Equilibrium Density Gradient Research Articles

Applications and Limitations of Equilibrium Density Gradient Analytical Ultracentrifugation for the Quantitative Characterization of Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) vectors are produced as a mixture of the desired particle (full particle, FP), which is filled with the designed DNA, product-related impurities such as particle without DNA (empty particle, EP), and aggregates. Cesium chloride or iodixanol equilibrium density gradient ultracentrifugation (DGE-UC) has been used for the purification of AAV vectors. DGE-UC can separate FP from impurities based on the difference in their buoyant densities. Here, we report the applications and limitations of equilibrium density gradient analytical ultracentrifugation (DGE-AUC) using a modern AUC instrument that employs DGE-UC principles for the characterization and quantitation of AAV vectors. We evaluated the quantitative ability of DGE-AUC in comparison with sedimentation velocity AUC (SV-AUC) or band sedimentation AUC (BS-AUC) using AAVs with different DNA lengths and different serotypes. DGE-AUC enabled the accurate quantification of the ratio of FP to EP when the AAV vector primarily contains these particles. Furthermore, we developed a new workflow to identify the components of separated peaks in addition to FP and EP. Ultraviolet absorption spectra obtained by multiwavelength detection can also support peak assignment following component identification. DGE-AUC experiments for AAV vectors have limitations with regard to minor components with low absorption at the detected wavelength or those with a density similar to that of major components of AAV vectors. DGE-AUC is the only analytical method that can evaluate particle density heterogeneity; therefore, SV-AUC or BS-AUC and DGE-AUC are complementary methods for reliable assessment of the purity of AAV vectors.

Optimizing high-throughput viral vector characterization with density gradient equilibrium analytical ultracentrifugation.

Viral vector-based gene therapies and vaccines require accurate characterization of capsid species. The current gold standard for assessing capsid loading of adeno-associated virus (AAV) is sedimentation velocity analytical ultracentrifugation (SV-AUC). However, routine SV-AUC analysis is often size-limited, especially without the use of advanced techniques (e.g., gravitational-sweep) or when acquiring the multiwavelength data needed for assessing the loading fraction of viral vectors, and requires analysis by specialized software packages. Density gradient equilibrium AUC (DGE-AUC) is a highly simplified analytical method that provides high-resolution separation of biologics of different densities (e.g., empty and full viral capsids). The analysis required is significantly simpler than SV-AUC, and larger viral particles such as adenovirus (AdV) are amenable to characterization by DGE-AUC using cesium chloride gradients. This method provides high-resolution data with significantly less sample (estimated 56-fold improvement in sensitivity compared to SV-AUC). Multiwavelength analysis can also be used without compromising data quality. Finally, DGE-AUC is serotype-agnostic and amenable to intuitive interpretation and analysis (not requiring specialized AUC software). Here, we present suggestions for optimizing DGE-AUC methods and demonstrate a high-throughput AdV packaging analysis with the AUC, running as many as 21 samples in 80min.

Determination of human DNA replication origin position and efficiency reveals principles of initiation zone organisation.

Replication of the human genome initiates within broad zones of ∼150 kb. The extent to which firing of individual DNA replication origins within initiation zones is spatially stochastic or localised at defined sites remains a matter of debate. A thorough characterisation of the dynamic activation of origins within initiation zones is hampered by the lack of a high-resolution map of both their position and efficiency. To address this shortcoming, we describe a modification of initiation site sequencing (ini-seq), based on density substitution. Newly replicated DNA is rendered ‘heavy-light’ (HL) by incorporation of BrdUTP while unreplicated DNA remains ‘light-light’ (LL). Replicated HL-DNA is separated from unreplicated LL-DNA by equilibrium density gradient centrifugation, then both fractions are subjected to massive parallel sequencing. This allows precise mapping of 23,905 replication origins simultaneously with an assignment of a replication initiation efficiency score to each. We show that origin firing within early initiation zones is not randomly distributed. Rather, origins are arranged hierarchically with a set of very highly efficient origins marking zone boundaries. We propose that these origins explain much of the early firing activity arising within initiation zones, helping to unify the concept of replication initiation zones with the identification of discrete replication origin sites.

