Getting Hold of the Tobamovirus Particle-Why and How? Purification Routes over Time and a New Customizable Approach.

This article develops a multi-perspective view on motivations and methods for tobamovirus purification through the ages and presents a novel, efficient, easy-to-use approach that can be well-adapted to different species of native and functionalized virions. We survey the various driving forces prompting researchers to enrich tobamoviruses, from the search for the causative agents of mosaic diseases in plants to their increasing recognition as versatile nanocarriers in biomedical and engineering applications. The best practices and rarely applied options for the serial processing steps required for successful isolation of tobamoviruses are then reviewed. Adaptations for distinct particle species, pitfalls, and 'forgotten' or underrepresented technologies are considered as well. The article is topped off with our own development of a method for virion preparation, rooted in historical protocols. It combines selective re-solubilization of polyethylene glycol (PEG) virion raw precipitates with density step gradient centrifugation in biocompatible iodixanol formulations, yielding ready-to-use particle suspensions. This newly established protocol and some considerations for perhaps worthwhile further developments could serve as putative stepping stones towards preparation procedures appropriate for routine practical uses of these multivalent soft-matter nanorods.

Challenges for Studying and Isolating Extracellular Vesicles from Cell-Conditioned Media.

Extracellular vesicles (EV) are small (100-1000 nm) particles that cells release into the extracellular space that have become increasingly famous for their potential in regenerative medicine and for their alterations in diseases such as cancer to promote disease progression, in particular for their potential for intercellular communication. However, studying EV can be challenging due to the broad diversity of both the EV themselves as well as the methods used to study them. This chapter aims to help investigators new to the EV field by describing challenges with studying EV, methods for enriching EV, and a simple EV enrichment protocol using differential ultracentrifugation.

Proteome-Wide Identification of RNA-Dependent Proteins in Lung Cancer Cells.

Following the concept of RNA dependence and exploiting its application in the R-DeeP screening approach, we have identified RNA-dependent proteins in A549 lung adenocarcinoma cells. RNA-dependent proteins are defined as proteins whose interactome depends on RNA and thus entails RNA-binding proteins (RBPs) as well as proteins in ribonucleoprotein complexes (RNPs) without direct RNA interaction. With this proteome-wide technique based on sucrose density gradient ultracentrifugation and fractionation followed by quantitative mass spectrometry and bioinformatic analysis, we have identified 1189 RNA-dependent proteins including 170 proteins which had never been linked to RNA before. R-DeeP provides quantitative information on the fraction of a protein being RNA-dependent as well as it allows the reconstruction of protein complexes based on co-segregation. The RNA dependence of three newly identified RNA-dependent proteins, DOCK5, ELMO2, also known as CED12A, and ABRAXAS1, also known as CCDC98, was validated using western blot analysis, and the direct RNA interaction was verified by iCLIP2 for the migration-related protein DOCK5 and the mitosis-related protein ABRAXAS1. The R-DeeP 2.0 database provides proteome-wide and cell line-specific information from A549 and HeLa S3 cells on proteins and their RNA dependence to contribute to understanding the functional role of RNA and RNA-binding proteins in cancer cells.

Density Gradients, Cellular Structure and Thermal Conductivity of High-Density Polyethylene Foams by Different Amounts of Chemical Blowing Agent.

LDPE (low-density polyethylene) foams were prepared using the improved compression moulding technique (ICM) with relative densities ranging from 0.3 to 0.7 and with different levels of chemical blowing agents (from 1% to 20%). The density gradients, cellular structure and thermal conductivity of the foams were characterized. The density and amount of CBA used were found to have a significant effect on the cellular structure both at the mesoscale (density gradients) and at the microscale (different cell sizes and cell densities). In addition, the thermal conductivity of the samples is very sensitive to the local structure where the heat flux is located. The technique used to measure this property, the Transient Plane Source method (TPS), makes it possible to detect the presence of density gradients. A simple method for determining these gradients based on thermal conductivity data was developed.

Purification of Leaf Mitochondria from Arabidopsis thaliana Using Percoll Density Gradients.

The study of plant mitochondria often requires isolation of mitochondria from plant tissues in intact and functional form. Here, we describe an effective procedure of mitochondrial isolation from leaf tissues and whole seedlings of the model dicot species Arabidopsis thaliana by using differential centrifugation and continuous Percoll density gradients.

Predictions of overloaded concentration profiles in supercritical fluid chromatography.

