scholarly journals Mechanistic Models of Inducible Synthetic Circuits for Joint Description of DNA Copy Number, Regulatory Protein Level, and Cell Load

Processes ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 119 ◽  
Author(s):  
Lorenzo Pasotti ◽  
Massimo Bellato ◽  
Davide De Marchi ◽  
Paolo Magni
Keyword(s):  
Protein Level ◽  
Copy Number ◽  
A Priori ◽  
Regulatory Protein ◽  
Inducible Promoter ◽  
Bottom Up ◽  
Protein Complex Formation ◽  
State Models ◽  
Preliminary Experimental Data ◽  
Circuit Output

Accurate predictive mathematical models are urgently needed in synthetic biology to support the bottom-up design of complex biological systems, minimizing trial-and-error approaches. The majority of models used so far adopt empirical Hill functions to describe activation and repression in exogenously-controlled inducible promoter systems. However, such equations may be poorly predictive in practical situations that are typical in bottom-up design, including changes in promoter copy number, regulatory protein level, and cell load. In this work, we derived novel mechanistic steady-state models of the lux inducible system, used as case study, relying on different assumptions on regulatory protein (LuxR) and cognate promoter (Plux) concentrations, inducer-protein complex formation, and resource usage limitation. We demonstrated that a change in the considered model assumptions can significantly affect circuit output, and preliminary experimental data are in accordance with the simulated activation curves. We finally showed that the models are identifiable a priori (in the analytically tractable cases) and a posteriori, and we determined the specific experiments needed to parametrize them. Although a larger-scale experimental validation is required, in the future the reported models may support synthetic circuits output prediction in practical situations with unprecedented details.

scholarly journals Nongenotoxic ABCB1 activator tetraphenylphosphonium can contribute to doxorubicin resistance in MX-1 breast cancer cell line

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Raimonda Kubiliute ◽  
Indre Januskeviciene ◽  
Ruta Urbanaviciute ◽  
Kristina Daniunaite ◽  
Monika Drobniene ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Protein Level ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Common Mechanism ◽  
Dna Hypomethylation ◽  
Mesenchymal Transition ◽  
Molecular Features

AbstractHyperactivation of ABC transporter ABCB1 and induction of epithelial–mesenchymal transition (EMT) are the most common mechanism of acquired cancer chemoresistance. This study describes possible mechanisms, that might contribute to upregulation of ABCB1 and synergistically boost the acquisition of doxorubicin (DOX) resistance in breast cancer MX-1 cell line. DOX resistance in MX-1 cell line was induced by a stepwise increase of drug concentration or by pretreatment of cells with an ABCB1 transporter activator tetraphenylphosphonium (TPP+) followed by DOX exposure. Transcriptome analysis of derived cells was performed by human gene expression microarrays and by quantitative PCR. Genetic and epigenetic mechanisms of ABCB1 regulation were evaluated by pyrosequencing and gene copy number variation analysis. Gradual activation of canonical EMT transcription factors with later activation of ABCB1 at the transcript level was observed in DOX-only treated cells, while TPP+ exposure induced considerable activation of ABCB1 at both, mRNA and protein level. The changes in ABCB1 mRNA and protein level were related to the promoter DNA hypomethylation and the increase in gene copy number. ABCB1-active cells were highly resistant to DOX and showed morphological and molecular features of EMT. The study suggests that nongenotoxic ABCB1 inducer can possibly accelerate development of DOX resistance.

scholarly journals Synergistic activation of ADH2 expression is sensitive to upstream activation sequence 2 (UAS2) orientation, copy number and UAS1-UAS2 helical phasing.

1995 ◽  
Vol 15 (6) ◽  
pp. 3442-3449 ◽  
Author(s):  
M S Donoviel ◽  
N Kacherovsky ◽  
E T Young
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Reporter Gene ◽  
Copy Number ◽  
Glucose Repression ◽  
Regulatory Protein ◽  
Regulatory Region ◽  
Upstream Regulatory Region ◽  
Specific Sequence

