Full Particles Research Articles

Radiation Belt Storm Probes: A New Mission for Space Weather Forecasting

Radiation Belt Storm Probes: A New Mission for Space Weather Forecasting

Molecular Simulations of Aqueous Electrolyte Solubility: 1. The Expanded-Ensemble Osmotic Molecular Dynamics Method for the Solution Phase

We have developed a molecular-level simulation technique called the expanded-ensemble osmotic molecular dynamics (EEOMD) method, for studying electrolyte solution systems. The EEOMD method performs simulations at a fixed number of solvent molecules, pressure, temperature, and overall electrolyte chemical potential. The method combines elements of constant pressure-constant temperature molecular dynamics and expanded-ensemble grand canonical Monte Carlo. The simulated electrolyte solution systems contain, in addition to solvent molecules, full and fractional ions and undissociated electrolyte molecular units. The fractional particles are coupled to the system via a coupling parameter that varies between 0 (no interaction between the fractional particle and the other particles in the system) and 1 (full interaction between the fractional particle and the other particles in the system). The time evolution of the system is governed by the constant pressure-constant temperature equations of motion and accompanied by random changes in the coupling parameter. The coupling-parameter changes are accepted with a probability derived from the expanded-ensemble osmotic partition function corresponding to the prescribed electrolyte chemical potential. The coupling-parameter changes mimic insertion/deletion of particles as in a crude grand canonical Monte Carlo simulation; if the coupling parameter becomes 0, the fractional particles disappear from the system, and as the coupling parameter reaches unity, the fractional particles become full particles. The method is demonstrated for a model of NaCl in water at ambient conditions. To test our approach, we first determine the chemical potential of NaCl in water by the thermodynamic integration technique and by the expanded-ensemble method. Then, we carry out EEOMD simulations for different specified values of the overall NaCl chemical potential and measure the concentration of ions resulting from the simulations. Both computations give consistent results, validating the EEOMD methodology.

A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini

The complex infection process of parvoviruses is not well understood so far. An important role has been attributed to a phospholipase A2 domain which is located within the unique N terminus of the capsid protein VP1. Based on the structural difference between adeno-associated virus type 2 wild-type capsids and capsids lacking VP1 or VP2, we show via electron cryomicroscopy that the N termini of VP1 and VP2 are involved in forming globules inside the capsids of empty and full particles. Upon limited heat shock, VP1 and possibly VP2 become exposed on the outsides of full but not empty capsids, which is correlated with the disappearance of the globules in the inner surfaces of the capsids. Using molecular modeling, we discuss the constraints on the release of the globularly organized VP1-unique N termini through the channels at the fivefold symmetry axes outside of the capsid.

The Infectivity and Lytic Activity of Minute Virus of Mice Wild-Type and Derived Vector Particles Are Strikingly Different

Gene therapy vectors have been developed from autonomous rodent parvoviruses that carry a therapeutic gene or a marker gene in place of the genes encoding the capsid proteins. These vectors are currently evaluated in preclinical experiments. The infectivity of the vector particles deriving from the fibroblastic strain of minute virus of mice (MVMp) (produced by transfection in human cells) was found to be far less (approximately 50-fold-less) infectious than that of wild-type virus particles routinely produced by infection of A9 mouse fibroblasts. Similarly, wild-type MVMp produced by transfection also had a low infectivity in mouse cells, indicating that the method and producer cells influence the infectivity of the virus produced. Interestingly, producer cells made as many full vector particles as wild-type particles, arguing against deficient packaging being responsible for the low infectivity of viruses recovered from transfected cells. The hurdle to infection with full particles produced through transfection was found to take place at an early step following entry and limiting viral DNA replication and gene expression. Infections with transfection or infection-derived virus stocks normalized for their replication ability yielded similar monomer and dimer DNA amplification and gene expression levels. Surprisingly, at equivalent replication units, the capacity of parvovirus vectors to kill tumor cells was lower than that of the parental wild-type virus produced under the same transfection conditions, suggesting that beside the viral nonstructural proteins, the capsid proteins, assembled capsids, or the corresponding coding region contribute to the lytic activity of these viruses.

