scholarly journals Complexation design of cationized gelatin and molecular beacon to visualize intracellular mRNA

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245899
Author(s):  
Sho Takehana ◽  
Yuki Murata ◽  
Jun-ichiro Jo ◽  
Yasuhiko Tabata
Keyword(s):  
Messenger Rna ◽  
Molecular Beacon ◽  
Fluorescent Protein ◽  
Apparent Size ◽  
Sequence Specificity ◽  
Fluorescent Intensity ◽  
Mixing Ratios ◽  
Preparation Conditions ◽  
Gapdh Mrna ◽  
Green Fluorescent

The objective of this study is to prepare cationized gelatin-molecular beacon (MB) complexes for the visualization of intracellular messenger RNA (mRNA). The complexes were prepared from cationized gelatins with different extents of cationization and different mixing ratios of MB to cationized gelatin. The apparent size of complexes was almost similar, while the zeta potential was different among the complexes. Irrespective of the preparation conditions, the complexes had a sequence specificity against the target oligonucleotides in hybridization. The cytotoxicity and the amount of complexes internalized into cells increased with an increase in the cationization extent and the concentration of cationized gelatin. After the incubation with complexes prepared from cationized gelatin with the highest extent of cationization and at mixing ratios of 10 and 20 pmole MB/μg cationized gelatin, a high fluorescent intensity was detected. On the other hand, the complex prepared with the mixing ratio at 20 pmole/μg did not show any cytotoxicity. The complex was the most effective to visualize the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA endogenously present. In addition, even for enhanced green fluorescent protein (EGFP) mRNA exogenously transfected, the complex permitted to effectively detect it as well. It is concluded that both the endogenous and exogenous mRNA can be visualized in living cells by use of cationized gelatin-MB complexes designed.

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 Elucidating the roles of Alzheimer disease-associated proteases and the signal-peptide peptidase-like 3 (SPPL3) in the shedding of glycosyltransferases

2018 ◽  
Author(s):  
Assou El-Battari ◽  
Sylvie Mathieu ◽  
Romain Sigaud ◽  
Maëlle Prorok-Hamon ◽  
L’Houcine Ouafik ◽  
...  
Keyword(s):  
Alzheimer Disease ◽  
Signal Peptide ◽  
Fluorescent Protein ◽  
Biological Fluids ◽  
Sequence Specificity ◽  
Embryonic Fibroblasts ◽  
Secretase Inhibitor ◽  
Membrane Bound ◽  
Green Fluorescent ◽  
St6gal I

ABSTRACTThe Golgi resident glycosyltransferases (GTs) are membrane-bound glycoproteins but are frequently found as soluble proteins in biological fluids where their function remains largely unknown. Previous studies have established that the release of these proteins involved Alzheimer disease-associated proteases such as β-secretases (BACE1 and BACE2) and the intramembrane-cleaving aspartyl proteases Presenilins 1 and 2. Recent studies have involved another intramembrane-cleaving enzyme, the signal peptide peptidese-like-3 (SPPL3). Except for the latter, the two former studies mostly addressed particular cases of GTs, namely ST6Gal-I (BACEs) or GnT-V (Presenilins). Therefore the question still remains as which of these secretases is truly responsible for the cleavage and secretion of GTs. We herein combined the 3 proteases in a single study with respect to their abilities to release 3 families of GTs encompassing three N-acetylglucosaminyltransferases, two fucosyltransferases and two sialyltransferases. Green fluorescent protein (gfp)-fused versions of these GTs were virally transduced in mouse embryonic fibroblasts devoid of BACEs, Presenilins or SPPL3. We found that neither BACE nor Presenilins are involved in the shedding of these glycosyltransferases, while SPPL3 was involved in the cleavage and release of some but not all GTs. Notably, the γ- secretase inhibitor DFK-167 was the only molecule capable of significantly decreasing glycosyltransferase secretion, suggesting the involvement of γ-secretase(s), yet different from Presenilins but comprising SPPL3 among other proteases still to be identified. Using confocal microscopy, we show that SPPL3 selectivity towards GTs relays not only on sequence specificity but also depends on how GTs distribute in the cell with respect SPPL3 during their cycling within and outside the Golgi.

