scholarly journals Plasmid hypermutation using a targeted artificial DNA replisome

2021 ◽  
Vol 7 (29) ◽  
pp. eabg8712
Author(s):  
Xiao Yi ◽  
Joleen Khey ◽  
Romas J. Kazlauskas ◽  
Michael Travisano
Keyword(s):  
Efflux Pump ◽  
Fold Increase ◽  
Mutation Rates ◽  
Live Cells ◽  
Biologic Drugs ◽  
Large Populations ◽  
Artificial Dna ◽  
Hard Problems ◽  
Target Plasmid

Extensive exploration of a protein’s sequence space for improved or new molecular functions requires in vivo evolution with large populations. But disentangling the evolution of a target protein from the rest of the proteome is challenging. Here, we designed a protein complex of a targeted artificial DNA replisome (TADR) that operates in live cells to processively replicate one strand of a plasmid with errors. It enhanced mutation rates of the target plasmid up to 2.3 × 105–fold with only a 78-fold increase in off-target mutagenesis. It was used to evolve itself to increase error rate and increase the efficiency of an efflux pump while simultaneously expanding the substrate repertoire. TADR enables multiple simultaneous substitutions to discover functions inaccessible by accumulating single substitutions, affording potential for solving hard problems in molecular evolution and developing biologic drugs and industrial catalysts.

scholarly journals Imaging mRNA Expression in Live Cells via PNA·DNA Strand Displacement-Activated Probes

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Zhenghui Wang ◽  
Ke Zhang ◽  
Karen L. Wooley ◽  
John-Stephen Taylor
Keyword(s):  
Mrna Expression ◽  
Effective Means ◽  
Fold Increase ◽  
Live Cells ◽  
Strand Displacement ◽  
Dna Strand Displacement ◽  
Dna Strand ◽  
Oxide Synthase ◽  
Average Fluorescence

Probes for monitoring mRNA expressionin vivoare of great interest for the study of biological and biomedical problems, but progress has been hampered by poor signal to noise and effective means for delivering the probes into live cells. Herein we report a PNA·DNA strand displacement-activated fluorescent probe that can image the expression of iNOS (inducible nitric oxide synthase) mRNA, a marker of inflammation. The probe consists of a fluorescein labeled antisense PNA annealed to a shorterDABCYLplus-labeled DNA which quenches the fluorescence, but when the quencher strand is displaced by the target mRNA the fluorescence is restored. DNA was used for the quencher strand to facilitate electrostatic binding of the otherwise netural PNA strand to a cationic shell crosslinked knedel-like (cSCK) nanoparticle which can deliver the PNA·DNA duplex probe into cells with less toxicity and greater efficiency than other transfection agents. RAW 264.7 mouse macrophage cells transfected with the iNOS PNA·DNA probe via the cSCK showed a 16 to 54-fold increase in average fluorescence per cell upon iNOS stimulation. The increase was 4 to 7-fold higher than that for a non-complementary probe, thereby validating the ability of a PNA·DNA strand displacement-activated probe to image mRNA expressionin vivo.

HPRT As a Selectable Safe Harbor for Transgenesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4796-4796
Author(s):  
Anthony Conway ◽  
Josée Laganière ◽  
David E Paschon ◽  
Katrin Hacke ◽  
Noriyuki Kasahara ◽  
...  
Keyword(s):  
Fold Increase ◽  
Selective Growth ◽  
Live Cells ◽  
Zinc Finger Nucleases ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Targeted Integration ◽  
Selection Of

