scholarly journals FISH-TAMB, a fixation-free mRNA fluorescent labeling technique, to target transcriptionally active members in microbial community

2021 ◽  
Author(s):  
Rachel L Harris ◽  
Maggie C.Y. Lau Vetter ◽  
Esta van Heerdeen ◽  
Errol Cason ◽  
Jan-G Vermeulen ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Keystone Species ◽  
Cell Penetrating Peptides ◽  
Methanosarcina Barkeri ◽  
Fluorescent Labeling ◽  
Molecular Beacons ◽  
Target Cells ◽  
Widespread Application ◽  
Mrna Targets ◽  
Hybridization Time

Abstract Keystone species or ecological engineers are vital to the health of an ecosystem, however, often their low abundance or biomass present challenges for their discovery, identification, visualization and selection. We report the development of fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB), a fixation-free protocol that is applicable to archaea and bacteria. The FISH-TAMB method differs from existing FISH methods by the absence of fixatives or surfactants in buffers, and the fast hybridization time of as short as 15 minutes at target cells’ growth temperature. Polyarginine cell-penetrating peptides are employed to deliver molecular beacons (MBs) across prokaryotic cell walls and membranes, fluorescently labeling cells when MBs hybridize to target mRNA sequences. Here, the detailed protocol of the preparation and application of FISH-TAMB is presented. To demonstrate FISH-TAMB’s ability to label intracellular mRNA targets, differentiate transcriptional states, detect active and rare taxa, and keep cell viability, labeling experiments were performed that targeted the messenger RNA (mRNA) of methyl-coenzyme M reductase A ( mcr A) expressed in 1) Escherichia coli containing a plasmid with a partial mcr A gene of the methanogen Methanosarcina barkeri ( E. coli mcr A + ); 2) M. barkeri ; and 3) an anaerobic methanotrophic (ANME) enrichment from a deep continental borehole. Although FISH-TAMB was initially envisioned for mRNA of any functional gene of interest without a requirement of prior knowledge of 16S ribosomal RNA (rRNA)-based taxonomy, FISH-TAMB has the potential for multiplexing and going beyond mRNA, thus is a versatile addition to the molecular ecologist’s toolkit, with potentially widespread application in the field of environmental microbiology.

scholarly journals FISH-TAMB, a Fixation-Free mRNA Fluorescent Labeling Technique to Target Transcriptionally Active Members in Microbial Communities

2021 ◽  
Author(s):  
Rachel L. Harris ◽  
Maggie C. Y. Lau Vetter ◽  
Esta van Heerden ◽  
Errol Cason ◽  
Jan-G Vermeulen ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Keystone Species ◽  
Cell Penetrating Peptides ◽  
Methanosarcina Barkeri ◽  
Fluorescent Labeling ◽  
Molecular Beacons ◽  
Target Cells ◽  
Widespread Application ◽  
Mrna Targets ◽  
Hybridization Time

AbstractKeystone species or ecological engineers are vital to the health of an ecosystem; however, often, their low abundance or biomass present challenges for their discovery, identification, visualization and selection. We report the development of fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB), a fixation-free protocol that is applicable to archaea and bacteria. The FISH-TAMB method differs from existing FISH methods by the absence of fixatives or surfactants in buffers, the fast hybridization time of as short as 15 min at target cells’ growth temperature, and the omission of washing steps. Polyarginine cell-penetrating peptides are employed to deliver molecular beacons (MBs) across prokaryotic cell walls and membranes, fluorescently labeling cells when MBs hybridize to target mRNA sequences. Here, the detailed protocol of the preparation and application of FISH-TAMB is presented. To demonstrate FISH-TAMB’s ability to label intracellular mRNA targets, differentiate transcriptional states, detect active and rare taxa, and keep cell viability, labeling experiments were performed that targeted the messenger RNA (mRNA) of methyl-coenzyme M reductase A (mcrA) expressed in (1) Escherichia coli containing a plasmid with a partial mcrA gene of the methanogen Methanosarcina barkeri (E. coli mcrA+); (2) M. barkeri; and (3) an anaerobic methanotrophic (ANME) enrichment from a deep continental borehole. Although FISH-TAMB was initially envisioned for mRNA of any functional gene of interest without a requirement of prior knowledge of 16S ribosomal RNA (rRNA)-based taxonomy, FISH-TAMB has the potential for multiplexing and going beyond mRNA and thus is a versatile addition to the molecular ecologist’s toolkit, with potentially widespread application in the field of environmental microbiology.

