scholarly journals C-tag TNF: a reporter system to study TNF shedding

2020 ◽  
Vol 295 (52) ◽  
pp. 18065-18075
Author(s):  
Francesca Pinci ◽  
Moritz M. Gaidt ◽  
Christophe Jung ◽  
Gunnar Kuut ◽  
Margaret A. Jackson ◽  
...  
Keyword(s):  
Single Cell ◽  
Inflammatory Diseases ◽  
Transmembrane Protein ◽  
Reporter System ◽  
Cell Level ◽  
C Terminus ◽  
Highly Sensitive ◽  
Cryptic Epitope ◽  
Complex Regulation ◽  
Exact Timing

TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow the live visualization of shedding events. Here, we generated C-tag TNF as a genetically encoded reporter to study TNF shedding at the single-cell level. The functionality of the C-tag TNF reporter is based on the exposure of a cryptic epitope on the C terminus of the transmembrane portion of pro-TNF on cleavage. In both denatured and nondenatured samples, this epitope can be detected by a nanobody in a highly sensitive and specific manner only upon TNF shedding. As such, C-tag TNF can successfully be used for the detection of TNF cleavage in flow cytometry and live-cell imaging applications. We furthermore demonstrate its applicability in a forward genetic screen geared toward the identification of genetic regulators of TNF maturation. In summary, the C-tag TNF reporter can be employed to gain novel insights into the complex regulation of ADAM-dependent TNF shedding.

Abstract 257: Clonal Analysis Of Multipotent Cardiovascular Progenitors That Generate Cardiomyocytes During Development

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Konstantina Ioanna Sereti ◽  
Paniz Kamran Rashani ◽  
Peng Zhao ◽  
Reza Ardehali
Keyword(s):  
Single Cell ◽  
Heart Development ◽  
Cardiac Development ◽  
Clonal Analysis ◽  
Cardiac Cells ◽  
Single Cell Level ◽  
Reporter System ◽  
Cell Level ◽  
Cardiac Progenitors

It has been proposed that cardiac development in lower vertebrates is driven by the proliferation of cardiomyocytes. Similarly, cycling myocytes have been suggested to direct cardiac regeneration in neonatal mice after injury. Although, the role of cardiomyocyte proliferation in cardiac tissue generation during development has been well documented, the extent of this contribution as well as the role of other cell types, such as progenitor cells, still remains controversial. Here we used a novel stochastic four-color Cre-dependent reporter system (Rainbow) that allows labeling at a single cell level and retrospective analysis of the progeny. Cardiac progenitors expressing Mesp1 or Nkx2.5 were shown to be a source of cardiomyocytes during embryonic development while the onset of αMHC expression marked the developmental stage where the capacity of cardiac cells to proliferate diminishes significantly. Through direct clonal analysis we provide strong evidence supporting that cardiac progenitors, as opposed to mature cardiomyocytes, are the main source of cardiomyocytes during cardiac development. Moreover, we have identified quadri-, tri-, bi, and uni-potent progenitors that at a single cell level can generate cardiomyocytes, fibroblasts, endothelial and smooth muscle cells. Although existing cardiomyocytes undergo limited proliferation, our data indicates that it is mainly the progenitors that contribute to heart development. Furthermore, we show that the limited proliferation capacity of cardiomyocytes observed during normal development was enhanced following neonatal cardiac injury allowing almost complete regeneration of the scared tissue. However, this ability was largely absent in adult injured hearts. Detailed characterization of dividing cardiomyocytes and proliferating progenitors would greatly benefit the development of novel therapeutic options for cardiovascular diseases.

scholarly journals NADH Fluorescence Lifetime Imaging Microscopy Reveals Selective Mitochondrial Dysfunction in Neurons Overexpressing Alzheimer’s Disease–Related Proteins

2021 ◽  
Vol 8 ◽  
Author(s):  
Moritz A. Niederschweiberer ◽  
Patrick M. Schaefer ◽  
Larry N. Singh ◽  
Ludwig Lausser ◽  
Devyani Bhosale ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Single Cell ◽  
Mitochondrial Function ◽  
Primary Neurons ◽  
Single Cell Level ◽  
Cell Level ◽  
Lifetime Imaging ◽  
Highly Sensitive ◽  
Related Proteins ◽  
Subcellular Level

