Escape Frequency Research Articles

A high-throughput Galectin-9 imaging assay for quantifying nanoparticle uptake, endosomal escape and functional RNA delivery

RNA-based therapies have great potential to treat many undruggable human diseases. However, their efficacy, in particular for mRNA, remains hampered by poor cellular delivery and limited endosomal escape. Development and optimisation of delivery vectors, such as lipid nanoparticles (LNPs), are impeded by limited screening methods to probe the intracellular processing of LNPs in sufficient detail. We have developed a high-throughput imaging-based endosomal escape assay utilising a Galectin-9 reporter and fluorescently labelled mRNA to probe correlations between nanoparticle-mediated uptake, endosomal escape frequency, and mRNA translation. Furthermore, this assay has been integrated within a screening platform for optimisation of lipid nanoparticle formulations. We show that Galectin-9 recruitment is a robust, quantitative reporter of endosomal escape events induced by different mRNA delivery nanoparticles and small molecules. We identify nanoparticles with superior escape properties and demonstrate cell line variances in endosomal escape response, highlighting the need for fine-tuning of delivery formulations for specific applications.

Bioinformatic analysis of post-transmission viral readaptation in Argentine patients with acute HIV-1 infection

During the acute phase of HIV-1 infection, a strong readaptation occurs in the viral population. Our objective was to analyze the post-transmission mutations associated with escape to the cytotoxic immune response and its relationship with the progression of the infection. In this study, a total of 17 patients were enrolled during acute/early primary HIV infection and 8 subjects that were the HIV positive partner resulting in 8 transmission pairs. Genotyping of the genetic polymorphisms of HLA class I A and B was performed using PCR-SSOP. Viral RNA extraction was from plasma. 570 single Gag-gene amplifications were obtained by limiting-dilution RT-PCR. Epitope prediction was performed with NetMHC CBS prediction server for the 19 HLA-A and B alleles. Cytotoxic response prediction was performed by using the IEDB Analysis Resource. From our results, we deduce that the transmitted CTL / gag escape frequency in the founder virus was at least double compared to the post-transmission events. Additionally, by means of an algorithm that combines these frequencies, we observed that the founder viruses better adapted to the HLA A / B alleles of the recipient could contribute to a greater progression of the infection. Our results suggest that there is a large adaptation of HIV-1 to the HLA A / B alleles prevalent in our population. However, despite this adaptive advantage, the virus needs to make “readjustments” through new escape and compensatory mutations. Interestingly, according to our results, this readaptation could have a role in the progression of the infection.

A General Strategy for Engineering Noncanonical Amino Acid Dependent Bacterial Growth.

Synthetic auxotrophy in which bacterial viability depends on the presence of a synthetic amino acid provides a robust strategy for the containment of genetically modified organisms and the development of safe, live vaccines. However, a simple, general strategy to evolve essential proteins to be dependent on synthetic amino acids is lacking. Using a temperature-sensitive selection system, we evolved an Escherichia coli (E. coli) sliding clamp variant with an orthogonal protein-protein interface, which contains a Leu273 to p-benzoylphenyl alanine (pBzF) mutation. The E. coli strain with this variant DNA clamp has a very low escape frequency (<10-10), and its growth is strictly dependent on the presence of pBzF. This selection strategy can be generally applied to create ncAA dependence of other organisms with DNA clamp homologues.

Development of a New Biocontainment Strategy in Model Cyanobacterium Synechococcus Strains.

Recent synthetic biology efforts have raised biosafety concerns for possible release of engineered cyanobacteria into natural environments. To address the issues, we developed a controllable metal ion induced biocontainment system for two model cyanobacteria. First, six ion-inducible promoters were respectively evaluated in both Synechococcus elongatus PCC 7942 and the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973, leading to the identification of an iron ion-repressed promoter PisiAB with low leakage and a reduction-fold of 5.4 and 7.9, respectively. Second, holin-endolysin and nuclease NucA systems were introduced, the inhibition rate of which against two Synechococcus strains varied from 61% to 86.4%. Third, two toxin/antitoxin modules were identified capable of inducing programmed suicide in both Synechococcus strains after induction. Furthermore, an escape experiment was conducted and the results showed that the system was able to achieve an escape frequency below the detection limit of 10-9 after 3 days' duration, demonstrating the strategy integrating iron ion-inducible promoter PisiAB and that toxin/antitoxin modules could be a useful tool for cyanobacterium biocontainment.