scholarly journals Labeling of prokaryotic mRNA in live cells using fluorescentin situhybridization of transcript-annealing molecular beacons (FISH-TAMB)

2017 ◽  
Author(s):  
Rachel L. Harris ◽  
Maggie C. Y. Lau ◽  
Esta van Heerden ◽  
Errol Cason ◽  
Jan-G Vermeulen ◽  
...  
Keyword(s):  
Messenger Rna ◽  
High Throughput Sequencing ◽  
Single Cells ◽  
Cell Penetrating Peptides ◽  
Methanosarcina Barkeri ◽  
Molecular Beacons ◽  
Live Cells ◽  
Target Cells ◽  
Growth Curve Analysis ◽  
Rare Biosphere

ABSTRACTHigh-throughput sequencing and cellular imaging have expanded our knowledge of microbial diversity and expression of cellular activity. However, it remains challenging to characterize low-abundance, slow-growing microorganisms that play key roles in biogeochemical cycling. With the goal of isolating transcriptionally active cells of these microorganisms from environmental samples, we developed fluorescentin situhybridization of transcript-annealing molecular beacons (FISH-TAMB) to label living prokaryotic cells. FISH-TAMB utilizes polyarginine cell-penetrating peptides to deliver molecular beacons across cell walls and membranes. Target cells are fluorescently labeled via hybridization between molecular beacons and messenger RNA of targeted functional genes. FISH-TAMB’s target specificity and deliverance into both bacterial and archaeal cells were demonstrated by labeling intracellular methyl-coenzyme M reductase A (mcrA) transcripts expressed byEscherichia colimcrA+,Methanosarcina barkeri,and a methanogenic enrichment of deep continental fracture fluid. Growth curve analysis supported sustained cellular viability following FISH-TAMB treatment. Flow cytometry and confocal microscopy detected labeled single cells and single cells in aggregates with unlabeled cells. As FISH-TAMB is amenable to target any functional gene of interest, when coupled with cell sorting, imaging, and sequencing techniques, FISH-TAMB will enable characterization of key uncharacterized rare biosphere microorganisms and of the syntrophically activated metabolic pathways between physically associated microorganisms.

An Overview of nano delivery systems for targeting RNA interference-based therapy in ulcerative colitis

2021 ◽  
Vol 27 ◽  
Author(s):  
Iman Alfagih ◽  
Basmah Aldosari ◽  
Bushra AlQuadeib ◽  
Alanood Almurshedi ◽  
Murtaza Tambuwala
Keyword(s):  
Ulcerative Colitis ◽  
Rna Interference ◽  
Protein Expression ◽  
Messenger Rna ◽  
Delivery Systems ◽  
Target Cells ◽  
Intracellular Space ◽  
Therapeutic Benefits ◽  
Negative Effect

: Ulcerative colitis (UC) is one of the main subtypes of inflammatory bowel disease. UC has a negative effect on patients’ quality of life, and it is an important risk factor for the development of colitis-associated cancer. Patients with UC need to take medications for their entire life because no permanent cure is available. Therefore, approaches that target messenger RNA (mRNA) of proinflammatory cytokines or anti-inflammatory cytokines are needed to improve the safety of UC therapy and promote intestinal mucosa recovery. The major challenge facing RNA interference-based therapy is the delivery of RNA molecules to the intracellular space of target cells. Moreover, nonspecific and systemic protein expression inhibition can result in adverse effects and less therapeutic benefits. Thus, it is important to develop an efficient delivery strategy targeting the cytoplasm of target cells to avoid side effects caused by off-target protein expression inhibition. This review focuses on the most recent advances in the targeted nano delivery systems of siRNAs and mRNA that have shown in vivo efficacy.

