Assembly Of Particles Research Articles

Stress accumulation versus shape flattening in frustrated, warped-jigsaw particle assemblies

Geometrically frustrated assembly has emerged as an attractive paradigm for understanding and engineering assemblies with self-limiting, finite equilibrium dimensions. We propose and study a novel 2D particle based on a so-called ‘warped jigsaw’ (WJ) shape design: directional bonds in a tapered particle favor curvature along multi-particle rows that frustrate 2D lattice order. We investigate how large-scale intra-assembly stress gradients emerge from the microscopic properties of the particles using a combination of numerical simulation and continuum elasticity. WJ particles can favor anisotropic ribbon assemblies, whose lateral width may be self-limiting depending on the relative strength of cohesive to elastic forces in the assembly, which we show to be controlled by the range of interactions and degree of shape misfit. The upper limits of self-limited size are controlled by the crossover between two elastic modes in assembly: the accumulation of shear with increasing width at small widths giving way to unbending of preferred row curvature, permitting assembly to grow to unlimited sizes. We show that the stiffness controlling distinct elastic modes is governed by combination and placement of repulsive and attractive binding regions, providing a means to extend the range of accumulating stress to sizes that are far in excess of the single particle size, which we corroborate via numerical studies of discrete particles of variable interactions. Lastly, we relate the ground-state energetics of the model to lower and upper limits on equilibrium assembly size control set by the fluctuations of width along the ribbon boundary.

A kinetic model for the impact of packaging signal mimics on genome encapsulation

Inspired by recent experiments on the spontaneous assembly of virus-like particles from a solution containing a synthetic coat protein and double-stranded DNA, we put forward a kinetic model that has as main ingredients a stochastic nucleation and a deterministic growth process. The efficiency and rate of DNA packaging strongly increase after tiling the DNA with CRISPR-Cas proteins at predesignated locations, mimicking assembly signals in viruses. Our model shows that treating these proteins as nucleation-inducing diffusion barriers is sufficient to explain the experimentally observed increase in encapsulation efficiency, but only if the nucleation rate is sufficiently high. We find an optimum in the encapsulation kinetics for conditions where the number of packaging signal mimics is equal to the number of nucleation events that can occur during the time required to fully encapsulate the DNA template, presuming that the nucleation events can only take place adjacent to a packaging signal. Our theory is in satisfactory agreement with the available experimental data.

Line Patterns and Fractured Coatings in Deposited Colloidal Hydrochar on Glass Substrates after Evaporation of Water

Patterns of assembled colloidal particles can form on substrates due to solvent evaporation, and here we studied such phenomena in the drying of monodispersed colloidal hydrochar dispersions prepared by the hydrothermal carbonization of glucose and purified by dialysis. During the evaporation of water, line patterns or, in some cases, mud-like patterns formed. The line formation was investigated as a function of the pH of the dispersion, substrate shape, particle concentration, and concentration of sodium dodecylsulfate (SDS). The lines comprised dense assemblies of hydrochar particles. The line width increased with the successive evaporation of water. Sharper lines formed with the addition of SDS, which was ascribed to the effects of solubilization or moderated interactions. At greater particle concentrations, we also observed a continuous layer of colloidal particles between the lines. A mechanism for the line pattern formation derived from the literature on other colloids was proposed. Mud-like patterns formed on the substrate in concentrated samples without SDS addition and were put in the context of the formation of cracks in the drying of colloidal coatings. Hydrochars belong to carbon-rich colloids, which are of fundamental and technological importance. This research could be useful for in situ line printing within microfluidic devices, for example.

Nucleation and growth of ellipsoidal crystals in a supercooled binary melt

When considering the evolution of particulate assemblages in supercooled and supersaturated liquids, the shape of crystals often differs from spherical. Existing experiments show that evolved crystals often are ellipsoidal. Motivated by important applications in materials physics, chemistry, and biophysics, we develop here a growth theory of a polydisperse ensemble of ellipsoidal crystals in a binary supercooled melt. An integro-differential model of kinetic and balance equations supplemented by initial and boundary conditions that describe desupercooling dynamics and particle-volume distribution function with allowance for the ellipsoidal shape of growing crystals is formulated and solved analytically using the saddle-point technique for a Laplace-type integral. The distribution function increases up to the maximal volume of particles and shifts to larger crystal volumes with time. The solute concentration substantially changes the evolution of a particulate assemblage. The melt supercooling decays faster and the particle-volume distribution function is lower with increasing the initial solute concentration. The theory under consideration generalizes previously developed growth theories for spherical crystals in a binary melt (Alexandrov 2014 J. Phys. A: Math. Theor. 47 125102) and ellipsoidal crystals in a single-component melt (Nikishina and Alexandrov 2021 Phil. Trans. R. Soc. A 379 20200306).