Optimized Method for Pseudomonas aeruginosa Integrative Filamentous Bacteriophage Propagation.

Filamentous bacteriophages frequently infect Pseudomonas aeruginosa and alter its phenotypic traits, including virulence factors. The first step in examination of these phages is to obtain suspensions with high virus titer, but as there are no methods for integrative filamentous phage multiplication, the aim was to design, describe, and compare two methods for this purpose. As models, three strains of Pseudomonas aeruginosa, containing (pro)phages Pf4, Pf5, and PfLES were used (PAO1, UCBPP-PA14, and LESB58, respectively). Method 1 comprised propagation of phages in 6 L of bacterial culture for 48 h, and method 2 applied 600 mL culture and incubation for 6 days with centrifugation and addition of new medium and inoculum at 2-day intervals. In method 1, phages were propagated by culture agitation, followed by centrifugation and filtration (0.45 and 0.22 μm), and in method 2, cultures were agitated and centrifuged several times to remove bacteria without filtration. Regardless of the propagation method, supernatants were subjected to concentration by PEG8000 and CsCl equilibrium density gradient centrifugation, and phage bands were removed after ultracentrifugation and dialyzed. In the obtained suspensions, phage titer was determined, and concentration of isolated ssDNA from virions was measured. When propagation method 2 was compared with method 1, the phage bands in CsCl were much thicker, phage number was 3.5–7.4 logs greater, and concentration of ssDNA was 7.6–22.4 times higher. When phage count was monitored from days 2 to 6, virion numbers increased for 1.8–5.6 logs, depending on phage. We also observed that filamentous phage plaques faded after 8 h of incubation when the double layer agar spot method was applied, whereas the plaques were visible for 24 h on single-layer agar. Finally, for the first time, we confirmed existence of replicative form and virions of PfLES (pro)phage as well as its ability to produce plaques. Similarly, for the first time, we confirmed plaque production of Pf5 (pro)phage present in P. aeruginosa strain UCBPP-PA14. The described method 2 has many advantages and can be further improved and adopted for filamentous phages of other hosts.

W7-X and the sawtooth instability: towards realistic simulations of current-driven magnetic reconnection

Magnetic reconnection in W7-X is studied by means of global numerical simulations in a series of models of increasing complexity. The magnetic geometry ranges from that of a cylinder to the full three-dimensional field of W7-X, and the equations solved range from ideal magnetohydrodynamics (MHD) to gyrokinetics. We simulate plasmas from the first operation phase with electron cyclotron current drive (ECCD). These are characterized by an equilibrium magnetic field featuring an ECCD-distorted ‘humped’ profile of the rotational transform ι, with ι = 1 in two radial locations. Such plasmas generally show sawtooth activity, hence motivating the present study. We pay particular attention to the role of equilibrium current density gradients in the destabilization of reconnecting modes. When the equilibrium temperature and density gradients are artificially suppressed (to eliminate the pressure gradient drive), the perturbed electrostatic potential is radially localized between the locations at which ι = 1. This is shown with a purely collisionless gyrokinetic model, in cylindrical geometry. In the real toroidal geometry of W7-X, for a non-ideal MHD model including a uniform resistivity, electron inertia and (numerical) viscosity, the same qualitative behaviour is observed. In particular, even if a resonant (m, n) = (1, −1) perturbation is initialized, the most unstable mode is the (m, n) = (−4, 4), where m and n are the poloidal and toroidal mode numbers, respectively. Other modes are destabilized due to geometric coupling. The growth rate of this instability scales as η 1/3, where η is the plasma resistivity, thus suggesting that ECCD drives ideal MHD stable W7-X plasmas towards non-ideal marginality. An ideal magnetohydrodynamic analysis confirms the result. A fluid-kinetic hybrid version of the EUTERPE code shows that gyrokinetic ions have a stabilizing effect on these modes. For W7-X relevant collisionalities, the growth rate scales linearly with the electron skin depth, d e. Implications of our results for sawtoothing W7-X operation are discussed.

