Enhanced stability of a chimeric hepatitis B core antigen virus-like-particle (HBcAg-VLP) by a C-terminal linker-hexahistidine-peptide.

BACKGROUND: Virus-like-particles (VLPs) are attractive nanoparticulate scaffolds for broad applications in material/biological sciences and medicine. Prior their functionalization, specific adaptations have to be carried out. These adjustments frequently lead to disordered particles, but the particle integrity is an essential factor for the VLP suitability. Therefore, major requirements for particle stabilization exist. The objective of this study was to evaluate novel stabilizing elements for functionalized chimeric hepatitis B virus core antigen virus-like particles (HBcAg-VLP), with beneficial characteristics for vaccine development, imaging or delivery. RESULTS: The effects of a carboxy-terminal polyhistidine-peptide and an intradimer disulfide-bridge on the stability of preclinically approved chimeric HBcAg-VLPs were assessed. We purified recombinant chimeric HBcAg-VLPs bearing different modified C-termini and compared their physical and chemical particle stability by quantitative protein-biochemical and biophysical techniques. We observed lower chemical resistance of T = 3- compared to T = 4-VLP (triangulation number) capsids and profound impairment of accessibility of hexahistidine-peptides in assembled VLPs. Histidines attached to the C-terminus were associated with superior mechanical and/or chemical particle stability depending on the number of histidine moieties. A molecular modeling approach based on cryo-electron microscopy and biolayer interferometry revealed the underlying structural mechanism for the strengthening of the integrity of VLPs. Interactions triggering capsid stabilization occur on a highly conserved residue on the basis of HBcAg-monomers as well as on hexahistidine-peptides of adjacent monomers. This new stabilization mechanism appears to mimic an evolutionary conserved stabilization concept for hepadnavirus core proteins. CONCLUSIONS: These findings establish the genetically simply transferable C-terminal polyhistidine-peptide as a general stabilizing element for chimeric HBcAg-VLPs to increase their suitability.

Highly stable Saccharomyces cerevisiae L-BC capsids with versatile packing potential.

Virus-like particles (VLPs) are promising nanoscaffolds in development of vaccines and nanodelivery systems. Along with efficient production in various expression systems, they also offer extensive functionalization options. Nevertheless, the ultimate integrity of VLPs is an important burden for the applicability in nanobiotechnology. In this study, we characterize the Saccharomyces cerevisiae L-BC VLPs synthesized and purified from Escherichia coli and Saccharomyces cerevisiae cells. The particles exhibited prominent size stability in buffers within a range of ionic strength conditions, pH environment and presence of magnesium ions during the long-term storage at temperatures up to 37°C. Bacteria-derived particles exhibited alleviated stability in acidic pH values, higher ionic strength and temperature compared to yeast-derived particles. Taking advantage of gene engineering, 120 copies of red fluorescent protein mCherry were successfully encapsulated into both preparations of L-BC VLPs, while passive diffusion enabled encapsulation of antimicrobial peptide nisin into the yeast-derived unmodified VLPs. Our findings indicate that L-BC VLPs generally exhibit high long-term stability under various conditions, while yeast-derived L-BC VLPs are more stable under the elevated temperatures than bacteria-derived particles. Stability studies and encapsulation of particles by different molecules involving alternative strategies delineate the L-BC VLP potential to be developed into versatile nanodelivery system.

Replication-independent change in the frequencies of distinct genome segments of a multipartite virus during its transit within aphid vectors.