Here, overloaded concentration profiles were predicted in supercritical fluid chromatography using a combined two-dimensional heat and mass transfer model. The heat balance equation provided the temperature and pressure profiles inside the column. From this the density, viscosity, and mobile phase velocity profiles in the column were calculated. The adsorption model is here expressed as a function of the density and temperature of the mobile phase. The model system consisted of a Kromasil Diol column packed with 2.2-µm particles (i.e., a UHPSFC column) and the solute was phenol eluted with neat carbon dioxide at three different outlet pressures and five different mobile phase flow rates. The proposed model successfully predicted the eluted concentration profiles in all experimental runs with good agreement even with high-density drops along the column. It could be concluded that the radial temperature and density gradients did not significantly influence the overloaded concentration elution profiles.

Colour-coded density-gradients stone mapping: A novel reporting system for stone density on non-contrast computed tomography and its clinical applications.

OBJECTIVE: To presents a novel clinically oriented system to report stone attenuation on non-contrast computed tomography (NCCT) using colour-coded density-gradients stone mapping and its clinical applications. PATIENTS AND METHODS: This study was performed on 50 patients with 63 stones. All patients had a recent history of failed shockwave lithotripsy (SWL) or failed dissolution therapy by alkalinisation of urine for radiolucent stones. A multi-detector NCCT examination of the abdomen and pelvis was performed in all patients. The stones were isolated and displayed in 'Volume Rendering Technique' using four-colour encoding. RESULTS: Eight patients with failed dissolution therapy for radiolucent stones showed an outer layer of >500 Hounsfield units (HU) or a heterogeneous composition. A total of 42 patients with failed SWL had mean attenuations of <1000 HU on NCCT. Subsequent colour-coded stone mapping showed a dense core in all stones (>1000 HU) that failed to be clearly demonstrated by the mean HU alone. CONCLUSION: The initial use of a colour-coded density-gradients stone mapping reporting system for stone density on NCCT is useful for explaining failure of SWL or failure of dissolution therapy for radiolucent stones in selected cases.Abbreviations: HU: Hounsfield units; MSD: mean stone density; NCCT: non-contrast computed tomography; PCNL: percutaneous nephrolithotomy; SWL: shockwave lithotripsy; VRT: Volume Rendering Technique.

A rapid and sensitive method to detect Toxoplasma gondii oocysts in soil samples.

Documenting the extent of soil contamination by Toxoplasma gondii oocysts is a key issue to prevent the worldwide infection caused by this protozoan. Our aim was to improve the practicability and sensitivity of a low-cost method to detect T. gondii DNA in soil samples developed a few years ago. Various parameters of the reference protocol were modified to determine their effect on the detection of T. gondii DNA in soil samples ("natural soil" and "sand") spiked with oocysts. We tested i) filtration using stomacher bags, ii) Tween 80, Tween 20, SDS and Triton X100 as dispersion solutions, iii) sucrose solution, zinc chloride solution, Optiprep and Percoll as density gradients, iv) freeze/thaw versus mechanical grinding as lysis methods, and v) Qiagen versus Fastprep as extraction kits The optimized protocol is quicker and easier to use than the previous one, and includes the following items: 0.1% Tween80/PBS for dispersion, sucrose solution for flotation, mechanical grinding, and FastDNA spin kit for extraction. It accurately detects T. gondii DNA in both fresh and frozen soil samples and displays a detection limit below 1 oocyst/g of fresh soil.

Mitochondria-Associated Membranes (MAMs) are involved in Bax mitochondrial localization and cytochrome c release.

The distribution of the pro-apoptotic protein Bax in the outer mi-tochondrial membrane (OMM) is a central point of regulation of apoptosis. It is now widely recognized that parts of the endoplasmic reticulum (ER) are closely associated to the OMM, and are actively involved in different signaling processes. We addressed a possible role of these domains, called Mitochon-dria-Associated Membranes (MAMs) in Bax localization and function, by ex-pressing the human protein in a yeast mutant deleted of MDM34, a ERMES (ER-Mitochondria Encounter Structure) component. By affecting MAMs stabil-ity, the deletion of MDM34 altered Bax mitochondrial localization, and de-creased its capacity to release cytochrome c. Furthermore, the deletion of MDM34 decreased the size of an incompletely released, MAMs-associated pool of cytochrome c.

Nonuniform Composition Profiles in Amorphous Multimetal Oxide Thin Films Deposited from Aqueous Solution.