Suppression of turbulence in the drift-resistive plasma where zonal flow changes direction

The edge region of quasi-adiabatic plasma is pedagogically simulated by the dynamics between the electric potential $\unicode[STIX]{x1D711}$ and the electron density $n$ whose equilibrium density gradient is negative and held fixed. The zonal flow (ZF) $V$ is either enforced sinusoidally or generated self-consistently from the turbulence. Cross-phase $\unicode[STIX]{x1D6FF}$ between $\unicode[STIX]{x1D711}$ and $n$ , which is important in the determination of the turbulence level and the transport, is strongly influenced by the ZF. In the region near $V=0$ , $\unicode[STIX]{x1D6FF}$ becomes negative due to the large gradient of the ZF. It is found that the instabilities are quenched there, and the fluctuations decay. The ZF thus works as a transport barrier in the region where the ZF changes direction with large gradient.

Validity limits of the passive treatment of impurities in gyrokinetic tokamak simulations

In gyrokinetic simulations of turbulent impurity transport, trace impurity species are often treated as passive species, in the sense that they are not included in Maxwell equations. This is consistent with the assumption that impurities with low enough concentrations are impacted by turbulence generated by electrons and main ions, but do not impact it significantly in return. In this work, we relax this assumption, and investigate the active impacts of impurity on impurity transport as a function of its concentration, in the presence of trapped-particle-driven turbulence. We focus on W40+ tungsten, which is relevant for modern tokamaks, and adopt a reduced gyrokinetic bounce-averaged model for trapped particles in a simplified tokamak geometry. The impacts depend on the relationship between equilibrium density gradient and temperature gradient. When these gradients are equal, we observe that tungsten can be treated as a passive species for concentrations below . Above this concentration, the impurity significantly impacts both density and heat transport, essentially quenching them for concentrations above 10−3. This quenching occurs as electric potential fluctuations become in phase with impurity density fluctuations.

Plasma instability of magnetically enhanced vacuum arc thruster

A two-fluid flowing plasma model is applied to describe the plasma rotation and resulted instability evolution in magnetically enhanced vacuum arc thruster (MEVAT). Typical experimental parameters are employed, including plasma density, equilibrium magnetic field, ion and electron temperatures, cathode materials, axial streaming velocity, and azimuthal rotation frequency. It is found that the growth rate of plasma instability increases with growing rotation frequency and field strength, and with descending electron temperature and atomic weight, for which the underlying physics are explained. The radial structure of density fluctuation is compared with that of equilibrium density gradient, and the radial locations of their peak magnitudes are very close, implying that the mode may be driven by density gradient. Temporal evolution of perturbed mass flow in the cross section of plasma column is also presented, which behaves in the form of clockwise rotation (direction of electron diamagnetic drift) at edge and anti-clockwise rotation (direction of ion diamagnetic drift) in the core, separated by a mode transition layer from n = 0 to n = 1. This work, to our best knowledge, is the first treatment of plasma instability caused by rotation and axial flow in MEVAT, and is also of great practical interest for other electric thrusters where rotating plasma is concerned for long-time stable operation and propulsion efficiency optimization.

Arrowtail RNA for Ligand Display on Ginger Exosome-like Nanovesicles to Systemic Deliver siRNA for Cancer Suppression

Exosomes have shown increasing potential as delivery vesicles for therapy, but challenges like cost/yield, drug payload, and targeting specificity still exist. Plant derived exosome-like nanoparticles have been reported as a promising substitution and exhibit biocompatibility through oral, intranasal administration; however, systemic delivery of siRNA by exosome-like nanoparticles directly isolated from plants has not been reported. Recently, we reported the control of RNA orientation to decorate human derived exosome with cell targeting ligands for specific delivery of siRNA to tumors. Here, we expand to the application of arrowtail RNA nanoparticles for displaying ligands on ginger derived exosome-like nanovesicles (GDENs) for siRNA delivery and tumor inhibition through IV administration. Cushion ultracentrifugation coupled with equilibrium density gradient ultracentrifugation were used for purifying GDENs that displayed size, density, and morphology similar to human derived exosomes. Folic acid (FA), as a ligand, was displayed on the surface of GDENs for targeted delivery of survivin siRNA to KB cancer models. In vitro gene knockdown efficacy by FA-3WJ/GDENs/siRNA complex was comparable to transfection. We observed inhibition of tumor growth on a xenograft model by intravenous administration, which reveals the potential of GDENs as an economic delivery system for siRNA.