Multipartite viruses exhibit a fragmented genome composed of several nucleic acid segments individually packaged in distinct viral particles. The genome of all species of the genus Nanovirus holds eight segments, which accumulate at a very specific and reproducible relative frequency in the host plant tissues. In a given host species, the steady state pattern of the segments' relative frequencies is designated the genome formula and is thought to have an adaptive function through the modulation of gene expression. Nanoviruses are aphid-transmitted circulative non-propagative viruses, meaning that the virus particles are internalized into the midgut cells, transferred to the hemolymph, and then to the saliva, with no replication during this transit. Unexpectedly, a previous study on the faba bean necrotic stunt virus revealed that the genome formula changes after ingestion by aphids. We investigate here the possible mechanism inducing this change by first comparing the relative segment frequencies in different compartments of the aphid. We show that changes occur both in the midgut lumen and in the secreted saliva but not in the gut, salivary gland, or hemolymph. We further establish that the viral particles differentially resist physicochemical variations, in particular pH, ionic strength, and/or type of salt, depending on the encapsidated segment. We thus propose that the replication-independent genome formula changes within aphids are not adaptive, contrary to changes occurring in plants, and most likely reflect a fortuitous differential degradation of virus particles containing distinct segments when passing into extra-cellular media such as gastric fluid or saliva. IMPORTANCE: The genome of multipartite viruses is composed of several segments individually packaged into distinct viral particles. Each segment accumulates at a specific frequency that depends on the host plant species and regulates gene expression. Intriguingly, the relative frequencies of the genome segments also change when the octopartite faba bean necrotic stunt virus (FBNSV) is ingested by aphid vectors, despite the present view that this virus travels through the aphid gut and salivary glands without replicating. By monitoring the genomic composition of FBNSV populations during the transit in aphids, we demonstrate here that the changes take place extracellularly in the gut lumen and in the saliva. We further show that physicochemical factors induce differential degradation of viral particles depending on the encapsidated segment. We propose that the replication-independent changes within the insect vector are not adaptive and result from the differential stability of virus particles containing distinct segments according to environmental parameters.

Size, shape, and stability of organic particles formed during freeze-thaw cycles: Model experiments with tannic acid.

HYPOTHESIS: Freeze-thaw cycles (FTC) in soils can cause the aggregation of dissolved organic matter but controlling factors are little understood. EXPERIMENTS: In freeze-thaw experiments with tannic acid (TA) as model substance, we studied the effect of TA concentration, pH, electrolytes (NaCl, CaCl2, AlCl3), and number of FTC on particle formation. Tannic acid (0.005 to 10 g L-1) was exposed to 1-20 FTC at pH 3 and 6. The size and shape of particles was determined by confocal laser scanning microscopy. Particle stability was deduced from the equivalent circle diameter (ECD) obtained in dry state and the hydrodynamic diameter measured in thawing solutions. FINDINGS: Tannic acid particles occurred as plates and veins, resembling the morphology of ice grain boundaries. Low pH and presence of electrolytes favored the formation of large particles. The freeze-concentration effect was most intense at low TA concentrations and increased with the number of FTC. While ECD of particles formed at low TA concentrations were smaller than at high concentrations, it was vice versa in the thawed state. At low TA concentrations, higher crystallization pressure of ice caused enhanced stability of large particles. We conclude that FTC can strongly alter the physical state of dissolved organic matter, with likely consequences for its bioavailability.

Interplay of HCPro and CP in the Regulation of Potato Virus A RNA Expression and Encapsidation.

Potyviral coat protein (CP) and helper component-proteinase (HCPro) play key roles in both the regulation of viral gene expression and the formation of viral particles. We investigated the interplay between CP and HCPro during these viral processes. While the endogenous HCPro and a heterologous viral suppressor of gene silencing both complemented HCPro-less potato virus A (PVA) expression, CP stabilization connected to particle formation could be complemented only by the cognate PVA HCPro. We found that HCPro relieves CP-mediated inhibition of PVA RNA expression likely by enabling HCPro-mediated sequestration of CPs to particles. We addressed the question about the role of replication in formation of PVA particles and gained evidence for encapsidation of non-replicating PVA RNA. The extreme instability of these particles substantiates the need for replication in the formation of stable particles. During replication, viral protein genome linked (VPg) becomes covalently attached to PVA RNA and can attract HCPro, cylindrical inclusion protein and host proteins. Based on the results of the current study and our previous findings we propose a model in which a large ribonucleoprotein complex formed around VPg at one end of PVA particles is essential for their integrity.

Seasonal formation and stability of dissolved metal particles in mining-impacted, lacustrine sediments.

