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https://www.readbyqxmd.com/read/28748464/the-use-of-bacterial-repair-endonucleases-in-the-comet-assay
#1
Andrew R Collins
The comet assay is a sensitive electrophoretic method for measuring DNA breaks at the level of single cells, used widely in genotoxicity experiments, in biomonitoring, and in fundamental research. Its sensitivity and range of application are increased by the incorporation of an extra step, after lysis of agarose-embedded cells, in which the DNA is digested with lesion-specific endonucleases (DNA repair enzymes of bacterial or phage origin). Enzymes with specificity for oxidized purines, oxidized pyrimidines, alkylated bases, UV-induced cyclobutane pyrimidine dimers, and misincorporated uracil have been employed...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28748462/in-vivo-rat-t-lymphocyte-pig-a-assay-detection-and-expansion-of-cells-deficient-in-the-gpi-anchored-cd48-surface-marker-for-analysis-of-mutation-in-the-endogenous-pig-a-gene
#2
Vasily N Dobrovolsky, Javier Revollo, Dayton M Petibone, Robert H Heflich
The Pig-a assay is being developed as an in vivo gene mutation assay for regulatory safety assessments. The assay is based on detecting mutation in the endogenous Pig-a gene of treated rats by using flow cytometry to measure changes in cell surface markers of peripheral blood cells. Here we present a methodology for demonstrating that phenotypically mutant rat T-cells identified by flow cytometry contain mutations in the Pig-a gene, an important step for validating the assay. In our approach, the mutant phenotype T-cells are sorted into individual wells of 96-well plates and expanded into clones...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28748452/single-cell-transcriptomics-reveals-gene-signatures-and-alterations-associated-with-aging-in-distinct-neural-stem-progenitor-cell-subpopulations
#3
Zhanping Shi, Yanan Geng, Jiping Liu, Huina Zhang, Liqiang Zhou, Quan Lin, Juehua Yu, Kunshan Zhang, Jie Liu, Xinpei Gao, Chunxue Zhang, Yinan Yao, Chong Zhang, Yi E Sun
Aging associated cognitive decline has been linked to dampened neural stem/progenitor cells (NSC/NPCs) activities manifested by decreased proliferation, reduced propensity to produce neurons, and increased differentiation into astrocytes. While gene transcription changes objectively reveal molecular alterations of cells undergoing various biological processes, the search for molecular mechanisms underlying aging of NSC/NPCs has been confronted by the enormous heterogeneity in cellular compositions of the brain and the complex cellular microenvironment where NSC/NPCs reside...
July 26, 2017: Protein & Cell
https://www.readbyqxmd.com/read/28748182/mycobacteria-modify-their-cell-size-control-under-sub-optimal-carbon-sources
#4
Miles Priestman, Philipp Thomas, Brian D Robertson, Vahid Shahrezaei
The decision to divide is the most important one that any cell must make. Recent single cell studies suggest that most bacteria follow an "adder" model of cell size control, incorporating a fixed amount of cell wall material before dividing. Mycobacteria, including the causative agent of tuberculosis Mycobacterium tuberculosis, are known to divide asymmetrically resulting in heterogeneity in growth rate, doubling time, and other growth characteristics in daughter cells. The interplay between asymmetric cell division and adder size control has not been extensively investigated...
2017: Frontiers in Cell and Developmental Biology
https://www.readbyqxmd.com/read/28747891/proliferative-role-of-kv11-channels-in-murine-arteries
#5
Vincenzo Barrese, Pilar Cidad, Shuk Y Yeung, Jose R López-López, Alister J McNeish, Susumu Ohya, Maria T Pérez-García, Iain A Greenwood
K(+) channels encoded by the ether-a-go-go related gene (ERG1 or KCNH2) are important determinants of the cardiac action potential. Expression of both cardiac isoforms (ERG1a and ERG1b) were identified in murine portal vein and distinctive voltage-gated K(+) currents were recorded from single myocytes. The aim of the present study was to ascertain the expression and functional impact of ERG channels in murine arteries. Methods: Quantitative RT-PCR was undertaken on RNA extracted from a number of murine arteries...