Perennial Prescience.

Perennial Prescience.

Tripolar vortices in ion-temperature-gradient mode with non-Maxwellian electrons in an inhomogeneous magnetoplasma

We consider a low frequency plasma comprising of Kappa distributed electrons and Maxwellian ions embedded in an external magnetic field in toroidal ion-temperature-gradient driven modes. A set of nonlinear equations are derived in the presence of equilibrium density, temperature, and magnetic field gradients. In the nonlinear regime, solutions in the form of tripolar vortices are derived by using Braginskii’s transport equations. It has been observed that the scale lengths over which the nonlinear vortex structures form get modified in the presence of Kappa distributed electrons. In tokamak the present study is applicable where non-Maxwellian population has been observed in electron cyclotron heating experiments and resonant frequency heating.

Isolation of Endoplasmic Reticulum and Its Membrane.

The association of ribosomes with the rough endoplasmic reticulum (ER) is dependent on Mg2+. The ribosomes can be stripped from the ER by removal of Mg2+ from the medium, resulting in a reduction in the ER membrane density and a diagnostic shift in migration when ER vesicles are analyzed by equilibrium density gradient centrifugation. Here, I describe the isolation of microsomes from Arabidopsis, followed by the use of the density shift approach in conjunction with equilibrium density gradient centrifugation as a means to diagnose whether a protein is associated with the ER. The same approach can also be used as a means to enrich for ER membranes.

DNA 형태 적응을 거쳐 P2sir-관련 도움파지 비효율성을 극복하는 박테리오파지 P4 sid<sup>+</sup> 유도체 정성 연구

A certain size of DNA (28-29 kb long) to be packaged into P2-size head and the mutation in sid gene of bacteriophage P4 are the major factors to overcome sir-associated helper inefficiency. To clarify whether the presence of sid mutation is essential to overcome sir-associated helper inefficiency or not, we tested the P4 derivative, P4 delRI::kmr, which is and whose genome size supposed to be 28.5 kb long in the case of being packaged into -sized large head. As P4 delRI::kmr showed the low EOP with P2 sir3 lysogen, P4 delRI::kmr phage stock was prepared in P2 sir3 lysogen host to increase the EOP with P2 sir3 lysogen. Through this process, P4 delRI::kmr had been adapted for P2 sir3 lysogen. With a CsCl buoyant equilibrium density gradient experiment and gel electrophoresis of the isolated DNA, it was evident that the adaptation of P4 delRI::kmr for P2 sir3 lysogen was caused by the conformational change of DNA to be packaged into large head. The burst size determination experiments with P4 delRI::kmr phage stock adapted for P2 sir3 lysogen and normal P4 delRI::kmr phage stock showed that not the sid mutation but the size of DNA to be packaged (28-29 kb long) was essential to overcome sir-associated helper inefficiency.

Stabilization of electron-scale turbulence by electron density gradient in national spherical torus experiment

Theory and experiments have shown that electron temperature gradient (ETG) turbulence on the electron gyro-scale, k⊥ρe ≲ 1, can be responsible for anomalous electron thermal transport in NSTX. Electron scale (high-k) turbulence is diagnosed in NSTX with a high-k microwave scattering system [D. R. Smith et al., Rev. Sci. Instrum. 79, 123501 (2008)]. Here we report on stabilization effects of the electron density gradient on electron-scale density fluctuations in a set of neutral beam injection heated H-mode plasmas. We found that the absence of high-k density fluctuations from measurements is correlated with large equilibrium density gradient, which is shown to be consistent with linear stabilization of ETG modes due to the density gradient using the analytical ETG linear threshold in F. Jenko et al. [Phys. Plasmas 8, 4096 (2001)] and linear gyrokinetic simulations with GS2 [M. Kotschenreuther et al., Comput. Phys. Commun. 88, 128 (1995)]. We also found that the observed power of electron-scale turbulence (when it exists) is anti-correlated with the equilibrium density gradient, suggesting density gradient as a nonlinear stabilizing mechanism. Higher density gradients give rise to lower values of the plasma frame frequency, calculated based on the Doppler shift of the measured density fluctuations. Linear gyrokinetic simulations show that higher values of the electron density gradient reduce the value of the real frequency, in agreement with experimental observation. Nonlinear electron-scale gyrokinetic simulations show that high electron density gradient reduces electron heat flux and stiffness, and increases the ETG nonlinear threshold, consistent with experimental observations.