Formation of dissolved metal particles (<450 nm) in mining-impacted environments is a concern because of their potential for greater mobility and ecotoxicity compared to free ion and(or) sediment-bound metals. Metal-contaminated environments may produce soluble metal(loid) particles whose stability and transportability are determined by environmental conditions and particle composition. The Coeur d'Alene River Basin of northern Idaho, USA, is impacted by legacy mine waste-estimated 56 million tonnes of waste rock containing 900,000 t of Pb and 700,000 t of Zn were discharged into the Coeur d'Alene River and its tributaries during mining of argentiferous galena-sphalerite deposits. These legacy disposal practices resulted in substantial metal contamination-including As, Cd, Fe, Pb, Mn, and Zn-of floodplain sediments. Monthly lakewater samples and sediment cores were collected along the shoreline of a metal-contaminated lateral lake of the Coeur d'Alene River. Porewater was extracted from upper and lower sediments to evaluate the formation and stability of dissolved metal particles during seasonal changes. Substantial concentrations of Fe, Pb, Mn, and Zn were present in 450-nm filtered porewater during each month, with variable increases and decreases of metal concentrations in filtered lakewater according to seasonal changes. Dissolved metal particles with an average diameter of 180 ±â€¯115 nm were present in the porewater of the upper and lower sediments with size increases in early spring and decreases in fall. Particles in the lower sediment porewater were typically more stable, as indicated by more negative ζ potential values, and the greatest particle stability occurred during summer. Differences between upper and lower porewater metal particles correspond to changes in sediment S speciation and bond relocation resulting from an input of oxygenated groundwater. Transport of the dissolved metal particles in and from the sediments likely occurs with a change in the hydraulic gradient during a spring-to-summer transition that induces redox changes and increases particle stability. The presence of mining-related minerals and seasonal changes in environmental conditions allow for formation of dissolved metal particles, but the limited stability of the particles and/or low permeability of the sediments appear to limit, but not fully restrict, possible transport of metal particles to the overlying lakewater.

Targeted Modification of the Foot-And-Mouth Disease Virus Genome for Quick Cell Culture Adaptation.

Foot-and-mouth disease virus (FMDV) causes the highly contagious foot-and-mouth disease, which is characterized by the appearance of vesicles in and around the mouth and feet of cloven-hoofed animals. BHK-21 cells are the cell line of choice for the propagation of FMDV for vaccine production worldwide but vary in their susceptibility for different FMDV strains. Previous studies showed that the FMDV resistance of a certain BHK cell line can be overcome by using a closely related but permissive cell line for the pre-adaptation of the virus, but the adapted strains were found to harbor several capsid mutations. In this study, these adaptive mutations were introduced into the original Asia-1 Shamir isolate individually or in combination to create a panel of 17 Asia-1 mutants by reverse genetics and examine the effects of the mutations on receptor usage, viral growth, immunogenicity and stability. A single amino acid exchange from glutamic acid to lysine at position 202 in VP1 turned out to be of major importance for productive infection of the suspension cell line BHK-2P. In consequence, two traditionally passage-derived strains and two recombinant viruses with a minimum set of mutations were tested in vivo. While the passaged-derived viruses showed a reduced particle stability, the genetically modified viruses were more stable but did not confer a protective immune response against the original virus isolate.

Impact of pH on the stability, dissolution and aggregation kinetics of silver nanoparticles.

Widespread usage of silver nanoparticles (AgNPs) in consumer products has resulted in their presence in the aquatic environment. The evolution of the properties of AgNPs with changes in pH and time in terms of colloidal stability, dissolution and aggregation were investigated in a series of short and long-term experiments using freshly synthesized uncoated AgNPs. The solution pH modifies the surface charge and the oxidative dissolution of AgNPs. As a result, the particle behavior varied in acidic and alkaline conditions. The particle size decreased with the increasing pH at a given time frame resulting in lower aggregation in the higher pH regime and increased particle stability. These results have been further proved with the direct evidence obtained using time resolved in situ imaging acquired through Liquid cell transmission electron microscopy (LCTEM). Furthermore, the magnitude of the impact of the pH on the particle properties is higher than the impact of the dissolved oxygen concentration. The derived empirical formulae reflect that the AgNP oxidation depends on both dissolved oxygen and protons while the AgNP dissolution increasing with the increase of either of these. Overall, our results highlight the impact of the solution pH on the evolution of the properties of AgNPs over the time and provide an insight to confidently extend the results to predict the environmental transformation of AgNPs from ideal systems to the real.

Influence of aggregation on nanoscale titanium dioxide (nTiO2) deposition to quartz sand.

