

Single cells often generate precise responses by involving dissipative out-of-thermodynamic-equilibrium processes in signaling networks. The available free energy to fuel these processes could become limited depending on the metabolic state of an individual cell. How does limiting dissipation affect the kinetics of high-precision responses in single cells? I address this question in the context of a kinetic proofreading scheme used in a simple model of early-time T cell signaling. Using exact analytical calculations and numerical simulations, I show that limiting dissipation qualitatively changes the kinetics in single cells marked by emergence of slow kinetics, large cell-to-cell variations of copy numbers, temporally correlated stochastic events (dynamic facilitation), and ergodicity breaking. Thus, constraints in energy dissipation, in addition to negatively affecting ligand discrimination in T cells, can create a fundamental difficulty in determining single-cell kinetics from cell-population results. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Tumor Cell Gene Expression Changes Following Short-term In vivo Exposure to Single Agent Chemotherapeutics are Related to Survival in Multiple Myeloma

PubMed Central

Burington, Bart; Barlogie, Bart; Zhan, Fenghuang; Crowley, John; Shaughnessy, John D.

2013-01-01

Changes in global gene expression patterns in tumor cells following in vivo therapy may vary by treatment and provide added or synergistic prognostic power over pretherapy gene expression profiles (GEP). This molecular readout of drug-cell interaction may also point to mechanisms of action/resistance. In newly diagnosed patients with multiple myeloma (MM), microarray data were obtained on tumor cells prior to and 48 hours after in vivo treatment using dexamethasone (n = 45) or thalidomide (n = 42); in the case of relapsed MM, microarray data were obtained prior to (n = 36) and after (n = 19) lenalidomide administration. Dexamethasone and thalidomide induced both common and unique GEP changes in tumor cells. Combined baseline and 48-hour changes in GEP in a subset of genes, many related to oxidative stress and cytoskeletal dynamics, were predictive of outcome in newly diagnosed MM patients receiving tandem transplants. Thalidomide-altered genes also changed following lenalidomide exposure and predicted event-free and overall survival in relapsed patients receiving lenalidomide as a single agent. Combined with baseline molecular features, changes in GEP following short-term single-agent exposure may help guide treatment decisions for patients with MM. Genes whose drug-altered expression were found to be related to survival may point to molecular switches related to response and/or resistance to different classes of drugs. PMID:18676754

Cell Electrical Impedance as a Novel Approach for Studies on Senescence Not Based on Biomarkers

PubMed Central

Cha, Jung-Joon; Park, Yangkyu; Yun, Joho; Kim, Hyeon Woo; Park, Chang-Ju; Kang, Giseok; Jung, Minhyun; Pak, Boryeong; Jin, Suk-Won

2016-01-01

Senescence of cardiac myocytes is frequently associated with heart diseases. To analyze senescence in cardiac myocytes, a number of biomarkers have been isolated. However, due to the complex nature of senescence, multiple markers are required for a single assay to accurately depict complex physiological changes associated with senescence. In single cells, changes in both cytoplasm and cell membrane during senescence can affect the changes in electrical impedance. Based on this phenomenon, we developed MEDoS, a novel microelectrochemical impedance spectroscopy for diagnosis of senescence, which allows us to precisely measure quantitative changes in electrical properties of aging cells. Using cardiac myocytes isolated from 3-, 6-, and 18-month-old isogenic zebrafish, we examined the efficacy of MEDoS and showed that MEDoS can identify discernible changes in electrical impedance. Taken together, our data demonstrated that electrical impedance in cells at different ages is distinct with quantitative values; these results were comparable with previously reported ones. Therefore, we propose that MEDoS be used as a new biomarker-independent methodology to obtain quantitative data on the biological senescence status of individual cells. PMID:27812531

Single-Cell Sequencing of the Healthy and Diseased Heart Reveals Ckap4 as a New Modulator of Fibroblasts Activation.

PubMed

Gladka, Monika M; Molenaar, Bas; de Ruiter, Hesther; van der Elst, Stefan; Tsui, Hoyee; Versteeg, Danielle; Lacraz, Grègory P A; Huibers, Manon M H; van Oudenaarden, Alexander; van Rooij, Eva

2018-01-31

Background -Genome-wide transcriptome analysis has greatly advanced our understanding of the regulatory networks underlying basic cardiac biology and mechanisms driving disease. However, so far, the resolution of studying gene expression patterns in the adult heart has been limited to the level of extracts from whole tissues. The use of tissue homogenates inherently causes the loss of any information on cellular origin or cell type-specific changes in gene expression. Recent developments in RNA amplification strategies provide a unique opportunity to use small amounts of input RNA for genome-wide sequencing of single cells. Methods -Here, we present a method to obtain high quality RNA from digested cardiac tissue from adult mice for automated single-cell sequencing of both the healthy and diseased heart. Results -After optimization, we were able to perform single-cell sequencing on adult cardiac tissue under both homeostatic conditions and after ischemic injury. Clustering analysis based on differential gene expression unveiled known and novel markers of all main cardiac cell types. Based on differential gene expression we were also able to identify multiple subpopulations within a certain cell type. Furthermore, applying single-cell sequencing on both the healthy and the injured heart indicated the presence of disease-specific cell subpopulations. As such, we identified cytoskeleton associated protein 4 ( Ckap4 ) as a novel marker for activated fibroblasts that positively correlates with known myofibroblast markers in both mouse and human cardiac tissue. Ckap4 inhibition in activated fibroblasts treated with TGFβ triggered a greater increase in the expression of genes related to activated fibroblasts compared to control, suggesting a role of Ckap4 in modulating fibroblast activation in the injured heart. Conclusions -Single-cell sequencing on both the healthy and diseased adult heart allows us to study transcriptomic differences between cardiac cells, as well as cell type-specific changes in gene expression during cardiac disease. This new approach provides a wealth of novel insights into molecular changes that underlie the cellular processes relevant for cardiac biology and pathophysiology. Applying this technology could lead to the discovery of new therapeutic targets relevant for heart disease.

Temporal Variation in Single-Cell Power-Law Rheology Spans the Ensemble Variation of Cell Population.

PubMed

Cai, PingGen; Takahashi, Ryosuke; Kuribayashi-Shigetomi, Kaori; Subagyo, Agus; Sueoka, Kazuhisa; Maloney, John M; Van Vliet, Krystyn J; Okajima, Takaharu

2017-08-08

Changes in the cytoskeletal organization within cells can be characterized by large spatial and temporal variations in rheological properties of the cell (e.g., the complex shear modulus G ∗ ). Although the ensemble variation in G ∗ of single cells has been elucidated, the detailed temporal variation of G ∗ remains unknown. In this study, we investigated how the rheological properties of individual fibroblast cells change under a spatially confined environment in which the cell translational motion is highly restricted and the whole cell shape remains unchanged. The temporal evolution of single-cell rheology was probed at the same measurement location within the cell, using atomic force microscopy-based oscillatory deformation. The measurements reveal that the temporal variation in the power-law rheology of cells is quantitatively consistent with the ensemble variation, indicating that the cell system satisfies an ergodic hypothesis in which the temporal statistics are identical to the ensemble statistics. The autocorrelation of G ∗ implies that the cell mechanical state evolves in the ensemble of possible states with a characteristic timescale. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Nanosecond pulsed electric field induced changes in cell surface charge density.

PubMed

Dutta, Diganta; Palmer, Xavier-Lewis; Asmar, Anthony; Stacey, Michael; Qian, Shizhi

2017-09-01

This study reports that the surface charge density changes in Jurkat cells with the application of single 60 nanosecond pulse electric fields, using atomic force microscopy. Using an atomic force microscope tip and Jurkat cells on silica in a 0.01M KCl ionic concentration, we were able to measure the interfacial forces, while also predicting surface charge densities of both Jurkat cell and silica surfaces. The most important finding is that the pulsing conditions varyingly reduced the cells' surface charge density. This offers a novel way in which to examine cellular effects of pulsed electric fields that may lead to the identification of unique mechanical responses. Compared to a single low field strength NsPEF (15kV/cm) application, exposure of Jurkat cells to a single high field strength NsPEF (60kV/cm) resulted in a further reduction in charge density and major morphological changes. The structural, physical, and chemical properties of biological cells immensely influence their electrostatic force; we were able to investigate this through the use of atomic force microscopy by measuring the surface forces between the AFM's tip and the Jurkat cells under different pulsing conditions as well as the interfacial forces in ionic concentrations. Copyright © 2017 Elsevier Ltd. All rights reserved.

Single-cell transcriptomes identify human islet cell signatures and reveal cell-type–specific expression changes in type 2 diabetes

PubMed Central

Bolisetty, Mohan; Kursawe, Romy; Sun, Lili; Sivakamasundari, V.; Kycia, Ina

2017-01-01

Blood glucose levels are tightly controlled by the coordinated action of at least four cell types constituting pancreatic islets. Changes in the proportion and/or function of these cells are associated with genetic and molecular pathophysiology of monogenic, type 1, and type 2 (T2D) diabetes. Cellular heterogeneity impedes precise understanding of the molecular components of each islet cell type that govern islet (dys)function, particularly the less abundant delta and gamma/pancreatic polypeptide (PP) cells. Here, we report single-cell transcriptomes for 638 cells from nondiabetic (ND) and T2D human islet samples. Analyses of ND single-cell transcriptomes identified distinct alpha, beta, delta, and PP/gamma cell-type signatures. Genes linked to rare and common forms of islet dysfunction and diabetes were expressed in the delta and PP/gamma cell types. Moreover, this study revealed that delta cells specifically express receptors that receive and coordinate systemic cues from the leptin, ghrelin, and dopamine signaling pathways implicating them as integrators of central and peripheral metabolic signals into the pancreatic islet. Finally, single-cell transcriptome profiling revealed genes differentially regulated between T2D and ND alpha, beta, and delta cells that were undetectable in paired whole islet analyses. This study thus identifies fundamental cell-type–specific features of pancreatic islet (dys)function and provides a critical resource for comprehensive understanding of islet biology and diabetes pathogenesis. PMID:27864352

Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects.

PubMed

Zhang, Xiaoyan; Marjani, Sadie L; Hu, Zhaoyang; Weissman, Sherman M; Pan, Xinghua; Wu, Shixiu

2016-03-15

Advances in genomic technology have enabled the faithful detection and measurement of mutations and the gene expression profile of cancer cells at the single-cell level. Recently, several single-cell sequencing methods have been developed that permit the comprehensive and precise analysis of the cancer-cell genome, transcriptome, and epigenome. The use of these methods to analyze cancer cells has led to a series of unanticipated discoveries, such as the high heterogeneity and stochastic changes in cancer-cell populations, the new driver mutations and the complicated clonal evolution mechanisms, and the novel identification of biomarkers of variant tumors. These methods and the knowledge gained from their utilization could potentially improve the early detection and monitoring of rare cancer cells, such as circulating tumor cells and disseminated tumor cells, and promote the development of personalized and highly precise cancer therapy. Here, we discuss the current methods for single cancer-cell sequencing, with a strong focus on those practically used or potentially valuable in cancer research, including single-cell isolation, whole genome and transcriptome amplification, epigenome profiling, multi-dimensional sequencing, and next-generation sequencing and analysis. We also examine the current applications, challenges, and prospects of single cancer-cell sequencing. ©2016 American Association for Cancer Research.

Imaging mRNA In Vivo, from Birth to Death.

PubMed

Tutucci, Evelina; Livingston, Nathan M; Singer, Robert H; Wu, Bin

2018-05-20

RNA is the fundamental information transfer system in the cell. The ability to follow single messenger RNAs (mRNAs) from transcription to degradation with fluorescent probes gives quantitative information about how the information is transferred from DNA to proteins. This review focuses on the latest technological developments in the field of single-mRNA detection and their usage to study gene expression in both fixed and live cells. By describing the application of these imaging tools, we follow the journey of mRNA from transcription to decay in single cells, with single-molecule resolution. We review current theoretical models for describing transcription and translation that were generated by single-molecule and single-cell studies. These methods provide a basis to study how single-molecule interactions generate phenotypes, fundamentally changing our understating of gene expression regulation.

Complex dynamics of selection and cellular memory in adaptation to a changing environment

NASA Astrophysics Data System (ADS)

Kussell, Edo; Lin, Wei-Hsiang

We study a synthetic evolutionary system in bacteria in which an antibiotic resistance gene is controlled by a stochastic on/off switching promoter. At the population level, this system displays all the basic ingredients for evolutionary selection, including diversity, fitness differences, and heritability. At the single cell level, physiological processes can modulate the ability of selection to act. We expose the stochastic switching strains to pulses of antibiotics of different durations in periodically changing environments using microfluidics. Small populations are tracked over a large number of periods at single cell resolution, allowing the visualization and quantification of selective sweeps and counter-sweeps at the population level, as well as detailed single cell analysis. A simple model is introduced to predict long-term population growth rates from single cell measurements, and reveals unexpected aspects of population dynamics, including cellular memory that acts on a fast timescale to modulate growth rates. This work is supported by NIH Grant No. R01-GM097356.

In Vivo Single-Cell Fluorescence and Size Scaling of Phytoplankton Chlorophyll Content.

PubMed

Álvarez, Eva; Nogueira, Enrique; López-Urrutia, Ángel

2017-04-01

In unicellular phytoplankton, the size scaling exponent of chlorophyll content per cell decreases with increasing light limitation. Empirical studies have explored this allometry by combining data from several species, using average values of pigment content and cell size for each species. The resulting allometry thus includes phylogenetic and size scaling effects. The possibility of measuring single-cell fluorescence with imaging-in-flow cytometry devices allows the study of the size scaling of chlorophyll content at both the inter- and intraspecific levels. In this work, the changing allometry of chlorophyll content was estimated for the first time for single phytoplankton populations by using data from a series of incubations with monocultures exposed to different light levels. Interspecifically, our experiments confirm previous modeling and experimental results of increasing size scaling exponents with increasing irradiance. A similar pattern was observed intraspecifically but with a larger variability in size scaling exponents. Our results show that size-based processes and geometrical approaches explain variations in chlorophyll content. We also show that the single-cell fluorescence measurements provided by imaging-in-flow devices can be applied to field samples to understand the changes in the size dependence of chlorophyll content in response to environmental variables affecting primary production. IMPORTANCE The chlorophyll concentrations in phytoplankton register physiological adjustments in cellular pigmentation arising mainly from changes in light conditions. The extent of these adjustments is constrained by the size of the phytoplankton cells, even within single populations. Hence, variations in community chlorophyll derived from photoacclimation are also dependent on the phytoplankton size distribution. Copyright © 2017 American Society for Microbiology.

In Vivo Single-Cell Fluorescence and Size Scaling of Phytoplankton Chlorophyll Content

PubMed Central

Nogueira, Enrique; López-Urrutia, Ángel

2017-01-01

ABSTRACT In unicellular phytoplankton, the size scaling exponent of chlorophyll content per cell decreases with increasing light limitation. Empirical studies have explored this allometry by combining data from several species, using average values of pigment content and cell size for each species. The resulting allometry thus includes phylogenetic and size scaling effects. The possibility of measuring single-cell fluorescence with imaging-in-flow cytometry devices allows the study of the size scaling of chlorophyll content at both the inter- and intraspecific levels. In this work, the changing allometry of chlorophyll content was estimated for the first time for single phytoplankton populations by using data from a series of incubations with monocultures exposed to different light levels. Interspecifically, our experiments confirm previous modeling and experimental results of increasing size scaling exponents with increasing irradiance. A similar pattern was observed intraspecifically but with a larger variability in size scaling exponents. Our results show that size-based processes and geometrical approaches explain variations in chlorophyll content. We also show that the single-cell fluorescence measurements provided by imaging-in-flow devices can be applied to field samples to understand the changes in the size dependence of chlorophyll content in response to environmental variables affecting primary production. IMPORTANCE The chlorophyll concentrations in phytoplankton register physiological adjustments in cellular pigmentation arising mainly from changes in light conditions. The extent of these adjustments is constrained by the size of the phytoplankton cells, even within single populations. Hence, variations in community chlorophyll derived from photoacclimation are also dependent on the phytoplankton size distribution. PMID:28115378

Precision toxicology based on single cell sequencing: an evolving trend in toxicological evaluations and mechanism exploration.

PubMed

Zhang, Boyang; Huang, Kunlun; Zhu, Liye; Luo, Yunbo; Xu, Wentao

2017-07-01

In this review, we introduce a new concept, precision toxicology: the mode of action of chemical- or drug-induced toxicity can be sensitively and specifically investigated by isolating a small group of cells or even a single cell with typical phenotype of interest followed by a single cell sequencing-based analysis. Precision toxicology can contribute to the better detection of subtle intracellular changes in response to exogenous substrates, and thus help researchers find solutions to control or relieve the toxicological effects that are serious threats to human health. We give examples for single cell isolation and recommend laser capture microdissection for in vivo studies and flow cytometric sorting for in vitro studies. In addition, we introduce the procedures for single cell sequencing and describe the expected application of these techniques to toxicological evaluations and mechanism exploration, which we believe will become a trend in toxicology.

Single-cell paired-end genome sequencing reveals structural variation per cell cycle

PubMed Central

Voet, Thierry; Kumar, Parveen; Van Loo, Peter; Cooke, Susanna L.; Marshall, John; Lin, Meng-Lay; Zamani Esteki, Masoud; Van der Aa, Niels; Mateiu, Ligia; McBride, David J.; Bignell, Graham R.; McLaren, Stuart; Teague, Jon; Butler, Adam; Raine, Keiran; Stebbings, Lucy A.; Quail, Michael A.; D’Hooghe, Thomas; Moreau, Yves; Futreal, P. Andrew; Stratton, Michael R.; Vermeesch, Joris R.; Campbell, Peter J.

2013-01-01

The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants. By applying the methods, we capture DNA copy number changes acquired over one cell cycle in breast cancer cells and in blastomeres derived from a human zygote after in vitro fertilization. Furthermore, we were able to discover and fine-map a heritable inter-chromosomal rearrangement t(1;16)(p36;p12) by sequencing a single blastomere. The methods will expedite applications in basic genome research and provide a stepping stone to novel approaches for clinical genetic diagnosis. PMID:23630320

Synchrotron FTIR microspectroscopy reveals early adipogenic differentiation of human mesenchymal stem cells at single-cell level

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

Liu, Zhixiao; University of Chinese Academy of Science, Beijing 100049; Tang, Yuzhao

Human mesenchymal stem cells (hMSCs) have been used as an ideal in vitro model to study human adipogenesis. However, little knowledge of the early stage differentiation greatly hinders our understanding on the mechanism of the adipogenesis processes. In this study, synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy was applied to track the global structural and compositional changes of lipids, proteins and nucleic acids inside individual hMSCs along the time course. The multivariate analysis of the SR-FTIR spectra distinguished the dynamic and significant changes of the lipids and nucleic acid at early differentiation stage. Importantly, changes of lipid structure during early daysmore » (Day 1–3) of differentiation might serve as a potential biomarker in identifying the state in early differentiation at single cell level. These results proved that SR-FTIR is a powerful tool to study the stem cell fate determination and early lipogenesis events. - Highlights: • Molecular events occur in the early adipogenic differentiation stage of hMSCs are studied by SR-FTIR. • SR-FTIR data suggest that lipids may play an important role in hMSCs determination. • As potential biomarkers, lipids peaks can identify the state of cell in early differentiation stage at single-cell level.« less

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.

A Minimally Invasive Method for Retrieving Single Adherent Cells of Different Types from Cultures

PubMed Central

Zeng, Jia; Mohammadreza, Aida; Gao, Weimin; Merza, Saeed; Smith, Dean; Kelbauskas, Laimonas; Meldrum, Deirdre R.

2014-01-01

The field of single-cell analysis has gained a significant momentum over the last decade. Separation and isolation of individual cells is an indispensable step in almost all currently available single-cell analysis technologies. However, stress levels introduced by such manipulations remain largely unstudied. We present a method for minimally invasive retrieval of selected individual adherent cells of different types from cell cultures. The method is based on a combination of mechanical (shear flow) force and biochemical (trypsin digestion) treatment. We quantified alterations in the transcription levels of stress response genes in individual cells exposed to varying levels of shear flow and trypsinization. We report optimal temperature, RNA preservation reagents, shear force and trypsinization conditions necessary to minimize changes in the stress-related gene expression levels. The method and experimental findings are broadly applicable and can be used by a broad research community working in the field of single cell analysis. PMID:24957932

Digital Quantification of Proteins and mRNA in Single Mammalian Cells.

PubMed

Albayrak, Cem; Jordi, Christian A; Zechner, Christoph; Lin, Jing; Bichsel, Colette A; Khammash, Mustafa; Tay, Savaş

2016-03-17

Absolute quantification of macromolecules in single cells is critical for understanding and modeling biological systems that feature cellular heterogeneity. Here we show extremely sensitive and absolute quantification of both proteins and mRNA in single mammalian cells by a very practical workflow that combines proximity ligation assay (PLA) and digital PCR. This digital PLA method has femtomolar sensitivity, which enables the quantification of very small protein concentration changes over its entire 3-log dynamic range, a quality necessary for accounting for single-cell heterogeneity. We counted both endogenous (CD147) and exogenously expressed (GFP-p65) proteins from hundreds of single cells and determined the correlation between CD147 mRNA and the protein it encodes. Using our data, a stochastic two-state model of the central dogma was constructed and verified using joint mRNA/protein distributions, allowing us to estimate transcription burst sizes and extrinsic noise strength and calculate the transcription and translation rate constants in single mammalian cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Fundamental trade-offs between information flow in single cells and cellular populations.

PubMed

Suderman, Ryan; Bachman, John A; Smith, Adam; Sorger, Peter K; Deeds, Eric J

2017-05-30

Signal transduction networks allow eukaryotic cells to make decisions based on information about intracellular state and the environment. Biochemical noise significantly diminishes the fidelity of signaling: networks examined to date seem to transmit less than 1 bit of information. It is unclear how networks that control critical cell-fate decisions (e.g., cell division and apoptosis) can function with such low levels of information transfer. Here, we use theory, experiments, and numerical analysis to demonstrate an inherent trade-off between the information transferred in individual cells and the information available to control population-level responses. Noise in receptor-mediated apoptosis reduces information transfer to approximately 1 bit at the single-cell level but allows 3-4 bits of information to be transmitted at the population level. For processes such as eukaryotic chemotaxis, in which single cells are the functional unit, we find high levels of information transmission at a single-cell level. Thus, low levels of information transfer are unlikely to represent a physical limit. Instead, we propose that signaling networks exploit noise at the single-cell level to increase population-level information transfer, allowing extracellular ligands, whose levels are also subject to noise, to incrementally regulate phenotypic changes. This is particularly critical for discrete changes in fate (e.g., life vs. death) for which the key variable is the fraction of cells engaged. Our findings provide a framework for rationalizing the high levels of noise in metazoan signaling networks and have implications for the development of drugs that target these networks in the treatment of cancer and other diseases.

Spatial and temporal single-cell volume estimation by a fluorescence imaging technique with application to astrocytes in primary culture

NASA Astrophysics Data System (ADS)

Khatibi, Siamak; Allansson, Louise; Gustavsson, Tomas; Blomstrand, Fredrik; Hansson, Elisabeth; Olsson, Torsten

1999-05-01

Cell volume changes are often associated with important physiological and pathological processes in the cell. These changes may be the means by which the cell interacts with its surrounding. Astroglial cells change their volume and shape under several circumstances that affect the central nervous system. Following an incidence of brain damage, such as a stroke or a traumatic brain injury, one of the first events seen is swelling of the astroglial cells. In order to study this and other similar phenomena, it is desirable to develop technical instrumentation and analysis methods capable of detecting and characterizing dynamic cell shape changes in a quantitative and robust way. We have developed a technique to monitor and to quantify the spatial and temporal volume changes in a single cell in primary culture. The technique is based on two- and three-dimensional fluorescence imaging. The temporal information is obtained from a sequence of microscope images, which are analyzed in real time. The spatial data is collected in a sequence of images from the microscope, which is automatically focused up and down through the specimen. The analysis of spatial data is performed off-line and consists of photobleaching compensation, focus restoration, filtering, segmentation and spatial volume estimation.

Cell Deformation by Single-beam Acoustic Trapping: A Promising Tool for Measurements of Cell Mechanics

PubMed Central

Hwang, Jae Youn; Kim, Jihun; Park, Jin Man; Lee, Changyang; Jung, Hayong; Lee, Jungwoo; Shung, K. Kirk

2016-01-01

We demonstrate a noncontact single-beam acoustic trapping method for the quantification of the mechanical properties of a single suspended cell with label-free. Experimentally results show that the single-beam acoustic trapping force results in morphological deformation of a trapped cell. While a cancer cell was trapped in an acoustic beam focus, the morphological changes of the immobilized cell were monitored using bright-field imaging. The cell deformability was then compared with that of a trapped polystyrene microbead as a function of the applied acoustic pressure for a better understanding of the relationship between the pressure and degree of cell deformation. Cell deformation was found to become more pronounced as higher pressure levels were applied. Furthermore, to determine if this acoustic trapping method can be exploited in quantifying the cell mechanics in a suspension and in a non-contact manner, the deformability levels of breast cancer cells with different degrees of invasiveness due to acoustic trapping were compared. It was found that highly-invasive breast cancer cells exhibited greater deformability than weakly-invasive breast cancer cells. These results clearly demonstrate that the single-beam acoustic trapping technique is a promising tool for non-contact quantitative assessments of the mechanical properties of single cells in suspensions with label-free. PMID:27273365

Oxygen depletion speeds and simplifies diffusion in HeLa cells.

PubMed

Edwald, Elin; Stone, Matthew B; Gray, Erin M; Wu, Jing; Veatch, Sarah L

2014-10-21

Many cell types undergo a hypoxic response in the presence of low oxygen, which can lead to transcriptional, metabolic, and structural changes within the cell. Many biophysical studies to probe the localization and dynamics of single fluorescently labeled molecules in live cells either require or benefit from low-oxygen conditions. In this study, we examine how low-oxygen conditions alter the mobility of a series of plasma membrane proteins with a range of anchoring motifs in HeLa cells at 37°C. Under high-oxygen conditions, diffusion of all proteins is heterogeneous and confined. When oxygen is reduced with an enzymatic oxygen-scavenging system for ≥ 15 min, diffusion rates increase by > 2-fold, motion becomes unconfined on the timescales and distance scales investigated, and distributions of diffusion coefficients are remarkably consistent with those expected from Brownian motion. More subtle changes in protein mobility are observed in several other laboratory cell lines examined under both high- and low-oxygen conditions. Morphological changes and actin remodeling are observed in HeLa cells placed in a low-oxygen environment for 30 min, but changes are less apparent in the other cell types investigated. This suggests that changes in actin structure are responsible for increased diffusion in hypoxic HeLa cells, although superresolution localization measurements in chemically fixed cells indicate that membrane proteins do not colocalize with F-actin under either experimental condition. These studies emphasize the importance of controls in single-molecule imaging measurements, and indicate that acute response to low oxygen in HeLa cells leads to dramatic changes in plasma membrane structure. It is possible that these changes are either a cause or consequence of phenotypic changes in solid tumor cells associated with increased drug resistance and malignancy.

P53 Gene Mutagenesis in Breast Cancer

DTIC Science & Technology

2005-03-01

the wild type T peak. 12 Table 1. Sonic ntations dected by SINtA Individual Cell Sequence Amino Acid Species Conservation 3 ID’ ID Change2 Change... differences in the content of toxic substances in the diet (Biggs et al., 1993; Blaszyk et al., 1996). The development of this p53 mutation load...Changes in the P53 Gene in Single Cells Individual Sequence Amino acid Species conservation ’ ID’ Cell ID change’ change Monkey Mouse Rat Chicken

Pinched-flow hydrodynamic stretching of single-cells.

PubMed

Dudani, Jaideep S; Gossett, Daniel R; Tse, Henry T K; Di Carlo, Dino

2013-09-21

Reorganization of cytoskeletal networks, condensation and decondensation of chromatin, and other whole cell structural changes often accompany changes in cell state and can reflect underlying disease processes. As such, the observable mechanical properties, or mechanophenotype, which is closely linked to intracellular architecture, can be a useful label-free biomarker of disease. In order to make use of this biomarker, a tool to measure cell mechanical properties should accurately characterize clinical specimens that consist of heterogeneous cell populations or contain small diseased subpopulations. Because of the heterogeneity and potential for rare populations in clinical samples, single-cell, high-throughput assays are ideally suited. Hydrodynamic stretching has recently emerged as a powerful method for carrying out mechanical phenotyping. Importantly, this method operates independently of molecular probes, reducing cost and sample preparation time, and yields information-rich signatures of cell populations through significant image analysis automation, promoting more widespread adoption. In this work, we present an alternative mode of hydrodynamic stretching where inertially-focused cells are squeezed in flow by perpendicular high-speed pinch flows that are extracted from the single inputted cell suspension. The pinched-flow stretching method reveals expected differences in cell deformability in two model systems. Furthermore, hydraulic circuit design is used to tune stretching forces and carry out multiple stretching modes (pinched-flow and extensional) in the same microfluidic channel with a single fluid input. The ability to create a self-sheathing flow from a single input solution should have general utility for other cytometry systems and the pinched-flow design enables an order of magnitude higher throughput (65,000 cells s(-1)) compared to our previously reported deformability cytometry method, which will be especially useful for identification of rare cell populations in clinical body fluids in the future.

Divergent cytosine DNA methylation patterns in single-cell, soybean root hairs.

PubMed

Hossain, Md Shakhawat; Kawakatsu, Taiji; Kim, Kyung Do; Zhang, Ning; Nguyen, Cuong T; Khan, Saad M; Batek, Josef M; Joshi, Trupti; Schmutz, Jeremy; Grimwood, Jane; Schmitz, Robert J; Xu, Dong; Jackson, Scott A; Ecker, Joseph R; Stacey, Gary

2017-04-01

Chromatin modifications, such as cytosine methylation of DNA, play a significant role in mediating gene expression in plants, which affects growth, development, and cell differentiation. As root hairs are single-cell extensions of the root epidermis and the primary organs for water uptake and nutrients, we sought to use root hairs as a single-cell model system to measure the impact of environmental stress. We measured changes in cytosine DNA methylation in single-cell root hairs as compared with multicellular stripped roots, as well as in response to heat stress. Differentially methylated regions (DMRs) in each methylation context showed very distinct methylation patterns between cell types and in response to heat stress. Intriguingly, at normal temperature, root hairs were more hypermethylated than were stripped roots. However, in response to heat stress, both root hairs and stripped roots showed hypomethylation in each context, especially in the CHH context. Moreover, expression analysis of mRNA from similar tissues and treatments identified some associations between DMRs, genes and transposons. Taken together, the data indicate that changes in DNA methylation are directly or indirectly associated with expression of genes and transposons within the context of either specific tissues/cells or stress (heat). © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Alterations in cell migration and cell viability of wounded human skin fibroblasts following visible red light exposure

NASA Astrophysics Data System (ADS)

Prabhu, Vijendra; Rao, Bola Sadashiva S.; Mahato, Krishna Kishore

2014-02-01

The present study intended to examine the effect of visible red light on structural and cellular parameters on wounded skin fibroblast cells. To achieve the stated objective, uniform scratch was created on confluent monolayered human skin fibroblast cells, and were exposed to single dose of He-Ne laser (15 mm spot, 6.6808 mWcm-2) at 1, 2, 3, 4, 5, 6 and 7 Jcm-2 in the presence and absence of 10% fetal bovine serum (FBS). Beam profile measurements of the expanded laser beam were conducted to ensure the beam uniformity. The influence of laser dose on the change in temperature was recorded using sensitive temperature probe. Additionally, following laser exposure cell migration and cell survival were documented at different time intervals on wounded human skin fibroblast cells grown in vitro. Beam profile measurements indicated more or less uniform power distribution over the whole beam area. Temperature monitoring of sham irradiated control and laser treatment groups displayed negligible temperature change indicating the absence of thermal effect at the tested laser doses. In the absence of 10% FBS, single exposure of different laser doses failed to produce any significant effects on cell migration or cell survival. However, in the presence of serum single exposure of 5 J/cm2 on wounded skin fibroblasts significantly enhanced the cell migration (P<0.05) compared to the other tested doses (1, 2, 3, 4, 6 and 7 J/cm2) and sham irradiated controls. In conclusion, the LLLT acts by improving cell migration and cell proliferation to produce measurable changes in wounded fibroblast cells.

Morphological changes in cultured bovine lymphoid cell lines associated with bovine viral diarrhea virus (BVDV) single and dual infections with bovine leukemia virus (BLV)

USDA-ARS?s Scientific Manuscript database

Currently, American Type Culture Collection (ATCC) makes available two cell lines derived from the same lymphoblast-like suspension cell that have been confirmed by next-generation sequencing and RT-PCR to have either a single contaminate of BVDV2a (CRL-8037) or dual contaminates of both BVDV and BL...

Imaging tumor microscopic viscosity in vivo using molecular rotors

PubMed Central

Shimolina, Lyubov’ E.; Izquierdo, Maria Angeles; López-Duarte, Ismael; Bull, James A.; Shirmanova, Marina V.; Klapshina, Larisa G.; Zagaynova, Elena V.; Kuimova, Marina K.

2017-01-01

The microscopic viscosity plays an essential role in cellular biophysics by controlling the rates of diffusion and bimolecular reactions within the cell interior. While several approaches have emerged that have allowed the measurement of viscosity and diffusion on a single cell level in vitro, the in vivo viscosity monitoring has not yet been realized. Here we report the use of fluorescent molecular rotors in combination with Fluorescence Lifetime Imaging Microscopy (FLIM) to image microscopic viscosity in vivo, both on a single cell level and in connecting tissues of subcutaneous tumors in mice. We find that viscosities recorded from single tumor cells in vivo correlate well with the in vitro values from the same cancer cell line. Importantly, our new method allows both imaging and dynamic monitoring of viscosity changes in real time in live animals and thus it is particularly suitable for diagnostics and monitoring of the progress of treatments that might be accompanied by changes in microscopic viscosity. PMID:28134273

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

Single-cell MALDI-MS as an analytical tool for studying intrapopulation metabolic heterogeneity of unicellular organisms.

PubMed

Amantonico, Andrea; Urban, Pawel L; Fagerer, Stephan R; Balabin, Roman M; Zenobi, Renato

2010-09-01

Heterogeneity is a characteristic feature of all populations of living organisms. Here we make an attempt to validate a single-cell mass spectrometric method for detection of changes in metabolite levels occurring in populations of unicellular organisms. Selected metabolites involved in central metabolism (ADP, ATP, GTP, and UDP-Glucose) could readily be detected in single cells of Closterium acerosum by means of negative-mode matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The analytical capabilities of this approach were characterized using standard compounds. The method was then used to study populations of individual cells with different levels of the chosen metabolites. With principal component analysis and support vector machine algorithms, it was possible to achieve a clear separation of individual C. acerosum cells in different metabolic states. This study demonstrates the suitability of mass spectrometric analysis of metabolites in single cells to measure cell-population heterogeneity.

Atomic Force Microscopy in Characterizing Cell Mechanics for Biomedical Applications: A Review.

PubMed

Li, Mi; Dang, Dan; Liu, Lianqing; Xi, Ning; Wang, Yuechao

2017-09-01

Cell mechanics is a novel label-free biomarker for indicating cell states and pathological changes. The advent of atomic force microscopy (AFM) provides a powerful tool for quantifying the mechanical properties of single living cells in aqueous conditions. The wide use of AFM in characterizing cell mechanics in the past two decades has yielded remarkable novel insights in understanding the development and progression of certain diseases, such as cancer, showing the huge potential of cell mechanics for practical applications in the field of biomedicine. In this paper, we reviewed the utilization of AFM to characterize cell mechanics. First, the principle and method of AFM single-cell mechanical analysis was presented, along with the mechanical responses of cells to representative external stimuli measured by AFM. Next, the unique changes of cell mechanics in two types of physiological processes (stem cell differentiation, cancer metastasis) revealed by AFM were summarized. After that, the molecular mechanisms guiding cell mechanics were analyzed. Finally the challenges and future directions were discussed.

Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular carcinomas

PubMed Central

Hou, Yu; Guo, Huahu; Cao, Chen; Li, Xianlong; Hu, Boqiang; Zhu, Ping; Wu, Xinglong; Wen, Lu; Tang, Fuchou; Huang, Yanyi; Peng, Jirun

2016-01-01

Single-cell genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple omics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations (CNVs), DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs generally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells. PMID:26902283

A novel method for multiparameter physiological phenotype characterization at the single-cell level

NASA Astrophysics Data System (ADS)

Kelbauskas, Laimonas; Ashili, Shashanka; Houkal, Jeff; Smith, Dean; Mohammadreza, Aida; Lee, Kristen; Kumar, Ashok; Anis, Yasser; Paulson, Tom; Youngbull, Cody; Tian, Yanqing; Johnson, Roger; Holl, Mark; Meldrum, Deirdre

2011-02-01

Non-genetic intercellular heterogeneity has been increasingly recognized as one of the key factors in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis and drug resistance. Many diseases, including cancer, originate in a single or a few cells. Early detection and characterization of these abnormal cells can provide new insights into the pathogenesis and serve as a tool for better disease diagnosis and treatment. We report on a novel technology for multiparameter physiological phenotype characterization at the single-cell level. It is based on real-time measurements of concentrations of several metabolites by means of extracellular optical sensors in microchambers of sub-nL volume containing single cells. In its current configuration, the measurement platform features the capability to detect oxygen consumption rate and pH changes under normoxic and hypoxic conditions at the single-cell level. We have conceived, designed and developed a semi-automated method for single-cell manipulation and loading into microwells utilizing custom, high-precision fluid handling at the nanoliter scale. We present the results of a series of measurements of oxygen consumption rates (OCRs) of single human metaplastic esophageal epithelial cells. In addition, to assess the effects of cell-to-cell interactions, we have measured OCRs of two and three cells placed in a single well. The major advantages of the approach are a) multiplexed characterization of cell phenotype at the single-cell level, b) minimal invasiveness due to the distant positioning of sensors, and c) flexibility in terms of accommodating measurements of other metabolites or biomolecules of interest.

Chemical imaging of molecular changes in a hydrated single cell by dynamic secondary ion mass spectrometry and super-resolution microscopy

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

Hua, Xin; Szymanski, Craig; Wang, Zhaoying

2016-01-01

Chemical imaging of single cells is important in capturing biological dynamics. Single cell correlative imaging is realized between structured illumination microscopy (SIM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) using System for Analysis at the Liquid Vacuum Interface (SALVI), a multimodal microreactor. SIM characterized cells and guided subsequent ToF-SIMS analysis. Dynamic ToF-SIMS provided time- and space-resolved cell molecular mapping. Lipid fragments were identified in the hydrated cell membrane. Principal component analysis was used to elucidate chemical component differences among mouse lung cells that uptake zinc oxide nanoparticles. Our results provided submicron chemical spatial mapping for investigations of cell dynamics atmore » the molecular level.« less

A statistical framework for multiparameter analysis at the single-cell level.

PubMed

Torres-García, Wandaliz; Ashili, Shashanka; Kelbauskas, Laimonas; Johnson, Roger H; Zhang, Weiwen; Runger, George C; Meldrum, Deirdre R

2012-03-01

Phenotypic characterization of individual cells provides crucial insights into intercellular heterogeneity and enables access to information that is unavailable from ensemble averaged, bulk cell analyses. Single-cell studies have attracted significant interest in recent years and spurred the development of a variety of commercially available and research-grade technologies. To quantify cell-to-cell variability of cell populations, we have developed an experimental platform for real-time measurements of oxygen consumption (OC) kinetics at the single-cell level. Unique challenges inherent to these single-cell measurements arise, and no existing data analysis methodology is available to address them. Here we present a data processing and analysis method that addresses challenges encountered with this unique type of data in order to extract biologically relevant information. We applied the method to analyze OC profiles obtained with single cells of two different cell lines derived from metaplastic and dysplastic human Barrett's esophageal epithelium. In terms of method development, three main challenges were considered for this heterogeneous dynamic system: (i) high levels of noise, (ii) the lack of a priori knowledge of single-cell dynamics, and (iii) the role of intercellular variability within and across cell types. Several strategies and solutions to address each of these three challenges are presented. The features such as slopes, intercepts, breakpoint or change-point were extracted for every OC profile and compared across individual cells and cell types. The results demonstrated that the extracted features facilitated exposition of subtle differences between individual cells and their responses to cell-cell interactions. With minor modifications, this method can be used to process and analyze data from other acquisition and experimental modalities at the single-cell level, providing a valuable statistical framework for single-cell analysis.

Live cell and immuno-labeling techniques to study gravitational effects on single plant cells.

PubMed

Chebli, Youssef; Geitmann, Anja

2015-01-01

The constant force of gravity plays a primordial role in the ontogeny of all living organisms. Plants, for example, develop their roots and shoots in accordance with the direction of the gravitational vector. Any change in the magnitude and/or the direction of gravity has an important impact on the development of tissues and cells. In order to understand how the gravitational force affects plant cell growth and differentiation, we established two complementary experimental procedures with which the effect of hyper-gravity on single plant cell development can be assessed. The single model cell system we used is the pollen tube or male gametophyte which, because of its rapid growth behavior, is known for its instant response to external stresses. The physiological response of the pollen tube can be assessed in a quantitative manner based on changes in the composition and spatial distribution of its cell wall components and in the precisely defined pattern of its very dynamic cytoplasmic streaming. Here, we provide a detailed description of the steps required for the immuno-localization of various cell wall components using microwave-assisted techniques and we explain how live imaging of the intracellular traffic can be achieved under hyper-gravity conditions.

Thermal phase transition behavior of lipid layers on a single human corneocyte cell.

PubMed

Imai, Tomohiro; Nakazawa, Hiromitsu; Kato, Satoru

2013-09-01

We have improved the selected area electron diffraction method to analyze the dynamic structural change in a single corneocyte cell non-invasively stripped off from human skin surface. The improved method made it possible to obtain reliable diffraction images to trace the structural change in the intercellular lipid layers on a single corneocyte cell during heating from 24°C to 100°C. Comparison of the results with those of synchrotron X-ray diffraction experiments on human stratum corneum sheets revealed that the intercellular lipid layers on a corneocyte cell exhibit essentially the same thermal phase transitions as those in a stratum corneum sheet. These results suggest that the structural features of the lipid layers are well preserved after the mechanical stripping of the corneocyte cell. Moreover, electron diffraction analyses of the thermal phase transition behaviors of the corneocyte cells that had the lipid layers with different distributions of orthorhombic and hexagonal domains at 24°C suggested that small orthorhombic domains interconnected with surrounding hexagonal domains transforms in a continuous manner into new hexagonal domains. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Single-cell imaging techniques for the real-time detection of IP₃ in live cells.

PubMed

Nelson, Carl P

2013-01-01

Inositol 1,4,5-trisphosphate (IP(3)) is a ubiquitous second messenger, derived from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)) by enzymes of the phospholipase C (PLC) family. Binding of IP(3) to its cognate receptor in the endoplasmic reticulum membrane leads to release of Ca(2+) into the cytoplasm, which is involved in the regulation of an array of cellular functions. Traditional techniques for the detection of IP(3) have required the extraction of a large number of cells, with limitations in the time resolution of changes in IP(3) and an inability to obtain detailed information on the dynamics of this second messenger in single cells. Recent progress in this field has led to the development of a number of genetically encoded fluorescent biosensors, which upon recombinant expression are able selectively to detect real-time changes in IP(3) in single live cells. In this chapter, I detail protocols for the expression, visualization (by confocol or fluorescence microscopy), and interpretation of data obtained with such biosensors expressed in mammalian cells.

Stress tolerance and biocontrol performance of the yeast antagonist, Candida diversa, change with morphology transition.

PubMed

Li, Guangkun; Chi, Mengshan; Chen, Huizhen; Sui, Yuan; Li, Yan; Liu, Yongsheng; Zhang, Xiaojing; Sun, Zhiqiang; Liu, Guoqing; Wang, Qi; Liu, Jia

2016-02-01

As an eco-friendly management method, biological control of postharvest diseases, utilizing antagonistic yeasts, is a research topic receiving considerable attention. Detailed knowledge on the biology of yeast antagonists is crucial when considering their potential application and development as biocontrol products. Changes in the growth form, such as single-cell to pseudohyphae, have been associated with the mode of action in postharvest biocontrol yeasts. In this study, the antagonistic yeast, Candida diversa, reversibly shifted from a single-cell morphology on yeast peptone dextrose (YPD) medium with 2 % agar to a pseudohyphal morphology on YPD with 0.3 % agar. The tolerance of the pseudohyphal form to heat and oxidative stresses, as well as the biocontrol efficacy against Botrytis cinerea on apple and kiwifruit stored at 25 and 4 °C, was significantly higher as compared to the single-cell form. This study provides new information on the ability of C. diversa to change its morphology and the impact of the morphology shift on stress tolerance and biocontrol performance.

Genome-wide maps of nuclear lamina interactions in single human cells.

PubMed

Kind, Jop; Pagie, Ludo; de Vries, Sandra S; Nahidiazar, Leila; Dey, Siddharth S; Bienko, Magda; Zhan, Ye; Lajoie, Bryan; de Graaf, Carolyn A; Amendola, Mario; Fudenberg, Geoffrey; Imakaev, Maxim; Mirny, Leonid A; Jalink, Kees; Dekker, Job; van Oudenaarden, Alexander; van Steensel, Bas

2015-09-24

Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization. VIDEO ABSTRACT. Copyright © 2015 Elsevier Inc. All rights reserved.

Quantifying the effect of electric current on cell adhesion studied by single-cell force spectroscopy.

PubMed

Jaatinen, Leena; Young, Eleanore; Hyttinen, Jari; Vörös, János; Zambelli, Tomaso; Demkó, László

2016-03-20

This study presents the effect of external electric current on the cell adhesive and mechanical properties of the C2C12 mouse myoblast cell line. Changes in cell morphology, viability, cytoskeleton, and focal adhesion structure were studied by standard staining protocols, while single-cell force spectroscopy based on the fluidic force microscopy technology provided a rapid, serial quantification and detailed analysis of cell adhesion and its dynamics. The setup allowed measurements of adhesion forces up to the μN range, and total detachment distances over 40 μm. Force-distance curves have been fitted with a simple elastic model including a cell detachment protocol in order to estimate the Young's modulus of the cells, as well as to reveal changes in the dynamic properties as functions of the applied current dose. While the cell spreading area decreased monotonously with increasing current doses, small current doses resulted only in differences related to cell elasticity. Current doses above 11 As/m(2), however, initiated more drastic changes in cell morphology, viability, cellular structure, as well as in properties related to cell adhesion. The observed differences, eventually leading to cell death toward higher doses, might originate from both the decrease in pH and the generation of reactive oxygen species.

Single-cell axotomy of cultured hippocampal neurons integrated in neuronal circuits.

PubMed

Gomis-Rüth, Susana; Stiess, Michael; Wierenga, Corette J; Meyn, Liane; Bradke, Frank

2014-05-01

An understanding of the molecular mechanisms of axon regeneration after injury is key for the development of potential therapies. Single-cell axotomy of dissociated neurons enables the study of the intrinsic regenerative capacities of injured axons. This protocol describes how to perform single-cell axotomy on dissociated hippocampal neurons containing synapses. Furthermore, to axotomize hippocampal neurons integrated in neuronal circuits, we describe how to set up coculture with a few fluorescently labeled neurons. This approach allows axotomy of single cells in a complex neuronal network and the observation of morphological and molecular changes during axon regeneration. Thus, single-cell axotomy of mature neurons is a valuable tool for gaining insights into cell intrinsic axon regeneration and the plasticity of neuronal polarity of mature neurons. Dissociation of the hippocampus and plating of hippocampal neurons takes ∼2 h. Neurons are then left to grow for 2 weeks, during which time they integrate into neuronal circuits. Subsequent axotomy takes 10 min per neuron and further imaging takes 10 min per neuron.

Gene expression profiling of single cells on large-scale oligonucleotide arrays

PubMed Central

Hartmann, Claudia H.; Klein, Christoph A.

2006-01-01

Over the last decade, important insights into the regulation of cellular responses to various stimuli were gained by global gene expression analyses of cell populations. More recently, specific cell functions and underlying regulatory networks of rare cells isolated from their natural environment moved to the center of attention. However, low cell numbers still hinder gene expression profiling of rare ex vivo material in biomedical research. Therefore, we developed a robust method for gene expression profiling of single cells on high-density oligonucleotide arrays with excellent coverage of low abundance transcripts. The protocol was extensively tested with freshly isolated single cells of very low mRNA content including single epithelial, mature and immature dendritic cells and hematopoietic stem cells. Quantitative PCR confirmed that the PCR-based global amplification method did not change the relative ratios of transcript abundance and unsupervised hierarchical cluster analysis revealed that the histogenetic origin of an individual cell is correctly reflected by the gene expression profile. Moreover, the gene expression data from dendritic cells demonstrate that cellular differentiation and pathway activation can be monitored in individual cells. PMID:17071717

In vivo imaging of T cell lymphoma infiltration process at the colon.

PubMed

Ueda, Yoshibumi; Ishiwata, Toshiyuki; Shinji, Seiichi; Arai, Tomio; Matsuda, Yoko; Aida, Junko; Sugimoto, Naotoshi; Okazaki, Toshiro; Kikuta, Junichi; Ishii, Masaru; Sato, Moritoshi

2018-03-05

The infiltration and proliferation of cancer cells in the secondary organs are of great interest, since they contribute to cancer metastasis. However, cancer cell dynamics in the secondary organs have not been elucidated at single-cell resolution. In the present study, we established an in vivo model using two-photon microscopy to observe how infiltrating cancer cells form assemblages from single T-cell lymphomas, EL4 cells, in the secondary organs. Using this model, after inoculation of EL4 cells in mice, we discovered that single EL4 cells infiltrated into the colon. In the early stage, sporadic elongated EL4 cells became lodged in small blood vessels. Real-time imaging revealed that, whereas more than 70% of EL4 cells did not move during a 1-hour observation, other EL4 cells irregularly moved even in small vessels and dynamically changed shape upon interacting with other cells. In the late stages, EL4 cells formed small nodules composed of several EL4 cells in blood vessels as well as crypts, suggesting the existence of diverse mechanisms of nodule formation. The present in vivo imaging system is instrumental to dissect cancer cell dynamics during metastasis in other organs at the single-cell level.

Study of living single cells in culture: automated recognition of cell behavior.

PubMed

Bodin, P; Papin, S; Meyer, C; Travo, P

1988-07-01

An automated system capable of analyzing the behavior, in real time, of single living cells in culture, in a noninvasive and nondestructive way, has been developed. A large number of cell positions in single culture dishes were recorded using a computer controlled, robotized microscope. During subsequent observations, binary images obtained from video image analysis of the microscope visual field allowed the identification of the recorded cells. These cells could be revisited automatically every few minutes. Long-term studies of the behavior of cells make possible the analysis of cellular locomotary and mitotic activities as well as determination of cell shape (chosen from a defined library) for several hours or days in a fully automated way with observations spaced up to 30 minutes. Short-term studies of the behavior of cells permit the study, in a semiautomatic way, of acute effects of drugs (5 to 15 minutes) on changes of surface area and length of cells.

Noise and Epigenetic Inheritance of Single-Cell Division Times Influence Population Fitness.

PubMed

Cerulus, Bram; New, Aaron M; Pougach, Ksenia; Verstrepen, Kevin J

2016-05-09

The fitness effect of biological noise remains unclear. For example, even within clonal microbial populations, individual cells grow at different speeds. Although it is known that the individuals' mean growth speed can affect population-level fitness, it is unclear how or whether growth speed heterogeneity itself is subject to natural selection. Here, we show that noisy single-cell division times can significantly affect population-level growth rate. Using time-lapse microscopy to measure the division times of thousands of individual S. cerevisiae cells across different genetic and environmental backgrounds, we find that the length of individual cells' division times can vary substantially between clonal individuals and that sublineages often show epigenetic inheritance of division times. By combining these experimental measurements with mathematical modeling, we find that, for a given mean division time, increasing heterogeneity and epigenetic inheritance of division times increases the population growth rate. Furthermore, we demonstrate that the heterogeneity and epigenetic inheritance of single-cell division times can be linked with variation in the expression of catabolic genes. Taken together, our results reveal how a change in noisy single-cell behaviors can directly influence fitness through dynamics that operate independently of effects caused by changes to the mean. These results not only allow a better understanding of microbial fitness but also help to more accurately predict fitness in other clonal populations, such as tumors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time.

PubMed

Wollman, Adam J M; Leake, Mark C

2015-01-01

We present a single-molecule tool called the CoPro (concentration of proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrate its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism of S. cerevisiae is a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal integration at physiological expression levels in living S. cerevisiae cells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a ∼4-fold shift towards higher values in the concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, and which is almost exclusively localized in the nucleus under high and low external glucose levels. CoPro facilitates time-resolved quantification of protein concentrations in single functional cells, and enables the distributions of concentrations across a cell population to be measured. This could be useful in investigating several cellular processes that are mediated by proteins, especially where changes in protein concentration in a single cell in response to changes in the extracellular chemical environment are subtle and rapid and may be smaller than the variability across a cell population.

Raman and Autofluorescence Spectrum Dynamics along the HRG-Induced Differentiation Pathway of MCF-7 Cells

PubMed Central

Morita, Shin-ichi; Takanezawa, Sota; Hiroshima, Michio; Mitsui, Toshiyuki; Ozaki, Yukihiro; Sako, Yasushi

2014-01-01

Cellular differentiation proceeds along complicated pathways, even when it is induced by extracellular signaling molecules. One of the major reasons for this complexity is the highly multidimensional internal dynamics of cells, which sometimes causes apparently stochastic responses in individual cells to extracellular stimuli. Therefore, to understand cell differentiation, it is necessary to monitor the internal dynamics of cells at single-cell resolution. Here, we used a Raman and autofluorescence spectrum analysis of single cells to detect dynamic changes in intracellular molecular components. MCF-7 cells are a human cancer-derived cell line that can be induced to differentiate into mammary-gland-like cells with the addition of heregulin (HRG) to the culture medium. We measured the spectra in the cytoplasm of MCF-7 cells during 12 days of HRG stimulation. The Raman scattering spectrum, which was the major component of the signal, changed with time. A multicomponent analysis of the Raman spectrum revealed that the dynamics of the major components of the intracellular molecules, including proteins and lipids, changed cyclically along the differentiation pathway. The background autofluorescence signals of Raman scattering also provided information about the differentiation process. Using the total information from the Raman and autofluorescence spectra, we were able to visualize the pathway of cell differentiation in the multicomponent phase space. PMID:25418290

The Effect of Single Pyramidal Neuron Firing Within Layer 2/3 and Layer 4 in Mouse V1.

PubMed

Meyer, Jochen F; Golshani, Peyman; Smirnakis, Stelios M

2018-01-01

The influence of cortical cell spiking activity on nearby cells has been studied extensively in vitro . Less is known, however, about the impact of single cell firing on local cortical networks in vivo . In a pioneering study, Kwan and Dan (Kwan and Dan, 2012) reported that in mouse layer 2/3 (L2/3), under anesthesia , stimulating a single pyramidal cell recruits ~2.1% of neighboring units. Here we employ two-photon calcium imaging in layer 2/3 of mouse V1, in conjunction with single-cell patch clamp stimulation in layer 2/3 or layer 4, to probe, in both the awake and lightly anesthetized states , how (i) activating single L2/3 pyramidal neurons recruits neighboring units within L2/3 and from layer 4 (L4) to L2/3, and whether (ii) activating single pyramidal neurons changes population activity in local circuit. To do this, it was essential to develop an algorithm capable of quantifying how sensitive the calcium signal is at detecting effectively recruited units ("followers"). This algorithm allowed us to estimate the chance of detecting a follower as a function of the probability that an epoch of stimulation elicits one extra action potential (AP) in the follower cell. Using this approach, we found only a small fraction (<0.75%) of L2/3 cells to be significantly activated within a radius of ~200 μm from a stimulated neighboring L2/3 pyramidal cell. This fraction did not change significantly in the awake vs. the lightly anesthetized state, nor when stimulating L2/3 vs. underlying L4 pyramidal neurons. These numbers are in general agreement with, though lower than, the percentage of neighboring cells (2.1% pyramidal cells and interneurons combined) reported by Kwan and Dan to be activated upon stimulating single L2/3 pyramidal neurons under anesthesia (Kwan and Dan, 2012). Interestingly, despite the small number of individual units found to be reliably driven, we did observe a modest but significant elevation in aggregate population responses compared to sham stimulation. This underscores the distributed impact that single cell stimulation has on neighboring microcircuit responses, revealing only a small minority of relatively strongly connected partners. Patch-clamp stimulation in conjunction with 2-photon imaging shows that activating single layer-2/3 or layer-4 pyramidal neurons produces few (<1% of local units) reliable single-cell followers in L2/3 of mouse area V1, either under light anesthesia or in quiet wakefulness: instead, single cell stimulation was found to elevate aggregate population activity in a weak but highly distributed fashion.

Fitness landscape transformation through a single amino acid change in the rho terminator.

PubMed

Freddolino, Peter L; Goodarzi, Hani; Tavazoie, Saeed

2012-05-01

Regulatory networks allow organisms to match adaptive behavior to the complex and dynamic contingencies of their native habitats. Upon a sudden transition to a novel environment, the mismatch between the native behavior and the new niche provides selective pressure for adaptive evolution through mutations in elements that control gene expression. In the case of core components of cellular regulation and metabolism, with broad control over diverse biological processes, such mutations may have substantial pleiotropic consequences. Through extensive phenotypic analyses, we have characterized the systems-level consequences of one such mutation (rho*) in the global transcriptional terminator Rho of Escherichia coli. We find that a single amino acid change in Rho results in a massive change in the fitness landscape of the cell, with widely discrepant fitness consequences of identical single locus perturbations in rho* versus rho(WT) backgrounds. Our observations reveal the extent to which a single regulatory mutation can transform the entire fitness landscape of the cell, causing a massive change in the interpretation of individual mutations and altering the evolutionary trajectories which may be accessible to a bacterial population.

Single Cell Detection with Driven Magnetic Beads

NASA Astrophysics Data System (ADS)

McNaughton, B. H.; Agayan, R. R.; Stoica, V. A.; Clarke, R.; Kopelman, R.

Shifts in the nonlinear rotational frequency of magnetic beads (microspheres) offer a new and dynamic approach for the detection of single cells. We present the first demonstration of this capability by measuring the changes in the nonlinear rotational frequency of magnetic beads driven by an external magnetic field. The presence of an Escherichia coli bacterium on the surface of a 2.0 μm magnetic bead affects the drag of the system, thus changing the nonlinear rotation rate. Measurement of this rotational frequency is straight-forward utilizing standard microscopy techniques.

Germination and Outgrowth of Single Spores of Saccharomyces cerevisiae Viewed by Scanning Electron and Phase-Contrast Microscopy

PubMed Central

Rousseau, Paul; Halvorson, Harlyn O.; Bulla, Lee A.; Julian, Grant St.

1972-01-01

Single spores of Saccharomyces cerevisiae were examined during germination and outgrowth by scanning electron and phase-contrast microscopy. Also determined were changes in cell weight and light absorbance, trehalose utilization, and synthesis of protein and KOH-soluble carbohydrates. These studies reveal that development of the vegetative cell from a spore follows a definite sequence of events involving dramatic physical and chemical modifications. These changes are: initial rapid loss in cellular absorbance followed later by an abrupt gain in absorbance; reduction in cell weight and a subsequent progressive increase; modification of the spore surface with concomitant diminution in refractility; elongation of the cell and augmentation of surface irregularities; rapid decline in trehalose content of the cell accompanied by extensive formation of KOH-soluble carbohydrates; and bud formation. Images PMID:4551750

Automated assembling of single fuel cell units for use in a fuel cell stack

NASA Astrophysics Data System (ADS)

Jalba, C. K.; Muminovic, A.; Barz, C.; Nasui, V.

2017-05-01

The manufacturing of PEMFC stacks (POLYMER ELEKTROLYT MEMBRAN Fuel Cell) is nowadays still done by hand. Over hundreds of identical single components have to be placed accurate together for the construction of a fuel cell stack. Beside logistic problems, higher total costs and disadvantages in weight the high number of components produce a higher statistic interference because of faulty erection or material defects and summation of manufacturing tolerances. The saving of costs is about 20 - 25 %. Furthermore, the total weight of the fuel cells will be reduced because of a new sealing technology. Overall a one minute cycle time has to be aimed per cell at the manufacturing of these single components. The change of the existing sealing concept to a bonded sealing is one of the important requisites to get an automated manufacturing of single cell units. One of the important steps for an automated gluing process is the checking of the glue application by using of an image processing system. After bonding the single fuel cell the sealing and electrical function can be checked, so that only functional and high qualitative cells can get into further manufacturing processes.

Quantitative Analysis of Protein Translocations by Microfluidic Total Internal Reflection Fluorescence Flow Cytometry

PubMed Central

Wang, Jun; Fei, Bei; Geahlen, Robert L.

2010-01-01

Protein translocation, or the change in a protein’s location between different subcellular compartments, is a critical process by which intracellular proteins carry out their cellular functions. Aberrant translocation events contribute to various diseases ranging from metabolic disorders to cancer. In this study, we demonstrate the use of a newly developed single-cell tool, microfluidic total internal reflection fluorescence flow cytometry (TIRF-FC), for detecting both cytosol to plasma membrane and cytosol to nucleus translocations using the tyrosine kinase Syk and the transcription factor NF-κB as models. This technique detects fluorescent molecules at the plasma membrane and in the membrane-proximal cytosol in single cells. We were able to record quantitatively changes in the fluorescence density in the evanescent field associated with these translocation processes for large cell populations with single cell resolution. We envision that TIRF-FC will provide a new approach to explore the molecular biology and clinical relevance of protein translocations. PMID:20820633

Visible micro-Raman spectroscopy of single human mammary epithelial cells exposed to x-ray radiation.

PubMed

Delfino, Ines; Perna, Giuseppe; Lasalvia, Maria; Capozzi, Vito; Manti, Lorenzo; Camerlingo, Carlo; Lepore, Maria

2015-03-01

A micro-Raman spectroscopy investigation has been performed in vitro on single human mammary epithelial cells after irradiation by graded x-ray doses. The analysis by principal component analysis (PCA) and interval-PCA (i-PCA) methods has allowed us to point out the small differences in the Raman spectra induced by irradiation. This experimental approach has enabled us to delineate radiation-induced changes in protein, nucleic acid, lipid, and carbohydrate content. In particular, the dose dependence of PCA and i-PCA components has been analyzed. Our results have confirmed that micro-Raman spectroscopy coupled to properly chosen data analysis methods is a very sensitive technique to detect early molecular changes at the single-cell level following exposure to ionizing radiation. This would help in developing innovative approaches to monitor radiation cancer radiotherapy outcome so as to reduce the overall radiation dose and minimize damage to the surrounding healthy cells, both aspects being of great importance in the field of radiation therapy.

MEMS squeezer for the measurement of single cell rupture force, stiffness change, and hysteresis

NASA Astrophysics Data System (ADS)

Barazani, B.; Warnat, S.; Fine, A.; Hubbard, T.

2017-02-01

A MEMS squeezer able to compress single living cells underwater until rupture was designed and tested. The relatively large motion range of the device in aqueous media (~2.5 µm) allows provoking cell disruption while measuring cell mechanical properties before and after membrane rupture. An AC driven electrothermal micro actuator with mechanical amplification pressed single cells against a reference back spring. Deformations of the cell and the reference spring were measured with nanoscale resolution using optical Fourier transform techniques. The motion of the reference spring divided by the cell deformation provides the cell stiffness relative to the reference spring constant. An abrupt change in the cell stiffness and the appearance of cracks indicated the cell wall rupture force was reached. A total of 22 baker’s yeast cells (Saccharomyces cerevisiae) were squeezed with the micro device. The average force necessary to rupture the cell membrane was 0.47  ±  0.1 µN. Before rupture the cells had an average stiffness of 9.3  ±  3.1 N m-1 the post-rupture stiffness dropped to 0.94  ±  0.57 N m-1. Cell hysteresis was also measured: cells squeezed and released before reaching the rupture force showed residual deformations below 100 nm, while cells squeezed past the rupture force and then released showed residual deformations between 490 and 990 nm.

Early transcriptional and epigenetic regulation of CD8+ T cell differentiation revealed by single-cell RNA-seq

PubMed Central

Kakaradov, Boyko; Arsenio, Janilyn; Widjaja, Christella E.; He, Zhaoren; Aigner, Stefan; Metz, Patrick J.; Yu, Bingfei; Wehrens, Ellen J.; Lopez, Justine; Kim, Stephanie H.; Zuniga, Elina I.; Goldrath, Ananda W.; Chang, John T.; Yeo, Gene W.

2017-01-01

SUMMARY During microbial infection, responding CD8+ T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA sequencing approach and analyzed individual CD8+ T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants controlling CD8+ T lymphocyte fate specification. These findings suggest a model of terminal effector cell differentiation initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, highlighting the power and necessity of single-cell approaches. PMID:28218746

Prospective guidance in a free-swimming cell.

PubMed

Delafield-Butt, Jonathan T; Pepping, Gert-Jan; McCaig, Colin D; Lee, David N

2012-07-01

A systems theory of movement control in animals is presented in this article and applied to explaining the controlled behaviour of the single-celled Paramecium caudatum in an electric field. The theory-General Tau Theory-is founded on three basic principles: (i) all purposive movement entails prospectively controlling the closure of action-gaps (e.g. a distance gap when reaching, or an angle gap when steering); (ii) the sole informational variable required for controlling gaps is the relative rate of change of the gap (the time derivative of the gap size divided by the size), which can be directly sensed; and (iii) a coordinated movement is achieved by keeping the relative rates of change of gaps in a constant ratio. The theory is supported by studies of controlled movement in mammals, birds and insects. We now show for the first time that it is also supported by single-celled paramecia steering to the cathode in a bi-polar electric field. General Tau Theory is deployed to explain this guided steering by the cell. This article presents the first computational model of prospective perceptual control in a non-neural, single-celled system.

Effects of methotrexate on the viscoelastic properties of single cells probed by atomic force microscopy.

PubMed

Li, Mi; Liu, Lianqing; Xiao, Xiubin; Xi, Ning; Wang, Yuechao

2016-10-01

Methotrexate is a commonly used anti-cancer chemotherapy drug. Cellular mechanical properties are fundamental parameters that reflect the physiological state of a cell. However, so far the role of cellular mechanical properties in the actions of methotrexate is still unclear. In recent years, probing the behaviors of single cells with the use of atomic force microscopy (AFM) has contributed much to the field of cell biomechanics. In this work, with the use of AFM, the effects of methotrexate on the viscoelastic properties of four types of cells were quantitatively investigated. The inhibitory and cytotoxic effects of methotrexate on the proliferation of cells were observed by optical and fluorescence microscopy. AFM indenting was used to measure the changes of cellular viscoelastic properties (Young's modulus and relaxation time) by using both conical tip and spherical tip, quantitatively showing that the stimulation of methotrexate resulted in a significant decrease of both cellular Young's modulus and relaxation times. The morphological changes of cells induced by methotrexate were visualized by AFM imaging. The study improves our understanding of methotrexate action and offers a novel way to quantify drug actions at the single-cell level by measuring cellular viscoelastic properties, which may have potential impacts on developing label-free methods for drug evaluation.

Emerging Imaging and Genomic Tools for Developmental Systems Biology.

