Ring-like Particles Research Articles

Synthesis of stimuli-responsive nanosized ring-like colloids and cyclic polymers via a dual-template approach.

Ring-like particles have received considerable attention due to their unique interior cavity and properties. However, the preparation of stimuli-responsive nanosized rings with internal size smaller than 100nm remains unexplored likely due to the challenges encountered in their synthesis. The successful fulfillment of this target will not only significantly enrich the family of ring-like nanoparticles but also build a connection that bridges ring-like nanoparticles and cyclic polymers. For this purpose, we report in this study a controlled synthesis of stimuli-responsive ring-like colloids and cyclic polymers using both star-shaped polymers and β-cyclodextrin (β-CD) as the dual templates. The first template comprising star-shaped polymers generated a ring-like structure and adoption of β-CD as the second template further restricted the ring thickness to the height of a β-CD, leading to the generation of stimuli-responsive nanosized ring-like colloids with ring thickness less than 1 nm, which shifted the ring-like structure to cyclic polymers with reversible cross-linked disulfide bridges. The reported "dual-template" approach is thus a valuable alternative to the current synthetic strategies toward stimuli-responsive ring-like colloids and cyclic polymers.

Initiation, extension, and termination of RNA synthesis by a paramyxovirus polymerase.

Paramyxoviruses represent a family of RNA viruses causing significant human diseases. These include measles virus, the most infectious virus ever reported, in addition to parainfluenza virus, and other emerging viruses. Paramyxoviruses likely share common replication machinery but their mechanisms of RNA biosynthesis activities and details of their complex polymerase structures are unknown. Mechanistic and functional details of a paramyxovirus polymerase would have sweeping implications for understanding RNA virus replication and for the development of new antiviral medicines. To study paramyxovirus polymerase structure and function, we expressed an active recombinant Nipah virus (NiV) polymerase complex assembled from the multifunctional NiV L protein bound to its phosphoprotein cofactor. NiV is an emerging highly pathogenic virus that causes severe encephalitis and has been declared a global public health concern due to its high mortality rate. Using negative-stain electron microscopy, we demonstrated NiV polymerase forms ring-like particles resembling related RNA polymerases. We identified conserved sequence elements driving recognition of the 3′-terminal genomic promoter by NiV polymerase, and leading to initiation of RNA synthesis, primer extension, and transition to elongation mode. Polyadenylation resulting from NiV polymerase stuttering provides a mechanistic basis for transcription termination. It also suggests a divergent adaptation in promoter recognition between pneumo- and paramyxoviruses. The lack of available antiviral therapy for NiV prompted us to identify the triphosphate forms of R1479 and GS-5734, two clinically relevant nucleotide analogs, as substrates and inhibitors of NiV polymerase activity by delayed chain termination. Overall, these findings provide low-resolution structural details and the mechanism of an RNA polymerase from a previously uncharacterized virus family. This work illustrates important functional differences yet remarkable similarities between the polymerases of nonsegmented negative-strand RNA viruses.

Formation of metastable phases of ferrous sulfide via pulsed Nd:YAG laser deposition: Experimental and theoretical study

Highly polymorphic ferrous sulfide exhibits attractive optical, semiconducting, magnetic and biocatalytic properties related to its phase modification. Nd:YAG laser ablation of ferrous sulfide (FeS) in vaccum results in noncongruent deposition of nanostructured FeS1-x thin films. Deposits have been carried out on Ta, Al and Cu substrates and achieved thin films were analyzed using scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM) and electron diffraction in order to characterize morphology, chemical composition and phase transformation induced by ablative process. Round-shaped and ring-like particles, shapeless agglomerates as well as flat discontinuous areas have been observed for all the coats deposited on various substrates. However, using HRTEM, in agreement with electron diffraction, different phase compositions on various substrates have been detected. Cubic pyrite phase (FeS2) has been detected on Ta substrate. Metastable rhombohedral smythite Fe9S11 and cubic pyrite FeS2 have been found on Al substrate. And cubic pyrite FeS2, metastable rhombohedral smythite Fe9S11 and metastable orthorhombic marcasite FeS2m have been revealed on Cu substrate. The detected crystalline nanograins in all deposits were surrounded by amorphous phase. Furthermore, to gain deep insight into the electronic structure of obtained stable (cubic pyrite) and less known unstable phases (orthorhombic marcasite and rhombohedral smythite) the density functional theory is employed and important characteristics such as band gap values have been calculated.