Toroidal ion-temperature-gradient driven vortices in an inhomogeneous magnetoplasma with non-Maxwellian electrons

Nonlinear equations which govern the dynamics of low-frequency toroidal ion-temperature-gradient driven modes (i.e., ω≪ωci, where ωci is the ion gyro-frequency) are derived in the presence of equilibrium density, temperature, and magnetic field gradients. In the nonlinear case, solutions in the form of dipolar vortices and vortex street are presented for a plasma comprising of Maxwellian ions and nonthermal electrons that are embedded in an external magnetic field. By using Braginskii's transport equations for the Maxwellian ions and Kappa distributed electrons, the coupled mode equations for the system under consideration are derived. The results have been applied in Tokamak plasmas, and it has been observed that the scale lengths over which the nonlinear vortex structures form get modified in the presence of Kappa distributed electrons. The present study is also applicable to tokamaks and stellarators where non-Maxwellian population has been observed in resonant frequency heating, electron cyclotron heating experiments, and in runaway electrons.

P2 <sub>sir3</sub>-sized head에 packaging 되기 용이한 크기의 박테리오파지 P4 유도체 조성과 정성 연구

The term “P2 sir-associated helper inefficiency” has been used to define the inefficient helper capability of P2 sir mutants for their satellite bacteriophage P4. The aim of this study was to investigate the factors overcoming P2 sir-associated helper inefficiency. At first, we verified whether the P2 cos region containing P4 sid71 cosP2 could overcome P2 sir-associated helper inefficiency with P2 sir3. The result was that P4 sid71 cosP2 could not overcome P2 sir-associated helper inefficiency with P2 sir3. Instead of cos region of P2, the size of the DNA packaged into a P2 sir -sized head seems to be important for overcoming P2 sir-associated helper inefficiency. In the present work, three kinds of P4 derivatives with packaged DNA sizes between those of P4 ost1 and P4 ost2, were constructed through DNA manipulation. In one P4 derivative, P4 sid71 delRI::apr, the size of the packaged DNA was identified with a CsCl buoyant equilibrium density gradient experiment. According to the burst sizes of the P4 derivatives, they could overcome P2 sir3-associated helper inefficiency. The size of the P4 derivative DNA suitable for packaging into a P2 sir3 -sized head was 28-29 kb.

Large scale purification of RNA nanoparticles by preparative ultracentrifugation.

Purification of large quantities of supramolecular RNA complexes is of paramount importance due to the large quantities of RNA needed and the purity requirements for in vitro and in vivo assays. Purification is generally carried out by liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), or agarose gel electrophoresis (AGE). Here, we describe an efficient method for the large-scale purification of RNA prepared by in vitro transcription using T7 RNA polymerase by cesium chloride (CsCl) equilibrium density gradient ultracentrifugation and the large-scale purification of RNA nanoparticles by sucrose gradient rate-zonal ultracentrifugation or cushioned sucrose gradient rate-zonal ultracentrifugation.