Analysis of elastic bulk waves in functionally graded triclinic nanoplates using a quasi-3D bi-Helmholtz nonlocal strain gradient model

Elastic bulk waves of functionally graded triclinic nanoplates are studied using a quasi-3D shear deformation theory and nonlocal strain gradient theory. The present non-classical model includes three-fine scale parameters in addition to the elastic constants of the anisotropic material. The governing equations are obtained for a triclinic nanoplates based on Hamilton's principle. Subsequently, we employ an analytical based method using harmonic series to determine the wave frequency, phase velocity as well as the escape frequency. The accuracy of the current non-classical method is validated with experimental data for wave dispersion of graphene sheets and good agreement is achieved. Furthermore, we study the influence of the fine-scale parameters, the exponential factor, and wave number in detail. We also test the influence of the anisotropy by replacing the anisotropic model with an isotropic one. The results for phase velocity and escape frequency show that the differences between the two approaches of modeling grow up to 2% in some conditions.

Quantifying Virus Escape from T Cells in the Latent HIV Reservoir

Abstract There is no cure for HIV, largely because HIV establishes a small but sustained pool of latently infected cells that are not cleared by antiretroviral therapy (ART). Current strategies include firstly reactivating the latent HIV reservoir and then using T cell immunotherapy to clear reactivated cells. However, pre-ART CD8 T cell escape variants have been reported in the HIV reservoir, which may limit CD8 T cell recognition and clearance of HIV-infected cells. The extent of virus escape from T cells in the latent reservoir is unclear. HIV-specific T cell responses were comprehensively mapped across the Clade B HIV proteome by IFN-g ELISpot in 25 ART-suppressed participants. In parallel, replication competent viruses derived from supernatants of autologous resting CD4 T cells following mitogenic reactivation were sequenced. Peptides spanning virus variants within reactive T cell epitopes were synthesized and examined by ELISpot for evidence of escape, defined as ≥50% difference in T cell magnitude between peptide variants. No correlations were observed between the size of the latent HIV reservoir and either HIV-specific T cell breadth (1–19 epitopes) or magnitude (156–2855 SFU/M PBMCs). T cell escape was assessed in 17 participants. 39% of reactive T cell epitopes (48/124) harbored ≥1 amino acid variants in the sequenced latent reservoir. Of those 48, 20 afforded T cell escape, providing an overall escape frequency in the reservoir of 16% (20/124). These data show that the majority of replication competent latent HIV viruses do not harbor CD8 T cell escape mutants, suggesting that immunotherapy approaches that boost CD8 T cell responses can successfully target the latent reservoir in HIV curative and or remission strategies.

Biobased plastics with insect‐repellent functionality

Natural insecticides/repellents, such as pyrethrum (derived from chrysanthemum plants), and insect repellent N,N‐Diethyl‐meta‐toluamide (DEET) were added to poly(lactic acid) (PLA) fibers through extrusion and spray coating on the PLA fabrics. Contact irritancy assay (CIA) showed that DEET‐treated PLA fabrics caused the lowest relative escape response of mosquitoes with an escape frequency of 33.3% ± 3.3%, indicating that DEET was less effect compared with natural insecticides/repellents. This was followed by the extruded natural pyrethrum‐treated PLA fabric with an escape frequency of 80% ± 6.3%. Finally, the PLA fabrics spray‐coated with natural pyrethrum caused the highest escape frequency of 98.3% ± 1.7%. Thus, it was found that pyrethrum/PLA fabrics functioned as a mosquito repellent better than DEET/PLA fabrics. In addition, TGA and tensile testing results demonstrated that pyrethrum was sufficiently thermally stable to be extrusion compounded with PLA. GPC results showed that DEET promoted de‐polymerization of PLA when co‐extruded. The results demonstrated that pyrethrum can be a viable additive for PLA to produce fibers that function as mosquito repellent to produce temporary garments that are compostable. The potential use of the developed biobased fibers with natural insect repellents is for single use of personal protection equipment (PPE) garments. POLYM. ENG. SCI., 59:E460–E467, 2019. © 2019 Society of Plastics Engineers

Viscoelastic wave propagation analysis of axially motivated double-layered graphene sheets via nonlocal strain gradient theory

ABSTRACTThis paper contains a nonlocal strain gradient-based theory to survey viscoelastic wave dispersion characteristics of axially loaded double-layered graphene sheets (DLGSs) resting on the viscoelastic substrate. Actually, a comprehensive size-dependent analysis is performed in which both amplifying and minimizing effects are covered. Also, the kinematic relations have been derived by the means of a one-variable classical plate theory. Besides, the final nonlocal governing equations can be developed using the Hamilton’s principle. These equations will be finally solved utilizing an analytical solution to obtain wave frequency, phase velocity and escape frequency of DLGSs. Last section is allocated to study the effects of various terms including wave number, nonlocal parameter, length scale parameter, structural damping coefficient, Winkler coefficient, Pasternak coefficient, damping coefficient and axial load on the wave propagation behaviors of DLGSs.

