scholarly journals Comparison between Effects of Retroactivity and Resource Competition upon Change in Downstream Reporter Genes of Synthetic Genetic Circuits

Life ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 30 ◽  
Author(s):  
Takefumi Moriya ◽  
Tomohiro Yamaoka ◽  
Yuki Wakayama ◽  
Shotaro Ayukawa ◽  
Zicong Zhang ◽  
...  
Keyword(s):  
In Silico ◽  
Fluorescent Protein ◽  
Resource Competition ◽  
Regulatory Protein ◽  
Reporter Genes ◽  
Genetic Circuits ◽  
In Vivo Experiments ◽  
Reporter Promoter ◽  
The Stability

Reporter genes have contributed to advancements in molecular biology. Binding of an upstream regulatory protein to a downstream reporter promoter allows quantification of the activity of the upstream protein produced from the corresponding gene. In studies of synthetic biology, analyses of reporter gene activities ensure control of the cell with synthetic genetic circuits, as achieved using a combination of in silico and in vivo experiments. However, unexpected effects of downstream reporter genes on upstream regulatory genes may interfere with in vivo observations. This phenomenon is termed as retroactivity. Using in silico and in vivo experiments, we found that a different copy number of regulatory protein-binding sites in a downstream gene altered the upstream dynamics, suggesting retroactivity of reporters in this synthetic genetic oscillator. Furthermore, by separating the two sources of retroactivity (titration of the component and competition for degradation), we showed that, in the dual-feedback oscillator, the level of the fluorescent protein reporter competing for degradation with the circuits’ components is important for the stability of the oscillations. Altogether, our results indicate that the selection of reporter promoters using a combination of in silico and in vivo experiments is essential for the advanced design of genetic circuits.

In-Silico Analysis to Identify Potential Inhibitors Against the Protein NSP12 of SARS-CoV-2

2021 ◽  
Vol 6 (3) ◽  
pp. 48-60
Author(s):  
Hima Vyshnavi ◽  
Gayathri S. S. ◽  
Shahanas Naisam ◽  
Suvanish Kumar ◽  
Nidhin Sreekumar
Keyword(s):  
In Silico ◽  
Interaction Analysis ◽  
In Silico Analysis ◽  
Drug Efficacy ◽  
Drug Candidate ◽  
In Vivo Analysis ◽  
The Stability ◽  
Potential Inhibitors ◽  
Silico Analysis

In this pandemic condition, a drug candidate which is effective against COVID-19 is very much desired. This study initiates an in silico analysis to screen small molecules such as phytochemicals, drug metabolites, and natural metabolites against Nsp12 (a catalytic unit for RNA transcription and replication). Molecular interaction analysis of 6M71 was carried out against 2,860 ligands using Schrodinger Glide software. After docking analysis, the top 10 molecules (Glide score) were subjected to MD simulation for validating the stability. It resulted in top 10 compounds with high binding affinities with the target molecule NSP 12. Out of these, top 3 compounds including PSID_08_LIG3 (HMDB0133544), PSID_08_LIG4 (HMDB0132898), and PSID_08_LIG9 (HMDB0128199) show better Glide scores, better H-bond interaction, better MMGBSA value and stability on dynamic simulation after analysis of the results. The suggested ligands can be postulated as effective antiviral drugs against COVID-19. Further in vivo analysis is needed for validating the drug efficacy.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 200010
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Emir Bora Akmeriç ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

scholarly journals Tissue-specific expression of ferritin H regulates cellular iron homoeostasis in vivo

2006 ◽  
Vol 395 (3) ◽  
pp. 501-507 ◽  
Author(s):  
John Wilkinson ◽  
Xiumin Di ◽  
Kai Schönig ◽  
Joan L. Buss ◽  
Nancy D. Kock ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Regulatory Protein ◽  
Inducible Promoter ◽  
Protein Activity ◽  
Specific Expression ◽  
Cellular Iron ◽  
Tissue Iron ◽  
Green Fluorescent ◽  
Ferritin H