Effect of dietary Sophora alopecuroides supplementation on differential expression of intramuscular fat-related genes of Ningxia Tan sheep as determined by transcriptome sequencing

The present study investigated the effect of different dietary levels (0%, 0.25%, 0.5%, 1%, 2%) of Sophora alopecuroides (S. alopecuroides) on intramuscular fat (IMF) content in Ningxia Tan sheep. Candidate genes affecting IMF deposition were screened by comparing the RNA-Seq profiles of test (2% S. alopecuroides) and control (no S. alopecuroides) groups. The results showed that the intramuscular fat content of the diet supplemented with S. alopecuroides was significantly lower than that of the control group, and the intramuscular fat content decreased significantly with the increase of the dietary content of S. alopecuroides (P < 0.05). The highest IMF content of the control group (group A) was 4.21%, which was significantly higher than that of the other four experimental groups (B, C, D, E) (P < 0.05). Moreover, 66 genes were significantly upregulated and 202 genes were significantly downregulated in the test group compared to the control. Gene Ontology functional annotation revealed that the differentially expressed genes were closely related to plasma lipoprotein particle assembly, plasma lipoprotein particle organization, protein-lipid complex subunit organization, and other biological processes. KEGG pathway analysis showed that the differentially expressed genes belonged to 147 metabolic pathways. Finally, four differential genes which may be related to IMF deposition were screened out in PPAR signalling pathway, fatty acid biosynthesis, fatty acid degradation, and fatty acid metabolism. The quantitative analysis of four differential genes including ACSL3, PLIN2, ABCA1 and ANGPTL4 was carried out by RT-qPCR method, and the results were basically consistent with those of RNA-seq. Among them, the quantitative results of PLIN2 gene were slightly different from the sequencing results, but the difference was not significant, and they may be false positive. This study lays the foundation for understanding the molecular mechanism regulating mutton quality, and provides a theoretical basis for the study of S. alopecuroides as a feed additive to improve mutton meat quality and impact the gene function.

Assembly of aluminum oxide particles with lignin-acrylic acid polymer in saline systems

The flocculation process is widely used for separating particles from suspension systems. To make this process more environmentally friendly, a biobased flocculant was proposed to be used in this work. This study involves the experimental evaluation of a flocculation system of aluminum oxide particles (with two different sizes) and a kraft lignin-acrylic acid (KL-AA) polymer, as a biobased-flocculant, with advanced tools, such as focused beam reflectance measurement, vertical scanning analyzer, and gravitational scanning analyzer. A statistical framework was adapted to model the experimental results based on the mixture distribution theory, which led to a more in-depth analysis of the shape and size of flocs in the heterogeneous suspension system. The results revealed that the flocculation process of aluminum oxide suspension was altered by the salinity of the medium as well as the size of aluminum oxide particles. It was found that the flocs formed with KL-AA at a higher NaCl concentration had smaller sizes and exhibited higher shear resistance than the flocs formed in the absence of salt. Furthermore, by increasing the concentration of NaCl, the particles tended to possess more spherical shapes. The results also showed that the fractal dimension for flocculated particles increased by concentrating NaCl in the suspensions. Under gravity and centrifugal forces, the impact of KL-AA in flocculating particles was insignificant in the presence of salt.

Wafer-Scale Particle Assembly in Connected and Isolated Micromachined Pockets via PDMS Rubbing.

The present contribution reports on a study aiming to find the most suitable rubbing method for filling arrays of separated and interconnected micromachined pockets with individual microspheres on rigid, uncoated silicon substrates without breaking the particles or damaging the substrate. The explored dry rubbing methods generally yielded unsatisfactory results, marked by very large percentages of empty pockets and misplaced particles. On the other hand, the combination of wet rubbing with a patterned rubbing tool provided excellent results (typically <1% of empty pockets and <5% of misplaced particles). The wet method also did not leave any damage marks on the silicon substrate or the particles. When the pockets were aligned in linear grooves, markedly the best results were obtained when the ridge pattern of the rubbing tool was moved under a 45° angle with respect to the direction of the grooves. The method was tested for both silica and polystyrene particles. The proposed assembly method can be used in the production of medical devices, antireflective coatings, and microfluidic devices with applications in chemical analysis and/or catalysis.