Rigid ring-shaped particles that align in simple shear flow

AbstractMost rigid, torque-free, low-Reynolds-number, axisymmetric particles undergo a time-periodic tumbling motion in a simple shear flow, with their axes of symmetry following a set of closed Jeffery orbits. We have identified a class of rigid, ring-like particles whose axes of symmetry instead reach a permanent alignment near the velocity gradient direction with the plane of the particle aligning near the flow–vorticity plane. An asymptotic analysis for small particle aspect ratio (ratio of length parallel to the axis of symmetry to diameter perpendicular to the axis) shows that an appropriate asymmetry of the ring cross-section with a thinner outer edge and thicker inner edge leads to a tendency to rotate in a direction opposite to the vorticity; this tendency can balance the usual rotation rate associated with the finite thickness of the particle. Boundary integral computations for finite particle aspect ratios are used to determine the conditions of aspect ratio and degree of asymmetry that lead to the aligning behaviour and the final orientation of the axis of symmetry of the aligned particles. The aligning particle follows an equation of motion similar to the Leslie–Erickson equation for the director of a small-molecule nematic liquid crystal. However, whereas the alignment of the director arises from intermolecular interactions, the ring-like particle aligns solely due to its intrinsic rotational motion in a low-Reynolds-number flow.

Poliovirus 2C Protein Forms Homo-oligomeric Structures Required for ATPase Activity

The poliovirus protein 2C plays an essential role in viral RNA replication, although its precise biochemical activities or structural requirements have not been elucidated. The protein has several distinctive properties, including ATPase activity and membrane and RNA binding, that are conserved among orthologs of many positive-strand RNA viruses. Sequence alignments have placed these proteins in the SF3 helicase family, a subset of the AAA+ ATPase superfamily. A feature common to AAA+ proteins is the formation of oligomeric rings that are essential for their catalytic functions. Here we show that a recombinant protein, MBP-2C, in which maltose-binding protein was fused to 2C, formed soluble oligomers and that ATPase activity was restricted to oligomer-containing fractions from gel-filtration chromatography. The active fraction was visualized by negative-staining electron microscopy as ring-like particles composed of 5-8 protomers. This conclusion was confirmed by mass measurements obtained by scanning transmission electron microscopy. Mutation of amino acid residues in the 2C nucleotide-binding domain demonstrated that loss of the ability to bind or hydrolyze ATP did not affect oligomerization. Co-expression of active MBP-2C and inactive mutant proteins generated mixed oligomers that exhibited little ATPase activity, suggesting that incorporation of inactive subunits eliminates the function of the entire particle. Finally, deletion of the N-terminal 38 amino acids blocked oligomerization of the fusion protein and eliminated ATPase activity, despite retention of an unaltered nucleotide-binding domain.

Synthesis of Layered Double Hydroxides with Novel Morphologies

Generally, the hexagonal plate morphology of Mg-Al layered double hydroxides (LDHs) is more favorable, in this article, we report the synthesis of Mg-Al LDHs with unique morphologies, such as ring-like, fiber-like, and sphere-like particles, respectively, using simple traditional coprecipitation method. This synthesis of MgAl-LDHs were carried out with sodium dodecylsulfate together with Na2-edta, Na2-edta, and sodium tartrate, respectively, as assistants to favor the precipitation process. The obtained products were characterized by transmission electron microscopy, x-ray diffraction, and FTIR spectroscopy. The studied results confirm that morphology of LDHs might be controlled by a soft-template effect of assistants in traditional coprecipitation method.