Ferritin is a ubiquitously distributed iron-binding protein. Cell culture studies have demonstrated that ferritin plays a role in maintenance of iron homoeostasis and in the protection against cytokine- and oxidant-induced stress. To test whether FerH (ferritin H) can regulate tissue iron homoeostasis in vivo, we prepared transgenic mice that conditionally express FerH and EGFP (enhanced green fluorescent protein) from a bicistronic tetracycline-inducible promoter. Two transgenic models were explored. In the first, the FerH and EGFP transgenes were controlled by the tTACMV (Tet-OFF) (where tTA and CMV are tet transactivator protein and cytomegalovirus respectively). In skeletal muscle of mice bearing the FerH/EGFP and tTACMV transgenes, FerH expression was increased 6.0±1.1-fold (mean±S.D.) compared with controls. In the second model, the FerH/EGFP transgenes were controlled by an optimized Tet-ON transactivator, rtTA2S-S2LAP (where rtTA is reverse tTA and LAP is liver activator protein), resulting in expression predominantly in the kidney and liver. In mice expressing these transgenes, doxycycline induced FerH in the kidney by 14.2±4.8-fold (mean±S.D.). Notably, increases in ferritin in overexpressers versus control littermates were accompanied by an elevation of IRP (iron regulatory protein) activity of 2.3±0.9-fold (mean±S.D.), concurrent with a 4.5±2.1-fold (mean±S.D.) increase in transferrin receptor, indicating that overexpression of FerH is sufficient to elicit a phenotype of iron depletion. These results demonstrate that FerH not only responds to changes in tissue iron (its classic role), but can actively regulate overall tissue iron balance.

In Silico Testing and in Vivo Experiments with Closed-Loop Control of Blood Glucose in Diabetes

2008 ◽  
Vol 41 (2) ◽  
pp. 4234-4239 ◽  
Author(s):  
B. Kovatchev ◽  
D.M. Raimondo ◽  
M. Breton ◽  
S. Patek ◽  
C. Cobelli
Keyword(s):  
Blood Glucose ◽  
In Silico ◽  
Closed Loop ◽  
Closed Loop Control ◽  
Loop Control ◽  
In Vivo Experiments

scholarly journals In-silico Exploration of Mouse Brain Dynamics by Stimulation explains Functional Networks and Sensory Processing

2019 ◽  
Author(s):  
Andreas Spiegler ◽  
Javad Karimi Abadchi ◽  
Majid Mohajerani ◽  
Viktor K. Jirsa
Keyword(s):  
Mouse Brain ◽  
In Silico ◽  
Dynamic Properties ◽  
Brain Activity ◽  
Parameter Tuning ◽  
Functional Networks ◽  
Direct Stimulation ◽  
Systematic Exploration ◽  
The Stability

ABSTRACTSensory and direct stimulation of the brain probes its functional repertoire and the information processing capacity of networks. However, a systematic exploration can only be performed in silico. Stimulation takes the system out of its attractor states and samples the environment of the flow to gain insight into the stability and multiplicity of trajectories. It is the only means of obtaining a complete understanding of the healthy brain network’s dynamic properties. We built a whole mouse brain model with connectivity derived from tracer studies. We systematically varied the stimulation location, the ratio of long- to short-range interactions, and the range of short connections. Functional networks appeared in the spatial motifs of simulated brain activity. Several motifs included the default mode network, suggesting a junction of functional networks. The model explains processing in sensory systems and replicates the in vivo dynamics after stimulation without parameter tuning, emphasizing the role of connectivity.

scholarly journals Light-Regulated Transcription of a Mitochondrial-Targeted K+ Channel

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2507
Author(s):  
Anja J. Engel ◽  
Laura-Marie Winterstein ◽  
Marina Kithil ◽  
Markus Langhans ◽  
Anna Moroni ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Regulatory Protein ◽  
K Channel ◽  
Physiological Changes ◽  
Channel Activity ◽  
Cryptochrome 2 ◽  
Green Fluorescent ◽  
Gfp Tag

The inner membranes of mitochondria contain several types of K+ channels, which modulate the membrane potential of the organelle and contribute in this way to cytoprotection and the regulation of cell death. To better study the causal relationship between K+ channel activity and physiological changes, we developed an optogenetic platform for a light-triggered modulation of K+ conductance in mitochondria. By using the light-sensitive interaction between cryptochrome 2 and the regulatory protein CIB1, we can trigger the transcription of a small and highly selective K+ channel, which is in mammalian cells targeted into the inner membrane of mitochondria. After exposing cells to very low intensities (≤0.16 mW/mm2) of blue light, the channel protein is detectable as an accumulation of its green fluorescent protein (GFP) tag in the mitochondria less than 1 h after stimulation. This system allows for an in vivo monitoring of crucial physiological parameters of mitochondria, showing that the presence of an active K+ channel causes a substantial depolarization compatible with the effect of an uncoupler. Elevated K+ conductance also results in a decrease in the Ca2+ concentration in the mitochondria but has no impact on apoptosis.

scholarly journals Short artificial hairpins sequester splicing signals and inhibit yeast pre-mRNA splicing.