Although extensive research has been conducted to investigate nTiO2 aggregation and deposition, effects of aggregation on concurrent/subsequent deposition of nTiO2, which has important implications to the fate and transport of nTiO2 in groundwater, has received only limited attention. The objective of this study was to investigate how pH, dissolved organic matter (DOM), and valence of background solution cation influence aggregation and concurrent/subsequent deposition of nTiO2. Experiments were performed to examine nTiO2 aggregation and deposition onto quartz sand with co-present illite, kaolinite, and montmorillonite colloids under various geochemical conditions. Results showed that nTiO2 formed hetero-aggregates (i.e., nTiO2-clay aggregates) at low pH when nTiO2 and clay colloids carried opposite charges, and the hetero-aggregates may either deposit or remain suspended depending on their interactions with quartz sand and Fe/Al oxyhydroxide coatings. Deposition of nTiO2 and/or nTiO2-clay aggregates occurred as a result of electrostatic attraction, secondary minimum, and potentially Mg2+ bridging. Humic acid prevented nTiO2 aggregation and deposition under most conditions. In MgCl2 solutions, however, it facilitated deposition by adsorbing to nTiO2 and Fe/Al oxyhydroxides, thereby enabling Mg2+ bridging. This study demonstrated the important and complex roles of pH, DOM, cation valence, and clay colloids in controlling aggregation and subsequent deposition of nTiO2.

Toxicity and accumulation of Copper oxide (CuO) nanoparticles in different life stages of Artemia salina.

Metal nanoparticles production rate and its applications have raised concerns about their release and toxicity to the aquatic and terrestrial organisms. The primary size of Copper Oxide nanoparticles (CuO NP's) was found to be 114±36nm using Scanning Electron Microscopy (SEM) and a significant increase in the hydrodynamic diameter of CuO NP was seen within 1h of interaction. The median lethal concentration (LC50) values obtained from the acute toxicity studies on different life stages of Artemia salina was found to be 61.4, 35, 12.2 and 175.2mg/L for 1d, 2d, 7d old and adult, respectively. The toxicity associated changes in biochemical markers such as Catalase, Reduced glutathione and Glutathione-S-Transferase were evident. The accumulation of Cu nanoparticles into the gut of Artemia salina was the major reason for toxicity. This study demonstrate the toxicity of CuO NPs to Artemia salina, and the obtained results necessitate the detailed investigation on the possible eco-toxicological implication of these nanomaterials.

Agar agar-stabilized milled zerovalent iron particles for in situ groundwater remediation.

Submicron-scale milled zerovalent iron (milled ZVI) particles produced by grinding macroscopic raw materials could provide a cost-effective alternative to nanoscale zerovalent iron (nZVI) particles for in situ degradation of chlorinated aliphatic hydrocarbons in groundwater. However, the aggregation and settling of bare milled ZVI particles from suspension presents a significant obstacle to their in situ application for groundwater remediation. In our investigations we reduced the rapid aggregation and settling rate of bare milled ZVI particles from suspension by stabilization with a "green" agar agar polymer. The transport potential of stabilized milled ZVI particle suspensions in a diverse array of natural heterogeneous porous media was evaluated in a series of well-controlled laboratory column experiments. The impact of agar agar on trichloroethene (TCE) removal by milled ZVI particles was assessed in laboratory-scale batch reactors. The use of agar agar significantly enhanced the transport of milled ZVI particles in all of the investigated porous media. Reactivity tests showed that the agar agar-stabilized milled ZVI particles were reactive towards TCE, but that their reactivity was an order of magnitude less than that of bare, non-stabilized milled ZVI particles. Our results suggest that milled ZVI particles could be used as an alternative to nZVI particles as their potential for emplacement into contaminated zone, their reactivity, and expected longevity are beneficial for in situ groundwater remediation.

Stability of nTiO2 particles and their attachment to sand: Effects of humic acid at different pH.