Alzheimer’s disease (AD), the most prevalent form of dementia, affects globally more than 30 million people suffering from cognitive deficits and neuropsychiatric symptoms. Substantial evidence for the involvement of mitochondrial dysfunction in the development and/or progression of AD has been shown in addition to the pathological hallmarks amyloid beta (Aβ) and tau. Still, the selective vulnerability and associated selective mitochondrial dysfunction cannot even be resolved to date. We aimed at optically quantifying mitochondrial function on a single-cell level in primary hippocampal neuron models of AD, unraveling differential involvement of cell and mitochondrial populations in amyloid precursor protein (APP)-associated mitochondrial dysfunction. NADH lifetime imaging is a highly sensitive marker-free method with high spatial resolution. However, deciphering cellular bioenergetics of complex cells like primary neurons has still not succeeded yet. To achieve this, we combined highly sensitive NADH lifetime imaging with respiratory inhibitor treatment, allowing characterization of mitochondrial function down to even the subcellular level in primary neurons. Measuring NADH lifetime of the same neuron before and after respiratory treatment reveals the metabolic delta, which can be taken as a surrogate for cellular redox capacity. Correlating NADH lifetime delta with overexpression strength of Aβ-related proteins on the single-cell level, we could verify the important role of intracellular Aβ-mediated mitochondrial toxicity. Subcellularly, we could demonstrate a higher respiration in neuronal somata in general than dendrites, but a similar impairment of somatic and dendritic mitochondria in our AD models. This illustrates the power of NADH lifetime imaging in revealing mitochondrial function on a single and even subcellular level and its potential to shed light into bioenergetic alterations in neuropsychiatric diseases and beyond.

scholarly journals Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Nicolas Lemus-Diaz ◽  
Kai O. Böker ◽  
Ignacio Rodriguez-Polo ◽  
Michael Mitter ◽  
Jasmin Preis ◽  
...  
Keyword(s):  
Single Cell ◽  
Mirna Gene ◽  
Gene Repression ◽  
Single Cell Level ◽  
Reporter System ◽  
Cell Level ◽  
Fluorescent Reporter

High resolution FTIR imaging provides automated discrimination and detection of single malaria parasite infected erythrocytes on glass

2016 ◽  
Vol 187 ◽  
pp. 341-352 ◽  
Author(s):  
David Perez-Guaita ◽  
Dean Andrew ◽  
Philip Heraud ◽  
James Beeson ◽  
David Anderson ◽  
...  
Keyword(s):  
Single Cell ◽  
Numerical Aperture ◽  
Single Cell Level ◽  
Cell Level ◽  
High Quality ◽  
Pixel Resolution ◽  
Ftir Microscopy ◽  
Synchrotron Source ◽  
Average Spectrum ◽  
Highly Sensitive

New highly sensitive tools for malaria diagnostics are urgently needed to enable the detection of infection in asymptomatic carriers and patients with low parasitemia. In pursuit of a highly sensitive diagnostic tool that can identify parasite infections at the single cell level, we have been exploring Fourier transform infrared (FTIR) microscopy using a Focal Plane Array (FPA) imaging detector. Here we report for the first time the application of a new optic configuration developed by Agilent that incorporates 25× condenser and objective Cassegrain optics with a high numerical aperture (NA = 0.81) along with additional high magnification optics within the microscope to provide 0.66 micron pixel resolution (total IR system magnification of 61×) to diagnose malaria parasites at the single cell level on a conventional glass microscope slide. The high quality images clearly resolve the parasite's digestive vacuole demonstrating sub-cellular resolution using this approach. Moreover, we have developed an algorithm that first detects the cells in the infrared image, and secondly extracts the average spectrum. The average spectrum is then run through a model based on Partial Least Squares-Discriminant Analysis (PLS-DA), which diagnoses unequivocally the infected from normal cells. The high quality images, and the fact this measurement can be achieved without a synchrotron source on a conventional glass slide, shows promise as a potential gold standard for malaria detection at the single cell level.

scholarly journals A new triple fluorescence reporter system for discrimination of Apobec1 and Apobec3 C-to-U RNA editing activities and editing-dependent protein expression

2021 ◽  
Author(s):  
Barbara Schweissthal ◽  
Kea Brunken ◽  
Julia Brach ◽  
Leonie Emde ◽  
Florian Hetsch ◽  
...  
Keyword(s):  
Protein Expression ◽  
Single Cell ◽  
Rna Editing ◽  
Bulk Material ◽  
Splice Donor Site ◽  
Single Cell Level ◽  
Reporter System ◽  
Cell Level ◽  
Application Systems ◽  
New System

AbstractThe human body is composed of many different cell types which communicate with each other. In particular, the brain consists of billions of neurons and non-neuronal cells which are interconnected and require tight and precise regulation of cellular processes. RNA editing is a cellular process that diversifies gene function by enzymatic deamination of cytidine or adenine. This can result in changes of protein structure and function. Altered RNA editing is becoming increasingly associated with all kind of disease, but most approaches use advanced sequencing technologies to analyze bulk material. However, it is also becoming progressively evident that changes in RNA editing have to be analyzed and considered in a cell type specific way. We present here a triple fluorescence reporter system that discriminates between Apobec1- and Apobec3-dependent C-to-U RNA editing at the single cell level. In particular, the Apobec3 reporter enables C-to-U RNA editing inducible protein expression through generation of a RNA splice donor site. We used the new system here to analyze Apobec1- and Apobec3-dependent RNA editing in primary neuron culture. The results reveal a large heterogeneity of C-to-U RNA editing in neurons and glia cells, and they show that GABAergic neurons are not able to perform Apobec1-dependent RNA editing, but Apobec3-dependent editing. Altogether, the new system can be the foundation of therapeutic application systems that counteract changes in Apobec3-dependent RNA editing in disease while simultaneously monitoring Apobec1-dependent RNA editing at the single cell level.