A current transient method for trap analysis in BiFeO3 thin films

We present a method to study the trap characteristics in BiFeO3 thin films, which display a resistance switching effect caused by trapping/detrapping of charge carriers. The method consists of measuring the current transients for the detrapping of charge carriers from trap sites and subsequent analysis of the time constant from the current transient curve. Using this method, the energy level of the traps was identified to be 0.71 ± 0.06 eV. The parameters of the trapping pulses (voltage and time duration) were found to affect the time constant, indicating a close relationship between the escape frequency of the charge carriers and the structure ordering caused by polarization and the filling of the traps.

Endosomal Size and Membrane Leakiness Influence Proton Sponge-Based Rupture of Endosomal Vesicles.

In gene therapy, endosomal escape represents a major bottleneck since nanoparticles often remain entrapped inside endosomes and are trafficked toward the lysosomes for degradation. A detailed understanding of the endosomal barrier would be beneficial for developing rational strategies to improve transfection and endosomal escape. By visualizing individual endosomal escape events in live cells, we obtain insight into mechanistic factors that influence proton sponge-based endosomal escape. In a comparative study, we found that HeLa cells treated with JetPEI/pDNA polyplexes have a 3.5-fold increased endosomal escape frequency compared to ARPE-19 cells. We found that endosomal size has a major impact on the escape capacity. The smaller HeLa endosomes are more easily ruptured by the proton sponge effect than the larger ARPE-19 endosomes, a finding supported by a mathematical model based on the underlying physical principles. Still, it remains intriguing that even in the small HeLa endosomes, <10% of the polyplex-containing endosomes show endosomal escape. Further experiments revealed that the membrane of polyplex-containing endosomes becomes leaky to small compounds, preventing effective buildup of osmotic pressure, which in turn prevents endosomal rupture. Analysis of H1299 and A549 cells revealed that endosomal size determines endosomal escape efficiency when cells have comparable membrane leakiness. However, at high levels of membrane leakiness, buildup of osmotic pressure is no longer possible, regardless of endosomal size. Based on our findings that both endosomal size and membrane leakiness have a high impact on proton sponge-based endosomal rupture, we provide important clues toward further improvement of this escape strategy.

On wave dispersion characteristics of double-layered graphene sheets in thermal environments

In this research, it is tried to inquire the wave propagation problem of a double-layered graphene sheet (DLGS) undergoing thermal loading for the first time. Here, uniform and linear temperature distributions are included to enucleate the effect of each one in comparison with the other. A classical plate theory is employed to derive the kinematic relations of each layer of DLGS. On the other hand, the non-local elasticity theory is introduced to account for the nanoscale effects. In addition, attachment of graphene sheets to a fixed surface is modeled by a visco-Pasternak foundation and the interactions between two layers are simulated utilizing van der Waals (vdW) model. By the means of Hamilton’s principle, the non-local governing equations are derived. Also, in the framework of an analytical procedure, the wave frequency and phase velocity values are obtained. Eventually, a complex of various diagrams is organized to separately investigate the influence of each parameter on the wave frequency, phase velocity, and escape frequency of DLGSs.

Wave propagation analysis of smart strain gradient piezo-magneto-elastic nonlocal beams

This study presents the investigation of wave dispersion characteristics of a magneto-electro-elastic functionally graded (MEE-FG) nanosize beam utilizing nonlocal strain gradient theory (NSGT). In this theory, a material length scale parameter is propounded to show the influence of strain gradient stress field, and likewise, a nonlocal parameter is nominated to emphasize on the importance of elastic stress field effects. The material properties of heterogeneous nanobeam are supposed to vary smoothly through the thickness direction based on power-law form. Applying Hamilton\'s principle, the nonlocal governing equations of MEE-FG nanobeam are derived. Furthermore, to derive the wave frequency, phase velocity and escape frequency of MEE-FG nanobeam, an analytical solution is employed. The validation procedure is performed by comparing the results of present model with results exhibited by previous papers. Results are rendered in the framework of an exact parametric study by changing various parameters such as wave number, nonlocal parameter, length scale parameter, gradient index, magnetic potential and electric voltage to show their influence on the wave frequency, phase velocity and escape frequency of MEE-FG nanobeams.