scholarly journals SAMD9L autoinflammatory or ataxia pancytopenia disease mutations activate cell-autonomous translational repression

2021 ◽  
Vol 118 (34) ◽  
pp. e2110190118
Author(s):  
Amanda J. Russell ◽  
Paul E. Gray ◽  
John B. Ziegler ◽  
Yae Jean Kim ◽  
Sandy Smith ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Autoinflammatory Disease ◽  
Fluorescent Protein ◽  
Single Cell Analysis ◽  
De Novo ◽  
Protein Translation ◽  
Translational Repression ◽  
Translational Repressor ◽  
Activate Cell ◽  
Green Fluorescent

Sterile α motif domain-containing protein 9-like (SAMD9L) is encoded by a hallmark interferon-induced gene with a role in controlling virus replication that is not well understood. Here, we analyze SAMD9L function from the perspective of human mutations causing neonatal-onset severe autoinflammatory disease. Whole-genome sequencing of two children with leukocytoclastic panniculitis, basal ganglia calcifications, raised blood inflammatory markers, neutrophilia, anemia, thrombocytopaenia, and almost no B cells revealed heterozygous de novo SAMD9L mutations, p.Asn885Thrfs*6 and p.Lys878Serfs*13. These frameshift mutations truncate the SAMD9L protein within a domain a region of homology to the nucleotide-binding and oligomerization domain (NOD) of APAF1, ∼80 amino acids C-terminal to the Walker B motif. Single-cell analysis of human cells expressing green fluorescent protein (GFP)-SAMD9L fusion proteins revealed that enforced expression of wild-type SAMD9L repressed translation of red fluorescent protein messenger RNA and globally repressed endogenous protein translation, cell autonomously and in proportion to the level of GFP-SAMD9L in each cell. The children’s truncating mutations dramatically exaggerated translational repression even at low levels of GFP-SAMD9L per cell, as did a missense Arg986Cys mutation reported recurrently as causing ataxia pancytopenia syndrome. Autoinflammatory disease associated with SAMD9L truncating mutations appears to result from an interferon-induced translational repressor whose activity goes unchecked by the loss of C-terminal domains that may normally sense virus infection.

Development of a Novel In Vivo Assay for Assessment of Tissue Viability: A Cryosurgery Application

2000 ◽  
Author(s):  
G. D. Elliott ◽  
D. Fron ◽  
J. J. McGrath ◽  
C. Seip ◽  
E. Crockett-Torabi
Keyword(s):  
Fluorescent Protein ◽  
Skin Flap ◽  
Isolated Cells ◽  
Fluorescent Intensity ◽  
Recent Success ◽  
Viability Assay ◽  
Thaw Cycle ◽  
Green Fluorescent ◽  
Observation Period

Abstract Our recent success with applying a novel Green Fluorescent Protein (GFP) viability assay to isolated cells after freezing has suggested possible utility for assessing viability of cells within tissues, both in situ, and intravitally. The current work seeks to establish that following freezing, changes in the fluorescent intensity of tumors grown from GFP-transfected cells will correlate with tissue damage as assessed by histological methods, thereby evaluating the use of a transfection-based assay for in vivo purposes. GFP-fluorescing R3230AC tumors were grown inside implanted dorsal skin flap chambers, and treated cryosurgically after 6-9 days of growth. Fluorescent images of the tumors were acquired at the center of the viewing area before, during, and for 6 hours following freezing. Following the observation period the animal was immediately euthanized and the tissue within the chamber was fixed in formalin and stained for histological analysis. The results of intravital microscopy in a centrally located test area revealed a large reduction in fluorescent intensity within the tumor tissue immediately following the final freeze-thaw cycle, with little subsequent change during the subsequent 6 hour observation period. In H&E section significant damage was observed in the central test areas. Taken together, this evidence suggests that the GFP assay is not limited to isolated cells, but can serve as the basis for intravital assessment of viability.