Abstract A current limitation in gene therapy is obtaining a sufficient number of modified cells to produce a therapeutic effect in vivo. In several diseases, correction of a mutant allele confers a selective growth advantage to the modified cells, thus enhancing efficacy with moderate initial modification. For most diseases, however, there is no selective advantage to the corrected cells. One potential strategy to address this limitation is in vivo selection of modified cells using pharmacological agents. It has previously been shown that 6-thioguanine (6-TG), an FDA-approved chemotherapeutic small molecule, is cytotoxic to cells expressing the enzyme HPRT, allowing for selective growth of HPRT knockout cells. Knockout of HPRT can be achieved by creating a nonsense mutation in an upstream exon, or by terminating splicing by introducing a large transgene into an intron. To allow for selectable transgenesis of only cells which have undergone targeted integration (TI), engineered zinc-finger nucleases (ZFNs) were used to insert a virally-delivered transgene into an HPRT intron. After two weeks of in vitro 6-TG selection following genome modification, a 95-fold increase in TI was observed in pooled K562 cell populations to a final level of 72% TI, whereas a 30-fold increase in transgene-expressing live cells was seen in peripheral blood-mobilized primary CD34+ cells resulting in 90% transgene-positive live cells. Furthermore, a 72-fold increase in transgene mRNA transcript was observed after two weeks of erythroid differentiation and 6-TG selection of CD34+ cells compared to unselected genome-modified controls. These results represent an important step in developing hematopoietic stem cell (HSC)-based gene therapies, as well as a platform technology for creating gene-modified HSC populations with high proportions of therapeutic transgene expression via precise, targeted integration of a transgene of interest. Disclosures Conway: Sangamo Biosciences: Employment. Paschon:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment. Cost:Sangamo Biosciences: Employment.

scholarly journals The Efflux Pump Inhibitor Timcodar Improves the Potency of Antimycobacterial Agents

2014 ◽  
Vol 59 (3) ◽  
pp. 1534-1541 ◽  
Author(s):  
Trudy H. Grossman ◽  
Carolyn M. Shoen ◽  
Steven M. Jones ◽  
Peter L. Jones ◽  
Michael H. Cynamon ◽  
...  
Keyword(s):  
Mouse Model ◽  
Efflux Pump ◽  
Fold Increase ◽  
Broth Culture ◽  
Efflux Pump Inhibitor ◽  
Content Type ◽  
Combination Studies ◽  
Drug Dependent

ABSTRACTPrevious studies indicated that inhibition of efflux pumps augments tuberculosis therapy. In this study, we used timcodar (formerly VX-853) to determine if this efflux pump inhibitor could increase the potency of antituberculosis (anti-TB) drugs againstMycobacterium tuberculosisinin vitroandin vivocombination studies. When used alone, timcodar weakly inhibitedM. tuberculosisgrowth in broth culture (MIC, 19 μg/ml); however, it demonstrated synergism in drug combination studies with rifampin, bedaquiline, and clofazimine but not with other anti-TB agents. WhenM. tuberculosiswas cultured in host macrophage cells, timcodar had about a 10-fold increase (50% inhibitory concentration, 1.9 μg/ml) in the growth inhibition ofM. tuberculosisand demonstrated synergy with rifampin, moxifloxacin, and bedaquiline. In a mouse model of tuberculosis lung infection, timcodar potentiated the efficacies of rifampin and isoniazid, conferring 1.0 and 0.4 log10reductions in bacterial burden in lung, respectively, compared to the efficacy of each drug alone. Furthermore, timcodar reduced the likelihood of a relapse infection when evaluated in a mouse model of long-term, chronic infection with treatment with a combination of rifampin, isoniazid, and timcodar. Although timcodar had no effect on the pharmacokinetics of rifampin in plasma and lung, it did increase the plasma exposure of bedaquiline. These data suggest that the antimycobacterial drug-potentiating activity of timcodar is complex and drug dependent and involves both bacterial and host-targeted mechanisms. Further study of the improvement of the potency of antimycobacterial drugs and drug candidates when used in combination with timcodar is warranted.

scholarly journals Semimechanistic Pharmacokinetic-Pharmacodynamic Model with Adaptation Development for Time-Kill Experiments of Ciprofloxacin against Pseudomonasaeruginosa

2010 ◽  
Vol 54 (6) ◽  
pp. 2379-2384 ◽  
Author(s):  
Nicolas Grégoire ◽  
Sophie Raherison ◽  
Claire Grignon ◽  
Emmanuelle Comets ◽  
Manuella Marliat ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Fold Increase ◽  
Multidrug Efflux ◽  
Multiple Dosing ◽  
Initial Inoculum ◽  
Dosing Regimens ◽  
Moderate Resistance ◽  
Time Kill