2017: Frontiers in Physiology
https://www.readbyqxmd.com/read/28747668/deformability-based-cell-sorting-using-microfluidic-ratchets-enabling-phenotypic-separation-of-leukocytes-directly-from-whole-blood
#6
Quan Guo, Simon P Duffy, Kerryn Matthews, Emel Islamzada, Hongshen Ma
The separation of leukocytes from whole blood is a prerequisite for many biological assays. Traditional methods require significant sample volumes and are often undesirable because they expose leukocytes to harsh physical or chemical treatment. Existing microfluidic approaches can work with smaller volumes, but lack selectivity. In particular, the selectivity of microfluidic systems based on microfiltration is limited by fouling due to clogging. Here, we developed a method to separate leukocytes from whole blood using the microfluidic ratchet mechanism, which filters the blood sample using a matrix of micrometer-scale tapered constrictions...
July 26, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28747643/generation-of-ribosome-imprinted-polymers-for-sensitive-detection-of-translational-responses
#7
Helen A King, Hazim F El-Sharif, Ana M Matia-González, Valentina Iadevaia, Adeola Fowotade, Subrayal M Reddy, André P Gerber
Whilst the profiling of the transcriptome and proteome even of single-cells becomes feasible, the analysis of the translatome, which refers to all messenger RNAs (mRNAs) engaged with ribosomes for protein synthesis, is still an elaborate procedure requiring millions of cells. Herein, we report the generation and use of "smart materials", namely molecularly imprinted polymers (MIPs) to facilitate the isolation of ribosomes and translated mRNAs from merely 1,000 cells. In particular, we show that a hydrogel-based ribosome imprinted polymer could recover ribosomes and associated mRNAs from human, simian and mice cellular extracts, but did not selectively enrich yeast ribosomes, thereby demonstrating selectivity...
July 26, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28747624/number-of-infection-events-per-cell-during-hiv-1-cell-free-infection
#8
Yusuke Ito, Azaria Remion, Alexandra Tauzin, Keisuke Ejima, Shinji Nakaoka, Yoh Iwasa, Shingo Iwami, Fabrizio Mammano
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...
July 26, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28747472/disentangling-sources-of-variation-in-ssu-rdna-sequences-from-single-cell-analyses-of-ciliates-impact-of-copy-number-variation-and-experimental-error
#9
Chundi Wang, Tengteng Zhang, Yurui Wang, Laura A Katz, Feng Gao, Weibo Song
Small subunit ribosomal DNA (SSU rDNA) is widely used for phylogenetic inference, barcoding and other taxonomy-based analyses. Recent studies indicate that SSU rDNA of ciliates may have a high level of sequence variation within a single cell, which impacts the interpretation of rDNA-based surveys. However, sequence variation can come from a variety of sources including experimental errors, especially the mutations generated by DNA polymerase in PCR. In the present study, we explore the impact of four DNA polymerases on sequence variation and find that low-fidelity polymerases exaggerate the estimates of single-cell sequence variation...
July 26, 2017: Proceedings. Biological Sciences
https://www.readbyqxmd.com/read/28747440/aligned-fibers-direct-collective-cell-migration-to-engineer-closing-and-non-closing-wound-gaps
#10
Puja Sharma, Colin Ng, Aniket Jana, Abinash Padhi, Paige Szymanski, Jerry S H Lee, Bahareh Behkam, Amrinder S Nain
Cell emergence onto damaged or organized fibrous extracellular matrix (ECM) is a crucial precursor to collective cell migration in wound closure and cancer metastasis, respectively. However, there is a fundamental gap in our quantitative understanding of the role of local ECM size and arrangement in cell emergence based migration and local gap closure. Here, using ECM-mimicking nanofibers bridging cell monolayers, we describe a method to recapitulate and quantitatively describe these in vivo behaviors over multi spatial (single cell to cell sheets) and temporal (minutes-weeks) scales...