Mesh generation of complex three‐dimensional geometries for beam‐particle modeling of fracture

AbstractIn this article, we propose a three‐dimensional meshing algorithm for discrete models combining the lattice approach with the polyhedral particle approach. Our aim is to be able to leverage readily available, well‐supported meshers to handle various geometries. We use them to generate a tetrahedral mesh, which our mesher then converts to a polyhedral mesh. The input mesh serves as geometrical support to generate the nodes of the discrete mesh. The desired shape is obtained with an assembly of convex polyhedral particles—without having to clip them. We show that this approach enables meshing convex or concave geometries with sharp edges, curved features, and more. Several three‐dimensional geometries are presented to support this claim and illustrate the capabilities of the mesher. We provide a detailed analysis of its isotropy. The geometric isotropy is studied by analyzing the orientation of the generated beams. The mechanical isotropy is verified by assessing the properties of the elasticity tensor. Finally, we show that the new mesh retains its ability to be a good support for the generation of realistic cracking patterns.

Human Retrovirus Genomic RNA Packaging.

Two non-covalently linked copies of the retrovirus genome are specifically recruited to the site of virus particle assembly and packaged into released particles. Retroviral RNA packaging requires RNA export of the unspliced genomic RNA from the nucleus, translocation of the genome to virus assembly sites, and specific interaction with Gag, the main viral structural protein. While some aspects of the RNA packaging process are understood, many others remain poorly understood. In this review, we provide an update on recent advancements in understanding the mechanism of RNA packaging for retroviruses that cause disease in humans, i.e., HIV-1, HIV-2, and HTLV-1, as well as advances in the understanding of the details of genomic RNA nuclear export, genome translocation to virus assembly sites, and genomic RNA dimerization.

Generalized coupled dipole method for thermal far-field radiation

We introduce a many-body theory for thermal far-field emission of dipolar dielectric and metallic nanoparticles in the vicinity of a substrate within the framework of fluctuational electrodynamics. Our theoretical model includes the possibility to define the temperatures of each nanoparticle, the substrate temperature, and the temperature of the background thermal radiation, separately. To demonstrate the versatility of our method, we apply it in an exemplary way by discussing the thermal radiation of four particle assemblies of SiC and Ag nanoparticles above a planar SiC and Ag substrate. Furthermore, we use discrete dipole approximation to determine the thermal emission of a spherical nanoparticle in free space and close to a substrate. Finally, the calculation of the thermal far-field radiation of a sharp Si tip close to a SiC substrate using the discrete dipole approximation including the near-field scattering by the tip as well as the thermal emission of the tip and the contribution of the substrate which is partially blocked by the tip serves as another example.

Actin dynamics regulate proteasome homeostasis

Cells require thousands of unique proteins to be in the right place, at the right time, in the right amounts and with the right modifications. They do this through several processes collectively known as protein homeostasis. TORC1 is a principal regulator of protein homeostasis, coordinating protein synthesis and degradation. The proteasome, composed of a core particle and one or two regulatory particles, degrades unwanted protein. Diverse stresses cause a protein homeostasis imbalance: inhibiting TORC1 and the misfolded/damaged protein load. Following TORC1 inhibition, proteasome regulatory particle assembly chaperone (RPAC) translation is increased and thus cells assemble more proteasomes to degrade the damaged and misfolded proteins, thereby restoring protein homeostasis. Using yeast, we identify an endocytic protein, Ede1, that interacts with and is critical for translation of RPAC mRNA following TORC1 inhibition. We find two further endocytic proteins important for RPAC translation regulation. Mutants of these proteins cause altered Arp2/3 activity, and hence altered formation of actin patches/endocytic sites. We show that RPAC mRNA is transported on actin cables and interacts with actin patches. TORC1 inhibition depolarises the actin cytoskeleton, causing RPAC mRNA accumulation on actin patches concurrent with translation. We demonstrate Ede1 is essential for RPAC mRNA localisation regulation following rapamycin treatment. This work shows that, upon actin depolarisation, RPAC mRNA is recruited to actin patches, likely by Ede1, and translation occurs. Actin regulation is therefore a key element of proteasome (and therefore protein) homeostasis.