Abstract 15113: A Novel Purification Method for Cardiomyocytes and Endothelial Cells Derived From Human Pluripotent Stem Cells Using Microrna-responsive Messenger Rna

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Kenji Miki ◽  
Seiya Takahashi ◽  
Shunsuke Funakoshi ◽  
Shinya Yamanaka ◽  
Hirohide Saito ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Messenger Rna ◽  
Pluripotent Stem Cell ◽  
Troponin T ◽  
Target Cells ◽  
Mouse Heart ◽  
Effective Manner ◽  
High Purification

MicroRNAs (miRNAs) are small non-coding RNA that controls gene expression through translational regulation or mRNA cleavage. Recent progress in synthetic biology demonstrated that synthetic “RNA switches” enable to regulate gene expression depending on the intracellular environment. Here we show that synthetic mRNA technologies that modulate reporter protein expression by sensing miRNA activities enable purification of target cells in a safe and effective manner. To select cardiac-specific miRNA candidates, we performed miRNA microarray analyses using human induced pluripotent stem cell (hiPSC) line integrated MYH6 promoter driven EGFP cassette. We found 14 candidates, and synthesized individually these 14 miRNA-responsive OFF switch mRNAs (miR-switches) containing the sequence of blue fluorescent protein (TagBFP). We individually transfected them with EGFP mRNA into differentiated cells including cardiomyocytes derived from human pluripotent stem cell (hPSCs). We found that several cells transfected with miR-1- or miR-208-switches diminished TagBFP intensity, and sorting of these cells yielded high populations of troponin T-positive cardiomyocytes reproducibly and significantly (e.g., up to >98% in five hPSC lines). In addition, we developed a non-sorting system for purification of cardiomyocytes using miR-switch that code apoptosis-inducible protein (up to >97%). These cardiomyocytes purified by miR-switches were successfully engrafted in mouse heart and did not form tumor even when injected into testes of SCID mice. We next examined whether the system can be applied to purify endothelial cells, and confirmed that several miR-switches enabled high purification of CD31+ cells from differentiated derivatives of hPSCs (up to >98%), which have the function of tube formation like HUVEC. Moreover, the use of multiple miRNA-responsive mRNAs enabled simultaneous and high purification of cardiomyocytes and endothelial cells from heterogeneous population derived from hPSCs induced by single differentiation method (up to >98%). Our results suggest that miR-switch technology can be used for a variety of purposes including purification in any type of cells.

Live Cell Detection of Specific Messenger RNA for Molecular Analysis of Plaque Formation

2007 ◽  
Author(s):  
Wonjong Rhee ◽  
Hanjoong Jo ◽  
Gang Bao
Keyword(s):  
Shear Flow ◽  
Molecular Analysis ◽  
Messenger Rna ◽  
Molecular Beacon ◽  
Mrna Level ◽  
Live Cell ◽  
Molecular Beacons ◽  
Cell Detection ◽  
Unstable Flow ◽  
Fluorescent Intensity

The ability to visualize mRNA in single living cells and monitor in real-time the changes of mRNA level and localization in response to shear flow can provide unprecedented opportunities for the molecular analysis of atherosclerosis. We carried out an extensive study of the design of molecular beacons to target BMP-4 mRNA, which plays important roles in proatherogenic development in response to unstable flow conditions. Specifically, we selected an optimal molecular beacon design, and found that the fluorescent intensity from targeting BMP-4 mRNA correlated well with the GFP signal after up-regulating BMP-4 and co-expressing GFP using adenovirus. The knock-down of BMP-4 mRNA using siRNA significantly reduced the beacon signal, further demonstrating detection specificity. We found that, due to target accessibility, molecular beacons designed with different target sequences gave very different signal levels, and establishing molecular beacon design rules has significant implications to live cell mRNA detections, especially to the studies of BMP-4 mRNA in endothelial cells under shear flow.

scholarly journals Cell-Penetrating Peptides as a Tool for the Cellular Uptake of a Genetically Modified Nitroreductase for use in Directed Enzyme Prodrug Therapy

2019 ◽  
Vol 10 (4) ◽  
pp. 45 ◽  
Author(s):  
Anderson ◽  
Hobbs ◽  
Gwenin ◽  
Ball ◽  
Bennie ◽  
...  
Keyword(s):  
Magnetic Nanoparticle ◽  
Genetically Modified ◽  
Cell Penetrating Peptides ◽  
Target Cells ◽  
Tumour Site ◽  
Enzyme Prodrug Therapy ◽  
Delivery Vector ◽  
Cell Penetrating ◽  
Subsequent Activation ◽  
Potential Use

