scholarly journals Rapid generation of sequence-diverse terminator libraries and their parameterization using quantitative Term-Seq

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Andrew J Hudson ◽  
Hans-Joachim Wieden
Keyword(s):  
Rational Design ◽  
Design Tools ◽  
Sequence Structure ◽  
E Coli ◽  
Quantitative Term ◽  
Additional Sequence ◽  
The Common ◽  
Rapid Generation ◽  
Reliable Design

Abstract Synthetic biology and the rational design and construction of biological devices require vast numbers of characterized biological parts, as well as reliable design tools to build increasingly complex, multigene architectures. Design principles for intrinsic terminators have been established; however, additional sequence-structure studies are needed to refine parameters for termination-based genetic devices. We report a rapid single-pot method to generate libraries of thousands of randomized bidirectional intrinsic terminators and a modified quantitative Term-Seq (qTerm-Seq) method to simultaneously identify terminator sequences and measure their termination efficiencies (TEs). Using qTerm-Seq, we characterize hundreds of additional strong terminators (TE > 90%) with some terminators reducing transcription read-through by up to 1000-fold in Escherichia coli. Our terminator library and qTerm-Seq pipeline constitute a flexible platform enabling identification of terminator parts that can achieve transcription termination not only over a desired range but also to investigate their sequence-structure features, including for specific genetic and application contexts beyond the common in vivo systems such as E. coli.

An Autonomous In Vivo Dual Selection Protocol for Boolean Genetic Circuits

2015 ◽  
Vol 21 (2) ◽  
pp. 247-260 ◽  
Author(s):  
David Beneš ◽  
Petr Sosík ◽  
Alfonso Rodríguez-Patón
Keyword(s):  
Rational Design ◽  
Selection Process ◽  
Logic Gates ◽  
Genetic Circuit ◽  
Evolutionary Engineering ◽  
Genetic Circuits ◽  
E Coli ◽  
Search And Selection ◽  
Selection Of

Success in synthetic biology depends on the efficient construction of robust genetic circuitry. However, even the direct engineering of the simplest genetic elements (switches, logic gates) is a challenge and involves intense lab work. As the complexity of biological circuits grows, it becomes more complicated and less fruitful to rely on the rational design paradigm, because it demands many time-consuming trial-and-error cycles. One of the reasons is the context-dependent behavior of small assembly parts (like BioBricks), which in a complex environment often interact in an unpredictable way. Therefore, the idea of evolutionary engineering (artificial directed in vivo evolution) based on screening and selection of randomized combinatorial genetic circuit libraries became popular. In this article we build on the so-called dual selection technique. We propose a plasmid-based framework using toxin-antitoxin pairs together with the relaxase conjugative protein, enabling an efficient autonomous in vivo evolutionary selection of simple Boolean circuits in bacteria (E. coli was chosen for demonstration). Unlike previously reported protocols, both on and off selection steps can run simultaneously in various cells in the same environment without human intervention; and good circuits not only survive the selection process but are also horizontally transferred by conjugation to the neighbor cells to accelerate the convergence rate of the selection process. Our directed evolution strategy combines a new dual selection method with fluorescence-based screening to increase the robustness of the technique against mutations. As there are more orthogonal toxin-antitoxin pairs in E. coli, the approach is likely to be scalable to more complex functions. In silico experiments based on empirical data confirm the high search and selection capability of the protocol.

scholarly journals In Vivo Pharmacodynamic Activities of Two Glycylcyclines (GAR-936 and WAY 152,288) against Various Gram-Positive and Gram-Negative Bacteria

2000 ◽  
Vol 44 (4) ◽  
pp. 943-949 ◽  
Author(s):  
M. L. van Ogtrop ◽  
D. Andes ◽  
T. J. Stamstad ◽  
B. Conklin ◽  
W. J. Weiss ◽  
...  
Keyword(s):  
Rational Design ◽  
Dose Finding ◽  
Maximum Effect ◽  
Resistant Bacteria ◽  
Infection Model ◽  
Pharmacokinetic Studies ◽  
Time Curve ◽  
E Coli ◽  
Elimination Half