PubMed

Liu, Zhe; Keller, Philipp J

2016-03-21

Animal development is a complex and dynamic process orchestrated by exquisitely timed cell lineage commitment, divisions, migration, and morphological changes at the single-cell level. In the past decade, extensive genetic, stem cell, and genomic studies provided crucial insights into molecular underpinnings and the functional importance of genetic pathways governing various cellular differentiation processes. However, it is still largely unknown how the precise coordination of these pathways is achieved at the whole-organism level and how the highly regulated spatiotemporal choreography of development is established in turn. Here, we discuss the latest technological advances in imaging and single-cell genomics that hold great promise for advancing our understanding of this intricate process. We propose an integrated approach that combines such methods to quantitatively decipher in vivo cellular dynamic behaviors and their underlying molecular mechanisms at the systems level with single-cell, single-molecule resolution. Copyright © 2016 Elsevier Inc. All rights reserved.

Single-cell analysis of population context advances RNAi screening at multiple levels

PubMed Central

Snijder, Berend; Sacher, Raphael; Rämö, Pauli; Liberali, Prisca; Mench, Karin; Wolfrum, Nina; Burleigh, Laura; Scott, Cameron C; Verheije, Monique H; Mercer, Jason; Moese, Stefan; Heger, Thomas; Theusner, Kristina; Jurgeit, Andreas; Lamparter, David; Balistreri, Giuseppe; Schelhaas, Mario; De Haan, Cornelis A M; Marjomäki, Varpu; Hyypiä, Timo; Rottier, Peter J M; Sodeik, Beate; Marsh, Mark; Gruenberg, Jean; Amara, Ali; Greber, Urs; Helenius, Ari; Pelkmans, Lucas

2012-01-01

Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image-based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single-cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome-wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell-based screens at this depth reveals widespread RNAi-induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell-to-cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large-scale RNAi screens are increasingly performed to reach a systems-level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single-cell microenvironment. PMID:22531119

The interplay between genetic and bioelectrical signaling permits a spatial regionalisation of membrane potentials in model multicellular ensembles

PubMed Central

Cervera, Javier; Meseguer, Salvador; Mafe, Salvador

2016-01-01

The single cell-centred approach emphasises ion channels as specific proteins that determine individual properties, disregarding their contribution to multicellular outcomes. We simulate the interplay between genetic and bioelectrical signals in non-excitable cells from the local single-cell level to the long range multicellular ensemble. The single-cell genetic regulation is based on mean-field kinetic equations involving the mRNA and protein concentrations. The transcription rate factor is assumed to depend on the absolute value of the cell potential, which is dictated by the voltage-gated cell ion channels and the intercellular gap junctions. The interplay between genetic and electrical signals may allow translating single-cell states into multicellular states which provide spatio-temporal information. The model results have clear implications for biological processes: (i) bioelectric signals can override slightly different genetic pre-patterns; (ii) ensembles of cells initially at the same potential can undergo an electrical regionalisation because of persistent genetic differences between adjacent spatial regions; and (iii) shifts in the normal cell electrical balance could trigger significant changes in the genetic regulation. PMID:27731412

The interplay between genetic and bioelectrical signaling permits a spatial regionalisation of membrane potentials in model multicellular ensembles.

PubMed

Cervera, Javier; Meseguer, Salvador; Mafe, Salvador

2016-10-12

The single cell-centred approach emphasises ion channels as specific proteins that determine individual properties, disregarding their contribution to multicellular outcomes. We simulate the interplay between genetic and bioelectrical signals in non-excitable cells from the local single-cell level to the long range multicellular ensemble. The single-cell genetic regulation is based on mean-field kinetic equations involving the mRNA and protein concentrations. The transcription rate factor is assumed to depend on the absolute value of the cell potential, which is dictated by the voltage-gated cell ion channels and the intercellular gap junctions. The interplay between genetic and electrical signals may allow translating single-cell states into multicellular states which provide spatio-temporal information. The model results have clear implications for biological processes: (i) bioelectric signals can override slightly different genetic pre-patterns; (ii) ensembles of cells initially at the same potential can undergo an electrical regionalisation because of persistent genetic differences between adjacent spatial regions; and (iii) shifts in the normal cell electrical balance could trigger significant changes in the genetic regulation.

Improved single-cell culture achieved using micromolding in capillaries technology coupled with poly (HEMA).

PubMed

Ye, Fang; Jiang, Jin; Chang, Honglong; Xie, Li; Deng, Jinjun; Ma, Zhibo; Yuan, Weizheng

2015-07-01

Cell studies at the single-cell level are becoming more and more critical for understanding the complex biological processes. Here, we present an optimization study investigating the positioning of single cells using micromolding in capillaries technology coupled with the cytophobic biomaterial poly (2-hydroxyethyl methacrylate) (poly (HEMA)). As a cytophobic biomaterial, poly (HEMA) was used to inhibit cells, whereas the glass was used as the substrate to provide a cell adhesive background. The poly (HEMA) chemical barrier was obtained using micromolding in capillaries, and the microchannel networks used for capillarity were easily achieved by reversibly bonding the polydimethylsiloxane mold and the glass. Finally, discrete cell adhesion regions were presented on the glass surface. This method is facile and low cost, and the reagents are commercially available. We validated the cytophobic abilities of the poly (HEMA), optimized the channel parameters for higher quality and more stable poly (HEMA) patterns by investigating the effects of changing the aspect ratio and the width of the microchannel on the poly (HEMA) grid pattern, and improved the single-cell occupancy by optimizing the dimensions of the cell adhesion regions.

Global Effects of DDX3 Inhibition on Cell Cycle Regulation Identified by a Combined Phosphoproteomics and Single Cell Tracking Approach.

PubMed

Heerma van Voss, Marise R; Kammers, Kai; Vesuna, Farhad; Brilliant, Justin; Bergman, Yehudit; Tantravedi, Saritha; Wu, Xinyan; Cole, Robert N; Holland, Andrew; van Diest, Paul J; Raman, Venu

2018-06-01

DDX3 is an RNA helicase with oncogenic properties. The small molecule inhibitor RK-33 is designed to fit into the ATP binding cleft of DDX3 and hereby block its activity. RK-33 has shown potent activity in preclinical cancer models. However, the mechanism behind the antineoplastic activity of RK-33 remains largely unknown. In this study we used a dual phosphoproteomic and single cell tracking approach to evaluate the effect of RK-33 on cancer cells. MDA-MB-435 cells were treated for 24 hours with RK-33 or vehicle control. Changes in phosphopeptide abundance were analyzed with quantitative mass spectrometry using isobaric mass tags (Tandem Mass Tags). At the proteome level we mainly observed changes in mitochondrial translation, cell division pathways and proteins related to cell cycle progression. Analysis of the phosphoproteome indicated decreased CDK1 activity after RK-33 treatment. To further evaluate the effect of DDX3 inhibition on cell cycle progression over time, we performed timelapse microscopy of Fluorescent Ubiquitin Cell Cycle Indicators labeled cells after RK-33 or siDDX3 exposure. Single cell tracking indicated that DDX3 inhibition resulted in a global delay in cell cycle progression in interphase and mitosis. In addition, we observed an increase in endoreduplication. Overall, we conclude that DDX3 inhibition affects cells in all phases and causes a global cell cycle progression delay. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Analysis of gene expression in single live neurons.

PubMed Central

Eberwine, J; Yeh, H; Miyashiro, K; Cao, Y; Nair, S; Finnell, R; Zettel, M; Coleman, P

1992-01-01

We present here a method for broadly characterizing single cells at the molecular level beyond the more common morphological and transmitter/receptor classifications. The RNA from defined single cells is amplified by microinjecting primer, nucleotides, and enzyme into acutely dissociated cells from a defined region of rat brain. Further processing yields amplified antisense RNA. A second round of amplification results in greater than 10(6)-fold amplification of the original starting material, which is adequate for analysis--e.g., use as a probe, making of cDNA libraries, etc. We demonstrate this method by constructing expression profiles of single live cells from rat hippocampus. This profiling suggests that cells that appear to be morphologically similar may show marked differences in patterns of expression. In addition, we characterize several mRNAs from a single cell, some of which were previously undescribed, perhaps due to "rarity" when averaged over many cell types. Electrophysiological analysis coupled with molecular biology within the same cell will facilitate a better understanding of how changes at the molecular level are manifested in functional properties. This approach should be applicable to a wide variety of studies, including development, mutant models, aging, and neurodegenerative disease. Images PMID:1557406

Point-of-Care Assay of Telomerase Activity at Single-Cell Level via Gas Pressure Readout.

PubMed

Wang, Yanjun; Yang, Luzhu; Li, Baoxin; Yang, Chaoyong James; Jin, Yan

2017-08-15

Detection of telomerase activity at the single-cell level is one of the central challenges in cancer diagnostics and therapy. Herein, we describe a facile and reliable point-of-care testing (POCT) strategy for detection of telomerase activity via a portable pressure meter. Telomerase primer (TS) was immobilized onto the surface of magnetic beads (MBs), and then was elongated to a long single-stranded DNA by telomerase. The elongated (TTAGGG) n repeat unit hybridized with several short PtNP-functionalized complementary DNA (PtNPs-cDNA), which specifically enriched PtNPs onto the surfaces of magnetic beads (MBs), which were separated using a magnet. Then, nanoparticle-catalyzed gas-generation reaction converted telomerase activity into significant change in gas pressure. Because of the self-amplification of telomerase and enrichment by magnetic separation, the diluted telomerase equivalent to a single HeLa cell was facilely detected. More importantly, the telomerase in the lysate of 1 HeLa cell can be reliably detected by monitoring change in gas pressure, indicating that it is feasible and possible to study differences between individual cells. The difference in relative activity between different kinds of cancer cells was easily and sensitively studied. Study of inhibition of telomerase activity demonstrated that our method has great potential in screening of telomerase-targeted antitumor drugs as well as in clinical diagnosis.

Determining Physical Mechanisms of Gene Expression Regulation from Single Cell Gene Expression Data.

PubMed

Ezer, Daphne; Moignard, Victoria; Göttgens, Berthold; Adryan, Boris

2016-08-01

Many genes are expressed in bursts, which can contribute to cell-to-cell heterogeneity. It is now possible to measure this heterogeneity with high throughput single cell gene expression assays (single cell qPCR and RNA-seq). These experimental approaches generate gene expression distributions which can be used to estimate the kinetic parameters of gene expression bursting, namely the rate that genes turn on, the rate that genes turn off, and the rate of transcription. We construct a complete pipeline for the analysis of single cell qPCR data that uses the mathematics behind bursty expression to develop more accurate and robust algorithms for analyzing the origin of heterogeneity in experimental samples, specifically an algorithm for clustering cells by their bursting behavior (Simulated Annealing for Bursty Expression Clustering, SABEC) and a statistical tool for comparing the kinetic parameters of bursty expression across populations of cells (Estimation of Parameter changes in Kinetics, EPiK). We applied these methods to hematopoiesis, including a new single cell dataset in which transcription factors (TFs) involved in the earliest branchpoint of blood differentiation were individually up- and down-regulated. We could identify two unique sub-populations within a seemingly homogenous group of hematopoietic stem cells. In addition, we could predict regulatory mechanisms controlling the expression levels of eighteen key hematopoietic transcription factors throughout differentiation. Detailed information about gene regulatory mechanisms can therefore be obtained simply from high throughput single cell gene expression data, which should be widely applicable given the rapid expansion of single cell genomics.

Evolution of New cis-Regulatory Motifs Required for Cell-Specific Gene Expression in Caenorhabditis

PubMed Central

Félix, Marie-Anne

2016-01-01

Patterning of C. elegans vulval cell fates relies on inductive signaling. In this induction event, a single cell, the gonadal anchor cell, secretes LIN-3/EGF and induces three out of six competent precursor cells to acquire a vulval fate. We previously showed that this developmental system is robust to a four-fold variation in lin-3/EGF genetic dose. Here using single-molecule FISH, we find that the mean level of expression of lin-3 in the anchor cell is remarkably conserved. No change in lin-3 expression level could be detected among C. elegans wild isolates and only a low level of change—less than 30%—in the Caenorhabditis genus and in Oscheius tipulae. In C. elegans, lin-3 expression in the anchor cell is known to require three transcription factor binding sites, specifically two E-boxes and a nuclear-hormone-receptor (NHR) binding site. Mutation of any of these three elements in C. elegans results in a dramatic decrease in lin-3 expression. Yet only a single E-box is found in the Drosophilae supergroup of Caenorhabditis species, including C. angaria, while the NHR-binding site likely only evolved at the base of the Elegans group. We find that a transgene from C. angaria bearing a single E-box is sufficient for normal expression in C. elegans. Even a short 58 bp cis-regulatory fragment from C. angaria with this single E-box is able to replace the three transcription factor binding sites at the endogenous C. elegans lin-3 locus, resulting in the wild-type expression level. Thus, regulatory evolution occurring in cis within a 58 bp lin-3 fragment, results in a strict requirement for the NHR binding site and a second E-box in C. elegans. This single-cell, single-molecule, quantitative and functional evo-devo study demonstrates that conserved expression levels can hide extensive change in cis-regulatory site requirements and highlights the evolution of new cis-regulatory elements required for cell-specific gene expression. PMID:27588814

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.

Optical visualisation of thermogenesis in stimulated single-cell brown adipocytes.

PubMed

Kriszt, Rókus; Arai, Satoshi; Itoh, Hideki; Lee, Michelle H; Goralczyk, Anna G; Ang, Xiu Min; Cypess, Aaron M; White, Andrew P; Shamsi, Farnaz; Xue, Ruidan; Lee, Jung Yeol; Lee, Sung-Chan; Hou, Yanyan; Kitaguchi, Tetsuya; Sudhaharan, Thankiah; Ishiwata, Shin'ichi; Lane, E Birgitte; Chang, Young-Tae; Tseng, Yu-Hua; Suzuki, Madoka; Raghunath, Michael

2017-05-03

The identification of brown adipose deposits in adults has led to significant interest in targeting this metabolically active tissue for treatment of obesity and diabetes. Improved methods for the direct measurement of heat production as the signature function of brown adipocytes (BAs), particularly at the single cell level, would be of substantial benefit to these ongoing efforts. Here, we report the first application of a small molecule-type thermosensitive fluorescent dye, ERthermAC, to monitor thermogenesis in BAs derived from murine brown fat precursors and in human brown fat cells differentiated from human neck brown preadipocytes. ERthermAC accumulated in the endoplasmic reticulum of BAs and displayed a marked change in fluorescence intensity in response to adrenergic stimulation of cells, which corresponded to temperature change. ERthermAC fluorescence intensity profiles were congruent with mitochondrial depolarisation events visualised by the JC-1 probe. Moreover, the averaged fluorescence intensity changes across a population of cells correlated well with dynamic changes such as thermal power, oxygen consumption, and extracellular acidification rates. These findings suggest ERthermAC as a promising new tool for studying thermogenic function in brown adipocytes of both murine and human origins.

Single-Cell RNA Sequencing of Human T Cells.

PubMed

Villani, Alexandra-Chloé; Shekhar, Karthik

2017-01-01

Understanding how populations of human T cells leverage cellular heterogeneity, plasticity, and diversity to achieve a wide range of functional flexibility, particularly during dynamic processes such as development, differentiation, and antigenic response, is a core challenge that is well suited for single-cell analysis. Hypothesis-free evaluation of cellular states and subpopulations by transcriptional profiling of single T cells can identify relationships that may be obscured by targeted approaches such as FACS sorting on cell-surface antigens, or bulk expression analysis. While this approach is relevant to all cell types, it is of particular interest in the study of T cells for which classical phenotypic criteria are now viewed as insufficient for distinguishing different T cell subtypes and transitional states, and defining the changes associated with dysfunctional T cell states in autoimmunity and tumor-related exhaustion. This unit describes a protocol to generate single-cell transcriptomic libraries of human blood CD4 + and CD8 + T cells, and also introduces the basic bioinformatic steps to process the resulting sequence data for further computational analysis. We show how cellular subpopulations can be identified from transcriptional data, and derive characteristic gene expression signatures that distinguish these states. We believe single-cell RNA-seq is a powerful technique to study the cellular heterogeneity in complex tissues, a paradigm that will be of great value for the immune system.

Spearhead Nanometric Field-Effect Transistor Sensors for Single-Cell Analysis.

PubMed

Zhang, Yanjun; Clausmeyer, Jan; Babakinejad, Babak; Córdoba, Ainara López; Ali, Tayyibah; Shevchuk, Andrew; Takahashi, Yasufumi; Novak, Pavel; Edwards, Christopher; Lab, Max; Gopal, Sahana; Chiappini, Ciro; Anand, Uma; Magnani, Luca; Coombes, R Charles; Gorelik, Julia; Matsue, Tomokazu; Schuhmann, Wolfgang; Klenerman, David; Sviderskaya, Elena V; Korchev, Yuri

2016-03-22

Nanometric field-effect-transistor (FET) sensors are made on the tip of spear-shaped dual carbon nanoelectrodes derived from carbon deposition inside double-barrel nanopipettes. The easy fabrication route allows deposition of semiconductors or conducting polymers to comprise the transistor channel. A channel from electrodeposited poly pyrrole (PPy) exhibits high sensitivity toward pH changes. This property is exploited by immobilizing hexokinase on PPy nano-FETs to give rise to a selective ATP biosensor. Extracellular pH and ATP gradients are key biochemical constituents in the microenvironment of living cells; we monitor their real-time changes in relation to cancer cells and cardiomyocytes. The highly localized detection is possible because of the high aspect ratio and the spear-like design of the nano-FET probes. The accurately positioned nano-FET sensors can detect concentration gradients in three-dimensional space, identify biochemical properties of a single living cell, and after cell membrane penetration perform intracellular measurements.

Spearhead Nanometric Field-Effect Transistor Sensors for Single-Cell Analysis

PubMed Central

Córdoba, Ainara López; Ali, Tayyibah; Shevchuk, Andrew; Takahashi, Yasufumi; Novak, Pavel; Edwards, Christopher; Lab, Max; Gopal, Sahana; Chiappini, Ciro; Anand, Uma; Magnani, Luca; Coombes, R. Charles; Gorelik, Julia; Matsue, Tomokazu; Schuhmann, Wolfgang; Klenerman, David; Sviderskaya, Elena V.; Korchev, Yuri

2016-01-01

Nanometric field-effect-transistor (FET) sensors are made on the tip of spear-shaped dual carbon nanoelectrodes derived from carbon deposition inside double-barrel nanopipettes. The easy fabrication route allows deposition of semiconductors or conducting polymers to comprise the transistor channel. A channel from electrodeposited poly pyrrole (PPy) exhibits high sensitivity toward pH changes. This property is exploited by immobilizing hexokinase on PPy nano-FETs to give rise to a selective ATP biosensor. Extracellular pH and ATP gradients are key biochemical constituents in the microenvironment of living cells; we monitor their real-time changes in relation to cancer cells and cardiomyocytes. The highly localized detection is possible because of the high aspect ratio and the spear-like design of the nano-FET probes. The accurately positioned nano-FET sensors can detect concentration gradients in three-dimensional space, identify biochemical properties of a single living cell, and after cell membrane penetration perform intracellular measurements. PMID:26816294

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

Population Distribution Analyses Reveal a Hierarchy of Molecular Players Underlying Parallel Endocytic Pathways

PubMed Central

Gupta, Gagan D.; Howes, Mark T.; Chandran, Ruma; Das, Anupam; Menon, Sindhu; Parton, Robert G.; Sowdhamini, R.; Thattai, Mukund; Mayor, Satyajit

2014-01-01

Single-cell-resolved measurements reveal heterogeneous distributions of clathrin-dependent (CD) and -independent (CLIC/GEEC: CG) endocytic activity in Drosophila cell populations. dsRNA-mediated knockdown of core versus peripheral endocytic machinery induces strong changes in the mean, or subtle changes in the shapes of these distributions, respectively. By quantifying these subtle shape changes for 27 single-cell features which report on endocytic activity and cell morphology, we organize 1072 Drosophila genes into a tree-like hierarchy. We find that tree nodes contain gene sets enriched in functional classes and protein complexes, providing a portrait of core and peripheral control of CD and CG endocytosis. For 470 genes we obtain additional features from separate assays and classify them into early- or late-acting genes of the endocytic pathways. Detailed analyses of specific genes at intermediate levels of the tree suggest that Vacuolar ATPase and lysosomal genes involved in vacuolar biogenesis play an evolutionarily conserved role in CG endocytosis. PMID:24971745

Novel approaches in function-driven single-cell genomics.

PubMed

Doud, Devin F R; Woyke, Tanja

2017-07-01

Deeper sequencing and improved bioinformatics in conjunction with single-cell and metagenomic approaches continue to illuminate undercharacterized environmental microbial communities. This has propelled the 'who is there, and what might they be doing' paradigm to the uncultivated and has already radically changed the topology of the tree of life and provided key insights into the microbial contribution to biogeochemistry. While characterization of 'who' based on marker genes can describe a large fraction of the community, answering 'what are they doing' remains the elusive pinnacle for microbiology. Function-driven single-cell genomics provides a solution by using a function-based screen to subsample complex microbial communities in a targeted manner for the isolation and genome sequencing of single cells. This enables single-cell sequencing to be focused on cells with specific phenotypic or metabolic characteristics of interest. Recovered genomes are conclusively implicated for both encoding and exhibiting the feature of interest, improving downstream annotation and revealing activity levels within that environment. This emerging approach has already improved our understanding of microbial community functioning and facilitated the experimental analysis of uncharacterized gene product space. Here we provide a comprehensive review of strategies that have been applied for function-driven single-cell genomics and the future directions we envision. © FEMS 2017.

Novel approaches in function-driven single-cell genomics

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

Doud, Devin F. R.; Woyke, Tanja

Deeper sequencing and improved bioinformatics in conjunction with single-cell and metagenomic approaches continue to illuminate undercharacterized environmental microbial communities. This has propelled the 'who is there, and what might they be doing' paradigm to the uncultivated and has already radically changed the topology of the tree of life and provided key insights into the microbial contribution to biogeochemistry. While characterization of 'who' based on marker genes can describe a large fraction of the community, answering 'what are they doing' remains the elusive pinnacle for microbiology. Function-driven single-cell genomics provides a solution by using a function-based screen to subsample complex microbialmore » communities in a targeted manner for the isolation and genome sequencing of single cells. This enables single-cell sequencing to be focused on cells with specific phenotypic or metabolic characteristics of interest. Recovered genomes are conclusively implicated for both encoding and exhibiting the feature of interest, improving downstream annotation and revealing activity levels within that environment. This emerging approach has already improved our understanding of microbial community functioning and facilitated the experimental analysis of uncharacterized gene product space. Here we provide a comprehensive review of strategies that have been applied for function-driven single-cell genomics and the future directions we envision.« less

Novel approaches in function-driven single-cell genomics

DOE PAGES

Doud, Devin F. R.; Woyke, Tanja

2017-06-07

Deeper sequencing and improved bioinformatics in conjunction with single-cell and metagenomic approaches continue to illuminate undercharacterized environmental microbial communities. This has propelled the 'who is there, and what might they be doing' paradigm to the uncultivated and has already radically changed the topology of the tree of life and provided key insights into the microbial contribution to biogeochemistry. While characterization of 'who' based on marker genes can describe a large fraction of the community, answering 'what are they doing' remains the elusive pinnacle for microbiology. Function-driven single-cell genomics provides a solution by using a function-based screen to subsample complex microbialmore » communities in a targeted manner for the isolation and genome sequencing of single cells. This enables single-cell sequencing to be focused on cells with specific phenotypic or metabolic characteristics of interest. Recovered genomes are conclusively implicated for both encoding and exhibiting the feature of interest, improving downstream annotation and revealing activity levels within that environment. This emerging approach has already improved our understanding of microbial community functioning and facilitated the experimental analysis of uncharacterized gene product space. Here we provide a comprehensive review of strategies that have been applied for function-driven single-cell genomics and the future directions we envision.« less

Decipher the Molecular Response of Plant Single Cell Types to Environmental Stresses

DOE PAGES

Nourbakhsh-Rey, Mehrnoush; Libault, Marc

2016-01-01

The analysis of the molecular response of entire plants or organs to environmental stresses suffers from the cellular complexity of the samples used. Specifically, this cellular complexity masks cell-specific responses to environmental stresses and logically leads to the dilution of the molecular changes occurring in each cell type composing the tissue/organ/plant in response to the stress. Therefore, to generate a more accurate picture of these responses, scientists are focusing on plant single cell type approaches. Several cell types are now considered as models such as the pollen, the trichomes, the cotton fiber, various root cell types including the root hairmore » cell, and the guard cell of stomata. Among them, several have been used to characterize plant response to abiotic and biotic stresses. Lastly, in this review, we are describing the various -omic studies performed on these different plant single cell type models to better understand plant cell response to biotic and abiotic stresses.« less

Decipher the Molecular Response of Plant Single Cell Types to Environmental Stresses

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

Nourbakhsh-Rey, Mehrnoush; Libault, Marc

The analysis of the molecular response of entire plants or organs to environmental stresses suffers from the cellular complexity of the samples used. Specifically, this cellular complexity masks cell-specific responses to environmental stresses and logically leads to the dilution of the molecular changes occurring in each cell type composing the tissue/organ/plant in response to the stress. Therefore, to generate a more accurate picture of these responses, scientists are focusing on plant single cell type approaches. Several cell types are now considered as models such as the pollen, the trichomes, the cotton fiber, various root cell types including the root hairmore » cell, and the guard cell of stomata. Among them, several have been used to characterize plant response to abiotic and biotic stresses. Lastly, in this review, we are describing the various -omic studies performed on these different plant single cell type models to better understand plant cell response to biotic and abiotic stresses.« less

Extreme heterogeneity of influenza virus infection in single cells

PubMed Central

Russell, Alistair B; Trapnell, Cole

2018-01-01

Viral infection can dramatically alter a cell’s transcriptome. However, these changes have mostly been studied by bulk measurements on many cells. Here we use single-cell mRNA sequencing to examine the transcriptional consequences of influenza virus infection. We find extremely wide cell-to-cell variation in the productivity of viral transcription – viral transcripts comprise less than a percent of total mRNA in many infected cells, but a few cells derive over half their mRNA from virus. Some infected cells fail to express at least one viral gene, but this gene absence only partially explains variation in viral transcriptional load. Despite variation in viral load, the relative abundances of viral mRNAs are fairly consistent across infected cells. Activation of innate immune pathways is rare, but some cellular genes co-vary in abundance with the amount of viral mRNA. Overall, our results highlight the complexity of viral infection at the level of single cells. PMID:29451492

Quantifying Stochastic Noise in Cultured Circadian Reporter Cells

DOE PAGES

John, Peter C.; Doyle, III, Francis J.

2015-11-20

We report that stochastic noise at the cellular level has been shown to play a fundamental role in circadian oscillations, influencing how groups of cells entrain to external cues and likely serving as the mechanism by which cell-autonomous rhythms are generated. Despite this importance, few studies have investigated how clock perturbations affect stochastic noise—even as increasing numbers of high-throughput screens categorize how gene knockdowns or small molecules can change clock period and amplitude. This absence is likely due to the difficulty associated with measuring cell-autonomous stochastic noise directly, which currently requires the careful collection and processing of single-cell data. Inmore » this study, we show that the damping rate of population-level bioluminescence recordings can serve as an accurate measure of overall stochastic noise, and one that can be applied to future and existing high-throughput circadian screens. Using cell-autonomous fibroblast data, we first show directly that higher noise at the single-cell results in faster damping at the population level. Next, we show that the damping rate of cultured cells can be changed in a dose-dependent fashion by small molecule modulators, and confirm that such a change can be explained by single-cell noise using a mathematical model. We further demonstrate the insights that can be gained by applying our method to a genome-wide siRNA screen, revealing that stochastic noise is altered independently from period, amplitude, and phase. Finally, we hypothesize that the unperturbed clock is highly optimized for robust rhythms, as very few gene perturbations are capable of simultaneously increasing amplitude and lowering stochastic noise. Ultimately, this study demonstrates the importance of considering the effect of circadian perturbations on stochastic noise, particularly with regard to the development of small-molecule circadian therapeutics.« less

Single-Cell Mass Spectrometry Reveals Changes in Lipid and Metabolite Expression in RAW 264.7 Cells upon Lipopolysaccharide Stimulation

NASA Astrophysics Data System (ADS)

Yang, Bo; Patterson, Nathan Heath; Tsui, Tina; Caprioli, Richard M.; Norris, Jeremy L.

2018-05-01

It has been widely recognized that individual cells that exist within a large population of cells, even if they are genetically identical, can have divergent molecular makeups resulting from a variety of factors, including local environmental factors and stochastic processes within each cell. Presently, numerous approaches have been described that permit the resolution of these single-cell expression differences for RNA and protein; however, relatively few techniques exist for the study of lipids and metabolites in this manner. This study presents a methodology for the analysis of metabolite and lipid expression at the level of a single cell through the use of imaging mass spectrometry on a high-performance Fourier transform ion cyclotron resonance mass spectrometer. This report provides a detailed description of the overall experimental approach, including sample preparation as well as the data acquisition and analysis strategy for single cells. Applying this approach to the study of cultured RAW264.7 cells, we demonstrate that this method can be used to study the variation in molecular expression with cell populations and is sensitive to alterations in that expression that occurs upon lipopolysaccharide stimulation. [Figure not available: see fulltext.

Single-Cell Mass Spectrometry Reveals Changes in Lipid and Metabolite Expression in RAW 264.7 Cells upon Lipopolysaccharide Stimulation

NASA Astrophysics Data System (ADS)

Yang, Bo; Patterson, Nathan Heath; Tsui, Tina; Caprioli, Richard M.; Norris, Jeremy L.

2018-03-01

It has been widely recognized that individual cells that exist within a large population of cells, even if they are genetically identical, can have divergent molecular makeups resulting from a variety of factors, including local environmental factors and stochastic processes within each cell. Presently, numerous approaches have been described that permit the resolution of these single-cell expression differences for RNA and protein; however, relatively few techniques exist for the study of lipids and metabolites in this manner. This study presents a methodology for the analysis of metabolite and lipid expression at the level of a single cell through the use of imaging mass spectrometry on a high-performance Fourier transform ion cyclotron resonance mass spectrometer. This report provides a detailed description of the overall experimental approach, including sample preparation as well as the data acquisition and analysis strategy for single cells. Applying this approach to the study of cultured RAW264.7 cells, we demonstrate that this method can be used to study the variation in molecular expression with cell populations and is sensitive to alterations in that expression that occurs upon lipopolysaccharide stimulation. [Figure not available: see fulltext.

An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced.

PubMed

Morinaga, Takao; Nguyễn, Thảo Thi Thanh; Zhong, Boya; Hanazono, Michiko; Shingyoji, Masato; Sekine, Ikuo; Tada, Yuji; Tatsumi, Koichiro; Shimada, Hideaki; Hiroshima, Kenzo; Tagawa, Masatoshi

2017-11-10

Genetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity. We used type 5 Ad in which the expression of E1A gene was activated by 5'-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses. The replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level. Image cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection.

Inferring fitness landscapes and selection on phenotypic states from single-cell genealogical data

PubMed Central

Kussell, Edo

2017-01-01

Recent advances in single-cell time-lapse microscopy have revealed non-genetic heterogeneity and temporal fluctuations of cellular phenotypes. While different phenotypic traits such as abundance of growth-related proteins in single cells may have differential effects on the reproductive success of cells, rigorous experimental quantification of this process has remained elusive due to the complexity of single cell physiology within the context of a proliferating population. We introduce and apply a practical empirical method to quantify the fitness landscapes of arbitrary phenotypic traits, using genealogical data in the form of population lineage trees which can include phenotypic data of various kinds. Our inference methodology for fitness landscapes determines how reproductivity is correlated to cellular phenotypes, and provides a natural generalization of bulk growth rate measures for single-cell histories. Using this technique, we quantify the strength of selection acting on different cellular phenotypic traits within populations, which allows us to determine whether a change in population growth is caused by individual cells’ response, selection within a population, or by a mixture of these two processes. By applying these methods to single-cell time-lapse data of growing bacterial populations that express a resistance-conferring protein under antibiotic stress, we show how the distributions, fitness landscapes, and selection strength of single-cell phenotypes are affected by the drug. Our work provides a unified and practical framework for quantitative measurements of fitness landscapes and selection strength for any statistical quantities definable on lineages, and thus elucidates the adaptive significance of phenotypic states in time series data. The method is applicable in diverse fields, from single cell biology to stem cell differentiation and viral evolution. PMID:28267748

High-resolution Myogenic Lineage Mapping by Single-Cell Mass Cytometry

PubMed Central

Porpiglia, Ermelinda; Samusik, Nikolay; Ho, Andrew Tri Van; Cosgrove, Benjamin D.; Mai, Thach; Davis, Kara L.; Jager, Astraea; Nolan, Garry P.; Bendall, Sean C.; Fantl, Wendy J.; Blau, Helen M.

2017-01-01

Muscle regeneration is a dynamic process during which cell state and identity change over time. A major roadblock has been a lack of tools to resolve a myogenic progression in vivo. Here we capitalize on a transformative technology, single-cell mass cytometry (CyTOF), to identify in vivo skeletal muscle stem cell and previously unrecognized progenitor populations that precede differentiation. We discovered two cell surface markers, CD9 and CD104, whose combined expression enabled in vivo identification and prospective isolation of stem and progenitor cells. Data analysis using the X-shift algorithm paired with single-cell force directed layout visualization, defined a molecular signature of the activated stem cell state (CD44+/CD98+/MyoD+) and delineated a myogenic trajectory during recovery from acute muscle injury. Our studies uncover the dynamics of skeletal muscle regeneration in vivo and pave the way for the elucidation of the regulatory networks that underlie cell-state transitions in muscle diseases and aging. PMID:28414312

Single molecule and single cell epigenomics.

PubMed

Hyun, Byung-Ryool; McElwee, John L; Soloway, Paul D

2015-01-15

Dynamically regulated changes in chromatin states are vital for normal development and can produce disease when they go awry. Accordingly, much effort has been devoted to characterizing these states under normal and pathological conditions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most widely used method to characterize where in the genome transcription factors, modified histones, modified nucleotides and chromatin binding proteins are found; bisulfite sequencing (BS-seq) and its variants are commonly used to characterize the locations of DNA modifications. Though very powerful, these methods are not without limitations. Notably, they are best at characterizing one chromatin feature at a time, yet chromatin features arise and function in combination. Investigators commonly superimpose separate ChIP-seq or BS-seq datasets, and then infer where chromatin features are found together. While these inferences might be correct, they can be misleading when the chromatin source has distinct cell types, or when a given cell type exhibits any cell to cell variation in chromatin state. These ambiguities can be eliminated by robust methods that directly characterize the existence and genomic locations of combinations of chromatin features in very small inputs of cells or ideally, single cells. Here we review single molecule epigenomic methods under development to overcome these limitations, the technical challenges associated with single molecule methods and their potential application to single cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Single Molecule and Single Cell Epigenomics

PubMed Central

Hyun, Byung-Ryool; McElwee, John L.; Soloway, Paul D.

2014-01-01

Dynamically regulated changes in chromatin states are vital for normal development and can produce disease when they go awry. Accordingly, much effort has been devoted to characterizing these states under normal and pathological conditions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most widely used method to characterize where in the genome transcription factors, modified histones, modified nucleotides and chromatin binding proteins are found; bisulfite sequencing (BS-seq) and its variants are commonly used to characterize the locations of DNA modifications. Though very powerful, these methods are not without limitations. Notably, they are best at characterizing one chromatin feature at a time, yet chromatin features arise and function in combination. Investigators commonly superimpose separate ChIP-seq or BS-seq datasets, and then infer where chromatin features are found together. While these inferences might be correct, they can be misleading when the chromatin source has distinct cell types, or when a given cell type exhibits any cell to cell variation in chromatin state. These ambiguities can be eliminated by robust methods that directly characterize the existence and genomic locations of combinations of chromatin features in very small inputs of cells or ideally, single cells. Here we review single molecule epigenomic methods under development to overcome these limitations, the technical challenges associated with single molecule methods and their potential application to single cells. PMID:25204781

Multiplexed droplet single-cell RNA-sequencing using natural genetic variation.

PubMed

Kang, Hyun Min; Subramaniam, Meena; Targ, Sasha; Nguyen, Michelle; Maliskova, Lenka; McCarthy, Elizabeth; Wan, Eunice; Wong, Simon; Byrnes, Lauren; Lanata, Cristina M; Gate, Rachel E; Mostafavi, Sara; Marson, Alexander; Zaitlen, Noah; Criswell, Lindsey A; Ye, Chun Jimmie

2018-01-01

Droplet single-cell RNA-sequencing (dscRNA-seq) has enabled rapid, massively parallel profiling of transcriptomes. However, assessing differential expression across multiple individuals has been hampered by inefficient sample processing and technical batch effects. Here we describe a computational tool, demuxlet, that harnesses natural genetic variation to determine the sample identity of each droplet containing a single cell (singlet) and detect droplets containing two cells (doublets). These capabilities enable multiplexed dscRNA-seq experiments in which cells from unrelated individuals are pooled and captured at higher throughput than in standard workflows. Using simulated data, we show that 50 single-nucleotide polymorphisms (SNPs) per cell are sufficient to assign 97% of singlets and identify 92% of doublets in pools of up to 64 individuals. Given genotyping data for each of eight pooled samples, demuxlet correctly recovers the sample identity of >99% of singlets and identifies doublets at rates consistent with previous estimates. We apply demuxlet to assess cell-type-specific changes in gene expression in 8 pooled lupus patient samples treated with interferon (IFN)-β and perform eQTL analysis on 23 pooled samples.

Robust organelle size extractions from elastic scattering measurements of single cells (Conference Presentation)

NASA Astrophysics Data System (ADS)

Cannaday, Ashley E.; Draham, Robert; Berger, Andrew J.

2016-04-01

The goal of this project is to estimate non-nuclear organelle size distributions in single cells by measuring angular scattering patterns and fitting them with Mie theory. Simulations have indicated that the large relative size distribution of organelles (mean:width≈2) leads to unstable Mie fits unless scattering is collected at polar angles less than 20 degrees. Our optical system has therefore been modified to collect angles down to 10 degrees. Initial validations will be performed on polystyrene bead populations whose size distributions resemble those of cell organelles. Unlike with the narrow bead distributions that are often used for calibration, we expect to see an order-of-magnitude improvement in the stability of the size estimates as the minimum angle decreases from 20 to 10 degrees. Scattering patterns will then be acquired and analyzed from single cells (EMT6 mouse cancer cells), both fixed and live, at multiple time points. Fixed cells, with no changes in organelle sizes over time, will be measured to determine the fluctuation level in estimated size distribution due to measurement imperfections alone. Subsequent measurements on live cells will determine whether there is a higher level of fluctuation that could be attributed to dynamic changes in organelle size. Studies on unperturbed cells are precursors to ones in which the effects of exogenous agents are monitored over time.

[Prediction of the molecular response to pertubations from single cell measurements].

PubMed

Remacle, Françoise; Levine, Raphael D

2014-12-01

The response of protein signalization networks to perturbations is analysed from single cell measurements. This experimental approach allows characterizing the fluctuations in protein expression levels from cell to cell. The analysis is based on an information theoretic approach grounded in thermodynamics leading to a quantitative version of Le Chatelier principle which allows to predict the molecular response. Two systems are investigated: human macrophages subjected to lipopolysaccharide challenge, analogous to the immune response against Gram-negative bacteria and the response of the proteins involved in the mTOR signalizing network of GBM cancer cells to changes in partial oxygen pressure. © 2014 médecine/sciences – Inserm.

Fluorescence from a single Symbiodinium cell

NASA Astrophysics Data System (ADS)

Guzman, Christine; Han, Xue; Shoguchi, Eiichi; Chormaic, Síle Nic

2018-07-01

The partnership between coral and its algal symbionts, Symbiodinium, is crucial to the global environment. Yet, the regulatory process within the photosynthetic machinery of Symbiodinium is still not clearly understood. Here, we studied the influence of light stress from focussed red and blue lasers on single Symbiodinium cells. Fluorescence signals were measured to show cell response. Increasing the incident laser power or the exposure time resulted in an increase followed by a decline in fluorescence intensity. The trend of fluorescence intensity changes was associated with mechanisms of light use efficiency, non-photochemical quenching, photoinhibition, and repair of the cell. Our study provides new approaches to studying the photobiology and physiology of Symbiodinium cells.

Early dynamic fate changes in haemogenic endothelium characterized at the single-cell level

NASA Astrophysics Data System (ADS)

Swiers, Gemma; Baumann, Claudia; O'Rourke, John; Giannoulatou, Eleni; Taylor, Stephen; Joshi, Anagha; Moignard, Victoria; Pina, Cristina; Bee, Thomas; Kokkaliaris, Konstantinos D.; Yoshimoto, Momoko; Yoder, Mervin C.; Frampton, Jon; Schroeder, Timm; Enver, Tariq; Göttgens, Berthold; de Bruijn, Marella F. T. R.

2013-12-01

Haematopoietic stem cells (HSCs) are the founding cells of the adult haematopoietic system, born during ontogeny from a specialized subset of endothelium, the haemogenic endothelium (HE) via an endothelial-to-haematopoietic transition (EHT). Although recently imaged in real time, the underlying mechanism of EHT is still poorly understood. We have generated a Runx1 +23 enhancer-reporter transgenic mouse (23GFP) for the prospective isolation of HE throughout embryonic development. Here we perform functional analysis of over 1,800 and transcriptional analysis of 268 single 23GFP+ HE cells to explore the onset of EHT at the single-cell level. We show that initiation of the haematopoietic programme occurs in cells still embedded in the endothelial layer, and is accompanied by a previously unrecognized early loss of endothelial potential before HSCs emerge. Our data therefore provide important insights on the timeline of early haematopoietic commitment.

Single-nucleus analysis of accessible chromatin in developing mouse forebrain reveals cell-type-specific transcriptional regulation.

PubMed

Preissl, Sebastian; Fang, Rongxin; Huang, Hui; Zhao, Yuan; Raviram, Ramya; Gorkin, David U; Zhang, Yanxiao; Sos, Brandon C; Afzal, Veena; Dickel, Diane E; Kuan, Samantha; Visel, Axel; Pennacchio, Len A; Zhang, Kun; Ren, Bing

2018-03-01

Analysis of chromatin accessibility can reveal transcriptional regulatory sequences, but heterogeneity of primary tissues poses a significant challenge in mapping the precise chromatin landscape in specific cell types. Here we report single-nucleus ATAC-seq, a combinatorial barcoding-assisted single-cell assay for transposase-accessible chromatin that is optimized for use on flash-frozen primary tissue samples. We apply this technique to the mouse forebrain through eight developmental stages. Through analysis of more than 15,000 nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell types. We further define cell-type-specific transcriptional regulatory sequences, infer potential master transcriptional regulators and delineate developmental changes in forebrain cellular composition. Our results provide insight into the molecular and cellular dynamics that underlie forebrain development in the mouse and establish technical and analytical frameworks that are broadly applicable to other heterogeneous tissues.

Dynamic mechanical measurement of the viscoelasticity of single adherent cells

NASA Astrophysics Data System (ADS)

Corbin, Elise A.; Adeniba, Olaoluwa O.; Ewoldt, Randy H.; Bashir, Rashid

2016-02-01

Many recent studies on the viscoelasticity of individual cells link mechanics with cellular function and health. Here, we introduce a measurement of the viscoelastic properties of individual human colon cancer cells (HT-29) using silicon pedestal microelectromechanical systems (MEMS) resonant sensors. We demonstrate that the viscoelastic properties of single adherent cells can be extracted by measuring a difference in vibrational amplitude of our resonant sensor platform. The magnitude of vibration of the pedestal sensor is measured using a laser Doppler vibrometer (LDV). A change in amplitude of the sensor, compared with the driving amplitude (amplitude ratio), is influenced by the mechanical properties of the adhered cells. The amplitude ratio of the fixed cells was greater than the live cells, with a p-value <0.0001. By combining the amplitude shift with the resonant frequency shift measure, we determined the elastic modulus and viscosity values of 100 Pa and 0.0031 Pa s, respectively. Our method using the change in amplitude of resonant MEMS devices can enable the determination of a refined solution space and could improve measuring the stiffness of cells.

Step Inside NIH's Sickle Cell Branch

MedlinePlus

... attack it. This is gene transfer, which requires gene editing. Sickle cell disease is caused by a single ... can reverse that mutation in the DNA using gene editing, we could change that one mutant gene back ...

Effect of temperature on the formation of creep substructure in sodium chloride single crystals

NASA Technical Reports Server (NTRS)

Raj, Sai V.; Pharr, George M.

1992-01-01

The effect of temperature on the substructure morphology and the cell and subgrain size was investigated experimentally in NaCl single crystals under creep in the temperature range 573-873 K. It is found that the effect of temperature on the cell and subgrain sizes is weak in comparison with the effect of stress. However, there was a qualitative change in the substructure morphology with temperature, with the cells and subgrains better defined at higher temperatures. The volume fraction of the cell boundaries decreased with increasing temperature, thereby indicating a refinement of the microstructure at higher temperatures.

Single-Cell Analysis Reveals Early Manifestation of Cancerous Phenotype in Pre-Malignant Esophageal Cells

PubMed Central

Wang, Jiangxin; Shi, Xu; Johnson, Roger H.; Kelbauskas, Laimonas; Zhang, Weiwen; Meldrum, Deirdre R.

2013-01-01

Cellular heterogeneity plays a pivotal role in a variety of functional processes in vivo including carcinogenesis. However, our knowledge about cell-to-cell diversity and how differences in individual cells manifest in alterations at the population level remains very limited mainly due to the lack of appropriate tools enabling studies at the single-cell level. We present a study on changes in cellular heterogeneity in the context of pre-malignant progression in response to hypoxic stress. Utilizing pre-malignant progression of Barrett’s esophagus (BE) as a disease model system we studied molecular mechanisms underlying the progression from metaplastic to dysplastic (pre-cancerous) stage. We used newly developed methods enabling measurements of cell-to-cell differences in copy numbers of mitochondrial DNA, expression levels of a set of mitochondrial and nuclear genes involved in hypoxia response pathways, and mitochondrial membrane potential. In contrast to bulk cell studies reported earlier, our study shows significant differences between metaplastic and dysplastic BE cells in both average values and single-cell parameter distributions of mtDNA copy numbers, mitochondrial function, and mRNA expression levels of studied genes. Based on single-cell data analysis, we propose that mitochondria may be one of the key factors in pre-malignant progression in BE. PMID:24116039

Microfluidic platform for single cell analysis under dynamic spatial and temporal stimulation.

PubMed

Song, Jiyoung; Ryu, Hyunryul; Chung, Minhwan; Kim, Youngtaek; Blum, Yannick; Lee, Sung Sik; Pertz, Olivier; Jeon, Noo Li

2018-05-01

Recent research on cellular responses is shifting from static observations recorded under static stimuli to real-time monitoring in a dynamic environment. Since cells sense and interact with their surrounding microenvironment, an experimental platform where dynamically changing cellular microenvironments should be recreated in vitro. There has been a lack of microfluidic devices to support spatial and temporal stimulations in a simple and robust manner. Here, we describe a microfluidic device that generates dynamic chemical gradients and pulses in both space and time using a single device. This microfluidic device provides at least 12h of continuous stimulations that can be used to observe responses from mammalian cells. Combination of the microfluidic de-vice with live-cell imaging facilitates real-time observation of dynamic cellular response at single cell level. Using stable HEK cells with biosensors, ERK (Extracellular signal-Regulated Kinase) activities were observed un-der the pulsatile and ramping stimulations of EGF (Epidermal Growth Factor). We quantified ERK activation even at extremely low EGF concentration (0.0625µg/ml), which can not be observed using conventional techniques such as western blot. Cytoskeleton re-arrangement of the 3T3 fibroblast (stable transfection with Lifeact-GFP) was compared under abrupt and gradually changing gradient of PDGF. Copyright © 2017 Elsevier B.V. All rights reserved.

The value of cultures to modern microbiology.

PubMed

Austin, Brian

2017-10-01

Since the late nineteenth century, pure cultures have been regarded as the cornerstone of bacteriology. However, not all bacteria will multiply sufficiently to produce visible colonies on solid media; some cells will produce micro-colonies that are invisible to the naked eye. Moreover, the proportion of culturable cells that produce visible growth will vary according to the species and the state of the cells-are they actively growing or comparatively inactive? The latter have a poorer rate of recovery in terms of cultivability. It is unclear whether or not an individual colony is always derived from a single cell; it is possible that organisms in close proximity to each other may multiply and come together to produce single colonies. Then, the resultant growth will most certainly be derived from more than one initial cell. Although it is generally assumed that streaking and re-streaking on fresh media will purify any culture, there is evidence for microbial consortia interacting to form what appear to be single pure cultures. As so-called pure cultures underpin traditional microbiology, it is relevant to understand that the culture does not necessarily contain clones of identical bacteria, but that there may be variation in the genetic potential of the component cells, i.e. the cells are not homogeneous. Certainly, many bacteria change rapidly upon culturing, with some becoming bigger and less active. It is difficult to be sure if these changes reflect a loss or change of DNA or whether standard culturing methods select faster growing cells that are effectively not representative of the environment from which they were derived. These concepts are reviewed with an emphasis on bacterial fish pathogens.

Transportation of single cell and microbubbles by phase-shift introduced to standing leaky surface acoustic waves

PubMed Central

Meng, Long; Cai, Feiyan; Zhang, Zidong; Niu, Lili; Jin, Qiaofeng; Yan, Fei; Wu, Junru; Wang, Zhanhui; Zheng, Hairong

2011-01-01

A microfluidic device was developed to precisely transport a single cell or multiple microbubbles by introducing phase-shifts to a standing leaky surface acoustic wave (SLSAW). The device consists of a polydimethyl-siloxane (PDMS) microchannel and two phase-tunable interdigital transducers (IDTs) for the generation of the relative phase for the pair of surface acoustic waves (SAW) propagating along the opposite directions forming a standing wave. When the SAW contacts the fluid medium inside the microchannel, some of SAW energy is coupled to the fluid and the SAW becomes the leaky surface wave. By modulating the relative phase between two IDTs, the positions of pressure nodes of the SLSAW in the microchannel change linearly resulting in the transportation of a single cell or microbubbles. The results also reveal that there is a good linear relationship between the relative phase and the displacement of a single cell or microbubbles. Furthermore, the single cell and the microbubbles can be transported over a predetermined distance continuously until they reach the targeted locations. This technique has its distinct advantages, such as precise position-manipulation, simple to implement, miniature size, and noninvasive character, which may provide an effective method for the position-manipulation of a single cell and microbubbles in many biological and biomedical applications. PMID:22662056

Mechanisms for Selective Single-Cell Reactivation during Offline Sharp-Wave Ripples and Their Distortion by Fast Ripples.

PubMed

Valero, Manuel; Averkin, Robert G; Fernandez-Lamo, Ivan; Aguilar, Juan; Lopez-Pigozzi, Diego; Brotons-Mas, Jorge R; Cid, Elena; Tamas, Gabor; Menendez de la Prida, Liset

2017-06-21

Memory traces are reactivated selectively during sharp-wave ripples. The mechanisms of selective reactivation, and how degraded reactivation affects memory, are poorly understood. We evaluated hippocampal single-cell activity during physiological and pathological sharp-wave ripples using juxtacellular and intracellular recordings in normal and epileptic rats with different memory abilities. CA1 pyramidal cells participate selectively during physiological events but fired together during epileptic fast ripples. We found that firing selectivity was dominated by an event- and cell-specific synaptic drive, modulated in single cells by changes in the excitatory/inhibitory ratio measured intracellularly. This mechanism collapses during pathological fast ripples to exacerbate and randomize neuronal firing. Acute administration of a use- and cell-type-dependent sodium channel blocker reduced neuronal collapse and randomness and improved recall in epileptic rats. We propose that cell-specific synaptic inputs govern firing selectivity of CA1 pyramidal cells during sharp-wave ripples. Copyright © 2017 Elsevier Inc. All rights reserved.

Neutral competition of stem cells is skewed by proliferative changes downstream of Hh and Hpo.

PubMed

Amoyel, Marc; Simons, Benjamin D; Bach, Erika A

2014-10-16

Neutral competition, an emerging feature of stem cell homeostasis, posits that individual stem cells can be lost and replaced by their neighbors stochastically, resulting in chance dominance of a clone at the niche. A single stem cell with an oncogenic mutation could bias this process and clonally spread the mutation throughout the stem cell pool. The Drosophila testis provides an ideal system for testing this model. The niche supports two stem cell populations that compete for niche occupancy. Here, we show that cyst stem cells (CySCs) conform to the paradigm of neutral competition and that clonal deregulation of either the Hedgehog (Hh) or Hippo (Hpo) pathway allows a single CySC to colonize the niche. We find that the driving force behind such behavior is accelerated proliferation. Our results demonstrate that a single stem cell colonizes its niche through oncogenic mutation by co-opting an underlying homeostatic process. © 2014 The Authors.

DTWscore: differential expression and cell clustering analysis for time-series single-cell RNA-seq data.

PubMed

Wang, Zhuo; Jin, Shuilin; Liu, Guiyou; Zhang, Xiurui; Wang, Nan; Wu, Deliang; Hu, Yang; Zhang, Chiping; Jiang, Qinghua; Xu, Li; Wang, Yadong

2017-05-23

The development of single-cell RNA sequencing has enabled profound discoveries in biology, ranging from the dissection of the composition of complex tissues to the identification of novel cell types and dynamics in some specialized cellular environments. However, the large-scale generation of single-cell RNA-seq (scRNA-seq) data collected at multiple time points remains a challenge to effective measurement gene expression patterns in transcriptome analysis. We present an algorithm based on the Dynamic Time Warping score (DTWscore) combined with time-series data, that enables the detection of gene expression changes across scRNA-seq samples and recovery of potential cell types from complex mixtures of multiple cell types. The DTWscore successfully classify cells of different types with the most highly variable genes from time-series scRNA-seq data. The study was confined to methods that are implemented and available within the R framework. Sample datasets and R packages are available at https://github.com/xiaoxiaoxier/DTWscore .

Time-lapse electrical impedance spectroscopy for monitoring the cell cycle of single immobilized S. pombe cells.

PubMed

Zhu, Zhen; Frey, Olivier; Haandbaek, Niels; Franke, Felix; Rudolf, Fabian; Hierlemann, Andreas

2015-11-26

As a complement and alternative to optical methods, wide-band electrical impedance spectroscopy (EIS) enables multi-parameter, label-free and real-time detection of cellular and subcellular features. We report on a microfluidics-based system designed to reliably capture single rod-shaped Schizosaccharomyces pombe cells by applying suction through orifices in a channel wall. The system enables subsequent culturing of immobilized cells in an upright position, while dynamic changes in cell-cycle state and morphology were continuously monitored through EIS over a broad frequency range. Besides measuring cell growth, clear impedance signals for nuclear division have been obtained. The EIS system has been characterized with respect to sensitivity and detection limits. The spatial resolution in measuring cell length was 0.25 μm, which corresponds to approximately a 5-min interval of cell growth under standard conditions. The comprehensive impedance data sets were also used to determine the occurrence of nuclear division and cytokinesis. The obtained results have been validated through concurrent confocal imaging and plausibilized through comparison with finite-element modeling data. The possibility to monitor cellular and intracellular features of single S. pombe cells during the cell cycle at high spatiotemporal resolution renders the presented microfluidics-based EIS system a suitable tool for dynamic single-cell investigations.

Time-lapse electrical impedance spectroscopy for monitoring the cell cycle of single immobilized S. pombe cells

PubMed Central

Zhu, Zhen; Frey, Olivier; Haandbaek, Niels; Franke, Felix; Rudolf, Fabian; Hierlemann, Andreas

2015-01-01

As a complement and alternative to optical methods, wide-band electrical impedance spectroscopy (EIS) enables multi-parameter, label-free and real-time detection of cellular and subcellular features. We report on a microfluidics-based system designed to reliably capture single rod-shaped Schizosaccharomyces pombe cells by applying suction through orifices in a channel wall. The system enables subsequent culturing of immobilized cells in an upright position, while dynamic changes in cell-cycle state and morphology were continuously monitored through EIS over a broad frequency range. Besides measuring cell growth, clear impedance signals for nuclear division have been obtained. The EIS system has been characterized with respect to sensitivity and detection limits. The spatial resolution in measuring cell length was 0.25 μm, which corresponds to approximately a 5-min interval of cell growth under standard conditions. The comprehensive impedance data sets were also used to determine the occurrence of nuclear division and cytokinesis. The obtained results have been validated through concurrent confocal imaging and plausibilized through comparison with finite-element modeling data. The possibility to monitor cellular and intracellular features of single S. pombe cells during the cell cycle at high spatiotemporal resolution renders the presented microfluidics-based EIS system a suitable tool for dynamic single-cell investigations. PMID:26608589

Single-organelle tracking by two-photon conversion

NASA Astrophysics Data System (ADS)

Watanabe, Wataru; Shimada, Tomoko; Matsunaga, Sachihiro; Kurihara, Daisuke; Fukui, Kiichi; Shin-Ichi Arimura, Shin-Ichi; Tsutsumi, Nobuhiro; Isobe, Keisuke; Itoh, Kazuyoshi

2007-03-01

Spatial and temporal information about intracellular objects and their dynamics within a living cell are essential for dynamic analysis of such objects in cell biology. A specific intracellular object can be discriminated by photoactivatable fluorescent proteins that exhibit pronounced light-induced spectral changes. Here, we report on selective labeling and tracking of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. We performed selective labeling of a single mitochondrion in a living tobacco BY-2 cell using two-photon photoconversion of Kaede. Using this technique, we demonstrated that, in plants, the directed movement of individual mitochondria along the cytoskeletons was mediated by actin filaments, whereas microtubules were not required for the movement of mitochondria. This single-organelle labeling technique enabled us to track the dynamics of a single organelle, revealing the mechanisms involved in organelle dynamics. The technique has potential application in direct tracking of selective cellular and intracellular structures.

Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process.

PubMed

Laranjeiro, Ricardo; Tamai, T Katherine; Letton, William; Hamilton, Noémie; Whitmore, David

2018-04-01

Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles.

Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process

PubMed Central

Tamai, T. Katherine; Letton, William; Hamilton, Noémie; Whitmore, David

2018-01-01

Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles. PMID:29444612

Space-saving tips to lower stress

NASA Astrophysics Data System (ADS)

Gordon, Vernita D.

2018-03-01

Understanding how some single cells evolved into multicellular life means figuring out how they overcome the stresses associated with crowding as they multiply. New insights from yeast suggest that changes in the shape of cells may provide an answer.

Second-site changes affect viability of amphotropic/ecotropic chimeric enveloped murine leukemia viruses.

PubMed

O'Reilly, L; Roth, M J

2000-01-01

Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells.

Second-Site Changes Affect Viability of Amphotropic/Ecotropic Chimeric Enveloped Murine Leukemia Viruses

PubMed Central

O'Reilly, Lucille; Roth, Monica J.

2000-01-01

Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells. PMID:10623753

Impact of jamming on collective cell migration

NASA Astrophysics Data System (ADS)

Nnetu, Kenechukwu David; Knorr, Melanie; Pawlizak, Steve; Fuhs, Thomas; Zink, Mareike; KäS, Josef A.

2012-02-01

Multi-cellular migration plays an important role in physiological processes such as embryogenesis, cancer metastasis and tissue repair. During migration, single cells undergo cycles of extension, adhesion and retraction resulting in morphological changes. In a confluent monolayer, there are inter-cellular interactions and crowding, however, the impact of these interactions on the dynamics and elasticity of the monolayer at the multi-cellular and single cell level is not well understood. Here we study the dynamics of a confluent epithelial monolayer by simultaneously measuring cell motion at the multi-cellular and single cell level for various cell densities and tensile elasticity. At the multi-cellular level, the system exhibited spatial kinetic transitions from isotropic to anisotropic migration on long times and the velocity of the monolayer decreased with increasing cell density. Moreover, the dynamics was spatially and temporally heterogeneous. Interestingly, the dynamics was also heterogeneous in wound-healing assays and the correlation length was fitted by compressed exponential. On the single cell scale, we observed transient caging effects with increasing cage rearrangement times as the system age due to an increase in density. Also, the density dependent elastic modulus of the monolayer scaled as a weak power law. Together, these findings suggest that caging effects at the single cell level initiates a slow and heterogeneous dynamics at the multi-cellular level which is similar to the glassy dynamics of deformable colloidal systems.

Fragile X mental retardation protein knockdown in the developing Xenopus tadpole optic tectum results in enhanced feedforward inhibition and behavioral deficits.

PubMed

Truszkowski, Torrey L S; James, Eric J; Hasan, Mashfiq; Wishard, Tyler J; Liu, Zhenyu; Pratt, Kara G; Cline, Hollis T; Aizenman, Carlos D

2016-08-08

Fragile X Syndrome is the leading monogenetic cause of autism and most common form of intellectual disability. Previous studies have implicated changes in dendritic spine architecture as the primary result of loss of Fragile X Mental Retardation Protein (FMRP), but recent work has shown that neural proliferation is decreased and cell death is increased with either loss of FMRP or overexpression of FMRP. The purpose of this study was to investigate the effects of loss of FMRP on behavior and cellular activity. We knocked down FMRP expression using morpholino oligos in the optic tectum of Xenopus laevis tadpoles and performed a series of behavioral and electrophysiological assays. We investigated visually guided collision avoidance, schooling, and seizure propensity. Using single cell electrophysiology, we assessed intrinsic excitability and synaptic connectivity of tectal neurons. We found that FMRP knockdown results in decreased swimming speed, reduced schooling behavior and decreased seizure severity. In single cells, we found increased inhibition relative to excitation in response to sensory input. Our results indicate that the electrophysiological development of single cells in the absence of FMRP is largely unaffected despite the large neural proliferation defect. The changes in behavior are consistent with an increase in inhibition, which could be due to either changes in cell number or altered inhibitory drive, and indicate that FMRP can play a significant role in neural development much earlier than previously thought.

Poly(3-hydroxybutyrate) anabolism in Cupriavidus necator cultivated at various carbon-to-nitrogen ratios: insights from single-cell Raman spectroscopy

NASA Astrophysics Data System (ADS)

Tao, Zhanhua; Zhang, Pengfei; Qin, Zhaojun; Li, Yong-Qing; Wang, Guiwen

2016-09-01

Cupriavidus necator accumulates large amounts of poly(3-hydroxybutyrate) (PHB), a biodegradable substitute for petroleum-based plastics, under certain nutrient conditions. Conventional solvent-extraction-based methods for PHB quantification only obtain average information from cell populations and, thus, mask the heterogeneity among individual cells. Laser tweezers Raman spectroscopy (LTRS) was used to monitor dynamic changes in the contents of PHB, nucleic acids, and proteins in C. necator at the population and single-cell levels when the microorganism cells were cultivated at various carbon-to-nitrogen ratios. The biosynthetic activities of nucleic acids and proteins were maintained at high levels, and only a small amount of PHB was produced when the bacterial cells were cultured under balanced growth conditions. By contrast, the syntheses of nucleic acids and proteins were blocked, and PHB was accumulated in massive amount inside the microbial cells under nitrogen-limiting growth circumstances. Single-cell analysis revealed a relatively high heterogeneity in PHB level at the early stage of the bacterial growth. Additionally, bacterial cells in populations at certain cultivation stages were composed of two or three subpopulations on the basis of their PHB abundance. Overall, LTRS is a reliable single-cell analysis tool that can provide insights into PHB fermentation.

Nanotechnology Approaches to Studying Epigenetic Changes in Cancer

NASA Astrophysics Data System (ADS)

Riehn, Robert

2011-03-01

Placing polyelectrolytes into confined geometries has a profound effect on their molecular configuration. For instance, placing long DNA molecules into channels with a cross-section of about 100 nm 2 stretches them out to about 70% of their contour length. We are using this effect to map epigenetic changes on single DNA and chromatin strands. This mapping on single molecules becomes central in the study of the heterogeneity of cell population in cancer, since rapid change of epigenetic makeup, propagated through rare cancer stem cells, is a hallmark of its progression. We demonstrate the basic building blocks for the single-molecule epigenetic analysis of genomic sized DNA. In particular, we have achieved the mapping of methylated regions in DNA with heterogeneous 5-methyl cytosine modification using a specific fluorescent marker. We further show that chromatin with an intact histone structure can be stretched similar to DNA, and that the epigenetic state of histone tails can be detected using fluorescent antibodies.

Coupling between apical tension and basal adhesion allow epithelia to collectively sense and respond to substrate topography over long distances.

PubMed

Broaders, Kyle E; Cerchiari, Alec E; Gartner, Zev J

2015-12-01

Epithelial sheets fold into complex topographies that contribute to their function in vivo. Cells can sense and respond to substrate topography in their immediate vicinity by modulating their interfacial mechanics, but the extent to which these mechanical properties contribute to their ability to sense substrate topography across length scales larger than a single cell has not been explored in detail. To study the relationship between the interfacial mechanics of single cells and their collective behavior as tissues, we grew cell-sheets on substrates engraved with surface features spanning macroscopic length-scales. We found that many epithelial cell-types sense and respond to substrate topography, even when it is locally nearly planar. Cells clear or detach from regions of local negative curvature, but not from regions with positive or no curvature. We investigated this phenomenon using a finite element model where substrate topography is coupled to epithelial response through a balance of tissue contractility and adhesive forces. The model correctly predicts the focal sites of cell-clearing and epithelial detachment. Furthermore, the model predicts that local tissue response to substrate curvature is a function of the surrounding topography of the substrate across long distances. Analysis of cell-cell and cell-substrate contact angles suggests a relationship between these single-cell interfacial properties, epithelial interfacial properties, and collective epithelial response to substrate topography. Finally, we show that contact angles change upon activation of oncogenes or inhibition of cell-contractility, and that these changes correlate with collective epithelial response. Our results demonstrate that in mechanically integrated epithelial sheets, cell contractility can be transmitted through multiple cells and focused by substrate topography to affect a behavioral response at distant sites.

Understanding Papillary Renal Cell Carcinoma | Center for Cancer Research

Cancer.gov

Renal cell carcinoma (RCC), the most common form of kidney cancer in adults, is not a single disease but rather a collection of different tumor types driven by distinct genetic changes that arise within the same tissue.

Ultrafast single molecule technique for the study of force dependent kinetics and conformational changes of actin-protein interaction involved in mechanotransduction

NASA Astrophysics Data System (ADS)

Sergides, M.; Arbore, C.; Pavone, F. S.; Capitanio, M.

2018-02-01

Mechanical signals occurring at the interface between cell membrane and extracellular matrix and at intercellular junctions trigger biochemical signals that are fundamental for cell growth, development and regulation. Adaptor proteins, which link the cell membrane to the actin cytoskeleton, seem to partake in this process of mechanotransduction. In particular, catenins play a key role in intercellular junctions, where they act as a bridge between the cell membrane and actin. Studies suggest that α-catenin contains a domain that normally masks vinculin binding sites, which can become accessible after a conformational change induced by an external force. Here we demonstrate a single-molecule technique for investigating actin-protein interactions at different forces (up to 17 pN) with adequate temporal resolution (sub-ms). This system is based on the ultrafast force-clamp spectroscopy technique that has been recently developed by our group and is adapted to study and measure force-dependent kinetics of the catenin-actin interaction, as well as the amplitude of the expected conformational changes such as force-induced protein unfolding.