Anisotropic Thermally Conductive Composites Enabled by Acoustophoresis and Stereolithography

AbstractOpportunities to improve thermal management in electronic devices are currently hindered by processing constraints that limit thermal conductivity in polymer‐matrix composites. Active patterning of filler particles is a promising route to improve conductivity while retaining processability by improving particle contact density and directing heat along optimized pathways. This study employs acoustic patterning to align and compact filler particles into stripes during stereolithographic 3D printing. This approach produces polymer‐based composite materials with highly efficient embedded heat transport pathways which reach 95 vol% particle utilization (relative to the parallel conduction upper limit). These composites exhibit anisotropic thermal conductivity up to 300% higher than unpatterned composites, with in‐plane anisotropy ratios of up to 350%. Combining this high conductivity with 3D printing enables materials with engineered heat networks that optimize transport from hot spots to heatsinks while maintaining low viscosity for fast particle patterning and for infiltration around electronic components. Finally, numerical simulations of acoustic assembly of particles with varied geometry, when compared to experimentally characterized particle packing, illuminate pathways for further improving conductivity by optimizing particle geometry for alignment and stacking of particles with maximum contact surface area.

Assembly of alumina particles in aqueous suspensions induced by high‐frequency AC electric field

AbstractThe role of high‐frequency alternating current (AC) electric field in the assembly of alumina particles in aqueous media was investigated. Field–particle interactions were in situ investigated for coarse and fine powder particles in very dilute suspensions. For both coarse and fine particles, AC field‐induced assembly led to the formation of chains of particles within a minute, which were aligned in the field direction. However, a much finer network of particle chains evolved in fine particle suspensions. Threshold field strength for chain formation was also lower for fine particles (28 V/mm) than for coarse particles (50 V/mm), suggesting stronger interactions for finer particles. Chain length increased with both field strength and field duration. Chain formation was attributed to mutual dielectrophoretic (DEP) interaction forces. Increase in DEP forces with field strength resulted in enhanced interactions. For finer particles, decreasing interparticle distance might have favored stronger interactions. Suspension microstructure was disrupted as soon as the field was removed. However, higher field duration was associated with an improved pattern stability and retention following the field removal. Finally, particle motion was studied in deliberately applied spatially nonuniform AC field, which revealed different mechanisms of chain formation for coarse (negative‐DEP) and fine (positive‐DEP) particles.

A Hybrid Particle/Finite Element Model with Surface Roughness for Stone Masonry Analysis

Circular and spherical particle models are a class of discrete elements (DEM) that have been increasingly applied to fracture studies of quasi-brittle materials, such as rock and concrete, due to their proven ability to simulate fracture processes through random particle assemblies representing quasi-brittle materials at the grain scale. More recently, DEM models have been applied to old stone masonry fracture studies. In order to extend its applicability to structures of larger dimensions, an enhanced hybrid particle model is proposed here that allows finite elements with a given surface roughness, provided by the discretization of the element boundary with particles, to interact with the particulate media in which they are embedded. The performance of the hybrid model is compared with that of a traditional all-particle model under uniaxial testing. It is shown that similar results are obtained, namely, in the elastic phase, figures of rupture and pre-peak and post-peak behavior, while the hybrid model allows for a significant computational run time reduction of 20% to 25% in the coarse particle assemblies. Finally, the proposed hybrid model is applied in the simulation of shear tests of stone masonry walls and dry and mortared joints, providing reasonably good agreement with both the experimental results and predictions. For the rubble masonry tests, the hybrid model allows for a computation run time reduction of around 40% when compared with an all-particle model.

Marine Sesquiterpenes for Plant Protection: Discovery of Laurene Sesquiterpenes and Their Derivatives as Novel Antiviral and Antiphytopathogenic Fungal Agents.