July 26, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28747214/culturing-and-transcriptome-profiling-of-progenitor-like-colonies-derived-from-adult-mouse-pancreas
#11
Dongshen Ma, Shanshan Tang, Jing Song, Qiong Wu, Fangfang Zhang, Yun Xing, Yi Pan, Yanfeng Zhang, Jingwei Jiang, Yubin Zhang, Liang Jin
BACKGROUND: Transplantation of insulin-producing cells is considered an important diabetes therapy. Many research studies have shown that insulin-producing cells can be derived from the in-vitro cultured pancreatic colonies with self-renewal ability and multilineage potential. Even though these progenitor-like colonies have been prepared from adult pancreas cells, the efficient culture method is hardly established and regulation of the colonies is rarely known. We confirmed previously that single cells acquired from adult mouse pancreas could form cyst-like colonies in a 3D semi-solid system containing Matrigel and methylcellulose...
July 26, 2017: Stem Cell Research & Therapy
https://www.readbyqxmd.com/read/28746881/mass-cytometric-analysis-of-hiv-entry-replication-and-remodeling-in-tissue-cd4-t-cells
#12
Marielle Cavrois, Trambak Banerjee, Gourab Mukherjee, Nandhini Raman, Rajaa Hussien, Brandon Aguilar Rodriguez, Joshua Vasquez, Matthew H Spitzer, Nicole H Lazarus, Jennifer J Jones, Christina Ochsenbauer, Joseph M McCune, Eugene C Butcher, Ann M Arvin, Nandini Sen, Warner C Greene, Nadia R Roan
To characterize susceptibility to HIV infection, we phenotyped infected tonsillar T cells by single-cell mass cytometry and created comprehensive maps to identify which subsets of CD4+ T cells support HIV fusion and productive infection. By comparing HIV-fused and HIV-infected cells through dimensionality reduction, clustering, and statistical approaches to account for viral perturbations, we identified a subset of memory CD4+ T cells that support HIV entry but not viral gene expression. These cells express high levels of CD127, the IL-7 receptor, and are believed to be long-lived lymphocytes...
July 25, 2017: Cell Reports
https://www.readbyqxmd.com/read/28746843/the-role-of-the-microenvironment-in-the-biophysics-of-cancer
#13
REVIEW
Carmela Rianna, Prem Kumar, Manfred Radmacher
During the last decades, cell mechanics has been recognized as a quantitative measure to discriminate between many physiological and pathological states of single cells. In the field of biophysics of cancer, a large body of research has been focused on the comparison between normal and cancer mechanics and slowly the hypothesis that cancer cells are softer than their normal counterparts has been accepted, even though in situ tumor tissue is usually stiffer than the surrounding normal tissue. This corroborates the idea that the extra cellular matrix (ECM) has a critical role in regulating tumor cell properties and behavior...
July 23, 2017: Seminars in Cell & Developmental Biology
https://www.readbyqxmd.com/read/28746390/homogenized-modeling-methodology-for-18650-lithium-ion-battery-module-under-large-deformation
#14
Liang Tang, Jinjie Zhang, Pengle Cheng
Effective lithium-ion battery module modeling has become a bottleneck for full-size electric vehicle crash safety numerical simulation. Modeling every single cell in detail would be costly. However, computational accuracy could be lost if the module is modeled by using a simple bulk material or rigid body. To solve this critical engineering problem, a general method to establish a computational homogenized model for the cylindrical battery module is proposed. A single battery cell model is developed and validated through radial compression and bending experiments...