Loss of Parafollicular Cells during Gravitational Changes (Microgravity, Hypergravity) and the Secret Effect of Pleiotrophin

PubMed Central

Albi, Elisabetta; Curcio, Francesco; Spelat, Renza; Lazzarini, Andrea; Lazzarini, Remo; Cataldi, Samuela; Loreti, Elisabetta; Ferri, Ivana; Ambesi-Impiombato, Francesco Saverio

2012-01-01

It is generally known that bone loss is one of the most important complications for astronauts who are exposed to long-term microgravity in space. Changes in blood flow, systemic hormones, and locally produced factors were indicated as important elements contributing to the response of osteoblastic cells to loading, but research in this field still has many questions. Here, the possible biological involvement of thyroid C cells is being investigated. The paper is a comparison between a case of a wild type single mouse and a over-expressing pleiotrophin single mouse exposed to hypogravity conditions during the first animal experiment of long stay in International Space Station (91 days) and three similar mice exposed to hypergravity (2Gs) conditions. We provide evidence that both microgravity and hypergravity induce similar loss of C cells with reduction of calcitonin production. Pleiotrophin over-expression result in some protection against negative effects of gravity change. Potential implication of the gravity mechanic forces in the regulation of bone homeostasis via thyroid equilibrium is discussed. PMID:23284618

Aerobiological Stabilities of Different Species of Gram-Negative Bacteria, Including Well-Known Biothreat Simulants, in Single-Cell Particles and Cell Clusters of Different Compositions

PubMed Central

Skogan, Gunnar

2017-01-01

ABSTRACT The ability to perform controlled experiments with bioaerosols is a fundamental enabler of many bioaerosol research disciplines. A practical alternative to using hazardous biothreat agents, e.g., for detection equipment development and testing, involves using appropriate model organisms (simulants). Several species of Gram-negative bacteria have been used or proposed as biothreat simulants. However, the appropriateness of different bacterial genera, species, and strains as simulants is still debated. Here, we report aerobiological stability characteristics of four species of Gram-negative bacteria (Pantoea agglomerans, Serratia marcescens, Escherichia coli, and Xanthomonas arboricola) in single-cell particles and cell clusters produced using four spray liquids (H2O, phosphate-buffered saline[PBS], spent culture medium[SCM], and a SCM-PBS mixture). E. coli showed higher stability in cell clusters from all spray liquids than the other species, but it showed similar or lower stability in single-cell particles. The overall stability was higher in cell clusters than in single-cell particles. The highest overall stability was observed for bioaerosols produced using SCM-containing spray liquids. A key finding was the observation that stability differences caused by particle size or compositional changes frequently followed species-specific patterns. The results highlight how even moderate changes to one experimental parameter, e.g., bacterial species, spray liquid, or particle size, can strongly affect the aerobiological stability of Gram-negative bacteria. Taken together, the results highlight the importance of careful and informed selection of Gram-negative bacterial biothreat simulants and also the accompanying particle size and composition. The outcome of this work contributes to improved selection of simulants, spray liquids, and particle size for use in bioaerosol research. IMPORTANCE The outcome of this work contributes to improved selection of simulants, spray liquids, and particle size for use in bioaerosol research. Taken together, the results highlight the importance of careful and informed selection of Gram-negative bacterial biothreat simulants and also the accompanying particle size and composition. The results highlight how even moderate changes to one experimental parameter, e.g., bacterial species, spray liquid, or particle size, can strongly affect the aerobiological stability of Gram-negative bacteria. A key finding was the observation that stability differences caused by particle size or compositional changes frequently followed species-specific patterns. PMID:28687646

Single-cell analysis of intercellular heteroplasmy of mtDNA in Leber hereditary optic neuropathy

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

Kobayashi, Y.; Sharpe, H.; Brown, N.

1994-07-01

The authors have investigated the distribution of mutant mtDNA molecules in single cells from a patient with Leber hereditary optic neuropathy (LHON). LHON is a maternally inherited disease that is characterized by a sudden-onset bilateral loss of central vision, which typically occurs in early adulthood. More than 50% of all LHON patients carry an mtDNA mutation at nucleotide position 11778. This nucleotide change converts a highly conserved arginine residue to histidine at codon 340 in the NADH-ubiquinone oxidoreductase subunit 4 (ND4) gene of mtDNA. In the present study, the authors used PCR amplification of mtDNA from lymphocytes to investigate mtDNAmore » heteroplasmy at the single-cell level in a LHON patient. They found that most cells were either homoplasmic normal or homoplasmic mutant at nucleotide position 11778. Some (16%) cells contained both mutant and normal mtDNA.« less

On the holographic 3D tracking of in vitro cells characterized by a highly-morphological change.

PubMed

Memmolo, Pasquale; Iannone, Maria; Ventre, Maurizio; Netti, Paolo Antonio; Finizio, Andrea; Paturzo, Melania; Ferraro, Pietro

2012-12-17

Digital Holography (DH) in microscopic configuration is a powerful tool for the imaging of micro-objects contained into a three dimensional (3D) volume, by a single-shot image acquisition. Many studies report on the ability of DH to track particle, microorganism and cells in 3D. However, very few investigations are performed with objects that change severely their morphology during the observation period. Here we study DH as a tool for 3D tracking an osteosarcoma cell line for which extensive changes in cell morphology are associated to cell motion. Due to the great unpredictable morphological change, retrieving cell's position in 3D can become a complicated issue. We investigate and discuss in this paper how the tridimensional position can be affected by the continuous change of the cells. Moreover we propose and test some strategies to afford the problems and compare it with others approaches. Finally, results on the 3D tracking and comments are reported and illustrated.

Integrated Raman and angular scattering microscopy reveals chemical and morphological differences between activated and nonactivated CD8+ T lymphocytes

PubMed Central

Smith, Zachary J.; Wang, Jyh-Chiang E.; Quataert, Sally A.; Berger, Andrew J.

2010-01-01

Integrated Raman and angular-scattering microscopy (IRAM) is a multimodal platform capable of noninvasively probing both the chemistry and morphology of a single cell without prior labeling. Using this system, we are able to detect activation-dependent changes in the Raman and elastic-scattering signals from CD8+ T cells stimulated with either Staphylococcal enterotoxin B (SEB) or phorbol myristate acetate (PMA). In both cases, results obtained from the IRAM instrument correlate well with results obtained from traditional fluorescence-based flow cytometry for paired samples. SEB-mediated activation was distinguished from resting state in CD8+ T cells by an increase in the number and mean size of small (∼500-nm) elastic scatterers as well as a decrease in Raman bands, indicating changes in nuclear content. PMA-mediated activation induced a different profile in CD8+ T cells from SEB, showing a similar increase in small elastic scatterers but a different Raman change, with elevation of cellular protein and lipid bands. These results suggest the potential of this multimodal, label-free optical technique for studying processes in single cells. PMID:20615023

Single unit activity in the medial prefrontal cortex during Pavlovian heart rate conditioning: Effects of peripheral autonomic blockade.

PubMed

Powell, D A; Ginsberg, Jay P

2005-11-01

Electrical activity was recorded from single neurons in the medial prefrontal cortex of rabbits during differential Pavlovian heart rate (HR) conditioning. A heterogeneous population of cells were found, some of which showed CS-evoked increases and others CS-evoked decreases in discharge, while some cells were biphasic. A subset of cells also showed trial-related changes in discharge that were related to acquisition of the HR discrimination between the reinforced CS+ and non-reinforced CS-. Administration of the peripheral cholinergic antagonist, methylscopolamine, and the andrenergic antagonist, atenolol, either increased or decreased maintained baseline activity of many cells, but had little or no effect on the CS-evoked activity of these cells. Waveform changes also did not result from administration of these drugs. This finding suggests that CS-evoked mPFC activity is not being driven by cardiac afferent input to CNS cardiac control centers. Previous studies have shown that ibotenic acid lesions of this area greatly decreases the magnitude of decelerative heart rate conditioned responses; the latter finding, plus the results of the present study, suggest that processing of CS/US contingencies by the prefrontal cortex contributes to the acquisition of autonomic changes during Pavlovian conditioning.

Single-cell optoporation and transfection using femtosecond laser and optical tweezers.

PubMed

Waleed, Muhammad; Hwang, Sun-Uk; Kim, Jung-Dae; Shabbir, Irfan; Shin, Sang-Mo; Lee, Yong-Gu

2013-01-01

In this paper, we demonstrate a new single-cell optoporation and transfection technique using a femtosecond Gaussian laser beam and optical tweezers. Tightly focused near-infrared (NIR) femtosecond laser pulse was employed to transiently perforate the cellular membrane at a single point in MCF-7 cancer cells. A distinct technique was developed by trapping the microparticle using optical tweezers to focus the femtosecond laser precisely on the cell membrane to puncture it. Subsequently, an external gene was introduced in the cell by trapping and inserting the same plasmid-coated microparticle into the optoporated cell using optical tweezers. Various experimental parameters such as femtosecond laser exposure power, exposure time, puncture hole size, exact focusing of the femtosecond laser on the cell membrane, and cell healing time were closely analyzed to create the optimal conditions for cell viability. Following the insertion of plasmid-coated microparticles in the cell, the targeted cells exhibited green fluorescent protein (GFP) under the fluorescent microscope, hence confirming successful transfection into the cell. This new optoporation and transfection technique maximizes the level of selectivity and control over the targeted cell, and this may be a breakthrough method through which to induce controllable genetic changes in the cell.

Micro-Raman Spectroscopy of Silver Nanoparticle Induced Stress on Optically-Trapped Stem Cells

PubMed Central

Bankapur, Aseefhali; Krishnamurthy, R. Sagar; Zachariah, Elsa; Santhosh, Chidangil; Chougule, Basavaraj; Praveen, Bhavishna; Valiathan, Manna; Mathur, Deepak

2012-01-01

We report here results of a single-cell Raman spectroscopy study of stress effects induced by silver nanoparticles in human mesenchymal stem cells (hMSCs). A high-sensitivity, high-resolution Raman Tweezers set-up has been used to monitor nanoparticle-induced biochemical changes in optically-trapped single cells. Our micro-Raman spectroscopic study reveals that hMSCs treated with silver nanoparticles undergo oxidative stress at doping levels in excess of 2 µg/ml, with results of a statistical analysis of Raman spectra suggesting that the induced stress becomes more dominant at nanoparticle concentration levels above 3 µg/ml. PMID:22514708

Population transcriptomics with single-cell resolution: a new field made possible by microfluidics: a technology for high throughput transcript counting and data-driven definition of cell types.

PubMed

Plessy, Charles; Desbois, Linda; Fujii, Teruo; Carninci, Piero

2013-02-01

Tissues contain complex populations of cells. Like countries, which are comprised of mixed populations of people, tissues are not homogeneous. Gene expression studies that analyze entire populations of cells from tissues as a mixture are blind to this diversity. Thus, critical information is lost when studying samples rich in specialized but diverse cells such as tumors, iPS colonies, or brain tissue. High throughput methods are needed to address, model and understand the constitutive and stochastic differences between individual cells. Here, we describe microfluidics technologies that utilize a combination of molecular biology and miniaturized labs on chips to study gene expression at the single cell level. We discuss how the characterization of the transcriptome of each cell in a sample will open a new field in gene expression analysis, population transcriptomics, that will change the academic and biomedical analysis of complex samples by defining them as quantified populations of single cells. Copyright © 2013 WILEY Periodicals, Inc.

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

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.

Salicylate-induced changes in spontaneous activity of single units in the inferior colliculus of the guinea pig.

PubMed

Jastreboff, P J; Sasaki, C T

1986-11-01

Changes in spontaneous neuronal activity of the inferior colliculus in albino guinea pigs before and after administration of sodium salicylate were analyzed. Animals were anesthetized with pentobarbital, and two microelectrodes separated by a few hundred microns were driven through the inferior colliculus. After collecting a sufficiently large sample of cells, sodium salicylate (450 mg/kg) was injected i.p. and recordings again made 2 h after the injection. Comparison of spontaneous activity recorded before and after salicylate administration revealed highly statistically significant differences (p less than 0.001). After salicylate, the mean rate of the cell population increased from 29 to 83 Hz and the median from 26 to 74 Hz. Control experiments in which sodium salicylate was replaced by saline injection revealed no statistically significant differences in cell discharges. Recordings made during the same experiments from lobulus V of the cerebellar vermis revealed no changes in response to salicylate. The observed changes in single-unit activity due to salicylate administration may represent the first systematic evidence of a tinnituslike phenomenon in animals.

TCF21 hypermethylation in genetically quiescent clear cell sarcoma of the kidney | Office of Cancer Genomics

Cancer.gov

Clear Cell Sarcoma of the Kidney (CCSK) is a rare childhood tumor whose molecular pathogenesis remains poorly understood. We analyzed a discovery set of 13 CCSKs for changes in chromosome copy number, mutations, rearrangements, global gene expression and global DNA methylation. No recurrent segmental chromosomal copy number changes or somatic variants (single nucleotide or small insertion/deletion) were identified.

Spontaneous activity of single neurones in the hypothalamus of rabbits during sleep and waking

PubMed Central

Findlay, Alan L. R.; Hayward, James N.

1969-01-01

1. A method is described for recording from single cells in the hypothalamus of unanaesthetized freely moving rabbits. Behaviour, bodily movement, skin and brain temperatures and e.e.g. were monitored. 2. Patterns of unit firing during slow sleep, paradoxical sleep and waking were studied in several regions of the hypothalamus, thalamus and in the septum. 3. Of the 144 cells analysed from waking to slow sleep, fifty-six (39%) decreased mean firing rates, thirty (21%) increased spike discharges and fifty-eight (40%) showed no marked change. Dorsal hypothalamic and massa intermedia thalamic cells fired in brief high frequency clusters during slow sleep with a characteristic `bimodal' interspike interval histogram. Waking and paradoxical sleep abolished these cluster discharges with a concomitant change to an `asymmetric' histogram. 4. Of the thirty-two cells observed during the three states of waking, slow sleep and paradoxical sleep, a majority (twenty-five or 78%) showed their highest rates of spontaneous discharge during paradoxical sleep. Discharge rates of cells sometimes changed in the course of paradoxical sleep according to the presence or absence of phasic events such as myoclonic motor activity. Two hypothalmic cells were almost totally arrested during paradoxical sleep. 5. Analysis of unit firing rates during spontaneous rises in brain temperature during waking and paradoxical sleep revealed that a majority of the neurones (22/24) changed their discharge rates in relation to behaviour rather than to brain temperature. Two cells did appear to respond specifically to the central thermal stimulus. 6. Hypothalamic cells do not behave as a homogeneous population in relation to changes in the state of arousal of the rabbit. Spontaneous changes in cell discharge related to sleep-waking behaviour must be considered in any interpretation of hypothalamic unit activity as related to neuroendocrine or autonomic mechanisms. PMID:4304342

The nesprin-cytoskeleton interface probed directly on single nuclei is a mechanically rich system.

PubMed

Balikov, Daniel A; Brady, Sonia K; Ko, Ung Hyun; Shin, Jennifer H; de Pereda, Jose M; Sonnenberg, Arnoud; Sung, Hak-Joon; Lang, Matthew J

2017-09-03

The cytoskeleton provides structure and plays an important role in cellular function such as migration, resisting compression forces, and transport. The cytoskeleton also reacts to physical cues such as fluid shear stress or extracellular matrix remodeling by reorganizing filament associations, most commonly focal adhesions and cell-cell cadherin junctions. These mechanical stimuli can result in genome-level changes, and the physical connection of the cytoskeleton to the nucleus provides an optimal conduit for signal transduction by interfacing with nuclear envelope proteins, called nesprins, within the LINC (linker of the nucleus to the cytoskeleton) complex. Using single-molecule on single nuclei assays, we report that the interactions between the nucleus and the cytoskeleton, thought to be nesprin-cytoskeleton interactions, are highly sensitive to force magnitude and direction depending on whether cells are historically interfaced with the matrix or with cell aggregates. Application of ∼10-30 pN forces to these nesprin linkages yielded structural transitions, with a base transition size of 5-6 nm, which are speculated to be associated with partial unfoldings of the spectrin domains of the nesprins and/or structural changes of histones within the nucleus.

Development of Nano/Micro Probes for Femtoliter Volume and Single Cell Measurements

NASA Astrophysics Data System (ADS)

Gao, Yang

Single cell analysis has recently emerged as an important field of biomedical re- search. It is now clear that heterogeneity of cell metabolism functions in complex biological systems is correlated to changes in biological function and disease processes. A variety of nano/micro probes were developed to enable investigation of cells properties such as membrane stiffness, pH value. However, very few designs were focused on single cell metabolic function studies. There is a critical need for technologies that provide analysis of heterogeneity of cell metabolic functions, especially on metabolism. Nevertheless, the few existing approaches suffer from fundamental defects and need to be improved. This work focused on developing nano/micro probes that are suitable for single cell functionality investigation. Both types of probes are designed to measure cell-to-cell/time-to-time heterogeneity in metabolic functions over a long period of time. Lab-made carbon nanoprobes were developed especially for electro-physiological measurement. The unique structure of the carbon nanoprobes makes them suitable for important intracellular applications like trans-membrane potential measurements and various electrochemical measurement for cell function studies. While it is important of have ability to carry out intracellular measure, there are also occasions where the information of a cell as a whole is collected. One of the most important indicator of a cells metabolic functions is cell respiration rate/oxygen consumption rate. A micro-perfusion based multi-functional single cell sensing probe was the developed to carry out measurements on cell as a whole. Formed by a double-barrel theta pipette, the perfusion flow enables the direct measurement of the metabolic flux for example oxygen consumption rate. In conclusion, this work developed nano/micro-probes as novel single cell investigation tools. The data acquired from these tools could provide valuable assistance on applications including cell metabolism studies, cancer diagnoses, and therapy evaluations.

Microplasma Induced Cell Morphological Changes and Apoptosis of Ex Vivo Cultured Human Anterior Lens Epithelial Cells – Relevance to Capsular Opacification

PubMed Central

Hojnik, Nataša; Filipič, Gregor; Lazović, Saša; Vesel, Alenka; Primc, Gregor; Mozetič, Miran; Hawlina, Marko; Petrovski, Goran; Cvelbar, Uroš

2016-01-01

Inducing selective or targeted cell apoptosis without affecting large number of neighbouring cells remains a challenge. A plausible method for treatment of posterior capsular opacification (PCO) due to remaining lens epithelial cells (LECs) by reactive chemistry induced by localized single electrode microplasma discharge at top of a needle-like glass electrode with spot size ~3 μm is hereby presented. The focused and highly-localized atmospheric pressure microplasma jet with electrode discharge could induce a dose-dependent apoptosis in selected and targeted individual LECs, which could be confirmed by real-time monitoring of the morphological and structural changes at cellular level. Direct cell treatment with microplasma inside the medium appeared more effective in inducing apoptosis (caspase 8 positivity and DNA fragmentation) at a highly targeted cell level compared to treatment on top of the medium (indirect treatment). Our results show that single cell specific micropipette plasma can be used to selectively induce demise in LECs which remain in the capsular bag after cataract surgery and thus prevent their migration (CXCR4 positivity) to the posterior lens capsule and PCO formation. PMID:27832099

Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin.

PubMed

Maciaszek, Jamie L; Partola, Kostyantyn; Zhang, Jing; Andemariam, Biree; Lykotrafitis, George

2014-12-18

Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding. Copyright © 2014 Elsevier Ltd. All rights reserved.

Transcription factors and stress response gene alterations in human keratinocytes following Solar Simulated Ultra Violet Radiation.

PubMed

Marais, Thomas L Des; Kluz, Thomas; Xu, Dazhong; Zhang, Xiaoru; Gesumaria, Lisa; Matsui, Mary S; Costa, Max; Sun, Hong

2017-10-19

Ultraviolet radiation (UVR) from sunlight is the major effector for skin aging and carcinogenesis. However, genes and pathways altered by solar-simulated UVR (ssUVR), a mixture of UVA and UVB, are not well characterized. Here we report global changes in gene expression as well as associated pathways and upstream transcription factors in human keratinocytes exposed to ssUVR. Human HaCaT keratinocytes were exposed to either a single dose or 5 repetitive doses of ssUVR. Comprehensive analyses of gene expression profiles as well as functional annotation were performed at 24 hours post irradiation. Our results revealed that ssUVR modulated genes with diverse cellular functions changed in a dose-dependent manner. Gene expression in cells exposed to a single dose of ssUVR differed significantly from those that underwent repetitive exposures. While single ssUVR caused a significant inhibition in genes involved in cell cycle progression, especially G2/M checkpoint and mitotic regulation, repetitive ssUVR led to extensive changes in genes related to cell signaling and metabolism. We have also identified a panel of ssUVR target genes that exhibited persistent changes in gene expression even at 1 week after irradiation. These results revealed a complex network of transcriptional regulators and pathways that orchestrate the cellular response to ssUVR.

Pinus Monophylla (Single Needled Pinyon Pine) show morphological changes in needle cell size and stomata over the past 100 years of rising CO2 in Western Arid Ecosystems.

NASA Astrophysics Data System (ADS)

Van De Water, P. K.

2016-12-01

The size, frequency, and morphology of leaf surface stomata is used to reconstruct past levels of atmospheric carbon dioxide over geologic time. This technique relies on measuring cell and cell-clusters to correlate with changes of known carbon dioxide levels in the atmosphere. Unfortunately, not all plants are suitable because the occurrence and placement of stomatal cell-complexes differ significantly between plant families. Monocot and dicot angiosperms exhibit different types of stomata and stomatal complexes that lack order and thus are unsuitable. But, in gymnosperms, the number and distribution of stomata and pavement cells is formalized and can be used to reconstruct past atmospheric carbon dioxide levels. However, characteristic of each plant species must still be considered. For example, conifers are useful but are divided into two-needle to five-needle pines, or have irregular surface morphology (Pseudotsuga sp. and Tsuga sp. needles). This study uses Pinus monophylla an undivided needle morphology, that being a cylinder has no interior surface cells. Pinus monophylla (single needle pinyon) needles were collected along Geiger Grade (Nevada State Highway 341, Reno) in 2005 and 2013 from 1500m to 2195m. Herbarium samples were also collected from 13 historic collections made between 1911 and 1994. The study determined changes with elevation and/or over time using in these populations. Using Pinus monophylla, insured needles represented a single surface with stomata, stomatal complex cells, and co-occurring pavement cell types. Results show decreased stomatal densities (stomata/area), stomatal index (stomata/stomata + epidermal cells) and stable stomata per row (stomata/row) . Epidermal cell density (Epidermal Cells /Area), and Pavement cell density (Pavement cell/area) track stomatal density similarly. Data comparison, using elevation in the 2005 and 2013 collections showed no-significant trends. Individual stomatal complexes show no differences in the size and shape over time or with elevation. Stomata morphology and the stomatal pores appear conservative. However some complex cells show a morphology suggesting they are not fully formed and functional. These characteristics appear often in the modern material suggesting some stomata never fully develop.

Ca2+ and MgATP2- dependence of shortening in skinned single smooth muscle cells.

PubMed

Warshaw, D M; McBride, W J; Hubbard, M S

1987-04-01

Most studies of skinned smooth muscle have been performed in whole tissue preparations. In this study, we report the development of a chemically skinned single smooth muscle cell preparation from the toad, Bufo marinus, stomach. Isolated smooth muscle cells were skinned using saponin. The effect of various ionic environments (i.e., changing free Ca2+ and MgATP2-) on skinned cell contractile response was assessed by measuring cell lengths from populations of cells using a computer-assisted length-measuring system. Comparison of cell length histograms were used to determine the extent of cell shortening in response to a given ionic perturbation. Once skinned, the single cells shortened with a sensitivity to free calcium (ED50 = 1.5 microM Ca2+) that was three orders of magnitude lower than potassium depolarized cells (ED50 = 1.5 mM Ca2+). In addition to the calcium sensitivity, the effect of free MgATP2- on the extent of cell shortening was investigated. The extent of cell shortening was dependent on free MgATP2- with the maximum shortening response occurring at MgATP2- greater than 1 mM.

In Vivo Biochemistry: Single-Cell Dynamics of Cyclic Di-GMP in Escherichia coli in Response to Zinc Overload.

PubMed

Yeo, Jongchan; Dippel, Andrew B; Wang, Xin C; Hammond, Ming C

2018-01-09

Intracellular signaling enzymes drive critical changes in cellular physiology and gene expression, but their endogenous activities in vivo remain highly challenging to study in real time and for individual cells. Here we show that flow cytometry can be performed in complex media to monitor single-cell population distributions and dynamics of cyclic di-GMP signaling, which controls the bacterial colonization program. These in vivo biochemistry experiments are enabled by our second-generation RNA-based fluorescent (RBF) biosensors, which exhibit high fluorescence turn-on in response to cyclic di-GMP. Specifically, we demonstrate that intracellular levels of cyclic di-GMP in Escherichia coli are repressed with excess zinc, but not with other divalent metals. Furthermore, in both flow cytometry and fluorescence microscopy setups, we monitor the dynamic increase in cellular cyclic di-GMP levels upon zinc depletion and show that this response is due to de-repression of the endogenous diguanylate cyclase DgcZ. In the presence of zinc, cells exhibit enhanced cell motility and increased sensitivity to antibiotics due to inhibited biofilm formation. Taken together, these results showcase the application of RBF biosensors in visualizing single-cell dynamic changes in cyclic di-GMP signaling in direct response to environmental cues such as zinc and highlight our ability to assess whether observed phenotypes are related to specific signaling enzymes and pathways.

Mutagenic consequences of a single G-quadruplex demonstrate mitotic inheritance of DNA replication fork barriers

PubMed Central

Lemmens, Bennie; van Schendel, Robin; Tijsterman, Marcel

2015-01-01

Faithful DNA replication is vital to prevent disease-causing mutations, chromosomal aberrations and malignant transformation. However, accuracy conflicts with pace and flexibility and cells rely on specialized polymerases and helicases to ensure effective and timely replication of genomes that contain DNA lesions or secondary structures. If and how cells can tolerate a permanent barrier to replication is, however, unknown. Here we show that a single unresolved G-quadruplexed DNA structure can persist through multiple mitotic divisions without changing conformation. Failed replication across a G-quadruplex causes single-strand DNA gaps that give rise to DNA double-strand breaks in subsequent cell divisions, which are processed by polymerase theta (POLQ)-mediated alternative end joining. Lineage tracing experiments further reveal that persistent G-quadruplexes cause genetic heterogeneity during organ development. Our data demonstrate that a single lesion can cause multiple unique genomic rearrangements, and that alternative end joining enables cells to proliferate in the presence of mitotically inherited replication blocks. PMID:26563448

Mutagenic consequences of a single G-quadruplex demonstrate mitotic inheritance of DNA replication fork barriers.

PubMed

Lemmens, Bennie; van Schendel, Robin; Tijsterman, Marcel

2015-11-13

Faithful DNA replication is vital to prevent disease-causing mutations, chromosomal aberrations and malignant transformation. However, accuracy conflicts with pace and flexibility and cells rely on specialized polymerases and helicases to ensure effective and timely replication of genomes that contain DNA lesions or secondary structures. If and how cells can tolerate a permanent barrier to replication is, however, unknown. Here we show that a single unresolved G-quadruplexed DNA structure can persist through multiple mitotic divisions without changing conformation. Failed replication across a G-quadruplex causes single-strand DNA gaps that give rise to DNA double-strand breaks in subsequent cell divisions, which are processed by polymerase theta (POLQ)-mediated alternative end joining. Lineage tracing experiments further reveal that persistent G-quadruplexes cause genetic heterogeneity during organ development. Our data demonstrate that a single lesion can cause multiple unique genomic rearrangements, and that alternative end joining enables cells to proliferate in the presence of mitotically inherited replication blocks.

Single-cell topological RNA-Seq analysis reveals insights into cellular differentiation and development

PubMed Central

Rizvi, Abbas H.; Camara, Pablo G.; Kandror, Elena K.; Roberts, Thomas J.; Schieren, Ira; Maniatis, Tom; Rabadan, Raul

2017-01-01

Transcriptional programs control cellular lineage commitment and differentiation during development. Understanding cell fate has been advanced by studying single-cell RNA-seq, but is limited by the assumptions of current analytic methods regarding the structure of data. We present single-cell topological data analysis (scTDA), an algorithm for topology-based computational analyses to study temporal, unbiased transcriptional regulation. Compared to other methods, scTDA is a non-linear, model-independent, unsupervised statistical framework that can characterize transient cellular states. We applied scTDA to the analysis of murine embryonic stem cell (mESC) differentiation in vitro in response to inducers of motor neuron differentiation. scTDA resolved asynchrony and continuity in cellular identity over time, and identified four transient states (pluripotent, precursor, progenitor, and fully differentiated cells) based on changes in stage-dependent combinations of transcription factors, RNA-binding proteins and long non-coding RNAs. scTDA can be applied to study asynchronous cellular responses to either developmental cues or environmental perturbations. PMID:28459448

Revealing Early Steps of α2β1 Integrin-mediated Adhesion to Collagen Type I by Using Single-Cell Force Spectroscopy

PubMed Central

Taubenberger, Anna; Cisneros, David A.; Friedrichs, Jens; Puech, Pierre-Henri; Muller, Daniel J.

2007-01-01

We have characterized early steps of α2β1 integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of α2β1 as a collagen type I receptor, α2β1-expressing Chinese hamster ovary (CHO)-A2 cells spread rapidly on the matrix, whereas α2β1-negative CHO wild-type cells adhered poorly. Probing CHO-A2 cell detachment forces over a contact time range of 600 s revealed a nonlinear adhesion response. During the first 60 s, cell adhesion increased slowly, and forces associated with the smallest rupture events were consistent with the breakage of individual integrin–collagen bonds. Above 60 s, a fraction of cells rapidly switched into an activated adhesion state marked by up to 10-fold increased detachment forces. Elevated overall cell adhesion coincided with a rise of the smallest rupture forces above the value required to break a single-integrin–collagen bond, suggesting a change from single to cooperative receptor binding. Transition into the activated adhesion mode and the increase of the smallest rupture forces were both blocked by inhibitors of actomyosin contractility. We therefore propose a two-step mechanism for the establishment of α2β1-mediated adhesion as weak initial, single-integrin–mediated binding events are superseded by strong adhesive interactions involving receptor cooperativity and actomyosin contractility. PMID:17314408

Gravity Research on Plants: Use of Single-Cell Experimental Models

PubMed Central

Chebli, Youssef; Geitmann, Anja

2011-01-01

Future space missions and implementation of permanent bases on Moon and Mars will greatly depend on the availability of ambient air and sustainable food supply. Therefore, understanding the effects of altered gravity conditions on plant metabolism and growth is vital for space missions and extra-terrestrial human existence. In this mini-review we summarize how plant cells are thought to perceive changes in magnitude and orientation of the gravity vector. The particular advantages of several single-celled model systems for gravity research are explored and an overview over recent advancements and potential use of these systems is provided. PMID:22639598

Cell-cell bioelectrical interactions and local heterogeneities in genetic networks: a model for the stabilization of single-cell states and multicellular oscillations.

PubMed

Cervera, Javier; Manzanares, José A; Mafe, Salvador

2018-04-04

Genetic networks operate in the presence of local heterogeneities in single-cell transcription and translation rates. Bioelectrical networks and spatio-temporal maps of cell electric potentials can influence multicellular ensembles. Could cell-cell bioelectrical interactions mediated by intercellular gap junctions contribute to the stabilization of multicellular states against local genetic heterogeneities? We theoretically analyze this question on the basis of two well-established experimental facts: (i) the membrane potential is a reliable read-out of the single-cell electrical state and (ii) when the cells are coupled together, their individual cell potentials can be influenced by ensemble-averaged electrical potentials. We propose a minimal biophysical model for the coupling between genetic and bioelectrical networks that associates the local changes occurring in the transcription and translation rates of an ion channel protein with abnormally low (depolarized) cell potentials. We then analyze the conditions under which the depolarization of a small region (patch) in a multicellular ensemble can be reverted by its bioelectrical coupling with the (normally polarized) neighboring cells. We show also that the coupling between genetic and bioelectric networks of non-excitable cells, modulated by average electric potentials at the multicellular ensemble level, can produce oscillatory phenomena. The simulations show the importance of single-cell potentials characteristic of polarized and depolarized states, the relative sizes of the abnormally polarized patch and the rest of the normally polarized ensemble, and intercellular coupling.

Single cell-type analysis of cellular lipid remodelling in response to salinity in the epidermal bladder cells of the model halophyte Mesembryanthemum crystallinum.

PubMed

Barkla, Bronwyn J; Garibay-Hernández, Adriana; Melzer, Michael; Rupasinghe, Thusitha W T; Roessner, Ute

2018-05-29

Salt stress causes dramatic changes in the organization and dynamic properties of membranes, however, little is known about the underlying mechanisms involved. Modified trichomes, known as epidermal bladder cells (EBC), on the leaves and stems of the halophyte Mesembryanthemum crystallinum can be successfully exploited as a single-cell-type system to investigate salt-induced changes to cellular lipid composition. In this study alterations in key molecular species from different lipid classes highlighted an increase in phospholipid species, particularly those from phosphatidylcholine (PC) and phosphatidic acid (PA), where the latter is central to the synthesis of membrane lipids. Triacylglycerol (TG) species decreased during salinity, while there was little change in plastidic galactolipids. EBC transcriptomic and proteomic data mining revealed changes in genes and proteins involved in lipid metabolism and the upregulation of transcripts for PIPKIB, PI5PII, PIPKIII, and PLDδ, suggested the induction of signalling processes mediated by phosphoinositides and PA. TEM and flow cytometry showed the dynamic nature of lipid droplets in these cells under salt stress. Altogether, this work indicates the metabolism of TG might play an important role in EBC response to salinity as either an energy reserve for sodium accumulation and/or driving membrane biosynthesis for EBC expansion. This article is protected by copyright. All rights reserved.

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells.

PubMed

Banáth, J P; Bañuelos, C A; Klokov, D; MacPhail, S M; Lansdorp, P M; Olive, P L

2009-05-01

Pluripotent mouse embryonic stem cells (mES cells) exhibit approximately 100 large gammaH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gammaH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gammaH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gammaH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gammaH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media.

PubMed

Kelley, Rebecca L; Gardner, David K

2017-05-01

Single embryo culture is suboptimal compared with group culture, but necessary for embryo monitoring, and culture systems should be improved for single embryos. Pronucleate mouse embryos were used to assess the effect of culture conditions on single embryo development. Single culture either before or after compaction reduced cell numbers (112.2 ± 3.1; 110.2 ± 3.5) compared with group culture throughout (127.0 ± 3.4; P < 0.05). Reduction of media volume from 20 µl to 2 µl increased blastocyst cell numbers in single embryos cultured in 5% oxygen (84.4 ± 3.2 versus 97.8 ± 2.8; P < 0.05), but not in 20% oxygen (55.2 ± 2.9 versus 57.1 ± 2.8). Culture in microwell plates for the EmbryoScope and Primo Vision time-lapse systems changed cleavage timings and increased inner cell mass cell number (24.1 ± 1.0; 23.4 ± 1.2) compared with a 2 µl microdrop (18.4 ± 1.0; P < 0.05). Addition of embryo-conditioned media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P < 0.01). Single culture before or after compaction is therefore detrimental; oxygen, media volume and microwells influence single embryo development; and embryo-conditioned media may substitute for group culture. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Visible light-sensitive APTES-bound ZnO nanowire toward a potent nanoinjector sensing biomolecules in a living cell

NASA Astrophysics Data System (ADS)

Lee, Jooran; Choi, Sunyoung; Bae, Seon Joo; Yoon, Seok Min; Choi, Joon Sig; Yoon, Minjoong

2013-10-01

Nanoscale cell injection techniques combined with nanoscopic photoluminescence (PL) spectroscopy have been important issues in high-resolution optical biosensing, gene and drug delivery and single-cell endoscopy for medical diagnostics and therapeutics. However, the current nanoinjectors remain limited for optical biosensing and communication at the subwavelength level, requiring an optical probe such as semiconductor quantum dots, separately. Here, we show that waveguided red emission is observed at the tip of a single visible light-sensitive APTES-modified ZnO nanowire (APTES-ZnO NW) and it exhibits great enhancement upon interaction with a complementary sequence-based double stranded (ds) DNA, whereas it is not significantly affected by non-complementary ds DNA. Further, the tip of a single APTES-ZnO NW can be inserted into the subcellular region of living HEK 293 cells without significant toxicity, and it can also detect the enhancement of the tip emission from subcellular regions with high spatial resolution. These results indicate that the single APTES-ZnO NW would be useful as a potent nanoinjector which can guide visible light into intracellular compartments of mammalian cells, and can also detect nanoscopic optical signal changes induced by interaction with the subcellular specific target biomolecules without separate optical probes.Nanoscale cell injection techniques combined with nanoscopic photoluminescence (PL) spectroscopy have been important issues in high-resolution optical biosensing, gene and drug delivery and single-cell endoscopy for medical diagnostics and therapeutics. However, the current nanoinjectors remain limited for optical biosensing and communication at the subwavelength level, requiring an optical probe such as semiconductor quantum dots, separately. Here, we show that waveguided red emission is observed at the tip of a single visible light-sensitive APTES-modified ZnO nanowire (APTES-ZnO NW) and it exhibits great enhancement upon interaction with a complementary sequence-based double stranded (ds) DNA, whereas it is not significantly affected by non-complementary ds DNA. Further, the tip of a single APTES-ZnO NW can be inserted into the subcellular region of living HEK 293 cells without significant toxicity, and it can also detect the enhancement of the tip emission from subcellular regions with high spatial resolution. These results indicate that the single APTES-ZnO NW would be useful as a potent nanoinjector which can guide visible light into intracellular compartments of mammalian cells, and can also detect nanoscopic optical signal changes induced by interaction with the subcellular specific target biomolecules without separate optical probes. Electronic supplementary information (ESI) available: Synthesis of APTES-modified ZnO nanowires, DNA functionalization and spectroscopic measurements with additional fluorescence image ad fluorescence decay times, cell culture, injection of a single nanowire into living cells, subcellular imaging and determination of cytotoxicity. See DOI: 10.1039/c3nr03042c

Parallel changes in intracellular water volume and pH induced by NH(3)/NH(4)(+) exposure in single neuroblastoma cells.

PubMed

Blanco, Víctor M; Márquez, Martín S; Alvarez-Leefmans, Francisco J

2013-01-01

Increased blood levels of ammonia (NH3) and ammonium (NH4(+)), i.e. hyperammonemia, leads to cellular brain edema in humans with acute liver failure. The pathophysiology of this edema is poorly understood. This is partly due to incomplete understanding of the osmotic effects of the pair NH3/NH4(+) at the cellular and molecular levels. Cell exposure to solutions containing NH3/NH4(+) elicits changes in intracellular pH (pHi), which can in turn affect cell water volume (CWV) by activating transport mechanisms that produce net gain or loss of solutes and water. The occurrence of CWV changes caused by NH3/NH4(+) has long been suspected, but the mechanisms, magnitude and kinetics of these changes remain unknown. Using fluorescence imaging microscopy we measured, in real time, parallel changes in pHi and CWV caused by brief exposure to NH3/NH4(+) of single cells (N1E-115 neuroblastoma or NG-108 neuroblastoma X glioma ) loaded with the fluorescent indicator BCECF. Changes in CWV were measured by exciting BCECF at its intracellular isosbestic wavelength (∼438 nm), and pHi was measured ratiometrically. Brief exposure to isosmotic solutions (i.e. having the same osmolality as that of control solutions) containing NH4Cl (0.5- 30 mM) resulted in a rapid, dose-dependent swelling, followed by isosmotic regulatory volume decrease (iRVD). NH4Cl solutions in which either extracellular [NH3] or [NH4(+)] was kept constant while the other was changed by varying the pH of the solution, demonstrated that [NH3]o rather than [NH4(+)]o is the main determinant of the NH4Cl-induced swelling. The iRVD response was sensitive to the anion channel blocker NPPB, and partly dependent on external Ca(2+). Upon removal of NH4Cl, cells shrank and displayed isosmotic regulatory volume increase (iRVI). Regulatory volume responses could not be activated by comparable CWV changes produced by anisosmotic solutions, suggesting that membrane stretch or contraction by themselves are not sufficient to trigger these responses. Inhibition of glutamine synthetase partially blocked the NH4Cl-induced swelling. A quantitative description of the osmotic changes produced by exposure to NH3/NH4(+) in single neurons and glial cells shows that ∼35 to 45% of the initial cell swelling can be explained by intracellular accumulation of NH4(+) due to rapid permeation and protonation of NH3. Another∼23% of the swelling can be accounted for by rapid glutamine accumulation. The results are discussed in terms of basic cell physiology and their potential relevance to the pathophysiology of hyperammonemic cellular brain edema. © 2014 S. Karger AG, Basel.

Sensitivity of continental United States atmospheric budgets of oxidized and reduced nitrogen to dry deposition parametrizations

PubMed Central

Dennis, Robin L.; Schwede, Donna B.; Bash, Jesse O.; Pleim, Jon E.; Walker, John T.; Foley, Kristen M.

2013-01-01

Reactive nitrogen (Nr) is removed by surface fluxes (air–surface exchange) and wet deposition. The chemistry and physics of the atmosphere result in a complicated system in which competing chemical sources and sinks exist and impact that removal. Therefore, uncertainties are best examined with complete regional chemical transport models that simulate these feedbacks. We analysed several uncertainties in regional air quality model resistance analogue representations of air–surface exchange for unidirectional and bi-directional fluxes and their effect on the continental Nr budget. Model sensitivity tests of key parameters in dry deposition formulations showed that uncertainty estimates of continental total nitrogen deposition are surprisingly small, 5 per cent or less, owing to feedbacks in the chemistry and rebalancing among removal pathways. The largest uncertainties (5%) occur with the change from a unidirectional to a bi-directional NH3 formulation followed by uncertainties in bi-directional compensation points (1–4%) and unidirectional aerodynamic resistance (2%). Uncertainties have a greater effect at the local scale. Between unidirectional and bi-directional formulations, single grid cell changes can be up to 50 per cent, whereas 84 per cent of the cells have changes less than 30 per cent. For uncertainties within either formulation, single grid cell change can be up to 20 per cent, but for 90 per cent of the cells changes are less than 10 per cent. PMID:23713122

Sensitivity of continental United States atmospheric budgets of oxidized and reduced nitrogen to dry deposition parametrizations.

PubMed

Dennis, Robin L; Schwede, Donna B; Bash, Jesse O; Pleim, Jon E; Walker, John T; Foley, Kristen M

2013-07-05

Reactive nitrogen (Nr) is removed by surface fluxes (air-surface exchange) and wet deposition. The chemistry and physics of the atmosphere result in a complicated system in which competing chemical sources and sinks exist and impact that removal. Therefore, uncertainties are best examined with complete regional chemical transport models that simulate these feedbacks. We analysed several uncertainties in regional air quality model resistance analogue representations of air-surface exchange for unidirectional and bi-directional fluxes and their effect on the continental Nr budget. Model sensitivity tests of key parameters in dry deposition formulations showed that uncertainty estimates of continental total nitrogen deposition are surprisingly small, 5 per cent or less, owing to feedbacks in the chemistry and rebalancing among removal pathways. The largest uncertainties (5%) occur with the change from a unidirectional to a bi-directional NH3 formulation followed by uncertainties in bi-directional compensation points (1-4%) and unidirectional aerodynamic resistance (2%). Uncertainties have a greater effect at the local scale. Between unidirectional and bi-directional formulations, single grid cell changes can be up to 50 per cent, whereas 84 per cent of the cells have changes less than 30 per cent. For uncertainties within either formulation, single grid cell change can be up to 20 per cent, but for 90 per cent of the cells changes are less than 10 per cent.

Long term effects of lipopolysaccharide on satellite glial cells in mouse dorsal root ganglia

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

Blum, E.; Procacci, P.; Conte, V.

Lipopolysaccharide (LPS) has been used extensively to study neuroinflammation, but usually its effects were examined acutely (24 h<). We have shown previously that a single intraperitoneal LPS injection activated satellite glial cells (SGCs) in mouse dorsal root ganglia (DRG) and altered several functional parameters in these cells for at least one week. Here we asked whether the LPS effects would persist for 1 month. We injected mice with a single LPS dose and tested pain behavior, assessed SGCs activation in DRG using glial fibrillary acidic protein (GFAP) immunostaining, and injected a fluorescent dye intracellularly to study intercellular coupling. Electron microscopymore » was used to quantitate changes in gap junctions. We found that at 30 days post-LPS the threshold to mechanical stimulation was lower than in controls. GFAP expression, as well as the magnitude of dye coupling among SGCs were greater than in controls. Electron microscopy analysis supported these results, showing a greater number of gap junctions and an abnormal growth of SGC processes. These changes were significant, but less prominent than at 7 days post-LPS. We conclude that a single LPS injection exerts long-term behavioral and cellular changes. The results are consistent with the idea that SGC activation contributes to hyperalgesia. - Highlights: • A single lipopolysaccharides injection activated glia in mouse dorsal root ganglia for 30 days. • This was accompanied by increased communications by gap junctions among glia and by hyperalgesia. • Glial activation and coupling may contribute to chronic pain.« less

The Impact of Non-Lethal Single-Dose Radiation on Tumor Invasion and Cytoskeletal Properties.

PubMed

Hohmann, Tim; Grabiec, Urszula; Vogel, Carolin; Ghadban, Chalid; Ensminger, Stephan; Bache, Matthias; Vordermark, Dirk; Dehghani, Faramarz

2017-09-18

Irradiation is the standard therapy for glioblastoma multiforme. Glioblastoma are highly resistant to radiotherapy and the underlying mechanisms remain unclear. To better understand the biological effects of irradiation on glioblastoma cells, we tested whether nonlethal irradiation influences the invasiveness, cell stiffness, and actin cytoskeleton properties. Two different glioblastoma cell lines were irradiated with 2 Gy and changes in mechanical and migratory properties and alterations in the actin structure were measured. The invasiveness of cell lines was determined using a co-culture model with organotypic hippocampal slice cultures. Irradiation led to changes in motility and a less invasive phenotype in both investigated cell lines that were associated with an increase in a "generalized stiffness" and changes in the actin structure. In this study we demonstrate that irradiation can induce changes in the actin cytoskeleton and motility, which probably results in reduced invasiveness of glioblastoma cell lines. Furthermore, "generalized stiffness" was shown to be a profound marker of the invasiveness of a tumor cell population in our model.

Chemotaxis of Dictyostelium discoideum: Collective Oscillation of Cellular Contacts

PubMed Central

Schäfer, Edith; Tarantola, Marco; Polo, Elena; Westendorf, Christian; Oikawa, Noriko; Bodenschatz, Eberhard; Geil, Burkhard; Janshoff, Andreas

2013-01-01

Chemotactic responses of Dictyostelium discoideum cells to periodic self-generated signals of extracellular cAMP comprise a large number of intricate morphological changes on different length scales. Here, we scrutinized chemotaxis of single Dictyostelium discoideum cells under conditions of starvation using a variety of optical, electrical and acoustic methods. Amebas were seeded on gold electrodes displaying impedance oscillations that were simultaneously analyzed by optical video microscopy to relate synchronous changes in cell density, morphology, and distance from the surface to the transient impedance signal. We found that starved amebas periodically reduce their overall distance from the surface producing a larger impedance and higher total fluorescence intensity in total internal reflection fluorescence microscopy. Therefore, we propose that the dominant sources of the observed impedance oscillations observed on electric cell-substrate impedance sensing electrodes are periodic changes of the overall cell-substrate distance of a cell. These synchronous changes of the cell-electrode distance were also observed in the oscillating signal of acoustic resonators covered with amebas. We also found that periodic cell-cell aggregation into transient clusters correlates with changes in the cell-substrate distance and might also contribute to the impedance signal. It turned out that cell-cell contacts as well as cell-substrate contacts form synchronously during chemotaxis of Dictyostelium discoideum cells. PMID:23349816

Single-Cell Growth Rates in Photoautotrophic Populations Measured by Stable Isotope Probing and Resonance Raman Microspectrometry

PubMed Central

Taylor, Gordon T.; Suter, Elizabeth A.; Li, Zhuo Q.; Chow, Stephanie; Stinton, Dallyce; Zaliznyak, Tatiana; Beaupré, Steven R.

2017-01-01

A new method to measure growth rates of individual photoautotrophic cells by combining stable isotope probing (SIP) and single-cell resonance Raman microspectrometry is introduced. This report explores optimal experimental design and the theoretical underpinnings for quantitative responses of Raman spectra to cellular isotopic composition. Resonance Raman spectra of isogenic cultures of the cyanobacterium, Synechococcus sp., grown in 13C-bicarbonate revealed linear covariance between wavenumber (cm−1) shifts in dominant carotenoid Raman peaks and a broad range of cellular 13C fractional isotopic abundance. Single-cell growth rates were calculated from spectra-derived isotopic content and empirical relationships. Growth rates among any 25 cells in a sample varied considerably; mean coefficient of variation, CV, was 29 ± 3% (σ/x¯), of which only ~2% was propagated analytical error. Instantaneous population growth rates measured independently by in vivo fluorescence also varied daily (CV ≈ 53%) and were statistically indistinguishable from single-cell growth rates at all but the lowest levels of cell labeling. SCRR censuses of mixtures prepared from Synechococcus sp. and T. pseudonana (a diatom) populations with varying 13C-content and growth rates closely approximated predicted spectral responses and fractional labeling of cells added to the sample. This approach enables direct microspectrometric interrogation of isotopically- and phylogenetically-labeled cells and detects as little as 3% changes in cellular fractional labeling. This is the first description of a non-destructive technique to measure single-cell photoautotrophic growth rates based on Raman spectroscopy and well-constrained assumptions, while requiring few ancillary measurements. PMID:28824580

Process change evaluation framework for allogeneic cell therapies: impact on drug development and commercialization.

PubMed

Hassan, Sally; Huang, Hsini; Warren, Kim; Mahdavi, Behzad; Smith, David; Jong, Simcha; Farid, Suzanne S

2016-04-01

Some allogeneic cell therapies requiring a high dose of cells for large indication groups demand a change in cell expansion technology, from planar units to microcarriers in single-use bioreactors for the market phase. The aim was to model the optimal timing for making this change. A development lifecycle cash flow framework was created to examine the implications of process changes to microcarrier cultures at different stages of a cell therapy's lifecycle. The analysis performed under assumptions used in the framework predicted that making this switch earlier in development is optimal from a total expected out-of-pocket cost perspective. From a risk-adjusted net present value view, switching at Phase I is economically competitive but a post-approval switch can offer the highest risk-adjusted net present value as the cost of switching is offset by initial market penetration with planar technologies. The framework can facilitate early decision-making during process development.

Apparatus and method for measuring single cell and sub-cellular photosynthetic efficiency

DOEpatents

Davis, Ryan Wesley; Singh, Seema; Wu, Huawen

2013-07-09

Devices for measuring single cell changes in photosynthetic efficiency in algal aquaculture are disclosed that include a combination of modulated LED trans-illumination of different intensities with synchronized through objective laser illumination and confocal detection. Synchronization and intensity modulation of a dual illumination scheme were provided using a custom microcontroller for a laser beam block and constant current LED driver. Therefore, single whole cell photosynthetic efficiency, and subcellular (diffraction limited) photosynthetic efficiency measurement modes are permitted. Wide field rapid light scanning actinic illumination is provided for both by an intensity modulated 470 nm LED. For the whole cell photosynthetic efficiency measurement, the same LED provides saturating pulses for generating photosynthetic induction curves. For the subcellular photosynthetic efficiency measurement, a switched through objective 488 nm laser provides saturating pulses for generating photosynthetic induction curves. A second near IR LED is employed to generate dark adapted states in the system under study.

Characteristics of single Ca(2+) channel kinetics in feline hypertrophied ventricular myocytes.

PubMed

Yang, Xiangjun; Hui, Jie; Jiang, Tingbo; Song, Jianping; Liu, Zhihua; Jiang, Wenping

2002-04-01

To explore the mechanism underlying the prolongation of action potential and delayed inactivation of the L-type Ca(2+) (I(Ca, L)) current in a feline model of left ventricular system hypertension and concomitant hypertrophy. Single Ca(2+) channel properties in myocytes isolated from normal and pressure overloaded cat left ventricles were studied, using patch-clamp techniques. Left ventricular pressure overload was induced by partial ligation of the ascending aorta for 4 - 6 weeks. The amplitude of single Ca(2+) channel current evoked by depolarizing pulses from -40 mV to 0 mV was 1.02 +/- 0.03 pA in normal cells and 1.05 +/- 0.03 pA in hypertrophied cells, and there was no difference in single channel current-voltage relationships between the groups since slope conductance was 26.2 +/- 1.0 pS in normal and hypertrophied cells, respectively. Peak amplitudes of the ensemble-averaged single Ca(2+) channel currents were not different between the two groups of cells. However, the amplitude of this averaged current at the end of the clamp pulse was significantly larger in hypertrophied cells than in normal cells. Open-time histograms revealed that open-time distribution was fitted by a single exponential function in channels of normal cells and by a two exponential function in channels of hypertrophied cells. The number of long-lasting openings was increased in channels of hypertrophied cells, and therefore the calculated mean open time of the channel was significantly longer compared to normal controls. Kinetic changes in the Ca(2+) channel may underlie both hypertrophy-associated delayed inactivation of the Ca(2+) current and, in part, the pressure overload-induced action potential lengthening in this cat model of ventricular left systolic hypertension and hypertrophy.

Investigation of Ruthenium Dissolution in Advanced Membrane Electrode Assemblies for Direct Methanol Based Fuel Cell Stacks

NASA Technical Reports Server (NTRS)

Valdez, Thomas I.; Firdosy, S.; Koel, B. E.; Narayanan, S. R.

2005-01-01

Dissolution of ruthenium was observed in the 80-cell stack. Duration testing was performed in single cell MEAs to determine the pathway of cell degradation. EDAX analysis on each of the single cell MEAs has shown that the Johnson Matthey commercial catalyst is stable in DMFC operation for 250 hours, no ruthenium dissolution was observed. Changes in the hydrophobicity of the cathode backing papers was minimum. Electrode polarization analysis revealed that the MEA performance loss is attributed to changes in the cathode catalyst layer. Ruthenium migration does not seem to occur during cell operation but can occur when methanol is absent from the anode compartment, the cathode compartment has access to air, and the cells in the stack are electrically connected to a load (Shunt Currents). The open-to-air cathode stack design allowed for: a) The MEAs to have continual access to oxygen; and b) The stack to sustain shunt currents. Ruthenium dissolution in a DMFC stack can be prevented by: a) Developing an internally manifolded stacks that seal reactant compartments when not in operation; b) Bringing the cell voltages to zero quickly when not in operation; and c) Limiting the total number of cells to 25 in an effort to limit shunt currents.

Accurate analytic solution of chemical master equations for gene regulation networks in a single cell

NASA Astrophysics Data System (ADS)

Huang, Guan-Rong; Saakian, David B.; Hu, Chin-Kun

2018-01-01

Studying gene regulation networks in a single cell is an important, interesting, and hot research topic of molecular biology. Such process can be described by chemical master equations (CMEs). We propose a Hamilton-Jacobi equation method with finite-size corrections to solve such CMEs accurately at the intermediate region of switching, where switching rate is comparable to fast protein production rate. We applied this approach to a model of self-regulating proteins [H. Ge et al., Phys. Rev. Lett. 114, 078101 (2015), 10.1103/PhysRevLett.114.078101] and found that as a parameter related to inducer concentration increases the probability of protein production changes from unimodal to bimodal, then to unimodal, consistent with phenotype switching observed in a single cell.

Continuous bioluminescent monitoring of cytoplasmic ATP in single isolated rat hepatocytes during metabolic poisoning.

PubMed Central

Koop, A; Cobbold, P H

1993-01-01

We have devised a technique for monitoring cytoplasmic ATP continuously in single hepatocytes. Single isolated rat hepatocytes were injected with the ATP-dependent luminescent protein firefly luciferase, and then superfused with 45 microM luciferin in air-equilibrated medium. Signals of approx. 10-200 photoelectron counts per second could be recorded from individual healthy cells for up to 3 h. The response of the luminescent signal to chemical hypoxia (2-5 mM CN- and 5-10 mM 2-deoxyglucose) was monitored. We found a great cell-to-cell variability in the time course of the ATP decline in response to CN-, 2-deoxyglucose or to their combination; the time for the signal to fall to 10% of the original (corresponding to approx. 100 microM ATP) ranged from approx. 20 to 75 min. This resistance of the cytoplasmic ATP concentration to depletion after blockade of oxidative phosphorylation and glycolysis could be abolished by pretreatment of the cells with etomoxir, which blocks mitochondrial beta-oxidation. Etomoxir alone had no effect on the luciferase signal, but etomoxir-pre-treated cells showed a prompt fall in the luciferase signal starting within 1-2 min of application of cyanide and 2-deoxyglucose and falling to 10% of the original signal in approx. 6-10 min. The technique allows cytoplasmic ATP changes to be monitored in single hepatocytes at concentrations of 1 mM or lower, but more precise calibration of the signal will require correction for the effects of cytoplasmic pH changes. PMID:8216212

Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution

PubMed Central

Ludlow, Andrew T.; Robin, Jerome D.; Sayed, Mohammed; Litterst, Claudia M.; Shelton, Dawne N.; Shay, Jerry W.; Wright, Woodring E.

2014-01-01

The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect ∼2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase. PMID:24861623

The effect of a histone deacetylase inhibitor - valproic acid - on nucleoli in human leukaemic myeloblasts.

PubMed

Smetana, K; Zápotocký, M

2010-01-01

The present study was undertaken to provide more information on nucleolar changes induced by a histone deacetylase inhibitor such as valproic acid in leukaemic myeloblasts at the single-cell level. For this study, RNA in nucleoli was visualized by a simple but sensitive cytochemical procedure in unfixed cytospins of short-term bone marrow cultures from patients suffering from acute myeloid leukaemia. Valproic acid in leukaemic myeloblasts markedly reduced the nucleolar size and also produced significant transformation of "active" to "resting" and "inactive" nucleoli that reflected the alteration of the nucleolar transcription in sensitive myeloblasts. On this occasion it should be added that valproic acid significantly increased the incidence of altered myeloblasts that changed to apoptotic cells or apoptotic bodies and cell ghosts. In contrast to the above-mentioned decreased nucleolar size, the nucleolar RNA concentration, expressed by computerassisted RNA image densitometry in valproic acidtreated myeloblasts, was not significantly changed. The results of the present study clearly indicated that the nucleolar size and transformation of "active" to "sleeping" or "inactive" nucleoli are convenient markers of the sensitivity and alteration of leukaemic myeloblasts produced by a histone deacetylase inhibitor, valproic acid, at the single-cell level.

Exacerbated in vivo metabolic changes suggestive of a spontaneous muscular vaso-occlusive crisis in exercising muscle of a sickle cell mouse.

PubMed

Chatel, Benjamin; Messonnier, Laurent A; Bendahan, David

2017-06-01

While sickle cell disease (SCD) is characterized by frequent vaso-occlusive crisis (VOC), no direct observation of such an event in skeletal muscle has been performed in vivo. The present study reported exacerbated in vivo metabolic changes suggestive of a spontaneous muscular VOC in exercising muscle of a sickle cell mouse. Using magnetic resonance spectroscopy of phosphorus 31, phosphocreatine and inorganic phosphate concentrations and intramuscular pH were measured throughout two standardized protocols of rest - exercise - recovery at two different intensities in ten SCD mice. Among these mice, one single mouse presented divergent responses. A statistical analysis (based on confidence intervals) revealed that this single mouse presented slower phosphocreatine resynthesis and inorganic phosphate disappearance during the post-stimulation recovery of one of the protocols, what could suggest an ischemia. This study described, for the first time in a sickle cell mouse in vivo, exacerbated metabolic changes triggered by an exercise session that would be suggestive of a live observation of a muscular VOC. However, no evidence of a direct cause-effect relationship between exercise and VOC has been put forth. Copyright © 2017 Elsevier Inc. All rights reserved.

The pathological anatomy of acute experimental baryllium poisoning peripheral blood changes resulting from intravenous administration of beryllium sulphate

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

Scott, J.E.

1947-09-01

The lesions produced in the organs following a single intravenous administration of hydrated beryllium sulfate are described. The lesions of the lungs and eyes of animals exposed to the above compound are reviewed. When the baryllium sulfate is given intravenously, midsonal focal necrosis of the liver cells, necrosis of cells of the distal one-third of the proximal convoluted tubules of the kidney and generative changes in the cells of the hemopoietic system are produced. Following exposure of animals to beryllium sulfate dust (100 mg/m{sup 3}, 8 hours daily for eleven days), inflammatory pulmonary lesions are produced which vary in intensitymore » with different species. Pulmonary edema, a terminal bronchitis, and focal atelectasis are the most commonly observed lesions. The eyes of some species exposed to this dust develop conjunctivitis, heratitis, and corneal ulcers. Following a single intravenous administration of beryllium sulfate, rather sharp changes occur in the elements of the peripheral blood. These consist of a secondary anemia (probably resulting from intravascular lysis of red cells), a leukocytosis, and an increase in the number of circulating platelets. 29 figs.« less

Stress Modulus of Cancer Cells

NASA Astrophysics Data System (ADS)

Bonin, Keith; Guthold, Martin; Guo, Xinyi; Sigley, Justin

2012-02-01

Our main goal is to study the different physical and mechanical properties of cells as they advance through different stages of neoplastic transformation from normal to the metastatic state. Since recent reports indicate there is significant ambiguity about how these properties change for different cancer cells, we plan to measure these properties for a single line of cells, and to determine whether the changes vary for different cellular components: i.e. whether the change in physical properties is due to a change in the cytoskeleton, the cell membrane, the cytoplasm, or a combination of these elements. Here we expect to present data on the stress modulus of cancer cells at different stages: normal, mortal cancerous, immortal cancerous, and tumorigenic. The cells are Weinberg cell line Human Mammary Epithelial (HME) cells. Atomic force microscope (AFM) probes with different diameters are used to push on the cell membrane to measure the local, regional and global cell stress modulus. Preliminary results on normal HME cells suggests a stress modulus of 1.5 ± 0.8 kPa when pushing with 7 μm spherical probes. We anticipate reporting an improved value for the modulus as well as results for some of the Weinberg cancer cells.

Spike-timing dependent plasticity in primate corticospinal connections induced during free behavior

PubMed Central

Nishimura, Yukio; Perlmutter, Steve I.; Eaton, Ryan W.; Fetz, Eberhard E.

2014-01-01

Motor learning and functional recovery from brain damage involve changes in the strength of synaptic connections between neurons. Relevant in vivo evidence on the underlying cellular mechanisms remains limited and indirect. We found that the strength of neural connections between motor cortex and spinal cord in monkeys can be modified with an autonomous recurrent neural interface that delivers electrical stimuli in the spinal cord triggered by action potentials of corticospinal cells during free behavior. The activity-dependent stimulation modified the strength of the terminal connections of single corticomotoneuronal cells, consistent with a bidirectional spike-timing dependent plasticity rule previously derived from in vitro experiments. For some cells the changes lasted for days after the end of conditioning, but most effects eventually reverted to preconditioning levels. These results provide the first direct evidence of corticospinal synaptic plasticity in vivo at the level of single neurons induced by normal firing patterns during free behavior. PMID:24210907

Peering into Cells One Molecule at a Time: Single-molecule and plasmon-enhanced fluorescence super-resolution imaging

NASA Astrophysics Data System (ADS)

Biteen, Julie

2013-03-01

Single-molecule fluorescence brings the resolution of optical microscopy down to the nanometer scale, allowing us to unlock the mysteries of how biomolecules work together to achieve the complexity that is a cell. This high-resolution, non-destructive method for examining subcellular events has opened up an exciting new frontier: the study of macromolecular localization and dynamics in living cells. We have developed methods for single-molecule investigations of live bacterial cells, and have used these techniques to investigate thee important prokaryotic systems: membrane-bound transcription activation in Vibrio cholerae, carbohydrate catabolism in Bacteroides thetaiotaomicron, and DNA mismatch repair in Bacillus subtilis. Each system presents unique challenges, and we will discuss the important methods developed for each system. Furthermore, we use the plasmon modes of bio-compatible metal nanoparticles to enhance the emissivity of single-molecule fluorophores. The resolution of single-molecule imaging in cells is generally limited to 20-40 nm, far worse than the 1.5-nm localization accuracies which have been attained in vitro. We use plasmonics to improve the brightness and stability of single-molecule probes, and in particular fluorescent proteins, which are widely used for bio-imaging. We find that gold-coupled fluorophores demonstrate brighter, longer-lived emission, yielding an overall enhancement in total photons detected. Ultimately, this results in increased localization accuracy for single-molecule imaging. Furthermore, since fluorescence intensity is proportional to local electromagnetic field intensity, these changes in decay intensity and rate serve as a nm-scale read-out of the field intensity. Our work indicates that plasmonic substrates are uniquely advantageous for super-resolution imaging, and that plasmon-enhanced imaging is a promising technique for improving live cell single-molecule microscopy.

Single Targeted Exon Mutation Creates a True Congenic Mouse for Competitive Hematopoietic Stem Cell Transplantation: The C57BL/6-CD45.1(STEM) Mouse.

PubMed

Mercier, Francois E; Sykes, David B; Scadden, David T

2016-06-14

Defining the molecular regulators of hematopoietic stem and progenitor cells (HSPCs) requires in vivo functional analyses. Competitive bone marrow transplants (BMTs) compare control and test HSPCs to demonstrate the functional role of a genetic change or chemical perturbation. Competitive BMT is enabled by antibodies that specifically recognize hematopoietic cells from congenic mouse strains due to variants of the cell surface protein CD45, designated CD45.1 and CD45.2. The current congenic competitor strain, B6.SJL-Ptprc(a) Pepc(b)/BoyJ (CD45.1), has a substantial inherent disadvantage in competition against the C57BL/6 (CD45.2) strain, confounding experimental interpretation. Despite backcrossing, the congenic interval over which the B6.SJL-Ptprc(a) Pepc(b)/BoyJ strain differs is almost 40 Mb encoding ∼300 genes. Here, we demonstrate that a single amino acid change determines the CD45.1 epitope. Further, we report on the single targeted exon mutant (STEM) mouse strain, CD45.1(STEM), which is functionally equivalent to CD45.2 cells in competitive BMT. This strain will permit the precise definition of functional roles for candidate genes using in vivo HSPC assays. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Biochemical signatures of in vitro radiation response in human lung, breast and prostate tumour cells observed with Raman spectroscopy

NASA Astrophysics Data System (ADS)

Matthews, Q.; Jirasek, A.; Lum, J. J.; Brolo, A. G.

2011-11-01

This work applies noninvasive single-cell Raman spectroscopy (RS) and principal component analysis (PCA) to analyze and correlate radiation-induced biochemical changes in a panel of human tumour cell lines that vary by tissue of origin, p53 status and intrinsic radiosensitivity. Six human tumour cell lines, derived from prostate (DU145, PC3 and LNCaP), breast (MDA-MB-231 and MCF7) and lung (H460), were irradiated in vitro with single fractions (15, 30 or 50 Gy) of 6 MV photons. Remaining live cells were harvested for RS analysis at 0, 24, 48 and 72 h post-irradiation, along with unirradiated controls. Single-cell Raman spectra were acquired from 20 cells per sample utilizing a 785 nm excitation laser. All spectra (200 per cell line) were individually post-processed using established methods and the total data set for each cell line was analyzed with PCA using standard algorithms. One radiation-induced PCA component was detected for each cell line by identification of statistically significant changes in the PCA score distributions for irradiated samples, as compared to unirradiated samples, in the first 24-72 h post-irradiation. These RS response signatures arise from radiation-induced changes in cellular concentrations of aromatic amino acids, conformational protein structures and certain nucleic acid and lipid functional groups. Correlation analysis between the radiation-induced PCA components separates the cell lines into three distinct RS response categories: R1 (H460 and MCF7), R2 (MDA-MB-231 and PC3) and R3 (DU145 and LNCaP). These RS categories partially segregate according to radiosensitivity, as the R1 and R2 cell lines are radioresistant (SF2 > 0.6) and the R3 cell lines are radiosensitive (SF2 < 0.5). The R1 and R2 cell lines further segregate according to p53 gene status, corroborated by cell cycle analysis post-irradiation. Potential radiation-induced biochemical response mechanisms underlying our RS observations are proposed, such as (1) the regulated synthesis and degradation of structured proteins and (2) the expression of anti-apoptosis factors or other survival signals. This study demonstrates the utility of RS for noninvasive radiobiological analysis of tumour cell radiation response, and indicates the potential for future RS studies designed to investigate, monitor or predict radiation response.