Metal oxide thin films are ubiquitous in technological applications. Often, multiple metal components are used to achieve desired film properties for specific functions. Solution deposition offers an attractive route for producing these multimetal oxides because it allows for careful control of film composition through the manipulation of precursor stoichiometry. Although it has been generally assumed that homogeneous precursor solutions yield homogeneous thin films, we recently reported evidence of nonuniform electron density profiles in aqueous-deposited films. Herein, we show that nonuniform electron densities in lanthanum zirconium oxide (LZO) thin films are the result of inhomogeneous distributions of metal components. Specifically, La aggregates at the film surface, whereas Zr is relatively evenly distributed throughout single-layer films. This inhomogeneous metal distribution persists in stacked multilayer films, resulting in La-rich interfaces between the sequentially deposited layers. Testing of metal-insulator-semiconductor devices fabricated from single and multilayer LZO films shows that multilayer films have higher dielectric constants, indicating that La-rich interfaces in multilayer films do not detrimentally impact film properties. We attribute the enhanced dielectric properties of multilayer films to greater condensation and densification relative to single-layer films, and these results suggest that multilayer films may be preferred for device applications despite the presence of layering artifacts.

Leucoplast Isolation and Subfractionation.

Leucoplasts are colorless plastids of nonphotosynthetic plant tissues that support a variety of anabolic roles, particularly the biosynthesis of long-chain fatty acids in storage tissues of developing oil seeds. They also perform other important metabolic functions including the biosynthesis of amino acids and tetrapyrrole compounds. Leucoplasts use a complex set of membrane carriers and channels to actively translocate nuclear-encoded precursor proteins from the cytosol, while exchanging various metabolites with the cytosol. Leucoplast purification is a necessary prerequisite for detailed studies of their soluble (stromal) and membrane (envelope) (phospho)proteomes, as well as for achieving a detailed understanding of their metabolic capabilities, transport processes, and biogenesis. This chapter describes protocols for leucoplast purification from endosperm of developing castor oil seeds, and their subsequent subfractionation into envelope membrane and soluble stromal compartments for biochemical analysis.

Cell Growth on ("Janus") Density Gradients of Bifunctional Zeolite L Crystals.

Nanoparticle density gradients on surfaces have attracted interest as two-dimensional material surfaces that can mimic the complex nano-/microstructure of the native extracellular matrix, including its chemical and physical gradients, and can therefore be used to systematically study cell-material interactions. In this respect, we report the preparation of density gradients made of bifunctional zeolite L crystals on glass surfaces and the effects of the density gradient and biopolymer functionalization of zeolite L crystals on cell adhesion. We also describe how we created "Janus" density gradient surfaces by gradually depositing two different types of zeolite L crystals that were functionalized and loaded with different chemical groups and guest molecules onto the two distinct sides of the same glass substrate. Our results show that more cells adhered on the density gradient of biopolymer-coated zeolites than on uncoated ones. The number of adhered cells increased up to a certain surface coverage of the glass by the zeolite L crystals, but then it decreased beyond the zeolite density at which a higher surface coverage decreased fibroblast cell adhesion and spreading. Additionally, cell experiments showed that cells gradually internalized the guest-molecule-loaded zeolite L crystals from the underlying density gradient containing bifunctional zeolite L crystals.

The symbiotic relationship of sediment and biofilm dynamics at the sediment water interface of oil sands industrial tailings ponds.

Within the oil sands industry, tailings ponds are used as a means of retaining tailings until a reclamation technology such as end pit lakes (EPLs) can be developed and optimized to remediate such tailings with a water cap (although dry-land strategies for tailing reclamation are also being developed). EPLs have proven successful for other mining ventures (e.g. metal rock mines) in eventually mitigating contaminant loads to receiving waters once biochemical remediation has taken place (although the duration for this to occur may be decades). While the biological interactions at the sediment water interface of tailings ponds or EPLs have been shown to control biogeochemical processes (i.e. chemical fluxes and redox profiles), these have often been limited to static microcosm conditions. Results from such experiments may not tell the whole story given that the sediment water interface often represents a dynamic environment where erosion and deposition may be occurring in association with microbial growth and decay. Mobilization of sediments and associated contaminants may therefore have a profound effect on remediation rates and, as such, may decrease the effectiveness of EPLs as viable reclamation strategies for mining industries. Using a novel core erosion system (U-GEMS), this paper examines how the microbial community can influence sediment water interface stability and how the biofilm community may change with tailings age and after disturbance (biofilm reestablishment). Shear strength, eroded mass measurements, density gradients, high-resolution microscopy, and microbial community analyses were made on 2 different aged tailings (fresh and ∼38 years) under biotic and abiotic conditions. The same experiments were repeated as duplicates with both sets of experiments having consolidation/biostabilization periods of 21 days. Results suggest that the stability of the tailings varies between types and conditions with the fresh biotic tailings experiencing up to 75% more biostabilization than the same abiotic tailings. Further, greater microbial diversity in the aged pond could be a contributing factor to the overall increase in stability of this material over the fresh tailings source.