Directed enzyme prodrug therapy (DEPT) involves the delivery of a prodrug-activating enzyme to a solid tumour site, followed by the subsequent activation of an administered prodrug. One of the most studied enzyme–prodrug combinations is the nitroreductase from Escherichia coli (NfnB) with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitro-benzamide]. One of the major issues faced by DEPT is the ability to successfully internalize the enzyme into the target cells. NfnB has previously been genetically modified to contain cysteine residues (NfnB-Cys) which bind to gold nanoparticles for a novel DEPT therapy called magnetic nanoparticle directed enzyme prodrug therapy (MNDEPT). One cellular internalisation method is the use of cell-penetrating peptides (CPPs), which aid cellular internalization of cargo. Here the cell-penetrating peptides: HR9 and Pep-1 were tested for their ability to conjugate with NfnB-Cys. The conjugates were further tested for their potential use in MNDEPT, as well as conjugating with the delivery vector intended for use in MNDEPT and tested for the vectors capability to penetrate into cells.

scholarly journals Tuning the Immunostimulation Properties of Cationic Lipid Nanocarriers for Nucleic Acid Delivery

2021 ◽  
Vol 12 ◽  
Author(s):  
Arindam K. Dey ◽  
Adrien Nougarède ◽  
Flora Clément ◽  
Carole Fournier ◽  
Evelyne Jouvin-Marche ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Messenger Rna ◽  
Cationic Lipid ◽  
Lipid Nanoparticles ◽  
Cationic Lipids ◽  
Fine Tuning ◽  
Target Cells ◽  
Substantial Impact ◽  
Lipid Nanocarriers ◽  
The Impact

Nonviral systems, such as lipid nanoparticles, have emerged as reliable methods to enable nucleic acid intracellular delivery. The use of cationic lipids in various formulations of lipid nanoparticles enables the formation of complexes with nucleic acid cargo and facilitates their uptake by target cells. However, due to their small size and highly charged nature, these nanocarrier systems can interact in vivo with antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. As this might prove to be a safety concern for developing therapies based on lipid nanocarriers, we sought to understand how they could affect the physiology of APCs. In the present study, we investigate the cellular and metabolic response of primary macrophages or DCs exposed to the neutral or cationic variant of the same lipid nanoparticle formulation. We demonstrate that macrophages are the cells affected most significantly and that the cationic nanocarrier has a substantial impact on their physiology, depending on the positive surface charge. Our study provides a first model explaining the impact of charged lipid materials on immune cells and demonstrates that the primary adverse effects observed can be prevented by fine-tuning the load of nucleic acid cargo. Finally, we bring rationale to calibrate the nucleic acid load of cationic lipid nanocarriers depending on whether immunostimulation is desirable with the intended therapeutic application, for instance, gene delivery or messenger RNA vaccines.

scholarly journals Tuning the immunostimulation properties of cationic lipid nanocarriers for nucleic acid delivery

2021 ◽  
Author(s):  
Arindam K Dey ◽  
Adrien Nougarede ◽  
Flora Clement ◽  
Carole Fournier ◽  
Evelyne Jouvin-Marche ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Messenger Rna ◽  
Cationic Lipid ◽  
Lipid Nanoparticles ◽  
Cationic Lipids ◽  
Fine Tuning ◽  
Target Cells ◽  
Substantial Impact ◽  
Lipid Nanocarriers ◽  
The Impact

Nonviral systems, such as lipid nanoparticles, have emerged as reliable methods to enable nucleic acid intracellular delivery. The use of cationic lipids in various formulations of lipid nanoparticles enables the formation of complexes with nucleic acid cargo and facilitates their uptake by target cells. However, due to their small size and highly charged nature, these nanocarrier systems can interact in vivo with antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. As this might prove to be a safety concern for developing therapies based on lipid nanocarriers, we sought to understand how they could affect the physiology of APCs. In the present study, we investigate the cellular and metabolic response of primary macrophages or DCs exposed to the neutral or cationic variant of the same lipid nanoparticle formulation. We demonstrate that macrophages are the cells affected most significantly and that the cationic nanocarrier has a substantial impact on their physiology, depending on the positive surface charge. Our study provides a first model explaining the impact of charged lipid materials on immune cells and demonstrates that the primary adverse effects observed can be prevented by fine-tuning the load of nucleic acid cargo. Finally, we bring rationale to calibrate the nucleic acid load of cationic lipid nanocarriers depending on whether immunostimulation is desirable with the intended therapeutic application, for instance, gene delivery or messenger RNA vaccines.