Cell cycle dependent changes in the plasma membrane organization of mammalian cells.

PubMed

Denz, Manuela; Chiantia, Salvatore; Herrmann, Andreas; Mueller, Peter; Korte, Thomas; Schwarzer, Roland

2017-03-01

Lipid membranes are major structural elements of all eukaryotic and prokaryotic organisms. Although many aspects of their biology have been studied extensively, their dynamics and lateral heterogeneity are still not fully understood. Recently, we observed a cell-to-cell variability in the plasma membrane organization of CHO-K1 cells (Schwarzer et al., 2014). We surmised that cell cycle dependent changes of the individual cells from our unsynchronized cell population account for this phenomenon. In the present study, this hypothesis was tested. To this aim, CHO-K1 cells were arrested in different cell cycle phases by chemical treatments, and the order of their plasma membranes was determined by various fluorescent lipid analogues using fluorescence lifetime imaging microscopy. Our experiments exhibit significant differences in the membrane order of cells arrested in the G2/M or S phase compared to control cells. Our single-cell analysis also enabled the specific selection of mitotic cells, which displayed a significant increase of the membrane order compared to the control. In addition, the lipid raft marker GPImYFP was used to study the lateral organization of cell cycle arrested cells as well as mitotic cells and freely cycling samples. Again, significant differences were found between control and arrested cells and even more pronounced between control and mitotic cells. Our data demonstrate a direct correlation between cell cycle progression and plasma membrane organization, underlining that cell-to-cell heterogeneities of membrane properties have to be taken into account in cellular studies especially at the single-cell level. Copyright © 2016 Elsevier B.V. All rights reserved.

Phloretin-induced changes of lipophilic ion transport across the plasma membrane of mammalian cells.

PubMed Central

Sukhorukov, V L; Kürschner, M; Dilsky, S; Lisec, T; Wagner, B; Schenk, W A; Benz, R; Zimmermann, U

2001-01-01

The adsorption of the hydrophobic anion [W(CO)(5)CN](-) to human lymphoid Jurkat cells gave rise to an additional anti-field peak in the rotational spectra of single cells, indicating that the cell membrane displayed a strong dielectric dispersion in the kilohertz to megahertz frequency range. The surface concentration of the adsorbed anion and its translocation rate constant between the two membrane boundaries could be evaluated from the rotation spectra of cells by applying the previously proposed mobile charge model. Similar single-cell electrorotation experiments were performed to examine the effect of phloretin, a dipolar molecule known to influence the dipole potential of membranes, on the transport of [W(CO)(5)CN](-) across the plasma membrane of mammalian cells. The adsorption of [W(CO)(5)CN](-) was significantly reduced by phloretin, which is in reasonable agreement with the known phloretin-induced effects on artificial and biological membranes. The IC(50) for the effect of phloretin on the transport parameters of the lipophilic ion was approximately 10 microM. The results of this study are consistent with the assumption that the binding of phloretin reduces the intrinsic dipole potential of the plasma membrane. The experimental approach developed here allows the quantification of intrinsic dipole potential changes within the plasma membrane of living cells. PMID:11463642

Identification of stepped changes of binding affinity during interactions between the disintegrin rhodostomin and integrin αIIbβ3 in living cells using optical tweezers

NASA Astrophysics Data System (ADS)

Hsieh, Chia-Fen; Chang, Bo-Jui; Pai, Chyi-Huey; Chen, Hsuan-Yi; Chi, Sien; Hsu, Long; Tsai, Jin-Wu; Lin, Chi-Hung

2004-10-01

Integrin receptors serve as both mechanical links and signal transduction mediators between the cell and its environment. Experimental evidence demonstrates that conformational changes and lateral clustering of the integrin proteins may affect their binding to ligands and regulate downstream cellular responses; however, experimental links between the structural and functional correlations of the ligand-receptor interactions are not yet elucidated. In the present report, we utilized optical tweezers to measure the dynamic binding between the snake venom rhodostomin, coated on a microparticle and functioned as a ligand, and the membrane receptor integrin alpha(IIb)beta(3) expressed on a Chinese Hamster Ovary (CHO) cell. A progressive increase of total binding affinity was found between the bead and CHO cell in the first 300 sec following optical tweezers-guided contact. Further analysis of the cumulative data revealed the presence of "unit binding force" presumably exerted by a single rhodostomin-integrin pair. Interestingly, two such units were found. Among the measurements of less total binding forces, presumably taken at the early stage of ligand-receptor interactions, a unit of 4.15 pN per molecule pair was derived. This unit force dropped to 2.54 pN per molecule pair toward the later stage of interactions when the total binding forces were relatively large. This stepped change of single molecule pair binding affinity was not found when mutant rhodostomin proteins were used as ligands (a single unit of 1.81 pN per pair was found). These results were interpreted along with the current knowledge about the conformational changes of integrins during the "molecule activation" process.

Neurons as sensors: individual and cascaded chemical sensing.

PubMed

Prasad, Shalini; Zhang, Xuan; Yang, Mo; Ozkan, Cengiz S; Ozkan, Mihrimah

2004-07-15

A single neuron sensor has been developed based on the interaction of gradient electric fields and the cell membrane. Single neurons are rapidly positioned over individual microelectrodes using positive dielectrophoretic traps. This enables the continuous extracellular electrophysiological measurements from individual neurons. The sensor developed using this technique provides the first experimental method for determining single cell sensitivity; the speed of response and the associated physiological changes to a broad spectrum of chemical agents. Binding of specific chemical agents to a specific combination of receptors induces changes to the extracellular membrane potential of a single neuron, which can be translated into unique "signature patterns" (SP), which function as identification tags. Signature patterns are derived using Fast Fourier Transformation (FFT) analysis and Wavelet Transformation (WT) analysis of the modified extracellular action potential. The validity and the sensitivity of the system are demonstrated for a variety of chemical agents ranging from behavior altering chemicals (ethanol), environmentally hazardous agents (hydrogen peroxide, EDTA) to physiologically harmful agents (pyrethroids) at pico- and femto-molar concentrations. The ability of a single neuron to selectively identify specific chemical agents when injected in a serial manner is demonstrated in "cascaded sensing".

A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells.

PubMed

Volovitz, Ilan; Shapira, Netanel; Ezer, Haim; Gafni, Aviv; Lustgarten, Merav; Alter, Tal; Ben-Horin, Idan; Barzilai, Ori; Shahar, Tal; Kanner, Andrew; Fried, Itzhak; Veshchev, Igor; Grossman, Rachel; Ram, Zvi

2016-06-01

Conducting research on the molecular biology, immunology, and physiology of brain tumors (BTs) and primary brain tissues requires the use of viably dissociated single cells. Inadequate methods for tissue dissociation generate considerable loss in the quantity of single cells produced and in the produced cells' viability. Improper dissociation may also demote the quality of data attained in functional and molecular assays due to the presence of large quantities cellular debris containing immune-activatory danger associated molecular patterns, and due to the increased quantities of degraded proteins and RNA. Over 40 resected BTs and non-tumorous brain tissue samples were dissociated into single cells by mechanical dissociation or by mechanical and enzymatic dissociation. The quality of dissociation was compared for all frequently used dissociation enzymes (collagenase, DNase, hyaluronidase, papain, dispase) and for neutral protease (NP) from Clostridium histolyticum. Single-cell-dissociated cell mixtures were evaluated for cellular viability and for the cell-mixture dissociation quality. Dissociation quality was graded by the quantity of subcellular debris, non-dissociated cell clumps, and DNA released from dead cells. Of all enzymes or enzyme combinations examined, NP (an enzyme previously not evaluated on brain tissues) produced dissociated cell mixtures with the highest mean cellular viability: 93 % in gliomas, 85 % in brain metastases, and 89 % in non-tumorous brain tissue. NP also produced cell mixtures with significantly less cellular debris than other enzymes tested. Dissociation using NP was non-aggressive over time-no changes in cell viability or dissociation quality were found when comparing 2-h dissociation at 37 °C to overnight dissociation at ambient temperature. The use of NP allows for the most effective dissociation of viable single cells from human BTs or brain tissue. Its non-aggressive dissociative capacity may enable ambient-temperature shipping of tumor pieces in multi-center clinical trials, meanwhile being dissociated. As clinical grade NP is commercially available it can be easily integrated into cell-therapy clinical trials in neuro-oncology. The high quality viable cells produced may enable investigators to conduct more consistent research by avoiding the experimental artifacts associated with the presence dead cells or cellular debris.

Rapid learning in visual cortical networks.

PubMed

Wang, Ye; Dragoi, Valentin

2015-08-26

Although changes in brain activity during learning have been extensively examined at the single neuron level, the coding strategies employed by cell populations remain mysterious. We examined cell populations in macaque area V4 during a rapid form of perceptual learning that emerges within tens of minutes. Multiple single units and LFP responses were recorded as monkeys improved their performance in an image discrimination task. We show that the increase in behavioral performance during learning is predicted by a tight coordination of spike timing with local population activity. More spike-LFP theta synchronization is correlated with higher learning performance, while high-frequency synchronization is unrelated with changes in performance, but these changes were absent once learning had stabilized and stimuli became familiar, or in the absence of learning. These findings reveal a novel mechanism of plasticity in visual cortex by which elevated low-frequency synchronization between individual neurons and local population activity accompanies the improvement in performance during learning.

Cell mechanics and human disease states

NASA Astrophysics Data System (ADS)

Suresh, Subra

2006-03-01

This presentation will provide summary of our very recent studies exploring the effects of biochemical factors, influenced by foreign organisms or in vivo processes, on intracellular structural reorganization, single-cell mechanical response and motility of a population of cells in the context of two human diseases: malaria induced by Plasmodium falciparum merozoites that invade red blood cells, and gastrointestinal cancer metastasis involving epithelial cells. In both cases, particular attention will be devoted to systematic changes induced in specific molecular species in response to controlled alterations in disease state. The role of critical proteins in influencing the mechanical response of human red bloods during the intra-erythrocytic development of P. falciparum merozoites has also been assessed quantitatively using specific protein knock-out experiments by recourse to gene inactivation methods. Single-cell mechanical response characterization entails such tools as optical tweezers and mechanical plate stretchers whereas cell motility assays and cell-population biorheology characterization involves microfluidic channels. The experimental studies are accompanied by three-dimensional computational simulations at the continuum and mesoscopic scales of cell deformation. An outcome of such combined experimental and computational biophysical studies is the realization of how chemical factors influence single-cell mechanical response, cytoadherence, the biorheology of a large population of cells through microchannels representative of in vivo conditions, and the onset and progression of disease states.

Water Triple-Point Comparisons: Plateau Averaging or Peak Value?

NASA Astrophysics Data System (ADS)

Steur, P. P. M.; Dematteis, R.

2014-04-01

With a certain regularity, national metrology institutes conduct comparisons of water triple-point (WTP) cells. The WTP is the most important fixed point for the International Temperature Scale of 1990 (ITS-90). In such comparisons, it is common practice to simply average all the single measured temperature points obtained on a single ice mantle. This practice is quite reasonable whenever the measurements show no time dependence in the results. Ever since the first Supplementary Information for the International Temperature Scale of 1990, published by the Bureau International des Poids et Mesures in Sèvres, it was strongly suggested to wait at least 1 day before taking measurements (now up to 10 days), in order for a newly created ice mantle to stabilize. This stabilization is accompanied by a change in temperature with time. A recent improvement in the sensitivity of resistance measurement enabled the Istituto Nazionale di Ricerca Metrologica to detect more clearly the (possible) change in temperature with time of the WTP on a single ice mantle, as for old borosilicate cells. A limited investigation was performed where the temperature of two cells was monitored day-by-day, from the moment of mantle creation, where it was found that with (old) borosilicate cells it may be counterproductive to wait the usual week before starting measurements. The results are presented and discussed, and it is suggested to adapt the standard procedure for comparisons of WTP cells allowing for a different data treatment with (old) borosilicate cells, because taking the temperature dependence into account will surely reduce the reported differences between cells.

Single or group housing altered hormonal physiology and affected pituitary and interstitial cell kinetics

EPA Science Inventory

A significant negative correlation between testicular interstitial cell tumors and pituitary tumors in control male F344 rats has been reported associated with the number of animals per cage. Change in numbers of animals per cage may cause stress and increased serum corticosteroi...

Cancer cell metabolism: one hallmark, many faces.

PubMed

Cantor, Jason R; Sabatini, David M

2012-10-01

Cancer cells must rewire cellular metabolism to satisfy the demands of growth and proliferation. Although many of the metabolic alterations are largely similar to those in normal proliferating cells, they are aberrantly driven in cancer by a combination of genetic lesions and nongenetic factors such as the tumor microenvironment. However, a single model of altered tumor metabolism does not describe the sum of metabolic changes that can support cell growth. Instead, the diversity of such changes within the metabolic program of a cancer cell can dictate by what means proliferative rewiring is driven, and can also impart heterogeneity in the metabolic dependencies of the cell. A better understanding of this heterogeneity may enable the development and optimization of therapeutic strategies that target tumor metabolism.

Performance evaluation of thermally treated graphite felt electrodes for vanadium redox flow battery and their four-point single cell characterization

NASA Astrophysics Data System (ADS)

Mazúr, P.; Mrlík, J.; Beneš, J.; Pocedič, J.; Vrána, J.; Dundálek, J.; Kosek, J.

2018-03-01

In our contribution we study the electrocatalytic effect of oxygen functionalization of thermally treated graphite felt on kinetics of electrode reactions of vanadium redox flow battery. Chemical and morphological changes of the felts are analysed by standard physico-chemical characterization techniques. A complex method four-point method is developed and employed for characterization of the felts in a laboratory single-cell. The method is based on electrochemical impedance spectroscopy and load curves measurements of positive and negative half-cells using platinum wire pseudo-reference electrodes. The distribution of ohmic and faradaic losses within a single-cell is evaluated for both symmetric and asymmetric electrode set-up with respect to the treatment conditions. Positive effect of oxygen functionalization is observed only for negative electrode, whereas kinetics of positive electrode reaction is almost unaffected by the treatment. This is in a contradiction to the results of typically employed cyclovoltammetric characterization which indicate that both electrodes are enhanced by the treatment to a similar extent. The developed four-point characterization method can be further used e.g., for the component screening and in-situ durability studies on single-cell scale redox flow batteries of various chemistries.

Quantifying Hydrostatic Pressure in Plant Cells by Using Indentation with an Atomic Force Microscope

PubMed Central

Beauzamy, Léna; Derr, Julien; Boudaoud, Arezki

2015-01-01

Plant cell growth depends on a delicate balance between an inner drive—the hydrostatic pressure known as turgor—and an outer restraint—the polymeric wall that surrounds a cell. The classical technique to measure turgor in a single cell, the pressure probe, is intrusive and cannot be applied to small cells. In order to overcome these limitations, we developed a method that combines quantification of topography, nanoindentation force measurements, and an interpretation using a published mechanical model for the pointlike loading of thin elastic shells. We used atomic force microscopy to estimate the elastic properties of the cell wall and turgor pressure from a single force-depth curve. We applied this method to onion epidermal peels and quantified the response to changes in osmolality of the bathing solution. Overall our approach is accessible and enables a straightforward estimation of the hydrostatic pressure inside a walled cell. PMID:25992723

New technologies for the human cytome project.

PubMed

Tárnok, A

2004-01-01

Cytomes or cell systems are composed of various kinds of single-cells and constitute the elementary building units of organs and organisms. Their individualised (cytomic) analysis overcomes the problem of averaged results from cell and tissue homogenates where molecular changes in low frequency cell populations may be hidden and wrongly interpreted. Analysis of the cytome is of pivotal importance in basic research for the understanding of cells and their interrelations in complex environments like tissues and in predictive medicine where it is a prerequisite for individualised preventive therapy. Analysis of molecular phenotypes requires instrumentation that on the one hand provides high-throughput measurement of individual cells and is on the other hand highly multiplexed, enabling the simultaneous acquisition of many parameters on the single cell level. Upcoming technology suitable to this task, such as slide based cytometry is available or under development. The realisation of cytomic technology is important for the realisation of the human cytome project.

Real-time Molecular Study of Bystander Effects of Low dose Low LET radiation Using Living Cell Imaging and Nanoparticale Optics

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

Natarajan, Mohan; Xu, Nancy R; Mohan, Sumathy

2013-06-03

In this study two novel approaches are proposed to investigate precisely the low dose low LET radiation damage and its effect on bystander cells in real time. First, a flow shear model system, which would provide us a near in vivo situation where endothelial cells in the presence of extra cellular matrix experiencing continuous flow shear stress, will be used. Endothelial cells on matri-gel (simulated extra cellular matrix) will be subjected to physiological flow shear (that occurs in normal blood vessels). Second, a unique tool (Single nano particle/single live cell/single molecule microscopy and spectroscopy; Figure A) will be used tomore » track the molecular trafficking by single live cell imaging. Single molecule chemical microscopy allows one to single out and study rare events that otherwise might be lost in assembled average measurement, and monitor many target single molecules simultaneously in real-time. Multi color single novel metal nanoparticle probes allow one to prepare multicolor probes (Figure B) to monitor many single components (events) simultaneously and perform multi-complex analysis in real-time. These nano-particles resist to photo bleaching and hence serve as probes for unlimited timeframe of analysis. Single live cell microscopy allows one to image many single cells simultaneously in real-time. With the combination of these unique tools, we will be able to study under near-physiological conditions the cellular and sub-cellular responses (even subtle changes at one molecule level) to low and very low doses of low LET radiation in real time (milli-second or nano-second) at sub-10 nanometer spatial resolution. This would allow us to precisely identify, at least in part, the molecular mediators that are responsible of radiation damage in the irradiated cells and the mediators that are responsible for initiating the signaling in the neighboring cells. Endothelial cells subjected to flow shear (2 dynes/cm2 or 16 dynes/cm2) and exposed to 0.1, 1 and 10 cGy on coverslips will be examined for (a) low LET radiation-induced alterations of cellular function and its physiological relevance in real time; and (b) radiation damage triggered bystander effect on the neighboring unirradiated cells. First, to determine the low LET radiation induced alteration of cellular function we will examine: (i) the real time transformation of single membrane transporters in single living cells; (ii) the pump efficiency of membrane efflux pump of live cells in real time at the molecular level; (iii) the kinetics of single-ligand receptor interaction on single live cell surface (Figure C); and (iv) alteration in chromosome replication in living cell. Second, to study the radiation triggered bystander responses, we will examine one of the key signaling pathway i.e. TNF- alpha/NF-kappa B mediated signaling. TNF-alpha specific nano particle sensors (green) will be developed to detect the releasing dynamics, transport mechanisms and ligand-receptor binding on live cell surface in real time. A second sensor (blue) will be developed to simultaneously monitor the track of NF-kB inside the cell. The proposed nano-particle optics approach would complement our DOE funded study on biochemical mechanisms of TNF-alpha- NF-kappa B-mediated bystander effect.« less

Imaging the cell surface and its organization down to the level of single molecules.

PubMed

Klenerman, David; Shevchuk, Andrew; Novak, Pavel; Korchev, Yuri E; Davis, Simon J

2013-02-05

Determining the organization of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signalling, is a major challenge in cell biology and requires methods with nanoscale spatial resolution and high temporal resolution. Here, we review biophysical tools, based on scanning ion conductance microscopy and single-molecule fluorescence and the combination of both of these methods, which have recently been developed to address these issues. We then give examples of how these methods have been be applied to provide new insights into cell membrane organization and function, and discuss some of the issues that will need to be addressed to further exploit these methods in the future.

Taking the ruler to the jungle: single-molecule FRET for understanding biomolecular structure and dynamics in live cells.

PubMed

Sustarsic, Marko; Kapanidis, Achillefs N

2015-10-01

Single-molecule Förster resonance energy transfer (smFRET) serves as a molecular ruler that is ideally posed to study static and dynamic heterogeneity in living cells. Observing smFRET in cells requires appropriately integrated labeling, internalization and imaging strategies, and significant progress has been made towards that goal. Pioneering studies have demonstrated smFRET detection in both prokaryotic and eukaryotic systems, using both wide-field and confocal microscopies, and have started to answer exciting biological questions. We anticipate that future technical developments will open the door to smFRET for the study of structure, conformational changes and kinetics of biomolecules in living cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming.

PubMed

West, Jason A; Cook, April; Alver, Burak H; Stadtfeld, Matthias; Deaton, Aimee M; Hochedlinger, Konrad; Park, Peter J; Tolstorukov, Michael Y; Kingston, Robert E

2014-08-27

Chromatin structure determines DNA accessibility. We compare nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs) and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach to identify regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. Surprisingly, most chromatin remains unchanged; a majority of rearrangements appear to affect a single nucleosome. RoDs are enriched at genes and regulatory elements, including enhancers associated with pluripotency and differentiation. RoDs co-localize with binding sites of key developmental regulators, including the reprogramming factors Klf4, Oct4/Sox2 and c-Myc. Nucleosomal landscapes in ESC enhancers are extensively altered, exhibiting lower nucleosome occupancy in pluripotent cells than in somatic cells. Most changes are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of cell differentiation and reprogramming and likely identify regulatory regions essential for these processes.

Direct Correlation between Motile Behavior and Protein Abundance in Single Cells

PubMed Central

Gillet, Sébastien; Frankel, Nicholas W.; Weibel, Douglas B.

2016-01-01

Understanding how stochastic molecular fluctuations affect cell behavior requires the quantification of both behavior and protein numbers in the same cells. Here, we combine automated microscopy with in situ hydrogel polymerization to measure single-cell protein expression after tracking swimming behavior. We characterized the distribution of non-genetic phenotypic diversity in Escherichia coli motility, which affects single-cell exploration. By expressing fluorescently tagged chemotaxis proteins (CheR and CheB) at different levels, we quantitatively mapped motile phenotype (tumble bias) to protein numbers using thousands of single-cell measurements. Our results disagreed with established models until we incorporated the role of CheB in receptor deamidation and the slow fluctuations in receptor methylation. Beyond refining models, our central finding is that changes in numbers of CheR and CheB affect the population mean tumble bias and its variance independently. Therefore, it is possible to adjust the degree of phenotypic diversity of a population by adjusting the global level of expression of CheR and CheB while keeping their ratio constant, which, as shown in previous studies, confers functional robustness to the system. Since genetic control of protein expression is heritable, our results suggest that non-genetic diversity in motile behavior is selectable, supporting earlier hypotheses that such diversity confers a selective advantage. PMID:27599206

Mechanisms for the epigenetic inheritance of stress response in single cells.

PubMed

Xue, Yuan; Acar, Murat

2018-05-30

Cells have evolved to dynamically respond to different types of environmental and physiological stress conditions. The information about a previous stress stimulus experience by a mother cell can be passed to its descendants, allowing them to better adapt to and survive in new environments. In recent years, live-cell imaging combined with cell-lineage tracking approaches has elucidated many important principles that guide stress inheritance at the single-cell and population level. In this review, we summarize different strategies that cells can employ to pass the 'memory' of previous stress responses to their descendants. Among these strategies, we focus on a recent discovery of how specific features of Msn2 nucleo-cytoplasmic shuttling dynamics could be inherited across cell lineages. We also discuss how stress response can be transmitted to progenies through changes in chromatin and through partitioning of anti-stress factors and/or damaged macromolecules between mother and daughter cells during cell division. Finally, we highlight how emergent technologies will help address open questions in the field.

Microfluidic Cell-based Assays in Stem Cell and Other Rare Cell Type Research

DOE PAGES

Wu, Meiye

2015-03-23

Microfluidics is a technology defined by the engineered precise manipulation of minute amount of liquids through channels with dimensions in the micron scale. Much of microfluidic devices used for biomedical purposes are produced in the form of so called “lab-on-a-chip” format, where multiple steps of conventional biochemical analyses such as staining, washing, and signal collection are miniaturized and integrated into chips fabricated from polymer or glass. Cell-based microfluidic lab-on-achip technology provides some obvious advantages: 1) drastically reduced sample and reagent requirement, and 2) separation and detection with improved sensitivity due to fluid properties at the microscale, i.e. laminar flow. Basedmore » on these two advantages, the obvious place where microfluidic cell assays will provide the most benefit is wherescientists must gather much information from precious little sample. Stem cells and other precious cell types such as circulating tumor cells (CTCs), and rare immune subsets are the perfect match for microfluidic multiplex assays. The recent demonstration that multiple cellular changes such as surface receptor activation, protein translocation, long and short RNA, and DNA changes can all be extracted from intact single cells paves the way to systems level understanding of cellular states during development or disease. Finally, with the ability to preserve cell integrity in a microfluidic device during multiplexed analysis, one also preserves the single cell resolution, where information regarding the cell-to-cell heterogeneity during differentiation or response to stimuli is vitally important.« less

Sequential glycan profiling at single cell level with the microfluidic lab-in-a-trench platform: a new era in experimental cell biology.

PubMed

O'Connell, Tríona M; King, Damien; Dixit, Chandra K; O'Connor, Brendan; Walls, Dermot; Ducrée, Jens

2014-09-21

It is now widely recognised that the earliest changes that occur on a cell when it is stressed or becoming diseased are alterations in its surface glycosylation. Current state-of-the-art technologies in glycoanalysis include mass spectrometry, protein microarray formats, techniques in cytometry and more recently, glyco-quantitative polymerase chain reaction (Glyco-qPCR). Techniques for the glycoprofiling of the surfaces of single cells are either limited to the analysis of large cell populations or are unable to handle multiple and/or sequential probing. Here, we report a novel approach of single live cell glycoprofiling enabled by the microfluidic "Lab-in-a-Trench" (LiaT) platform for performing capture and retention of cells, along with shear-free reagent loading and washing. The significant technical improvement on state-of-the-art is the demonstration of consecutive, spatio-temporally profiling of glycans on a single cell by sequential elution of the previous lectin probe using their corresponding free sugar. We have qualitatively analysed glycan density on the surface of individual cells. This has allowed us to qualitatively co-localise the observed glycans. This approach enables exhaustive glycoprofiling and glycan mapping on the surface of individual live cells with multiple lectins. The possibility of sequentially profiling glycans on cells will be a powerful new tool to add to current glycoanalytical techniques. The LiaT platform will enable cell biologists to perform many high sensitivity assays and also will also make a significant impact on biomarker research.

Importance of Interaction between Integrin and Actin Cytoskeleton in Suspension Adaptation of CHO cells.

PubMed

Walther, Christa G; Whitfield, Robert; James, David C

2016-04-01

The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions.

Short-term desensitization of fast escape behavior associated with suppression of Mauthner cell activity in larval zebrafish.

PubMed

Takahashi, Megumi; Inoue, Maya; Tanimoto, Masashi; Kohashi, Tsunehiko; Oda, Yoichi

2017-08-01

Escape is among the simplest animal behaviors employed to study the neural mechanisms underlying learning. Teleost fishes exhibit behavioral learning of fast escape initiated with a C-shaped body bend (C-start). C-starts are subdivided into short-latency (SLC) and long-latency (LLC) types in larval zebrafish. Whether these two can be separately modified, and the neural correlates of this modification, however, remains undetermined. We thus performed Ca 2+ imaging of Mauthner (M-) cells, a pair of giant hindbrain neurons constituting a core element of SLC circuit, during behavioral learning in larval zebrafish. The Ca 2+ response corresponding to a single spiking of the M-cells was coupled with SLCs but not LLCs. Conditioning with a repeated weak sound at subthreshold intensity to elicit C-starts selectively suppressed SLC occurrence for 10min without affecting LLC responsiveness. The short-term desensitization of SLC was associated with the suppression of M-cell activity, suggesting that changes in single neuron responsiveness mediate behavioral learning. The conditioning did not affect the acoustically evoked mechanotransduction of inner ear hair cells, further suggesting plastic change in transmission efficacy within the auditory input circuit between the hair cells and the M-cell. Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

Chromosomal aberrations and deoxyribonucleic acid single-strand breaks in adipose-derived stem cells during long-term expansion in vitro.

PubMed

Froelich, Katrin; Mickler, Johannes; Steusloff, Gudrun; Technau, Antje; Ramos Tirado, Mario; Scherzed, Agmal; Hackenberg, Stephan; Radeloff, Andreas; Hagen, Rudolf; Kleinsasser, Norbert

2013-07-01

Adipose-derived stem cells (ASCs) are a promising mesenchymal cell source for tissue engineering approaches. To obtain an adequate cell amount, in vitro expansion of the cells may be required in some cases. To monitor potential contraindications for therapeutic applications in humans, DNA strand breaks and chromosomal aberrations in ASCs during in vitro expansion were examined. After isolation of ASC from human lipoaspirates of seven patients, in vitro expansion over 10 passages was performed. Cells from passages 1, 2, 3, 5 and 10 were used for the alkaline single-cell microgel electrophoresis (comet) assay to detect DNA single-strand breaks and alkali labile as well as incomplete excision repair sites. Chromosomal changes were examined by means of the chromosomal aberration test. During in vitro expansion, ASC showed no DNA single-strand breaks in the comet assay. With the chromosomal aberration test, however, a significant increase in chromosomal aberrations were detected. The study showed that although no DNA fragmentation could be determined, the safety of ASC cannot be ensured with respect to chromosome stability during in vitro expansion. Thus, reliable analyses for detecting ASC populations, which accumulate chromosomal aberrations or even undergo malignant transformation during extensive in vitro expansion, must be implemented as part of the safety evaluation of these cells for stem cell-based therapy. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

The Impact of Non-Lethal Single-Dose Radiation on Tumor Invasion and Cytoskeletal Properties

PubMed Central

Hohmann, Tim; Grabiec, Urszula; Vogel, Carolin; Ghadban, Chalid; Ensminger, Stephan; Bache, Matthias; Vordermark, Dirk; Dehghani, Faramarz

2017-01-01

Irradiation is the standard therapy for glioblastoma multiforme. Glioblastoma are highly resistant to radiotherapy and the underlying mechanisms remain unclear. To better understand the biological effects of irradiation on glioblastoma cells, we tested whether nonlethal irradiation influences the invasiveness, cell stiffness, and actin cytoskeleton properties. Two different glioblastoma cell lines were irradiated with 2 Gy and changes in mechanical and migratory properties and alterations in the actin structure were measured. The invasiveness of cell lines was determined using a co-culture model with organotypic hippocampal slice cultures. Irradiation led to changes in motility and a less invasive phenotype in both investigated cell lines that were associated with an increase in a ”generalized stiffness” and changes in the actin structure. In this study we demonstrate that irradiation can induce changes in the actin cytoskeleton and motility, which probably results in reduced invasiveness of glioblastoma cell lines. Furthermore, “generalized stiffness” was shown to be a profound marker of the invasiveness of a tumor cell population in our model. PMID:28926987

Simultaneous measurement of sensor-protein dynamics and motility of a single cell by on-chip microcultivation system

PubMed Central

Inoue, Ippei; Shiomi, Daisuke; Kawagishi, Ikuro; Yasuda, Kenji

2004-01-01

Measurement of the correlation between sensor-protein expression, motility and environmental change is important for understanding the adaptation process of cells during their change of generation. We have developed a novel assay exploiting the on-chip cultivation system, which enabled us to observe the change of the localization of expressed sensor-protein and the motility for generations. Localization of the aspartate sensitive sensor protein at two poles in Escherichia coli decreased quickly after the aspartate was added into the cultivation medium. However, it took more than three generations for recovering the localization after the removal of aspartate from the medium. Moreover, the tumbling frequency was strongly related to the localization of the sensor protein in a cell. The results indicate that the change of the spatial localization of sensor protein, which was inherited for more than three generations, may contribute to cells, motility as the inheritable information. PMID:15119953

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells

PubMed Central

Eroglu, Emrah; Rost, Rene; Bischof, Helmut; Blass, Sandra; Schreilechner, Anna; Gottschalk, Benjamin; Depaoli, Maria R.; Klec, Christiane; Charoensin, Suphachai; Madreiter-Sokolowski, Corina T.; Ramadani, Jeta; Waldeck-Weiermair, Markus; Graier, Wolfgang F.; Malli, Roland

2017-01-01

Nitric Oxide (NO•) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO• in vivo and in vitro, the real-time monitoring of NO• at the single-cell level is very challenging. The physiological or pathological effects of NO• are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO• are highly desirable. Recently, we expanded the pallet of NO• indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO•) probes (geNOps) that directly respond to cellular NO• fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO• signals in response to two different chemical NO•-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO• levels as compared with the inorganic NO• donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO• formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO• signals in diverse experimental setups. PMID:28362417

Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution.

PubMed

Ludlow, Andrew T; Robin, Jerome D; Sayed, Mohammed; Litterst, Claudia M; Shelton, Dawne N; Shay, Jerry W; Wright, Woodring E

2014-07-01

The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect ∼ 2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Finite Element Analysis of Single Cell Stiffness Measurements Using PZT-Integrated Buckling Nanoneedles.

PubMed

Rad, Maryam Alsadat; Tijjani, Auwal Shehu; Ahmad, Mohd Ridzuan; Auwal, Shehu Muhammad

2016-12-23

This paper proposes a new technique for real-time single cell stiffness measurement using lead zirconate titanate (PZT)-integrated buckling nanoneedles. The PZT and the buckling part of the nanoneedle have been modelled and validated using the ABAQUS software. The two parts are integrated together to function as a single unit. After calibration, the stiffness, Young's modulus, Poisson's ratio and sensitivity of the PZT-integrated buckling nanoneedle have been determined to be 0.7100 N·m -1 , 123.4700 GPa, 0.3000 and 0.0693 V·m·N -1 , respectively. Three Saccharomyces cerevisiae cells have been modelled and validated based on compression tests. The average global stiffness and Young's modulus of the cells are determined to be 10.8867 ± 0.0094 N·m -1 and 110.7033 ± 0.0081 MPa, respectively. The nanoneedle and the cell have been assembled to measure the local stiffness of the single Saccharomyces cerevisiae cells The local stiffness, Young's modulus and PZT output voltage of the three different size Saccharomyces cerevisiae have been determined at different environmental conditions. We investigated that, at low temperature the stiffness value is low to adapt to the change in the environmental condition. As a result, Saccharomyces cerevisiae becomes vulnerable to viral and bacterial attacks. Therefore, the proposed technique will serve as a quick and accurate process to diagnose diseases at early stage in a cell for effective treatment.

Acute toxic effects of single dose dacarbazine: hematological and histological changes in an animal model.

PubMed

Milijašević, B; Stefanović, D; Lalić-Popović, M; Tomić, Z; Kolarović, J; Lalošević, D; Mikov, M

2014-11-01

Treatment of advanced soft tissue sarcoma usually includes dacarbazine (DTIC), an alkylating agent that methylates DNA and is active during all phases of the cell cycle. Common side effects of DTIC include nausea, vomiting, impaired liver and kidney function, myelosuppression, and pneumonia. There are no accounts, however, of histological and hematological changes caused by DTIC. We investigated acute hematological and morphological changes in different organs and in tumors that were caused by a single dose of DTIC. Adult Syrian golden hamsters were inoculated with a suspension of tumorigenic baby hamster kidney (BHK) cells by subcutaneous injection. On day 14 after inoculation, doses of 1.4, 1.6, 1.8 or 2.0 g/m(2) DTIC were injected intraperitoneally into the hamsters. Hamsters in the control group were injected with physiological saline in the same way. Seven days after drug or saline injection the animals were sacrificed and samples of blood, heart, kidney, liver, lungs, spleen, small intestine and tumor were excised, processed and analyzed. Mitoses were counted using an ocular extension with engraved frame. Anemia, thrombocytopenia and leukocytosis were found in the control group of hamsters with fibrosarcoma, whereas animals with fibrosarcoma treated with DTIC developed anemia, thrombocytopenia and leukopenia. Severe pneumonia and moderate hepatitis were detected in all DTIC treated groups. Effects of DTIC on tumor cells included rounding and enlargement of nuclei and rarefaction of chromatin. The number of mitoses was reduced with increasing doses of DTIC. Hepatitis, myelosuppression, pneumonia, and dose-related inhibition of tumor cell proliferation were observed after a single dose of DTIC.

Cellular and molecular alterations in human epithelial cells transformed by high let radiation

NASA Astrophysics Data System (ADS)

Hei, T. K.; Piao, C. Q.; Sutter, T.; Willey, J. C.; Suzuki, K.

An understanding of the radiobiological effects of high LET radiation is essential for human risk estimation and radiation protection. In the present study, we show that a single, 30 cGy dose of 150 keV/mum ^4He ions can malignantly transform human papillomavirus immortalized human bronchial epithelial [BEP2D] cells. Transformed cells produce progressively growing tumors in nude mice. The transformation frequency by the single dose of alpha particles is estimated to be approximately 4 x 10^-7. Based on the average cross-sectional area of BEP2D cells, it can be calculated that a mean traversal of 1.4 particles per cell is sufficient to induce tumorigenic conversion of these cells 3 to 4 months post-irradiation. Tumorigenic BEP2D cells overexpress mutated p53 tumor suppressor oncoproteins in addition to the cell cycle control gene cyclin D1 and D2. This model provides an opportunity to study the cellular and molecular changes at the various stages in radiation carcinogenesis involving human cells.

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

Bai, Rui; Yi, Shaoqiong; Zhang, Xuejie

Highlights: • We evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations. • AFM was used to measure single-molecule binding ability on living cells. • The SNP of ICAM-1 may induce changes in expressions rather than single-molecule binding ability. - Abstract: Atherosclerosis (As) is characterized by chronic inflammation and is a major cause of human mortality. ICAM-1-mediated adhesion of leukocytes in vessel walls plays an important role in the pathogenesis of atherosclerosis. Two single nucleotide polymorphisms (SNPs) of human intercellular adhesion molecule-1 (ICAM-1), G241R and K469E, are associated with a number of inflammatory diseases. SNP inducedmore » changes in ICAM-1 function rely not only on the expression level but also on the single-molecule binding ability which may be affected by single molecule conformation variations such as protein splicing and folding. Previous studies have shown associations between G241R/K469E polymorphisms and ICAM-1 gene expression. Nevertheless, few studies have been done that focus on the single-molecule forces of the above SNPs and their ligands. In the current study, we evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations – GK (G241/K469), GE (G241/E469), RK (R241/K469) and RE (R241/E469). No difference in adhesion ability was observed via cell adhesion assay or atomic force microscopy (AFM) measurement when comparing the GK, GE, RK, or RE genotypes of ICAM-1 to each other. On the other hand, flow cytometry suggested that there was significantly higher expression of GE genotype of ICAM-1 on transfected CHO cells. Thus, we concluded that genetic susceptibility to diseases related to ICAM-1 polymorphisms, G241R or K469E, might be due to the different expressions of ICAM-1 variants rather than to the single-molecule binding ability of ICAM-1.« less

Fundamental limits on dynamic inference from single-cell snapshots

PubMed Central

Weinreb, Caleb; Tusi, Betsabeh K.; Socolovsky, Merav

2018-01-01

Single-cell expression profiling reveals the molecular states of individual cells with unprecedented detail. Because these methods destroy cells in the process of analysis, they cannot measure how gene expression changes over time. However, some information on dynamics is present in the data: the continuum of molecular states in the population can reflect the trajectory of a typical cell. Many methods for extracting single-cell dynamics from population data have been proposed. However, all such attempts face a common limitation: for any measured distribution of cell states, there are multiple dynamics that could give rise to it, and by extension, multiple possibilities for underlying mechanisms of gene regulation. Here, we describe the aspects of gene expression dynamics that cannot be inferred from a static snapshot alone and identify assumptions necessary to constrain a unique solution for cell dynamics from static snapshots. We translate these constraints into a practical algorithmic approach, population balance analysis (PBA), which makes use of a method from spectral graph theory to solve a class of high-dimensional differential equations. We use simulations to show the strengths and limitations of PBA, and then apply it to single-cell profiles of hematopoietic progenitor cells (HPCs). Cell state predictions from this analysis agree with HPC fate assays reported in several papers over the past two decades. By highlighting the fundamental limits on dynamic inference faced by any method, our framework provides a rigorous basis for dynamic interpretation of a gene expression continuum and clarifies best experimental designs for trajectory reconstruction from static snapshot measurements. PMID:29463712

Ultrastructural changes in the receptor parts of retinal rods in experimental alloxan-induced diabetes in rabbits.

PubMed

Zarebska, A; Łańcut, M; Bakiera, K; Matejko, E; Czerny, K; Kiś, G; Wójtowicz, Z

2001-01-01

Mature rabbits were administered a single dose of alloxan at the dose 100 mg/kg b.m. After 3 and 6 weeks and after 3 and 6 months, the samples of the retina were taken from the areas immediate to the papilla of the optic nerve. Ultrathin sections were dyed according to the Reynold's method, and the receptive parts of the rods were examined under electron microscope BS-500 Tesla. After 6 weeks following alloxan administration, distinct morphological changes in the form of enlargement of certain discs in the receptive parts of rod cells were observed. After 3 months the majority of the discs was damaged, and after 6 months only single, quite well preserved rod cells were found to be present in the retina.

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

PubMed Central

Etheridge, Thomas J.; Boulineau, Rémi L.; Herbert, Alex; Watson, Adam T.; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter; Palayret, Matthieu; Lando, David; Laue, Ernest; Osborne, Mark A.; Klenerman, David; Lee, Steven F.; Carr, Antony M.

2014-01-01

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds. PMID:25106872

Cell and molecular mechanics of biological materials

NASA Astrophysics Data System (ADS)

Bao, G.; Suresh, S.

2003-11-01

Living cells can sense mechanical forces and convert them into biological responses. Similarly, biological and biochemical signals are known to influence the abilities of cells to sense, generate and bear mechanical forces. Studies into the mechanics of single cells, subcellular components and biological molecules have rapidly evolved during the past decade with significant implications for biotechnology and human health. This progress has been facilitated by new capabilities for measuring forces and displacements with piconewton and nanometre resolutions, respectively, and by improvements in bio-imaging. Details of mechanical, chemical and biological interactions in cells remain elusive. However, the mechanical deformation of proteins and nucleic acids may provide key insights for understanding the changes in cellular structure, response and function under force, and offer new opportunities for the diagnosis and treatment of disease. This review discusses some basic features of the deformation of single cells and biomolecules, and examines opportunities for further research.

Unraveling the genetic driving forces enabling antibiotic resistance at the single cell level

NASA Astrophysics Data System (ADS)

Bos, Julia

Bacteria are champions at finding ways to quickly respond and adapt to environments like the human gut, known as the epicentre of antibiotic resistance. How do they do it? Combining molecular biology tools to microfluidic and fluorescence microscopy technologies, we monitor the behavior of bacteria at the single cell level in the presence of non-toxic doses of antibiotics. By tracking the chromosome dynamics of Escherichia coli cells upon antibiotic treatment, we examine the changes in the number, localization and content of the chromosome copies within one cell compartment or between adjacent cells. I will discuss how our work pictures the bacterial genomic plasticity as a driving force in evolution and how it provides access to the mechanisms controlling the subtle balance between genetic diversity and stability in the development of antibiotic resistance.

Changes of heterogeneous cell populations in the Ishikawa cell line during long-term culture: Proposal for an in vitro clonal evolution model of tumor cells.

PubMed

Kasai, Fumio; Hirayama, Noriko; Ozawa, Midori; Iemura, Masashi; Kohara, Arihiro

2016-06-01

Genomic changes in tumor cell lines can occur during culture, leading to differences between cell lines carrying the same name. In this study, genome profiles between low and high passages were investigated in the Ishikawa 3-H-12 cell line (JCRB1505). Cells contained between 43 and 46 chromosomes and the modal number changed from 46 to 45 during culture. Cytogenetic analysis revealed that a translocation t(9;14), observed in all metaphases, is a robust marker for this cell line. Single-nucleotide polymorphism microarrays showed a heterogeneous copy number in the early passages and distinct profiles at late passages. These results demonstrate that cell culture can lead to elimination of ancestral clones by sequential selection, resulting in extensive replacement with a novel clone. Our observations on Ishikawa cells in vitro are different from the in vivo heterogeneity in which ancestral clones are often retained during tumor evolution and suggest a model for in vitro clonal evolution. Copyright © 2016 Elsevier Inc. All rights reserved.

A disposable picolitre bioreactor for cultivation and investigation of industrially relevant bacteria on the single cell level.

PubMed

Grünberger, Alexander; Paczia, Nicole; Probst, Christopher; Schendzielorz, Georg; Eggeling, Lothar; Noack, Stephan; Wiechert, Wolfgang; Kohlheyer, Dietrich

2012-05-08

In the continuously growing field of industrial biotechnology the scale-up from lab to industrial scale is still a major hurdle to develop competitive bioprocesses. During scale-up the productivity of single cells might be affected by bioreactor inhomogeneity and population heterogeneity. Currently, these complex interactions are difficult to investigate. In this report, design, fabrication and operation of a disposable picolitre cultivation system is described, in which environmental conditions can be well controlled on a short time scale and bacterial microcolony growth experiments can be observed by time-lapse microscopy. Three exemplary investigations will be discussed emphasizing the applicability and versatility of the device. Growth and analysis of industrially relevant bacteria with single cell resolution (in particular Escherichia coli and Corynebacterium glutamicum) starting from one single mother cell to densely packed cultures is demonstrated. Applying the picolitre bioreactor, 1.5-fold increased growth rates of C. glutamicum wild type cells were observed compared to typical 1 litre lab-scale batch cultivation. Moreover, the device was used to analyse and quantify the morphological changes of an industrially relevant l-lysine producer C. glutamicum after artificially inducing starvation conditions. Instead of a one week lab-scale experiment, only 1 h was sufficient to reveal the same information. Furthermore, time lapse microscopy during 24 h picolitre cultivation of an arginine producing strain containing a genetically encoded fluorescence sensor disclosed time dependent single cell productivity and growth, which was not possible with conventional methods.

Intercellular signaling through secreted proteins induces free-energy gradient-directed cell movement.

PubMed

Kravchenko-Balasha, Nataly; Shin, Young Shik; Sutherland, Alex; Levine, R D; Heath, James R

2016-05-17

Controlling cell migration is important in tissue engineering and medicine. Cell motility depends on factors such as nutrient concentration gradients and soluble factor signaling. In particular, cell-cell signaling can depend on cell-cell separation distance and can influence cellular arrangements in bulk cultures. Here, we seek a physical-based approach, which identifies a potential governed by cell-cell signaling that induces a directed cell-cell motion. A single-cell barcode chip (SCBC) was used to experimentally interrogate secreted proteins in hundreds of isolated glioblastoma brain cancer cell pairs and to monitor their relative motions over time. We used these trajectories to identify a range of cell-cell separation distances where the signaling was most stable. We then used a thermodynamics-motivated analysis of secreted protein levels to characterize free-energy changes for different cell-cell distances. We show that glioblastoma cell-cell movement can be described as Brownian motion biased by cell-cell potential. To demonstrate that the free-energy potential as determined by the signaling is the driver of motion, we inhibited two proteins most involved in maintaining the free-energy gradient. Following inhibition, cell pairs showed an essentially random Brownian motion, similar to the case for untreated, isolated single cells.

Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors

PubMed Central

Patsialou, Antonia; Bravo-Cordero, Jose Javier; Wang, Yarong; Entenberg, David; Liu, Huiping; Clarke, Michael; Condeelis, John S.

2014-01-01

Metastasis is the main cause of death in breast cancer patients. Cell migration is an essential component of almost every step of the metastatic cascade, especially the early step of invasion inside the primary tumor. In this report, we have used intravital multiphoton microscopy to visualize the different migration patterns of human breast tumor cells in live primary tumors. We used xenograft tumors of MDA-MB-231 cells as well as a low passage xenograft tumor from orthotopically injected patient-derived breast tumor cells. Direct visualization of human tumor cells in vivo shows two patterns of high-speed migration inside primary tumors: a. single cells and b. multicellular streams (i.e., cells following each other in a single file but without cohesive cell junctions). Critically, we found that only streaming and not random migration of single cells was significantly correlated with proximity to vessels, with intravasation and with numbers of elevated circulating tumor cells in the bloodstream. Finally, although the two human tumors were derived from diverse genetic backgrounds, we found that their migratory tumor cells exhibited coordinated gene expression changes that led to the same end-phenotype of enhanced migration involving activating actin polymerization and myosin contraction. Our data are the first direct visualization and assessment of in vivo migration within a live patient-derived breast xenograft tumor. PMID:25013744

Geometry and network connectivity govern the mechanics of stress fibers.

PubMed

Kassianidou, Elena; Brand, Christoph A; Schwarz, Ulrich S; Kumar, Sanjay

2017-03-07

Actomyosin stress fibers (SFs) play key roles in driving polarized motility and generating traction forces, yet little is known about how tension borne by an individual SF is governed by SF geometry and its connectivity to other cytoskeletal elements. We now address this question by combining single-cell micropatterning with subcellular laser ablation to probe the mechanics of single, geometrically defined SFs. The retraction length of geometrically isolated SFs after cutting depends strongly on SF length, demonstrating that longer SFs dissipate more energy upon incision. Furthermore, when cell geometry and adhesive spacing are fixed, cell-to-cell heterogeneities in SF dissipated elastic energy can be predicted from varying degrees of physical integration with the surrounding network. We apply genetic, pharmacological, and computational approaches to demonstrate a causal and quantitative relationship between SF connectivity and mechanics for patterned cells and show that similar relationships hold for nonpatterned cells allowed to form cell-cell contacts in monolayer culture. Remarkably, dissipation of a single SF within a monolayer induces cytoskeletal rearrangements in cells long distances away. Finally, stimulation of cell migration leads to characteristic changes in network connectivity that promote SF bundling at the cell rear. Our findings demonstrate that SFs influence and are influenced by the networks in which they reside. Such higher order network interactions contribute in unexpected ways to cell mechanics and motility.

Pseudotemporal Ordering of Single Cells Reveals Metabolic Control of Postnatal β Cell Proliferation.

PubMed

Zeng, Chun; Mulas, Francesca; Sui, Yinghui; Guan, Tiffany; Miller, Nathanael; Tan, Yuliang; Liu, Fenfen; Jin, Wen; Carrano, Andrea C; Huising, Mark O; Shirihai, Orian S; Yeo, Gene W; Sander, Maike

2017-05-02

Pancreatic β cell mass for appropriate blood glucose control is established during early postnatal life. β cell proliferative capacity declines postnatally, but the extrinsic cues and intracellular signals that cause this decline remain unknown. To obtain a high-resolution map of β cell transcriptome dynamics after birth, we generated single-cell RNA-seq data of β cells from multiple postnatal time points and ordered cells based on transcriptional similarity using a new analytical tool. This analysis captured signatures of immature, proliferative β cells and established high expression of amino acid metabolic, mitochondrial, and Srf/Jun/Fos transcription factor genes as their hallmark feature. Experimental validation revealed high metabolic activity in immature β cells and a role for reactive oxygen species and Srf/Jun/Fos transcription factors in driving postnatal β cell proliferation and mass expansion. Our work provides the first high-resolution molecular characterization of state changes in postnatal β cells and paves the way for the identification of novel therapeutic targets to stimulate β cell regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Manipulating motions of targeted single cells in solution by an integrated double-ring magnetic tweezers imaging microscope.

PubMed

Wu, Meiling; Yadav, Rajeev; Pal, Nibedita; Lu, H Peter

2017-07-01

Controlling and manipulating living cell motions in solution hold a high promise in developing new biotechnology and biological science. Here, we developed a magnetic tweezers device that employs a combination of two permanent magnets in up-down double-ring configuration axially fitting with a microscopic objective, allowing a picoNewton (pN) bidirectional force and motion control on the sample beyond a single upward pulling direction. The experimental force calibration and magnetic field simulation using finite element method magnetics demonstrate that the designed magnetic tweezers covers a linear-combined pN force with positive-negative polarization changes in a tenability of sub-pN scale, which can be utilized to further achieve motion manipulation by shifting the force balance. We demonstrate an application of the up-down double-ring magnetic tweezers for single cell manipulation, showing that the cells with internalized paramagnetic beads can be selectively picked up and guided in a controlled fine motion.

Manipulating motions of targeted single cells in solution by an integrated double-ring magnetic tweezers imaging microscope

NASA Astrophysics Data System (ADS)

Wu, Meiling; Yadav, Rajeev; Pal, Nibedita; Lu, H. Peter

2017-07-01

Controlling and manipulating living cell motions in solution hold a high promise in developing new biotechnology and biological science. Here, we developed a magnetic tweezers device that employs a combination of two permanent magnets in up-down double-ring configuration axially fitting with a microscopic objective, allowing a picoNewton (pN) bidirectional force and motion control on the sample beyond a single upward pulling direction. The experimental force calibration and magnetic field simulation using finite element method magnetics demonstrate that the designed magnetic tweezers covers a linear-combined pN force with positive-negative polarization changes in a tenability of sub-pN scale, which can be utilized to further achieve motion manipulation by shifting the force balance. We demonstrate an application of the up-down double-ring magnetic tweezers for single cell manipulation, showing that the cells with internalized paramagnetic beads can be selectively picked up and guided in a controlled fine motion.

Conformational plasticity of JRAB/MICAL-L2 provides "law and order" in collective cell migration.

PubMed

Sakane, Ayuko; Yoshizawa, Shin; Nishimura, Masaomi; Tsuchiya, Yuko; Matsushita, Natsuki; Miyake, Kazuhisa; Horikawa, Kazuki; Imoto, Issei; Mizuguchi, Chiharu; Saito, Hiroyuki; Ueno, Takato; Matsushita, Sachi; Haga, Hisashi; Deguchi, Shinji; Mizuguchi, Kenji; Yokota, Hideo; Sasaki, Takuya

2016-10-15

In fundamental biological processes, cells often move in groups, a process termed collective cell migration. Collectively migrating cells are much better organized than a random assemblage of individual cells. Many molecules have been identified as factors involved in collective cell migration, and no one molecule is adequate to explain the whole picture. Here we show that JRAB/MICAL-L2, an effector protein of Rab13 GTPase, provides the "law and order" allowing myriad cells to behave as a single unit just by changing its conformation. First, we generated a structural model of JRAB/MICAL-L2 by a combination of bioinformatic and biochemical analyses and showed how JRAB/MICAL-L2 interacts with Rab13 and how its conformational change occurs. We combined cell biology, live imaging, computational biology, and biomechanics to show that impairment of conformational plasticity in JRAB/MICAL-L2 causes excessive rigidity and loss of directionality, leading to imbalance in cell group behavior. This multidisciplinary approach supports the concept that the conformational plasticity of a single molecule provides "law and order" in collective cell migration. © 2016 Sakane et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Measurement of In Vitro Single Cell Temperature by Novel Thermocouple Nanoprobe in Acute Lung Injury Models.

PubMed

Wang, Xing; Chen, Qiuhua; Tian, Wenjuan; Wang, Jianqing; Cheng, Lu; Lu, Jun; Chen, Mingqi; Pei, Yinhao; Li, Can; Chen, Gong; Gu, Ning

2017-01-01

Energy metabolism may alter pattern differences in acute lung injury (ALI) as one of the causes but the detailed features at single-cellular level remain unclear. Changes in intercellular temperature and adenosine triphosphate (ATP) concentration within the single cell may help to understand the role of energy metabolism in causing ALI. ALI in vitro models were established by treating mice lung epithelial (MLE-12) cells with lipopolysaccharide (LPS), hydrogen peroxide (H2O2), hydrochloric acid (HCl) and cobalt chloride (CoCl2, respectively. 100 nm micro thermocouple probe (TMP) was inserted into the cytosol by micromanipulation system and thermoelectric readings were recorded to calculate the intracellular temperature based on standard curve. The total ATP contents for the MLE-12 cells were evaluated at different time intervals after treatments. A significant increase of intracellular temperature was observed after 10 or 20 μg/L LPS and HCl treatments. The HCl increased the temperature in a dose-dependent manner. On the contrary, H2O2 induced a significant decline of intracellular temperature after treatment. No significant difference in intracellular temperature was observed after CoCl2 exposure. The intracellular ATP levels decreased in a time-dependent manner after treatment with H2O2 and HCl, while the LPS and CoCl2 had no significant effect on ATP levels. The intracellular temperature responses varied in different ALI models. The concentration of ATP in the MLE-12 cells played part in the intracellular temperature changes. No direct correlation was observed between the intracellular temperature and concentration of ATP in the MLE-12 cells.

Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy

PubMed Central

Aguet, François; Upadhyayula, Srigokul; Gaudin, Raphaël; Chou, Yi-ying; Cocucci, Emanuele; He, Kangmin; Chen, Bi-Chang; Mosaliganti, Kishore; Pasham, Mithun; Skillern, Wesley; Legant, Wesley R.; Liu, Tsung-Li; Findlay, Greg; Marino, Eric; Danuser, Gaudenz; Megason, Sean; Betzig, Eric; Kirchhausen, Tom

2016-01-01

Membrane remodeling is an essential part of transferring components to and from the cell surface and membrane-bound organelles and for changes in cell shape, which are particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging and mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and noninvasive illumination of the newly developed lattice light-sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and found that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and was relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light-sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and multicellular assemblies. PMID:27535432

Single cell-type comparative metabolomics of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum

PubMed Central

Barkla, Bronwyn J.; Vera-Estrella, Rosario

2015-01-01

One of the remarkable adaptive features of the halophyte Mesembryanthemum crystallinum are the specialized modified trichomes called epidermal bladder cells (EBC) which cover the leaves, stems, and peduncle of the plant. They are present from an early developmental stage but upon salt stress rapidly expand due to the accumulation of water and sodium. This particular plant feature makes it an attractive system for single cell type studies, with recent proteomics and transcriptomics studies of the EBC establishing that these cells are metabolically active and have roles other than sodium sequestration. To continue our investigation into the function of these unusual cells we carried out a comprehensive global analysis of the metabolites present in the EBC extract by gas chromatography Time-of-Flight mass spectrometry (GC-TOF) and identified 194 known and 722 total molecular features. Statistical analysis of the metabolic changes between control and salt-treated samples identified 352 significantly differing metabolites (268 after correction for FDR). Principal components analysis provided an unbiased evaluation of the data variance structure. Biochemical pathway enrichment analysis suggested significant perturbations in 13 biochemical pathways as defined in KEGG. More than 50% of the metabolites that show significant changes in the EBC, can be classified as compatible solutes and include sugars, sugar alcohols, protein and non-protein amino acids, and organic acids, highlighting the need to maintain osmotic homeostasis to balance the accumulation of Na+ and Cl− ions. Overall, the comparison of metabolic changes in salt treated relative to control samples suggests large alterations in M. crystallinum epidermal bladder cells. PMID:26113856

Single cell-type comparative metabolomics of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum.

PubMed

Barkla, Bronwyn J; Vera-Estrella, Rosario

2015-01-01

One of the remarkable adaptive features of the halophyte Mesembryanthemum crystallinum are the specialized modified trichomes called epidermal bladder cells (EBC) which cover the leaves, stems, and peduncle of the plant. They are present from an early developmental stage but upon salt stress rapidly expand due to the accumulation of water and sodium. This particular plant feature makes it an attractive system for single cell type studies, with recent proteomics and transcriptomics studies of the EBC establishing that these cells are metabolically active and have roles other than sodium sequestration. To continue our investigation into the function of these unusual cells we carried out a comprehensive global analysis of the metabolites present in the EBC extract by gas chromatography Time-of-Flight mass spectrometry (GC-TOF) and identified 194 known and 722 total molecular features. Statistical analysis of the metabolic changes between control and salt-treated samples identified 352 significantly differing metabolites (268 after correction for FDR). Principal components analysis provided an unbiased evaluation of the data variance structure. Biochemical pathway enrichment analysis suggested significant perturbations in 13 biochemical pathways as defined in KEGG. More than 50% of the metabolites that show significant changes in the EBC, can be classified as compatible solutes and include sugars, sugar alcohols, protein and non-protein amino acids, and organic acids, highlighting the need to maintain osmotic homeostasis to balance the accumulation of Na(+) and Cl(-) ions. Overall, the comparison of metabolic changes in salt treated relative to control samples suggests large alterations in M. crystallinum epidermal bladder cells.

Geometry and network connectivity govern the mechanics of stress fibers

PubMed Central

Kassianidou, Elena; Brand, Christoph A.; Kumar, Sanjay

2017-01-01

Actomyosin stress fibers (SFs) play key roles in driving polarized motility and generating traction forces, yet little is known about how tension borne by an individual SF is governed by SF geometry and its connectivity to other cytoskeletal elements. We now address this question by combining single-cell micropatterning with subcellular laser ablation to probe the mechanics of single, geometrically defined SFs. The retraction length of geometrically isolated SFs after cutting depends strongly on SF length, demonstrating that longer SFs dissipate more energy upon incision. Furthermore, when cell geometry and adhesive spacing are fixed, cell-to-cell heterogeneities in SF dissipated elastic energy can be predicted from varying degrees of physical integration with the surrounding network. We apply genetic, pharmacological, and computational approaches to demonstrate a causal and quantitative relationship between SF connectivity and mechanics for patterned cells and show that similar relationships hold for nonpatterned cells allowed to form cell–cell contacts in monolayer culture. Remarkably, dissipation of a single SF within a monolayer induces cytoskeletal rearrangements in cells long distances away. Finally, stimulation of cell migration leads to characteristic changes in network connectivity that promote SF bundling at the cell rear. Our findings demonstrate that SFs influence and are influenced by the networks in which they reside. Such higher order network interactions contribute in unexpected ways to cell mechanics and motility. PMID:28213499

Alterations in neuronal activity in basal ganglia-thalamocortical circuits in the parkinsonian state

PubMed Central

Galvan, Adriana; Devergnas, Annaelle; Wichmann, Thomas

2015-01-01

In patients with Parkinson’s disease and in animal models of this disorder, neurons in the basal ganglia and related regions in thalamus and cortex show changes that can be recorded by using electrophysiologic single-cell recording techniques, including altered firing rates and patterns, pathologic oscillatory activity and increased inter-neuronal synchronization. In addition, changes in synaptic potentials or in the joint spiking activities of populations of neurons can be monitored as alterations in local field potentials (LFPs), electroencephalograms (EEGs) or electrocorticograms (ECoGs). Most of the mentioned electrophysiologic changes are probably related to the degeneration of diencephalic dopaminergic neurons, leading to dopamine loss in the striatum and other basal ganglia nuclei, although degeneration of non-dopaminergic cell groups may also have a role. The altered electrical activity of the basal ganglia and associated nuclei may contribute to some of the motor signs of the disease. We here review the current knowledge of the electrophysiologic changes at the single cell level, the level of local populations of neural elements, and the level of the entire basal ganglia-thalamocortical network in parkinsonism, and discuss the possible use of this information to optimize treatment approaches to Parkinson’s disease, such as deep brain stimulation (DBS) therapy. PMID:25698937

Lineage Tracking for Probing Heritable Phenotypes at Single-Cell Resolution.

PubMed

Cottinet, Denis; Condamine, Florence; Bremond, Nicolas; Griffiths, Andrew D; Rainey, Paul B; de Visser, J Arjan G M; Baudry, Jean; Bibette, Jérôme

2016-01-01

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes-via growth-over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology.

A DNA methylation map of human cancer at single base-pair resolution.

PubMed

Vidal, E; Sayols, S; Moran, S; Guillaumet-Adkins, A; Schroeder, M P; Royo, R; Orozco, M; Gut, M; Gut, I; Lopez-Bigas, N; Heyn, H; Esteller, M

2017-10-05

Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination.

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

Magnetic field exposure stiffens regenerating plant protoplast cell walls.

PubMed

Haneda, Toshihiko; Fujimura, Yuu; Iino, Masaaki

2006-02-01

Single suspension-cultured plant cells (Catharanthus roseus) and their protoplasts were anchored to a glass plate and exposed to a magnetic field of 302 +/- 8 mT for several hours. Compression forces required to produce constant cell deformation were measured parallel to the magnetic field by means of a cantilever-type force sensor. Exposure of intact cells to the magnetic field did not result in any changes within experimental error, while exposure of regenerating protoplasts significantly increased the measured forces and stiffened regenerating protoplasts. The diameters of intact cells or regenerating protoplasts were not changed after exposure to the magnetic field. Measured forces for regenerating protoplasts with and without exposure to the magnetic field increased linearly with incubation time, with these forces being divided into components based on the elasticity of synthesized cell walls and cytoplasm. Cell wall synthesis was also measured using a cell wall-specific fluorescent dye, and no changes were noted after exposure to the magnetic field. Analysis suggested that exposure to the magnetic field roughly tripled the Young's modulus of the newly synthesized cell wall without any lag.

Imaging and Force Recognition of Single Molecular Behaviors Using Atomic Force Microscopy

PubMed Central

Li, Mi; Dang, Dan; Liu, Lianqing; Xi, Ning; Wang, Yuechao

2017-01-01

The advent of atomic force microscopy (AFM) has provided a powerful tool for investigating the behaviors of single native biological molecules under physiological conditions. AFM can not only image the conformational changes of single biological molecules at work with sub-nanometer resolution, but also sense the specific interactions of individual molecular pair with piconewton force sensitivity. In the past decade, the performance of AFM has been greatly improved, which makes it widely used in biology to address diverse biomedical issues. Characterizing the behaviors of single molecules by AFM provides considerable novel insights into the underlying mechanisms guiding life activities, contributing much to cell and molecular biology. In this article, we review the recent developments of AFM studies in single-molecule assay. The related techniques involved in AFM single-molecule assay were firstly presented, and then the progress in several aspects (including molecular imaging, molecular mechanics, molecular recognition, and molecular activities on cell surface) was summarized. The challenges and future directions were also discussed. PMID:28117741

Functionalization of nanotextured substrates for enhanced identification of metastatic breast cancer cells

NASA Astrophysics Data System (ADS)

Mansur, Nuzhat; Raziul Hasan, Mohammad; Kim, Young-tae; Iqbal, Samir M.

2017-09-01

Metastasis is the major cause of low survival rates among cancer patients. Once cancer cells metastasize, it is extremely difficult to contain the disease. We report on a nanotextured platform for enhanced detection of metastatic cells. We captured metastatic (MDA-MDB-231) and non-metastatic (MCF-7) breast cancer cells on anti-EGFR aptamer modified plane and nanotextured substrates. Metastatic cells were seen to change their morphology at higher rates when captured on nanotextured substrates than on plane substrates. Analysis showed statistically different morphological behaviors of metastatic cells that were very pronounced on the nanotextured substrates. Several distance matrices were calculated to quantify the dissimilarity of cell shape change. Nanotexturing increased the dissimilarity of the metastatic cells and as a result the contrast between metastatic and non-metastatic cells increased. Jaccard distance measurements found that the shape change ratio of the non-metastatic and metastatic cells was enhanced from 1:1.01 to 1:1.81, going from plane to nanotextured substrates. The shape change ratio of the non-metastatic to metastatic cells improved from 1:1.48 to 1:2.19 for the Hausdorff distance and from 1:1.87 to 1:4.69 for the Mahalanobis distance after introducing nanotexture. Distance matrix analysis showed that nanotexture increased the shape change ratios of non-metastatic and metastatic cells. Hence, the detectability of metastatic cells increased. These calculated matrices provided clear and explicit measures to discriminate single cells for their metastatic state on functional nanotextured substrates.