1993 ◽  
Vol 13 (11) ◽  
pp. 6841-6848 ◽  
Author(s):  
V Goguel ◽  
Y Wang ◽  
M Rosbash
Keyword(s):  
Splice Site ◽  
Major Effect ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spliceosome Assembly ◽  
In Vivo Experiments ◽  
Small Nuclear Ribonucleoprotein ◽  
Sequence Elements ◽  
The Stability

To examine the stability of yeast (Saccharomyces cerevisiae) pre-mRNA structures, we inserted a series of small sequence elements that generated potential RNA hairpins at the 5' splice site and branch point regions. We analyzed spliceosome assembly and splicing in vitro as well as splicing and nuclear pre-mRNA retention in vivo. Surprisingly, the inhibition of in vivo splicing approximately paralleled that of in vitro splicing. Even a 6-nucleotide hairpin could be shown to inhibit splicing, and a 15-nucleotide hairpin gave rise to almost complete inhibition. The in vitro results indicate that hairpins that sequester the 5' splice site have a major effect on the early steps of spliceosome assembly, including U1 small nuclear ribonucleoprotein binding. The in vivo experiments lead to comparable conclusions as the sequestering hairpins apparently result in the transport of pre-mRNA to the cytoplasm. The observations are compared with previous data from both yeast and mammalian systems and suggest an important effect of pre-mRNA structure on in vivo splicing.

Potential Candidates against COVID-19 Targeting RNA-Dependent RNA Polymerase: A Comprehensive Review

2021 ◽  
Vol 22 ◽  
Author(s):  
Neetu Agrawal ◽  
Ahsas Goyal
Keyword(s):  
Rna Polymerase ◽  
In Silico ◽  
Host Cells ◽  
Rna Dependent Rna Polymerase ◽  
In Vivo Experiments ◽  
Contagious Nature ◽  
In Silico Studies ◽  
Viral Enzyme

: Due to the extremely contagious nature of SARS-COV-2, it presents a significant threat to humans worldwide. A plethora of studies are going on all over the world to discover the drug to fight SARS-COV-2. One of the most promising targets is RNA-dependent RNA polymerase (RdRp), responsible for viral RNA replication in host cells. Since RdRp is a viral enzyme with no host cell homologs, it allows the development of selective SARS-COV-2 RdRp inhibitors. A variety of studies used in silico approaches for virtual screening, molecular docking, and repurposing of already existing drugs and phytochemicals against SARS-COV-2 RdRp. This review focuses on collating compounds possessing the potential to inhibit SARS-COV-2 RdRp based on in silico studies to give medicinal chemists food for thought so that the existing drugs can be repurposed for the control and treatment of ongoing COVID-19 pandemic after performing in vitro and in vivo experiments.

Potential Papain-like Protease Inhibitors against COVID-19: A Comprehensive In Silico Based Review

2021 ◽  
Vol 25 ◽  
Author(s):  
Neetu Agrawal ◽  
Shilpi Pathak ◽  
Ahsas Goyal
Keyword(s):  
In Silico ◽  
Chemical Compounds ◽  
Potential Candidate ◽  
In Vivo Experiments ◽  
Drug Databases ◽  
In Silico Studies ◽  
Approved Drugs ◽  
Fda Approved Drugs

: The entire world has been in a battle against the COVID-19 pandemic since its first appearance in December 2019. Thus researchers are desperately working to find an effective and safe therapeutic agent for its treatment. The multifunctional coronavirus enzyme papain-like protease (PLpro) is a potential target for drug discovery to combat the ongoing pandemic responsible for cleavage of the polypeptide, deISGylation, and suppression of host immune response. The present review collates the in silico studies performed on various FDA-approved drugs, chemical compounds, and phytochemicals from various drug databases and represents the compounds possessing the potential to inhibit PLpro. Thus this review can provide quick access to a potential candidate to medicinal chemists to perform in vitro and in vivo experiments who are thriving to find the effective agents for the treatment of COVID-19.

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