The alcohol dehydrogenase 2 (ADH2) gene of Saccharomyces cerevisiae is under stringent glucose repression. Two cis-acting upstream activation sequences (UAS) that function synergistically in the derepression of ADH2 gene expression have been identified. UAS1 is the binding site for the transcriptional regulator Adr1p. UAS2 has been shown to be important for ADH2 expression and confers glucose-regulated, ADR1-independent activity to a heterologous reporter gene. An analysis of point mutations within UAS2, in the context of the entire ADH2 upstream regulatory region, showed that the specific sequence of UAS2 is important for efficient derepression of ADH2, as would be expected if UAS2 were the binding site for a transcriptional regulatory protein. In the context of the ADH2 upstream regulatory region, including UAS1, working in concert with the ADH2 basal promoter elements, UAS2-dependent gene activation was dependent on orientation, copy number, and helix phase. Multimerization of UAS2, or its presence in reversed orientation, resulted in a decrease in ADH2 expression. In contrast, UAS2-dependent expression of a reporter gene containing the ADH2 basal promoter and coding sequence was enhanced by multimerization of UAS2 and was independent of UAS2 orientation. The reduced expression caused by multimerization of UAS2 in the native promoter was observed only in the presence of ADR1. Inhibition of UAS2-dependent gene expression by Adr1p was also observed with a UAS2-dependent ADH2 reporter gene. This inhibition increased with ADR1 copy number and required the DNA-binding activity of Adr1p. Specific but low-affinity binding of Adr1p to UAS2 in vitro was demonstrated, suggesting that the inhibition of UAS2-dependent gene expression observed in vivo could be a direct effect due to Adr1p binding to UAS2.

scholarly journals An historical framework for psychiatric nosology

2009 ◽  
Vol 39 (12) ◽  
pp. 1935-1941 ◽  
Author(s):  
K. S. Kendler
Keyword(s):  
Psychiatric Illness ◽  
Theoretical Orientation ◽  
A Priori ◽  
Early History ◽  
Top Down ◽  
Species Definition ◽  
Psychiatric Nosology ◽  
Bottom Up ◽  
Starting Point ◽  
History Of

This essay, which seeks to provide an historical framework for our efforts to develop a scientific psychiatric nosology, begins by reviewing the classificatory approaches that arose in the early history of biological taxonomy. Initial attempts at species definition used top-down approaches advocated by experts and based on a few essential features of the organism chosena priori. This approach was subsequently rejected on both conceptual and practical grounds and replaced by bottom-up approaches making use of a much wider array of features. Multiple parallels exist between the beginnings of biological taxonomy and psychiatric nosology. Like biological taxonomy, psychiatric nosology largely began with ‘expert’ classifications, typically influenced by a few essential features, articulated by one or more great 19th-century diagnosticians. Like biology, psychiatry is struggling toward more soundly based bottom-up approaches using diverse illness characteristics. The underemphasized historically contingent nature of our current psychiatric classification is illustrated by recounting the history of how ‘Schneiderian’ symptoms of schizophrenia entered into DSM-III. Given these historical contingencies, it is vital that our psychiatric nosologic enterprise be cumulative. This can be best achieved through a process of epistemic iteration. If we can develop a stable consensus in our theoretical orientation toward psychiatric illness, we can apply this approach, which has one crucial virtue. Regardless of the starting point, if each iteration (or revision) improves the performance of the nosology, the eventual success of the nosologic process, to optimally reflect the complex reality of psychiatric illness, is assured.

scholarly journals Comparative Biology of Two Natural Variants of the IncQ-2 Family Plasmids, pRAS3.1 and pRAS3.2

2009 ◽  
Vol 191 (20) ◽  
pp. 6436-6446 ◽  
Author(s):  
Wesley Loftie-Eaton ◽  
Douglas E. Rawlings
Keyword(s):  
Copy Number ◽  
Promoter Region ◽  
Plasmid Stability ◽  
Inducible Promoter ◽  
Open Reading Frame ◽  
Comparative Biology ◽  
Incompatibility Group ◽  
Reading Frame ◽  
Repeat Sequences ◽  
Natural Variants