The fate and transport of nano-scale or micro-scale titanium dioxide particles (nTiO2) in subsurface environments are strongly influenced by the stability of nTiO2 and their attachment to sediment grains. nTiO2 may carry either positive or negative charges in natural water, therefore, environmental factors such as pH, humic substances, and Fe oxyhydroxide coatings on sediment grains, which are known to control the stability and transport of negatively-charged colloids, may influence nTiO2 in different manners. The objective of this study is to investigate the effects of pH and humic acid (HA) on the stability and attachment of nTiO2 to sand at HA concentrations that are relevant to typical groundwater conditions, so that mechanisms that control nTiO2 immobilization and transport in natural systems can be elucidated. Stability and attachment of nTiO2 to quartz sand and Fe oxyhydroxide coated quartz sand are experimentally measured under a range of HA concentrations at pH5 and 9. Results show that at pH5, negatively-charged HA strongly adsorbs to positively-charged nTiO2 and Fe oxyhydroxide, which, at low HA concentrations, partially neutralizes the positive charges on nTiO2 and Fe oxyhydroxide, and therefore decreases the repulsive electrostatic forces between the surfaces, resulting in nTiO2 aggregation and attachment. At high HA concentrations, adsorbed HA reverses the surface charges of nTiO2 and Fe oxyhydroxide, and makes nTiO2 and Fe oxyhydroxide strongly negatively charged, resulting in stable nTiO2 suspension and low nTiO2 attachment. At pH9, HA, nTiO2, and Fe oxyhydroxide are all negatively charged, and HA adsorption is low and does not have a strong impact on the stability and attachment of nTiO2. Overall, this study shows that changes in surface charges of nTiO2 and Fe oxyhydroxide coating caused by HA adsorption is a key factor that influences the stability and attachment of nTiO2.

The interaction of sterically stabilized magnetic nanoparticles with fresh human red blood cells.

Sterically stabilized superparamagnetic iron oxide nanoparticles (SPIONs) were incubated with fresh human erythrocytes (red blood cells [RBCs]) to explore their potential application as magnetic resonance imaging contrast agents. The chemical shift and linewidth of (133)Cs(+) resonances from inside and outside the RBCs in (133)Cs nuclear magnetic resonance spectra were monitored as a function of time. Thus, we investigated whether SPIONs of two different core sizes and with three different types of polymeric stabilizers entered metabolically active RBCs, consuming glucose at 37°C. The SPIONs broadened the extracellular (133)Cs(+) nuclear magnetic resonance, and brought about a small change in its chemical shift to a higher frequency; while the intracellular resonance remained unchanged in both amplitude and chemical shift. This situation pertained over incubation times of up to 90 minutes. If the SPIONs had entered the RBCs, the intracellular resonance would have become broader and possibly even shifted. Therefore, we concluded that our SPIONs did not enter the RBCs. In addition, the T 2 relaxivity of the small and large particles was 368 and 953 mM(-1) s(-1), respectively (three and nine times that of the most effective commercially available samples). This suggests that these new SPIONs will provide a superior performance to any others reported thus far as magnetic resonance imaging contrast agents.

Formation Mechanism for Stable Hybrid Clusters of Proteins and Nanoparticles.

Citrate-stabilized gold nanoparticles (AuNP) agglomerate in the presence of hemoglobin (Hb) at acidic pH. The extent of agglomeration strongly depends on the concentration ratio [Hb]/[AuNP]. Negligible agglomeration occurs at very low and very high [Hb]/[AuNP]. Full agglomeration and precipitation occur at [Hb]/[AuNP] corresponding to an Hb monolayer on the AuNP. Ratios above and below this value lead to the formation of an unexpected phase: stable, microscopic AuNP-Hb agglomerates. We investigated the kinetics of agglomeration with dynamic light scattering and the adsorption kinetics of Hb on planar gold with surface-acoustic wave-phase measurements. Comparing agglomeration and adsorption kinetics leads to an explanation of the complex behavior of this nanoparticle-protein mixture. Agglomeration is initiated either when Hb bridges AuNP or when the electrostatic repulsion between AuNP is neutralized by Hb. It is terminated when Hb has been depleted or when Hb forms multilayers on the agglomerates that stabilize microscopic clusters indefinitely.

Attractive Forces between Charged Colloidal Particles Induced by Multivalent Ions Revealed by Confronting Aggregation and Direct Force Measurements.

Interactions involving charged particles in the presence of multivalent ions are relevant in wide-range of phenomena, including condensation of nucleic acids, cement hardening, or water treatment. Here, we study such interactions by combining direct force measurements with atomic force microscopy (AFM) and aggregation studies with time-resolved light scattering for particles originating from the same colloidal suspension for the first time. Classical DLVO theory is found to be only applicable for monovalent and divalent ions. For ions of higher valence, charge inversion and additional non-DLVO attractive forces are observed. These attractive forces can be attributed to surface charge heterogeneities, which leads to stability ratios that are calculated from direct force measurements to be higher than the experimental ones. Ion-ion correlations are equally important as they induce the charge inversion in the presence of trivalent or tetravalent ions, and they enhance the surface charge heterogeneities. Such heterogeneities therefore play an essential role in controlling interactions in particle suspensions containing multivalent ions.