The unreasonable use or long-term use of a single variety of pesticide has led to drug resistance and made the pesticides ineffective. Therefore, the creation of new, efficient, and low-risk pesticides is imminent. Marine natural products play a vital role in serving as new lead compounds. In this work, we realized the efficient preparation of nine marine sesquiterpenes with the Stork-Danheiser reaction as the key step and designed and synthesized a series of their derivatives. The antiviral activity and antifungal activity research showed that these compounds exhibited good to excellent biological activities. Compounds 7b and 8e displayed significantly higher antiviral activities against tobacco mosaic virus (TMV) than ribavirin and could be used as new antiviral candidates. The antiviral mode of action research indicated that compound 8e inhibited the formation of the 20S protein disk by acting on the TMV coat protein and therefore inhibited the assembly of TMV particles. This work provides a new idea for the development of new pesticides based on marine sesquiterpenes.

Muramidase, nuclease, or hypothetical protein genes intervene between paired genes encoding DNA packaging terminase and portal proteins in Wolbachia phages and prophages.

Genomes of the obligate intracellular alpha proteobacterium Wolbachia pipientis often encode prophage-like regions, and in a few cases, purified particles have been recovered. Because the structure of a conserved WO phage genome has been difficult to establish, we examined paired terminase and portal genes in Wolbachia phages and prophages, relative to those encoded by the gene transfer agent RcGTA from the free-living alpha proteobacterium Rhodobacter capsulatus. Terminase and portal proteins from Wolbachia have higher similarity to orthologs encoded by RcGTA than to orthologs encoded by bacteriophage lambda. In lambdoid phages, these proteins play key roles in assembly of mature phage particles, while in less well-studied gene transfer agents, terminase and portal proteins package random fragments of bacterial DNA, which could confound elucidation of WO phage genomes. In WO phages and prophages, terminase genes followed by a short gpW gene may be separated from the downstream portal gene by open-reading frames encoding a GH_25 hydrolase/muramidase, a PD-(D/E)XK nuclease, a hypothetical protein and/or a RelE/ParE toxin-antitoxin module. These aspects of gene organization, coupled with evidence for a low, non-inducible yield of WO phages, and the small size of WO phage particles described in the literature raise the possibility that Wolbachia prophage regions participate in processes that extend beyond conventional bacteriophage lysogeny and lytic replication. These intervening genes, and their possible relation to functions associated with GTAs, may contribute to variability among WO phage genomes recovered from physical particles and impact the ability of WO phages to act as transducing agents.

Bioinspired Approaches to Self-Assembly of Virus-like Particles: From Molecules to Materials.

When viewed through the lens of materials science, nature provides a vast library of hierarchically organized structures that serve as inspiration and raw materials for new synthetic materials. The structural organization of complex bioarchitectures with advanced functions arises from the association of building blocks and is strongly supported by ubiquitous mechanisms of self-assembly, where interactions among components result in spontaneous assembly into defined structures. Viruses are exemplary, where a capsid structure, often formed from the self-assembly of many individual protein subunits, serves as a vehicle for the transport and protection of the viral genome. Higher-order assemblies of viral particles are also found in nature with unexpected collective behaviors. When the infectious aspect of viruses is removed, the self-assembly of viral particles and their potential for hierarchical assembly become an inspiration for the design and construction of a new class of functional materials at a range of different length scales.Salmonella typhimurium bacteriophage P22 is a well-studied model for understanding viral self-assembly and the construction of virus-like particle (VLP)-based materials. The formation of cage-like P22 VLP structures results from scaffold protein (SP)-directed self-assembly of coat protein (CP) subunits into icosahedral capsids with encapsulation of SP inside the capsid. Employing the CP-SP interaction during self-assembly, the encapsulation of guest protein cargos inside P22 VLPs can be achieved with control over the composition and the number of guest cargos. The morphology of cargo-loaded VLPs can be altered, along with changes in both the physical properties of the capsid and the cargo-capsid interactions, by mimicking aspects of the infectious P22 viral maturation. The structure of the capsid differentiates the inside cavity from the outside environment and serves as a protecting layer for the encapsulated cargos. Pores in the capsid shell regulate molecular exchange between inside and outside, where small molecules can traverse the capsid freely while the diffusion of larger molecules is limited by the pores. The interior cavity of the P22 capsid can be packed with hundreds of copies of cargo proteins (especially enzymes), enforcing intermolecular proximity, making this an ideal model system in which to study enzymatic catalysis in crowded and confined environments. These aspects highlight the development of functional nanomaterials from individual P22 VLPs, through biomimetic design and self-assembly, resulting in fabrication of nanoreactors with controlled catalytic behaviors.Individual P22 VLPs have been used as building blocks for the self-assembly of higher-order structures. This relies on a balance between the intrinsic interparticle repulsion and a tunable interparticle attraction. The ordering of VLPs within three-dimensional assemblies is dependent on the balance between repulsive and attractive interactions: too strong an attraction results in kinetically trapped disordered structures, while decreasing the attraction can lead to more ordered arrays. These higher-order assemblies display collective behavior of high charge density beyond those of the individual VLPs.The development of synthetic nanomaterials based on P22 VLPs demonstrates how the potential for hierarchical self-assembly can be applied to other self-assembling capsid structures across multiple length scales toward future bioinspired functional materials.