ABSTRACT Plasmids pRAS3.1 and pRAS3.2 are two closely related, natural variants of the IncQ-2 plasmid family that have identical plasmid backbones except for two differences. Plasmid pRAS3.1 has five 6-bp repeat sequences in the promoter region of the mobB gene and four 22-bp iterons in its oriV region, whereas pRAS3.2 has only four 6-bp repeats and three 22-bp iterons. Plasmid pRAS3.1 was found to have a higher copy number than pRAS3.2, and we show that the extra 6-bp repeat results in an increase in mobB and downstream mobA/repB expression. Placement of repB (primase) behind an arabinose-inducible promoter in trans resulted in an increase in repB expression and an approximately twofold increase in the copy number of plasmids with identical numbers of 22-bp iterons. The pRAS3 plasmids were shown to have a previously unrecognized toxin-antitoxin plasmid stability module within their replicons. The ability of the pRAS3 plasmids to mobilize the oriT regions of two other plasmids of the IncQ-2 family, pTF-FC2 and pTC-F14, suggested that the mobilization proteins pRAS3 are relaxed and can mobilize oriT regions with substantially different sequences. Plasmids pRAS3.1 and pRAS3.2 were highly incompatible with plasmids pTF-FC2 and pTC-F14, and this incompatibility was removed on inactivation of an open reading frame situated downstream of the mobCDE mobilization genes rather than being due to the 22-bp oriV-associated iterons. We propose that the pRAS3 plasmids represent a third, γ incompatibility group within the IncQ-2 family plasmids.

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.

Mapping seabed assemblages using comparative top-down and bottom-up classification approaches

2006 ◽  
Vol 63 (7) ◽  
pp. 1536-1548 ◽  
Author(s):  
Paul D Eastwood ◽  
Sami Souissi ◽  
Stuart I Rogers ◽  
Roger A Coggan ◽  
Craig J Brown
Keyword(s):  
Low Cost ◽  
A Priori ◽  
Ground Truth ◽  
Unsupervised Classification ◽  
Physical Structure ◽  
Unconsolidated Sediments ◽  
Top Down ◽  
Bottom Up ◽  
Infaunal Community ◽  
Acoustic Technologies

Acoustic technologies yield many benefits for mapping the physical structure of seabed environments but are not ideally suited to classifying associated biological assemblages. We tested this assumption using benthic infauna data collected off the south coast of England by applying top-down (supervised) and bottom-up (unsupervised) classification approaches. The top-down approach was based on an a priori acoustic classification of the seabed followed by characterization of the acoustic regions using ground-truth biological samples. By contrast, measures of similarity between the ground-truth infaunal community data formed the basis of the bottom-up approach to assemblage classification. For both approaches, individual assemblages were mapped by first computing Bayesian conditional probabilities for ground-truth stations to estimate the probability of each station belonging to an assemblage. Assemblage distributions were then interpolated over a regular grid and characterized using an indicator value index. While the two methods of classification yielded assemblages and output maps that were broadly comparable, the bottom-up approach arrived at a slightly better defined set of biological assemblages. This suggests that acoustically derived seabed data are not ideally suited to class ifying biological assemblages over unconsolidated sediments, despite offering considerable advantages in providing rapid and low-cost assessments of seabed physical structure.

scholarly journals MepR, a Repressor of the Staphylococcus aureus MATE Family Multidrug Efflux Pump MepA, Is a Substrate-Responsive Regulatory Protein

2006 ◽  
Vol 50 (4) ◽  
pp. 1276-1281 ◽  
Author(s):  
Glenn W. Kaatz ◽  
Carmen E. DeMarco ◽  
Susan M. Seo
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Regulatory Protein ◽  
Inducible Promoter ◽  
Multidrug Efflux ◽  
Recognition Sequence ◽  
Multidrug Efflux Pump ◽  
Mobility Shift ◽  
Repressive Effect ◽  
Operator Interaction

ABSTRACT The mepRAB gene cluster of Staphylococcus aureus encodes a MarR family repressor (MepR; known to repress mepA expression), a MATE family multidrug efflux pump (MepA), and a protein of unknown function (MepB). In this report, we show that MepR also is autoregulatory, repressing the expression of its own gene. Exposure of strains containing a mepR::lacZ fusion with mepR provided in trans under the control of an inducible promoter, or a mepA::lacZ fusion alone, to subinhibitory concentrations of MepA substrates resulted in variably increased expression mainly of mepA. Mobility shift assays revealed that MepR binds upstream of mepR and mepA, with an apparently higher affinity for the mepA binding site. MepA substrates abrogated MepR binding to each site in a differential manner, with the greatest effect observed on the MepR-mepA operator interaction. DNase I footprinting identified precise binding sites which included promoter motifs, inverted repeats, and transcription start sites for mepR and mepA, as well as a conserved GTTAG motif, which may be a signature recognition sequence for MepR. Analogous to other multidrug efflux pump regulatory proteins such as QacR, the substrate-MepR interaction likely results in its dissociation from its mepA, and in a more limited fashion its mepR, operator sites and relief of its repressive effect. The enhanced effect of substrates on mepA compared to mepR expression, and on the MepR-mepA operator interaction, results in significant relief of mepA and relative maintenance of mepR repression, leading to increased MepA protein unimpeded by MepR when the need for detoxification exists.