424. Development of a Scalable Column Chromatography-Based Purification Process To Manufacure Empty Capsid-Free AAV Vectors

Recombinant adeno-associated virus (rAAV) has shown great promise as a DNA-based biopharmaceutical agent to treat serious human diseases. In conjunction with efforts in pre-clinical and clinical development, there is a significant need to develop and implement manufacturing processes capable of consistently providing vectors of optimal purity, potency and safety. We have previously reported the development and implementation of a fully scalable rAAV purification process that enabled us to manufacture AAV vectors at a scale to support our clinical development programs. However, like many other reported column-based purification processes, the vectors purified in our second generation purification method contained significant levels of empty capsids. In consideration of the possible deleterious effects of empty capsids, a product-related impurity, on vector efficacy and safety, we have developed a scalable method to separate empty capsids from vector particles. While this separation has been historically achieved using density gradient ultracentrifugation, such methods are not preferred for scalable, cost-effective vector manufacturing. Instead, we have developed a column chromatograpy-based technique to separate empty capsids from DNA containing vector particles. AAV vectors expressing human coagulation factor IX (AAV-hFIX16) were produced using helper virus-free triple transfection of HEK293 cells in roller bottles, and purified using anion exchange chromatography. This empty capsid-containing material was used as the starting point to screen various ion exchange resins to identify conditions that could differentiate between empty and full AAV2 particles. These studies indicated that the surface characteristics of the two particle types are very similar. However, identification of subtle, pH-dependent differences in binding to selected resins, in conjunction the selective use of chaotropic agents, allowed us to separate these two populations effectively. Our data indicated that more than 90% of the empty particles were removed from the purified vector using this newly developed column chromatography technique, with approximately 60% recovery of the DNA-containing vector particles. This new column step, in combination with the column-based scalable method that we previously reported, now enables us to efficiently manufacture empty capsid-free recombinant AAV using a fully scalable process.

Performance limits of isothermal packed bed and perforated monolithic bed reactors operated under laminar flow conditions. I. General optimization analysis

A global optimization analysis of a general class of perforated monolithic bed reactors is presented for the case of an isothermal first-order reaction and for laminar flow conditions. The resulting design rules indicate how a given amount of catalyst material should best be perforated or distributed in space as a function of the available inlet pressure. It is shown that the influence of the different process variables ( k,Δ P, V cata, C in/ C out, D mol, S reac) on the reactor productivity and on the optimal bed design can be grouped into a single dimensionless number E. This number also allows to discuss the sensitive relation between the total and the volumetric productivity of single bed reactors in a very general, compact manner. Two different perforated monolithic bed designs, a slit pore bed (SPB) and a cylindrical pore bed (CPB) are considered. It is found that, when there is an excess inlet pressure (i.e., for E≪1), the optimal catalyst layer thickness is given by φ=0.3–0.5 and that the optimal pore diameter is in both cases 1.4–1.45 times smaller than the catalyst layer, independently of the internal catalyst diffusivity ( D int) and the other process variables. When the available inlet pressure is limiting ( E>10 −4), and when the absolute reactor productivity is more important than the volumetric productivity, it is found that much more open structures, with much wider pores are needed, i.e., perforated beds show the same behavior as packed beds, where the occurrence of a pressure-drop limitation also induces a shift from the use of full particles to the use of hollow extrudates.

Structure determination of adeno-associated virus 2: three complete virus particles per asymmetric unit.

The atomic structure of adeno-associated virus 2 (AAV-2) has been determined to 3.0 A resolution. AAV-2 crystallized in space group P1, with unit-cell parameters a = 249.7, b = 249.7, c = 644.8 A, alpha = 90.0, beta = 101.2, gamma = 120.0 degrees. The crystals contained three full virus particles in the asymmetric unit, allowing 180-fold non-crystallographic symmetry averaging. The particle orientations were determined using the self-rotation function and found to have similar but resolvably different orientations. Approximate alignment of icosahedral and interparticle threefold screw symmetry led to a native Patterson that was interpretable in terms of approximate particle positions. Accurate positions required a Patterson correlation search that was constrained to be consistent with non-crystallographic threefold projection symmetry evident in the diffraction intensities. Initial phases to 15.0 A resolution were calculated by molecular replacement using the known structure of a distantly related homolog (23% sequence identity). Real-space averaging was performed and phases were extended from 15.0 to 3.0 A. An atomic model was fitted and refined using a simulated-annealing real-space procedure.