Optical imaging of radiation-induced metabolic changes in radiation-sensitive and resistant cancer cells

NASA Astrophysics Data System (ADS)

Alhallak, Kinan; Jenkins, Samir V.; Lee, David E.; Greene, Nicholas P.; Quinn, Kyle P.; Griffin, Robert J.; Dings, Ruud P. M.; Rajaram, Narasimhan

2017-06-01

Radiation resistance remains a significant problem for cancer patients, especially due to the time required to definitively determine treatment outcome. For fractionated radiation therapy, nearly 7 to 8 weeks can elapse before a tumor is deemed to be radiation-resistant. We used the optical redox ratio of FAD/(FAD+NADH) to identify early metabolic changes in radiation-resistant lung cancer cells. These radiation-resistant human A549 lung cancer cells were developed by exposing the parental A549 cells to repeated doses of radiation (2 Gy). Although there were no significant differences in the optical redox ratio between the parental and resistant cell lines prior to radiation, there was a significant decrease in the optical redox ratio of the radiation-resistant cells 24 h after a single radiation exposure (p=0.01). This change in the redox ratio was indicative of increased catabolism of glucose in the resistant cells after radiation and was associated with significantly greater protein content of hypoxia-inducible factor 1 (HIF-1α), a key promoter of glycolytic metabolism. Our results demonstrate that the optical redox ratio could provide a rapid method of determining radiation resistance status based on early metabolic changes in cancer cells.

CHANGES IN THE MORPHOLOGY AND POLYSACCHARIDE CONTENT OF MICROCYSTIS AERUGINOSA (CYANOBACTERIA) DURING FLAGELLATE GRAZING(1).

PubMed

Yang, Zhou; Kong, Fanxiang; Shi, Xiaoli; Zhang, Min; Xing, Peng; Cao, Huansheng

2008-06-01

To investigate the changes in the morphology and polysaccharide content of Microcystis aeruginosa (Kütz.) Kütz. during flagellate grazing, cultures of M. aeruginosa were exposed to grazing Ochromonas sp. for a period of 9 d under controlled laboratory conditions. M. aeruginosa responded actively to flagellate grazing and formed colonies, most of which were made up of several or dozens of cells, suggesting that flagellate grazing may be one of the biotic factors responsible for colony formation in M. aeruginosa. When colonies were formed, the cell surface ultrastructure changed, and the polysaccharide layer on the surface of the cell wall became thicker. This change indicated that synthesis and secretion of extracellular polysaccharide (EPS) of M. aeruginosa cells increased under flagellate grazing pressure. The contents of soluble extracellular polysaccharide (sEPS), bound extracellular polysaccharide (bEPS), and total polysaccharide (TPS) in colonial cells of M. aeruginosa increased significantly compared with those in single cells. This finding suggested that the increased amount of EPS on the cell surface may play a role in keeping M. aeruginosa cells together to form colonies. © 2008 Phycological Society of America.

Single cell active force generation under dynamic loading - Part I: AFM experiments.

PubMed

Weafer, P P; Reynolds, N H; Jarvis, S P; McGarry, J P

2015-11-01

A novel series of experiments are performed on single cells using a bespoke AFM system where the response of cells to dynamic loading at physiologically relevant frequencies is uncovered. Measured forces for the untreated cells are dramatically different to cytochalasin-D (cyto-D) treated cells, indicating that the contractile actin cytoskeleton plays a critical role in the response of cells to dynamic loading. Following a change in applied strain magnitude, while maintaining a constant applied strain rate, the compression force for contractile cells recovers to 88.9±7.8% of the steady state force. In contrast, cyto-D cell compression forces recover to only 38.0±6.7% of the steady state force. Additionally, untreated cells exhibit strongly negative (pulling) forces during unloading half-cycles when the probe is retracted. In comparison, negligible pulling forces are measured for cyto-D cells during probe retraction. The current study demonstrates that active contractile forces, generated by actin-myosin cross-bridge cycling, dominate the response of single cells to dynamic loading. Such active force generation is shown to be independent of applied strain magnitude. Passive forces generated by the applied deformation are shown to be of secondary importance, exhibiting a high dependence on applied strain magnitude, in contrast to the active forces in untreated cells. A novel series of experiments are performed on single cells using a bespoke AFM system where the response of cells to dynamic loading at physiologically relevant frequencies is uncovered. Contractile cells, which contain the active force generation machinery of the actin cytoskeleton, are shown to be insensitive to applied strain magnitude, exhibiting high resistance to dynamic compression and stretching. Such trends are not observed for cells in which the actin cytoskeleton has been chemically disrupted. These biomechanical insights have not been previously reported. This detailed characterisation of single cell active and passive stress during dynamic loading has important implications for tissue engineering strategies, where applied deformation has been reported to significantly affect cell mechanotransduction and matrix synthesis. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

How well can morphology assess cell death modality? A proteomics study

PubMed Central

Chernobrovkin, Alexey L; Zubarev, Roman A

2016-01-01

While the focus of attempts to classify cell death programs has finally shifted in 2010s from microscopy-based morphological characteristics to biochemical assays, more recent discoveries have put the underlying assumptions of many such assays under severe stress, mostly because of the limited specificity of the assays. On the other hand, proteomics can quantitatively measure the abundances of thousands of proteins in a single experiment. Thus proteomics could develop a modern alternative to both semiquantitative morphology assessment as well as single-molecule biochemical assays. Here we tested this hypothesis by analyzing the proteomes of cells dying after been treated with various chemical agents. The most striking finding is that, for a multivariate model based on the proteome changes in three cells lines, the regulation patterns of the 200–500 most abundant proteins typically attributed to household type more accurately reflect that of the proteins directly interacting with the drug than any other protein subset grouped by common function or biological process, including cell death. This is in broad agreement with the 'rigid cell death mechanics' model where drug action mechanism and morphological changes caused by it are bijectively linked. This finding, if confirmed, will open way for a broad use of proteomics in death modality assessment. PMID:27752363

Evolution of early embryogenesis in rhabditid nematodes

PubMed Central

Brauchle, Michael; Kiontke, Karin; MacMenamin, Philip; Fitch, David H. A.; Piano, Fabio

2009-01-01

The cell biological events that guide early embryonic development occur with great precision within species but can be quite diverse across species. How these cellular processes evolve and which molecular components underlie evolutionary changes is poorly understood. To begin to address these questions, we systematically investigated early embryogenesis, from the one- to the four-cell embryo, in 34 nematode species related to C. elegans. We found 40 cell-biological characters that captured the phenotypic differences between these species. By tracing the evolutionary changes on a molecular phylogeny, we found that these characters evolved multiple times and independently of one another. Strikingly, all these phenotypes are mimicked by single-gene RNAi experiments in C. elegans. We use these comparisons to hypothesize the molecular mechanisms underlying the evolutionary changes. For example, we predict that a cell polarity module was altered during the evolution of the Protorhabditis group and show that PAR-1, a kinase localized asymmetrically in C. elegans early embryos, is symmetrically localized in the one-cell stage of Protorhabditis group species. Our genome-wide approach identifies candidate molecules—and thereby modules—associated with evolutionary changes in cell-biological phenotypes. PMID:19643102

Radiation-Induced Glycogen Accumulation Detected by Single Cell Raman Spectroscopy Is Associated with Radioresistance that Can Be Reversed by Metformin

PubMed Central

Matthews, Quinn; Isabelle, Martin; Harder, Samantha J.; Smazynski, Julian; Beckham, Wayne; Brolo, Alexandre G.; Jirasek, Andrew; Lum, Julian J.

2015-01-01

Altered cellular metabolism is a hallmark of tumor cells and contributes to a host of properties associated with resistance to radiotherapy. Detection of radiation-induced biochemical changes can reveal unique metabolic pathways affecting radiosensitivity that may serve as attractive therapeutic targets. Using clinically relevant doses of radiation, we performed label-free single cell Raman spectroscopy on a series of human cancer cell lines and detected radiation-induced accumulation of intracellular glycogen. The increase in glycogen post-irradiation was highest in lung (H460) and breast (MCF7) tumor cells compared to prostate (LNCaP) tumor cells. In response to radiation, the appearance of this glycogen signature correlated with radiation resistance. Moreover, the buildup of glycogen was linked to the phosphorylation of GSK-3β, a canonical modulator of cell survival following radiation exposure and a key regulator of glycogen metabolism. When MCF7 cells were irradiated in the presence of the anti-diabetic drug metformin, there was a significant decrease in the amount of radiation-induced glycogen. The suppression of glycogen by metformin following radiation was associated with increased radiosensitivity. In contrast to MCF7 cells, metformin had minimal effects on both the level of glycogen in H460 cells following radiation and radiosensitivity. Our data demonstrate a novel approach of spectral monitoring by Raman spectroscopy to assess changes in the levels of intracellular glycogen as a potential marker and resistance mechanism to radiation therapy. PMID:26280348

Improved Time-Lapsed Angular Scattering Microscopy of Single Cells

NASA Astrophysics Data System (ADS)

Cannaday, Ashley E.

By measuring angular scattering patterns from biological samples and fitting them with a Mie theory model, one can estimate the organelle size distribution within many cells. Quantitative organelle sizing of ensembles of cells using this method has been well established. Our goal is to develop the methodology to extend this approach to the single cell level, measuring the angular scattering at multiple time points and estimating the non-nuclear organelle size distribution parameters. The diameters of individual organelle-size beads were successfully extracted using scattering measurements with a minimum deflection angle of 20 degrees. However, the accuracy of size estimates can be limited by the angular range detected. In particular, simulations by our group suggest that, for cell organelle populations with a broader size distribution, the accuracy of size prediction improves substantially if the minimum angle of detection angle is 15 degrees or less. The system was therefore modified to collect scattering angles down to 10 degrees. To confirm experimentally that size predictions will become more stable when lower scattering angles are detected, initial validations were performed on individual polystyrene beads ranging in diameter from 1 to 5 microns. We found that the lower minimum angle enabled the width of this delta-function size distribution to be predicted more accurately. Scattering patterns were then acquired and analyzed from single mouse squamous cell carcinoma cells at multiple time points. The scattering patterns exhibit angular dependencies that look unlike those of any single sphere size, but are well-fit by a broad distribution of sizes, as expected. To determine the fluctuation level in the estimated size distribution due to measurement imperfections alone, formaldehyde-fixed cells were measured. Subsequent measurements on live (non-fixed) cells revealed an order of magnitude greater fluctuation in the estimated sizes compared to fixed cells. With our improved and better-understood approach to single cell angular scattering, we are now capable of reliably detecting changes in organelle size predictions due to biological causes above our measurement error of 20 nm, which enables us to apply our system to future studies of the investigation of various single cell biological processes.

Single-cell analysis of pyroptosis dynamics reveals conserved GSDMD-mediated subcellular events that precede plasma membrane rupture.

PubMed

de Vasconcelos, Nathalia M; Van Opdenbosch, Nina; Van Gorp, Hanne; Parthoens, Eef; Lamkanfi, Mohamed

2018-04-17

Pyroptosis is rapidly emerging as a mechanism of anti-microbial host defense, and of extracellular release of the inflammasome-dependent cytokines interleukin (IL)-1β and IL-18, which contributes to autoinflammatory pathology. Caspases 1, 4, 5 and 11 trigger this regulated form of necrosis by cleaving the pyroptosis effector gasdermin D (GSDMD), causing its pore-forming amino-terminal domain to oligomerize and perforate the plasma membrane. However, the subcellular events that precede pyroptotic cell lysis are ill defined. In this study, we triggered primary macrophages to undergo pyroptosis from three inflammasome types and recorded their dynamics and morphology using high-resolution live-cell spinning disk confocal laser microscopy. Based on quantitative analysis of single-cell subcellular events, we propose a model of pyroptotic cell disintegration that is initiated by opening of GSDMD-dependent ion channels or pores that are more restrictive than recently proposed GSDMD pores, followed by osmotic cell swelling, commitment of mitochondria and other membrane-bound organelles prior to sudden rupture of the plasma membrane and full permeability to intracellular proteins. This study provides a dynamic framework for understanding cellular changes that occur during pyroptosis, and charts a chronological sequence of GSDMD-mediated subcellular events that define pyroptotic cell death at the single-cell level.

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.

Conceptual Challenges of the Systemic Approach in Understanding Cell Differentiation.

PubMed

Paldi, Andras

2018-01-01

The cells of a multicellular organism are derived from a single zygote and genetically identical. Yet, they are phenotypically very different. This difference is the result of a process commonly called cell differentiation. How the phenotypic diversity emerges during ontogenesis or regeneration is a central and intensely studied but still unresolved issue in biology. Cell biology is facing conceptual challenges that are frequently confused with methodological difficulties. How to define a cell type? What stability or change means in the context of cell differentiation and how to deal with the ubiquitous molecular variations seen in the living cells? What are the driving forces of the change? We propose to reframe the problem of cell differentiation in a systemic way by incorporating different theoretical approaches. The new conceptual framework is able to capture the insights made at different levels of cellular organization and considered previously as contradictory. It also provides a formal strategy for further experimental studies.

Imaging of single retinal ganglion cell with differential interference contrast microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Oh, Juyeong; Kim, Yu Jeong; Kim, Chul-Ki; Lee, Taik Jin; Seo, Mina; Lee, Seok; Woo, Deok Ha; Jun, Seong Chan; Park, Ki-Ho; Kim, Seok Hwan; Kim, Jae Hun

2017-02-01

Glaucoma is a progressive optic neuropathy, characterized by the selective loss of retinal ganglion cells (RGCs). Therefore, monitoring the change of number or morphology of RGC is essential for the early detection as well as investigation of pathophysiology of glaucoma. Since RGC layer is transparent and hyporeflective, the direct optical visualization of RGCs has not been successful so far. Therefore, glaucoma evaluation mostly depends on indirect diagnostic methods such as the evaluation of optic disc morphology or retinal nerve fiber layer thickness measurement by optical coherence tomography. We have previously demonstrated single photoreceptor cell imaging with differential interference contrast (DIC) microscopy. Herein, we successfully visualized single RGC using DIC microscopy. Since RGC layer is much less reflective than photoreceptor layer, various techniques including the control of light wavelength and bandwidth using a tunable band pass filter were adopted to reduce the chromatic aberration in z-axis for higher and clearer resolution. To verify that the imaged cells were the RGCs, the flat-mounted retina of Sprague-Dawley rat, in which the RGCs were retrogradely labeled with fluorescence, was observed by both fluorescence and DIC microscopies for direct comparison. We have confirmed that the cell images obtained by fluorescence microscopy were perfectly matched with cell images by DIC microscopy. As conclusion, we have visualized single RGC with DIC microscopy, and confirmed with fluorescence microscopy.

Photothermal monitoring of interaction of carcinoma cells with cytostatic drugs in vitro

NASA Astrophysics Data System (ADS)

Lapotko, Dmitri; Hanna, Ehab; Cannon, Martin

2003-06-01

Background/problem. Monitoring of tumor response to cancer chemotherapy and dose optimization for specific patients are the key factors for successful application of anti-tumor drugs. Using patient's tumor cells for preliminary in vitro drug screening may allow optimal selection of drug type and dose. Method. Single cell state was studied with photothermal microscope. Carcinoma cells were irradiated at 427 nm with 8 ns laser pulse with energy 30 - 40 μJ. Cell photothermal (PT) response amplitude and shape from each cell were analyzed and amount of cells that produced specific PT response was used as PT parameter. Parallel experiment included cell viability control. Results were obtained for two cytotoxic chemotherapy agents -- Platinol-aq and Adrucil. Incubation of cell suspensions for 90 min at 20 and 37°C caused changes in cell PT parameters. Reaction of carcinoma cells to the drug was very similar to reaction of hepatocytes to respiratory chain inhibition and reaction of RBC to osmotic pressure decrease. PT effect was found to be dose-dependent. PT method allows detecting drug-induced changes before cell death or morphological changes and therefore can be fast and sensitive modality for control of chemotherapy.

Application of a genetically encoded biosensor for live cell imaging of L-valine production in pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum strains.

PubMed

Mustafi, Nurije; Grünberger, Alexander; Mahr, Regina; Helfrich, Stefan; Nöh, Katharina; Blombach, Bastian; Kohlheyer, Dietrich; Frunzke, Julia

2014-01-01

The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ΔaceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor's suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ΔaceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains.

Application of a Genetically Encoded Biosensor for Live Cell Imaging of L-Valine Production in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains

PubMed Central

Mahr, Regina; Helfrich, Stefan; Nöh, Katharina; Blombach, Bastian; Kohlheyer, Dietrich; Frunzke, Julia

2014-01-01

The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ΔaceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor's suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ΔaceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains. PMID:24465669

Transient analysis of a thermal storage unit involving a phase change material

NASA Technical Reports Server (NTRS)

Griggs, E. I.; Pitts, D. R.; Humphries, W. R.

1974-01-01

The transient response of a single cell of a typical phase change material type thermal capacitor has been modeled using numerical conductive heat transfer techniques. The cell consists of a base plate, an insulated top, and two vertical walls (fins) forming a two-dimensional cavity filled with a phase change material. Both explicit and implicit numerical formulations are outlined. A mixed explicit-implicit scheme which treats the fin implicity while treating the phase change material explicitly is discussed. A band algorithmic scheme is used to reduce computer storage requirements for the implicit approach while retaining a relatively fine grid. All formulations are presented in dimensionless form thereby enabling application to geometrically similar problems. Typical parametric results are graphically presented for the case of melting with constant heat input to the base of the cell.

The Temporal Pattern of Changes in Serum Biomarker Levels Reveals Complex and Dynamically Changing Pathologies after Exposure to a Single Low-Intensity Blast in Mice

PubMed Central

Ahmed, Farid; Plantman, Stefan; Cernak, Ibolja; Agoston, Denes V.

2015-01-01

Time-dependent changes in blood-based protein biomarkers can help identify the ­pathological processes in blast-induced traumatic brain injury (bTBI), assess injury severity, and monitor disease progression. We obtained blood from control and injured mice (exposed to a single, low-intensity blast) at 2-h, 1-day, 1–week, and 1-month post-injury. We then determined the serum levels of biomarkers related to metabolism (4-HNE, HIF-1α, ceruloplasmin), vascular function (AQP1, AQP4, VEGF, vWF, Flk-1), inflammation (OPN, CINC1, fibrinogen, MIP-1a, OX-44, p38, MMP-8, MCP-1 CCR5, CRP, galectin-1), cell adhesion and the extracellular matrix (integrin α6, TIMP1, TIMP4, Ncad, connexin-43), and axonal (NF-H, Tau), neuronal (NSE, CK-BB) and glial damage (GFAP, S100β, MBP) at various post-injury time points. Our findings indicate that the exposure to a single, low-intensity blast results in metabolic and vascular changes, altered cell adhesion, and axonal and neuronal injury in the mouse model of bTBI. Interestingly, serum levels of several inflammatory and astroglial markers were either unchanged or elevated only during the acute and subacute phases of injury. Conversely, serum levels of the majority of biomarkers related to metabolic and vascular functions, cell adhesion, as well as neuronal and axonal damage remained elevated at the termination of the experiment (1 month), indicating long-term systemic and cerebral alterations due to blast. Our findings show that the exposure to a single, low-intensity blast induces complex pathological processes with distinct temporal profiles. Hence, monitoring serum biomarker levels at various post-injury time points may provide enhanced diagnostics in blast-related neurological and multi-system deficits. PMID:26124743

Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells.

PubMed

Terao, Kyohei; Gel, Murat; Okonogi, Atsuhito; Fuke, Ariko; Okitsu, Teru; Tada, Takashi; Suzuki, Takaaki; Nagamatsu, Shinya; Washizu, Masao; Kotera, Hidetoshi

2014-02-18

In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca(2+)] change in the β-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of β-cells.

Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells

PubMed Central

Terao, Kyohei; Gel, Murat; Okonogi, Atsuhito; Fuke, Ariko; Okitsu, Teru; Tada, Takashi; Suzuki, Takaaki; Nagamatsu, Shinya; Washizu, Masao; Kotera, Hidetoshi

2014-01-01

In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca2+] change in the β-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of β-cells. PMID:24535122

Quantifying hydrostatic pressure in plant cells by using indentation with an atomic force microscope.

PubMed

Beauzamy, Léna; Derr, Julien; Boudaoud, Arezki

2015-05-19

Plant cell growth depends on a delicate balance between an inner drive-the hydrostatic pressure known as turgor-and an outer restraint-the polymeric wall that surrounds a cell. The classical technique to measure turgor in a single cell, the pressure probe, is intrusive and cannot be applied to small cells. In order to overcome these limitations, we developed a method that combines quantification of topography, nanoindentation force measurements, and an interpretation using a published mechanical model for the pointlike loading of thin elastic shells. We used atomic force microscopy to estimate the elastic properties of the cell wall and turgor pressure from a single force-depth curve. We applied this method to onion epidermal peels and quantified the response to changes in osmolality of the bathing solution. Overall our approach is accessible and enables a straightforward estimation of the hydrostatic pressure inside a walled cell. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Finite Element Analysis of Single Cell Stiffness Measurements Using PZT-Integrated Buckling Nanoneedles

PubMed Central

Rad, Maryam Alsadat; Tijjani, Auwal Shehu; Ahmad, Mohd Ridzuan; Auwal, Shehu Muhammad

2016-01-01

This paper proposes a new technique for real-time single cell stiffness measurement using lead zirconate titanate (PZT)-integrated buckling nanoneedles. The PZT and the buckling part of the nanoneedle have been modelled and validated using the ABAQUS software. The two parts are integrated together to function as a single unit. After calibration, the stiffness, Young’s modulus, Poisson’s ratio and sensitivity of the PZT-integrated buckling nanoneedle have been determined to be 0.7100 N·m−1, 123.4700 GPa, 0.3000 and 0.0693 V·m·N−1, respectively. Three Saccharomyces cerevisiae cells have been modelled and validated based on compression tests. The average global stiffness and Young’s modulus of the cells are determined to be 10.8867 ± 0.0094 N·m−1 and 110.7033 ± 0.0081 MPa, respectively. The nanoneedle and the cell have been assembled to measure the local stiffness of the single Saccharomyces cerevisiae cells The local stiffness, Young’s modulus and PZT output voltage of the three different size Saccharomyces cerevisiae have been determined at different environmental conditions. We investigated that, at low temperature the stiffness value is low to adapt to the change in the environmental condition. As a result, Saccharomyces cerevisiae becomes vulnerable to viral and bacterial attacks. Therefore, the proposed technique will serve as a quick and accurate process to diagnose diseases at early stage in a cell for effective treatment. PMID:28025571

The swimming behavior of flagellated bacteria in viscous and viscoelastic media

NASA Astrophysics Data System (ADS)

Qu, Zijie; Henderikx, Rene; Breuer, Kenneth

2016-11-01

The motility of bacteria E.coli in viscous and viscoelastic fluids has been widely studied although full understanding remains elusive. The swimming mode of wild-type E.coli is well-described by a run-and-tumble sequence in which periods of straight swimming at a constant speed are randomly interrupted by a tumble, defined as a sudden change of direction with a very low speed. Using a tracking microscope, we follow cells for extended periods of time and find that the swimming behavior can be more complex, and can include a wider variety of behaviors including a "slow random walk" in which the cells move at relatively low speed without the characteristic run. Significant variation between individual cells is observed, and furthermore, a single cell can change its motility during the course of a tracking event. Changing the viscosity and viscoelasticy of the swimming media also has profound effects on the average swimming speed and run-tumble nature of the cell motility, including changing the distribution, duration of tumbling and slow random walk events. The reasons for these changes are explained using a Purcell-style resistive force model for the cell and flagellar behavior as well as model for the changes in flagellar bundling in different fluid viscosities. National Science Foundation.

Acute adaptation of the vestibuloocular reflex: signal processing by floccular and ventral parafloccular Purkinje cells.

PubMed

Hirata, Y; Highstein, S M

2001-05-01

The gain of the vertical vestibuloocular reflex (VVOR), defined as eye velocity/head velocity was adapted in squirrel monkeys by employing visual-vestibular mismatch stimuli. VVOR gain, measured in the dark, could be trained to values between 0.4 and 1.5. Single-unit activity of vertical zone Purkinje cells was recorded from the flocculus and ventral paraflocculus in alert squirrel monkeys before and during the gain change training. Our goal was to evaluate the site(s) of learning of the gain change. To aid in the evaluation, a model of the vertical optokinetic reflex (VOKR) and VVOR was constructed consisting of floccular and nonfloccular systems divided into subsystems based on the known anatomy and input and output parameters. Three kinds of input to floccular Purkinje cells via mossy fibers were explicitly described, namely vestibular, visual (retinal slip), and efference copy of eye movement. The characteristics of each subsystem (gain and phase) were identified at different VOR gains by reconstructing single-unit activity of Purkinje cells during VOKR and VVOR with multiple linear regression models consisting of sensory input and motor output signals. Model adequacy was checked by evaluating the residual following the regressions and by predicting Purkinje cells' activity during visual-vestibular mismatch paradigms. As a result, parallel changes in identified characteristics with VVOR adaptation were found in the prefloccular/floccular subsystem that conveys vestibular signals and in the nonfloccular subsystem that conveys vestibular signals, while no change was found in other subsystems, namely prefloccular/floccular subsystems conveying efference copy or visual signals, nonfloccular subsystem conveying visual signals, and postfloccular subsystem transforming Purkinje cell activity to eye movements. The result suggests multiple sites for VVOR motor learning including both flocculus and nonflocculus pathways. The gain change in the nonfloccular vestibular subsystem was in the correct direction to cause VOR gain adaptation while the change in the prefloccular/floccular vestibular subsystem was incorrect (anti-compensatory). This apparent incorrect directional change might serve to prevent instability of the VOR caused by positive feedback via the efference copy pathway.

Assessment of changes in plasma hemoglobin and potassium levels in red cell units during processing and storage.

PubMed

Saini, Nishant; Basu, Sabita; Kaur, Ravneet; Kaur, Jasbinder

2015-06-01

Red cell units undergo changes during storage and processing. The study was planned to assess plasma potassium, plasma hemoglobin, percentage hemolysis during storage and to determine the effects of outdoor blood collection and processing on those parameters. Blood collection in three types of blood storage bags was done - single CPDA bag (40 outdoor and 40 in-house collection), triple CPD + SAGM bag (40 in-house collection) and quadruple CPD + SAGM bag with integral leukoreduction filter (40 in-house collection). All bags were sampled on day 0 (day of collection), day 1 (after processing), day 7, day 14 and day 28 for measurement of percentage hemolysis and potassium levels in the plasma of bag contents. There was significant increase in percentage hemolysis, plasma hemoglobin and plasma potassium level in all the groups during storage (p < 0.001). No significant difference was found between any parameter analyzed for outdoor and in-house collected single CPDA red cell units. There was significant lower percentage hemolysis (p < 0.001) and potassium (day 7 to day 14 - p < 0.05 and day 14 to day 28 - p < 0.001) in red cell units from day 7 onward until day 28 of storage in the leukoreduced quadruple bag as compared to the triple bag. The in-house single CPDA red cell units showed significantly more hemolysis (p < 0.001) as compared to the triple bags with SAGM additive solution after 28 days of storage. There is gradual increase in plasma hemoglobin and plasma potassium levels during the storage of red blood cells. Blood collection can be safely undertaken in outdoor blood donation camps even in hot summer months in monitored blood transport boxes. SAGM additive solution decreases the red cell hemolysis and allows extended storage of red cells. Prestorage leukoreduction decreases the red cell hemolysis and improves the quality of blood. Copyright © 2015 Elsevier Ltd. All rights reserved.

Experimental determination of the magnetic dipole moment of candidate magnetoreceptor cells in trout

NASA Astrophysics Data System (ADS)

Winklhofer, M.; Eder, S.; Cadioiu, H.; McNaughton, P. A.; Kirschvink, J. L.

2011-12-01

Based on histological, physiological, and physical evidence, Walker et al (1997) and Diebel et al (2000) have identified distinctive cells in the olfactory epithelium of the rainbow trout (Onchorynchus mykiss) that contain magnetite and are closely associated with neurons that respond to changes in magnetic field. To put biophysical constraints on the possible transduction mechanism of magnetic signals, and in particular, to find out if the intracellular magnet is free to rotate or rather firmly anchored within the cell body, we have studied the magneto-mechanical response of isolated candidate receptor cells in suspension using a light microscope equipped with two pairs of Helmholtz coils. From the characteristic re-orientation time of suspended cells after a change in magnetic field direction, we have determined the magnitude of the magnetic dipole moment of the cells in function of the external field strength (0.4 mT to 3.2 mT) in order to find out whether or not the natural magnetic moment is remanence-based or induced (i.e., single-domain vs. superparamagnetic/multi-domain). Results: 1) The mechanical response of isolated cells to a change in magnetic field direction was always immediate, irrespective of the direction of change, which implies that the intracellular magnet is not free to rotate in the cell, but rather rigidly attached, probably to the plasma membrane, which is also suggested by our confocal fluorescence-microscope studies. 2) The cellular dipole moment turned out to be independent of the external field strength. Thus, the natural magnetic dipole moment is based on magnetic remanence, which points to single-domain particles and corroborates the results by Diebel et al (2000), who obtained switching fields consistent with single-domain magnetite. 3). The magnetic dipole moment is found to be of the order of several tens of fAm2, which greatly exceeds previous estimates (0.5 fAm2), and thus is similar to values reported for the most strongly magnetic types of magnetotactic bacteria (Hanzlik et al. 2002). Our results demonstrate that the magnetically identified cells clearly meet the physical requirements for a magnetoreceptor capable of rapidly detecting small changes in the external magnetic field. Diebel, C.E., Proksch, R., Green, C.R., Neilson, P. & Walker, M.M. (2000) Magnetite defines a vertebrate magnetoreceptor. Nature 406, 299-302. Hanzlik, M., Winklhofer, M., Petersen, N. (2002) Pulsed-field-remanence measurements on individual magnetotactic bacteria, J. Magn. Magn. Mater., 248(2), 258-267. Walker, M.M., Diebel, C.E., Haugh, C.V., Pankhurst, P.M., Montgomery, J.C. & Green, C.R. (1997) Structure and function of the vertebrate magnetic sense. Nature 390, 371-376.

In vivo collective cell migration requires an LPAR2-dependent increase in tissue fluidity

PubMed Central

Kuriyama, Sei; Theveneau, Eric; Benedetto, Alexandre; Parsons, Maddy; Tanaka, Masamitsu; Charras, Guillaume; Kabla, Alexandre

2014-01-01

Collective cell migration (CCM) and epithelial–mesenchymal transition (EMT) are common to cancer and morphogenesis, and are often considered to be mutually exclusive in spite of the fact that many cancer and embryonic cells that have gone through EMT still cooperate to migrate collectively. Here we use neural crest (NC) cells to address the question of how cells that have down-regulated cell–cell adhesions can migrate collectively. NC cell dissociation relies on a qualitative and quantitative change of the cadherin repertoire. We found that the level of cell–cell adhesion is precisely regulated by internalization of N-cadherin downstream of lysophosphatidic acid (LPA) receptor 2. Rather than promoting the generation of single, fully mesenchymal cells, this reduction of membrane N-cadherin only triggers a partial mesenchymal phenotype. This intermediate phenotype is characterized by an increase in tissue fluidity akin to a solid-like–to–fluid-like transition. This change of plasticity allows cells to migrate under physical constraints without abolishing cell cooperation required for collectiveness. PMID:25002680

Quantitative phase imaging of biological cells and tissues using singleshot white light interference microscopy and phase subtraction method for extended range of measurement

NASA Astrophysics Data System (ADS)

Mehta, Dalip Singh; Sharma, Anuradha; Dubey, Vishesh; Singh, Veena; Ahmad, Azeem

2016-03-01

We present a single-shot white light interference microscopy for the quantitative phase imaging (QPI) of biological cells and tissues. A common path white light interference microscope is developed and colorful white light interferogram is recorded by three-chip color CCD camera. The recorded white light interferogram is decomposed into the red, green and blue color wavelength component interferograms and processed it to find out the RI for different color wavelengths. The decomposed interferograms are analyzed using local model fitting (LMF)" algorithm developed for reconstructing the phase map from single interferogram. LMF is slightly off-axis interferometric QPI method which is a single-shot method that employs only a single image, so it is fast and accurate. The present method is very useful for dynamic process where path-length changes at millisecond level. From the single interferogram a wavelength-dependent quantitative phase imaging of human red blood cells (RBCs) are reconstructed and refractive index is determined. The LMF algorithm is simple to implement and is efficient in computation. The results are compared with the conventional phase shifting interferometry and Hilbert transform techniques.

Strategies for Increasing Pancreatic Tumor Immunogenicity

PubMed Central

Johnson, Burles A.; Yarchoan, Mark; Lee, Valerie; Laheru, Daniel A.; Jaffee, Elizabeth M.

2017-01-01

Immunotherapy has changed the standard of care for multiple deadly cancers including lung, head and neck, gastric, and some colorectal cancers. However, single agent immunotherapy has had little effect in pancreatic adenocarcinoma (PDAC). Increasing evidence suggests that the PDAC microenvironment is comprised of an intricate network of signals between immune cells, PDAC cells, and stroma, resulting in an immunosuppressive environment resistant to single agent immunotherapies. In this review, we discuss differences between immunotherapy sensitive cancers and PDAC, the complex interactions between PDAC stroma and suppressive tumor infiltrating cells that facilitate PDAC development and progression, the immunologic targets within these complex networks that are drugable, and data supporting combination drug approaches that modulate multiple PDAC signals, which should lead to improved clinical outcomes. PMID:28373364

Studying molecular changes during gravity perception and response in a single cell.

PubMed

Cannon, Ashley E; Salmi, Mari L; Bushart, Thomas J; Roux, Stanley J

2015-01-01

Early studies revealed a highly predictable pattern of gravity-directed growth and development in Ceratopteris richardii spores. This makes the spores a valuable model system for the study of how a single cell senses and responds to the force of gravity. Gravity regulates both the direction and magnitude of a trans-cell calcium current in germinating spores, and the orientation of this current predicts the polarization of spore development. Molecular techniques have been developed to evaluate the transcriptomic and proteomic profiles of spores before and after gravity establishes the polarity of their development. Here we describe these techniques, along with protocols for sterilizing the spores, sowing them in a solid or liquid growth media, and evaluating germination.

Partial functional redundancy of MreB isoforms, MreB, Mbl and MreBH, in cell morphogenesis of Bacillus subtilis.

PubMed

Kawai, Yoshikazu; Asai, Kei; Errington, Jeffery

2009-08-01

MreB proteins are bacterial actin homologues thought to have a role in cell shape determination by positioning the cell wall synthetic machinery. Many bacteria, particularly Gram-positives, have more than one MreB isoform. Bacillus subtilis has three, MreB, Mbl and MreBH, which colocalize in a single helical structure. We now show that the helical pattern of peptidoglycan (PG) synthesis in the cylindrical part of the rod-shaped cell is governed by the redundant action of the three MreB isoforms. Single mutants for any one of mreB isoforms can still incorporate PG in a helical pattern and generate a rod shape. However, after depletion of MreB in an mbl mutant (or depletion of all three isoforms) lateral wall PG synthesis was impaired and the cells became spherical and lytic. Overexpression of any one of the MreB isoforms overcame the lethality as well as the defects in lateral PG synthesis and cell shape. Furthermore, MreB and Mbl can associate with the peptidoglycan biosynthetic machinery independently. However, no single MreB isoform was able to support normal growth under various stress conditions, suggesting that the multiple isoforms are used to allow cells to maintain proper growth and morphogenesis under changing and sometimes adverse conditions.

Monitoring single-cell bioenergetics via the coarsening of emulsion droplets

PubMed Central

Boitard, L.; Cottinet, D.; Kleinschmitt, C.; Bremond, N.; Baudry, J.; Yvert, G.; Bibette, J.

2012-01-01

Microorganisms are widely used to generate valuable products, and their efficiency is a major industrial focus. Bioreactors are typically composed of billions of cells, and available measurements only reflect the overall performance of the population. However, cells do not equally contribute, and process optimization would therefore benefit from monitoring this intrapopulation diversity. Such monitoring has so far remained difficult because of the inability to probe concentration changes at the single-cell level. Here, we unlock this limitation by taking advantage of the osmotically driven water flux between a droplet containing a living cell toward surrounding empty droplets, within a concentrated inverse emulsion. With proper formulation, excreted products are far more soluble within the continuous hydrophobic phase compared to initial nutrients (carbohydrates and salts). Fast diffusion of products induces an osmotic mismatch, which further relaxes due to slower diffusion of water through hydrophobic interfaces. By measuring droplet volume variations, we can deduce the metabolic activity down to isolated single cells. As a proof of concept, we present the first direct measurement of the maintenance energy of individual yeast cells. This method does not require any added probes and can in principle apply to any osmotically sensitive bioactivity, opening new routes for screening, and sorting large libraries of microorganisms and biomolecules. PMID:22538813

Hypericin-mediated photocytotoxic effect on HT-29 adenocarcinoma cells is reduced by light fractionation with longer dark pause between two unequal light doses.

PubMed

Sacková, Veronika; Kuliková, Lucia; Mikes, Jaromír; Kleban, Ján; Fedorocko, Peter

2005-01-01

The present study demonstrates the in vitro effect of hypericin-mediated PDT with fractionated light delivery. Cells were photosensitized with unequal light fractions separated by dark intervals (1 or 6 h). We compared the changes in viability, cell number, survival, apoptosis and cell cycle on HT-29 cells irradiated with a single light dose (12 J/cm(2)) to the fractionated light delivery (1 + 11 J/cm(2)) 24 and 48 h after photodynamic treatment. We found that a fractionated light regime with a longer dark period resulted in a decrease of hypericin cytotoxicity. Both cell number and survival were higher after light sensitization with a 6-h dark interval. DNA fragmentation occurred after a single light-dose application, but in contrast no apoptotic DNA formation was detected with a 6-h dark pause. After fractionation the percentage of cells in the G1 phase of the cell cycle was increased, while the proportion of cells in the G2 phase decreased as compared to a single light-dose application, i.e. both percentage of cells in the G1 and G2 phase of the cell cycle were near control levels. We presume that the longer dark interval after the irradiation of cells by first light dose makes them resistant to the effect of the second illumination. These findings confirm that the light application scheme together with other photodynamic protocol components is crucial for the photocytotoxicity of hypericin.

Automated multi-slice extracellular and patch-clamp experiments using the WinLTP data acquisition system with automated perfusion control

PubMed Central

Anderson, William W.; Fitzjohn, Stephen M.; Collingridge, Graham L.

2012-01-01

WinLTP is a data acquisition program for studying long-term potentiation (LTP) and other aspects of synaptic function. Earlier versions of WinLTP (J. Neurosci. Methods, 162:346–356, 2007) provided automated electrical stimulation and data acquisition capable of running nearly an entire synaptic plasticity experiment, with the primary exception that perfusion solutions had to be changed manually. This automated stimulation and acquisition was done by using ‘Sweep’, ‘Loop’ and ‘Delay’ events to build scripts using the ‘Protocol Builder’. However, this did not allow automatic changing of many solutions while running multiple slice experiments, or solution changing when this had to be performed rapidly and with accurate timing during patch-clamp experiments. We report here the addition of automated perfusion control to WinLTP. First, perfusion change between sweeps is enabled by adding the ‘Perfuse’ event to Protocol Builder scripting and is used in slice experiments. Second, fast perfusion changes during as well as between sweeps is enabled by using the Perfuse event in the protocol scripts to control changes between sweeps, and also by changing digital or analog output during a sweep and is used for single cell single-line perfusion patch-clamp experiments. The addition of stepper control of tube placement allows dual- or triple-line perfusion patch-clamp experiments for up to 48 solutions. The ability to automate perfusion changes and fully integrate them with the already automated stimulation and data acquisition goes a long way toward complete automation of multi-slice extracellularly recorded and single cell patch-clamp experiments. PMID:22524994

A single-cell correlative nanoelectromechanosensing approach to detect cancerous transformation: monitoring the function of F-actin microfilaments in the modulation of the ion channel activity

NASA Astrophysics Data System (ADS)

AbdolahadThe Authors With Same Contributions., Mohammad; Saeidi, Ali; Janmaleki, Mohsen; Mashinchian, Omid; Taghinejad, Mohammad; Taghinejad, Hossein; Azimi, Soheil; Mahmoudi, Morteza; Mohajerzadeh, Shams

2015-01-01

Cancerous transformation may be dependent on correlation between electrical disruptions in the cell membrane and mechanical disruptions of cytoskeleton structures. Silicon nanotube (SiNT)-based electrical probes, as ultra-accurate signal recorders with subcellular resolution, may create many opportunities for fundamental biological research and biomedical applications. Here, we used this technology to electrically monitor cellular mechanosensing. The SiNT probe was combined with an electrically activated glass micropipette aspiration system to achieve a new cancer diagnostic technique that is based on real-time correlation between mechanical and electrical behaviour of single cells. Our studies demonstrated marked changes in the electrical response following increases in the mechanical aspiration force in healthy cells. In contrast, such responses were extremely weak for malignant cells. Confocal microscopy results showed the impact of actin microfilament remodelling on the reduction of the electrical response for aspirated cancer cells due to the significant role of actin in modulating the ion channel activity in the cell membrane.Cancerous transformation may be dependent on correlation between electrical disruptions in the cell membrane and mechanical disruptions of cytoskeleton structures. Silicon nanotube (SiNT)-based electrical probes, as ultra-accurate signal recorders with subcellular resolution, may create many opportunities for fundamental biological research and biomedical applications. Here, we used this technology to electrically monitor cellular mechanosensing. The SiNT probe was combined with an electrically activated glass micropipette aspiration system to achieve a new cancer diagnostic technique that is based on real-time correlation between mechanical and electrical behaviour of single cells. Our studies demonstrated marked changes in the electrical response following increases in the mechanical aspiration force in healthy cells. In contrast, such responses were extremely weak for malignant cells. Confocal microscopy results showed the impact of actin microfilament remodelling on the reduction of the electrical response for aspirated cancer cells due to the significant role of actin in modulating the ion channel activity in the cell membrane. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr06102k

C. elegans STK39/SPAK ortholog-mediated inhibition of ClC anion channel activity is regulated by WNK-independent ERK kinase signaling

PubMed Central

Falin, Rebecca A.; Miyazaki, Hiroaki

2011-01-01

Mammalian Ste20-like proline/alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinases phosphorylate and regulate cation-coupled Cl− cotransporter activity in response to cell volume changes. SPAK and OSR1 are activated via phosphorylation by upstream with-no-lysine (WNK) kinases. In Caenorhabditis elegans, the SPAK/OSR1 ortholog germinal center kinase (GCK)-3 binds to and regulates the activity of the cell volume- and meiotic cell cycle-dependent ClC anion channel CLH-3b. We tested the hypothesis that WNK kinases function in the GCK-3/CLH-3b signaling cascade. CLH-3b heterologously expressed in human embryonic kidney (HEK) cells was unaffected by coexpression with the single C. elegans WNK kinase, WNK-1, or kinase-dead WNK-1 dominant-negative mutants. RNA interference (RNAi) knockdown of the single Drosophila WNK kinase had no effect on the activity of CLH-3b expressed in Drosophila S2 cells. Similarly, RNAi silencing of C. elegans WNK-1 had no effect on basal or cell volume-sensitive activity of CLH-3b expressed endogenously in worm oocytes. Previous yeast 2-hybrid studies suggested that ERK kinases may function upstream of GCK-3. Pharmacological inhibition of ERK signaling disrupted CLH-3b activity in HEK cells in a GCK-3-dependent manner. RNAi silencing of the C. elegans ERK kinase MPK-1 or the ERK phosphorylating/activating kinase MEK-2 constitutively activated native CLH-3b. MEK-2 and MPK-1 play important roles in regulating the meiotic cell cycle in C. elegans oocytes. Cell cycle-dependent changes in MPK-1 correlate with the pattern of CLH-3b activation observed during oocyte meiotic maturation. We postulate that MEK-2/MPK-1 functions upstream from GCK-3 to regulate its activity during cell volume and meiotic cell cycle changes. PMID:21160027

Elastic light scattering from single cells: orientational dynamics in optical trap.

PubMed

Watson, Dakota; Hagen, Norbert; Diver, Jonathan; Marchand, Philippe; Chachisvilis, Mirianas

2004-08-01

Light-scattering diagrams (phase functions) from single living cells and beads suspended in an optical trap were recorded with 30-ms time resolution. The intensity of the scattered light was recorded over an angular range of 0.5-179.5 degrees using an optical setup based on an elliptical mirror and rotating aperture. Experiments revealed that light-scattering diagrams from biological cells exhibit significant and complex time dependence. We have attributed this dependence to the cell's orientational dynamics within the trap. We have also used experimentally measured phase function information to calculate the time dependence of the optical radiation pressure force on the trapped particle and show how it changes depending on the orientation of the particle. Relevance of these experiments to potential improvement in the sensitivity of label-free flow cytometry is discussed.

Differential histone modification and protein expression associated with cell wall removal and regeneration in rice (Oryza sativa).

PubMed

Tan, Feng; Zhang, Kangling; Mujahid, Hana; Verma, Desh Pal S; Peng, Zhaohua

2011-02-04

The cell wall is a critical extracellular structure that provides protection and structural support in plant cells. To study the biological function of the cell wall and the regulation of cell wall resynthesis, we examined cellular responses to enzymatic removal of the cell wall in rice (Oryza sativa) suspension cells using proteomic approaches. We find that removal of cell wall stimulates cell wall synthesis from multiple sites in protoplasts instead of from a single site as in cytokinesis. Nucleus DAPI stain and MNase digestion further show that removal of the cell wall is concomitant with substantial chromatin reorganization. Histone post-translational modification studies using both Western blots and isotope labeling assisted quantitative mass spectrometry analyses reveal that substantial histone modification changes, particularly H3K18(AC) and H3K23(AC), are associated with the removal and regeneration of the cell wall. Label-free quantitative proteome analyses further reveal that chromatin associated proteins undergo dramatic changes upon removal of the cell wall, along with cytoskeleton, cell wall metabolism, and stress-response proteins. This study demonstrates that cell wall removal is associated with substantial chromatin change and may lead to stimulation of cell wall synthesis using a novel mechanism.

Connectivity of the intracytoplasmic membrane of Rhodobacter sphaeroides: a functional approach.

PubMed

Verméglio, André; Lavergne, Jérôme; Rappaport, Fabrice

2016-01-01

The photosynthetic apparatus in the bacterium Rhodobacter sphaeroides is mostly present in intracytoplasmic membrane invaginations. It has long been debated whether these invaginations remain in topological continuity with the cytoplasmic membrane, or form isolated chromatophore vesicles. This issue is revisited here by functional approaches. The ionophore gramicidin was used as a probe of the relative size of the electro-osmotic units in isolated chromatophores, spheroplasts, or intact cells. The decay of the membrane potential was monitored from the electrochromic shift of carotenoids. The half-time of the decay induced by a single channel in intact cells was about 6 ms, thus three orders of magnitude slower than in isolated chromatophores. In spheroplasts obtained by lysis of the cell wall, the single channel decay was still slower (~23 ms) and the sensitivity toward the gramicidin concentration was enhanced 1,000-fold with respect to isolated chromatophores. These results indicate that the area of the functional membrane in cells or spheroplasts is about three orders of magnitude larger than that of isolated chromatophores. Intracytoplasmic vesicles, if present, could contribute to at most 10% of the photosynthetic apparatus in intact cells of Rba. sphaeroides. Similar conclusions were obtained from the effect of a ∆pH-induced diffusion potential in intact cells. This caused a large electrochromic response of carotenoids, of similar amplitude as the light-induced change, indicating that most of the system is sensitive to a pH change of the external medium. A single internal membrane and periplasmic space may offer significant advantages concerning renewal of the photosynthetic apparatus and reallocation of the components shared with other bioenergetic pathways.

A genetically encoded tool kit for manipulating and monitoring membrane phosphatidylinositol 4,5-bisphosphate in intact cells.

PubMed

Hertel, Fabian; Switalski, Agathe; Mintert-Jancke, Elisa; Karavassilidou, Katharina; Bender, Kirsten; Pott, Lutz; Kienitz, Marie-Cécile

2011-01-01

Most ion channels are regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) in the cell membrane by diverse mechanisms. Important molecular tools to study ion channel regulation by PtdIns(4,5)P(2) in living cells have been developed in the past. These include fluorescent PH-domains as sensors for Förster resonance energy transfer (FRET), to monitor changes in plasma membrane(.) For controlled and reversible depletion of PtdIns(4,5)P(2), voltage-sensing phosphoinositide phosphatases (VSD) have been demonstrated as a superior tool, since they are independent of cellular signaling pathways. Combining these methods in intact cells requires multiple transfections. We used self-cleaving viral 2A-peptide sequences for adenovirus driven expression of the PH-domain of phospholipase-Cδ1 (PLCδ1) fused to ECFP and EYFP respectively and Ciona intestinalis VSP (Ci-VSP), from a single open reading frame (ORF) in adult rat cardiac myocytes. Expression and correct targeting of ECFP-PH-PLCδ1(,) EYFP-PH-PLCδ1, and Ci-VSP from a single tricistronic vector containing 2A-peptide sequences first was demonstrated in HEK293 cells by voltage-controlled FRET measurements and Western blotting. Adult rat cardiac myocytes expressed Ci-VSP and the two fluorescent PH-domains within 4 days after gene transfer using the vector integrated into an adenoviral construct. Activation of Ci-VSP by depolarization resulted in rapid changes in FRET ratio indicating depletion of PtdIns(4,5)P(2) in the plasma membrane. This was paralleled by inhibition of endogenous G protein activated K(+) (GIRK) current. By comparing changes in FRET and current, a component of GIRK inhibition by adrenergic receptors unrelated to depletion of PtdIns(4,5)P(2) was identified. Expression of a FRET sensor pair and Ci-VSP from a single ORF provides a useful approach to study regulation of ion channels by phosphoinositides in cell lines and transfection-resistant postmitotic cells. Generally, adenoviral constructs containing self-cleaving 2A-peptide sequences are highly suited for simultaneous transfer of multiple genes in adult cardiac myocytes.

Terbutaline causes immobilization of single β2-adrenergic receptor-ligand complexes in the plasma membrane of living A549 cells as revealed by single-molecule microscopy

NASA Astrophysics Data System (ADS)

Sieben, Anne; Kaminski, Tim; Kubitscheck, Ulrich; Häberlein, Hanns

2011-02-01

G-protein-coupled receptors are important targets for various drugs. After signal transduction, regulatory processes, such as receptor desensitization and internalization, change the lateral receptor mobility. In order to study the lateral diffusion of β2-adrenergic receptors (β2AR) complexed with fluorescently labeled noradrenaline (Alexa-NA) in plasma membranes of A549 cells, trajectories of single receptor-ligand complexes were monitored using single-particle tracking. We found that a fraction of 18% of all β2ARs are constitutively immobile. About 2/3 of the β2ARs moved with a diffusion constant of D2 = 0.03+/-0.001 μm2/s and about 17% were diffusing five-fold faster (D3 = 0.15+/-0.02 μm2/s). The mobile receptors moved within restricted domains and also showed a discontinuous diffusion behavior. Analysis of the trajectory lengths revealed two different binding durations with τ1 = 77+/-1 ms and τ2 = 388+/-11 ms. Agonistic stimulation of the β2AR-Alexa-NA complexes with 1 μM terbutaline caused immobilization of almost 50% of the receptors within 35 min. Simultaneously, the mean area covered by the mobile receptors decreased significantly. Thus, we demonstrated that agonistic stimulation followed by cell regulatory processes results in a change in β2AR mobility suggesting that different receptor dynamics characterize different receptor states.

Genotoxicity of endosseous implants using two cellular lineages in vitro.

PubMed

Matsumoto, Mariza; Filho, Hugo Nary; Ferrari, Raquel; Fernandes, Kristianne; Renno, Ana Claudia; Ribeiro, Daniel

2014-02-01

The genotoxic potential of corrosion eluates obtained from a single dental implant using murine fibroblasts or osteoblasts cells in vitro by the single-cell gel (comet) assay was examined. A single commercially available dental implant (Biotechnology) was eluted in a solution consisting of equal amounts of acetic acid and sodium chloride (0.1 M) for 1, 3, 7, 14, and 21 days. Murine fibroblast or osteoblast cultures were then exposed to all corrosion eluates obtained from endosseous dental implants for 30 minutes at 37°C. The results suggest that none of the eluates produced genotoxic changes in murine fibroblasts regardless of the length of exposure to the eluate. Similarly, no genotoxicity was found in osteoblasts. The results suggest that the dental implant eluates tested in this study did not induce genetic damage as depicted by the single-cell gel (comet) assay. Because DNA damage is an important event during oncogenesis, this study represents a relevant contribution to estimate the real risks to the cellular system induced by the corrosion products of a dental implant.

Viscoelastic properties of cell walls of single living plant cells determined by dynamic nanoindentation

PubMed Central

Hayot, Céline M.; Forouzesh, Elham; Goel, Ashwani; Avramova, Zoya; Turner, Joseph A.

2012-01-01

Plant development results from controlled cell divisions, structural modifications, and reorganizations of the cell wall. Thereby, regulation of cell wall behaviour takes place at multiple length scales involving compositional and architectural aspects in addition to various developmental and/or environmental factors. The physical properties of the primary wall are largely determined by the nature of the complex polymer network, which exhibits time-dependent behaviour representative of viscoelastic materials. Here, a dynamic nanoindentation technique is used to measure the time-dependent response and the viscoelastic behaviour of the cell wall in single living cells at a micron or sub-micron scale. With this approach, significant changes in storage (stiffness) and loss (loss of energy) moduli are captured among the tested cells. The results reveal hitherto unknown differences in the viscoelastic parameters of the walls of same-age similarly positioned cells of the Arabidopsis ecotypes (Col 0 and Ws 2). The technique is also shown to be sensitive enough to detect changes in cell wall properties in cells deficient in the activity of the chromatin modifier ATX1. Extensive computational modelling of the experimental measurements (i.e. modelling the cell as a viscoelastic pressure vessel) is used to analyse the influence of the wall thickness, as well as the turgor pressure, at the positions of our measurements. By combining the nanoDMA technique with finite element simulations quantifiable measurements of the viscoelastic properties of plant cell walls are achieved. Such techniques are expected to find broader applications in quantifying the influence of genetic, biological, and environmental factors on the nanoscale mechanical properties of the cell wall. PMID:22291130

Digital biology and chemistry.

PubMed

Witters, Daan; Sun, Bing; Begolo, Stefano; Rodriguez-Manzano, Jesus; Robles, Whitney; Ismagilov, Rustem F

2014-09-07

This account examines developments in "digital" biology and chemistry within the context of microfluidics, from a personal perspective. Using microfluidics as a frame of reference, we identify two areas of research within digital biology and chemistry that are of special interest: (i) the study of systems that switch between discrete states in response to changes in chemical concentration of signals, and (ii) the study of single biological entities such as molecules or cells. In particular, microfluidics accelerates analysis of switching systems (i.e., those that exhibit a sharp change in output over a narrow range of input) by enabling monitoring of multiple reactions in parallel over a range of concentrations of signals. Conversely, such switching systems can be used to create new kinds of microfluidic detection systems that provide "analog-to-digital" signal conversion and logic. Microfluidic compartmentalization technologies for studying and isolating single entities can be used to reconstruct and understand cellular processes, study interactions between single biological entities, and examine the intrinsic heterogeneity of populations of molecules, cells, or organisms. Furthermore, compartmentalization of single cells or molecules in "digital" microfluidic experiments can induce switching in a range of reaction systems to enable sensitive detection of cells or biomolecules, such as with digital ELISA or digital PCR. This "digitizing" offers advantages in terms of robustness, assay design, and simplicity because quantitative information can be obtained with qualitative measurements. While digital formats have been shown to improve the robustness of existing chemistries, we anticipate that in the future they will enable new chemistries to be used for quantitative measurements, and that digital biology and chemistry will continue to provide further opportunities for measuring biomolecules, understanding natural systems more deeply, and advancing molecular and cellular analysis. Microfluidics will impact digital biology and chemistry and will also benefit from them if it becomes massively distributed.

Modeling malaria infected cells in microcirculation

NASA Astrophysics Data System (ADS)

Raffiee, Amir Hossein; Dabiri, Sadegh; Motavalizadeh Ardekani, Arezoo

2016-11-01

Plasmodim (P.) falciparum is one of the deadliest types of malaria species that invades healthy red blood cells (RBC) in human blood flow. This parasite develops through 48-hour intra-RBC process leading to significant morphological and mechanical (e.g., stiffening) changes in RBC membrane. These changes have remarkable effects on blood circulation such as increase in flow resistance and obstruction in microcirculation. In this work a computational framework is developed to model RBC suspension in blood flow using front-tracking technique. The present study focuses on blood flow behavior under normal and infected circumstances and predicts changes in blood rheology for different levels of parasitemia and hematocrit. This model allows better understanding of blood flow circulation up to a single cell level and provides us with realistic and deep insight into hematologic diseases such as malaria.

Immunological changes in the intestines and skin after senna administration.

PubMed

Yamate, Yurika; Hiramoto, Keiichi; Yokoyama, Satoshi; Ooi, Kazuya

2015-06-01

It has been reported that chronic sennoside use is associated with the development of melanosis coli, colonic adenoma, and/or carcinomas. In this study, we investigated the immunological changes in the colon and skin after the administration of senna. In this study, we investigated the colon and epidermis of C57/BL6j mice after a single administration of 10 mg/kg of senna [Cassia angustifolia (Caesalpiniaceae); 3, 6, 12, and 24 h after administration] and after repeated once per week administrations (on days 3, 5, 7, 14, and 21 of administration). The LD50 and ED50 of senna used in this experiment were 165 mg/kg and 13 g/kg, respectively. We demonstrated that the DOPA-positive cells in the colon increased at 12 h after single administration and were further increased from at 5-28 d after repeated administration. We also studied the physiological changes of the small intestine using the charcoal meal test. We found that there was a tendency for peristalsis to be inhibited after repeated senna administration. In the epidermis, we investigated the number of Langerhans cells, because they are important immune cells of the skin. The number of these cells decreased, especially after repeated administration. The present findings suggested that it is necessary to pay attention to not only the intestine but also the skin, during long-term senna treatment.

Modeling cell-substrate de-adhesion dynamics under fluid shear

NASA Astrophysics Data System (ADS)

Maan, Renu; Rani, Garima; Menon, Gautam I.; Pullarkat, Pramod A.

2018-07-01

Changes in cell-substrate adhesion are believed to signal the onset of cancer metastasis, but such changes must be quantified against background levels of intrinsic heterogeneity between cells. Variations in cell-substrate adhesion strengths can be probed through biophysical measurements of cell detachment from substrates upon the application of an external force. Here, we investigate, theoretically and experimentally, the detachment of cells adhered to substrates when these cells are subjected to fluid shear. We present a theoretical framework within which we calculate the fraction of detached cells as a function of shear stress for fast ramps as well as the decay in this fraction at fixed shear stress as a function of time. Using HEK and 3T3 fibroblast cells as experimental model systems, we extract characteristic force scales for cell adhesion as well as characteristic detachment times. We estimate force-scales of  ∼500 pN associated to a single focal contact, and characteristic time-scales of s representing cell-spread-area dependent mean first passage times to the detached state at intermediate values of the shear stress. Variations in adhesion across cell types are especially prominent when cell detachment is probed by applying a time-varying shear stress. These methods can be applied to characterizing changes in cell adhesion in a variety of contexts, including metastasis.

CROSS-DISCIPLINARY PHYSICS AND RELATED AREAS OF SCIENCE AND TECHNOLOGY: Noise effect in metabolic networks

NASA Astrophysics Data System (ADS)

Li, Zheng-Yan; Xie, Zheng-Wei; Chen, Tong; Ouyang, Qi

2009-12-01

Constraint-based models such as flux balance analysis (FBA) are a powerful tool to study biological metabolic networks. Under the hypothesis that cells operate at an optimal growth rate as the result of evolution and natural selection, this model successfully predicts most cellular behaviours in growth rate. However, the model ignores the fact that cells can change their cellular metabolic states during evolution, leaving optimal metabolic states unstable. Here, we consider all the cellular processes that change metabolic states into a single term 'noise', and assume that cells change metabolic states by randomly walking in feasible solution space. By simulating a state of a cell randomly walking in the constrained solution space of metabolic networks, we found that in a noisy environment cells in optimal states tend to travel away from these points. On considering the competition between the noise effect and the growth effect in cell evolution, we found that there exists a trade-off between these two effects. As a result, the population of the cells contains different cellular metabolic states, and the population growth rate is at suboptimal states.

Single-cell dynamics of mast cell-CD4+ CD25+ regulatory T cell interactions.

PubMed

Frossi, Barbara; D'Incà, Federica; Crivellato, Enrico; Sibilano, Riccardo; Gri, Giorgia; Mongillo, Marco; Danelli, Luca; Maggi, Laura; Pucillo, Carlo E

2011-07-01

The biological behavior of immune cells is determined by their intrinsic properties and interactions with other cell populations within their microenvironment. Several studies have confirmed the existence of tight spatial interactions between mast cells (MCs) and Tregs in different settings. For instance, we have recently identified the functional cross-talk between MCs and Tregs, through the OX40L-OX40 axis, as a new mechanism of reciprocal influence. However, there is scant information regarding the single-cell dynamics of this process. In this study, time-lapse video microscopy revealed direct interactions between Tregs and MCs in both murine and human cell co-cultures, resulting in the inhibition of the MC degranulation response. MCs incubated with WT, but not OX40-deficient, Tregs mediated numerous and long-lasting interactions and displayed different morphological features lacking the classical signs of exocytosis. MC degranulation and Ca2+ mobilization upon activation were inhibited by Tregs on a single-cell basis, without affecting overall cytokine secretion. Transmission electron microscopy showed ultrastructural evidence of vesicle-mediated secretion reconcilable with the morphological pattern of piecemeal degranulation. Our results suggest that MC morphological and functional changes following MC-Treg interactions can be ascribed to cell-cell contact and represent a transversal, non-species-specific mechanism of immune response regulation. Further research, looking at the molecular composition of this interaction will broaden our understanding of its contribution to immunity. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evolution of power sources for implantable cardioverter defibrillators

NASA Astrophysics Data System (ADS)

Crespi, Ann M.; Somdahl, Sonja K.; Schmidt, Craig L.; Skarstad, Paul M.

The evolution of seven generations of power sources for implantable cardioverter defibrillators (ICD) is presented. The packaging efficiency of the power sources has steadily increased, resulting in smaller, lighter batteries while maintaining the required electrical characteristics. The main areas for improvement were reduction of headspace volume, reduction of separator volume, and a change from a two-cell battery to a single cell.

Discriminating cellular heterogeneity using microwell-based RNA cytometry

PubMed Central

Dimov, Ivan K.; Lu, Rong; Lee, Eric P.; Seita, Jun; Sahoo, Debashis; Park, Seung-min; Weissman, Irving L.; Lee, Luke P.

2014-01-01

Discriminating cellular heterogeneity is important for understanding cellular physiology. However, it is limited by the technical difficulties of single-cell measurements. Here, we develop a two-stage system to determine cellular heterogeneity. In the first stage, we perform multiplex single-cell RNA-cytometry in a microwell array containing over 60,000 reaction chambers. In the second stage, we use the RNA-cytometry data to determine cellular heterogeneity by providing a heterogeneity likelihood score. Moreover, we use Monte-Carlo simulation and RNA-cytometry data to calculate the minimum number of cells required for detecting heterogeneity. We applied this system to characterize the RNA distributions of aging related genes in a highly purified mouse hematopoietic stem cell population. We identified genes that reveal novel heterogeneity of these cells. We also show that changes in expression of genes such as Birc6 during aging can be attributed to the shift of relative portions of cells in the high-expressing subgroup versus low-expressing subgroup. PMID:24667995

V-band electronically reconfigurable metamaterial

NASA Astrophysics Data System (ADS)

Radisic, Vesna; Hester, Jimmy G.; Nguyen, Vinh N.; Caira, Nicholas W.; DiMarzio, Donald; Hilgeman, Theodore; Larouche, Stéphane; Kaneshiro, Eric; Gutierrez-Aitken, Augusto

2017-04-01

In this work, we report on a reconfigurable V-band metamaterial fabricated using an InP heterojunction bipolar transistor production process. As designed and fabricated, the implementation uses complementary split ring resonators (cSRRs) and Schottky diodes in both single unit cell and three unit cell monolithic microwave integrated circuits. Each unit cell has two diodes embedded within the gaps of the cSRRs. Reconfigurability is achieved by applying an external bias that turns the diodes on and off, which effectively controls the resonant property of the structure. In order to measure the metamaterial properties, the unit cells are fed and followed by transmission lines. Measured data show good agreement with simulations and demonstrate that the metamaterial structure exhibits resonance at around 65 GHz that can be switched on and off. The three-unit cell transmission line metamaterial shows a deeper resonance and a larger phase change than a single cell, as expected. These are the first reported reconfigurable metamaterials operating at the V-band using the InP high speed device fabrication process.

Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver

PubMed Central

Huch, Meritxell; Gehart, Helmuth; van Boxtel, Ruben; Hamer, Karien; Blokzijl, Francis; Verstegen, Monique M.A.; Ellis, Ewa; van Wenum, Martien; Fuchs, Sabine A.; de Ligt, Joep; van de Wetering, Marc; Sasaki, Nobuo; Boers, Susanne J.; Kemperman, Hans; de Jonge, Jeroen; Ijzermans, Jan N.M.; Nieuwenhuis, Edward E.S.; Hoekstra, Ruurdtje; Strom, Stephen; Vries, Robert R.G.; van der Laan, Luc J.W.; Cuppen, Edwin; Clevers, Hans

2015-01-01

Summary Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy. PMID:25533785

Acquired resistance to combination treatment through loss of synergy with MEK and PI3K inhibitors in colorectal cancer

PubMed Central

Bhattacharya, Bhaskar; Low, Sarah Hong Hui; Chong, Mei Ling; Chia, Dilys; Koh, King Xin; Sapari, Nur Sabrina; Kaye, Stanley; Hung, Huynh; Benoukraf, Touati; Soong, Richie

2016-01-01

Historically, understanding of acquired resistance (AQR) to combination treatment has been based on knowledge of resistance to its component agents. To test whether an altered drug interaction could be an additional factor in AQR to combination treatment, models of AQR to combination and single agent MEK and PI3K inhibitor treatment were generated. Combination indices indicated combination treatment of PI3K and MEK inhibitors remained synergistic in cells with AQR to single agent but not combination AQR cells. Differences were also observed between the models in cellular phenotypes, pathway signaling and drug cross-resistance. Genomics implicated TGFB2-EDN1 overexpression as candidate determinants in models of AQR to combination treatment. Supplementation of endothelin in parental cells converted synergism to antagonism. Silencing of TGFB2 or EDN1 in cells with AQR conferred synergy between PI3K and MEK inhibitor. These results highlight that AQR to combination treatment may develop through alternative mechanisms to those of single agent treatment, including a change in drug interaction. PMID:27081080

Auto-luminescent genetically encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level

NASA Astrophysics Data System (ADS)

Saito, Kenta; Kobayashi, Kentaro; Nagai, Takeharu

2011-12-01

Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. We have applied this strategy to develop an autoluminescent Ca2+ indicator, BRAC, which is composed of Ca2+-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. With this BRAC, we succeeded visualization of Ca2+ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca2+-independent signal drifts due to change in cell shape, focus shift, etc. Taking advantage of the bioluminescence imaging property that does not require external excitation light, BRAC might become a powerful tool applicable in conjunction with so-called optogenetic technology by which we can control cellular and protein function by light illumination.

Structural and functional changes in acute liver injury.

PubMed

Smuckler, E A

1976-06-01

Carbon tetrachloride produces liver cell injury in a variety of animal species. The first structurally recognizable changes occur in the endoplasmic reticulum, with alteration in ribosome-membrane interactions. Later there is an increase in intracellular fat, and the formation of tangled nets of the ergastoplasm. At no time are there changes in mitochondria or single membrane limited bodies in cells with intact plasmalemma, although a relative increase in cell sap may appear. In dead cells (those with plasmalemma discontinuties) crystalline deposits of calcium phosphatase may be noted. Functional changes are related to the endoplasmic reticulum and the plasma membrane. An early decrease in protein synthesis takes place; an accumulation of neutral lipid is related to this change. Later alterations in the ergastoplasmic functions (e.g., mixed function oxidation) occurs. Carbon tetrachloride is not the active agent; rather, a product of its metabolism, probably the CC1, free radical, is. The mechanisms of injury include macromolecular adduction and peroxide propagation. A third possibility includes a cascade effect with the production of secondary and tertiary products, also toxic in nature, with the ability to produce more widespread damage to intracellular structures.