ABSTRACT The objective of this study was to implement a semimechanistic pharmacokinetic-pharmacodynamic (PK-PD) model to describe the effects of ciprofloxacin against Pseudomonas aeruginosa in vitro. Time-kill curves were generated with an initial inoculum close to 5 × 106CFU/ml of P. aeruginosa PAO1 and constant ciprofloxacin concentrations between 0.12 and 4.0 μg/ml (corresponding to 0.5× and 16× MIC). To support the model, phenotypic experiments were conducted with the PAO7H mutant strain, which overexpresses the MexEF OprN efflux pump and phenyl arginine β-naphthylamide (PAβN), a known efflux inhibitor of main Mex multidrug efflux systems. A population approach was used for parameter estimation. At subinhibitory ciprofloxacin concentrations (0.12 and 0.25 μg/ml), an initial CFU decay followed by regrowth was observed, attesting to rapid emergence of bacteria with increased but moderate resistance (8-fold increase of MIC). This phenomenon was mainly due to an overexpression of the Mex protein efflux pumps, as shown by a 16-fold diminution of the MIC in the presence of PAβN in these strains with low-level resistance. A PK-PD model with adaptation development was successfully used to describe these data. However, additional experiments are required to validate the robustness of this model after longer exposure periods and multiple dosing regimens, as well as in vivo.

scholarly journals Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation

2019 ◽  
Author(s):  
Wei Shi ◽  
Guanghui Zong ◽  
Zhijian Hu ◽  
Sarah O’Keefe ◽  
Dale Tranter ◽  
...  
Keyword(s):  
Cell Lines ◽  
Protein Translocation ◽  
Chemical Space ◽  
Direct Interaction ◽  
Fold Increase ◽  
Cellular Level ◽  
Live Cells ◽  
Structural Class

The ipomoeassin family of natural resin glycosides is underexplored chemical space with potent antitumor activity revealed in the NCI-60 cell lines screen; however, its mode of action has so far remained unexplored. In this manuscript, we report our chemical proteomics and subsequent biology studies that transform our collective knowledge of the ipomoeassin glycolipids from Organic Synthesis and Medicinal Chemistry to biological mechanism and provide a step change in our understanding of its action at a cellular level. Hence, we created an ipomoeassin F-based biotin affinity probe and used it in live cells to isolate the ER membrane protein Sec61α as its presumptive molecular target. A direct interaction between Sec61α and ipomoeassin F was confirmed by cell imaging, pulldown from purified ER membranes and competition studies using a photo-crosslinking analogue of the cyclodepsipeptide cotransin, a known Sec61α inhibitor. Crucially, we then showed that ipomoeassin F binding has a profound effect on Sec61 function, using both in vitro and in vivo assays for protein translocation and protein secretion respectively. Although structurally quite distinct, the potency of ipomoeassin F is comparable to that of mycolactone, a recently identified and intensely studied inhibitor of Sec61. The ~1,000 fold increase in the ipomoeassin F resistance of two cell lines expressing mutant forms of Sec61α strongly supports our conclusion that the effect of the compound on Sec61α is the primary basis for its potent cytotoxicity. However, we also provide evidence that ipomoeassin F is mechanistically distinct from known Sec61α inhibitors, suggesting that it is a novel structural class that may offer new opportunities to explore the Sec61 protein translocation complex as a therapeutic target for drug discovery.

scholarly journals Human mismatch-repair protein MutL homologue 1 (MLH1) interacts with Escherichia coli MutL and MutS in vivo and in vitro: a simple genetic system to assay MLH1 function

2003 ◽  
Vol 371 (1) ◽  
pp. 183-189 ◽  
Author(s):  
Barbara QUARESIMA ◽  
Pietro ALIFANO ◽  
Pierfrancesco TASSONE ◽  
Enrico V. AVVEDIMENTO ◽  
Francesco S. COSTANZO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Fold Increase ◽  
Genetic System ◽  
Mutation Rates ◽  
Wild Type ◽  
Mismatched Dna ◽  
Repair Protein

A simple genetic system has been developed to test the effect of over-expression of wild-type or mutated human MutL homologue 1 (hMLH1) proteins on methyl-directed mismatch repair (MMR) in Escherichia coli. The system relies on detection of Lac+ revertants using MMR-proficient or MMR-deficient E. coli strains carrying a lac +1 frameshift mutation expressing hMLH1 proteins. We report that expression of wild-type hMLH1 protein causes an approx. 19-fold increase in mutation rates. The mutator phenotype was due to the ability of hMLH1 protein to interact with bacterial MutL and MutS proteins, thereby interfering with the formation of complexes between MMR proteins and mismatched DNA. Conversely, expression of proteins encoded by alleles deriving from hereditary-non-polyposis-colon-cancer (HNPCC) families decreases mutation rates, depending on the specific amino acid substitutions. These effects parallel the MutL-and MutS-binding and ATP-binding/hydrolysis activities of the mutated proteins.