2017: PloS One
https://www.readbyqxmd.com/read/28746386/tracmit-an-effective-pipeline-for-tracking-and-analyzing-cells-on-micropatterns-through-mitosis
#15
Olivier Burri, Benita Wolf, Arne Seitz, Pierre Gönczy
The use of micropatterns has transformed investigations of dynamic biological processes by enabling the reproducible analysis of live cells using time-lapse fluorescence microscopy. With micropatterns, thousands of individual cells can be efficiently imaged in parallel, rendering the approach well suited for screening projects. Despite being powerful, such screens remain challenging in terms of data handling and analysis. Typically, only a fraction of micropatterns is occupied in a manner suitable to monitor a given phenotypic output...
2017: PloS One
https://www.readbyqxmd.com/read/28746017/tumor-evolution-heterogeneity-and-therapy-for-our-patients-with-advanced-cancer-how-far-have-we-come
#16
Wafik S El-Deiry, Barry Taylor, Joel W Neal
The clinical and molecular heterogeneity of various cancer types is well documented. In the era of precision oncology whereby molecular profiling of tumors is incorporated into clinical care, both intra- and intertumoral molecular and genetic heterogeneity have been described. Together, they impact patient treatment and outcomes. Host genetics and the tumor microenvironment impact on tumor evolution and heterogeneity through variations in immune cell infiltration, stromal variations, and selection pressures from hypoxia or nutrient stress, among others...
2017: American Society of Clinical Oncology Educational Book
https://www.readbyqxmd.com/read/28745623/a-standardized-approach-for-multispecies-purification-of-mammalian-male-germ-cells-by-mechanical-tissue-dissociation-and-flow-cytometry
#17
Ana C Lima, Min Jung, Jannette Rusch, Abul Usmani, Alexandra M Lopes, Donald F Conrad
Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst)...
July 12, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28745517/it-is-all-about-location-how-to-pinpoint-microorganisms-and-their-functions-in-multispecies-biofilms
#18
Angela M Costa, Filipe J Mergulhão, Romain Briandet, Nuno F Azevedo
Multispecies biofilms represent the dominant mode of life for the vast majority of microorganisms. Bacterial spatial localization in such biostructures governs ecological interactions between different populations and triggers the overall community functions. Here, we discuss the pros and cons of fluorescence-based techniques used to decipher bacterial species patterns in biofilms at single cell level, including fluorescence in situ hybridization and the use of genetically modified bacteria that express fluorescent proteins, reporting the significant improvements of those techniques...
July 26, 2017: Future Microbiology
https://www.readbyqxmd.com/read/28745495/kinetic-switching-of-the-concentration-separation-behavior-of-microdroplets
#19
Mao Fukuyama, Akihide Hibara, Yumi Yoshida, Kohji Maeda
This work demonstrates that the solute concentration inside 100 μm-sized aqueous microdroplets can be controlled by adjusting the time required for the aqueous nanometer-sized droplets (nanodroplet) or reverse micelles to pass over the surface of the microdroplet. The kinetics of molecular transport between the microdroplets and nanodroplets was investigated by utilizing a microdroplet array, and based on these results, a control over the concentration selectivity of the contents of the microdroplet was achieved...
July 26, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28743737/real-time-visualization-of-chromatin-modification-in-isolated-nuclei
#20
Luca Sardo, Angel Lin, Svetlana Khakhina, Lucas Beckman, Luis Ricon, Weam Elbezanti, Tara Jaison, Harshad Vishwasrao, Hari Shroff, Christopher Janetopoulos, Zachary A Klase
Chromatin modification is traditionally queried by biochemical assays that provide average measurements of static events since the analysis requires components from many cells. Microscopy can visualize single cells, but the cell body and organelles can hamper staining and visualization of the nucleus. Normally, chromatin is visualized by immunostaining a fixed sample or by expressing exogenous fluorescently tagged proteins in a live cell. Alternative microscopy tools to observe changes of endogenous chromatin in real-time are needed...
July 25, 2017: Journal of Cell Science
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