Purification and Characterization of Nipah Virus Nucleocapsid Protein Produced in Insect Cells

The nucleocapsid (N) protein of Nipah virus (NiV) is a major constituent of the viral proteins which play a role in encapsidation, regulating the transcription and replication of the viral genome. To investigate the use of a fusion system to aid the purification of the recombinant N protein for structural studies and potential use as a diagnostic reagent, the NiV N gene was cloned into the pFastBacHT vector and the His-tagged fusion protein was expressed in Sf9 insect cells by recombinant baculovirus. Western blot analysis of the recombinant fusion protein with anti-NiV antibodies produced a band of approximately 62 kDa. A time course study showed that the highest level of expression was achieved after 3 days of incubation. Electron microscopic analysis of the NiV recombinant N fusion protein purified on a nickel-nitrilotriacetic acid resin column revealed different types of structures, including spherical, ring-like, and herringbone-like particles. The light-scattering measurements of the recombinant N protein also confirmed the polydispersity of the sample with hyrdrodynamic radii of small and large types. The optical density spectra of the purified recombinant fusion protein revealed a high A(260)/A(280) ratio, indicating the presence of nucleic acids. Western blotting and enzyme-linked immunosorbent assay results showed that the recombinant N protein exhibited the antigenic sites and conformation necessary for specific antigen-antibody recognition.

CbpA, a DnaJ Homolog, Is a DnaK Co-chaperone, and Its Activity Is Modulated by CbpM

The DnaK chaperone system, consisting of DnaK, DnaJ, and GrpE, remodels and refolds proteins during both normal growth and stress conditions. CbpA, one of several DnaJ analogs in Escherichia coli, is known to function as a multicopy suppressor for dnaJ mutations and to bind nonspecifically to DNA and preferentially to curved DNA. We found that CbpA functions as a DnaJ-like co-chaperone in vitro. CbpA acted in an ATP-dependent reaction with DnaK and GrpE to remodel inactive dimers of plasmid P1 RepA into monomers active in P1 DNA binding. Additionally, CbpA participated with DnaK in an ATP-dependent reaction to prevent aggregation of denatured rhodanese. The cbpA gene is in an operon with an open reading frame, yccD, which encodes a protein that has some homology to DafA of Thermus thermophilus. DafA is a protein required for the assembly of ring-like particles that contain trimers each of T. thermophilus DnaK, DnaJ, and DafA. The E. coli YccD was isolated because of its potential functional relationship to CbpA. Purified YccD specifically inhibited both the co-chaperone activity and the DNA binding activity of CbpA, suggesting that YccD modulates the activity of CbpA. We named the product of the yccD gene CbpM for "CbpA modulator."

A novel water-soluble nanoparticles of hypocrellin B and their interaction with a model protein: C-phycocyanin

A type of novel hypocrellin B gelatin nanoparticles (HB-G-NP), with size of 20-200 nm, was prepared and characterized. The nanoparticles are readily soluble in water or phosphate-buffered saline (PBS). The interaction between HB-G-NP and a fluorescence protein, C-phycocyanin (C-PC) from Spirulina platensis, was studied. It was found that the energy transfer from HB to C-PC was quite efficient, suggesting adsorption of C-PC on surface of the nanoparticles; secondly, the photosensitization of HB resulted in not only the photo-damage of C-PC but also the photobleaching of HB in the presence of oxygen while it did not in the absence of oxygen, suggesting that the movable reactive oxygen species, instead of the immovable anionic radicals of the photosensitizer, should be responsible for the photo-induced processes. Considering the short free diffusion path length of the reactive oxygen species, it can be deduced that smaller or ring-like particles should be more effective for photo-damage of biomolecules or target tissues.

Direct atomic scale analysis of the distribution of Cu valence states in Cu/γ-Al2O3 catalysts

In this paper, the microstructure of a 1wt.% Cu/γ-Al2O3 catalyst that was reduced in a 4% hydrogen/argon atmosphere at temperatures of 523, 773 and 1073K, is studied by Z-contrast imaging and electron energy-loss spectroscopy (EELS) in a scanning transmission electron microscope (STEM). Results show that the copper species are well dispersed when the catalyst is reduced below 523K. At 773K, separated Cu(I) and Cu(0) species are found existing as ring-like and bulk-like particles. This appears to indicate that the copper has not been reduced to its metallic form due to the interaction between the copper oxide and the support. Large spherical particles having core-shell structures with Cu(I) in the shells and Cu(0) in the cores are generated when the catalyst is reduced at 1073K. The formation of partially oxidized copper species upon reduction at 1073K is attributed to the metallic copper interaction with the alumina support. This study also demonstrates that high-spatial resolution Z-contrast imaging and EELS performed simultaneously can provide unique information on the morphology and chemistry of metal species in supported metal catalysts.