Solute Transport in Fractured Rock. Testing a New and Simple Aapproach

ABSTRACTA new and simple approach for modelling solute transport in fractured rock is presented, based on our previously used Channel Network Model (CNM). In our underlying concept, solute transport in fractured rocks takes place through undulating flat channels in the rock fractures. The channels form a 3-dimensional network. For non-interacting solutes the volume of the channels determines the residence time of a stream of water. For sorbing radionuclides the residence time is mainly determined by the ratio between the Flow Wetted Surface (FWS) and the flow rate in the channel (Q), in addition to the parameters that determine the interaction between the rock and the radionuclides. The main question explored in this paper is whether it is necessary to solve the flow problem for each studied case. From previous investigations, we have found that the flow rates in the channels where the solutes travel are closely related to the transmissivities of the channels. Simulations are presented in which streams of water are allowed to randomly select channels and trace out the paths where the path volume and its FWS can be assessed. The results obtained in this manner for the RTD (Residence Time Distribution) show rather good agreement with those obtained by solving the full flow problem and tracing particles in the network.

Quantification of adeno-associated virus particles and empty capsids by optical density measurement.

We show here that UV absorbance of denatured adeno-associated virus (AAV) vector provides a simple, rapid, and direct method for quantifying vector genomes and capsid proteins in solution. We determined the molar extinction coefficients of capsid protein to be 3.72 x 10(6) M(-1) cm(-1) at 260 nm and 6.61 x 10(6) M(-1) cm(-1) at 280 nm. For recombinant AAV vectors, extinction coefficients can be calculated by including the predicted absorbance of the vector DNA. Since the amount of empty capsids in purified vector preparations lowers the A(260)/A(280) ratio in a predictable manner, the vector genome (vg) and capsid particle (cp) titers in purified AAV vector preparations can be calculated from the absorbance at 260 nm and the A(260)/A(280) ratio. To validate this method, the vg and cp titers calculated by UV absorbance were compared with titers determined by quantitative (Q)-PCR and capsid ELISA. The vg titers determined by absorbance agreed well with titers determined by Q-PCR. The cp/vg ratio determined by the A(260)/A(280) method also correlated well with those determined by AAV capsid ELISA in conjunction with Q-PCR. This new method provides a simple and rapid means to determine AAV vg titers and the ratio of empty to full particles in purified virus preparations.

Functional interactions of HslV (ClpQ) with the ATPase HslU (ClpY).

HslVU is a bacterial homolog of the proteasome, where HslV is the protease that is activated by HslU, an ATPase and chaperone. Structures of singly and doubly capped HslVU particles have been reported, and different binding modes have been observed. Even among HslVU structures with I-domains distal to HslV, no consensus mode of activation has emerged. A feature in the Haemophilus influenzae HslVU structure, insertion of the C termini of HslU into pockets in HslV, was not seen in all other structures of the enzyme. Here we report site-directed mutagenesis, peptide activation, and fluorescence experiments that strongly support the functional relevance of the C terminus insertion mechanism: we find that mutations in HslV that disrupt the interaction with the C termini of HslU invariably lead to inactive enzyme. Conversely, synthetic peptides derived from the C terminus of HslU bind to HslV with 10(-5) M affinity and can functionally replace full HslU particles for both peptide and casein degradation but fail to support degradation of a folded substrate. Thus, the data can be taken as evidence for separate substrate unfoldase and protease stimulation activities in HslU. Enhanced HslV proteolysis could be due to the opening of a gated channel or allosteric activation of the active sites. To distinguish between these possibilities, we have mutated a series of residues that line the entrance channel into the HslV particle. Our mutational and fluorescence experiments demonstrate that allosteric activation of the catalytic sites is required in HslV, but they do not exclude the possibility of channel opening taking place as well. The present data support the conclusion that the H. influenzae structure with I-domains distal to HslV captures the active species and point to significant differences in the activation mechanism of HslV, ClpP, and the proteasome.