Influence of immobilized quaternary ammonium group surface density on antimicrobial efficacy and cytotoxicity.

Bacterial colonization of medical devices causes infections and is a significant problem in healthcare. The use of antibacterial coatings is considered as a potential solution to this problem and has attracted a great deal of attention. Using concentration density gradients of immobilized quaternary ammonium compounds it was demonstrated that a specific threshold of surface concentration is required to induce significant bacterial death. It was determined that this threshold was 4.18% NR4(+) bonded nitrogen with a surface potential of + 120.4 mV. Furthermore, it is shown for the first time that adhesion of constituents of the culture medium to the quaternary ammonium modified surface eliminated any cytotoxicity towards eukaryotic cells such as primary human fibroblasts. The implications of this type of surface fouling on the antimicrobial efficacy of surface coatings are also discussed.

Controllable Assembly and Separation of Colloidal Nanoparticles through a One-Tube Synthesis Based on Density Gradient Centrifugation.

Self-assembly of gold nanoparticles into one-dimensional (1D) nanostructures with finite primary units was achieved by introducing a thin salt (NaCl) solution layer into density gradient before centrifugation. The electrostatic interactions between Au nanoparticles would be affected and cause 1D assembly upon passing through the salt layer. A negatively charged polymer such as poly(acrylic acid) was used as an encapsulation/stabilization layer to help the formation of 1D Au assemblies, which were subsequently sorted according to unit numbers at succeeding separation zones. A centrifugal field was introduced as the external field to overcome the random Brownian motion of NPs and benefit the assembly effect. Such a facile "one-tube synthesis" approach couples assembly and separation in one centrifuge tube by centrifuging once. The method can be tuned by changing the concentration of interference salt layer, encapsulation layer, and centrifugation rate. Furthermore, positively charged fluorescent polymers such as perylenediimide-poly(N,N-diethylaminoethyl methacrylate) could encapsulate the assemblies to give tunable fluorescence properties.

Separación de poblaciones de glóbulos rojos senescentes por gradientes preformados de Percoll / Separation of populations of senescent red blood cells by Percoll performed gradients

Los mecanismos que determinan la senescencia de los eritrocitos han sido extensamente estudiados, sin embargo, no se han logrado conclusiones definitivas debido a la ausencia de una técnica que permita el aislamiento de grupos etáreos bien definidos. Los métodos más comúnmente empleados se basan en el aumento de densidad de los eritrocitos durante el envejecimiento. En este trabajo desarrollamos una técnica para la separación de glóbulos rojos de distintas edades empleando gradientes preformados de Percoll, un polímero sintético con propiedades fisicoquímicas adecuadas para trabajar con células vivas. En las suspensiones eritrocitarias obtenidas se realizaron determinaciones hematológicas, actividades de enzimas antioxidantes y el ensayo de eritrofagocitosis. Los valores de los parámetros hematológicos evaluados fueron significativamente mayores en las suspensiones de glóbulos rojos jóvenes. Las actividades enzimáticas mostraron una disminución de la capacidad antioxidante en las poblaciones de eritrocitos senescentes. Este proceso favorecería la interacción de los hematíes envejecidos con las células fagocíticas, demostrada median­te el ensayo de eritrofagocitosis. Los resultados obtenidos indican que el método de gradientes de Percoll permite una adecuada separación de las suspensiones eritrocitarias de distintas edades, con una eficiencia comparable a la observada en la técnica de centrifugación diferencial considerada de referencia.

The mechanisms that determine the senescence of the erythrocytes have been extensively studied; however. definitive conclusions have not been achieved mainly because of the lack of a technique that allows the isolation of well-defined etarian groups. The methods most commonly used for separating erythrocytes from different ages are based on the increase in density that these cells present during their aging. In the present work we have developed a technique for obtaining red blood cells from different ages using Percoll preformed gradients, a synthetic polymer with adequate physic-chemic properties to work with lives cells. In the erythrocytes suspensions we have made hematological determinations. activities of antioxidants enzymes and the essay of erythrophagocytosis. The values of the hematological parameters were significantly higher in the suspensions of young red blood cells. In the measurements of the enzymatic activity we observed a decrease of the antioxidant capacity in the populations of senescent erythrocytes. This process would promote the interaction between the old erythrocytes and the phagocyte cells, demonstrated by the erythrophagocytosis essay. The results obtained indicate that the method Percoll density gradients allows an appropriate separation of the erythrocytes suspensions of different ages with a comparable efficiency to that observed in the technique differential centrifugation, considered as reference.