scholarly journals Association Between Side Effects and Blood microRNA Expression Levels and Their Targeted Pathways in Patients With Major Depressive Disorder Treated by a Selective Serotonin Reuptake Inhibitor, Escitalopram: A CAN-BIND-1 Report

2019 ◽  
Vol 23 (2) ◽  
pp. 88-95
Author(s):  
Antoine Yrondi ◽  
Laura M Fiori ◽  
Benicio N Frey ◽  
Raymond W Lam ◽  
Glenda M MacQueen ◽  
...  
Keyword(s):  
Major Depressive Disorder ◽  
Side Effects ◽  
Depressive Disorder ◽  
Messenger Rna ◽  
Antidepressant Drugs ◽  
Target Prediction ◽  
Major Depressive ◽  
Mrna Targets ◽  
Downstream Effects

Abstract Introduction Antidepressant drugs are effective therapies for major depressive disorder; however, they are frequently associated with side effects. Although there is some evidence for a relationship between genetic variation and side effects, little is known regarding the role of dynamic molecular factors as moderators of side effects. The aim of this study was to assess microRNA (miRNA) changes associated with side effects during escitalopram treatment and their downstream effects on target gene expression. Methods A total 160 patients with major depressive disorder from the CAN-BIND-1 cohort were included. Side effects were assessed with the Toronto Side Effect Scale after 2 weeks of treatment with escitalopram. We assessed the relationship between side effects and changes in peripheral expression of miRNAs between baseline and week 2. For miRNA whose expression changed, we used target prediction algorithms to identify putative messenger RNA (mRNA) targets and assessed their expression. Results Nausea was experienced by 42.5% of patients. We identified 45 miRNAs whose expression changed on initiation of escitalopram treatment, of which 10 displayed a negative association with intensity of nausea (miR15b-5p, miR17-5p, miR20a-5p, miR20b-5p, miR103a-3p, miR103b, miR106a-5p, miR182-5p, miR185-5p, and miR660-5p). Additionally, we found negative associations between 4 microRNAs (miR20a-5p, miR106a-5p, miR185-5p, miR660-5p) and mRNA targets. The expression of the miR185-5p target, CAMK2δ was significantly decreased [log 2 mean = −0.048 (0.233)] between weeks 0 and 2 (P = .01)]. Conclusions We identified an overexpression of miR185-5p during escitalopram treatment of major depressive disorder, which was negatively associated with intensity of nausea, and identified a potential mRNA target that may mediate this effect.

scholarly journals Improved Fluorescent In Situ Hybridization Method for Detection of Bacteria from Activated Sludge and River Water by Using DNA Molecular Beacons and Flow Cytometry

2007 ◽  
Vol 73 (6) ◽  
pp. 2020-2023 ◽  
Author(s):  
Jeremy Lenaerts ◽  
Hilary M. Lappin-Scott ◽  
Jonathan Porter
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Activated Sludge ◽  
River Water ◽  
Environmental Samples ◽  
Fluorescent In Situ Hybridization ◽  
Molecular Beacons ◽  
Target Cells ◽  
Linear Dna

ABSTRACT Fluorescent in situ hybridization (FISH) remains a key technique in microbial ecology. Molecular beacons (MBs) are self-reporting probes that have potential advantages over linear probes for FISH. MB-FISH strategies have been described using both DNA-based and peptide nucleic acid (PNA)-based approaches. Although recent reports have suggested that PNA MBs are superior, DNA MBs have some advantages, most notably cost. The data presented here demonstrate that DNA MBs are suitable for at least some FISH applications in complex samples, providing superior discriminatory power compared to that of corresponding linear DNA-FISH probes. The use of DNA MBs for flow cytometric detection of Pseudomonas putida resulted in approximately double the signal-to-noise ratio of standard linear DNA probes when using laboratory-grown cultures and yielded improved discrimination of target cells in spiked environmental samples, without a need for separate washing steps. DNA MBs were also effective for the detection and cell sorting of both spiked and indigenous P. putida from activated sludge and river water samples. The use of DNA MB-FISH presents another increase in sensitivity, allowing the detection of bacteria in environmental samples without the expense of PNA MBs or multilaser flow cytometry.

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