ABSTRACT The in vivo pharmacodynamic activities of two glycylcyclines (GAR-936 and WAY 152,288) were assessed in an experimental murine thigh infection model in neutropenic mice. Mice were infected with one of several strains of Streptococcus pneumoniae,Staphylococcus aureus, Escherichia coli, orKlebsiella pneumoniae. Most infections were treated with a twice-daily dosing schedule, with administration of 0.75 to 192 mg of GAR-936 or WAY 152,288 per kg of body weight. A maximum-effect dose-response model was used to calculate the dose that produced a net bacteriostatic effect over 24 h of therapy. This dose was called the bacteriostatic dose. More extensive dosing studies were performed with S. pneumoniae 1199, E. coli ATCC 25922, and K. pneumoniae ATCC 43816, with doses being given as one, two, four, or eight equal doses over a period of 24 h. The dosing schedules were designed in order to minimize the interrelationship between the various pharmacokinetic and pharmacodynamic parameters studied. These parameters were time above 0.03 to 32 times the MIC, area under the concentration-time curve (AUC), and maximum concentration of drug in serum (C max). The bacteriostatic dose remained essentially the same, irrespective of the dosing frequency, for S. pneumoniae 1199 (0.3 to 0.9 mg/kg/day). For E. coli ATCC 25922 and K. pneumoniae ATCC 43816, however, more frequent dosing led to lower bacteriostatic doses. Pharmacokinetic studies demonstrated dose-dependent elimination half-lives of 1.05 to 2.34 and 1.65 to 3.36 h and serum protein bindings of 59 and 71% for GAR-936 and WAY 152,288, respectively. GAR-936 and WAY 152,288 were similarly effective against the microorganisms studied, with small differences in maximum effect and 50% effective dose. The glycylcyclines were also similarly effective against tetracycline-sensitive and tetracycline-resistant bacteria. Time above a certain factor (range, 0.5 to 4 times) of the MIC was a better predictor of in vivo efficacy than C maxor AUC for most organism-drug combinations. The results demonstrate that in order to achieve 80% maximum efficacy, the concentration of unbound drug in serum should be maintained above the MIC for at least 50% of the time for GAR-936 and for at least 75% of the time for WAY 152,288. The results of these experiments will aid in the rational design of dose-finding studies for these glycylcyclines in humans.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Production and Preliminary In Vivo Evaluations of a Novel in silico-designed L2-based Potential HPV Vaccine

2020 ◽  
Vol 21 (4) ◽  
pp. 316-324
Author(s):  
Manica Negahdaripour ◽  
Navid Nezafat ◽  
Reza Heidari ◽  
Nasrollah Erfani ◽  
Nasim Hajighahramani ◽  
...  
Keyword(s):  
In Silico ◽  
Group Versus ◽  
Tlr Agonists ◽  
E Coli ◽  
Metal Affinity ◽  
Th1 And Th2 Cytokines ◽  
Ifn Γ ◽  
Humoral Responses ◽  
Future Work

Background: L2-based Human Papillomavirus (HPV) prophylactic vaccines, containing epitopes from HPV minor capsid proteins, are under investigation as second-generation HPV vaccines. No such vaccine has passed clinical trials yet, mainly due to the low immunogenicity of peptide vaccines; so efforts are being continued. A candidate vaccine composed of two HPV16 L2 epitopes, flagellin and a Toll-Like Receptor (TLR) 4 agonist (RS09) as adjuvants, and two universal T-helper epitopes was designed in silico in our previous researches. Methods: The designed vaccine construct was expressed in E. coli BL21 (DE3) and purified through metal affinity chromatography. Following mice vaccination, blood samples underwent ELISA and flow cytometry analyses for the detection of IgG and seven Th1 and Th2 cytokines. Results: Following immunization, Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5, IL-10) type cytokines, as well as IgG, were induced significantly compared with the PBS group. Significant increases in IFN-γ, IL-2, and IL-5 levels were observed in the vaccinated group versus Freund’s adjuvant group. Conclusion: The obtained cytokine induction profile implied both cellular and humoral responses, with a more Th-1 favored trend. However, an analysis of specific antibodies against L2 is required to confirm humoral responses. No significant elevation in inflammatory cytokines, (IL-6 and TNF-α), suggested a lack of unwanted inflammatory side effects despite using a combination of two TLR agonists. The designed construct might be capable of inducing adaptive and innate immunity; nevertheless, comprehensive immune tests were not conducted at this stage and will be a matter of future work.

