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

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 Watching antibiotics in action: Exploiting time-lapse microfluidic microscopy as a tool for target-drug interaction studies inMycobacterium

2018 ◽  
Author(s):  
Damian Trojanowski ◽  
Marta Kołodziej ◽  
Joanna Hołówka ◽  
Rolf Müller ◽  
Jolanta Zakrzewska-Czerwińska
Keyword(s):  
Single Cell ◽  
Nalidixic Acid ◽  
Bacterial Resistance ◽  
Bacterial Species ◽  
Single Cell Level ◽  
Cell Level ◽  
Fluorescent Reporter ◽  
Resistance To Antibiotics ◽  
Animal Pathogens ◽  
Multiple Drugs

AbstractSpreading resistance to antibiotics and the emergence of multidrug-resistant strains have become frequent in many bacterial species, including mycobacteria. The genusMycobacteriumencompasses both human and animal pathogens that cause severe diseases and have profound impacts on global health and the world economy. Here, we used a novel system of microfluidics, fluorescence microscopy and target-tagged fluorescent reporter strains ofM.smegmatisto perform real-time monitoring of replisome and chromosome dynamics following the addition of replication-altering drugs (novobiocin, nalidixic acid and griselimycin) at the single-cell level. We found that novobiocin stalled replication forks and caused relaxation of the nucleoid, nalidixic acid triggered rapid replisome collapse and compaction of the nucleoid, and griselimycin caused replisome instability with subsequent over-initiation of chromosome replication and over-relaxation of the nucleoid. This work is an example of using a microscopy-based approach to evaluate the activity of potential replication inhibitors and provides mechanistic insights into their modes of action. Our system also enabled us to observe how the tested antibiotics affected the physiology of mycobacterial cells (i.e., growth, chromosome segregation, etc.). Because proteins involved in the DNA replication are well conserved among bacteria (including mycobacterial species), the properties of various replication inhibitors observed here in fast-growingM. smegmatismay be easily extrapolated to slow-growing pathogenic tubercle bacilli, such asM. tuberculosis.SignificanceThe growing problem of bacterial resistance to antibiotics and the emergence of new strains that are resistant to multiple drugs raise the need to explore new antibiotics and re-evaluate the existing options. Here, we present a system that allows the action of antibiotics to be monitored at the single-cell level. Such studies are important in the light of bacterial heterogeneity, which may be enhanced in unfavorable conditions, such as under antibiotic treatment. Moreover, our studies provide mechanistic insights into the action modes of the tested compounds. As combined therapies have recently gained increased interest, it is also notable that our described system may help researchers identify the best combination of antimicrobials for use against infections caused by a variety of bacteria.

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.

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