A possible radiative model for microniccarbonaceous particle sizing based on time-resolved laser-induced incandescence

A numerical model of time-resolved laser-induced incandescence(TIRE-LII) for different kinds of large carbonaceous particleis developed in this paper. Theoretical temporal LII signalswere computed for the following classes of particles: soot,the well known hollow particles called cenospheres (tens ofmicrons) and full carbonaceous particles called by analogypleospheres (a few microns), pleo- from the Greek pleos(solid). The large carbonaceous particles have a characteristictime for internal gradient dissipation much longer than soot,thus a temperature profile establishes inside them. On thebasis of these observations, a spatial time-dependent model forcenospheres and pleospheres was considered, i.e. a system ofpartial differential equations based on mass and energybalances.The computed LII signals pointed out the sensitivity of themodel to the different kinds of particle considered in thispaper. The two classes of large carbonaceous particles showeddifferent thermal behaviours. Furthermore, the analysis of thecombined effects on the total theoretical LII signal of thedifferent particles highlighted the sensitivity of the time decayof the LII profiles to the mean particle size distributionsand to the relative particle number ratios inside thecontrol volume. These results may constitute a basis of astrategy for a comprehensive discrimination between soot andlarge particles, and for their identification, as in heavy-oilcombustion.

Mass-Transfer Resistances in the Catalytic Hydrodechlorination of Polychlorobiphenyls. Experimental Results of 2-Chlorobiphenyl Hydrodechlorination in a Slurry Reactor and in a Rotating Basket Reactor

The hydrodechlorination of 2-chlorobiphenyl promoted by a commercial Ni−Mo catalyst has been investigated in a stirred batch reactor (T = 300 °C and P = 20 bar) in order to analyze the mass-transfer resistances which might influence the performance of an industrial reactor. Powdered (diameter ≅ 100 μm) and full size particles (cylinders diameter = 3 mm; length = 4.5 mm) catalysts have been used. In the second case, a rotating basket reactor has been set up. The results of the investigation indicate that external mass-transfer resistances are not relevant in the majority of the cases. When catalyst pellets are adopted, the diffusion in the catalyst porosity plays a significant role and values of the effectiveness factor in the range 0.05−0.12 have been calculated.

Fecal shedding of hepatitis A virus in Indian patients with hepatitis A and in experimentally infected Rhesus monkey

Hepatitis A is highly endemic in India. The surveillance reports for the disease from this region are primarily based on the demonstration of hepatitis A virus (HAV) specific serum IgM and IgG antibodies. The present study was conducted to assess the presence and duration of fecal shedding of HAV in patients with hepatitis A and in an experimentally infected rhesus monkey. Nested reverse transcription polymerase chain reaction (RT-PCR) was applied to fecal specimens from 67 sporadic cases of hepatitis A. Recent infection with HAV in these cases was evidenced by the presence of serum anti-HAV IgM. Fecal HAV RNA positivity was observed in nearly 40% patients. The proportion of HAV RNA positivity in fecal specimens obtained within the first week (36.58%) was not different from those collected in 2–12 weeks post onset (42.42%) ( P>0.05). A significant number of HAV RNA positive stool specimens showed presence of full virus particles by immune electron microscopy (IEM). Extended fecal shedding of HAV could be a major contributory factor for high circulation of virus thereby maintaining hyperendemicity of the disease. One of the IEM positive samples was inoculated into an anti-HAV negative rhesus monkey. Serum alanine amino transferase levels of the monkey remained within the normal limits. However, HAV RNA positivity in the feces was noted from 3 to 50 days post inoculation. The monkey seroconverted to anti-HAV IgM on day 31. This study records prolonged excretion of HAV in humans as well as in experimentally infected rhesus monkey.

Hepatitis B virus Dane particles bind to human plasma apolipoprotein H

Human apolipoprotein H (apo H) was found to bind specifically to hepatitis B surface antigen (HBsAg) from hepatitis B virus (HBV)-infected individuals. We used recombinant HBsAg proteins to analyze HBV domains recognized by apo H. We showed that the myristylated pre-S1 domain of HBsAg strongly interacted with apo H. This binding involved phospholipid components of the HBV envelope because their removal by detergent prevented apo H-HBsAg interaction. The opposite effects of iron and zinc metal ions on binding suggest that the oxidation of phospholipids also affects apo H-HBsAg interaction. After fractionation of viral particles on a sucrose gradient, and their addition to microtiter plates coated with apo H or anti-HBsAg, we observed that the maximal anti-HBsAg capture activity corresponded to a sucrose concentration of 36%, whereas the maximal apo H capture activity corresponded to a concentration of 39%. Electron microscopy and polymerase chain reaction (PCR) Southern blot studies of these fractions showed that the fraction with maximal apo H binding predominantly contained full Dane particles. Finally, we studied apo H-HBsAg binding relative to the presence of hepatitis B virus markers and observed that apo H binding activity for HBsAg was higher in sera from patients in the active virus replication phase. (H EPATOLOGY 2001;33:207-217.)