[Atomic force microscopy: a tool to analyze the viral cycle].

PubMed

Bernaud, Julien; Castelnovo, Martin; Muriaux, Delphine; Faivre-Moskalenko, Cendrine

2015-05-01

Each step of the HIV-1 life cycle frequently involves a change in the morphology and/or mechanical properties of the viral particle or core. The atomic force microscope (AFM) constitutes a powerful tool for characterizing these physical changes at the scale of a single virus. Indeed, AFM enables the visualization of viral capsids in a controlled physiological environment and to probe their mechanical properties by nano-indentation. Finally, AFM force spectroscopy allows to characterize the affinities between viral envelope proteins and cell receptors at the single molecule level. © 2015 médecine/sciences – Inserm.

High performance static latches with complete single event upset immunity

DOEpatents

Corbett, Wayne T.; Weaver, Harry T.

1994-01-01

An asymmetric response latch providing immunity to single event upset without loss of speed. The latch has cross-coupled inverters having a hardened logic state and a soft state, wherein the logic state of the first inverter can only be changed when the voltage on the coupling node of that inverter is low and the logic state of the second inverter can only be changed when the coupling of that inverter is high. One of more of the asymmetric response latches may be configured into a memory cell having complete immunity, which protects information rather than logic states.

Microfluidic Blood Cell Preparation: Now and Beyond

PubMed Central

Yu, Zeta Tak For; Yong, Koh Meng Aw; Fu, Jianping

2014-01-01

Blood plays an important role in homeostatic regulation with each of its cellular components having important therapeutic and diagnostic uses. Therefore, separation and sorting of blood cells has been of a great interest to clinicians and researchers. However, while conventional methods of processing blood have been successful in generating relatively pure fractions, they are time consuming, labor intensive, and are not optimal for processing small volume blood samples. In recent years, microfluidics has garnered great interest from clinicians and researchers as a powerful technology for separating blood into different cell fractions. As microfluidics involves fluid manipulation at the microscale level, it has the potential for achieving high-resolution separation and sorting of blood cells down to a single-cell level, with an added benefit of integrating physical and biological methods for blood cell separation and analysis on the same single chip platform. This paper will first review the conventional methods of processing and sorting blood cells, followed by a discussion on how microfluidics is emerging as an efficient tool to rapidly change the field of blood cell sorting for blood-based therapeutic and diagnostic applications. PMID:24515899

Model-based cell number quantification using online single-oxygen sensor data for tissue engineering perfusion bioreactors.

PubMed

Lambrechts, T; Papantoniou, I; Sonnaert, M; Schrooten, J; Aerts, J-M

2014-10-01

Online and non-invasive quantification of critical tissue engineering (TE) construct quality attributes in TE bioreactors is indispensable for the cost-effective up-scaling and automation of cellular construct manufacturing. However, appropriate monitoring techniques for cellular constructs in bioreactors are still lacking. This study presents a generic and robust approach to determine cell number and metabolic activity of cell-based TE constructs in perfusion bioreactors based on single oxygen sensor data in dynamic perfusion conditions. A data-based mechanistic modeling technique was used that is able to correlate the number of cells within the scaffold (R(2)  = 0.80) and the metabolic activity of the cells (R(2)  = 0.82) to the dynamics of the oxygen response to step changes in the perfusion rate. This generic non-destructive measurement technique is effective for a large range of cells, from as low as 1.0 × 10(5) cells to potentially multiple millions of cells, and can open-up new possibilities for effective bioprocess monitoring. © 2014 Wiley Periodicals, Inc.

Modification of response functions of cat visual cortical cells by spatially congruent perturbing stimuli.

PubMed

Kabara, J F; Bonds, A B

2001-12-01

Responses of cat striate cortical cells to a drifting sinusoidal grating were modified by the superimposition of a second, perturbing grating (PG) that did not excite the cell when presented alone. One consequence of the presence of a PG was a shift in the tuning curves. The orientation tuning of all 41 cells exposed to a PG and the spatial frequency tuning of 83% of the 23 cells exposed to a PG showed statistically significant dislocations of both the response function peak and center of mass from their single grating values. As found in earlier reports, the presence of PGs suppressed responsiveness. However, reductions measured at the single grating optimum orientation or spatial frequency were on average 1.3 times greater than the suppression found at the peak of the response function modified by the presence of the PG. Much of the loss in response seen at the single grating optimum is thus a result of a shift in the tuning function rather than outright suppression. On average orientation shifts were repulsive and proportional (approximately 0.10 deg/deg) to the angle between the perturbing stimulus and the optimum single grating orientation. Shifts in the spatial frequency response function were both attractive and repulsive, resulting in an overall average of zero. For both simple and complex cells, PGs generally broadened orientation response function bandwidths. Similarly, complex cell spatial frequency response function bandwidths broadened. Simple cell spatial frequency response functions usually did not change, and those that did broadened only 4% on average. These data support the hypothesis that additional sinusoidal components in compound stimuli retune cells' response functions for orientation and spatial frequency.

Effects of Low Level Radiation exposure on Neurogenesis and Cognitive Function: Mechanisms and Prevention

DTIC Science & Technology

2005-09-01

precursor cells in culture with uX-lipoic acid reverses the density dependent changes observed in culture; this compound may provide an effective means...inhibited growth of precursor cells in vitro; - Antioxidant treatment of neural precursor cells in culture with a-lipoic acid (ALA) reverses the...with a single lO-Gy dose, and tissues avidin-biotinylated pemxidase complex; GFAP, glial fibrillary acidic protein; DAB, 3,3’- were collected from 6 to

Consequence of a sudden wind event on the dynamics of a coastal phytoplankton community: an insight into specific population growth rates using a single cell high frequency approach

PubMed Central

Dugenne, Mathilde; Thyssen, Melilotus; Nerini, David; Mante, Claude; Poggiale, Jean-Christophe; Garcia, Nicole; Garcia, Fabrice; Grégori, Gérald J.

2014-01-01

Phytoplankton is a key component in marine ecosystems. It is responsible for most of the marine primary production, particularly in eutrophic lagoons, where it frequently blooms. Because they are very sensitive to their environment, the dynamics of these microbial communities has to be observed over different time scales, however, assessment of short term variability is often out of reach of traditional monitoring methods. To overcome these limitations, we set up a Cytosense automated flow cytometer (Cytobuoy b.v.), designed for high frequency monitoring of phytoplankton composition, abundance, cell size, and pigment content, in one of the largest Mediterranean lagoons, the Berre lagoon (South-Eastern France). During October 2011, it recorded the cell optical properties of 12 groups of pico-, nano-, and microphytoplankton. Daily variations in the cluster optical properties were consistent with individual changes observed using microscopic imaging, during the cell cycle. We therefore used an adaptation of the size-structured matrix population model, developed by Sosik et al. (2003) to process the single cell analysis of the clusters and estimate the division rates of 2 dinoflagellate populations before, during, and after a strong wind event. The increase in the estimated in situ daily cluster growth rates suggest that physiological changes in the cells can prevail over the response of abundance. PMID:25309523

MiRAR-miRNA Activity Reporter for Living Cells.

PubMed

Turk, Matthew A; Chung, Christina Z; Manni, Emad; Zukowski, Stephanie A; Engineer, Anish; Badakhshi, Yasaman; Bi, Yumin; Heinemann, Ilka U

2018-06-19

microRNA (miRNA) activity and regulation are of increasing interest as new therapeutic targets. Traditional approaches to assess miRNA levels in cells rely on RNA sequencing or quantitative PCR. While useful, these approaches are based on RNA extraction and cannot be applied in real-time to observe miRNA activity with single-cell resolution. We developed a green fluorescence protein (GFP)-based reporter system that allows for a direct, real-time readout of changes in miRNA activity in live cells. The miRNA activity reporter (MiRAR) consists of GFP fused to a 3′ untranslated region containing specific miRNA binding sites, resulting in miRNA activity-dependent GFP expression. Using qPCR, we verified the inverse relationship of GFP fluorescence and miRNA levels. We demonstrated that this novel optogenetic reporter system quantifies cellular levels of the tumor suppressor miRNA let-7 in real-time in single Human embryonic kidney 293 (HEK 293) cells. Our data shows that the MiRAR can be applied to detect changes in miRNA levels upon disruption of miRNA degradation pathways. We further show that the reporter could be adapted to monitor another disease-relevant miRNA, miR-122. With trivial modifications, this approach could be applied across the miRNome for quantification of many specific miRNA in cell cultures, tissues, or transgenic animal models.

Single Cell Assay for Analyzing Single Cell Exosome and Endocrine Secretion and Cancer Markers

NASA Astrophysics Data System (ADS)

Chiu, Yu-Jui

To understand the inhomogeneity of cells in biological systems, there is a growing demand for the capability to characterize the properties of individual single cells. Since single cell studies require continuous monitoring of the cell behaviors instead of a snapshot test at a single time point, an effective single-cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single-cell technologies cannot provide proper environments to sustain cell growth and cannot provide, for appropriate cell types, proliferation of single cells and convenient, non-invasive tests of single cell behaviors from molecular markers. In this dissertation, I present a highly versatile single-cell assay that can accommodate different cellular types, enable easy and efficient single cell loading and culturing, and be suitable for the study of effects of in-vitro environmental factors in combination with drug screening. The salient features of the assay are the non-invasive collection and surveying of single cell secretions at different time points and massively parallel translocation of single cells by user defined criteria, producing very high compatibility to the downstream process such as single cell qPCR and sequencing. Above all, the acquired information is quantitative -- for example, one of the studies is measured by the number of exosomes each single cell secretes for a given time period. Therefore, our single-cell assay provides a convenient, low-cost, and enabling tool for quantitative, time lapsed studies of single cell properties.

PKA-regulated VASP phosphorylation promotes extrusion of transformed cells from the epithelium

PubMed Central

Anton, Katarzyna A.; Sinclair, John; Ohoka, Atsuko; Kajita, Mihoko; Ishikawa, Susumu; Benz, Peter M.; Renne, Thomas; Balda, Maria; Matter, Karl; Fujita, Yasuyuki

2014-01-01

ABSTRACT At the early stages of carcinogenesis, transformation occurs in single cells within tissues. In an epithelial monolayer, such mutated cells are recognized by their normal neighbors and are often apically extruded. The apical extrusion requires cytoskeletal reorganization and changes in cell shape, but the molecular switches involved in the regulation of these processes are poorly understood. Here, using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry, we have identified proteins that are modulated in transformed cells upon their interaction with normal cells. Phosphorylation of VASP at serine 239 is specifically upregulated in RasV12-transformed cells when they are surrounded by normal cells. VASP phosphorylation is required for the cell shape changes and apical extrusion of Ras-transformed cells. Furthermore, PKA is activated in Ras-transformed cells that are surrounded by normal cells, leading to VASP phosphorylation. These results indicate that the PKA–VASP pathway is a crucial regulator of tumor cell extrusion from the epithelium, and they shed light on the events occurring at the early stage of carcinogenesis. PMID:24963131

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.

Morphological Changes of Human Corneal Endothelial Cells after Rho-Associated Kinase Inhibitor Eye Drop (Ripasudil) Administration: A Prospective Open-Label Clinical Study

PubMed Central

Okumura, Naoki; Suganami, Hideki; Kinoshita, Shigeru

2015-01-01

Purpose To investigate the effect and safety of a selective Rho kinase inhibitor, ripasudil 0.4% eye drops, on corneal endothelial cells of healthy subjects. Design Prospective, interventional case series. Methods In this study, 6 healthy subjects were administered ripasudil 0.4% in the right eye twice daily for 1 week. Morphological changes and corneal endothelial cell density were examined by noncontact and contact specular microscopy. Central corneal thickness and corneal volume of 5 mm-diameter area of center cornea were analyzed by Pentacam Scheimpflug topography. All the above measurements were conducted in both eyes before administration, 1.5 and 6 hours after the initial administration on day 0; and in the same manner after the final administration on day 7. Results By noncontact specular microscopy, indistinct cell borders with pseudo guttae were observed, but by contact specular microscopy, morphological changes of corneal endothelial cells were mild and pseudo guttae was not observed after single and repeated administration of ripasudil in all subjects. These changes resolved prior to the next administration, and corneal endothelial cell density, central corneal thickness and corneal volume were not changed throughout the study period. Conclusion Transient morphological changes of corneal endothelial cells such as indistinct cell borders with pseudo guttae were observed by noncontact specular microscopy in healthy subjects after ripasudil administration. Corneal edema was not observed and corneal endothelial cell density did not decrease after 1 week repetitive administration. These morphological changes were reversible and corneal endothelial cell morphology returned to normal prior to the next administration. Trial Registration JAPIC Clinical Trials Information 142705 PMID:26367375

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides.

PubMed

Rivera-Torres, Natalia; Banas, Kelly; Bialk, Pawel; Bloh, Kevin M; Kmiec, Eric B

2017-01-01

CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and CRISPR/Cas9 ribonucleoprotein complex.

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides

PubMed Central

Rivera-Torres, Natalia; Bialk, Pawel; Bloh, Kevin M.; Kmiec, Eric B.

2017-01-01

CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and CRISPR/Cas9 ribonucleoprotein complex. PMID:28052104

[Study on garlic oil combined with 5-FU induced apoptosis of adenoid cystic carcinoma cell line ACC-M].

PubMed

Wu, Fayin; Zhou, Hefeng; Fan, Zhiying; Zhu, Yawen; Li, Yongye; Yao, Yukun; Ran, Dan

2014-02-01

To observe the effect of garlic oil combined with 5-FU induced apoptosis of adenoid cystic carcinoma cell line ACC-M. Human salivary in adenoid cystic carcinoma cell line AC-M was cultured, divided into the experimental group (5-FU group, garlic oil group, garlic oil + 5-FU group) and the control group, to observe the growth activity of tumor cells by MTT methods; to analyse the changes of cell cycle and apoptosis rate by flow cytometry. MTT experiments showed that 5-FU, garlic oil, garlic oil and 5-FU on ACC-M cells have inhibition in different concentration, with the increase of concentration and action time of the rise; Cell cycle analysis showed significant changes in flow cytometry. With the increase of concentration and the acting time, the G0/G1, phase of the cell ratio increased, S had no significant change, but G2/M phase cells decreased. Apoptosis rate display showed garlic oil combined with 5-FU induced apoptosis of ACC-M cells was significantly stronger than single group. Garlic oil can effectively induce the apoptosis of adenoid cystic carcinoma cell line ACC-M. The effect of garlic oil combined with 5-FU on ACC-M cells was stronger than the garlic oil, 5-FU used alone.

Nuclear apoptotic volume decrease in individual cells: Confocal microscopy imaging and kinetic modeling.

PubMed

Khalo, Irina V; Konokhova, Anastasiya I; Orlova, Darya Y; Trusov, Konstantin V; Yurkin, Maxim A; Bartova, Eva; Kozubek, Stanislav; Maltsev, Valeri P; Chernyshev, Andrei V

2018-05-30

The dynamics of nuclear morphology changes during apoptosis remains poorly investigated and understood. Using 3D time-lapse confocal microscopy we performed a study of early-stage apoptotic nuclear morphological changes induced by etoposide in single living HepG2 cells. These observations provide a definitive evidence that nuclear apoptotic volume decrease (AVD) is occurring simultaneously with peripheral chromatin condensation (so called "apoptotic ring"). In order to describe quantitatively the dynamics of nuclear morphological changes in the early stage of apoptosis we suggest a general molecular kinetic model, which fits well the obtained experimental data in our study. Results of this work may clarify molecular mechanisms of nuclear morphology changes during apoptosis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Flagella and motility behaviour of square bacteria.

PubMed Central

Alam, M; Claviez, M; Oesterhelt, D; Kessel, M

1984-01-01

Square bacteria are shown to have right-handed helical (RH) flagella. They swim forward by clockwise (CW), and backwards by counterclockwise (CCW) rotation of their flagella. They are propelled by several or single filaments arising at several or single points on the cell surface. When there are several filaments a stable bundle is formed that does not fly apart during the change from clockwise to counterclockwise rotation or vice versa. In addition to the flagella attached to the cells, large amounts of detached flagella aggregated into thick super-flagella, can be observed at all phases of growth. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6526006

Impact of acute exposure to WTC dust on ciliated and goblet cells in lungs of rats

PubMed Central

Cohen, Mitchell D.; Vaughan, Joshua M.; Garrett, Brittany; Prophete, Colette; Horton, Lori; Sisco, Maureen; Ghio, Andrew; Zelikoff, Judith; Lung-chi, Chen

2015-01-01

Clinical studies and the World Trade Center (WTC) Health Registry have revealed increases in the incidence of chronic (non-cancer) lung disorders among first responders (FR) who were at Ground Zero during the initial 72 h after the collapse. Our previous analyses of rats exposed to building-derived WTC dusts using exposure scenarios/levels that mimicked FR mouth-breathing showed that a single WTC dust exposure led to changes in expression of genes whose products could be involved in the lung ailments, but few other significant pathologies. We concluded that rather than acting as direct inducers of many of the FR health effects, it was more likely inhaled WTC dusts instead may have impacted on toxicities induced by other rescue-related co-pollutants present in Ground Zero air. To allow for such effects to occur, we hypothesized that the alkaline WTC dusts induced damage to the normal ability of the lungs to clear inhaled particles. To validate this, rats were exposed on two consecutive days (2 h/d, by intratracheal inhalation) to WTC dust (collected 12–13 September 2001) and examined over a 1-yr period thereafter for changes in the presence of ciliated cells in the airways and hyperplastic goblet cells in the lungs. WTC dust levels in the lungs were assessed in parallel to verify that any changes in levels of these cells corresponded with decreases in host ability to clear the particles themselves. Image analyses of the rat lungs revealed a significant decrease in ciliated cells and increase in hyperplastic goblet cells due to the single series of WTC dust exposures. The study also showed there was only a nominal non-significant decrease (6–11%) in WTC dust burden over a 1-yr period after the final exposure. These results provide support for our current hypothesis that exposure to WTC dusts caused changes in airway morphology/cell composition; such changes could, in turn, have led to potential alterations in the clearance/toxicities of other pollutants inhaled at Ground Zero in the critical initial 72-h period. PMID:26194034

Impact of acute exposure to WTC dust on ciliated and goblet cells in lungs of rats.

PubMed

Cohen, Mitchell D; Vaughan, Joshua M; Garrett, Brittany; Prophete, Colette; Horton, Lori; Sisco, Maureen; Ghio, Andrew; Zelikoff, Judith; Lung-chi, Chen

2015-01-01

Clinical studies and the World Trade Center (WTC) Health Registry have revealed increases in the incidence of chronic (non-cancer) lung disorders among first responders (FR) who were at Ground Zero during the initial 72 h after the collapse. Our previous analyses of rats exposed to building-derived WTC dusts using exposure scenarios/levels that mimicked FR mouth-breathing showed that a single WTC dust exposure led to changes in expression of genes whose products could be involved in the lung ailments, but few other significant pathologies. We concluded that rather than acting as direct inducers of many of the FR health effects, it was more likely inhaled WTC dusts instead may have impacted on toxicities induced by other rescue-related co-pollutants present in Ground Zero air. To allow for such effects to occur, we hypothesized that the alkaline WTC dusts induced damage to the normal ability of the lungs to clear inhaled particles. To validate this, rats were exposed on two consecutive days (2 h/d, by intratracheal inhalation) to WTC dust (collected 12-13 September 2001) and examined over a 1-yr period thereafter for changes in the presence of ciliated cells in the airways and hyperplastic goblet cells in the lungs. WTC dust levels in the lungs were assessed in parallel to verify that any changes in levels of these cells corresponded with decreases in host ability to clear the particles themselves. Image analyses of the rat lungs revealed a significant decrease in ciliated cells and increase in hyperplastic goblet cells due to the single series of WTC dust exposures. The study also showed there was only a nominal non-significant decrease (6-11%) in WTC dust burden over a 1-yr period after the final exposure. These results provide support for our current hypothesis that exposure to WTC dusts caused changes in airway morphology/cell composition; such changes could, in turn, have led to potential alterations in the clearance/toxicities of other pollutants inhaled at Ground Zero in the critical initial 72-h period.

Predictive lethal proarrhythmic risk evaluation using a closed-loop-circuit cell network with human induced pluripotent stem cells derived cardiomyocytes

NASA Astrophysics Data System (ADS)

Nomura, Fumimasa; Hattori, Akihiro; Terazono, Hideyuki; Kim, Hyonchol; Odaka, Masao; Sugio, Yoshihiro; Yasuda, Kenji

2016-06-01

For the prediction of lethal arrhythmia occurrence caused by abnormality of cell-to-cell conduction, we have developed a next-generation in vitro cell-to-cell conduction assay, i.e., a quasi in vivo assay, in which the change in spatial cell-to-cell conduction is quantitatively evaluated from the change in waveforms of the convoluted electrophysiological signals from lined-up cardiomyocytes on a single closed loop of a microelectrode of 1 mm diameter and 20 µm width in a cultivation chip. To evaluate the importance of the closed-loop arrangement of cardiomyocytes for prediction, we compared the change in waveforms of convoluted signals of the responses in the closed-loop circuit arrangement with that of the response of cardiomyocyte clusters using a typical human ether a go-go related gene (hERG) ion channel blocker, E-4031. The results showed that (1) waveform prolongation and fluctuation both in the closed loops and clusters increased depending on the E-4031 concentration increase. However, (2) only the waveform signals in closed loops showed an apparent temporal change in waveforms from ventricular tachycardia (VT) to ventricular fibrillation (VF), which is similar to the most typical cell-to-cell conductance abnormality. The results indicated the usefulness of convoluted waveform signals of a closed-loop cell network for acquiring reproducible results acquisition and more detailed temporal information on cell-to-cell conduction.

In vivo collective cell migration requires an LPAR2-dependent increase in tissue fluidity.

PubMed

Kuriyama, Sei; Theveneau, Eric; Benedetto, Alexandre; Parsons, Maddy; Tanaka, Masamitsu; Charras, Guillaume; Kabla, Alexandre; Mayor, Roberto

2014-07-07

Collective cell migration (CCM) and epithelial-mesenchymal transition (EMT) are common to cancer and morphogenesis, and are often considered to be mutually exclusive in spite of the fact that many cancer and embryonic cells that have gone through EMT still cooperate to migrate collectively. Here we use neural crest (NC) cells to address the question of how cells that have down-regulated cell-cell adhesions can migrate collectively. NC cell dissociation relies on a qualitative and quantitative change of the cadherin repertoire. We found that the level of cell-cell adhesion is precisely regulated by internalization of N-cadherin downstream of lysophosphatidic acid (LPA) receptor 2. Rather than promoting the generation of single, fully mesenchymal cells, this reduction of membrane N-cadherin only triggers a partial mesenchymal phenotype. This intermediate phenotype is characterized by an increase in tissue fluidity akin to a solid-like-to-fluid-like transition. This change of plasticity allows cells to migrate under physical constraints without abolishing cell cooperation required for collectiveness. © 2014 Kuriyama et al.

Charged residues in the H-NS linker drive DNA binding and gene silencing in single cells.

PubMed

Gao, Yunfeng; Foo, Yong Hwee; Winardhi, Ricksen S; Tang, Qingnan; Yan, Jie; Kenney, Linda J

2017-11-21

Nucleoid-associated proteins (NAPs) facilitate chromosome organization in bacteria, but the precise mechanism remains elusive. H-NS is a NAP that also plays a major role in silencing pathogen genes. We used genetics, single-particle tracking in live cells, superresolution microscopy, atomic force microscopy, and molecular dynamics simulations to examine H-NS/DNA interactions in single cells. We discovered a role for the unstructured linker region connecting the N-terminal oligomerization and C-terminal DNA binding domains. In the present work we demonstrate that linker amino acids promote engagement with DNA. In the absence of linker contacts, H-NS binding is significantly reduced, although no change in chromosome compaction is observed. H-NS is not localized to two distinct foci; rather, it is scattered all around the nucleoid. The linker makes DNA contacts that are required for gene silencing, while chromosome compaction does not appear to be an important H-NS function.

Single-lineage transcriptome analysis reveals key regulatory pathways in primitive erythroid progenitors in the mouse embryo

PubMed Central

Isern, Joan; He, Zhiyong; Fraser, Stuart T.; Nowotschin, Sonja; Ferrer-Vaquer, Anna; Moore, Rebecca; Hadjantonakis, Anna-Katerina; Schulz, Vincent; Tuck, David; Gallagher, Patrick G.

2011-01-01

Primitive erythroid (EryP) progenitors are the first cell type specified from the mesoderm late in gastrulation. We used a transgenic reporter to image and purify the earliest blood progenitors and their descendants from developing mouse embryos. EryP progenitors exhibited remarkable proliferative capacity in the yolk sac immediately before the onset of circulation, when these cells comprise nearly half of all cells of the embryo. Global expression profiles generated at 24-hour intervals from embryonic day 7.5 through 2.5 revealed 2 abrupt changes in transcript diversity that coincided with the entry of EryPs into the circulation and with their late maturation and enucleation, respectively. These changes were paralleled by the expression of critical regulatory factors. Experiments designed to test predictions from these data demonstrated that the Wnt-signaling pathway is active in EryP progenitors, which display an aerobic glycolytic profile and the numbers of which are regulated by transforming growth factor-β1 and hypoxia. This is the first transcriptome assembled for a single hematopoietic lineage of the embryo over the course of its differentiation. PMID:21263157

A DNA methylation map of human cancer at single base-pair resolution

PubMed Central

Vidal, E; Sayols, S; Moran, S; Guillaumet-Adkins, A; Schroeder, M P; Royo, R; Orozco, M; Gut, M; Gut, I; Lopez-Bigas, N; Heyn, H; Esteller, M

2017-01-01

Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination. PMID:28581523

Higher Performance of DSSC with Dyes from Cladophora sp. as Mixed Cosensitizer through Synergistic Effect

PubMed Central

Haji Manaf, Noramaliyana; Tennakoon, Kushan; Chandrakanthi, R. L. N.; Lim, Linda Biaw Leng; Bandara, J. M. R. Sarath; Ekanayake, Piyasiri

2015-01-01

Chlorophyll and xanthophyll dyes extracted from a single source of filamentous freshwater green algae (Cladophora sp.) were used to sensitize dye sensitized solar cells and their performances were investigated. A more positive interaction is expected as the derived dyes come from a single natural source because they work mutually in nature. Cell sensitized with mixed chlorophyll and xanthophyll showed synergistic activity with improved cell performance of 1.5- to 2-fold higher than that sensitized with any individual dye. The effect of temperature and the stability of these dyes were also investigated. Xanthophyll dye was found to be more stable compared to chlorophyll that is attributed in the ability of xanthophyll to dissipate extra energy via reversible structural changes. Mixing the dyes resulted to an increase in effective electron life time and reduced the process of electron recombination during solar cell operation, hence exhibiting a synergistic effect. PMID:25688266

Higher Performance of DSSC with Dyes from Cladophora sp. as Mixed Cosensitizer through Synergistic Effect.

PubMed

Lim, Andery; Haji Manaf, Noramaliyana; Tennakoon, Kushan; Chandrakanthi, R L N; Lim, Linda Biaw Leng; Bandara, J M R Sarath; Ekanayake, Piyasiri

2015-01-01

Chlorophyll and xanthophyll dyes extracted from a single source of filamentous freshwater green algae (Cladophora sp.) were used to sensitize dye sensitized solar cells and their performances were investigated. A more positive interaction is expected as the derived dyes come from a single natural source because they work mutually in nature. Cell sensitized with mixed chlorophyll and xanthophyll showed synergistic activity with improved cell performance of 1.5- to 2-fold higher than that sensitized with any individual dye. The effect of temperature and the stability of these dyes were also investigated. Xanthophyll dye was found to be more stable compared to chlorophyll that is attributed in the ability of xanthophyll to dissipate extra energy via reversible structural changes. Mixing the dyes resulted to an increase in effective electron life time and reduced the process of electron recombination during solar cell operation, hence exhibiting a synergistic effect.

Surface enhanced Raman spectroscopy (SERS) for the discrimination of Arthrobacter strains based on variations in cell surface composition.

PubMed

Stephen, Kate E; Homrighausen, Darren; DePalma, Glen; Nakatsu, Cindy H; Irudayaraj, Joseph

2012-09-21

Surface enhanced Raman spectroscopy (SERS) is a rapid and highly sensitive spectroscopic technique that has the potential to measure chemical changes in bacterial cell surface in response to environmental changes. The objective of this study was to determine whether SERS had sufficient resolution to differentiate closely related bacteria within a genus grown on solid and liquid medium, and a single Arthrobacter strain grown in multiple chromate concentrations. Fourteen closely related Arthrobacter strains, based on their 16S rRNA gene sequences, were used in this study. After performing principal component analysis in conjunction with Linear Discriminant Analysis, we used a novel, adapted cross-validation method, which more faithfully models the classification of spectra. All fourteen strains could be classified with up to 97% accuracy. The hierarchical trees comparing SERS spectra from the liquid and solid media datasets were different. Additionally, hierarchical trees created from the Raman data were different from those obtained using 16S rRNA gene sequences (a phylogenetic measure). A single bacterial strain grown on solid media culture with three different chromate levels also showed significant spectral distinction at discrete points identified by the new Elastic Net regularized regression method demonstrating the ability of SERS to detect environmentally induced changes in cell surface composition. This study demonstrates that SERS is effective in distinguishing between a large number of very closely related Arthrobacter strains and could be a valuable tool for rapid monitoring and characterization of phenotypic variations in a single population in response to environmental conditions.

Design Flexibility of Redox Flow Systems. [for energy storage applications

NASA Technical Reports Server (NTRS)

Hagedorn, N. H.; Thaller, L. H.

1982-01-01

The characteristics inherent in Redox flow systems permit considerable latitude in designing systems for specific storage applications. The first of these characteristics is the absence of plating/deplating reactions with their attendant morphology changes at the electrodes. This permits a given Redox system to operate over a wide range of depths of discharge and charge/discharge rates. The second characteristic is the separation of power generating components (stacks) from the energy storage components (tanks). This results in cost effective system design, ease of system growth via modularization, and freedom from sizing restraints so that the whole spectrum of applications, from utilities down to single residence can be considered. The final characteristic is the commonality of the reactant fluids which assures that all cells at all times are receiving reactants at the same state of charge. Since no cell can be out of balance with respect to any other cell, it is possible for some cells to be charged while others are discharging, in effect creating a DC to DC transformer. It is also possible for various groups of cells to be connected to separate loads, thus supplying a range of output voltages. Also, trim cells can be used to maintain constant bus voltage as the load is changed or as the depth of discharge increases. The commonality of reactant fluids also permits any corrective measures such as rebalancing to occur at the system level instead of at the single cell level.

Revealing chemical processes and kinetics of drug action within single living cells via plasmonic Raman probes.

PubMed

Li, Shan-Shan; Guan, Qi-Yuan; Meng, Gang; Chang, Xiao-Feng; Wei, Ji-Wu; Wang, Peng; Kang, Bin; Xu, Jing-Juan; Chen, Hong-Yuan

2017-05-23

Better understanding the drug action within cells may extend our knowledge on drug action mechanisms and promote new drugs discovery. Herein, we studied the processes of drug induced chemical changes on proteins and nucleic acids in human breast adenocarcinoma (MCF-7) cells via time-resolved plasmonic-enhanced Raman spectroscopy (PERS) in combination with principal component analysis (PCA). Using three popular chemotherapy drugs (fluorouracil, cisplatin and camptothecin) as models, chemical changes during drug action process were clearly discriminated. Reaction kinetics related to protein denaturation, conformational modification, DNA damage and their associated biomolecular events were calculated. Through rate constants and reaction delay times, the different action modes of these drugs could be distinguished. These results may provide vital insights into understanding the chemical reactions associated with drug-cell interactions.

A Paper-Based Electrochromic Array for Visualized Electrochemical Sensing.

PubMed

Zhang, Fengling; Cai, Tianyi; Ma, Liang; Zhan, Liyuan; Liu, Hong

2017-01-31

We report a battery-powered, paper-based electrochromic array for visualized electrochemical sensing. The paper-based sensing system consists of six parallel electrochemical cells, which are powered by an aluminum-air battery. Each single electrochemical cell uses a Prussian Blue spot electrodeposited on an indium-doped tin oxide thin film as the electrochromic indicator. Each electrochemical cell is preloaded with increasing amounts of analyte. The sample activates the battery for the sensing. Both the preloaded analyte and the analyte in the sample initiate the color change of Prussian Blue to Prussian White. With a reaction time of 60 s, the number of electrochemical cells with complete color changes is correlated to the concentration of analyte in the sample. As a proof-of-concept analyte, lactic acid was detected semi-quantitatively using the naked eye.

Experimental characterization and numerical modeling of tissue electrical conductivity during pulsed electric fields for irreversible electroporation treatment planning.

PubMed

Neal, Robert E; Garcia, Paulo A; Robertson, John L; Davalos, Rafael V

2012-04-01

Irreversible electroporation is a new technique to kill cells in targeted tissue, such as tumors, through a nonthermal mechanism using electric pulses to irrecoverably disrupt the cell membrane. Treatment effects relate to the tissue electric field distribution, which can be predicted with numerical modeling for therapy planning. Pulse effects will change the cell and tissue properties through thermal and electroporation (EP)-based processes. This investigation characterizes these changes by measuring the electrical conductivity and temperature of ex vivo renal porcine tissue within a single pulse and for a 200 pulse protocol. These changes are incorporated into an equivalent circuit model for cells and tissue with a variable EP-based resistance, providing a potential method to estimate conductivity as a function of electric field and pulse length for other tissues. Finally, a numerical model using a human kidney volumetric mesh evaluated how treatment predictions vary when EP- and temperature-based electrical conductivity changes are incorporated. We conclude that significant changes in predicted outcomes will occur when the experimental results are applied to the numerical model, where the direction and degree of change varies with the electric field considered.

Progressive alterations in multipotent hematopoietic progenitors underlie lymphoid cell loss in aging.

PubMed

Young, Kira; Borikar, Sneha; Bell, Rebecca; Kuffler, Lauren; Philip, Vivek; Trowbridge, Jennifer J

2016-10-17

Declining immune function with age is associated with reduced lymphoid output of hematopoietic stem cells (HSCs). Currently, there is poor understanding of changes with age in the heterogeneous multipotent progenitor (MPP) cell compartment, which is long lived and responsible for dynamically regulating output of mature hematopoietic cells. In this study, we observe an early and progressive loss of lymphoid-primed MPP cells (LMPP/MPP4) with aging, concomitant with expansion of HSCs. Transcriptome and in vitro functional analyses at the single-cell level reveal a concurrent increase in cycling of aging LMPP/MPP4 with loss of lymphoid priming and differentiation potential. Impaired lymphoid differentiation potential of aged LMPP/MPP4 is not rescued by transplantation into a young bone marrow microenvironment, demonstrating cell-autonomous changes in the MPP compartment with aging. These results pinpoint an age and cellular compartment to focus further interrogation of the drivers of lymphoid cell loss with aging. © 2016 Young et al.

Nucleosome architecture throughout the cell cycle

PubMed Central

Deniz, Özgen; Flores, Oscar; Aldea, Martí; Soler-López, Montserrat; Orozco, Modesto

2016-01-01

Nucleosomes provide additional regulatory mechanisms to transcription and DNA replication by mediating the access of proteins to DNA. During the cell cycle chromatin undergoes several conformational changes, however the functional significance of these changes to cellular processes are largely unexplored. Here, we present the first comprehensive genome-wide study of nucleosome plasticity at single base-pair resolution along the cell cycle in Saccharomyces cerevisiae. We determined nucleosome organization with a specific focus on two regulatory regions: transcription start sites (TSSs) and replication origins (ORIs). During the cell cycle, nucleosomes around TSSs display rearrangements in a cyclic manner. In contrast to gap (G1 and G2) phases, nucleosomes have a fuzzier organization during S and M phases, Moreover, the choreography of nucleosome rearrangements correlate with changes in gene expression during the cell cycle, indicating a strong association between nucleosomes and cell cycle-dependent gene functionality. On the other hand, nucleosomes are more dynamic around ORIs along the cell cycle, albeit with tighter regulation in early firing origins, implying the functional role of nucleosomes on replication origins. Our study provides a dynamic picture of nucleosome organization throughout the cell cycle and highlights the subsequent impact on transcription and replication activity. PMID:26818620

SINGLE MOLECULE APPROACHES TO BIOLOGY, 2010 GORDON RESEARCH CONFERENCE, JUNE 27-JULY 2, 2010, ITALY

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

Professor William Moerner

2010-07-09

The 2010 Gordon Conference on Single-Molecule Approaches to Biology focuses on cutting-edge research in single-molecule science. Tremendous technical developments have made it possible to detect, identify, track, and manipulate single biomolecules in an ambient environment or even in a live cell. Single-molecule approaches have changed the way many biological problems are addressed, and new knowledge derived from these approaches continues to emerge. The ability of single-molecule approaches to avoid ensemble averaging and to capture transient intermediates and heterogeneous behavior renders them particularly powerful in elucidating mechanisms of biomolecular machines: what they do, how they work individually, how they work together,more » and finally, how they work inside live cells. The burgeoning use of single-molecule methods to elucidate biological problems is a highly multidisciplinary pursuit, involving both force- and fluorescence-based methods, the most up-to-date advances in microscopy, innovative biological and chemical approaches, and nanotechnology tools. This conference seeks to bring together top experts in molecular and cell biology with innovators in the measurement and manipulation of single molecules, and will provide opportunities for junior scientists and graduate students to present their work in poster format and to exchange ideas with leaders in the field. A number of excellent poster presenters will be selected for short oral talks. Topics as diverse as single-molecule sequencing, DNA/RNA/protein interactions, folding machines, cellular biophysics, synthetic biology and bioengineering, force spectroscopy, new method developments, superresolution imaging in cells, and novel probes for single-molecule imaging will be on the program. Additionally, the collegial atmosphere of this Conference, with programmed discussion sessions as well as opportunities for informal gatherings in the afternoons and evenings in the beauty of the Il Ciocco site in Tuscany, provides an avenue for scientists from different disciplines to interact and brainstorm and promotes cross-disciplinary collaborations directed toward compelling biological problems.« less

EphA2 cleavage by MT1-MMP triggers single cancer cell invasion via homotypic cell repulsion

PubMed Central

Sugiyama, Nami; Gucciardo, Erika; Tatti, Olga; Varjosalo, Markku; Hyytiäinen, Marko; Gstaiger, Matthias

2013-01-01

Changes in EphA2 signaling can affect cancer cell–cell communication and motility through effects on actomyosin contractility. However, the underlying cell–surface interactions and molecular mechanisms of how EphA2 mediates these effects have remained unclear. We demonstrate here that EphA2 and membrane-anchored membrane type-1 matrix metalloproteinase (MT1-MMP) were selectively up-regulated and coexpressed in invasive breast carcinoma cells, where, upon physical interaction in same cell–surface complexes, MT1-MMP cleaved EphA2 at its Fibronectin type-III domain 1. This cleavage, coupled with EphA2-dependent Src activation, triggered intracellular EphA2 translocation, as well as an increase in RhoA activity and cell junction disassembly, which suggests an overall repulsive effect between cells. Consistent with this, cleavage-prone EphA2-D359I mutant shifted breast carcinoma cell invasion from collective to rounded single-cell invasion within collagen and in vivo. Up-regulated MT1-MMP also codistributed with intracellular EphA2 in invasive cells within human breast carcinomas. These results reveal a new proteolytic regulatory mechanism of cell–cell signaling in cancer invasion. PMID:23629968

Potentials of single-cell biology in identification and validation of disease biomarkers.

PubMed

Niu, Furong; Wang, Diane C; Lu, Jiapei; Wu, Wei; Wang, Xiangdong

2016-09-01

Single-cell biology is considered a new approach to identify and validate disease-specific biomarkers. However, the concern raised by clinicians is how to apply single-cell measurements for clinical practice, translate the message of single-cell systems biology into clinical phenotype or explain alterations of single-cell gene sequencing and function in patient response to therapies. This study is to address the importance and necessity of single-cell gene sequencing in the identification and development of disease-specific biomarkers, the definition and significance of single-cell biology and single-cell systems biology in the understanding of single-cell full picture, the development and establishment of whole-cell models in the validation of targeted biological function and the figure and meaning of single-molecule imaging in single cell to trace intra-single-cell molecule expression, signal, interaction and location. We headline the important role of single-cell biology in the discovery and development of disease-specific biomarkers with a special emphasis on understanding single-cell biological functions, e.g. mechanical phenotypes, single-cell biology, heterogeneity and organization of genome function. We have reason to believe that such multi-dimensional, multi-layer, multi-crossing and stereoscopic single-cell biology definitely benefits the discovery and development of disease-specific biomarkers. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

PyClone: statistical inference of clonal population structure in cancer.

PubMed

Roth, Andrew; Khattra, Jaswinder; Yap, Damian; Wan, Adrian; Laks, Emma; Biele, Justina; Ha, Gavin; Aparicio, Samuel; Bouchard-Côté, Alexandre; Shah, Sohrab P

2014-04-01

We introduce PyClone, a statistical model for inference of clonal population structures in cancers. PyClone is a Bayesian clustering method for grouping sets of deeply sequenced somatic mutations into putative clonal clusters while estimating their cellular prevalences and accounting for allelic imbalances introduced by segmental copy-number changes and normal-cell contamination. Single-cell sequencing validation demonstrates PyClone's accuracy.

Validation of Flow Cytometry and Magnetic Bead-Based Methods to Enrich CNS Single Cell Suspensions for Quiescent Microglia.

PubMed

Volden, T A; Reyelts, C D; Hoke, T A; Arikkath, J; Bonasera, S J

2015-12-01

Microglia are resident mononuclear phagocytes within the CNS parenchyma that intimately interact with neurons and astrocytes to remodel synapses and extracellular matrix. We briefly review studies elucidating the molecular pathways that underlie microglial surveillance, activation, chemotaxis, and phagocytosis; we additionally place these studies in a clinical context. We describe and validate an inexpensive and simple approach to obtain enriched single cell suspensions of quiescent parenchymal and perivascular microglia from the mouse cerebellum and hypothalamus. Following preparation of regional CNS single cell suspensions, we remove myelin debris, and then perform two serial enrichment steps for cells expressing surface CD11b. Myelin depletion and CD11b enrichment are both accomplished using antigen-specific magnetic beads in an automated cell separation system. Flow cytometry of the resultant suspensions shows a significant enrichment for CD11b(+)/CD45(+) cells (perivascular microglia) and CD11b(+)/CD45(-) cells (parenchymal microglia) compared to starting suspensions. Of note, cells from these enriched suspensions minimally express Aif1 (aka Iba1), suggesting that the enrichment process does not evoke significant microglial activation. However, these cells readily respond to a functional challenge (LPS) with significant changes in the expression of molecules specifically associated with microglia. We conclude that methods employing a combination of magnetic-bead based sorting and flow cytometry produce suspensions highly enriched for microglia that are appropriate for a variety of molecular and cellular assays.

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.

[Changes in the chromatin structure of lymphoid cells under the influence of low-intensity extremely high-frequency electromagnetic radiation against the background of inflammatory process].

PubMed

Gapeev, A B; Romanova, N A; Chemeris, N K

2011-01-01

Using the alkaline single cell gel electrophoresis technique (comet assay), changes in chromatin structure of peripheral blood leukocytes and peritoneal neutrophils have been studied in mice exposed to low-intensity extremely high-frequency electromagnetic radiation (42.2 GHz, 0.1 mW/cm2, 20 min at 1 h after induction of inflammation) against the background of the systemic inflammatory process. It was revealed that the exposure of mice with the developing inflammation leads to a pronounced decrease in the level of DNA damage to peripheral blood leukocytes and peritoneal neutrophils. It is supposed that the changes in the chromatin structure of lymphoid cells have a genoprotective character in the inflammatory process and can underlie the mechanisms of realization of antiinflammatory effects of the electromagnetic radiation.

Uncovering homo-and hetero-interactions on the cell membrane using single particle tracking approaches

NASA Astrophysics Data System (ADS)

Torreno-Pina, Juan A.; Manzo, Carlo; Garcia-Parajo, Maria F.

2016-03-01

The plasma membrane of eukaryotic cells is responsible for a myriad of functions that regulate cell physiology and plays a crucial role in a multitude of processes that include adhesion, migration, signaling recognition and cell-cell communication. This is accomplished by specific interactions between different membrane components such as lipids and proteins on the lipid bilayer but also through interactions with the underlying cortical actin cytoskeleton on the intracellular side and the glycocalyx matrix in close proximity to the extracellular side. Advanced biophysical techniques, including single particle tracking (SPT) have revealed that the lateral diffusion of molecular components on the plasma membrane represents a landmark manifestation of such interactions. Indeed, by studying changes in the diffusivity of individual membrane molecules, including sub-diffusion, confined diffusion and/or transient arrest of molecules in membrane compartments, it has been possible to gain insight on the nature of molecular interactions and to infer on its functional role for cell response. In this review, we will revise some exciting results where SPT has been crucial to reveal homo- and hetero-interactions on the cell membrane.

Controlled Human Malaria Infection Leads to Long-Lasting Changes in Innate and Innate-like Lymphocyte Populations.

PubMed

Mpina, Maxmillian; Maurice, Nicholas J; Yajima, Masanao; Slichter, Chloe K; Miller, Hannah W; Dutta, Mukta; McElrath, M Juliana; Stuart, Kenneth D; De Rosa, Stephen C; McNevin, John P; Linsley, Peter S; Abdulla, Salim; Tanner, Marcel; Hoffman, Stephen L; Gottardo, Raphael; Daubenberger, Claudia A; Prlic, Martin

2017-07-01

Animal model studies highlight the role of innate-like lymphocyte populations in the early inflammatory response and subsequent parasite control following Plasmodium infection. IFN-γ production by these lymphocytes likely plays a key role in the early control of the parasite and disease severity. Analyzing human innate-like T cell and NK cell responses following infection with Plasmodium has been challenging because the early stages of infection are clinically silent. To overcome this limitation, we examined blood samples from a controlled human malaria infection (CHMI) study in a Tanzanian cohort, in which volunteers underwent CHMI with a low or high dose of Plasmodium falciparum sporozoites. The CHMI differentially affected NK, NKT (invariant NKT), and mucosal-associated invariant T cell populations in a dose-dependent manner, resulting in an altered composition of this innate-like lymphocyte compartment. Although these innate-like responses are typically thought of as short-lived, we found that changes persisted for months after the infection was cleared, leading to significantly increased frequencies of mucosal-associated invariant T cells 6 mo postinfection. We used single-cell RNA sequencing and TCR αβ-chain usage analysis to define potential mechanisms for this expansion. These single-cell data suggest that this increase was mediated by homeostatic expansion-like mechanisms. Together, these data demonstrate that CHMI leads to previously unappreciated long-lasting alterations in the human innate-like lymphocyte compartment. We discuss the consequences of these changes for recurrent parasite infection and infection-associated pathologies and highlight the importance of considering host immunity and infection history for vaccine design. Copyright © 2017 by The American Association of Immunologists, Inc.

Fine Structure of Changes Produced in Cultured Cells Sampled at Specified Intervals During a Single Growth Cycle of Polio Virus

PubMed Central

Kallman, Frances; Williams, Robley C.; Dulbecco, Renato; Vogt, Marguerite

1958-01-01

Primary suspended cultures of rhesus monkey kidney cells were infected with poliomyelitis virus, type 1 (Brunhilde strain). The release of virus from these cells over a one-step growth curve was correlated with their change in fine structure, as seen in the electron microscope. Most of the cells were infected nearly simultaneously, and morphological changes developed in the cells were sufficiently synchronous to be classified into three stages. The earliest change (stage I) became visible at a time when virus release into the culture fluid begins, some 3 hours after adsorption. Accentuation of the abnormal characteristics soon occurs, at 4 to 7 hours after adsorption, and results in stage II. Stage III represents the appearance of cells after their rate of virus release had passed its maximum, and probably the abnormal morphology of these cells reflects non-specific physiological damage. There seems to be consistency between the previously described cellular changes as seen under the light microscope and the finer scale changes reported here. Cytoplasmic bodies, called U bodies, were seen in large number at the time when the virus release was the most rapid (stage II). While these bodies are not of proper size to be considered polio virus, they seem to be specifically related to the infection. No evidence was found for the presence of particles that could even be presumptively identified with those of polio virus. PMID:13549502

Fibered Confocal Fluorescence Microscopy for the Noninvasive Imaging of Langerhans Cells in Macaques.

PubMed

Todorova, Biliana; Salabert, Nina; Tricot, Sabine; Boisgard, Raphaël; Rathaux, Mélanie; Le Grand, Roger; Chapon, Catherine

2017-01-01

We developed a new approach to visualize skin Langerhans cells by in vivo fluorescence imaging in nonhuman primates. Macaques were intradermally injected with a monoclonal, fluorescently labeled antibody against HLA-DR molecule and were imaged for up to 5 days by fibered confocal microscopy (FCFM). The network of skin Langerhans cells was visualized by in vivo fibered confocal fluorescence microscopy. Quantification of Langerhans cells revealed no changes to cell density with time. Ex vivo experiments confirmed that injected fluorescent HLA-DR antibody specifically targeted Langerhans cells in the epidermis. This study demonstrates the feasibility of single-cell, in vivo imaging as a noninvasive technique to track Langerhans cells in nontransgenic animals.

Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases.

PubMed

Moon, JoonHo; Lee, Choongil; Kim, Su Jin; Choi, Ji-Yei; Lee, Byeong Chun; Kim, Jin-Soo; Jang, Goo

2014-05-27

Although noncancerous immortalized cell lines have been developed by introducing genes into human and murine somatic cells, such cell lines have not been available in large domesticated animals like pigs. For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the human telomerase reverse transcriptase (hTERT) gene. After selecting cells with neomycin for 2 weeks, outgrowing colonized cells were picked up and subcultured for expansion. Immortalized cells were cultured for more than 9 months without changing their doubling time (~24 hours) or their diameter (< 20 µm) while control cells became replicatively senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to knockout the CMAH gene, designed plasmids encoding a transcription activator-like effector nuclease (TALENs) pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation-sensitive T7 endonuclease I assay, fluorescent PCR, and dideoxy sequencing to obtain three independent clonal populations of cells that contained biallelic modifications. One CMAH knockout clone was chosen and used for somatic cell nuclear transfer. Cloned embryos developed to the blastocyst stage. In conclusion, we demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene, and the TALENs enabled biallelic gene disruptions in these immortalized cells.

Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases

PubMed Central

Moon, JoonHo; Lee, Choongil; Kim, Su Jin; Choi, Ji-Yei; Lee, Byeong Chun; Kim, Jin-Soo; Jang, Goo

2014-01-01

Although noncancerous immortalized cell lines have been developed by introducing genes into human and murine somatic cells, such cell lines have not been available in large domesticated animals like pigs. For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the human telomerase reverse transcriptase (hTERT) gene. After selecting cells with neomycin for 2 weeks, outgrowing colonized cells were picked up and subcultured for expansion. Immortalized cells were cultured for more than 9 months without changing their doubling time (~24 hours) or their diameter (< 20 µm) while control cells became replicatively senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to knockout the CMAH gene, designed plasmids encoding a transcription activator-like effector nuclease (TALENs) pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation-sensitive T7 endonuclease I assay, fluorescent PCR, and dideoxy sequencing to obtain three independent clonal populations of cells that contained biallelic modifications. One CMAH knockout clone was chosen and used for somatic cell nuclear transfer. Cloned embryos developed to the blastocyst stage. In conclusion, we demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene, and the TALENs enabled biallelic gene disruptions in these immortalized cells. PMID:24866481

Monitoring of Dual CRISPR/Cas9-Mediated Steroidogenic Acute Regulatory Protein Gene Deletion and Cholesterol Accumulation Using High-Resolution Fluorescence In Situ Hybridization in a Single Cell

PubMed Central

Lee, Jinwoo; Jefcoate, Colin

2017-01-01

Recent advances in fluorescence microscopy, coupled with CRISPR/Cas9 gene editing technology, provide opportunities for understanding gene regulation at the single-cell level. The application of direct imaging shown here provides an in situ side-by-side comparison of CRISPR/Cas9-edited cells and adjacent unedited cells. We apply this methodology to the steroidogenic acute regulatory protein (StAR) gene in Y-1 adrenal cells and MA-10 testis cells. StAR is a gatekeeper protein that controls the access of cholesterol from the cytoplasm to the inner mitochondria. The loss of this mitochondrial cholesterol transfer mediator rapidly increases lipid droplets in cells, as seen in StAR−/− mice. Here, we describe a dual CRISPR/Cas9 strategy marked by GFP/mCherry expression that deletes StAR activity within 12 h. We used single-molecule fluorescence in situ hybridization (sm-FISH) imaging to directly monitor the time course of gene editing in single cells. We achieved StAR gene deletion at high efficiency dual gRNA targeting to the proximal promoter and exon 2. Seventy percent of transfected cells showed a slow DNA deletion as measured by PCR, and loss of Br-cAMP stimulated transcription. This DNA deletion was seen by sm-FISH in both loci of individual cells relative to non-target Cyp11a1 and StAR exon 7. sm-FISH also distinguishes two effects on stimulated StAR expression without this deletion. Br-cAMP stimulation of primary and spliced StAR RNA at the gene loci were removed within 4 h in this dual CRISPR/Cas9 strategy before any effect on cytoplasmic mRNA and protein occurred. StAR mRNA disappeared between 12 and 24 h in parallel with this deletion, while cholesterol ester droplets increased fourfold. These alternative changes match distinct StAR expression processes. This dual gRNA and sm-FISH approach to CRISPR/Cas9 editing facilitates rapid testing of editing strategies and immediate assessment of single-cell adaptation responses without the perturbation of clonal expansion procedures. PMID:29118738

Single-cell immunofluorescence assay for terminal transferase: human leukaemic and non-leukaemic cells.

PubMed

Okamura, S; Crane, F; Jamal, N; Messner, H A; Mak, T W

1980-02-01

The characteristics of a single-cell immunofluorescence assay for terminal deoxynucleotidyl transferase (terminal transferase, TdT) is described. The data indicate that the single-cell immunofluorescence assay is highly efficient and specific for the detection of cells containing TdT. Using this assay, we have examined 124 marrow or peripheral-blood samples from 104 patients with or without haematological malignancies. Results indicate that TdT(+) cells from 6% to 100% were found in the following patients: 34/40 samples from patients with ALL at the time of diagnosis or during relapse; 2/3 patients with acute undifferentiated leukaemia; 2/3 patients with acute myelomonocytic leukaemia; 1/24 patients with acute myeloblastic leukaemia; 1/5 patients with chronic myelocytic leukaemia (CML) in blastic crisis; and 2/2 patients with diffuse lymphoblastic lymphoma. In contrast less than 1% of TdT(+) cells were found in 20 marrow or peripheral-blood samples from ALL patients in complete remission; 8 patients with CML in chronic phase; 2 patients with myeloma; 1 sample from a patient with Hodgkin's disease, peripheral-blood samples from 7 normal donors and marrow samples from 6 patients without haematological malignancies. TdT(+) cells were also found in association with cells with lymphoblast morphology. The TdT(+) cells in marrow were shown to be directly correlated with the percentage of morphological lymphoblasts, with a Spearman rank coefficient of 0·81, significant at a 0·001 level. In 2 longitudinal studies of 2 ALL patients with TdT(+) cells at diagnosis, the percentage TdT(+) cells also changed in parallel with the proportion of lymphoblasts. However, studies of 2 other patients with morphologically diagnosed ALL with < 1% TdT(+) cells at diagnosis also showed < 1% TdT(+) cells throughout the period studied, indicating a stable phenotype of blast cells in these patients. The single-cell immunofluorescence assay for TdT, which requires < 0·1% of the cells used in a conventional biochemical assay, is highly specific, and could provide a technically more efficient alternative for use in clinics as well as in experimental investigations of subpopulations of leukaemic and normal marrow cells.

Alouatta trichromatic color vision: cone spectra and physiological responses studied with microspectrophotometry and single unit retinal electrophysiology.

PubMed

Silveira, Luiz Carlos L; Saito, Cézar A; da Silva Filho, Manoel; Kremers, Jan; Bowmaker, James K; Lee, Barry B

2014-01-01

The howler monkeys (Alouatta sp.) are the only New World primates to exhibit routine trichromacy. Both males and females have three cone photopigments. However, in contrast to Old World monkeys, Alouatta has a locus control region upstream of each opsin gene on the X-chromosome and this might influence the retinal organization underlying its color vision. Post-mortem microspectrophotometry (MSP) was performed on the retinae of two male Alouatta to obtain rod and cone spectral sensitivities. The MSP data were consistent with only a single opsin being expressed in each cone and electrophysiological data were consistent with this primate expressing full trichromacy. To study the physiological organization of the retina underlying Alouatta trichromacy, we recorded from retinal ganglion cells of the same animals used for MSP measurements with a variety of achromatic and chromatic stimulus protocols. We found MC cells and PC cells in the Alouatta retina with similar properties to those previously found in the retina of other trichromatic primates. MC cells showed strong phasic responses to luminance changes and little response to chromatic pulses. PC cells showed strong tonic response to chromatic changes and small tonic response to luminance changes. Responses to other stimulus protocols (flicker photometry; changing the relative phase of red and green modulated lights; temporal modulation transfer functions) were also similar to those recorded in other trichromatic primates. MC cells also showed a pronounced frequency double response to chromatic modulation, and with luminance modulation response saturation accompanied by a phase advance between 10-20 Hz, characteristic of a contrast gain mechanism. This indicates a very similar retinal organization to Old-World monkeys. Cone-specific opsin expression in the presence of a locus control region for each opsin may call into question the hypothesis that this region exclusively controls opsin expression.

Single-cell isolation by a modular single-cell pipette for RNA-sequencing.

PubMed

Zhang, Kai; Gao, Min; Chong, Zechen; Li, Ying; Han, Xin; Chen, Rui; Qin, Lidong

2016-11-29

Single-cell transcriptome sequencing highly requires a convenient and reliable method to rapidly isolate a live cell into a specific container such as a PCR tube. Here, we report a modular single-cell pipette (mSCP) consisting of three modular components, a SCP-Tip, an air-displacement pipette (ADP), and ADP-Tips, that can be easily assembled, disassembled, and reassembled. By assembling the SCP-Tip containing a hydrodynamic trap, the mSCP can isolate single cells from 5-10 cells per μL of cell suspension. The mSCP is compatible with microscopic identification of captured single cells to finally achieve 100% single-cell isolation efficiency. The isolated live single cells are in submicroliter volumes and well suitable for single-cell PCR analysis and RNA-sequencing. The mSCP possesses merits of convenience, rapidness, and high efficiency, making it a powerful tool to isolate single cells for transcriptome analysis.

Regulation of alternative splicing at the single-cell level.

PubMed

Faigenbloom, Lior; Rubinstein, Nimrod D; Kloog, Yoel; Mayrose, Itay; Pupko, Tal; Stein, Reuven

2015-12-28

Alternative splicing is a key cellular mechanism for generating distinct isoforms, whose relative abundances regulate critical cellular processes. It is therefore essential that inclusion levels of alternative exons be tightly regulated. However, how the precision of inclusion levels among individual cells is governed is poorly understood. Using single-cell gene expression, we show that the precision of inclusion levels of alternative exons is determined by the degree of evolutionary conservation at their flanking intronic regions. Moreover, the inclusion levels of alternative exons, as well as the expression levels of the transcripts harboring them, also contribute to this precision. We further show that alternative exons whose inclusion levels are considerably changed during stem cell differentiation are also subject to this regulation. Our results imply that alternative splicing is coordinately regulated to achieve accuracy in relative isoform abundances and that such accuracy may be important in determining cell fate. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

Architectural transitions in Vibrio cholerae biofilms at single-cell resolution

PubMed Central

Drescher, Knut; Dunkel, Jörn; Nadell, Carey D.; van Teeffelen, Sven; Grnja, Ivan; Wingreen, Ned S.; Stone, Howard A.; Bassler, Bonnie L.

2016-01-01

Many bacterial species colonize surfaces and form dense 3D structures, known as biofilms, which are highly tolerant to antibiotics and constitute one of the major forms of bacterial biomass on Earth. Bacterial biofilms display remarkable changes during their development from initial attachment to maturity, yet the cellular architecture that gives rise to collective biofilm morphology during growth is largely unknown. Here, we use high-resolution optical microscopy to image all individual cells in Vibrio cholerae biofilms at different stages of development, including colonies that range in size from 2 to 4,500 cells. From these data, we extracted the precise 3D cellular arrangements, cell shapes, sizes, and global morphological features during biofilm growth on submerged glass substrates under flow. We discovered several critical transitions of the internal and external biofilm architectures that separate the major phases of V. cholerae biofilm growth. Optical imaging of biofilms with single-cell resolution provides a new window into biofilm formation that will prove invaluable to understanding the mechanics underlying biofilm development. PMID:26933214

How half-coated janus particles enter cells.

PubMed

Gao, Yuan; Yu, Yan

2013-12-26

Janus particles possess functional asymmetry and directionality within a single entity and thus are predicted to enable many promising biomedical applications that are not offered by homogeneous particles. However, it remains elusive what role the Janus principle plays in Janus particle-cell interactions, particularly in cellular uptake. We studied how asymmetric distribution of ligands on half-coated Janus microparticles dictates the membrane dynamics during receptor-mediated particle uptake, and found key differences from those characteristic of homogeneous particles. Live-cell fluorescence imaging combined with single-particle level quantification of particle-cell membrane interactions shows that the asymmetric distribution of ligands leads to a three-step endocytic process: membrane cup formation on the ligand-coated hemisphere, stalling at the Janus interface, and rapid membrane protrusion on the ligand-absent hemisphere to complete the particle engulfment. The direct correlation between the spatial presentation of ligands on Janus particles and the temporal changes of membrane dynamics revealed in this work elucidates the potential of using the Janus principle to fine-tune particle-cell interactions.

Long Term Study of Protective Mechanisms of Human Adipose Derived Mesenchymal Stem Cells on Cisplatin Induced Kidney injury in Sprague-Dawley Rats.

PubMed

Elhusseini, Fatma M; Saad, Mohamed-Ahdy A A; Anber, Nahla; Elghannam, Doaa; Sobh, Mohamed-Ahmed; Alsayed, Aziza; El-Dusoky, Sara; Sheashaa, Hussein; Abdel-Ghaffar, Hassan; Sobh, Mohamed

2016-01-01

Long-term evaluation of cisplatin induced nephrotoxicity and the probable renal protective activities of stem cells are lacking up until now. We evaluated the early and long-term role of human adipose derived mesenchymal stem cells (ADMSCs) in prevention or amelioration of cisplatin induced acute kidney injury (AKI) in Sprague-Dawley rats. For this, we determined the kidney tissue level of oxidative stress markers in conjugation with a renal histopathological scoring system of both acute and chronic renal changes. This study used eighty Sprague-Dawley (SD) rats weighing 250-300g. They were assigned into four equal groups (each group n=20): (I) Negative control group, rats injected with single dose of 1 ml normal saline. (II) Positive control cisplatin, rats injected with a single dose of 5 mg/kg I.P in 1 ml saline. (III) Cisplatin and culture media group, rats injected with 0.5 ml of culture media single dose into the tail vein and (IV) Cisplatin and ADMSCs group, rats injected with a single dose of 0.5 ml of culture media containing 5 x10(6)ADMSCs into the tail vein one day after cisplatin administration. Each main group was further divided according to the timing of sacrifice into four subgroups (each subgroup n=5). Rats in the subgroup A were sacrificed after 4 days; subgroup B were sacrificed after 7 days; subgroup C were sacrificed after 11 days; and subgroup D were sacrificed after 30 days. Before sacrifice, 24 hrs.-urine was collected using a metabolic cage. Renal function was evaluated through blood urea nitrogen (BUN), serum creatinine and creatinine clearance. Kidney tissue homogenate oxidative stress parameters, Malondialdehyde (MDA), Superoxide dismutase (SOD) and Glutathione (GSH) were determined. In addition, histopathological analysis for active injury, regenerative and chronic changes was performed. ADMSCs were characterized and their capability of differentiation was proved. Cisplatin induced a significant increase in plasma creatinine and tissue MDA and induced a decrease in SOD, GSH and creatinine clearance. ADMSCs attenuated these changes. Cisplatin resulted in prominent histopathological changes in the term of tubular necrosis, atrophy, inflammatory cells infiltration and fibrosis. ADMSCs significantly lowered the injury score at day 4, 7, 11 and 30 with marked regenerative changes starting from day 4 and limited fibrotic score at day 30. ADMSCs have both protective and regenerative abilities with consequent limitation of the development of renal fibrosis after the cisplatin induced acute tubular necrosis, largely through an anti-oxidative activity.

Action Potential Waveform Variability Limits Multi-Unit Separation in Freely Behaving Rats

PubMed Central

Stratton, Peter; Cheung, Allen; Wiles, Janet; Kiyatkin, Eugene; Sah, Pankaj; Windels, François

2012-01-01

Extracellular multi-unit recording is a widely used technique to study spontaneous and evoked neuronal activity in awake behaving animals. These recordings are done using either single-wire or mulitwire electrodes such as tetrodes. In this study we have tested the ability of single-wire electrodes to discriminate activity from multiple neurons under conditions of varying noise and neuronal cell density. Using extracellular single-unit recording, coupled with iontophoresis to drive cell activity across a wide dynamic range, we studied spike waveform variability, and explored systematic differences in single-unit spike waveform within and between brain regions as well as the influence of signal-to-noise ratio (SNR) on the similarity of spike waveforms. We also modelled spike misclassification for a range of cell densities based on neuronal recordings obtained at different SNRs. Modelling predictions were confirmed by classifying spike waveforms from multiple cells with various SNRs using a leading commercial spike-sorting system. Our results show that for single-wire recordings, multiple units can only be reliably distinguished under conditions of high recording SNR (≥4) and low neuronal density (≈20,000/ mm3). Physiological and behavioural changes, as well as technical limitations typical of awake animal preparations, reduce the accuracy of single-channel spike classification, resulting in serious classification errors. For SNR <4, the probability of misclassifying spikes approaches 100% in many cases. Our results suggest that in studies where the SNR is low or neuronal density is high, separation of distinct units needs to be evaluated with great caution. PMID:22719894

From single molecule to single tubules

NASA Astrophysics Data System (ADS)

Guo, Chin-Lin

2012-02-01

Biological systems often make decisions upon conformational changes and assembly of single molecules. In vivo, epithelial cells (such as the mammary gland cells) can respond to extracellular matrix (ECM) molecules, type I collagen (COL), and switch their morphology from a lobular lumen (100-200 micron) to a tubular lumen (1mm-1cm). However, how cells make such a morphogenetic decision through interactions with each other and with COL is unclear. Using a temporal control of cell-ECM interaction, we find that epithelial cells, in response to a fine-tuned percentage of type I collagen (COL) in ECM, develop various linear patterns. Remarkably, these patterns allow cells to self-assemble into a tubule of length ˜ 1cm and diameter ˜ 400 micron in the liquid phase (i.e., scaffold-free conditions). In contrast with conventional thought, the linear patterns arise through bi-directional transmission of traction force, but not through diffusible biochemical factors secreted by cells. In turn, the transmission of force evokes a long-range (˜ 600 micron) intercellular mechanical interaction. A feedback effect is encountered when the mechanical interaction modifies cell positioning and COL alignment. Micro-patterning experiments further reveal that such a feedback is a novel cell-number-dependent, rich-get-richer process, which allows cells to integrate mechanical interactions into long-range (> 1mm) linear coordination. Our results suggest a mechanism cells can use to form and coordinate long-range tubular patterns, independent of those controlled by diffusible biochemical factors, and provide a new strategy to engineer/regenerate epithelial organs using scaffold-free self-assembly methods.