scholarly journals Effective Biophysical Modeling of Cell Free Transcription and Translation Processes

2020 ◽  
Vol 8 ◽  
Author(s):  
Abhinav Adhikari ◽  
Michael Vilkhovoy ◽  
Sandra Vadhin ◽  
Ha Eun Lim ◽  
Jeffrey D. Varner
Keyword(s):  
Green Fluorescent Protein ◽  
Messenger Rna ◽  
Fluorescent Protein ◽  
Regulatory Control ◽  
Parameter Estimates ◽  
Model Parameters ◽  
Biophysical Modeling ◽  
Modeling Approach ◽  
Synthetic Cell ◽  
Green Fluorescent

Transcription and translation are at the heart of metabolism and signal transduction. In this study, we developed an effective biophysical modeling approach to simulate transcription and translation processes. The model, composed of coupled ordinary differential equations, was tested by comparing simulations of two cell free synthetic circuits with experimental measurements generated in this study. First, we considered a simple circuit in which sigma factor 70 induced the expression of green fluorescent protein. This relatively simple case was then followed by a more complex negative feedback circuit in which two control genes were coupled to the expression of a third reporter gene, green fluorescent protein. Many of the model parameters were estimated from previous biophysical studies in the literature, while the remaining unknown model parameters for each circuit were estimated by minimizing the difference between model simulations and messenger RNA (mRNA) and protein measurements generated in this study. In particular, either parameter estimates from published studies were used directly, or characteristic values found in the literature were used to establish feasible ranges for the parameter estimation problem. In order to perform a detailed analysis of the influence of individual model parameters on the expression dynamics of each circuit, global sensitivity analysis was used. Taken together, the effective biophysical modeling approach captured the expression dynamics, including the transcription dynamics, for the two synthetic cell free circuits. While, we considered only two circuits here, this approach could potentially be extended to simulate other genetic circuits in both cell free and whole cell biomolecular applications as the equations governing the regulatory control functions are modular and easily modifiable. The model code, parameters, and analysis scripts are available for download under an MIT software license from the Varnerlab GitHub repository.

scholarly journals Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Fatemeh Sadat Shariati ◽  
Dariush Norouzian ◽  
Vahideh Valizadeh ◽  
Reza Ahangari Cohan ◽  
Malihe Keramati
Keyword(s):  
Fluorescent Protein ◽  
Limit Of Detection ◽  
Pearson Correlation ◽  
Expression System ◽  
Rapid Screening ◽  
Limit Of Quantification ◽  
Fluorescent Intensity ◽  
E Coli ◽  
Protein Model ◽  
Green Fluorescent

Abstract Background Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). Results Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze–thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. Conclusion The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales.

scholarly journals Reassembling green fluorescent protein for in vitro evaluation of trans-translation

2020 ◽  
Vol 48 (4) ◽  
pp. e22-e22
Author(s):  
Charlotte Guyomar ◽  
Marion Thépaut ◽  
Sylvie Nonin-Lecomte ◽  
Agnès Méreau ◽  
Renan Goude ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
Messenger Rna ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Chemical Compounds ◽  
Reporter System ◽  
New Antibiotics ◽  
Green Fluorescent

Abstract In order to discover new antibiotics with improved activity and selectivity, we created a reliable in vitro reporter system to detect trans-translation activity, the main mechanism for recycling ribosomes stalled on problematic messenger RNA (mRNA) in bacteria. This system is based on an engineered tmRNA variant that reassembles the green fluorescent protein (GFP) when trans-translation is active. Our system is adapted for high-throughput screening of chemical compounds by fluorescence.