Rational Design of Evolutionarily Stable Microbial Kill Switches

The evolutionary stability of synthetic genetic circuits is key to both the understanding and application of genetic control elements. One useful but challenging situation is a switch between life and death depending on environment. Here are presented "essentializer" and "cryodeath" circuits, which act as kill switches in Escherichia coli. The essentializer element induces cell death upon the loss of a bi-stable cI/Cro memory switch. Cryodeath makes use of a cold-inducible promoter to express a toxin. We employ rational design and a toxin/antitoxin titering approach to produce and screen a small library of potential constructs, in order to select for constructs that are evolutionarily stable. Both kill switches were shown to maintain functionality invitro for at least 140 generations. Additionally, cryodeath was shown to control the growth environment of a population, with an escape frequency of less than 1 in 105 after 10days of growth in the mammalian gut.

Wave dispersion characteristics of rotating heterogeneous magneto-electro-elastic nanobeams based on nonlocal strain gradient elasticity theory

The coupled influences of shear deformation and angular velocity of a FG rotary nanobeam are going to be analyzed in the existence of external magnetic and electric fields. The effective material properties seem to be defined by the means of power law formulation. Moreover, the influences of small scale are included precisely in the framework of a nonlocal strain gradient theory. The magneto-electric potentials are supposed to vary through the thickness with a combination of linear and cosine approximations. Employing Hamilton’s principle, the nonlocal governing equations of magneto-electro-elastic functionally graded (MEE-FG) rotary size-dependent beams are derived in terms of displacement fields. Afterwards, the obtained governing equations are solved analytically to gather wave frequency, phase velocity, and escape frequency of the MEE-FG rotary nanobeam. The obtained results are validated with those of former researches. At the end, a numerical study is performed to show the influence of involved parameters on the wave propagation behaviors of MEE-FG rotary nanobeams.

A Strategy for Creating Organisms Dependent on Noncanonical Amino Acids.

The use of noncanonical amino acids (ncAAs) to control the viability of an organism provides a strategy for the development of conditional "kill switches" for live vaccines or engineered human cells. We report an approach inspired by the posttranslational acetylation/deacetylation of lysine residues, in which a protein encoded by a gene with an in-frame nonsense codon at an essential lysine can be expressed in its native state only upon genetic incorporation of N-ϵ-acetyl-l-Lys (AcK), and subsequent enzymatic deacetylation in the host cell. We applied this strategy to two essential E. coli enzymes: the branched-chain aminotransferase BCAT and the DNA replication initiator protein DnaA. We also devised a barnase-based conditional suicide switch to further lower the escape frequency of the host cells. This strategy offers a number of attractive features for controlling host viability, including a single small-molecule-based kill switch, low escape frequency, and unaffected protein function.

A Strategy for Creating Organisms Dependent on Noncanonical Amino Acids

AbstractThe use of noncanonical amino acids (ncAAs) to control the viability of an organism provides a strategy for the development of conditional “kill switches” for live vaccines or engineered human cells. We report an approach inspired by the posttranslational acetylation/deacetylation of lysine residues, in which a protein encoded by a gene with an in‐frame nonsense codon at an essential lysine can be expressed in its native state only upon genetic incorporation of N‐ϵ‐acetyl‐l‐Lys (AcK), and subsequent enzymatic deacetylation in the host cell. We applied this strategy to two essential E. coli enzymes: the branched‐chain aminotransferase BCAT and the DNA replication initiator protein DnaA. We also devised a barnase‐based conditional suicide switch to further lower the escape frequency of the host cells. This strategy offers a number of attractive features for controlling host viability, including a single small‐molecule‐based kill switch, low escape frequency, and unaffected protein function.

