Enzymatic activity in single cells

For a better understanding of the evolution of antibiotic resistance, it is imperative to study the factors that determine the initial establishment of mutant resistance alleles. In addition to the antibiotic concentration, the establishment of resistance alleles may be affected by interactions with the surrounding susceptible cells from which they derive, for instance via the release of nutrients or removal of the antibiotic. Here, we investigate the effects of social interactions with surrounding susceptible cells on the establishment of Escherichia coli mutants with increasing β-lactamase activity (i.e. the capacity to hydrolyze β-lactam antibiotics) from single cells under the exposure of the antibiotic cefotaxime on agar plates. We find that mutant establishment probability is increased in the presence of susceptible cells due to the active breakdown of the antibiotic, but the rate of breakdown by the susceptible strain is much higher than expected based on its low enzymatic activity. A detailed theoretical model suggests that this observation can be explained by cell filamentation causing delayed lysis. While susceptible cells may hamper the spread of higher-resistant β-lactamase mutants at relatively high frequencies, our findings show that they could promote establishment during their emergence.

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Probing Enzymatic Activity Inside Single Cells

Analytical Chemistry ◽

10.1021/ac4013122 ◽

2013 ◽

Vol 85 (21) ◽

pp. 10126-10133 ◽

Cited By ~ 5

Author(s):

Jessica Olofsson ◽

Shijun Xu ◽

Gavin D. M. Jeffries ◽

Aldo Jesorka ◽

Helen Bridle ◽

...

Keyword(s):

Enzymatic Activity ◽

Single Cells

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Inferring Telomerase Enzymatic Activity from Expression Data

10.1101/2020.05.21.109249 ◽

2020 ◽

Cited By ~ 1

Author(s):

Nighat Noureen ◽

Shaofang Wu ◽

Yingli Lyu ◽

Juechen Yang ◽

WK Alfred Yung ◽

...

Keyword(s):

Stem Cells ◽

Enzymatic Activity ◽

Single Cells ◽

Gene Signature ◽

Telomerase Activity ◽

Expression Data ◽

Cancer Stemness ◽

Cancer Proliferation ◽

Computational Framework ◽

Cancer Cell Population

AbstractActive telomerase is essential for stem cells and most cancers to maintain telomeres. The enzymatic activity of telomerase is related but not equivalent to the expression of TERT, the catalytic subunit of the complex. Here we show that telomerase enzymatic activity can be robustly estimated from the expression of a 13-gene signature. We demonstrate the validity of the expression-based approach, named EXTEND, using cell lines, cancer samples, and non-neoplastic samples. When applied to over 9,000 tumors and single cells, we find a strong correlation between telomerase activity and cancer stemness. This correlation is largely driven by a small proliferating cancer cell population that exhibits both high telomerase activity and cancer stemness. This study establishes a novel computational framework for quantifying telomerase enzymatic activity and provides new insights into the relationships among telomerase, cancer proliferation, and stemness.

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Measuring the Enzymatic Activity of Clinically Important Proteins in Single Cells

Biophysical Journal ◽

10.1016/j.bpj.2010.12.1401 ◽

2011 ◽

Vol 100 (3) ◽

pp. 217a-218a

Author(s):

Christopher E. Sims ◽

Nancy L. Allbritton ◽

Dechen Jiang ◽

Shan Yang ◽

Angie Proctor ◽

...

Keyword(s):

Enzymatic Activity ◽

Single Cells

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Integrated analysis of telomerase enzymatic activity unravels an association with cancer stemness and proliferation

Nature Communications ◽

10.1038/s41467-020-20474-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Nighat Noureen ◽

Shaofang Wu ◽

Yingli Lv ◽

Juechen Yang ◽

W. K. Alfred Yung ◽

...

Keyword(s):

Stem Cells ◽

Enzymatic Activity ◽

Single Cells ◽

Gene Signature ◽

Telomerase Activity ◽

Small Population ◽

Integrated Analysis ◽

Cancer Stemness ◽

Cancer Proliferation ◽

Computational Framework

AbstractActive telomerase is essential for stem cells and most cancers to maintain telomeres. The enzymatic activity of telomerase is related but not equivalent to the expression of TERT, the catalytic subunit of the complex. Here we show that telomerase enzymatic activity can be robustly estimated from the expression of a 13-gene signature. We demonstrate the validity of the expression-based approach, named EXTEND, using cell lines, cancer samples, and non-neoplastic samples. When applied to over 9,000 tumors and single cells, we find a strong correlation between telomerase activity and cancer stemness. This correlation is largely driven by a small population of proliferating cancer cells that exhibits both high telomerase activity and cancer stemness. This study establishes a computational framework for quantifying telomerase enzymatic activity and provides new insights into the relationships among telomerase, cancer proliferation, and stemness.