scholarly journals Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation

2019 ◽  
Author(s):  
Wei Shi ◽  
Guanghui Zong ◽  
Zhijian Hu ◽  
Sarah O’Keefe ◽  
Dale Tranter ◽  
...  
Keyword(s):  
Cell Lines ◽  
Protein Translocation ◽  
Chemical Space ◽  
Direct Interaction ◽  
Fold Increase ◽  
Cellular Level ◽  
Live Cells ◽  
Structural Class

The ipomoeassin family of natural resin glycosides is underexplored chemical space with potent antitumor activity revealed in the NCI-60 cell lines screen; however, its mode of action has so far remained unexplored. In this manuscript, we report our chemical proteomics and subsequent biology studies that transform our collective knowledge of the ipomoeassin glycolipids from Organic Synthesis and Medicinal Chemistry to biological mechanism and provide a step change in our understanding of its action at a cellular level. Hence, we created an ipomoeassin F-based biotin affinity probe and used it in live cells to isolate the ER membrane protein Sec61α as its presumptive molecular target. A direct interaction between Sec61α and ipomoeassin F was confirmed by cell imaging, pulldown from purified ER membranes and competition studies using a photo-crosslinking analogue of the cyclodepsipeptide cotransin, a known Sec61α inhibitor. Crucially, we then showed that ipomoeassin F binding has a profound effect on Sec61 function, using both in vitro and in vivo assays for protein translocation and protein secretion respectively. Although structurally quite distinct, the potency of ipomoeassin F is comparable to that of mycolactone, a recently identified and intensely studied inhibitor of Sec61. The ~1,000 fold increase in the ipomoeassin F resistance of two cell lines expressing mutant forms of Sec61α strongly supports our conclusion that the effect of the compound on Sec61α is the primary basis for its potent cytotoxicity. However, we also provide evidence that ipomoeassin F is mechanistically distinct from known Sec61α inhibitors, suggesting that it is a novel structural class that may offer new opportunities to explore the Sec61 protein translocation complex as a therapeutic target for drug discovery.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

Plasminogen Activation In Vivo upon Intravenous Infusion of DDAVP

1992 ◽  
Vol 67 (01) ◽  
pp. 111-116 ◽  
Author(s):  
Marcel Levi ◽  
Jan Paul de Boer ◽  
Dorina Roem ◽  
Jan Wouter ten Cate ◽  
C Erik Hack
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Plasma Levels ◽  
Fold Increase ◽  
Fibrinolytic System ◽  
Plasminogen Activation ◽  
Plasminogen Activator Activity ◽  
Insight Into

SummaryInfusion of desamino-d-arginine vasopressin (DDAVP) results in an increase in plasma plasminogen activator activity. Whether this increase results in the generation of plasmin in vivo has never been established.A novel sensitive radioimmunoassay (RIA) for the measurement of the complex between plasmin and its main inhibitor α2 antiplasmin (PAP complex) was developed using monoclonal antibodies preferentially reacting with complexed and inactivated α2-antiplasmin and monoclonal antibodies against plasmin. The assay was validated in healthy volunteers and in patients with an activated fibrinolytic system.Infusion of DDAVP in a randomized placebo controlled crossover study resulted in all volunteers in a 6.6-fold increase in PAP complex, which was maximal between 15 and 30 min after the start of the infusion. Hereafter, plasma levels of PAP complex decreased with an apparent half-life of disappearance of about 120 min. Infusion of DDAVP did not induce generation of thrombin, as measured by plasma levels of prothrombin fragment F1+2 and thrombin-antithrombin III (TAT) complex.We conclude that the increase in plasminogen activator activity upon the infusion of DDAVP results in the in vivo generation of plasmin, in the absence of coagulation activation. Studying the DDAVP induced increase in PAP complex of patients with thromboembolic disease and a defective plasminogen activator response upon DDAVP may provide more insight into the role of the fibrinolytic system in the pathogenesis of thrombosis.

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