Petunia vein banding virus: Characterization of a New Tymovirus from Petunia × hybrida.

Petunia plants from a nursery in the State of Rio Grande do Sul, Brazil, showed pronounced vein banding and contained isometric particles with diameters of approximately 45 and 30 nm. The larger ones apparently represent a caulimovirus, while the smaller ones, which included both empty shells and full particles, were identified as those of a new tymovirus for which we propose the name Petunia vein banding virus (PetVBV). Originally, PetVBV was transmitted only with difficulty to healthy petunia plants. However, from an experimentally infected petu-nia, it was later readily transmitted also to Nicotiana benthamiana and Nicandra physalodes, but not to other species in the Solanaceae or other plant families. It produces cytopathic effects typical for tymovirus infections. Its coat protein shows approximately 65% amino acid sequence identity with those of Eggplant mosaic and Andean potato latent viruses, to which it is also serologically more closely related than to any other tymoviruses.

Cryomicroscopy of human cytomegalovirus virions reveals more densely packed genomic DNA than in herpes simplex virus type 1

All members of the herpesvirus family have a characteristic virion structure, comprising a DNA containing, icosahedral capsid, embedded in a proteinaceous layer (tegument) and surrounded by a lipid envelope. Human cytomegalovirus (HCMV, the prototypic β-herpesvirus) has a genome that is significantly larger (>50 %) than that of the α-herpesvirus HSV-1. Although the internal volume of the HCMV capsid is approximately 17 % larger than that of HSV-1, this slight increase in volume does not provide adequate space to encapsidate the full length HCMV genome at the same packing density as HSV-1. We have investigated the nature of DNA packing in HCMV and HSV-1 virions by electron-cryomicroscopy and image processing. Radial density profiles calculated from projection images of HCMV and HSV-1 capsids suggest that there is no increase in the volume of the HCMV capsid upon DNA packaging. Packing density of the viral DNA was assessed for both HCMV and HSV-1 by image analysis of both full and empty particles. Our results for packing density in HSV-1 are in good agreement with previously published measurements, showing an average inter-layer spacing of approximately 26 Å. Measurements taken from our HCMV images, however, suggest that the viral genomic DNA is more densely packed, with an average inter-layer spacing of approximately 23 Å. We propose therefore, that the combination of greater volume in HCMV capsids and increased packing density of viral DNA accounts for its ability to encapsidate a large genome.

Grand canonical molecular dynamics for TIP4P water systems

An algorithm was developed enabling implementation of a Nosé—Hoover thermostat within the framework of grand canonical molecular dynamics [Lynch, C. G. and Pettitt, B. M., 1997, J. chem. Phys., 107, 8594]. The proposed algorithm could readily be extended to mixtures of molecular species with different chemical potentials as shown in the paper. This algorithm was first applied to simulate a μVT ensemble of TIP4P water molecules at 298 K by means of a system comprising a number of full particles and a single scaled (fractional) particle, with the scaling factor considered as a dynamic variable in its own right and chemical potential a pre-set parameter. Our finding showed that the scheme with a single fractional particle tended to freeze in metastable states as well as failed to reproduce either the real-life (−24.05 kJmol−1) or the model-specific chemical potential of water (−23.0kJ mol−1). In order to overcome the inadequacy of a single fractional particle as a chemical potential ‘probe’ the treatment of Pettitt and co-workers was extended to introduce multiple fractional particles. The extended scheme (with 4 fractional particles) was able to reproduce the actual density of water for the driving chemical potential of -24.0k mo−1. The actual behaviour of the density as a function of the chemical potential also agreed quite well with both the results of thermo-dynamic integration and the findings of Pettitt and co-workers.