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Digital Assays Part II: Digital Protein and Cell Assays

SLAS TECHNOLOGY Translating Life Sciences Innovation ◽

10.1177/2472630317705681 ◽

2017 ◽

Vol 22 (4) ◽

pp. 387-405 ◽

Cited By ~ 3

Author(s):

Amar S. Basu

Keyword(s):

Enzymatic Activity ◽

Single Cell ◽

Single Cells ◽

Digital Pcr ◽

Surface Proteins ◽

Broad Perspective ◽

Antigenic Proteins ◽

Conventional Elisa ◽

Cell Assays ◽

Biological Entities

A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist’s toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays. Digital enzyme assays measure the kinetics of single proteins with enzymatic activity. Digital enzyme-linked immunoassays (ELISAs) quantify antigenic proteins with 2 to 3 log lower detection limit than conventional ELISA, making them well suited for low-abundance biomarkers. Digital cell assays probe single-cell genotype and phenotype, including gene expression, intracellular and surface proteins, metabolic activity, cytotoxicity, and transcriptomes (scRNA-seq). These methods exploit partitioning to 1) isolate single cells or proteins, 2) detect their activity via enzymatic amplification, and 3) tag them individually by coencapsulating them with molecular barcodes. When scaled, digital assays reveal stochastic differences between proteins or cells within a population, a key to understanding biological heterogeneity. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows.

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Effects of carbamylcholine on chick embryo aggregate retina cell cultures

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100103814 ◽

1988 ◽

Vol 46 ◽

pp. 350-351

Author(s):

Glenn M. Cohen ◽

Radharaman Ray

Keyword(s):

Cell Cultures ◽

Single Cells ◽

Retinal Cell ◽

Cell Aggregates ◽

High Concentration ◽

In Ovo ◽

Sterile Culture ◽

Retinal Tissue ◽

Synaptic Junctions ◽

And Control

Retinal,cell aggregates develop in culture in a pattern similar to the in ovo retina, forming neurites first and then synapses. In the present study, we continuously exposed chick retinal cell aggregates to a high concentration (1 mM) of carbamylcholine (carbachol), an acetylcholine (ACh) analog that resists hydrolysis by acetylcholinesterase (AChE). This situation is similar to organophosphorus anticholinesterase poisoning in which the ACh level is elevated at synaptic junctions due to inhibition of AChE, Our objective was to determine whether continuous carbachol exposure either damaged cholino- ceptive neurites, cell bodies, and synaptic elements of the aggregates or influenced (hastened or retarded) their development.The retinal tissue was isolated aseptically from 11 day embryonic White Leghorn chicks and then enzymatically (trypsin) and mechanically (trituration) dissociated into single cells. After washing the cells by repeated suspension and low (about 200 x G) centrifugation twice, aggregate cell cultures (about l0 cells/culture) were initiated in 1.5 ml medium (BME, GIBCO) in 35 mm sterile culture dishes and maintained as experimental (containing 10-3 M carbachol) and control specimens.

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Selective Staining of Acid Mucopolysaccharides By Ruthenium Red

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100099933 ◽

1968 ◽

Vol 26 ◽

pp. 38-39

Author(s):

J. H. Luft

Keyword(s):

Electron Microscope ◽

Light Microscopy ◽

Light Microscope ◽

Osmium Tetroxide ◽

Single Cells ◽

Ruthenium Red ◽

Collagen Fibers ◽

Selective Staining ◽

Pectic Substances ◽

Solid Tissues

Ruthenium red is one of the few completely inorganic dyes used to stain tissues for light microscopy. This novelty is enhanced by ignorance regarding its staining mechanism. However, its continued usefulness in botany for demonstrating pectic substances attests to selectivity of some sort. Whether understood or not, histochemists continue to be grateful for small favors.Ruthenium red can also be used with the electron microscope. If single cells are exposed to ruthenium red solution, sufficient mass can be bound to produce observable density in the electron microscope. Generally, this effect is not useful with solid tissues because the contrast is wasted on the damaged cells at the block surface, with little dye diffusing more than 25-50 μ into the interior. Although these traces of ruthenium red which penetrate between and around cells are visible in the light microscope, they produce negligible contrast in the electron microscope. However, its presence can be amplified by a reaction with osmium tetroxide, probably catalytically, to be easily visible by EM. Now the density is clearly seen to be extracellular and closely associated with collagen fibers (Fig. 1).

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Fluorescent potentiometric dyes for membrane-potential imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100168566 ◽

1994 ◽

Vol 52 ◽

pp. 166-167

Author(s):

Leslie M. Loew

Keyword(s):

Membrane Potential ◽

Single Cells ◽

Quantitative Imaging ◽

Optical Recording ◽

Biological Processes ◽

Major Focus ◽

The Past ◽

Excitable Systems ◽

Major Application ◽

The Impact

A major application of potentiometric dyes has been the multisite optical recording of electrical activity in excitable systems. After being championed by L.B. Cohen and his colleagues for the past 20 years, the impact of this technology is rapidly being felt and is spreading to an increasing number of neuroscience laboratories. A second class of experiments involves using dyes to image membrane potential distributions in single cells by digital imaging microscopy - a major focus of this lab. These studies usually do not require the temporal resolution of multisite optical recording, being primarily focussed on slow cell biological processes, and therefore can achieve much higher spatial resolution. We have developed 2 methods for quantitative imaging of membrane potential. One method uses dual wavelength imaging of membrane-staining dyes and the other uses quantitative 3D imaging of a fluorescent lipophilic cation; the dyes used in each case were synthesized for this purpose in this laboratory.

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