The Optical Absorption Force Allows Controlling Colloidal Assembly Morphology at an Interface

AbstractGaining control on particle–particle interactions and in this way on their (self)‐assembled structures is essentialfor colloidal and material sciences. Currently, different strategies are described to achieve such control, however, all of them lack the spatiotemporal resolution required at the microscale. In this work, the potential of combining optical trapping and resonant photoexcitation for modifying particle–particle interactions and subsequent assembling of dye‐doped particles at the solution interface is demonstrated. The particle assemblies prepared by nonresonant 1064 nm optical trapping undergo morphology changes after resonant photoexcitation of the embedded dye molecules. Depending on the physicochemical properties of interface, quick hexagonal close packing (HCP)‐rearrangement or explosive dispersion of assemblies is observed at air/solution (A/S) and glass/solution interfaces, respectively. By contrast, by resonant photoexcitation only, the dispersed dye‐doped particles are pushed toward the A/S interface, followed by association to yield HCP‐structured assemblies. The results are rationalized by considering the optical absorption force coupled with other nonoptical forces (e.g., capillary force, dipole–dipole or electrostatic repulsion) at the solution interface. Due to the inherent spatiotemporal properties of light and electronic transition of materials, absorption force is a unique element to control and modify the structural order of particle assemblies at interfaces.

Synthesis of the transmembrane domain of the accessory protein ORF7a of SARS‐CoV‐2 using solid phase peptide synthesis and analyzing the oligomerization state

This research project consists of synthesizing and characterizing the accessory protein ORF7a of SARS‐CoV‐2. ORF7a plays a role in viral assembly and inhibition of this protein would prevent viruses from assembling and spreading within a host’s cells. The goal of this project is to determine the oligomerization state of the protein through a fluorescence assay, which would determine the protein’s structure and how it folds. After determining the oligomerization state of the protein, potential inhibitors could be synthesized and tested for their efficacy at inhibiting the function of the protein. Synthesis of the protein was done using a solid phase peptide synthesis technique. Characterization and purification were done using High Performance Liquid Chromatography (HPLC) as well as Liquid Chromatography Mass Spectrometry (LCMS). Fluorescence assay was performed using a UV‐vis spectrometer. This presentation will highlight current progress with the oligomerization state with the goal of devising an effective inhibitor for ORF7 activity in SARS‐CoV‐2 particle assembly.

KSHV Manipulation of Antigen Presentation via the Ubiquitin Proteasome System

Kaposi Sarcoma‐associated herpesvirus (KSHV, HHV‐8) is a human oncovirus with a two‐stage life cycle consisting of latent and lytic replication. Lytic replication is characterized by the expression of late genes, the assembly of viral particles, and is initiated by the expression of RTA. In addition to being a transcription factor, RTA is an E3 ubiquitin ligase with the ability to target proteins for degradation via the ubiquitin‐proteasome system (UPS). Previously in our lab, through a comparative proteomics study, we identified proteins that displayed increased ubiquitination in the presence of RTA. Three proteins identified are known to be involved in the process of antigen presentation. We hypothesize KSHV RTA is targeting these proteins to maintain a persistent infection. Here we provide preliminary evidence that suggests that RTA targets the process of antigen processing and presentation. suggesting a novel mechanism for evasion of the immune response by KSHV RTA. We demonstrate degradation of HLA, PSMB3, and TAP2 cells expressing RTA. Cells expressing RTA exhibited decreased peptide loading on HLA. Our data suggests a novel mechanism for evasion of the host immune response by KSHV RTA.