scholarly journals High Sensitivity In Vivo Imaging of Cancer Metastasis Using a Near-Infrared Luciferin Analogue seMpai

2020 ◽  
Vol 21 (21) ◽  
pp. 7896
Author(s):  
Jun Nakayama ◽  
Ryohei Saito ◽  
Yusuke Hayashi ◽  
Nobuo Kitada ◽  
Shota Tamaki ◽  
...  
Keyword(s):  
Single Cell ◽  
In Vivo Imaging ◽  
Cancer Metastasis ◽  
Near Infrared ◽  
High Sensitivity ◽  
Single Cell Level ◽  
Background Signal ◽  
Cell Level ◽  
Highly Sensitive

Bioluminescence imaging (BLI) is useful to monitor cell movement and gene expression in live animals. However, D-luciferin has a short wavelength (560 nm) which is absorbed by tissues and the use of near-infrared (NIR) luciferin analogues enable high sensitivity in vivo BLI. The AkaLumine-AkaLuc BLI system (Aka-BLI) can detect resolution at the single-cell level; however, it has a clear hepatic background signal. Here, to enable the highly sensitive detection of bioluminescence from the surrounding liver tissues, we focused on seMpai (C15H16N3O2S) which has been synthesized as a luciferin analogue and has high luminescent abilities as same as AkaLumine. We demonstrated that seMpai BLI could detect micro-signals near the liver without any background signal. The solution of seMpai was neutral; therefore, seMpai imaging did not cause any adverse effect in mice. seMpai enabled a highly sensitive in vivo BLI as compared to previous techniques. Our findings suggest that the development of a novel mutated luciferase against seMpai may enable a highly sensitive BLI at the single-cell level without any background signal. Novel seMpai BLI system can be used for in vivo imaging in the fields of life sciences and medicine.

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

Lab on a Chip ◽  
2012 ◽  
Vol 12 (20) ◽  
pp. 3907 ◽  
Author(s):  
Zhi Zhu ◽  
Wenhua Zhang ◽  
Xuefei Leng ◽  
Mingxia Zhang ◽  
Zhichao Guan ◽  
...  
Keyword(s):  
Single Cell ◽  
Quantitative Detection ◽  
Single Cell Level ◽  
Cell Level ◽  
Emulsion Pcr ◽  
Highly Sensitive

scholarly journals A fluorescent protein-readout for transcriptional activity reveals regulation of APP nuclear signaling by phosphorylation sites

2019 ◽  
Vol 400 (9) ◽  
pp. 1191-1203 ◽  
Author(s):  
Uwe Konietzko ◽  
Manuel T. Gersbacher ◽  
Jeremy Streuli ◽  
Maik Krüger ◽  
Sarina Thöni ◽  
...  
Keyword(s):  
Single Cell ◽  
Transcriptional Activity ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Screening Methods ◽  
Single Cell Level ◽  
Cell Level ◽  
Transcription System ◽  
Nuclear Signaling ◽  
Highly Sensitive

Abstract Signaling pathways that originate at the plasma membrane, including regulated intramembrane proteolysis (RIP), enable extracellular cues to control transcription. We modified the yeast Gal4 transcription system to study the nuclear translocation of transcriptionally active complexes using the fluorescent protein citrine (Cit) as a reporter. This enabled highly sensitive quantitative analysis of transcription in situ at the single cell level. The Gal4/UAS-Cit transcription assay displayed a sigmoidal response limited by the number of integrated reporter cassettes. We validated the assay by analyzing nuclear translocation of the amyloid precursor protein (APP) intracellular domain (AICD) and confirmed the requirement of Fe65 for nuclear translocation of AICD. In addition to the strong on-off effects on transcriptional activity, the results of this assay establish that phosphorylation modifies nuclear signaling. The Y682F mutation in APP showed the strongest increase in Cit expression, underscoring its role in regulating Fe65 binding. Together, we established a highly sensitive fluorescent protein-based assay that can monitor transcriptional activity at the single cell level and demonstrate that AICD phosphorylation affects Fe65 nuclear activity. This assay also introduces a platform for future single cell-based drug screening methods for nuclear translocation.

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