The Application of the Cold Atmospheric Plasma-Activated Solutions in Cancer Treatment

2018 ◽  
Vol 18 (6) ◽  
pp. 769-775 ◽  
Author(s):  
Dayun Yan ◽  
Jonathan H. Sherman ◽  
Michael Keidar
Keyword(s):  
Cancer Treatment ◽  
Atmospheric Plasma ◽  
Current Status ◽  
Cold Atmospheric Plasma ◽  
Anti Cancer ◽  
Long Time ◽  
Key Topics ◽  
The Common

Background: Over the past five years, the cold atmospheric plasma-activated solutions (PAS) have shown their promissing application in cancer treatment. Similar as the common direct cold plasma treatment, PAS shows a selective anti-cancer capacity in vitro and in vivo. However, different from the direct cold atmospheric plasma (CAP) treatment, PAS can be stored for a long time and can be used without dependence on a CAP device. The research on PAS is gradually becoming a hot topic in plasma medicine. Objectives: In this review, we gave a concise but comprehensive summary on key topics about PAS including the development, current status, as well as the main conclusions about the anti-cancer mechanism achieved in past years. The approaches to make strong and stable PAS are also summarized.

PAF increases vascular permeability without increasing pulmonary arterial pressure in the rat

2001 ◽  
Vol 90 (1) ◽  
pp. 261-268 ◽  
Author(s):  
Leonardo C. Clavijo ◽  
Mary B. Carter ◽  
Paul J. Matheson ◽  
Mark A. Wilson ◽  
William B. Wead ◽  
...  
Keyword(s):  
Arterial Pressure ◽  
Pulmonary Edema ◽  
Pulmonary Arterial Pressure ◽  
Ex Vivo ◽  
Platelet Activating Factor ◽  
Microvascular Permeability ◽  
Blue Dye ◽  
E Coli ◽  
Pulmonary Arterial

In vivo pulmonary arterial catheterization was used to determine the mechanism by which platelet-activating factor (PAF) produces pulmonary edema in rats. PAF induces pulmonary edema by increasing pulmonary microvascular permeability (PMP) without changing the pulmonary pressure gradient. Rats were cannulated for measurement of pulmonary arterial pressure (Ppa) and mean arterial pressure. PMP was determined by using either in vivo fluorescent videomicroscopy or the ex vivo Evans blue dye technique. WEB 2086 was administered intravenously (IV) to antagonize specific PAF effects. Three experiments were performed: 1) IV PAF, 2) topical PAF, and 3) Escherichia coli bacteremia. IV PAF induced systemic hypotension with a decrease in Ppa. PMP increased after IV PAF in a dose-related manner. Topical PAF increased PMP but decreased Ppa only at high doses. Both PMP (88 ± 5%) and Ppa (50 ± 3%) increased during E. coli bacteremia. PAF-receptor blockade prevents changes in Ppa and PMP after both topical PAF and E. coli bacteremia. PAF, which has been shown to mediate pulmonary edema in prior studies, appears to act in the lung by primarily increasing microvascular permeability. The presence of PAF might be prerequisite for pulmonary vascular constriction during gram-negative bacteremia.

scholarly journals 99mTc Labelling Strategies for the Development of Potential Nitroimidazolic Hypoxia Imaging Agents

Inorganics ◽  
2019 ◽  
Vol 7 (11) ◽  
pp. 128 ◽  
Author(s):  
Giglio ◽  
Rey
Keyword(s):  
Rational Design ◽  
Biological Properties ◽  
Fine Tuning ◽  
Oxidation States ◽  
Hypoxia Imaging ◽  
Biological Target ◽  
99Mtc Radiopharmaceuticals ◽  
99Mtc Labelling

Technetium-99m has a rich coordination chemistry that offers many possibilities in terms of oxidation states and donor atom sets. Modifications in the structure of the technetium complexes could be very useful for fine tuning the physicochemical and biological properties of potential 99mTc radiopharmaceuticals. However, systematic study of the influence of the labelling strategy on the “in vitro” and “in vivo” behaviour is necessary for a rational design of radiopharmaceuticals. Herein we present a review of the influence of the Tc complexes’ molecular structure on the biodistribution and the interaction with the biological target of potential nitroimidazolic hypoxia imaging radiopharmaceuticals presented in the literature from 2010 to the present. Comparison with the gold standard [18F]Fluoromisonidazole (FMISO) is also presented.

scholarly journals Stu2p binds tubulin and undergoes an open-to-closed conformational change

2006 ◽  
Vol 172 (7) ◽  
pp. 1009-1022 ◽  
Author(s):  
Jawdat Al-Bassam ◽  
Mark van Breugel ◽  
Stephen C. Harrison ◽  
Anthony Hyman
Keyword(s):  
Conformational Change ◽  
Conformational Transition ◽  
Budding Yeast ◽  
Microtubule Dynamics ◽  
Open Structure ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

Sign in / Sign up

Export Citation Format

Share Document