Analyzing neural responses with vector fields.

PubMed

Buneo, Christopher A

2011-04-15

Analyzing changes in the shape and scale of single cell response fields is a key component of many neurophysiological studies. Typical analyses of shape change involve correlating firing rates between experimental conditions or "cross-correlating" single cell tuning curves by shifting them with respect to one another and correlating the overlapping data. Such shifting results in a loss of data, making interpretation of the resulting correlation coefficients problematic. The problem is particularly acute for two dimensional response fields, which require shifting along two axes. Here, an alternative method for quantifying response field shape and scale based on correlation of vector field representations is introduced. The merits and limitations of the methods are illustrated using both simulated and experimental data. It is shown that vector correlation provides more information on response field changes than scalar correlation without requiring field shifting and concomitant data loss. An extension of this vector field approach is also demonstrated which can be used to identify the manner in which experimental variables are encoded in studies of neural reference frames. Copyright © 2011 Elsevier B.V. All rights reserved.

Number of infection events per cell during HIV-1 cell-free infection.

PubMed

Ito, Yusuke; Remion, Azaria; Tauzin, Alexandra; Ejima, Keisuke; Nakaoka, Shinji; Iwasa, Yoh; Iwami, Shingo; Mammano, Fabrizio

2017-07-26

HIV-1 accumulates changes in its genome through both recombination and mutation during the course of infection. For recombination to occur, a single cell must be infected by two HIV strains. These coinfection events were experimentally demonstrated to occur more frequently than would be expected for independent infection events and do not follow a random distribution. Previous mathematical modeling approaches demonstrated that differences in target cell susceptibility can explain the non-randomness, both in the context of direct cell-to-cell transmission, and in the context of free virus transmission (Q. Dang et al., Proc. Natl. Acad. Sci. USA 101:632-7, 2004: K. M. Law et al., Cell reports 15:2711-83, 2016). Here, we build on these notions and provide a more detailed and extensive quantitative framework. We developed a novel mathematical model explicitly considering the heterogeneity of target cells and analysed datasets of cell-free HIV-1 single and double infection experiments in cell culture. Particularly, in contrast to the previous studies, we took into account the different susceptibility of the target cells as a continuous distribution. Interestingly, we showed that the number of infection events per cell during cell-free HIV-1 infection follows a negative-binomial distribution, and our model reproduces these datasets.

NF-κB dynamics show digital activation and analog information processing in cells

NASA Astrophysics Data System (ADS)

Tay, Savas; Hughey, Jake; Lee, Timothy; Lipniacki, Tomasz; Covert, Markus; Quake, Stephen

2010-03-01

Cells operate in ever changing environments using extraordinary communication capabilities. Cell-to-cell communication is mediated by signaling molecules that form spatiotemporal concentration gradients, which requires cells to respond to a wide range of signal intensities. We used high-throughput microfluidic cell culture, quantitative gene expression analysis and mathematical modeling to investigate how single mammalian cells respond to different concentrations of the signaling molecule TNF-α via the transcription factor NF-κB. We measured NF-κB activity in thousands of live cells under TNF-α doses covering four orders of magnitude. In contrast to population studies, the activation is a stochastic, switch-like process at the single cell level with fewer cells responding at lower doses. The activated cells respond fully and express early genes independent of the TNF-α concentration, while only high dose stimulation results in the expression of late genes. Cells also encode a set of analog parameters such as the NF-κB peak intensity, response time and number of oscillations to modulate the outcome. We developed a stochastic model that reproduces both the digital and analog dynamics as well as the gene expression profiles at all measured conditions, constituting a broadly applicable model for TNF-α induced NF-κB signaling in various types of cells.

Kinetic Modeling of ABCG2 Transporter Heterogeneity: A Quantitative, Single-Cell Analysis of the Side Population Assay

PubMed Central

Prasanphanich, Adam F.; White, Douglas E.; Gran, Margaret A.

2016-01-01

The side population (SP) assay, a technique used in cancer and stem cell research, assesses the activity of ABC transporters on Hoechst staining in the presence and absence of transporter inhibition, identifying SP and non-SP cell (NSP) subpopulations by differential staining intensity. The interpretation of the assay is complicated because the transporter-mediated mechanisms fail to account for cell-to-cell variability within a population or adequately control the direct role of transporter activity on staining intensity. We hypothesized that differences in dye kinetics at the single-cell level, such as ABCG2 transporter-mediated efflux and DNA binding, are responsible for the differential cell staining that demarcates SP/NSP identity. We report changes in A549 phenotype during time in culture and with TGFβ treatment that correlate with SP size. Clonal expansion of individually sorted cells re-established both SP and NSPs, indicating that SP membership is dynamic. To assess the validity of a purely kinetics-based interpretation of SP/NSP identity, we developed a computational approach that simulated cell staining within a heterogeneous cell population; this exercise allowed for the direct inference of the role of transporter activity and inhibition on cell staining. Our simulated SP assay yielded appropriate SP responses for kinetic scenarios in which high transporter activity existed in a portion of the cells and little differential staining occurred in the majority of the population. With our approach for single-cell analysis, we observed SP and NSP cells at both ends of a transporter activity continuum, demonstrating that features of transporter activity as well as DNA content are determinants of SP/NSP identity. PMID:27851764

Separation of cell survival, growth, migration, and mesenchymal transdifferentiation effects of fibroblast secretome on tumor cells of head and neck squamous cell carcinoma.

PubMed

Metzler, Veronika Maria; Pritz, Christian; Riml, Anna; Romani, Angela; Tuertscher, Raphaela; Steinbichler, Teresa; Dejaco, Daniel; Riechelmann, Herbert; Dudás, József

2017-11-01

Fibroblasts play a central role in tumor invasion, recurrence, and metastasis in head and neck squamous cell carcinoma. The aim of this study was to investigate the influence of tumor cell self-produced factors and paracrine fibroblast-secreted factors in comparison to indirect co-culture on cancer cell survival, growth, migration, and epithelial-mesenchymal transition using the cell lines SCC-25 and human gingival fibroblasts. Thereby, we particularly focused on the participation of the fibroblast-secreted transforming growth factor beta-1.Tumor cell self-produced factors were sufficient to ensure tumor cell survival and basic cell growth, but fibroblast-secreted paracrine factors significantly increased cell proliferation, migration, and epithelial-mesenchymal transition-related phenotype changes in tumor cells. Transforming growth factor beta-1 generated individually migrating disseminating tumor cell groups or single cells separated from the tumor cell nest, which were characterized by reduced E-cadherin expression. At the same time, transforming growth factor beta-1 inhibited tumor cell proliferation under serum-starved conditions. Neutralizing transforming growth factor beta antibody reduced the cell migration support of fibroblast-conditioned medium. Transforming growth factor beta-1 as a single factor was sufficient for generation of disseminating tumor cells from epithelial tumor cell nests, while other fibroblast paracrine factors supported tumor nest outgrowth. Different fibroblast-released factors might support tumor cell proliferation and invasion, as two separate effects.

Red Blood Cell Deformation Under Shear Flow: The Effect of Changing Cell Properties

NASA Astrophysics Data System (ADS)

Forsyth, Alison M.; Wan, Jiandi; Ristenpart, William D.; Stone, Howard A.

2008-11-01

The deformability of red blood cells plays a major role in the pathology of several diseases, including malaria, sickle cell anemia and spherocytosis. Moreover, deformations are believed to trigger the release of adenosine triphosphate, which helps regulate vascular tone and is consequently an important factor in various vascular diseases. Here we investigate single-cell viscoelastic responses to increased shear stress in poly(dimethylsiloxane) channels with a single constriction 2-4 times larger than a typical erythrocyte. These channels mimic arteriole-sized vessels, and have the advantage that the cell membrane is not in contact with the channel walls which have vastly different mechanical and material properties than living tissue. High-speed video and image analysis were used to quantify the trajectories and deformations of cells exposed to varied doses of diamide, a chemical known to ``rigidify'' erythrocytes. Our results show that (i) deformation is proportional to shear rate and (ii) the deformability of diamide-treated cells is greater than that of untreated cells. The latter is an unforeseen result because micropipette aspiration experiments have shown the opposite. We expect that the experimental procedure described here will be useful for characterizing the effect of different therapeutic agents on cellular deformability.

Cytotoxicity of doxrubicin loaded single-walled carbon nanotubes.

PubMed

Ünlü, Ayhan; Meran, Mehdi; Dinc, Bircan; Karatepe, Nilgün; Bektaş, Muhammet; Güner, F Seniha

2018-05-24

Carbon nanotube (CNTs) is a new alternative for efficient drug delivery and it has a great potential to change drug delivery system profile in pharmaceutical industry. One of the important advantage of CNTs is their needle-like, cylindrical shape. This shape provides a high surface area for multiple connections and adsorption onto for millions of therapeutic molecules. CNTs can be internalized by cells via endocytosis, passive diffusion and phagocytosis and release the drug with different effects like pH and temperature. The acidic nature of cancer cells and the susceptibility of CNTs to release the drug in the acidic environment have made it a promising area of research in cancer drug delivery. In this research, we investigated cell viability, cytotoxicity and drug delivery in breast cancer cell line by designing non-covalent single walled carbon nanotubes (SWNT)-doxorubicin (DOX) supramolecular complex that can be developed for cancer therapy. Applied high concentrations of DOX loaded SWNTs changed the actin structure of the cells and prevented the proliferation of the cells. It was showed that doxorubicin loaded SWNTs were more effective than free doxorubicin at relatively small concentrations. Once we applied same procedure for short and long (short: 1-1.3 µm; long: 2.5-4 µm) SWNTs and compared the results, more disrupted cell structure and reduction in cell proliferation were observed for long CNTs. DOX is bounded more to nanotubes in basic medium, less bound in acidic environment. Cancer cells were also examined for concentration at which they were effective by applying DOX and it was seen that 3.68 µM doxorubicin kills more than 55% of the cells.

Energy output of a single outer hair cell: Effect of resonance

NASA Astrophysics Data System (ADS)

Iwasa, Kuni H.

2018-05-01

The ability of the mammalian ear in processing high frequency sounds, up to ˜100 kHz, is based on the capability of outer hair cells (OHCs) in responding to stimulation at high frequencies. These cells show a unique motility in their cell body coupled with charge movement. With this motile element, voltage changes generated by stimuli at their hair bundles drive the cell body and that, in turn, amplifies the signal. In vitro experiments show that the movement of these charges significantly increases the membrane capacitance, limiting the motile activity by an additional attenuation of voltage changes. It was found, however, that such an effect is due to the absence of mechanical load. In the presence of mechanical load, particularly inertial load, such as under in vivo conditions, the movement of motile charges should reduce the membrane capacitance, enhancing the mechanical power output.

Time lapse microscopy observation of cellular structural changes and image analysis of drug treated cancer cells to characterize the cellular heterogeneity.

PubMed

Vaiyapuri, Periasamy S; Ali, Alshatwi A; Mohammad, Akbarsha A; Kandhavelu, Jeyalakshmi; Kandhavelu, Meenakshisundaram

2015-01-01

The effect of Calotropis gigantea latex (CGLX) on human mammary carcinoma cells is not well established. We present the results of this drug activity at total population and single cell level. CGLX inhibited the growth of MCF7 cancer cells at lower IC50 concentration (17 µL/mL). Microscopy of IC50 drug treated cells at 24 hr confirming the appearance of morphological characteristics of apoptotic and necrotic cells, associated with 70% of DNA damage. FACS analysis confirmed that, 10 and 20% of the disruption of cellular mitochondrial nature by at 24 and 48 h, respectively. Microscopic image analysis of total population level proved that MMP changes were statistically significant with P values. The cell to cell variation was confirmed by functional heterogeneity analysis which proves that CGLX was able to induce the apoptosis without the contribution of mitochondria. We conclude that CGLX inhibits cell proliferation, survival, and heterogeneity of pathways in human mammary carcinoma cells. © 2014 Wiley Periodicals, Inc.

High performance static latches with complete single event upset immunity

DOEpatents

Corbett, W.T.; Weaver, H.T.

1994-04-26

An asymmetric response latch providing immunity to single event upset without loss of speed is described. The latch has cross-coupled inverters having a hardened logic state and a soft state, wherein the logic state of the first inverter can only be changed when the voltage on the coupling node of that inverter is low and the logic state of the second inverter can only be changed when the coupling of that inverter is high. One of more of the asymmetric response latches may be configured into a memory cell having complete immunity, which protects information rather than logic states. 5 figures.

Collision of two action potentials in a single excitable cell.

PubMed

Fillafer, Christian; Paeger, Anne; Schneider, Matthias F

2017-12-01

It is a common incident in nature, that two waves or pulses run into each other head-on. The outcome of such an event is of special interest, because it allows conclusions about the underlying physical nature of the pulses. The present experimental study dealt with the head-on meeting of two action potentials (AP) in a single excitable plant cell (Chara braunii internode). The membrane potential was monitored with multiple sensors along a single excitable cell. In control experiments, an AP was excited electrically at either end of the cell cylinder. Subsequently, stimuli were applied simultaneously at both ends of the cell in order to generate two APs that met each other head-on. When two action potentials propagated into each other, the pulses did not penetrate but annihilated (N=26 experiments in n=10 cells). APs in excitable plant cells did not penetrate upon meeting head-on. In the classical electrical model, this behavior is specifically attributed to relaxation of ion channel proteins. From an acoustic point of view, annihilation can be viewed as a result of nonlinear material properties (e.g. a phase change). The present results suggest that APs in excitable animal and plant cells belong to a similar class of nonlinear phenomena. Intriguingly, other excitation waves in biology (intracellular waves, cortical spreading depression, etc.) also annihilate upon collision and are thus expected to follow the same underlying principles as the observed action potentials. Copyright © 2017 Elsevier B.V. All rights reserved.

The activation threshold of CD4+ T cells is defined by TCR/peptide-MHC class II interactions in the thymic medulla.

PubMed

Stephen, Tom Li; Tikhonova, Anastasia; Riberdy, Janice M; Laufer, Terri M

2009-11-01

Immature thymocytes that are positively selected based upon their response to self-peptide-MHC complexes develop into mature T cells that are not overtly reactive to those same complexes. Developmental tuning is the active process through which TCR-associated signaling pathways of single-positive thymocytes are attenuated to respond appropriately to the peptide-MHC molecules that will be encountered in the periphery. In this study, we explore the mechanisms that regulate the tuning of CD4(+) single-positive T cells to MHC class II encountered in the thymic medulla. Experiments with murine BM chimeras demonstrate that tuning can be mediated by MHC class II expressed by either thymic medullary epithelial cells or thymic dendritic cells. Tuning does not require the engagement of CD4 by MHC class II on stromal cells. Rather, it is mediated by interactions between MHC class II and the TCR. To understand the molecular changes that distinguish immature hyperactive T cells from tuned mature CD4(+) T cells, we compared their responses to TCR stimulation. The altered response of mature CD4 single-positive thymocytes is characterized by the inhibition of ERK activation by low-affinity self-ligands and increased expression of the inhibitory tyrosine phosphatase SHP-1. Thus, persistent TCR engagement by peptide-MHC class II on thymic medullary stroma inhibits reactivity to self-Ags and prevents autoreactivity in the mature repertoire.

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

Husain, Mainul, E-mail: mainul.husain@hc-sc.gc.ca; Kyjovska, Zdenka O., E-mail: zky@nrcwe.dk; Bourdon-Lacombe, Julie, E-mail: julie.bourdon-lacombe@hc-sc.gc.ca

Inhalation of carbon black nanoparticles (CBNPs) causes pulmonary inflammation; however, time course data to evaluate the detailed evolution of lung inflammatory responses are lacking. Here we establish a time-series of lung inflammatory response to CBNPs. Female C57BL/6 mice were intratracheally instilled with 162 μg CBNPs alongside vehicle controls. Lung tissues were examined 3 h, and 1, 2, 3, 4, 5, 14, and 42 days (d) post-exposure. Global gene expression and pulmonary inflammation were assessed. DNA damage was evaluated in bronchoalveolar lavage (BAL) cells and lung tissue using the comet assay. Increased neutrophil influx was observed at all time-points. DNA strandmore » breaks were increased in BAL cells 3 h post-exposure, and in lung tissues 2–5 d post-exposure. Approximately 2600 genes were differentially expressed (± 1.5 fold; p ≤ 0.05) across all time-points in the lungs of exposed mice. Altered transcript levels were associated with immune-inflammatory response and acute phase response pathways, consistent with the BAL profiles and expression changes found in common respiratory infectious diseases. Genes involved in DNA repair, apoptosis, cell cycle regulation, and muscle contraction were also differentially expressed. Gene expression changes associated with inflammatory response followed a biphasic pattern, with initial changes at 3 h post-exposure declining to base-levels by 3 d, increasing again at 14 d, and then persisting to 42 d post-exposure. Thus, this single CBNP exposure that was equivalent to nine 8-h working days at the current Danish occupational exposure limit induced biphasic inflammatory response in gene expression that lasted until 42 d post-exposure, raising concern over the chronic effects of CBNP exposure. - Highlights: • A single exposure to CBNPs induced expression changes in over 2600 genes in mouse lungs. • Altered genes were associated with immune-inflammatory and acute phase responses. • Several genes were involved in DNA repair, apoptosis, and muscle contraction. • Effects of a single exposure to CBNPs lasted until 42 d post-exposure. • A single exposure to CBNPs induced a biphasic inflammatory response in gene expression.« less

Ring-Shaped Microlanes and Chemical Barriers as a Platform for Probing Single-Cell Migration.

PubMed

Schreiber, Christoph; Segerer, Felix J; Wagner, Ernst; Roidl, Andreas; Rädler, Joachim O

2016-05-31

Quantification and discrimination of pharmaceutical and disease-related effects on cell migration requires detailed characterization of single-cell motility. In this context, micropatterned substrates that constrain cells within defined geometries facilitate quantitative readout of locomotion. Here, we study quasi-one-dimensional cell migration in ring-shaped microlanes. We observe bimodal behavior in form of alternating states of directional migration (run state) and reorientation (rest state). Both states show exponential lifetime distributions with characteristic persistence times, which, together with the cell velocity in the run state, provide a set of parameters that succinctly describe cell motion. By introducing PEGylated barriers of different widths into the lane, we extend this description by quantifying the effects of abrupt changes in substrate chemistry on migrating cells. The transit probability decreases exponentially as a function of barrier width, thus specifying a characteristic penetration depth of the leading lamellipodia. Applying this fingerprint-like characterization of cell motion, we compare different cell lines, and demonstrate that the cancer drug candidate salinomycin affects transit probability and resting time, but not run time or run velocity. Hence, the presented assay allows to assess multiple migration-related parameters, permits detailed characterization of cell motility, and has potential applications in cell biology and advanced drug screening.

Cell differentiation modeled via a coupled two-switch regulatory network

NASA Astrophysics Data System (ADS)

Schittler, D.; Hasenauer, J.; Allgöwer, F.; Waldherr, S.

2010-12-01

Mesenchymal stem cells can give rise to bone and other tissue cells, but their differentiation still escapes full control. In this paper we address this issue by mathematical modeling. We present a model for a genetic switch determining the cell fate of progenitor cells which can differentiate into osteoblasts (bone cells) or chondrocytes (cartilage cells). The model consists of two switch mechanisms and reproduces the experimentally observed three stable equilibrium states: a progenitor, an osteogenic, and a chondrogenic state. Conventionally, the loss of an intermediate (progenitor) state and the entailed attraction to one of two opposite (differentiated) states is modeled as a result of changing parameters. In our model in contrast, we achieve this by distributing the differentiation process to two functional switch parts acting in concert: one triggering differentiation and the other determining cell fate. Via stability and bifurcation analysis, we investigate the effects of biochemical stimuli associated with different system inputs. We employ our model to generate differentiation scenarios on the single cell as well as on the cell population level. The single cell scenarios allow to reconstruct the switching upon extrinsic signals, whereas the cell population scenarios provide a framework to identify the impact of intrinsic properties and the limiting factors for successful differentiation.

Development of porous carbon foam polymer electrolyte membrane fuel cell

NASA Astrophysics Data System (ADS)

Kim, Jin; Cunningham, Nicolas

In order to prove the feasibility of using porous carbon foam material in a polymer electrolyte membrane fuel cell (PEMFC), a single PEMFC is constructed with a piece of 80PPI (pores per linear inch) Reticulated Vitreous Carbon (RVC) foam at a thickness of 3.5 mm employed in the cathode flow-field. The cell performance of such design is compared with that of a conventional fuel cell with serpentine channel design in the cathode and anode flow-fields. Experimental results show that the RVC foam fuel cell not only produces comparative power density to, but also offers interesting benefits over the conventional fuel cell. A 250 h long term test conducted on a RVC foam fuel cell shows that the durability and performance stability of the material is deemed to be acceptable. Furthermore, a parametric study is conducted on single RVC foam fuel cells. Effect of geometrical and material parameters of the RVC foam such as PPI and thickness and operating conditions such as pressure, temperature, and stoichiometric ratio of the reactant gases on the cell performance is experimentally investigated in detail. The single cell with the 80PPI RVC foam exhibits the best performance, especially if the thinnest foam (3.5 mm) is used. The cell performance improves with increasing the operating gauge pressure from 0 kPa to 80 kPa and the operating temperature from 40 °C to 60 °C, but deteriorates as it further increases to 80 °C. The cell performance improves as the stoichiometric ratio of air increases from 1.5 to 4.5; however, the improvement becomes marginal when it is raised above 3.0. On the other hand, changing the stoichiometric ratio of hydrogen does not have a significant impact on the cell performance.

Biological responses of human solid tumor cells to X-ray irradiation within a 1.5-Tesla magnetic field generated by a magnetic resonance imaging-linear accelerator.

PubMed

Wang, Li; Hoogcarspel, Stan Jelle; Wen, Zhifei; van Vulpen, Marco; Molkentine, David P; Kok, Jan; Lin, Steven H; Broekhuizen, Roel; Ang, Kie-Kian; Bovenschen, Niels; Raaymakers, Bas W; Frank, Steven J

2016-10-01

Devices that combine magnetic resonance imaging with linear accelerators (MRL) represent a novel tool for MR-guided radiotherapy. However, whether magnetic fields (MFs) generated by these devices affect the radiosensitivity of tumors is unknown. We investigated the influence of a 1.5-T MF on cell viability and radioresponse of human solid tumors. Human head/neck cancer and lung cancer cells were exposed to single or fractionated 6-MV X-ray radiation; effects of the MF on cell viability were determined by cell plating efficiency and on radioresponsiveness by clonogenic cell survival. Doses needed to reduce the fraction of surviving cells to 37% of the initial value (D0s) were calculated for multiple exposures to MF and radiation. Results were analyzed using Student's t-tests. Cell viability was no different after single or multiple exposures to MRL than after exposure to a conventional linear accelerator (Linac, without MR-generated MF) in 12 of 15 experiments (all P > 0.05). Single or multiple exposures to MF had no influence on cell radioresponse (all P > 0.05). Cells treated up to four times with an MRL or a Linac further showed no changes in D0s with MF versus without MF (all P > 0.05). In conclusion, MF within the MRL does not seem to affect in vitro tumor radioresponsiveness as compared with a conventional Linac. Bioelectromagnetics. 37:471-480, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Aging yeast gain a competitive advantage on non-optimal carbon sources.

PubMed

Frenk, Stephen; Pizza, Grazia; Walker, Rachael V; Houseley, Jonathan

2017-06-01

Animals, plants and fungi undergo an aging process with remarkable physiological and molecular similarities, suggesting that aging has long been a fact of life for eukaryotes and one to which our unicellular ancestors were subject. Key biochemical pathways that impact longevity evolved prior to multicellularity, and the interactions between these pathways and the aging process therefore emerged in ancient single-celled eukaryotes. Nevertheless, we do not fully understand how aging impacts the fitness of unicellular organisms, and whether such cells gain a benefit from modulating rather than simply suppressing the aging process. We hypothesized that age-related loss of fitness in single-celled eukaryotes may be counterbalanced, partly or wholly, by a transition from a specialist to a generalist life-history strategy that enhances adaptability to other environments. We tested this hypothesis in budding yeast using competition assays and found that while young cells are more successful in glucose, highly aged cells outcompete young cells on other carbon sources such as galactose. This occurs because aged yeast divide faster than young cells in galactose, reversing the normal association between age and fitness. The impact of aging on single-celled organisms is therefore complex and may be regulated in ways that anticipate changing nutrient availability. We propose that pathways connecting nutrient availability with aging arose in unicellular eukaryotes to capitalize on age-linked diversity in growth strategy and that individual cells in higher eukaryotes may similarly diversify during aging to the detriment of the organism as a whole. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio

PubMed Central

Tantama, Mathew; Martínez-François, Juan Ramón; Mongeon, Rebecca; Yellen, Gary

2013-01-01

The ATP:ADP ratio is a critical parameter of cellular energy status that regulates many metabolic activities. Here we report an optimized genetically-encoded fluorescent biosensor, PercevalHR, that senses the ATP:ADP ratio. PercevalHR is tuned to the range of intracellular ATP:ADP expected in mammalian cells, and it can be used with one- or two-photon microscopy in live samples. We use PercevalHR to visualize activity-dependent changes in ATP:ADP when neurons are exposed to multiple stimuli, demonstrating that it is a sensitive reporter of physiological changes in energy consumption and production. We also use PercevalHR to visualize intracellular ATP:ADP while simultaneously recording currents from ATP-sensitive potassium (KATP) channels in single cells, showing that PercevalHR enables the study of coordinated variation in ATP:ADP and KATP channel open probability in intact cells. With its ability to monitor changes in cellular energetics within seconds, PercevalHR should be a versatile tool for metabolic research. PMID:24096541

Genetic Engineering of Optical Properties of Biomaterials

NASA Astrophysics Data System (ADS)

Gourley, Paul; Naviaux, Robert; Yaffe, Michael

2008-03-01

Baker's yeast cells are easily cultured and can be manipulated genetically to produce large numbers of bioparticles (cells and mitochondria) with controllable size and optical properties. We have recently employed nanolaser spectroscopy to study the refractive index of individual cells and isolated mitochondria from two mutant strains. Results show that biomolecular changes induced by mutation can produce bioparticles with radical changes in refractive index. Wild-type mitochondria exhibit a distribution with a well-defined mean and small variance. In striking contrast, mitochondria from one mutant strain produced a histogram that is highly collapsed with a ten-fold decrease in the mean and standard deviation. In a second mutant strain we observed an opposite effect with the mean nearly unchanged but the variance increased nearly a thousand-fold. Both histograms could be self-consistently modeled with a single, log-normal distribution. The strains were further examined by 2-dimensional gel electrophoresis to measure changes in protein composition. All of these data show that genetic manipulation of cells represents a new approach to engineering optical properties of bioparticles.

Culture History and Population Heterogeneity as Determinants of Bacterial Adaptation: the Adaptomics of a Single Environmental Transition

PubMed Central

Ryall, Ben; Eydallin, Gustavo

2012-01-01

Summary: Diversity in adaptive responses is common within species and populations, especially when the heterogeneity of the frequently large populations found in environments is considered. By focusing on events in a single clonal population undergoing a single transition, we discuss how environmental cues and changes in growth rate initiate a multiplicity of adaptive pathways. Adaptation is a comprehensive process, and stochastic, regulatory, epigenetic, and mutational changes can contribute to fitness and overlap in timing and frequency. We identify culture history as a major determinant of both regulatory adaptations and microevolutionary change. Population history before a transition determines heterogeneities due to errors in translation, stochastic differences in regulation, the presence of aged, damaged, cheating, or dormant cells, and variations in intracellular metabolite or regulator concentrations. It matters whether bacteria come from dense, slow-growing, stressed, or structured states. Genotypic adaptations are history dependent due to variations in mutation supply, contingency gene changes, phase variation, lateral gene transfer, and genome amplifications. Phenotypic adaptations underpin genotypic changes in situations such as stress-induced mutagenesis or prophage induction or in biofilms to give a continuum of adaptive possibilities. Evolutionary selection additionally provides diverse adaptive outcomes in a single transition and generally does not result in single fitter types. The totality of heterogeneities in an adapting population increases the chance that at least some individuals meet immediate or future challenges. However, heterogeneity complicates the adaptomics of single transitions, and we propose that subpopulations will need to be integrated into future population biology and systems biology predictions of bacterial behavior. PMID:22933562

The fluidity of Chinese hamster ovary cell and bull sperm membranes after cholesterol addition.

PubMed

Purdy, P H; Fox, M H; Graham, J K

2005-08-01

Cell plasma membrane fluidity is affected by membrane lipid and protein composition as well as temperature. Altering the cholesterol content of a membrane can change membrane fluidity at different temperatures and this may affect cell survival during cryopreservation. In these experiments, we examined the effect that adding cholesterol to the membranes of Chinese hamster ovary cells (CHO) and bull sperm had on cell plasma membrane fluidity and cell survival when cells were cooled to 5 degrees C or were cryopreserved. Cells were treated with 0, 1.5 or 5.0mg cholesterol-loaded cyclodextrin (CLC), stained with N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl)trimethylammonium-p-toluenesulfonate (TMAP-DPH) to evaluate membrane fluidity and with propidium iodide to evaluate cell viability, prior to analysis by flow cytometry at 23, 5 degrees C, and after cryopreservation. CHO cells exhibited a single cell population with all cells having similar membrane fluidity. Membrane fluidity did not change when temperature had been reduced and then returned to 23 degrees C (P<0.05), however, adding cholesterol to the cells induced membranes to become more rigid (P<0.05). Bull sperm samples consisted of two cell subpopulations, one having relatively higher membrane fluidity than the other, regardless of cholesterol treatment or temperature. In addition, cells possessing the highest membrane fluidity did not survive cooling or cryopreservation efficiently. CLC treatment did not significantly alter membrane fluidity after temperature changes, but did maintain higher percentages of spermatozoa surviving cooling to 5 degrees C and cryopreservation (P<0.05). In conclusion, adding cholesterol to cell resulted in detectable membrane fluidity changes in CHO cells and increased survival of bull sperm after cooling to 5 degrees C and after cryopreservation.

Mitochondrial DNA 3243A>G heteroplasmy is associated with changes in cytoskeletal protein expression and cell mechanics.

PubMed

Kandel, Judith; Picard, Martin; Wallace, Douglas C; Eckmann, David M

2017-06-01

Mitochondrial and mechanical alterations in cells have both been shown to be hallmarks of human disease. However, little research has endeavoured to establish connections between these two essential features of cells in both functional and dysfunctional situations. In this work, we hypothesized that a specific genetic alteration in mitochondrial function known to cause human disease would trigger changes in cell mechanics. Using a previously characterized set of mitochondrial cybrid cell lines, we examined the relationship between heteroplasmy for the mitochondrial DNA (mtDNA) 3243A>G mutation, the cell cytoskeleton, and resulting cellular mechanical properties. We found that cells with increasing mitochondrial dysfunction markedly differed from one another in gene expression and protein production of various co-regulated cytoskeletal elements. The intracellular positioning and organization of actin also differed across cell lines. To explore the relationship between these changes and cell mechanics, we then measured cellular mechanical properties using atomic force microscopy and found that cell stiffness correlated with gene expression data for known determinants of cell mechanics, γ-actin, α-actinin and filamin A. This work points towards a mechanism linking mitochondrial genetics to single-cell mechanical properties. The transcriptional and structural regulation of cytoskeletal components by mitochondrial function may explain why energetic and mechanical alterations often coexist in clinical conditions. © 2017 The Author(s).

Determining the impact of cell mixing on signaling during development.

PubMed

Uriu, Koichiro; Morelli, Luis G

2017-06-01

Cell movement and intercellular signaling occur simultaneously to organize morphogenesis during embryonic development. Cell movement can cause relative positional changes between neighboring cells. When intercellular signals are local such cell mixing may affect signaling, changing the flow of information in developing tissues. Little is known about the effect of cell mixing on intercellular signaling in collective cellular behaviors and methods to quantify its impact are lacking. Here we discuss how to determine the impact of cell mixing on cell signaling drawing an example from vertebrate embryogenesis: the segmentation clock, a collective rhythm of interacting genetic oscillators. We argue that comparing cell mixing and signaling timescales is key to determining the influence of mixing. A signaling timescale can be estimated by combining theoretical models with cell signaling perturbation experiments. A mixing timescale can be obtained by analysis of cell trajectories from live imaging. After comparing cell movement analyses in different experimental settings, we highlight challenges in quantifying cell mixing from embryonic timelapse experiments, especially a reference frame problem due to embryonic motions and shape changes. We propose statistical observables characterizing cell mixing that do not depend on the choice of reference frames. Finally, we consider situations in which both cell mixing and signaling involve multiple timescales, precluding a direct comparison between single characteristic timescales. In such situations, physical models based on observables of cell mixing and signaling can simulate the flow of information in tissues and reveal the impact of observed cell mixing on signaling. © 2017 Japanese Society of Developmental Biologists.

Effects of in vivo hydrocortisone on lymphocyte-mediated cytotoxicity

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

Katz, P.; Zaytoun, A.M.; Lee, J.H. Jr.

To examine the effects of in vivo hydrocortisone sodium succinate (HC) on natural killer (NK) cell and antibody-dependent cellular cytotoxicity (ADCC), 11 normal adults received a single intravenous bolus of 400 mg hydrocortisone. Lymphocytes were tested for NK activity and ADCC using 51chromium (51Cr)-release and single cell cytotoxicity assays against Molt-4 and sensitized RL O leads to target cells, respectively. Four hours after injection, both NK and ADCC activity were transiently increased in the 51Cr-release system. At 4 hours, there was a twofold increase in the relative frequency of potentially cytotoxic target binding cells but the absolute number of thesemore » cells did not change. However, the percentage lysis of bound targets at 4 hours was not altered. These data suggest that: 1) lymphocytes participating in NK and ADCC reactions are refractory to the kinetic and functional effects of HC; 2) the increased lytic activity observed at 4 hours is due to a selective depletion of noncytotoxic cells from the circulation; and 3) NK and ADCC activity did not differ in their responses to HC.« less

[Establishment and application of mechanical strain loading system of multi-channel cells].

PubMed

Li, Yongming; Wang, Hua; Zhang, Xiaodong; Tang, Lin

2012-02-01

Based on single-chip microcomputer, we have established a mechanical strain loading system with multi-channel to study the biological behavior of cultured cells in vitro under mechanical strain. We developed a multi-channel cell strain loading device controlled by single-chip microcomputer. We controlled the vacuum pump with vacuum chamber to make negative pressure changing periodically in the vacuum chamber. The tested cells were seeded on the surface of an elastic membrane mounted on the vacuum chamber, and could be strained or relaxed by cyclic pressure. Since the cells are attached to the surface of the membrane, they presumably experience the same deformation as that was applied to the membrane. The system was easy to carry and to operate, with deformation rate (1%-21%) and frequency (0-0. 5Hz) which could be adjusted correctly according to experimental requirement, and could compare different deformation rate of three channels at the same time. The system ran stably and completely achieved design aims, and provided a method to study the biological behavior of cultured cells attached to the surface of the elastic membrane under mechanical strain in vitro.

Electronic platform for real-time multi-parametric analysis of cellular behavior post-exposure to single-walled carbon nanotubes

PubMed Central

Eldawud, Reem; Wagner, Alixandra; Dong, Chenbo; Rojansakul, Yon; Dinu, Cerasela Zoica

2016-01-01

Single-walled carbon nanotubes (SWCNTs) implementation in a variety of biomedical applications from bioimaging, to controlled drug delivery and cellular-directed alignment for muscle myofiber fabrication, has raised awareness of their potential toxicity. Nanotubes structural aspects which resemble asbestos, as well as their ability to induce cyto and genotoxicity upon interaction with biological systems by generating reactive oxygen species or inducing membrane damage, just to name a few, have led to focused efforts aimed to assess associated risks prior their user implementation. In this study, we employed a non-invasive and real-time electric cell impedance sensing (ECIS) platform to monitor behavior of lung epithelial cells upon exposure to a library of SWCNTs with user-defined physicochemical properties. Using the natural sensitivity of the cells, we evaluated SWCNT-induced cellular changes in relation to cell attachment, cell–cell interactions and cell viability respectively. Our methods have the potential to lead to the development of standardized assays for risk assessment of other nanomaterials as well as risk differentiation based on the nanomaterials surface chemistry, purity and agglomeration state. PMID:25913448

Fabrication of 20.19% Efficient Single-Crystalline Silicon Solar Cell with Inverted Pyramid Microstructure.

PubMed

Zhang, Chunyang; Chen, Lingzhi; Zhu, Yingjie; Guan, Zisheng

2018-04-03

This paper reports inverted pyramid microstructure-based single-crystalline silicon (sc-Si) solar cell with a conversion efficiency up to 20.19% in standard size of 156.75 × 156.75 mm 2 . The inverted pyramid microstructures were fabricated jointly by metal-assisted chemical etching process (MACE) with ultra-low concentration of silver ions and optimized alkaline anisotropic texturing process. And the inverted pyramid sizes were controlled by changing the parameters in both MACE and alkaline anisotropic texturing. Regarding passivation efficiency, the textured sc-Si with normal reflectivity of 9.2% and inverted pyramid size of 1 μm was used to fabricate solar cells. The best batch of solar cells showed a 0.19% higher of conversion efficiency and a 0.22 mA cm -2 improvement in short-circuit current density, and the excellent photoelectric property surpasses that of the same structure solar cell reported before. This technology shows great potential to be an alternative for large-scale production of high efficient sc-Si solar cells in the future.

Single-cell sequencing unveils the lifestyle and CRISPR-based population history of Hydrotalea sp. in acid mine drainage.

PubMed

Medeiros, J D; Leite, L R; Pylro, V S; Oliveira, F S; Almeida, V M; Fernandes, G R; Salim, A C M; Araújo, F M G; Volpini, A C; Oliveira, G; Cuadros-Orellana, S

2017-10-01

Acid mine drainage (AMD) is characterized by an acid and metal-rich run-off that originates from mining systems. Despite having been studied for many decades, much remains unknown about the microbial community dynamics in AMD sites, especially during their early development, when the acidity is moderate. Here, we describe draft genome assemblies from single cells retrieved from an early-stage AMD sample. These cells belong to the genus Hydrotalea and are closely related to Hydrotalea flava. The phylogeny and average nucleotide identity analysis suggest that all single amplified genomes (SAGs) form two clades that may represent different strains. These cells have the genomic potential for denitrification, copper and other metal resistance. Two coexisting CRISPR-Cas loci were recovered across SAGs, and we observed heterogeneity in the population with regard to the spacer sequences, together with the loss of trailer-end spacers. Our results suggest that the genomes of Hydrotalea sp. strains studied here are adjusting to a quickly changing selective pressure at the microhabitat scale, and an important form of this selective pressure is infection by foreign DNA. © 2017 John Wiley & Sons Ltd.

DAPNe with micro-capillary separatory chemistry-coupled to MALDI-MS for the analysis of polar and non-polar lipid metabolism in one cell

NASA Astrophysics Data System (ADS)

Hamilton, Jason S.; Aguilar, Roberto; Petros, Robby A.; Verbeck, Guido F.

2017-05-01

The cellular metabolome is considered to be a representation of cellular phenotype and cellular response to changes to internal or external events. Methods to expand the coverage of the expansive physiochemical properties that makeup the metabolome currently utilize multi-step extractions and chromatographic separations prior to chemical detection, leading to lengthy analysis times. In this study, a single-step procedure for the extraction and separation of a sample using a micro-capillary as a separatory funnel to achieve analyte partitioning within an organic/aqueous immiscible solvent system is described. The separated analytes are then spotted for MALDI-MS imaging and distribution ratios are calculated. Initially, the method is applied to standard mixtures for proof of partitioning. The extraction of an individual cell is non-reproducible; therefore, a broad chemical analysis of metabolites is necessary and will be illustrated with the one-cell analysis of a single Snu-5 gastric cancer cell taken from a cellular suspension. The method presented here shows a broad partitioning dynamic range as a single-step method for lipid analysis demonstrating a decrease in ion suppression often present in MALDI analysis of lipids.

Mapping Cd²⁺-induced membrane permeability changes of single live cells by means of scanning electrochemical microscopy.

PubMed

Filice, Fraser P; Li, Michelle S M; Henderson, Jeffrey D; Ding, Zhifeng

2016-02-18

Scanning Electrochemical Microscopy (SECM) is a powerful, non-invasive, analytical methodology that can be used to investigate live cell membrane permeability. Depth scan SECM imaging allowed for the generation of 2D current maps of live cells relative to electrode position in the x-z or y-z plane. Depending on resolution, one depth scan image can contain hundreds of probe approach curves (PACs). Individual PACs were obtained by simply extracting vertical cross-sections from the 2D image. These experimental PACs were overlaid onto theoretically generated PACs simulated at specific geometry conditions. Simulations were carried out using 3D models in COMSOL Multiphysics to determine the cell membrane permeability coefficients at different locations on the surface of the cells. Common in literature, theoretical PACs are generated using a 2D axially symmetric geometry. This saves on both compute time and memory utilization. However, due to symmetry limitations of the model, only one experimental PAC right above the cell can be matched with simulated PAC data. Full 3D models in this article were developed for the SECM system of live cells, allowing all experimental PACs over the entire cell to become usable. Cd(2+)-induced membrane permeability changes of single human bladder (T24) cells were investigated at several positions above the cell, displaced from the central axis. The experimental T24 cells under study were incubated with Cd(2+) in varying concentrations. It is experimentally observed that 50 and 100 μM Cd(2+) caused a decrease in membrane permeability, which was uniform across all locations over the cell regardless of Cd(2+) concentration. The Cd(2+) was found to have detrimental effects on the cell, with cells shrinking in size and volume, and the membrane permeability decreasing. A mapping technique for the analysis of the cell membrane permeability under the Cd(2+) stress is realized by the methodology presented. Copyright © 2016 Elsevier B.V. All rights reserved.

Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.

PubMed

Hosokawa, Masahito; Nishikawa, Yohei; Kogawa, Masato; Takeyama, Haruko

2017-07-12

Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

NASA Astrophysics Data System (ADS)

Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

2016-09-01

Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.

Evaluation of a Microelectrode Arrays for Neurotoxicity Testing Using a Chemical Training Set

EPA Science Inventory

Microelectrode array (MEA) approaches have been proposed as a tool for detecting functional changes in electrically active cells, including neurons, exposed to drugs, chemicals, or particles. However, conventional single well MEA systems lack the throughput necessary for screenin...

Droplet Microarray Based on Superhydrophobic-Superhydrophilic Patterns for Single Cell Analysis.

PubMed

Jogia, Gabriella E; Tronser, Tina; Popova, Anna A; Levkin, Pavel A

2016-12-09

Single-cell analysis provides fundamental information on individual cell response to different environmental cues and is a growing interest in cancer and stem cell research. However, current existing methods are still facing challenges in performing such analysis in a high-throughput manner whilst being cost-effective. Here we established the Droplet Microarray (DMA) as a miniaturized screening platform for high-throughput single-cell analysis. Using the method of limited dilution and varying cell density and seeding time, we optimized the distribution of single cells on the DMA. We established culturing conditions for single cells in individual droplets on DMA obtaining the survival of nearly 100% of single cells and doubling time of single cells comparable with that of cells cultured in bulk cell population using conventional methods. Our results demonstrate that the DMA is a suitable platform for single-cell analysis, which carries a number of advantages compared with existing technologies allowing for treatment, staining and spot-to-spot analysis of single cells over time using conventional analysis methods such as microscopy.

Cell Fate Decision as High-Dimensional Critical State Transition

PubMed Central

Zhou, Joseph; Castaño, Ivan G.; Leong-Quong, Rebecca Y. Y.; Chang, Hannah; Trachana, Kalliopi; Giuliani, Alessandro; Huang, Sui

2016-01-01

Cell fate choice and commitment of multipotent progenitor cells to a differentiated lineage requires broad changes of their gene expression profile. But how progenitor cells overcome the stability of their gene expression configuration (attractor) to exit the attractor in one direction remains elusive. Here we show that commitment of blood progenitor cells to the erythroid or myeloid lineage is preceded by the destabilization of their high-dimensional attractor state, such that differentiating cells undergo a critical state transition. Single-cell resolution analysis of gene expression in populations of differentiating cells affords a new quantitative index for predicting critical transitions in a high-dimensional state space based on decrease of correlation between cells and concomitant increase of correlation between genes as cells approach a tipping point. The detection of “rebellious cells” that enter the fate opposite to the one intended corroborates the model of preceding destabilization of a progenitor attractor. Thus, early warning signals associated with critical transitions can be detected in statistical ensembles of high-dimensional systems, offering a formal theory-based approach for analyzing single-cell molecular profiles that goes beyond current computational pattern recognition, does not require knowledge of specific pathways, and could be used to predict impending major shifts in development and disease. PMID:28027308

Changes in Chromatin Compaction During the Cell Cycle Revealed by Micrometer-Scale Measurement of Molecular Flow in the Nucleus

PubMed Central

Hinde, Elizabeth; Cardarelli, Francesco; Digman, Michelle A.; Gratton, Enrico

2012-01-01

We present a quantitative fluctuation-based assay to measure the degree of local chromatin compaction and investigate how chromatin density regulates the diffusive path adopted by an inert protein in dividing cells. The assay uses CHO-K1 cells coexpressing untagged enhanced green fluorescent protein (EGFP) and histone H2B tagged mCherry. We measure at the single-cell level the EGFP localization and molecular flow patterns characteristic of each stage of chromatin compaction from mitosis through interphase by means of pair-correlation analysis. We find that the naturally occurring changes in chromatin organization impart a regulation on the spatial distribution and temporal dynamics of EGFP within the nucleus. Combined with the analysis of Ca2+ intracellular homeostasis during cell division, EGFP flow regulation can be interpreted as the result of controlled changes in chromatin compaction. For the first time, to our knowledge, we were able to probe chromatin compaction on the micrometer scale, where the regulation of molecular diffusion may become relevant for many cellular processes. PMID:22325293

Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice.

PubMed

Chinnasamy, Dhanalakshmi; Yu, Zhiya; Kerkar, Sid P; Zhang, Ling; Morgan, Richard A; Restifo, Nicholas P; Rosenberg, Steven A

2012-03-15

We investigated the feasibility of delivering the proinflammatory cytokine interleukin (IL)-12 into tumor using T cells genetically engineered to express a chimeric antigen receptor (CAR) against the VEGF receptor-2 (VEGFR-2). Two different strains of mice bearing five different established subcutaneous tumors were treated with syngeneic T cells cotransduced with an anti-VEGFR-2 CAR and a constitutively expressed single-chain murine IL-12 or an inducible IL-12 gene after host lymphodepletion. Tumor regression, survival of mice, and persistence of the transferred cells were evaluated. Adoptive transfer of syngeneic T cells cotransduced with an anti-VEGFR-2 CAR and a constitutively expressing single-chain IL-12 resulted in the regression of five different established tumors of different histologies without the need for IL-2 administration. T cells transduced with either anti-VEGFR-2 CAR or single-chain IL-12 alone did not alter the tumor growth indicating that both of them had to be expressed in the same cell to mediate tumor regression. Anti-VEGFR-2 CAR and IL-12-cotransduced T cells infiltrated the tumors, expanded, and persisted for prolonged periods. The antitumor effect did not require the presence of host T and B cells but was dependent on host IL-12R-expressing cells. The anti-VEGFR-2 CAR changed the immunosuppressive tumor environment by altering/reducing both the systemic and the intratumoral CD11b(+)Gr1(+) myeloid suppressor cell subsets that expressed VEGFR-2. These results suggest that targeted delivery of IL-12 into the tumor environment with T cells redirected against VEGFR-2 is a promising approach for treating patients with a variety of solid tumor types.

Complete Genome Sequences, before and after Mammalian Cell Culture, of Zika Virus Isolated from the Serum of a Symptomatic Male Patient from Oaxaca, Mexico.

PubMed

Boukadida, Celia; Torres-Flores, Jesús M; Yocupicio-Monroy, Martha; Piten-Isidro, Elvira; Rivero-Arrieta, Amaranta Y; Luna-Villalobos, Yara A; Martínez-Vargas, Liliane; Alcaraz-Estrada, Sofía L; Torres, Klintsy J; Lira, Rosalia; Reyes-Terán, Gustavo; Sevilla-Reyes, Edgar E

2017-03-23

Zika virus (ZIKV) is an emerging arthropod-borne flavivirus associated with severe congenital malformations and neurological complications. Although the ZIKV genome is well characterized, there is limited information regarding changes after cell isolation and culture adaptation. We isolated, and passaged in Vero cells, ZIKV from the serum of a symptomatic male patient and compared the viral genomes before and after culture. Single nucleotide polymorphisms were characteristic among serum-circulating genomes, while such diversity decreased after cell culture. Copyright © 2017 Boukadida et al.

Apparatus for combinatorial screening of electrochemical materials

DOEpatents

Kepler, Keith Douglas [Belmont, CA; Wang, Yu [Foster City, CA

2009-12-15

A high throughput combinatorial screening method and apparatus for the evaluation of electrochemical materials using a single voltage source (2) is disclosed wherein temperature changes arising from the application of an electrical load to a cell array (1) are used to evaluate the relative electrochemical efficiency of the materials comprising the array. The apparatus may include an array of electrochemical cells (1) that are connected to each other in parallel or in series, an electronic load (2) for applying a voltage or current to the electrochemical cells (1), and a device (3), external to the cells, for monitoring the relative temperature of each cell when the load is applied.

Colourful death: six-parameter classification of cell death by flow cytometry--dead cells tell tales.

PubMed

Munoz, Luis E; Maueröder, Christian; Chaurio, Ricardo; Berens, Christian; Herrmann, Martin; Janko, Christina

2013-08-01

The response of the immune system against dying and dead cells strongly depends on the cell death phenotype. Beside other forms of cell death, two clearly distinct populations, early apoptotic and secondary necrotic cells, have been shown to induce anti-inflammation/tolerance and inflammation/immune priming, respectively. Cytofluorometry is a powerful technique to detect morphological and phenotypical changes occurring during cell death. Here, we describe a new technique using AnnexinA5, propidiumiodide, DiIC1(5) and Hoechst 33342 to sub-classify populations of apoptotic and/or necrotic cells. The method allows the fast and reliable identification of several different phases and pathways of cell death by analysing the following cell death associated changes in a single tube: cellular granularity and shrinkage, phosphatidylserine exposure, ion selectivity of the plasma membrane, mitochondrial membrane potential, and DNA content. The clear characterisation of cell death is of major importance for instance in immunization studies, in experimental therapeutic settings, and in the exploration of cell-death associated diseases. It also enables the analysis of immunological properties of distinct populations of dying cells and the pathways involved in this process.

Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

PubMed

Feaster, Tromondae K; Cadar, Adrian G; Wang, Lili; Williams, Charles H; Chun, Young Wook; Hempel, Jonathan E; Bloodworth, Nathaniel; Merryman, W David; Lim, Chee Chew; Wu, Joseph C; Knollmann, Björn C; Hong, Charles C

2015-12-04

The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (INa). The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing. © 2015 American Heart Association, Inc.

A High-Throughput Microenvironment for Single-Cell Operations

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

Christian, A T; Buckley, P; Miles, R R

2003-01-07

This project was conducted as a feasibility study, in preparation for including this work in the forthcoming ''Instrumented Cell'' (IC) Strategic Initiative. The goal of the IC is to study individual cells; the goal of this feasibility study was to determine the best method for isolating large numbers of individual cells in a way that facilitates various types of environmental changes and intracellular measurements. We have the capability to do this with one cell, and sought to expand the number of cells that we could study simultaneously. Our specific goal for this feasibility study was to discover a way tomore » isolate individual cells, and impale them on a nanopipette. This would enable samples to be introduced into and removed from a cell.« less

Single-cell genomics reveals features of a Colwellia species that was dominant during the Deepwater Horizon oil spill.

PubMed

Mason, Olivia U; Han, James; Woyke, Tanja; Jansson, Janet K

2014-01-01

During the Deepwater Horizon (DWH) oil spill in the Gulf of Mexico a deep-sea hydrocarbon plume developed resulting in a rapid succession of bacteria. Colwellia eventually supplanted Oceanospirillales, which dominated the plume early in the spill. These successional changes may have resulted, in part, from the changing composition and abundance of hydrocarbons over time. Colwellia abundance peaked when gaseous and simple aromatic hydrocarbons increased, yet the metabolic pathway used by Colwellia in hydrocarbon disposition is unknown. Here we used single-cell genomics to gain insights into the genome properties of a Colwellia enriched during the DWH deep-sea plume. A single amplified genome (SAG) of a Colwellia cell isolated from a DWH plume, closely related (avg. 98% 16S rRNA gene similarity) to other plume Colwellia, was sequenced and annotated. The SAG was similar to the sequenced isolate Colwellia psychrerythraea 34H (84% avg. nucleotide identity). Both had genes for denitrification, chemotaxis, and motility, adaptations to cold environments and a suite of nutrient acquisition genes. The Colwellia SAG may be capable of gaseous and aromatic hydrocarbon degradation, which contrasts with a DWH plume Oceanospirillales SAG which encoded non-gaseous n-alkane and cycloalkane degradation pathways. The disparate hydrocarbon degradation pathways are consistent with hydrocarbons that were abundant at different times in the deep-sea plume; first, non-gaseous n-alkanes and cycloalkanes that could be degraded by Oceanospirillales, followed by gaseous, and simple aromatic hydrocarbons that may have been degraded by Colwellia. These insights into the genomic properties of a Colwellia species, which were supported by existing metagenomic sequence data from the plume and DWH contaminated sediments, help further our understanding of the successional changes in the dominant microbial players in the plume over the course of the DWH spill.

Single-cell RNA sequencing reveals an altered gene expression pattern as a result of CRISPR/cas9-mediated deletion of Gene 33/Mig6 and chronic exposure to hexavalent chromium in human lung epithelial cells.

PubMed

Park, Soyoung; Zhang, Xiaowen; Li, Cen; Yin, Changhong; Li, Jiangwei; Fallon, John T; Huang, Weihua; Xu, Dazhong

2017-09-01

Gene 33 (Mig6, ERRFI1) is an adaptor protein with multiple cellular functions. We recently reported that depletion of this protein promotes lung epithelial cell transformation induced by hexavalent chromium [Cr(VI)]. However, the early molecular events that mediate this process are not clear. In the present study, we used single-cell RNA sequencing to compare gene expression profiles between BEAS-2B lung epithelial cells chronically exposed to a sublethal dose of Cr(VI) with or without CRISPR/cas9-mediated deletion of Gene 33. Our data reveal 83 differentially expressed genes. The most notable changes are genes associated with cell adhesion, oxidative stresses, protein ubiquitination, epithelial-mesenchymal transition/metastasis, and WNT signaling. Up-regulation of some neuro-specific genes is also evident, particularly ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a deubiquitinase and potential biomarker for lung cancer. Gene 33 deletion and/or Cr(VI) exposure did not cause discernable changes in cell morphology. However, Gene 33 deletion led to a modest but significant reduction of cells in the G2/M phase of the cell cycle regardless of Cr(VI) exposure. Gene 33 deletion also significantly reduced cell proliferation. Interestingly, Cr(VI) exposure eliminated the difference in cell proliferation between the two genotypes. Gene 33 deletion also significantly elevated cell migration. Our data indicate that combined Gene 33 deletion and chronic Cr(VI) exposure produces a gene expression pattern and a phenotype resemble those of the transformed lung epithelial cells. Given the known association of UCHL1 with lung cancer, we propose that UCHL1 is an important player in the early stage of lung epithelial cell transformation and tumorigenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

[Effect of low-intensity 900 MHz frequency electromagnetic radiation on rat liver and blood serum enzyme activities].

PubMed

Nersesova, L S; Petrosian, M S; Gazariants, M G; Mkrtchian, Z S; Meliksetian, G O; Pogosian, L G; Akopian, Zh I

2014-01-01

The comparative analysis of the rat liver and blood serum creatine kinase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and purine nucleoside phosphorylase post-radiation activity levels after a total two-hour long single and fractional exposure of the animals to low-intensity 900 MHz frequency electromagnetic field showed that the most sensitive enzymes to the both schedules of radiation are the liver creatine kinase, as well as the blood serum creatine kinase and alkaline phosphatase. According to the comparative analysis of the dynamics of changes in the activity level of the liver and blood serum creatine kinase, alanine aminotransferase, aspartate aminotransferase and purine nucleoside phosphorylase, both single and fractional radiation schedules do not affect the permeability of a hepatocyte cell membrane, but rather cause changes in their energetic metabolism. The correlation analysis of the post-radiation activity level changes of the investigated enzymes did not reveal a clear relationship between them. The dynamics of post-radiation changes in the activity of investigated enzyme levels following a single and short-term fractional schedules of radiation did not differ essentially.

Correlation of Cell Surface Biomarker Expression Levels with Adhesion Contact Angle Measured by Lateral Microscopy.

PubMed

Walz, Jenna A; Mace, Charles R

2018-06-05

Immunophenotyping is typically achieved using flow cytometry, but any influence a biomarker may have on adhesion or surface recognition cannot be determined concurrently. In this manuscript, we demonstrate the utility of lateral microscopy for correlating cell surface biomarker expression levels with quantitative descriptions of cell morphology. With our imaging system, we observed single cells from two T cell lines and two B cell lines adhere to antibody-coated substrates and quantified this adhesion using contact angle measurements. We found that SUP-T1 and CEM CD4+ cells, both of which express similar levels of CD4, experienced average changes in contact angle that were not statistically different from one another on surfaces coated in anti-CD4. However, MAVER-1 and BJAB K20 cells, both of which express different levels of CD20, underwent average changes in contact angle that were significantly different from one another on surfaces coated in anti-CD20. Our results indicate that changes in cell contact angles on antibody-coated substrates reflect the expression levels of corresponding antigens on the surfaces of cells as determined by flow cytometry. Our lateral microscopy approach offers a more reproducible and quantitative alternative to evaluate adhesion compared to commonly used wash assays and can be extended to many additional immunophenotyping applications to identify cells of interest within heterogeneous populations.

Massively parallel nanowell-based single-cell gene expression profiling.

PubMed

Goldstein, Leonard D; Chen, Ying-Jiun Jasmine; Dunne, Jude; Mir, Alain; Hubschle, Hermann; Guillory, Joseph; Yuan, Wenlin; Zhang, Jingli; Stinson, Jeremy; Jaiswal, Bijay; Pahuja, Kanika Bajaj; Mann, Ishminder; Schaal, Thomas; Chan, Leo; Anandakrishnan, Sangeetha; Lin, Chun-Wah; Espinoza, Patricio; Husain, Syed; Shapiro, Harris; Swaminathan, Karthikeyan; Wei, Sherry; Srinivasan, Maithreyan; Seshagiri, Somasekar; Modrusan, Zora

2017-07-07

Technological advances have enabled transcriptome characterization of cell types at the single-cell level providing new biological insights. New methods that enable simple yet high-throughput single-cell expression profiling are highly desirable. Here we report a novel nanowell-based single-cell RNA sequencing system, ICELL8, which enables processing of thousands of cells per sample. The system employs a 5,184-nanowell-containing microchip to capture ~1,300 single cells and process them. Each nanowell contains preprinted oligonucleotides encoding poly-d(T), a unique well barcode, and a unique molecular identifier. The ICELL8 system uses imaging software to identify nanowells containing viable single cells and only wells with single cells are processed into sequencing libraries. Here, we report the performance and utility of ICELL8 using samples of increasing complexity from cultured cells to mouse solid tissue samples. Our assessment of the system to discriminate between mixed human and mouse cells showed that ICELL8 has a low cell multiplet rate (< 3%) and low cross-cell contamination. We characterized single-cell transcriptomes of more than a thousand cultured human and mouse cells as well as 468 mouse pancreatic islets cells. We were able to identify distinct cell types in pancreatic islets, including alpha, beta, delta and gamma cells. Overall, ICELL8 provides efficient and cost-effective single-cell expression profiling of thousands of cells, allowing researchers to decipher single-cell transcriptomes within complex biological samples.

Characterizing deformability and surface friction of cancer cells

PubMed Central

Byun, Sangwon; Son, Sungmin; Amodei, Dario; Cermak, Nathan; Shaw, Josephine; Kang, Joon Ho; Hecht, Vivian C.; Winslow, Monte M.; Jacks, Tyler; Mallick, Parag; Manalis, Scott R.

2013-01-01

Metastasis requires the penetration of cancer cells through tight spaces, which is mediated by the physical properties of the cells as well as their interactions with the confined environment. Various microfluidic approaches have been devised to mimic traversal in vitro by measuring the time required for cells to pass through a constriction. Although a cell’s passage time is expected to depend on its deformability, measurements from existing approaches are confounded by a cell's size and its frictional properties with the channel wall. Here, we introduce a device that enables the precise measurement of (i) the size of a single cell, given by its buoyant mass, (ii) the velocity of the cell entering a constricted microchannel (entry velocity), and (iii) the velocity of the cell as it transits through the constriction (transit velocity). Changing the deformability of the cell by perturbing its cytoskeleton primarily alters the entry velocity, whereas changing the surface friction by immobilizing positive charges on the constriction's walls primarily alters the transit velocity, indicating that these parameters can give insight into the factors affecting the passage of each cell. When accounting for cell buoyant mass, we find that cells possessing higher metastatic potential exhibit faster entry velocities than cells with lower metastatic potential. We additionally find that some cell types with higher metastatic potential exhibit greater than expected changes in transit velocities, suggesting that not only the increased deformability but reduced friction may be a factor in enabling invasive cancer cells to efficiently squeeze through tight spaces. PMID:23610435

Delayed suppression of hippocampal cell proliferation in rats following inescapable shocks.

PubMed

Fornal, Casimir A; Stevens, Joanne; Barson, Jessica R; Blakley, Gregory G; Patterson-Buckendahl, Patricia; Jacobs, Barry L

2007-01-26

Adult Sprague-Dawley rats were exposed to a single session of 100 inescapable tail shocks (IS). Bromodeoxyuridine (BrdU) was administered 1 h, 2 days or 7 days later and hippocampal cell proliferation (CP) was assessed after a 2-h survival period. Measures of plasma corticosterone (CORT) levels were also obtained. Despite a large increase in CORT immediately following IS, no associated change in CP was observed. In fact, the only significant change in CP was seen 7 days after IS, at a time when CORT was unchanged from control levels. These data raise questions about the general nature of the relationship between CORT and CP. They also suggest that, under some conditions, changes in hippocampal CP may emerge only after an "incubation period".

Automated live cell screening system based on a 24-well-microplate with integrated micro fluidics.

PubMed

Lob, V; Geisler, T; Brischwein, M; Uhl, R; Wolf, B

2007-11-01

In research, pharmacologic drug-screening and medical diagnostics, the trend towards the utilization of functional assays using living cells is persisting. Research groups working with living cells are confronted with the problem, that common endpoint measurement methods are not able to map dynamic changes. With consideration of time as a further dimension, the dynamic and networked molecular processes of cells in culture can be monitored. These processes can be investigated by measuring several extracellular parameters. This paper describes a high-content system that provides real-time monitoring data of cell parameters (metabolic and morphological alterations), e.g., upon treatment with drug compounds. Accessible are acidification rates, the oxygen consumption and changes in adhesion forces within 24 cell cultures in parallel. Addressing the rising interest in biomedical and pharmacological high-content screening assays, a concept has been developed, which integrates multi-parametric sensor readout, automated imaging and probe handling into a single embedded platform. A life-maintenance system keeps important environmental parameters (gas, humidity, sterility, temperature) constant.

Cell-Type-Specific Transcriptome Analysis in the Drosophila Mushroom Body Reveals Memory-Related Changes in Gene Expression.

PubMed

Crocker, Amanda; Guan, Xiao-Juan; Murphy, Coleen T; Murthy, Mala

2016-05-17

Learning and memory formation in Drosophila rely on a network of neurons in the mushroom bodies (MBs). Whereas numerous studies have delineated roles for individual cell types within this network in aspects of learning or memory, whether or not these cells can also be distinguished by the genes they express remains unresolved. In addition, the changes in gene expression that accompany long-term memory formation within the MBs have not yet been studied by neuron type. Here, we address both issues by performing RNA sequencing on single cell types (harvested via patch pipets) within the MB. We discover that the expression of genes that encode cell surface receptors is sufficient to identify cell types and that a subset of these genes, required for sensory transduction in peripheral sensory neurons, is not only expressed within individual neurons of the MB in the central brain, but is also critical for memory formation. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Highly sensitive detection of mutations in CHO cell recombinant DNA using multi-parallel single molecule real-time DNA sequencing.

PubMed

Cartwright, Joseph F; Anderson, Karin; Longworth, Joseph; Lobb, Philip; James, David C

2018-06-01

High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should therefore form part of the cell line development process and be an essential quality assurance metric for instances where synthetic/multi-component assemblies are utilized to engineer mammalian cells, such as the assessment of recombinant DNA fidelity or the mutability of single-site integration target loci. Based on Pacific Biosciences (Menlo Park, CA) single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology we developed a rDNA sequence analysis tool to process the multi-parallel sequencing of ∼40,000 single recombinant DNA molecules. After statistical filtering of raw sequencing data, we show that this analytical method is capable of detecting single point mutations in rDNA to a minimum single mutation frequency of 0.0042% (<1/24,000 bases). Using a stable CHO transfectant pool harboring a randomly integrated 5 kB plasmid construct encoding GFP we found that 28% of recombinant plasmid copies contained at least one low frequency (<0.3%) point mutation. These mutations were predominantly found in GC base pairs (85%) and that there was no positional bias in mutation across the plasmid sequence. There was no discernable difference between the mutation frequencies of coding and non-coding DNA. The putative ratio of non-synonymous and synonymous changes within the open reading frames (ORFs) in the plasmid sequence indicates that natural selection does not impact upon the prevalence of these mutations. Here we have demonstrated the abundance of mutations that fall outside of the reported range of detection of next generation sequencing (NGS) and second generation sequencing (SGS) platforms, providing a methodology capable of being utilized in cell line development platforms to identify the fidelity of recombinant genes throughout the production process. © 2018 Wiley Periodicals, Inc.

Controlled permeation of cell membrane by single bubble acoustic cavitation

PubMed Central

Zhou, Y.; Yang, K.; Cui, J.; Ye, J. Y.; Deng, C. X.

2011-01-01

Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustic, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency (7.44 MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5 MHz) ultrasound pulse (duration 13.3 or 40 µs) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d = 0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106 ± 0.032 µm (n = 70) for acoustic pressure of 1.5 MPa (duration 13.3 µs), and increased to 0.171 ± 0.030 µm (n = 125) for acoustic pressure of 1.7 MPa and to 0.182 ± 0.052 µm (n=112) for a pulse duration of 40 µs (1.5 MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level. PMID:21945682

Rapid Changes of Potassium Concentration at the Outer Surface of Exposed Single Neurons during Membrane Current Flow

PubMed Central

Neher, E.; Lux, H. D.