Fast sawtooth reconnection at realistic Lundquist numbers

Magnetic reconnection, a ubiquitous phenomenon in astrophysics, space science and magnetic confinement research, frequently proceeds much faster than predicted by simple resistive MHD theory. Acceleration can result from the break-up of the thin Sweet–Parker current sheet into plasmoids, or from two-fluid effects decoupling mass and magnetic flux transport over the ion inertial length or the drift scale depending on the absence or presence of a strong magnetic guide field. We describe new results on the modelling of sawtooth reconnection in a simple tokamak geometry (circular cylindrical equilibrium) pushed to realistic Lundquist numbers for present day tokamaks. For the resistive MHD case, the onset criteria and the influence of plasmoids on the reconnection process agree well with earlier results found in the case of vanishing magnetic guide fields. While plasmoids are also observed in two-fluid calculations, they do not dominate the reconnection process for the range of plasma parameters considered in this study. In the two-fluid case they form as a transient phenomenon only. The reconnection times become weakly dependent on the S-value and for the most complete model—including two-fluid effects and equilibrium temperature and density gradients—agree well with those experimentally found on ASDEX Upgrade

Low-density lipoprotein particle diameter and mortality: the Ludwigshafen Risk and Cardiovascular Health Study

The aim of the study was to examine whether differences in average diameter of low-density lipoprotein (LDL) particles were associated with total and cardiovascular mortality. We studied 1643 subjects referred to coronary angiography, who did not receive lipid-lowering drugs. During a median follow-up of 9.9 years, 398 patients died, of these 246 from cardiovascular causes. We calculated average particle diameters of LDL from the composition of LDL obtained by β-quantification. When LDL with intermediate average diameters (16.5-16.8 nm) were used as reference category, the hazard ratios (HRs) adjusted for cardiovascular risk factors for death from any cause were 1.71 (95% CI: 1.31-2.25) and 1.24 (95% CI: 0.95-1.63) in patients with large (>16.8 nm) or small LDL (<16.5 nm), respectively. Adjusted HRs for death from cardiovascular causes were 1.89 (95% CI: 1.32-2.70) and 1.54 (95% CI: 1.06-2.12) in patients with large or small LDL, respectively. Patients with large LDL had higher concentrations of the inflammatory markers interleukin (IL)-6 and C-reactive protein than patients with small or intermediate LDL. Equilibrium density gradient ultracentrifugation revealed characteristic and distinct profiles of LDL particles in persons with large (approximately even distribution of intermediate-density lipoproteins and LDL-1 through LDL-6) intermediate (peak concentration at LDL-4) or small (peak concentration at LDL-6) average LDL particle diameters. Calculated LDL particle diameters identify patients with different profiles of LDL subfractions. Both large and small LDL diameters are independently associated with increased risk mortality of all causes and, more so, due to cardiovascular causes compared with LDL of intermediate size.

Popeye Domain Containing 1 (Popdc1/Bves) Is a Caveolae-Associated Protein Involved in Ischemia Tolerance

Popeye domain containing1 (Popdc1), also named Bves, is an evolutionary conserved membrane protein. Despite its high expression level in the heart little is known about its membrane localization and cardiac functions. The study examined the hypothesis that Popdc1 might be associated with the caveolae and play a role in myocardial ischemia tolerance. To address these issues, we analyzed hearts and cardiomyocytes of wild type and Popdc1-null mice. Immunoconfocal microscopy revealed co-localization of Popdc1 with caveolin3 in the sarcolemma, intercalated discs and T-tubules and with costameric vinculin. Popdc1 was co-immunoprecipitated with caveolin3 from cardiomyocytes and from transfected COS7 cells and was co-sedimented with caveolin3 in equilibrium density gradients. Caveolae disruption by methyl-β-cyclodextrin or by ischemia/reperfusion (I/R) abolished the cellular co-localization of Popdc1 with caveolin3 and modified their density co-sedimentation. The caveolin3-rich fractions of Popdc1-null hearts redistributed to fractions of lower buoyant density. Electron microscopy showed a statistically significant 70% reduction in caveolae number and a 12% increase in the average diameter of the remaining caveolae in the mutant hearts. In accordance with these changes, Popdc1-null cardiomyocytes displayed impaired [Ca+2]i transients, increased vulnerability to oxidative stress and no pharmacologic preconditioning. In addition, induction of I/R injury to Langendorff-perfused hearts indicated a significantly lower functional recovery in the mutant compared with wild type hearts while their infarct size was larger. No improvement in functional recovery was observed in Popdc1-null hearts following ischemic preconditioning. The results indicate that Popdc1 is a caveolae-associated protein important for the preservation of caveolae structural and functional integrity and for heart protection.