Wave propagation analysis of smart rotating porous heterogeneous piezo-electric nanobeams

The present work is mainly focused on studying the influences of angular velocity on the wave propagation responses of functionally graded (FG) piezo-electric nanobeams. Moreover, the effects of porosity are also regarded in the wave propagation analysis of size-dependent FG beams. The distribution of electro-mechanical properties of a piezo-electric beam are precisely described employing power-law formulation. The nonlocal elasticity theory is utilized to account for the influences of small scale. Herein, a classical beam theory is expounded to derive the nonlocal governing equations of the nanobeam. Once the governing equations are completely derived, an analytical solution method is applied to obtain the dispersion relations of propagating waves. A comparison of this model with previous studies is then made to show the validity of the obtained results. Finally, the influences of various variants, such as wave number, nonlocal parameter, gradient index, electric voltage, volume fraction of porosity and angular velocity, are studied in detail to show how these parameters can affect the wave frequency, phase velocity and escape frequency of FG smart rotary porous nanobeams.

A Novel Biocontainment Strategy Makes Bacterial Growth and Survival Dependent on Phosphite

There is a growing demand to develop biocontainment strategies that prevent unintended proliferation of genetically modified organisms in the open environment. We found that the hypophosphite (H3PO2, HPt) transporter HtxBCDE from Pseudomonas stutzeri WM88 was also capable of transporting phosphite (H3PO3, Pt) but not phosphate (H3PO4, Pi), suggesting the potential for engineering a Pt/HPt-dependent bacterial strain as a biocontainment strategy. We disrupted all Pi and organic Pi transporters in an Escherichia coli strain expressing HtxABCDE and a Pt dehydrogenase, leaving Pt/HPt uptake and oxidation as the only means to obtain Pi. Challenge on non-permissive growth medium revealed that no escape mutants appeared for at least 21 days with a detection limit of 1.94 × 10−13 per colony forming unit. This represents, to the best of our knowledge, the lowest escape frequency among reported strategies. Since Pt/HPt are ecologically rare and not available in amounts sufficient for the growth of the Pt/HPt-dependent bacteria, this strategy offers a reliable and practical method for biocontainment.

Nonlocal strain gradient based wave dispersion behavior of smart rotating magneto-electro-elastic nanoplates

Main object of the present research is an exact investigation of wave propagation responses of smart rotating magneto-electro-elastic (MEE) graded nanoscale plates. In addition, effective material properties of functionally graded (FG) nanoplate are presumed to be calculated using the power-law formulations. Also, it has been tried to cover both softening and stiffness-hardening behaviors of nanostructures by the means of employing nonlocal strain gradient theory (NSGT). Due to increasing the accuracy of the presented model in predicting shear deformation effects, a refined higher-order plate theory is introduced. In order to cover the most enormous circumstances, maximum amount of load generated by plate’s rotation is considered. Furthermore, utilizing a developed form of Hamilton’s principle, containing magneto-electric effects, the nonlocal governing equations of MEE-FG rotating nanoplates are derived. An analytical solution is obtained to solve the governing equations and validity of the solution method is proven by comparing results from present method with those of former attempts. At last, outcomes are plotted in the framework of some figures to show the influences of various parameters such as wave number, nonlocality, length scale parameter, magnetic potential, electric voltage, gradient index and angular velocity on wave frequency, phase velocity and escape frequency of the examined nanoplate.

Wave propagation behavior of coupled viscoelastic FG-CNTRPC micro plates subjected to electro-magnetic fields surrounded by orthotropic visco-Pasternak foundation

Wave propagation in a coupled visco-elastic micro polyvinylidene fluoride (PVDF) plate reinforced by single-walled carbon nanotubes (SWCNTs) under an electro-magnetic field will be analyzed in this study. Quasi-3D sinusoidal shear deformation theory (SSDT) of plates is presumed in order to simulate plates. The system is surrounded by orthotropic visco-Pasternak foundation. Plates are assumed to have piezoelectric property and CNTs are applied to reinforce plates in different types so micro plates are functionally graded carbon nanotube-reinforced piezoelectric composite (FG-CNTRPC); therefore, electro-magnetic field can be used as a controlling parameter for system. At first, energy equation is written according to local piezoelasticity theory and strain energy, kinetic energy and work of external forces are calculated and motion equation of system will be calculated by applying Hamilton's principle. Finally, motion equation will be solved by using analytical solution in order to obtaining velocity of wave propagation, cut-off and escape frequency of system in different types of wave propagation in system, such as in-phase, out-of-phase and one of plate fixed. After that, the effects of magnetic field, voltage, structure damping and distributions of CNTs on velocity of system and the effects of three type of wave propagation on cut-off and escape frequency of system will be studied. Results show that the effect of out-of-phase type on phase velocity and frequency of system is more than in-phase and one of plate fixed types.