scholarly journals Characterization and Regulation of the gbuA Gene, Encoding Guanidinobutyrase in the Arginine Dehydrogenase Pathway of Pseudomonas aeruginosa PAO1

2002 ◽  
Vol 184 (12) ◽  
pp. 3377-3384 ◽  
Author(s):  
Yuji Nakada ◽  
Yoshifumi Itoh
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Regulatory Protein ◽  
Inducible Promoter ◽  
Northern Blotting ◽  
Gene Encoding ◽  
Insertional Inactivation ◽  
Gentamicin Resistance ◽  
Genomic Map ◽  
Hydrolysis Of

ABSTRACT The arginine dehydrogenase (or oxidase) pathway catabolically converts arginine to succinate via 2-ketoglutarate and 4-guanidinobutyrate (4-GB) with the concomitant formation of CO2 and urea. Guanidinobutyrase (GBase; EC 3.5.3.7) catalyzes the conversion of 4-guanidinobutyrate to 4-aminobutyrate and urea in this pathway. We investigated the structure and regulation of the gene for GBase (designated gbuA) of Pseudomonas aeruginosa PAO1 and characterized the gbuA product. The gbuA and the adjacent gbuR genes were cloned by functional complementation of a gbuA9005 mutant of strain PAO1 defective in 4-GB utilization. The deduced amino acid sequence of GbuA (319 amino acids; M r 34,695) assigned GBase to the arginase/agmatinase family of C-N hydrolases. Purified GbuA was a homotetramer of 140 kDa that catalyzed the specific hydrolysis of 4-GB with Km and K cat values of 49 mM and 1,012 s−1, respectively. The divergent gbuR gene, which shared the intergenic promoter region of 206 bp with gbuA, encoded a putative regulatory protein (297 amino acids; M r 33,385) homologous to the LysR family of proteins. Insertional inactivation of gbuR by a gentamicin resistance cassette caused a defect in 4-GB utilization. GBase and gbuA′::′lacZ fusion assays demonstrated that this gbuR mutation abolishes the inducible expression of gbuA by exogenous 4-GB, indicating that GbuR participates in the regulation of this gene. Northern blotting located an inducible promoter for gbuA in the intergenic region, and primer extension localized the transcription start site of this promoter at 40 bp upstream from the initiation codon of gbuA. The gbuRA genes at the genomic map position of 1547000 are unlinked to the 2-ketoarginine utilization gene kauB at 5983000, indicative of at least two separate genetic units involved in the arginine dehydrogenase pathway.

scholarly journals A Virtual Reality Ensemble Molecular Dynamics Workflow to Study Complex Conformational Changes in Proteins

2020 ◽  
Author(s):  
Jordi Juárez-Jiménez ◽  
Philip Tew ◽  
Michael o'connor ◽  
Salome Llabres ◽  
Rebecca Sage ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Virtual Reality ◽  
Conformational Changes ◽  
Large Scale ◽  
A Priori ◽  
Computational Cost ◽  
Md Simulations ◽  
Collective Variables ◽  
Sampling Protocols ◽  
State Models

<p>Molecular dynamics (MD) simulations are increasingly used to elucidate relationships between protein structure, dynamics and their biological function. Currently it is extremely challenging to perform MD simulations of large-scale structural rearrangements in proteins that occur on millisecond timescales or beyond, as this requires very significant computational resources, or the use of cumbersome ‘collective variable’ enhanced sampling protocols. Here we describe a framework that combines ensemble MD simulations and virtual-reality visualization (eMD-VR) to enable users to interactively generate realistic descriptions of large amplitude, millisecond timescale protein conformational changes in proteins. Detailed tests demonstrate that eMD-VR substantially decreases the computational cost of folding simulations of a WW domain, without the need to define collective variables <i>a priori</i>. We further show that eMD-VR generated pathways can be combined with Markov State Models to describe the thermodynamics and kinetics of large-scale loop motions in the enzyme cyclophilin A. Our results suggest eMD-VR is a powerful tool for exploring protein energy landscapes in bioengineering efforts. </p>

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