Studies of bovine enterovirus structure by ultraviolet resonance Raman spectroscopy

The structural comparison of bovine enterovirus MZ468 strain before and after the heat treatment was studied by ultraviolet resonance Raman (UVRR) spectra excited at both 235 and 251 nm. The difference between full, heated full and purified empty particles, which were expected as an in vitro model of uncoating, were demonstrated. At 235 nm excitation, the Raman bands of the capsid protein dominated in all the UVRR spectra. The UVRR spectra of the empty particles exhibited non-homogenious broadening for tryptophan W3 band and W7 Fermi doublet bands, which were characteristics of hydrophobic environment, when compared with those of the full particles. The results indicates that some Trp indole rings of the full particles were packaged inside the viral capsids and not strained by virion assembly. On the other hand, the Raman bands assigned to guanine residues of the single stranded-RNA genome were enhanced strongly in the 251-nm excited UVRR spectrum. The spectral differences between the packaged (full particles) and the unpackaged virions (heated full particles) indicates that some guanine residues had strong hydrogen bonds in the full particles.

Assaying for Structural Variation in the Parvovirus Capsid and Its Role in Infection

The capsid of canine parvovirus (CPV) was assayed for susceptibility to proteases and for structural variation. The natural cleavage of VP2 to VP3 in CPV full (DNA containing) particles recovered from tissue culture occurred within the sequence Arg-Asn-Glu-Arg↓Ala-Thr. Trypsin, chymotrypsin, bromelain, and cathepsin B all cleaved >90% of the VP2 to VP3 in full but not in empty capsids and did not digest the capsid further. Digestion with proteinase K, Pronase, papain, or subtilisin cleaved the VP2 to VP3 and also cleaved at additional internal sites, causing particle disintegration and protein degradation. Several partial digestion products produced by proteinase K or subtilisin were ∼31–32.5 kDa, indicating cleavage within loop 3 of the capsid protein as well as other sites. Protease treatment of capsids at pH 5.5 or 7.5 did not significantly alter their susceptibility to digestion. The isoelectric point of CPV empty capsids was pH 5.3, and full capsids were 0.3 pH more acidic, but after proteolysis of VP2 to VP3, the pI of the full capsids became the same as that of the empty capsids. Antibodies against various capsid protein sequences showed the amino termini of most VP2 molecules were on the outside of full but not empty particles, that the VP1-unique sequence was internal, and that the capsid could be disintegrated by heat or urea treatment to expose the internal sequences. Capsids added to cells were localized within the cell cytoplasm in vesicles that appeared to be lysosomes. Microinjected capsids remained primarily in the cytoplasm, although a small proportion was observed to be in the nucleus after 2 h. After CPV capsids labeled with [35S]methionine were bound to cells at 0°C and the cells warmed, little cleavage of VP1 or VP2 was observed even after prolonged incubation. Inoculation of cells with virus in the presence of proteinase inhibitors did not significantly reduce the infection.

The Polyhedra of the Occluded Baculoviruses of Marine Decapod Crustacea: A Unique Structure, Crystal Organization, and Proposed Model

The baculoviruses of marine penaeid shrimp, PmSNPV and PvSNPV (MBV type and BP type, respectively), have distinctly different occlusion bodies (OBs) from those of the insect baculoviruses. In contrast to insect baculovirus, the penaeid baculovirus OB is unenveloped and formed by large subunits (SuOBs), as observed by electron microscopy after negative staining. The polyhedrin subunits measure 17 to 23 nm in diameter and appear icosahedral, resembling full and empty viral particles. Although these SuOBs look similar in morphometrics to shrimp parvoviruses, their density, polypeptide composition, and UV spectra are more characteristic of proteins than nucleoproteins. Common to the two shrimp baculovirus OBs that were investigated is the aggregation of three icosahedral SuOBs into a triplet. The observed difference in their crystalline structure is directly related to the way in which triplets attach to each other to form the OB. In the BP-type OB, the triplets form alternating parallel rows in all three dimensions. On the other hand, in the MBV-type OB, four triplets form a hollow sphere which we call a “rosette,” the building blocks of the MBV-type OB. We assembled models for the penaeid baculovirus OB as an alternative to those hypothesized for insect baculovirus OBs.