scholarly journals Effective Biophysical Modeling of Cell Free Transcription and Translation Processes

2020 ◽  
Author(s):  
Abhinav Adhikari ◽  
Michael Vilkhovoy ◽  
Sandra Vadhin ◽  
Ha Eun Lim ◽  
Jeffrey D. Varner
Keyword(s):  
Green Fluorescent Protein ◽  
Messenger Rna ◽  
Fluorescent Protein ◽  
Parameter Estimates ◽  
Model Parameters ◽  
Biophysical Modeling ◽  
Genetic Circuits ◽  
Modeling Approach ◽  
Synthetic Cell ◽  
Green Fluorescent

AbstractTranscription and translation are at the heart of metabolism and signal transduction. In this study, we developed an effective biophysical modeling approach to simulate transcription and translation processes. We tested this approach by simulating the dynamics of two cell free synthetic circuits. First, we considered a simple circuit in which sigma factor 70 induced the expression of green fluorescent protein. This relatively simple case was then followed by a more complex negative feedback circuit in which two control genes were coupled to the expression of a third reporter gene, green fluorescent protein. While many of the model parameters were estimated from previous biophysical literature, the remaining unknown model parameters for each circuit were estimated from messenger RNA (mRNA) and protein measurements using multi-objective optimization. In particular, either the literature parameter estimates were used directly in the model simulations, or characteristic literature values were used to establish feasible ranges for the multiobjective parameter search. Next, global sensitivity analysis was used to determine the influence of individual model parameters on the expression dynamics. Taken together, the effective biophysical modeling approach captured the expression dynamics, including the transcription dynamics, for the two synthetic cell free circuits. While we considered only two circuits here, this approach could potentially be extended to simulate other genetic circuits in both cell free and whole cell biomolecular applications. The model code, parameters, and analysis scripts are available for download under an MIT software license from the Varnerlab GitHub repository.

scholarly journals Use of a Green Fluorescent Protein-Based Reporter Fusion for Detection of Nitric Oxide Produced by Denitrifiers

2003 ◽  
Vol 69 (7) ◽  
pp. 3938-3944 ◽  
Author(s):  
Shixue Yin ◽  
Mayuree Fuangthong ◽  
William P. Laratta ◽  
James P. Shapleigh
Keyword(s):  
Nitric Oxide ◽  
Green Fluorescent Protein ◽  
Fluorescence Intensity ◽  
Nitrite Reductase ◽  
Fluorescent Protein ◽  
Reductase Activity ◽  
Rrna Gene ◽  
Fluorescent Intensity ◽  
Fusion Analysis ◽  
Green Fluorescent

ABSTRACT To determine if green fluorescent protein could be used as a reporter for detecting nitric oxide production, gfp was fused to nnrS from Rhodobacter sphaeroides 2.4.3. nnrS was chosen because its expression requires nitric oxide. The presence of the fusion in R. sphaeroides 2.4.3 resulted in a significant increase in fluorescent intensity of the cells, but only when nitrite reductase was active. Cells lacking nitrite reductase activity and consequently the ability to generate nitric oxide were only weakly fluorescent when grown under denitrification-inducing conditions. One of the R. sphaeroides strains unable to generate nitric oxide endogenously was used as a reporter to detect exogenously produced nitric oxide. Incubation of this strain with sodium nitroprusside, a nitric oxide generator, significantly increased its fluorescence intensity. Mixing of known denitrifiers with the reporter strain also led to significant increases in fluorescence intensity, although the level varied depending on the denitrifier used. The reporter was tested on unknown isolates capable of growing anaerobically in the presence of nitrate, and one of these was able to induce expression of the fusion. Analysis of the 16S rRNA gene sequence of this isolate placed it within the Thauera aromatica subgroup, which is known to contain denitrifiers. These experiments demonstrate that this green fluorescent protein-based assay provides a useful method for assessing the ability of bacteria to produce nitric oxide.

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