1973-01-01

K+-sensitive liquid ion-exchanger microelectrodes are shown to be capable of measuring concentration changes which occur on a millisecond time scale. However, some quaternary ammonium ions, such as tetraethylammonium and acetylcholine, are able to block electrode function when present in concentrations as low as 10-4 to 10-3 M. Changes in extracellular potassium concentration caused by spike activity or voltage clamp pulses of exposed single neurons of the snail Helix pomatia may be measured by these electrodes. Quantitative analysis shows that the total amount of excess potassium found in the vicinity of the cell a short time after a clamp pulse, is in relatively good agreement with the amount of potassium carried by the membrane current. PMID:4689624

Bioorthogonal chemical imaging of metabolic changes during epithelial-mesenchymal transition of cancer cells by stimulated Raman scattering microscopy

NASA Astrophysics Data System (ADS)

Zhang, Luyuan; Min, Wei

2017-10-01

Study of metabolic changes during epithelial-mesenchymal transition (EMT) of cancer cells is important for basic understanding and therapeutic management of cancer progression. We here used metabolic labeling and stimulated Raman scattering (SRS) microscopy, a strategy of bioorthogonal chemical imaging, to directly visualize changes in anabolic metabolism during cancer EMT at a single-cell level. MCF-7 breast cancer cell is employed as a model system. Four types of metabolites (amino acids, glucose, fatty acids, and choline) are labeled with either deuterium or alkyne (C≡C) tag. Their intracellular incorporations into MCF-7 cells before or after EMT are visualized by SRS imaging targeted at the signature vibration frequency of C-D or C≡C bonds. Overall, after EMT, anabolism of amino acids, glucose, and choline is less active, reflecting slower protein and membrane synthesis in mesenchymal cells. Interestingly, we also observed less incorporation of glucose and palmitate acids into membrane lipids, but more of them into lipid droplets in mesenchymal cells. This result indicates that, although mesenchymal cells synthesize fewer membrane lipids, they are actively storing energy into lipid droplets, either through de novo lipogenesis from glucose or direct scavenging of exogenous free fatty acids. Hence, metabolic labeling coupled with SRS can be a straightforward method in imaging cancer metabolism.

A smart core-sheath nanofiber that captures and releases red blood cells from the blood

NASA Astrophysics Data System (ADS)

Shi, Q.; Hou, J.; Zhao, C.; Xin, Z.; Jin, J.; Li, C.; Wong, S.-C.; Yin, J.

2016-01-01

A smart core-sheath nanofiber for non-adherent cell capture and release is demonstrated. The nanofibers are fabricated by single-spinneret electrospinning of poly(N-isopropylacrylamide) (PNIPAAm), polycaprolactone (PCL) and nattokinase (NK) solution blends. The self-assembly of PNIPAAm and PCL blends during the electrospinning generates the core-sheath PCL/PNIPAAm nanofibers with PNIPAAm as the sheath. The PNIPAAm-based core-sheath nanofibers are switchable between hydrophobicity and hydrophilicity with temperature change and enhance stability in the blood. When the nanofibers come in contact with blood, the NK is released from the nanofibers to resist platelet adhesion on the nanofiber surface, facilitating the direct capture and isolation of red blood cells (RBCs) from the blood above phase-transition temperature of PNIPAAm. Meanwhile, the captured RBCs are readily released from the nanofibers with temperature stimuli in an undamaged manner. The release efficiency of up to 100% is obtained while maintaining cellular integrity and function. This work presents promising nanofibers to effectively capture non-adherent cells and release for subsequent molecular analysis and diagnosis of single cells.A smart core-sheath nanofiber for non-adherent cell capture and release is demonstrated. The nanofibers are fabricated by single-spinneret electrospinning of poly(N-isopropylacrylamide) (PNIPAAm), polycaprolactone (PCL) and nattokinase (NK) solution blends. The self-assembly of PNIPAAm and PCL blends during the electrospinning generates the core-sheath PCL/PNIPAAm nanofibers with PNIPAAm as the sheath. The PNIPAAm-based core-sheath nanofibers are switchable between hydrophobicity and hydrophilicity with temperature change and enhance stability in the blood. When the nanofibers come in contact with blood, the NK is released from the nanofibers to resist platelet adhesion on the nanofiber surface, facilitating the direct capture and isolation of red blood cells (RBCs) from the blood above phase-transition temperature of PNIPAAm. Meanwhile, the captured RBCs are readily released from the nanofibers with temperature stimuli in an undamaged manner. The release efficiency of up to 100% is obtained while maintaining cellular integrity and function. This work presents promising nanofibers to effectively capture non-adherent cells and release for subsequent molecular analysis and diagnosis of single cells. Electronic supplementary information (ESI) available: Electrospinning of polymer nanofibers; FTIR spectra and XPS spectra of PCL, PNIPAAm and PCL/PNIPAAm nanofibers; SEM images of PCL/PNIPAAm nanofibers with varied composition; PNIPAAm content on the sheath of nanofibers; stability of core-sheath PCL/PNIPAAm nanofibers. Platelet adhesion on the PCL/PNIPAAm nanofibers in the presence of NK; Protein adsorption on nanofibers. See DOI: 10.1039/c5nr07070h

Magnetic Resonance Microscopy (MRM) of Single Mammalian Myofibers and Myonuclei.

PubMed

Lee, Choong H; Bengtsson, Niclas; Chrzanowski, Stephen M; Flint, Jeremy J; Walter, Glenn A; Blackband, Stephen J

2017-01-03

Recently, the first magnetic resonance microscopy (MRM) images at the cellular level in isolated mammalian brain tissues were obtained using microsurface coils. These methods can elucidate the cellular origins of MR signals and describe how these signals change over the course of disease progression and therapy. In this work, we explore the capability of these microimaging techniques to visualize mouse muscle fibers and their nuclei. Isolated myofibers expressing lacZ were imaged with and without a stain for β-galactosidase activity (S-Gal + ferric ammonium citrate) that produces both optical and MR contrast. We found that MRM can be used to image single myofibers with 6-μm resolution. The ability to image single myofibers will serve as a valuable tool to study MR properties attributed to healthy and myopathic cells. The ability to image nuclei tagged with MR/Optical gene markers may also find wide use in cell lineage MRI studies.

Magnetic Resonance Microscopy (MRM) of Single Mammalian Myofibers and Myonuclei

PubMed Central

Lee, Choong H.; Bengtsson, Niclas; Chrzanowski, Stephen M.; Flint, Jeremy J.; Walter, Glenn A.; Blackband, Stephen J.

2017-01-01

Recently, the first magnetic resonance microscopy (MRM) images at the cellular level in isolated mammalian brain tissues were obtained using microsurface coils. These methods can elucidate the cellular origins of MR signals and describe how these signals change over the course of disease progression and therapy. In this work, we explore the capability of these microimaging techniques to visualize mouse muscle fibers and their nuclei. Isolated myofibers expressing lacZ were imaged with and without a stain for β-galactosidase activity (S-Gal + ferric ammonium citrate) that produces both optical and MR contrast. We found that MRM can be used to image single myofibers with 6-μm resolution. The ability to image single myofibers will serve as a valuable tool to study MR properties attributed to healthy and myopathic cells. The ability to image nuclei tagged with MR/Optical gene markers may also find wide use in cell lineage MRI studies. PMID:28045071

Catalysts for electrochemical generation of oxygen

NASA Technical Reports Server (NTRS)

Hagans, P.; Yeager, E.

1978-01-01

Single crystal surfaces of platinum and gold and transition metal oxides of the spinel type were studied to find more effective catalysts for the electrolytic evolution of oxygen and to understand the mechanism and kinetics for the electrocatalysis in relation to the surface electronic and lattice properties of the catalyst. The single crystal studies involve the use of low energy electron diffraction (LEED) and Auger electron spectroscopy as complementary tools to the electrochemical measurements. Modifications to the transfer system and to the thin-layer electrochemical cell used to facilitate the transfer between the ultrahigh vacuum environment of the electron surface physics equipment and the electrochemical environment with a minimal possibility of changes in the surface structure, are described. The electrosorption underpotential deposition of Pb onto the Au(111), (100) and (110) single crystal surfaces with the thin-layer cell-LEED-Auger system is discussed as well as the synthesis of spinels for oxygen evolution studies.

Single-cell-based breeding: Rational strategy for the establishment of cell lines from a single cell with the most favorable properties.

PubMed

Yoshimoto, Nobuo; Kuroda, Shun'ichi

2014-04-01

For efficient biomolecule production (e.g., antibodies, recombinant proteins), mammalian cells with high expression rates should be selected from cell libraries, propagated while maintaining a homogenous expression rate, and subsequently stabilized at their high expression rate. Clusters of isogenic cells (i.e., colonies) have been used for these processes. However, cellular heterogeneity makes it difficult to obtain cell lines with the highest expression rates by using single-colony-based breeding. Furthermore, even among the single cells in an isogenic cell population, the desired cell properties fluctuate stochastically during long-term culture. Therefore, although the molecular mechanisms underlying stochastic fluctuation are poorly understood, it is necessary to establish excellent cell lines in order to breed single cells to have higher expression, higher stability, and higher homogeneity while suppressing stochastic fluctuation (i.e., single-cell-based breeding). In this review, we describe various methods for manipulating single cells and facilitating single-cell analysis in order to better understand stochastic fluctuation. We demonstrated that single-cell-based breeding is practical and promising by using a high-throughput automated system to analyze and manipulate single cells. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Noninvasive High-Throughput Single-Cell Analysis of the Intracellular pH of Saccharomyces cerevisiae by Ratiometric Flow Cytometry

PubMed Central

Valkonen, Mari; Mojzita, Dominik; Penttilä, Merja

2013-01-01

The ability of cells to maintain pH homeostasis in response to environmental changes has elicited interest in basic and applied research and has prompted the development of methods for intracellular pH measurements. Many traditional methods provide information at population level and thus the average values of the studied cell physiological phenomena, excluding the fact that cell cultures are very heterogeneous. Single-cell analysis, on the other hand, offers more detailed insight into population variability, thereby facilitating a considerably deeper understanding of cell physiology. Although microscopy methods can address this issue, they suffer from limitations in terms of the small number of individual cells that can be studied and complicated image processing. We developed a noninvasive high-throughput method that employs flow cytometry to analyze large populations of cells that express pHluorin, a genetically encoded ratiometric fluorescent probe that is sensitive to pH. The method described here enables measurement of the intracellular pH of single cells with high sensitivity and speed, which is a clear improvement compared to previously published methods that either require pretreatment of the cells, measure cell populations, or require complex data analysis. The ratios of fluorescence intensities, which correlate to the intracellular pH, are independent of the expression levels of the pH probe, making the use of transiently or extrachromosomally expressed probes possible. We conducted an experiment on the kinetics of the pH homeostasis of Saccharomyces cerevisiae cultures grown to a stationary phase after ethanol or glucose addition and after exposure to weak acid stress and glucose pulse. Minor populations with pH homeostasis behaving differently upon treatments were identified. PMID:24038689

Noninvasive high-throughput single-cell analysis of the intracellular pH of Saccharomyces cerevisiae by ratiometric flow cytometry.

PubMed

Valkonen, Mari; Mojzita, Dominik; Penttilä, Merja; Bencina, Mojca

2013-12-01

The ability of cells to maintain pH homeostasis in response to environmental changes has elicited interest in basic and applied research and has prompted the development of methods for intracellular pH measurements. Many traditional methods provide information at population level and thus the average values of the studied cell physiological phenomena, excluding the fact that cell cultures are very heterogeneous. Single-cell analysis, on the other hand, offers more detailed insight into population variability, thereby facilitating a considerably deeper understanding of cell physiology. Although microscopy methods can address this issue, they suffer from limitations in terms of the small number of individual cells that can be studied and complicated image processing. We developed a noninvasive high-throughput method that employs flow cytometry to analyze large populations of cells that express pHluorin, a genetically encoded ratiometric fluorescent probe that is sensitive to pH. The method described here enables measurement of the intracellular pH of single cells with high sensitivity and speed, which is a clear improvement compared to previously published methods that either require pretreatment of the cells, measure cell populations, or require complex data analysis. The ratios of fluorescence intensities, which correlate to the intracellular pH, are independent of the expression levels of the pH probe, making the use of transiently or extrachromosomally expressed probes possible. We conducted an experiment on the kinetics of the pH homeostasis of Saccharomyces cerevisiae cultures grown to a stationary phase after ethanol or glucose addition and after exposure to weak acid stress and glucose pulse. Minor populations with pH homeostasis behaving differently upon treatments were identified.

Molecular mechanisms of conformational specificity: A study of Hox in vivo target DNA binding specificities and the structure of a Ure2p mutation that affects fibril formation rates

NASA Astrophysics Data System (ADS)

Bauer, William Joseph, Jr.

The fate of an individual cell, or even an entire organism, is often determined by minute, yet very specific differences in the conformation of a single protein species. Very often, proteins take on alternate folds or even side chain conformations to deal with different situations present within the cell. These differences can be as large as a whole domain or as subtle as the alteration of a single amino acid side chain. Yet, even these seemingly minor side chain conformational differences can determine the development of a cell type during differentiation or even dictate whether a cell will live or die. Two examples of situations where minor conformational differences within a specific protein could lead to major differences in the life cycle of a cell are described herein. The first example describes the variations seen in DNA conformations which can lead to slightly different Hox protein binding conformations responsible for recognizing biologically relevant regulatory sites. These specific differences occur in the minor groove of the bound DNA and are limited to the conformation of only two side chains. The conformation of the bound DNA, however, is not solely determined by the sequence of the DNA, as multiple sequences can result in the same DNA conformation. The second example takes place in the context of a yeast prion protein which contains a mutation that decreases the frequency at which fibrils form. While the specific interactions leading to this physiological change were not directly detected, it can be ascertained from the crystal structure that the structural changes are subtle and most likely involve another binding partner. In both cases, these conformational changes are very slight but have a profound effect on the downstream processes.

Antigenic properties of the human immunodeficiency virus envelope glycoprotein gp120 on virions bound to target cells.

PubMed

Mengistu, Meron; Ray, Krishanu; Lewis, George K; DeVico, Anthony L

2015-03-01

The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step. The elucidation of these epitope exposure patterns during viral entry will help clarify antibody-mediated inhibition of HIV-1 as it is measured in vitro and in vivo.

Chromosomal changes in cultured human epithelial cells transformed by low- and high-LET radiation

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

Yang, Tracy Chui-hsu; Craise, L.M; Prioleau, J.C.

1990-11-01

For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude nice. Neoplastic transformation was achieved by irradiation cells successively. Our results showed that radiogenic cell transformation is a multistep process and thatmore » a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level. 15 refs., 9 figs., 2 tabs.« less

Chromosomal changes in cultured human epithelial cells transformed by low- and high-let radiation

NASA Astrophysics Data System (ADS)

Chui-Hsu Yang, Tracy; Craise, Laurie M.; Prioleau, John C.; Stampfer, Martha R.; Rhim, Johng S.

1992-07-01

For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude mice. Neoplastic transformation was achieved by irradiating cells successively. Our results showed that radiogenic cell transformation is a multistep process and that a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level.

Chromosomal changes in cultured human epithelial cells transformed by low- and high-LET radiation

NASA Technical Reports Server (NTRS)

Craise, L. M.; Prioleau, J. C.; Stampfer, M. R.; Rhim, J. S.; Yang, TC-H (Principal Investigator)

1992-01-01

For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude mice. Neoplastic transformation was achieved by irradiating cells successively. Our results showed that radiogenic cell transformation is a multistep process and that a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level.

Single-cell mechanical phenotype is an intrinsic marker of reprogramming and differentiation along the mouse neural lineage.

PubMed

Urbanska, Marta; Winzi, Maria; Neumann, Katrin; Abuhattum, Shada; Rosendahl, Philipp; Müller, Paul; Taubenberger, Anna; Anastassiadis, Konstantinos; Guck, Jochen

2017-12-01

Cellular reprogramming is a dedifferentiation process during which cells continuously undergo phenotypical remodeling. Although the genetic and biochemical details of this remodeling are fairly well understood, little is known about the change in cell mechanical properties during the process. In this study, we investigated changes in the mechanical phenotype of murine fetal neural progenitor cells (fNPCs) during reprogramming to induced pluripotent stem cells (iPSCs). We find that fNPCs become progressively stiffer en route to pluripotency, and that this stiffening is mirrored by iPSCs becoming more compliant during differentiation towards the neural lineage. Furthermore, we show that the mechanical phenotype of iPSCs is comparable with that of embryonic stem cells. These results suggest that mechanical properties of cells are inherent to their developmental stage. They also reveal that pluripotent cells can differentiate towards a more compliant phenotype, which challenges the view that pluripotent stem cells are less stiff than any cells more advanced developmentally. Finally, our study indicates that the cell mechanical phenotype might be utilized as an inherent biophysical marker of pluripotent stem cells. © 2017. Published by The Company of Biologists Ltd.

Single-Cell Genomic Analysis in Plants

PubMed Central

Hu, Haifei; Scheben, Armin; Edwards, David

2018-01-01

Individual cells in an organism are variable, which strongly impacts cellular processes. Advances in sequencing technologies have enabled single-cell genomic analysis to become widespread, addressing shortcomings of analyses conducted on populations of bulk cells. While the field of single-cell plant genomics is in its infancy, there is great potential to gain insights into cell lineage and functional cell types to help understand complex cellular interactions in plants. In this review, we discuss current approaches for single-cell plant genomic analysis, with a focus on single-cell isolation, DNA amplification, next-generation sequencing, and bioinformatics analysis. We outline the technical challenges of analysing material from a single plant cell, and then examine applications of single-cell genomics and the integration of this approach with genome editing. Finally, we indicate future directions we expect in the rapidly developing field of plant single-cell genomic analysis. PMID:29361790

Strategies for the acquisition of transcriptional and epigenetic information in single cells.

PubMed

Li, Guang; Dzilic, Elda; Flores, Nick; Shieh, Alice; Wu, Sean M

2017-03-01

As the basic unit of living organisms, each single cell has unique molecular signatures and functions. Our ability to uncover the transcriptional and epigenetic signature of single cells has been hampered by the lack of tools to explore this area of research. The advent of microfluidic single cell technology along with single cell genome-wide DNA amplification methods had greatly improved our understanding of the expression variation in single cells. Transcriptional expression profile by multiplex qPCR or genome-wide RNA sequencing has enabled us to examine genes expression in single cells in different tissues. With the new tools, the identification of new cellular heterogeneity, novel marker genes, unique subpopulations, and spatial locations of each single cell can be acquired successfully. Epigenetic modifications for each single cell can also be obtained via similar methods. Based on single cell genome sequencing, single cell epigenetic information including histone modifications, DNA methylation, and chromatin accessibility have been explored and provided valuable insights regarding gene regulation and disease prognosis. In this article, we review the development of strategies to obtain single cell transcriptional and epigenetic data. Furthermore, we discuss ways in which single cell studies may help to provide greater understanding of the mechanisms of basic cardiovascular biology that will eventually lead to improvement in our ability to diagnose disease and develop new therapies.

Nanostructure and force spectroscopy analysis of human peripheral blood CD4+ T cells using atomic force microscopy.

PubMed

Hu, Mingqian; Wang, Jiongkun; Cai, Jiye; Wu, Yangzhe; Wang, Xiaoping

2008-09-12

To date, nanoscale imaging of the morphological changes and adhesion force of CD4(+) T cells during in vitro activation remains largely unreported. In this study, we used atomic force microscopy (AFM) to study the morphological changes and specific binding forces in resting and activated human peripheral blood CD4(+) T cells. The AFM images revealed that the volume of activated CD4(+) T cells increased and the ultrastructure of these cells also became complex. Using a functionalized AFM tip, the strength of the specific binding force of the CD4 antigen-antibody interaction was found to be approximately three times that of the unspecific force. The adhesion forces were not randomly distributed over the surface of a single activated CD4(+) T cell, indicated that the CD4 molecules concentrated into nanodomains. The magnitude of the adhesion force of the CD4 antigen-antibody interaction did not change markedly with the activation time. Multiple bonds involved in the CD4 antigen-antibody interaction were measured at different activation times. These results suggest that the adhesion force involved in the CD4 antigen-antibody interaction is highly selective and of high affinity.

Gene-specific changes in alpha-tubulin transcript accumulation in developing cotton fibers.

PubMed

Whittaker, D J; Triplett, B A

1999-09-01

The fibers of cotton (Gossypium hirsutum) are single-cell trichomes that undergo rapid and synchronous elongation. Cortical microtubules provide spatial information necessary for the alignment of cellulose microfibrils that confine and regulate cell elongation. We used gene-specific probes to investigate alpha-tubulin transcript levels in elongating cotton fibers. Two discrete patterns of transcript accumulation were observed. Whereas transcripts of alpha-tubulin genes GhTua2/3 and GhTua4 increased in abundance from 10 to 20 d post anthesis (DPA), GhTua1 and GhTua5 transcripts were abundant only through to 14 DPA, and dropped significantly at 16 DPA with the onset of secondary wall synthesis. This is the first report, to our knowledge, of gene-specific changes in tubulin transcript levels during the development of a terminally differentiated plant cell. The decrease in abundance of GhTua1 and GhTua5 transcripts was correlated with pronounced changes in cell wall structure, suggesting that alpha-tubulin isoforms may be functionally distinct in elongating fiber cells. Although total alpha-tubulin transcript levels were much higher in fiber than several other tissues, including the hypocotyl and pollen, none of the alpha-tubulins was specific to fiber cells.

Evaluation of Multi-Well Microelectrode Arrays for Neurotoxicity Screening Using a Chemical Training Set

EPA Science Inventory

Microelectrode array (MEA) approaches have been proposed as a tool for detecting functional changes in electrically-excitable cells, including neurons, exposed to drugs, chemical or particles. However, conventional single well-MEA systems lack the throughput necessary for screen...

Neuropeptide F neurons modulate sugar reward during associative olfactory learning of Drosophila larvae.

PubMed

Rohwedder, Astrid; Selcho, Mareike; Chassot, Bérénice; Thum, Andreas S

2015-12-15

All organisms continuously have to adapt their behavior according to changes in the environment in order to survive. Experience-driven changes in behavior are usually mediated and maintained by modifications in signaling within defined brain circuits. Given the simplicity of the larval brain of Drosophila and its experimental accessibility on the genetic and behavioral level, we analyzed if Drosophila neuropeptide F (dNPF) neurons are involved in classical olfactory conditioning. dNPF is an ortholog of the mammalian neuropeptide Y, a highly conserved neuromodulator that stimulates food-seeking behavior. We provide a comprehensive anatomical analysis of the dNPF neurons on the single-cell level. We demonstrate that artificial activation of dNPF neurons inhibits appetitive olfactory learning by modulating the sugar reward signal during acquisition. No effect is detectable for the retrieval of an established appetitive olfactory memory. The modulatory effect is based on the joint action of three distinct cell types that, if tested on the single-cell level, inhibit and invert the conditioned behavior. Taken together, our work describes anatomically and functionally a new part of the sugar reinforcement signaling pathway for classical olfactory conditioning in Drosophila larvae. © 2015 Wiley Periodicals, Inc.

The DNA methylation profile of oocytes in mice with hyperinsulinaemia and hyperandrogenism as detected by single-cell level whole genome bisulphite sequencing (SC-WGBS) technology.

PubMed

Li, Qian-Nan; Guo, Lei; Hou, Yi; Ou, Xiang-Hong; Liu, Zhonghua; Sun, Qing-Yuan

2018-06-22

Polycystic ovary syndrome (PCOS), a familial aggregation disease that causes anovulation in women, has well-recognised characteristics, two of which are hyperinsulinaemia and hyperandrogenaemia. To determine whether the DNA methylation status is altered in oocytes by high insulin and androgen levels, we generated a mouse model with hyperinsulinaemia and hyperandrogenaemia by injection of insulin and human chorionic gonadotrophin and investigated DNA methylation changes through single-cell level whole genome bisulphite sequencing. Our results showed that hyperinsulinaemia and hyperandrogenaemia had no significant effects on the global DNA methylation profile and different functional regions of genes, but did alter methylation status of some genes, which were significantly enriched in 17 gene ontology (GO) terms (P<0.05) by GO analysis. Among differently methylated genes, some were related to the occurrence of PCOS. Based on our results, we suggest that hyperinsulinaemia and hyperandrogenaemia may cause changes in some DNA methylation loci in oocytes.

Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor.

PubMed

Tantama, Mathew; Hung, Yin Pun; Yellen, Gary

2011-07-06

Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm that can be used for ratiometric imaging. The intensity ratio responds with an apparent pK(a) of 6.6 and a >10-fold dynamic range. Furthermore, pHRed has a pH-responsive fluorescence lifetime that changes by ~0.4 ns over physiological pH values and can be monitored with single-wavelength two-photon excitation. After characterizing the sensor, we tested pHRed's ability to monitor intracellular pH by imaging energy-dependent changes in cytosolic and mitochondrial pH.

Arabidopsis myrosinases link the glucosinolate-myrosinase system and the cuticle

PubMed Central

Ahuja, Ishita; de Vos, Ric C. H.; Rohloff, Jens; Stoopen, Geert M.; Halle, Kari K.; Ahmad, Samina Jam Nazeer; Hoang, Linh; Hall, Robert D.; Bones, Atle M.

2016-01-01

Both physical barriers and reactive phytochemicals represent two important components of a plant’s defence system against environmental stress. However, these two defence systems have generally been studied independently. Here, we have taken an exclusive opportunity to investigate the connection between a chemical-based plant defence system, represented by the glucosinolate-myrosinase system, and a physical barrier, represented by the cuticle, using Arabidopsis myrosinase (thioglucosidase; TGG) mutants. The tgg1, single and tgg1 tgg2 double mutants showed morphological changes compared to wild-type plants visible as changes in pavement cells, stomatal cells and the ultrastructure of the cuticle. Extensive metabolite analyses of leaves from tgg mutants and wild-type Arabidopsis plants showed altered levels of cuticular fatty acids, fatty acid phytyl esters, glucosinolates, and indole compounds in tgg single and double mutants as compared to wild-type plants. These results point to a close and novel association between chemical defence systems and physical defence barriers. PMID:27976683

Phosphatidylserine-targeting antibodies augment the anti-tumorigenic activity of anti-PD-1 therapy by enhancing immune activation and downregulating pro-oncogenic factors induced by T-cell checkpoint inhibition in murine triple-negative breast cancers.

PubMed

Gray, Michael J; Gong, Jian; Hatch, Michaela M S; Nguyen, Van; Hughes, Christopher C W; Hutchins, Jeff T; Freimark, Bruce D

2016-05-11

The purpose of this study was to investigate the potential of antibody-directed immunotherapy targeting the aminophospholipid phosphatidylserine, which promotes immunosuppression when exposed in the tumor microenvironment, alone and in combination with antibody treatment towards the T-cell checkpoint inhibitor PD-1 in breast carcinomas, including triple-negative breast cancers. Immune-competent mice bearing syngeneic EMT-6 or E0771 tumors were subjected to treatments comprising of a phosphatidylserine-targeting and an anti-PD-1 antibody either as single or combinational treatments. Anti-tumor effects were determined by tumor growth inhibition and changes in overall survival accompanying each treatment. The generation of a tumor-specific immune response in animals undergoing complete tumor regression was assessed by secondary tumor cell challenge and splenocyte-produced IFNγ in the presence or absence of irradiated tumor cells. Changes in the presence of tumor-infiltrating lymphocytes were assessed by flow cytometry, while mRNA-based immune profiling was determined using NanoString PanCancer Immune Profiling Panel analysis. Treatment by a phosphatidylserine-targeting antibody inhibits in-vivo growth and significantly enhances the anti-tumor activity of antibody-mediated PD-1 therapy, including providing a distinct survival advantage over treatment by either single agent. Animals in which complete tumor regression occurred with combination treatments were resistant to secondary tumor challenge and presented heightened expression levels of splenocyte-produced IFNγ. Combinational treatment by a phosphatidylserine-targeting antibody with anti-PD-1 therapy increased the number of tumor-infiltrating lymphocytes more than that observed with single-arm therapies. Finally, immunoprofiling analysis revealed that the combination of anti-phosphatidylserine targeting antibody and anti-PD-1 therapy enhanced tumor-infiltrating lymphocytes, and increased expression of pro-immunosurveillance-associated cytokines while significantly decreasing expression of pro-tumorigenic cytokines that were induced by single anti-PD-1 therapy. Our data suggest that antibody therapy targeting phosphatidylserine-associated immunosuppression, which has activity as a single agent, can significantly enhance immunotherapies targeting the PD-1 pathway in murine breast neoplasms, including triple-negative breast cancers.

SU-8 hollow cantilevers for AFM cell adhesion studies

NASA Astrophysics Data System (ADS)

Martinez, Vincent; Behr, Pascal; Drechsler, Ute; Polesel-Maris, Jérôme; Potthoff, Eva; Vörös, Janos; Zambelli, Tomaso

2016-05-01

A novel fabrication method was established to produce flexible, transparent, and robust tipless hollow atomic force microscopy (AFM) cantilevers made entirely from SU-8. Channels of 3 μm thickness and several millimeters length were integrated into 12 μm thick and 40 μm wide cantilevers. Connected to a pressure controller, the devices showed high sealing performance with no leakage up to 6 bars. Changing the cantilever lengths from 100 μm to 500 μm among the same wafer allowed the targeting of various spring constants ranging from 0.5 to 80 N m-1 within a single fabrication run. These hollow polymeric AFM cantilevers were operated in the optical beam deflection configuration. To demonstrate the performance of the device, single-cell force spectroscopy experiments were performed with a single probe detaching in a serial protocol more than 100 Saccharomyces cerevisiae yeast cells from plain glass and glass coated with polydopamine while measuring adhesion forces in the sub-nanoNewton range. SU-8 now offers a new alternative to conventional silicon-based hollow cantilevers with more flexibility in terms of complex geometric design and surface chemistry modification.

A single frame: imaging live cells twenty-five years ago.

PubMed

Fink, Rachel

2011-07-01

In the mid-1980s live-cell imaging was changed by the introduction of video techniques, allowing new ways to collect and store data. The increased resolution obtained by manipulating video signals, the ability to use time-lapse videocassette recorders to study events that happen over long time intervals, and the introduction of fluorescent probes and sensitive video cameras opened research avenues previously unavailable. The author gives a personal account of this evolution, focusing on cell migration studies at the Marine Biological Laboratory 25 years ago. Copyright © 2011 Wiley-Liss, Inc.

Get to Understand More from Single-Cells: Current Studies of Microfluidic-Based Techniques for Single-Cell Analysis.

PubMed

Lo, Shih-Jie; Yao, Da-Jeng

2015-07-23

This review describes the microfluidic techniques developed for the analysis of a single cell. The characteristics of microfluidic (e.g., little sample amount required, high-throughput performance) make this tool suitable to answer and to solve biological questions of interest about a single cell. This review aims to introduce microfluidic related techniques for the isolation, trapping and manipulation of a single cell. The major approaches for detection in single-cell analysis are introduced; the applications of single-cell analysis are then summarized. The review concludes with discussions of the future directions and opportunities of microfluidic systems applied in analysis of a single cell.

Get to Understand More from Single-Cells: Current Studies of Microfluidic-Based Techniques for Single-Cell Analysis

PubMed Central

Lo, Shih-Jie; Yao, Da-Jeng

2015-01-01

This review describes the microfluidic techniques developed for the analysis of a single cell. The characteristics of microfluidic (e.g., little sample amount required, high-throughput performance) make this tool suitable to answer and to solve biological questions of interest about a single cell. This review aims to introduce microfluidic related techniques for the isolation, trapping and manipulation of a single cell. The major approaches for detection in single-cell analysis are introduced; the applications of single-cell analysis are then summarized. The review concludes with discussions of the future directions and opportunities of microfluidic systems applied in analysis of a single cell. PMID:26213918

Sensitivity to sequencing depth in single-cell cancer genomics.

PubMed

Alves, João M; Posada, David

2018-04-16

Querying cancer genomes at single-cell resolution is expected to provide a powerful framework to understand in detail the dynamics of cancer evolution. However, given the high costs currently associated with single-cell sequencing, together with the inevitable technical noise arising from single-cell genome amplification, cost-effective strategies that maximize the quality of single-cell data are critically needed. Taking advantage of previously published single-cell whole-genome and whole-exome cancer datasets, we studied the impact of sequencing depth and sampling effort towards single-cell variant detection. Five single-cell whole-genome and whole-exome cancer datasets were independently downscaled to 25, 10, 5, and 1× sequencing depth. For each depth level, ten technical replicates were generated, resulting in a total of 6280 single-cell BAM files. The sensitivity of variant detection, including structural and driver mutations, genotyping, clonal inference, and phylogenetic reconstruction to sequencing depth was evaluated using recent tools specifically designed for single-cell data. Altogether, our results suggest that for relatively large sample sizes (25 or more cells) sequencing single tumor cells at depths > 5× does not drastically improve somatic variant discovery, characterization of clonal genotypes, or estimation of single-cell phylogenies. We suggest that sequencing multiple individual tumor cells at a modest depth represents an effective alternative to explore the mutational landscape and clonal evolutionary patterns of cancer genomes.

The substrate matters in the Raman spectroscopy analysis of cells

PubMed Central

Mikoliunaite, Lina; Rodriguez, Raul D.; Sheremet, Evgeniya; Kolchuzhin, Vladimir; Mehner, Jan; Ramanavicius, Arunas; Zahn, Dietrich R.T.

2015-01-01

Raman spectroscopy is a powerful analytical method that allows deposited and/or immobilized cells to be evaluated without complex sample preparation or labeling. However, a main limitation of Raman spectroscopy in cell analysis is the extremely weak Raman intensity that results in low signal to noise ratios. Therefore, it is important to seize any opportunity that increases the intensity of the Raman signal and to understand whether and how the signal enhancement changes with respect to the substrate used. Our experimental results show clear differences in the spectroscopic response from cells on different surfaces. This result is partly due to the difference in spatial distribution of electric field at the substrate/cell interface as shown by numerical simulations. We found that the substrate also changes the spatial location of maximum field enhancement around the cells. Moreover, beyond conventional flat surfaces, we introduce an efficient nanostructured silver substrate that largely enhances the Raman signal intensity from a single yeast cell. This work contributes to the field of vibrational spectroscopy analysis by providing a fresh look at the significance of the substrate for Raman investigations in cell research. PMID:26310910

Potential role of centrioles in determining the morphogenetic status of animal somatic cells.

PubMed

Tkemaladze, J; Chichinadze, K

2005-05-01

Irreversible differentiation (change of morphogenetic status) and programmed death (apoptosis) are observed only in somatic cells. Cell division is the only way by which the morphogenetic status of the offspring cells may be modified. It is known that there is a fixed limit to the number of possible cell divisions, the so-called 'Hayflick limit'. Existing links between cell division, differentiation and apoptosis make it possible to conclude that all these processes could be controlled by a single self-reproducing structure. Potential candidates for this replicable structure in a somatic cell are chromosomes, mitochondria (both contain DNA), and centrioles. Centrioles (diplosome) are the most likely unit that can fully regulate the processes of irreversible differentiation, determination and modification of the morphogenetic status. It may contain differently encoded RNA molecules stacked in a definite order. During mitosis, these RNA molecules are released one by one into the cytoplasm. In the presence of reverse transcriptase and endonuclease, RNA can be embedded in nuclear DNA. This process presumably changes the status of repressed and potentially active genes and, subsequently, the morphogenetic status of a cell.

The substrate matters in the Raman spectroscopy analysis of cells

NASA Astrophysics Data System (ADS)

Mikoliunaite, Lina; Rodriguez, Raul D.; Sheremet, Evgeniya; Kolchuzhin, Vladimir; Mehner, Jan; Ramanavicius, Arunas; Zahn, Dietrich R. T.

2015-08-01

Raman spectroscopy is a powerful analytical method that allows deposited and/or immobilized cells to be evaluated without complex sample preparation or labeling. However, a main limitation of Raman spectroscopy in cell analysis is the extremely weak Raman intensity that results in low signal to noise ratios. Therefore, it is important to seize any opportunity that increases the intensity of the Raman signal and to understand whether and how the signal enhancement changes with respect to the substrate used. Our experimental results show clear differences in the spectroscopic response from cells on different surfaces. This result is partly due to the difference in spatial distribution of electric field at the substrate/cell interface as shown by numerical simulations. We found that the substrate also changes the spatial location of maximum field enhancement around the cells. Moreover, beyond conventional flat surfaces, we introduce an efficient nanostructured silver substrate that largely enhances the Raman signal intensity from a single yeast cell. This work contributes to the field of vibrational spectroscopy analysis by providing a fresh look at the significance of the substrate for Raman investigations in cell research.

Primary Blast Injury Criteria for Animal/Human TBI Models using Field Validated Shock Tubes

DTIC Science & Technology

2017-09-01

differential pathological response, which depends on the local tissue composition, and the response is to insult depends upon the cell type. regions...Neuroinflammation A single blast induces cell-type dependent increase in NADPH oxidase isoforms We have performed characterization of the spatial variations and...uniformly distribute and affect the whole brain. However, pathophysiological outcomes (e.g., NOX changes) in response to bTBI depend on the differential

Increased Cell-Intrinsic Excitability Induces Synaptic Changes in New Neurons in the Adult Dentate Gyrus That Require Npas4

PubMed Central

Sim, Shuyin; Antolin, Salome; Lin, Chia-Wei; Lin, Ying-Xi

2013-01-01

Electrical activity regulates the manner in which neurons mature and form connections to each other. However, it remains unclear whether increased single-cell activity is sufficient to alter the development of synaptic connectivity of that neuron or whether a global increase in circuit activity is necessary. To address this question, we genetically increased neuronal excitability of in vivo individual adult-born neurons in the mouse dentate gyrus via expression of a voltage-gated bacterial sodium channel. We observed that increasing the excitability of new neurons in an otherwise unperturbed circuit leads to changes in both their input and axonal synapses. Furthermore, the activity-dependent transcription factor Npas4 is necessary for the changes in the input synapses of these neurons, but it is not involved in changes to their axonal synapses. Our results reveal that an increase in cell-intrinsic activity during maturation is sufficient to alter the synaptic connectivity of a neuron with the hippocampal circuit and that Npas4 is required for activity-dependent changes in input synapses. PMID:23637184

Natural single amino acid polymorphism (F19Y) in human galectin-8: detection of structural alterations and increased growth-regulatory activity on tumor cells.

PubMed

Ruiz, Federico M; Scholz, Barbara A; Buzamet, Eliza; Kopitz, Jürgen; André, Sabine; Menéndez, Margarita; Romero, Antonio; Solís, Dolores; Gabius, Hans-Joachim

2014-03-01

Natural amino acid substitution by single-site nucleotide polymorphism can become a valuable tool for structure-activity correlations, especially if evidence for association to disease parameters exists. Focusing on the F19Y change in human galectin-8, connected clinically to rheumatoid arthritis, we here initiate the study of consequences of a single-site substitution in the carbohydrate recognition domain of this family of cellular effectors. We apply a strategically combined set of structural and cell biological techniques for comparing properties of the wild-type and variant proteins. The overall hydrodynamic behavior of the full-length protein and of the separate N-domain is not noticeably altered, but displacements in the F0 β-strand of the β-sandwich fold in the N-domain are induced, as evidenced by protein crystallography. Analysis of thermal stability by circular dichroism spectroscopy revealed perceptible differences for the full-length proteins, pointing to an impact of the substitution beyond the N-domain. In addition, small differences in thermodynamic parameters of carbohydrate binding are detected. On the level of two types of tumor cells, characteristics of binding appeared rather similar. In further comparison of the influence on proliferation, the variant proved to be more active as growth regulator in the six tested lines of neuroblastoma, erythroleukemia and colon adenocarcinoma. The seemingly subtle structural change identified here thus has functional implications in vitro, encouraging further analysis in autoimmune regulation and, in a broad context, in work with other natural single-site variants, using the documented combined strategy. The atomic coordinates and structure factors (codes 4BMB, 4BME) have been deposited in the Protein Data Bank. © 2014 FEBS.

Robust Inference of Cell-to-Cell Expression Variations from Single- and K-Cell Profiling

PubMed Central

Narayanan, Manikandan; Martins, Andrew J.; Tsang, John S.

2016-01-01

Quantifying heterogeneity in gene expression among single cells can reveal information inaccessible to cell-population averaged measurements. However, the expression level of many genes in single cells fall below the detection limit of even the most sensitive technologies currently available. One proposed approach to overcome this challenge is to measure random pools of k cells (e.g., 10) to increase sensitivity, followed by computational “deconvolution” of cellular heterogeneity parameters (CHPs), such as the biological variance of single-cell expression levels. Existing approaches infer CHPs using either single-cell or k-cell data alone, and typically within a single population of cells. However, integrating both single- and k-cell data may reap additional benefits, and quantifying differences in CHPs across cell populations or conditions could reveal novel biological information. Here we present a Bayesian approach that can utilize single-cell, k-cell, or both simultaneously to infer CHPs within a single condition or their differences across two conditions. Using simulated as well as experimentally generated single- and k-cell data, we found situations where each data type would offer advantages, but using both together can improve precision and better reconcile CHP information contained in single- and k-cell data. We illustrate the utility of our approach by applying it to jointly generated single- and k-cell data to reveal CHP differences in several key inflammatory genes between resting and inflammatory cytokine-activated human macrophages, delineating differences in the distribution of ‘ON’ versus ‘OFF’ cells and in continuous variation of expression level among cells. Our approach thus offers a practical and robust framework to assess and compare cellular heterogeneity within and across biological conditions using modern multiplexed technologies. PMID:27438699

Identification of the gene for disaggregatase from Methanosarcina mazei.

PubMed

Osumi, Naoki; Kakehashi, Yoshihiro; Matsumoto, Shiho; Nagaoka, Kazunari; Sakai, Junichi; Miyashita, Kiyotaka; Kimura, Makoto; Asakawa, Susumu

2008-12-01

The gene sequences encoding disaggregatase (Dag), the enzyme responsible for dispersion of cell aggregates of Methanosarcina mazei to single cells, were determined for three strains of M. mazei (S-6(T), LYC and TMA). The dag genes of the three strains were 3234 bp in length and had almost the same sequences with 97% amino acid sequence identities. Dag was predicted to comprise 1077 amino acid residues and to have a molecular mass of 120 kDa containing three repeats of the DNRLRE domain in the C terminus, which is specific to the genus Methanosarcina and may be responsible for structural organization and cell wall function. Recombinant Dag was overexpressed in Escherichia coli and preparations of the expressed protein exhibited enzymatic activity. The RT-PCR analysis showed that dag was transcribed to mRNA in M. mazei LYC and indicated that the gene was expressed in vivo. This is the first time the gene involved in the morphological change of Methanosarcina spp. from aggregate to single cells has been identified.

Single-strand DNA binding protein SSB1 facilitates TERT recruitment to telomeres and maintains telomere G-overhangs

PubMed Central

Pandita, Raj K.; Chow, Tracy T.; Udayakumar, Durga; Bain, Amanda L.; Cubeddu, Liza; Hunt, Clayton R.; Shi, Wei; Horikoshi, Nobuo; Zhao, Yong; Wright, Woodring E.; Khanna, Kum Kum; Shay, Jerry W.; Pandita, Tej K.

2015-01-01

Proliferating mammalian stem and cancer cells express telomerase (TERT) in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA binding protein SSB1, which has a critical role in DNA double-strand break repair. Here we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacted with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduced TERT interaction with telomeres and lead to G-overhang loss. While SSB1 was recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1-TERT interaction relied upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. PMID:25589350

Dynamic physical properties of dissociated tumor cells revealed by dielectrophoretic field-flow fractionation

PubMed Central

Shim, Sangjo; Gascoyne, Peter; Noshari, Jamileh; Stemke Hale, Katherine

2013-01-01

Metastatic disease results from the shedding of cancer cells from a solid primary tumor, their transport through the cardiovascular system as circulating tumor cells (CTCs) and their engraftment and growth at distant sites. Little is known about the properties and fate of tumor cells as they leave their growth site and travel as single cells. We applied analytical dielectrophoretic field-flow fractionation (dFFF) to study the membrane capacitance, density and hydrodynamic properties together with the size and morphology of cultured tumor cells after they were harvested and placed into single cell suspensions. After detachment, the tumor cells exhibited biophysical properties that changed with time through a process of cytoplasmic shedding whereby membrane and cytoplasm were lost. This process appeared to be distinct from the cell death mechanisms of apoptosis, anoikis and necrosis and it may explain why multiple phenotypes are seen among CTCs isolated from patients and among the tumor cells obtained from ascitic fluid of patients. The implications of dynamic biophysical properties and cytoplasmic loss for CTC migration into small blood vessels in the circulatory system, survival and gene expression are discussed. Because the total capacitance of tumor cells remained higher than blood cells even after they had shed cytoplasm, dFFF offers a compelling, antibody-independent technology for isolating viable CTCs from blood even when they are no larger than peripheral blood mononuclear cells. PMID:21691666

Single-Cell and Single-Molecule Analysis of Gene Expression Regulation.

PubMed

Vera, Maria; Biswas, Jeetayu; Senecal, Adrien; Singer, Robert H; Park, Hye Yoon

2016-11-23

Recent advancements in single-cell and single-molecule imaging technologies have resolved biological processes in time and space that are fundamental to understanding the regulation of gene expression. Observations of single-molecule events in their cellular context have revealed highly dynamic aspects of transcriptional and post-transcriptional control in eukaryotic cells. This approach can relate transcription with mRNA abundance and lifetimes. Another key aspect of single-cell analysis is the cell-to-cell variability among populations of cells. Definition of heterogeneity has revealed stochastic processes, determined characteristics of under-represented cell types or transitional states, and integrated cellular behaviors in the context of multicellular organisms. In this review, we discuss novel aspects of gene expression of eukaryotic cells and multicellular organisms revealed by the latest advances in single-cell and single-molecule imaging technology.

Separation and Analysis of Adherent and Non-Adherent Cancer Cells Using a Single-Cell Microarray Chip.

PubMed

Yamamura, Shohei; Yamada, Eriko; Kimura, Fukiko; Miyajima, Kumiko; Shigeto, Hajime

2017-10-21

A new single-cell microarray chip was designed and developed to separate and analyze single adherent and non-adherent cancer cells. The single-cell microarray chip is made of polystyrene with over 60,000 microchambers of 10 different size patterns (31-40 µm upper diameter, 11-20 µm lower diameter). A drop of suspension of adherent carcinoma (NCI-H1650) and non-adherent leukocyte (CCRF-CEM) cells was placed onto the chip, and single-cell occupancy of NCI-H1650 and CCRF-CEM was determined to be 79% and 84%, respectively. This was achieved by controlling the chip design and surface treatment. Analysis of protein expression in single NCI-H1650 and CCRF-CEM cells was performed on the single-cell microarray chip by multi-antibody staining. Additionally, with this system, we retrieved positive single cells from the microchambers by a micromanipulator. Thus, this system demonstrates the potential for easy and accurate separation and analysis of various types of single cells.

Single-Frame Terrain Mapping Software for Robotic Vehicles

NASA Technical Reports Server (NTRS)

Rankin, Arturo L.

2011-01-01

This software is a component in an unmanned ground vehicle (UGV) perception system that builds compact, single-frame terrain maps for distribution to other systems, such as a world model or an operator control unit, over a local area network (LAN). Each cell in the map encodes an elevation value, terrain classification, object classification, terrain traversability, terrain roughness, and a confidence value into four bytes of memory. The input to this software component is a range image (from a lidar or stereo vision system), and optionally a terrain classification image and an object classification image, both registered to the range image. The single-frame terrain map generates estimates of the support surface elevation, ground cover elevation, and minimum canopy elevation; generates terrain traversability cost; detects low overhangs and high-density obstacles; and can perform geometry-based terrain classification (ground, ground cover, unknown). A new origin is automatically selected for each single-frame terrain map in global coordinates such that it coincides with the corner of a world map cell. That way, single-frame terrain maps correctly line up with the world map, facilitating the merging of map data into the world map. Instead of using 32 bits to store the floating-point elevation for a map cell, the vehicle elevation is assigned to the map origin elevation and reports the change in elevation (from the origin elevation) in terms of the number of discrete steps. The single-frame terrain map elevation resolution is 2 cm. At that resolution, terrain elevation from 20.5 to 20.5 m (with respect to the vehicle's elevation) is encoded into 11 bits. For each four-byte map cell, bits are assigned to encode elevation, terrain roughness, terrain classification, object classification, terrain traversability cost, and a confidence value. The vehicle s current position and orientation, the map origin, and the map cell resolution are all included in a header for each map. The map is compressed into a vector prior to delivery to another system.

Quantification of rat retinal growth and vascular population changes after single and split doses of proton irradiation: translational study using stereology methods

NASA Technical Reports Server (NTRS)

Mao, Xiao W.; Archambeau, John O.; Kubinova, Lucie; Boyle, Soames; Petersen, Georgia; Grove, Roger; Nelson, G. A. (Principal Investigator)

2003-01-01

This study quantified architectural and population changes in the rat retinal vasculature after proton irradiation using stereology. A 100 MeV conformal proton beam delivered 8, 14, 20 and 28 Gy as single and split doses to the whole eye. The vascular networks were prepared from retinal digests. Stereological methods were used to obtain the area of the retina and unbiased estimates of microvessel/artery/vein endothelial, pericyte and smooth muscle population, and vessel length. The retinal area increased progressively in the unirradiated, age-matched controls and in the retinas irradiated with 8 and 14 Gy, indicating uniform progressive retinal growth. No growth occurred after 20 and 28 Gy. Regression analysis of total endothelial cell number in all vessels (arteries, veins and capillaries) after irradiation documented a progressive time- and dose-dependent cell loss occurring over 15 to 24 months. The difference from controls was significant (P<0.01) after 28 Gy given in single and split doses and after 20 Gy given as a split dose (P<0.05). Total vessel length in microvessel was significantly shortened at 20 and 28 Gy compared to that of controls (P<0.05). No evident dose recovery was observed in the endothelial populations after split doses. At 10 Gy, the rate of endothelial cell loss, a dose parameter used to characterize the time- and dose-dependent loss of the endothelial population, was doubled.

Single cell digital polymerase chain reaction on self-priming compartmentalization chip

PubMed Central

Zhu, Qiangyuan; Qiu, Lin; Xu, Yanan; Li, Guang; Mu, Ying

2017-01-01

Single cell analysis provides a new framework for understanding biology and disease, however, an absolute quantification of single cell gene expression still faces many challenges. Microfluidic digital polymerase chain reaction (PCR) provides a unique method to absolutely quantify the single cell gene expression, but only limited devices are developed to analyze a single cell with detection variation. This paper describes a self-priming compartmentalization (SPC) microfluidic digital polymerase chain reaction chip being capable of performing single molecule amplification from single cell. The chip can be used to detect four single cells simultaneously with 85% of sample digitization. With the optimized protocol for the SPC chip, we first tested the ability, precision, and sensitivity of our SPC digital PCR chip by assessing β-actin DNA gene expression in 1, 10, 100, and 1000 cells. And the reproducibility of the SPC chip is evaluated by testing 18S rRNA of single cells with 1.6%–4.6% of coefficient of variation. At last, by detecting the lung cancer related genes, PLAU gene expression of A549 cells at the single cell level, the single cell heterogeneity was demonstrated. So, with the power-free, valve-free SPC chip, the gene copy number of single cells can be quantified absolutely with higher sensitivity, reduced labor time, and reagent. We expect that this chip will enable new studies for biology and disease. PMID:28191267

Single cell digital polymerase chain reaction on self-priming compartmentalization chip.

PubMed

Zhu, Qiangyuan; Qiu, Lin; Xu, Yanan; Li, Guang; Mu, Ying

2017-01-01

Single cell analysis provides a new framework for understanding biology and disease, however, an absolute quantification of single cell gene expression still faces many challenges. Microfluidic digital polymerase chain reaction (PCR) provides a unique method to absolutely quantify the single cell gene expression, but only limited devices are developed to analyze a single cell with detection variation. This paper describes a self-priming compartmentalization (SPC) microfluidic digital polymerase chain reaction chip being capable of performing single molecule amplification from single cell. The chip can be used to detect four single cells simultaneously with 85% of sample digitization. With the optimized protocol for the SPC chip, we first tested the ability, precision, and sensitivity of our SPC digital PCR chip by assessing β-actin DNA gene expression in 1, 10, 100, and 1000 cells. And the reproducibility of the SPC chip is evaluated by testing 18S rRNA of single cells with 1.6%-4.6% of coefficient of variation. At last, by detecting the lung cancer related genes, PLAU gene expression of A549 cells at the single cell level, the single cell heterogeneity was demonstrated. So, with the power-free, valve-free SPC chip, the gene copy number of single cells can be quantified absolutely with higher sensitivity, reduced labor time, and reagent. We expect that this chip will enable new studies for biology and disease.

A nonlinear HP-type complementary resistive switch

NASA Astrophysics Data System (ADS)

Radtke, Paul K.; Schimansky-Geier, Lutz

2016-05-01

Resistive Switching (RS) is the change in resistance of a dielectric under the influence of an external current or electric field. This change is non-volatile, and the basis of both the memristor and resistive random access memory. In the latter, high integration densities favor the anti-serial combination of two RS-elements to a single cell, termed the complementary resistive switch (CRS). Motivated by the irregular shape of the filament protruding into the device, we suggest a nonlinearity in the resistance-interpolation function, characterized by a single parameter p. Thereby the original HP-memristor is expanded upon. We numerically simulate and analytically solve this model. Further, the nonlinearity allows for its application to the CRS.

Dynamic photopatterning of cells in situ by Q-switched neodymium-doped yttrium ortho-vanadate laser.

PubMed

Deka, Gitanjal; Okano, Kazunori; Kao, Fu-Jen

2014-01-01

Cellular micropattering has been increasingly adopted in quantitative biological experiments. A Q-switched pulsed neodymium-doped yttrium ortho-vanadate (Nd∶YVO4) laser directed in-situ microfabrication technique for cell patterning is presented. A platform is designed uniquely to achieve laser ablation. The platform is comprised of thin gold coating over a glass surface that functions as a thermal transducer and is over-layered by a cell repellant polymer layer. Micropatterns are engraved on the platform, subsequently exposing specific cell adhesive micro-domains by ablating the gold-polymer coating photothermally. Experimental results indicate that the proposed approach is applicable under culture conditions, viable toward cells, and has a higher engraving speed. Possible uses in arraying isolated single cells on the platform are also shown. Additionally, based on those micro-patterns, dynamic cellular morphological changes and migrational speed in response to geometrical barriers are studied to demonstrate the potential applications of the proposed approach. Our results further demonstrate that cells in narrower geometry had elongated shapes and higher migrational speed than those in wider geometry. Importantly, the proposed approach will provide a valuable reference for efforts to study single cell dynamics and cellular migration related processes for areas such as cell division, wound healing, and cancer invasion.

Induction of cell self-organization on weakly positively charged surfaces prepared by the deposition of polyion complex nanoparticles of thermoresponsive, zwitterionic copolymers.

PubMed

Iwai, Ryosuke; Haruki, Ryota; Nemoto, Yasushi; Nakayama, Yasuhide

2017-07-01

We have developed inducible cell self-organization through weakly positively charged culture surfaces. In this study, a thermoresponsive and zwitterionic copolymer comprised of N,N-dimethylaminoethyl methacrylate (DMAEMA) and methacrylic acid (MA) (PDMAEMA-co-PMA; Mn: ∼9.7 × 10 4 g/mol; PDMAEMA/PMA ratio: 10) was designed for inducing cell self-organization. The copolymer formed single polymer-derived polyion complex (sPIC) nanoparticles following dissolution in an aqueous solution. The sPIC nanoparticles had a positive charge (ca. 25 mV). Self-organization occurred in adipose-derived vascular stromal cell monolayers cultivated on sPIC-deposited surfaces. There were dramatic morphological changes of these cells with the formation of capillary-like networks and single-cell aggregates with little cytotoxicity. This was a significant improvement compared with cells grown on previously developed surfaces deposited with PIC, a mixture of PDMAEMA and plasmid DNA. Thus, sPICs of PDMAEMA-co-PMA may allow for the accurate evaluation of a variety of cell behaviors with less cytotoxicity, and may facilitate additional potential medical applications such as cell-based therapy and drug discovery. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1009-1015, 2017. © 2016 Wiley Periodicals, Inc.

Development of Droplet Microfluidics Enabling High-Throughput Single-Cell Analysis.

PubMed

Wen, Na; Zhao, Zhan; Fan, Beiyuan; Chen, Deyong; Men, Dong; Wang, Junbo; Chen, Jian

2016-07-05

This article reviews recent developments in droplet microfluidics enabling high-throughput single-cell analysis. Five key aspects in this field are included in this review: (1) prototype demonstration of single-cell encapsulation in microfluidic droplets; (2) technical improvements of single-cell encapsulation in microfluidic droplets; (3) microfluidic droplets enabling single-cell proteomic analysis; (4) microfluidic droplets enabling single-cell genomic analysis; and (5) integrated microfluidic droplet systems enabling single-cell screening. We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on key performances of throughput, multifunctionality, and absolute quantification.

Effect of gel re-organization and tensional forces on alpha2beta1 integrin levels in dermal fibroblasts.

PubMed

Jenkins, G; Redwood, K L; Meadows, L; Green, M R

1999-07-01

Mechanical forces are known to play an important role in regulating cell function in a wide range of biological systems. This is of particular relevance to dermal fibroblast function, given that the skin is known to be held under an intrinsic natural tension. To understand more about the generation of force by dermal fibroblasts and their ability to respond to changes in it, we have studied the role of the beta1 integrin receptors expressed by dermal fibroblasts in their ability to generate tensional forces within a collagen type I matrix and the effect of altered tensional force on integrin expression by dermal fibroblasts. Using a purpose-built culture force monitor, function-blocking antibodies directed towards the beta1 receptors dramatically reduced the tensional forces generated by dermal fibroblasts in a 3D collagen I matrix. However, the specific involvement of alpha1 or alpha2 subunits could not be demonstrated. Analysis of cellular response demonstrated that cells isolated from contracting collagen gels expressed fourfold higher levels of alpha2 mRNA than cells isolated from fully restrained gels. The levels of beta1 messenger RNA were relatively unaffected by reductions in force. Cells exposed to single reductions in force, however, did not exhibit alterations in either alpha1 or beta1 mRNA levels. We propose, therefore that alpha2beta1 integrin receptor levels in dermal fibroblasts are not altered in response to single reductions of gel tension, but do change following a continual change in force and associated matrix re-organization

Classification of blood cells and tumor cells using label-free ultrasound and photoacoustics.

PubMed

Strohm, Eric M; Kolios, Michael C

2015-08-01

A label-free method that can identify cells in a blood sample using high frequency photoacoustic and ultrasound signals is demonstrated. When the wavelength of the ultrasound or photoacoustic wave is similar to the size of a single cell (frequencies of 100-500 MHz), unique periodic features occur within the ultrasound and photoacoustic power spectrum that depend on the cell size, structure, and morphology. These spectral features can be used to identify different cell types present in blood, such as red blood cells (RBCs), white blood cells (WBCs), and circulating tumor cells. Circulating melanoma cells are ideal for photoacoustic detection due to their endogenous optical absorption properties. Using a 532 nm pulsed laser and a 375 MHz transducer, the ultrasound and photoacoustic signals from RBCs, WBCs, and melanoma cells were individually measured in an acoustic microscope to examine how the signals change between cell types. A photoacoustic and ultrasound signal was detected from RBCs and melanoma cells; only an ultrasound signal was detected from WBCs. The different cell types were distinctly separated using the ultrasound and photoacoustic signal amplitude and power spectral periodicity. The size of each cell was also estimated from the spectral periodicity. For the first time, sound waves generated using pulse-echo ultrasound and photoacoustics have been used to identify and size single cells, with applications toward counting and identifying cells, including circulating melanoma cells. © 2015 International Society for Advancement of Cytometry.

Agent selection and protective effects during single droplet drying of bacteria.

PubMed

Khem, Sarim; Woo, Meng Wai; Small, Darryl M; Chen, Xiao Dong; May, Bee K

2015-01-01

The protective mechanisms of whey protein isolate (WPI), trehalose, lactose, and skim milk on Lactobacillus plantarum A17 during convective droplet drying has been explored. A single droplet drying technique was used to monitor cell survival, droplet temperature and corresponding changes in mass. WPI and skim milk provided the highest protection amongst the materials tested. In situ analysis of the intermediate stage of drying revealed that for WPI and skim milk, crust formation reduces the rate of sudden temperature increase thereby imparting less stress on the cells. Irreversible denaturation of the WPI components might have also contributed to the protection of the cells. Skim milk, however, 'loses' the protective behaviour towards the latter stages of drying. This indicates that the concentration of the WPI components could be another possible factor determining the sustained protective behaviour during the later stages of drying when the moisture content is low. Copyright © 2014 Elsevier Ltd. All rights reserved.

Fabrication of luminescent SrWO{sub 4} thin films by a novel electrochemical method

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

Chen Lianping; Gao Yuanhong

2007-10-02

Highly crystallized SrWO{sub 4} thin films with single scheelite structure were prepared within 60 min by a cell electrochemical method. X-ray diffraction analysis shows that SrWO{sub 4} thin films have a tetragonal structure. Scanning electron microscopy examinations reveal that SrWO{sub 4} grains grow well in tetragonal tapers and grains like flowers or bunches, which can usually form by using the electrolysis electrochemical method, have disappeared under cell electrochemical conditions. X-ray photoelectron spectra and energy dispersive X-ray microanalysis examinations demonstrate that the composition of the film is consistent with its stoichiometry. These SrWO{sub 4} films show a single blue emission peakmore » (located at 460 nm) using an excitation wave of 230 nm. The speed of cell electrochemical method can be controlled by changing temperature. The optimum treatment temperature is about 50-60 deg. C.« less

Diadenosine tetraphosphate-gating of recombinant pancreatic ATP-sensitive K(+) channels.

PubMed

Jovanovic, S; Jovanovic, A

2001-02-01

Diadenosine tetraphosphate (Ap4A) has been recently discovered in the pancreatic beta cells where targets ATP-sensitive K(+) (K(ATP)) channels, depolarizes the cell membrane and induces insulin secretion. However, whether Ap4A inhibit pancreatic K(ATP) channels by targeting protein channel complex itself was unknown. Therefore, we coexpressed pancreatic K(ATP) channel subunits, Kir6.2 and SUR1, in COS-7 cells and examined the effect of Ap4A on the single channel behavior using the inside-out configuration of the patch-clamp technique. Ap4A inhibited channel opening in a concentration-dependent manner. Analysis of single channels demonstrated that Ap4A did not change intraburst kinetic behavior of K(ATP) channels, but rather decreased burst duration and increased between-burst duration. It is concluded that Ap4A antagonizes K(ATP) channel opening by targeting channel subunits themselves and by keeping channels longer in closed interburst states.

Rapid Antibiotic Susceptibility Testing of Uropathogenic E. coli by Tracking Submicron Scale Motion of Single Bacterial Cells.

PubMed

Syal, Karan; Shen, Simon; Yang, Yunze; Wang, Shaopeng; Haydel, Shelley E; Tao, Nongjian

2017-08-25

To combat antibiotic resistance, a rapid antibiotic susceptibility testing (AST) technology that can identify resistant infections at disease onset is required. Current clinical AST technologies take 1-3 days, which is often too slow for accurate treatment. Here we demonstrate a rapid AST method by tracking sub-μm scale bacterial motion with an optical imaging and tracking technique. We apply the method to clinically relevant bacterial pathogens, Escherichia coli O157: H7 and uropathogenic E. coli (UPEC) loosely tethered to a glass surface. By analyzing dose-dependent sub-μm motion changes in a population of bacterial cells, we obtain the minimum bactericidal concentration within 2 h using human urine samples spiked with UPEC. We validate the AST method using the standard culture-based AST methods. In addition to population studies, the method allows single cell analysis, which can identify subpopulations of resistance strains within a sample.

Mass Spectrometry of Single GABAergic Somatic Motorneurons Identifies a Novel Inhibitory Peptide, As-NLP-22, in the Nematode Ascaris suum.

PubMed

Konop, Christopher J; Knickelbine, Jennifer J; Sygulla, Molly S; Wruck, Colin D; Vestling, Martha M; Stretton, Antony O W

2015-12-01

Neuromodulators have become an increasingly important component of functional circuits, dramatically changing the properties of both neurons and synapses to affect behavior. To explore the role of neuropeptides in Ascaris suum behavior, we devised an improved method for cleanly dissecting single motorneuronal cell bodies from the many other cell processes and hypodermal tissue in the ventral nerve cord. We determined their peptide content using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The reduced complexity of the peptide mixture greatly aided the detection of peptides; peptide levels were sufficient to permit sequencing by tandem MS from single cells. Inhibitory motorneurons, known to be GABAergic, contain a novel neuropeptide, As-NLP-22 (SLASGRWGLRPamide). From this sequence and information from the A. suum expressed sequence tag (EST) database, we cloned the transcript (As-nlp-22) and synthesized a riboprobe for in situ hybridization, which labeled the inhibitory motorneurons; this validates the integrity of the dissection method, showing that the peptides detected originate from the cells themselves and not from adhering processes from other cells (e.g., synaptic terminals). Synthetic As-NLP-22 has potent inhibitory activity on acetylcholine-induced muscle contraction as well as on basal muscle tone. Both of these effects are dose-dependent: the inhibitory effect on ACh contraction has an IC50 of 8.3 × 10(-9) M. When injected into whole worms, As-NLP-22 produces a dose-dependent inhibition of locomotory movements and, at higher levels, complete paralysis. These experiments demonstrate the utility of MALDI TOF/TOF MS in identifying novel neuromodulators at the single-cell level. Graphical Abstract ᅟ.

Mass Spectrometry of Single GABAergic Somatic Motorneurons Identifies a Novel Inhibitory Peptide, As-NLP-22, in the Nematode Ascaris suum

NASA Astrophysics Data System (ADS)

Konop, Christopher J.; Knickelbine, Jennifer J.; Sygulla, Molly S.; Wruck, Colin D.; Vestling, Martha M.; Stretton, Antony O. W.

2015-12-01

Neuromodulators have become an increasingly important component of functional circuits, dramatically changing the properties of both neurons and synapses to affect behavior. To explore the role of neuropeptides in Ascaris suum behavior, we devised an improved method for cleanly dissecting single motorneuronal cell bodies from the many other cell processes and hypodermal tissue in the ventral nerve cord. We determined their peptide content using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The reduced complexity of the peptide mixture greatly aided the detection of peptides; peptide levels were sufficient to permit sequencing by tandem MS from single cells. Inhibitory motorneurons, known to be GABAergic, contain a novel neuropeptide, As-NLP-22 (SLASGRWGLRPamide). From this sequence and information from the A. suum expressed sequence tag (EST) database, we cloned the transcript ( As-nlp-22) and synthesized a riboprobe for in situ hybridization, which labeled the inhibitory motorneurons; this validates the integrity of the dissection method, showing that the peptides detected originate from the cells themselves and not from adhering processes from other cells (e.g., synaptic terminals). Synthetic As-NLP-22 has potent inhibitory activity on acetylcholine-induced muscle contraction as well as on basal muscle tone. Both of these effects are dose-dependent: the inhibitory effect on ACh contraction has an IC50 of 8.3 × 10-9 M. When injected into whole worms, As-NLP-22 produces a dose-dependent inhibition of locomotory movements and, at higher levels, complete paralysis. These experiments demonstrate the utility of MALDI TOF/TOF MS in identifying novel neuromodulators at the single-cell level.

Revealing Abrupt and Spontaneous Ruptures of Protein Native Structure under picoNewton Compressive Force Manipulation.

PubMed

Chowdhury, S Roy; Cao, Jin; He, Yufan; Lu, H Peter

2018-03-27

Manipulating protein conformations for exploring protein structure-function relationship has shown great promise. Although protein conformational changes under pulling force manipulation have been extensively studied, protein conformation changes under a compressive force have not been explored quantitatively. The latter is even more biologically significant and relevant in revealing protein functions in living cells associated with protein crowdedness, distribution fluctuations, and cell osmotic stress. Here we report our experimental observations on abrupt ruptures of protein native structures under compressive force, demonstrated and studied by single-molecule AFM-FRET spectroscopic nanoscopy. Our results show that the protein ruptures are abrupt and spontaneous events occurred when the compressive force reaches a threshold of 12-75 pN, a force amplitude accessible from thermal fluctuations in a living cell. The abrupt ruptures are sensitive to local environment, likely a general and important pathway of protein unfolding in living cells.