Electrostatic Interactions of a String-Like Particle with a Charged Plate

The electrostatic interactions for a string-like particle with a charged plate are investigated on the basis of the Poisson–Boltzmann equation under the Debye–Hückel approximation. During the interaction, the plate is assumed to be with constant surface potential or constant surface charge density. The interaction energy is found to consist of two terms. The first term is simply the result of Gouy–Chapman theory, and it is the work required to insert a ghost particle into the undisturbed electric field established by the electrolyte solution and the charged wall. The second term represents the interaction between the particle and its image with respect to the plate. The interaction energy, which is nondimensionalized byQΨp0, depends on the distance between the particle and the plate, the contour length, and the orientation of the particle.Qis the total charge carried by the particle, and Ψp0is the undisturbed surface potential. At short distance, the relative importance of the image to the plate contribution grows with increasing κQ/Ψp0. ForQΨp0> 0, the interaction can become attractive if κQ/Ψp0is large enough. Here κ−1is the Debye screening length. The results have been applied to fiber- and ring-like particles. Analytical expressions are obtained in some limiting cases. The free energy contributions due to the plate and the image are maxima when the plane containing the fiber or the ring is perpendicular to the wall. The effect of nonuniform charge distribution of the fiber has also been discussed.

Are prosome particles formed by the disintegration of proteasomes?

Quail reticulocyte 19S "prosome" fractions isolated on sucrose density gradients contain two kinds of particles: cylindrical proteasomes and ferritin. When samples of this fraction are prepared for electron microscopy using the one-step stain protocol described in this paper, most of the particles have a rectangular image resembling the proteasome. However, when samples are prepared for electron microscopy using the two-step stain protocol described here, there are few rectangular images. Their place is taken by round particles that resemble the prosome. Thus it appears that the round, raspberry-shaped particles called prosomes and the ring-like proteasome particles may be artifacts of specimen preparation for electron microscopy. We propose that proteasome particles may disintegrate when prepared for electron microscopy by methods such as the two-step stain protocol and that prosome particles represent the component parts of the proteasome. Furthermore, based on the enhancement of proteasome images obtained using the one-step main protocol we propose that, instead of consisting of a stack of four rings, the proteasome is constructed of three components, i.e., a spherical central particle flanked by two flat hexagonal end caps.

In vitro assembly of the outer shell of bacteriophage ϕ6 nucleocapsid

Following dissociation of bacteriophage ϕ6 nucleocapsid (NC) by EDTA, a particle composed of protein P8 and corresponding to the outer shell of the NC was assembled in vitro in the presence of Ca 2+ and Mg 2+. Assembly was obtained from soluble protein constituents frove 100 μg/ml and was optimal within a temperature range of 22–30°. Assembly did not require the presence of genomic RNA. Crosslinking results of intact NCs and in vitro-assembled outer shells suggested that protein P8 dimers are the structural subunits of the shell. Analysis of the assembly kinetics by electron microscopy suggested that ring-like particles of uniform size, packed in flat hexagonal arrays, are intermediates in outer shell assembly.

Structural studies of the outer envelope of Chlamydia trachomatis by electron microscopy

Electron microscopic observations have been made on the outer cell envelopes of Chlamydia trachomatis, the agent of trachoma. It has been shown that they contain a hexagonal lattice with a unit cell of about 17·5 nm. Computer processing has been carried out on the hexagonal array and on ring-like particles occurring in fragmented envelopes. The structure of these rings can be related to the distribution of material in a three-dimensional model obtained for the hexagonal lattice. This model has been compared with biochemical data for the cell envelopes.

The selective release of proteins from a bovine enterovirus.

SUMMARY A bovine enterovirus (serotype VG-5-27) was grown in BHK cells and purified by sedimentation procedures. Virus particles were heated at 100° in the presence of urea-sodium dodecyl sulphate, urea or borate buffer at pH 10. Whereas complete degradation of the virus in the presence of urea-SDS showed four main polypeptides, namely VP1, VP2, VP3 and VP4, heating in the presence of urea or alkali alone resulted in the selective release of VP2 and VP4, predominantly. Electron-microscopic examination of the residual complexes showed evidence for unstable intermediate ring-like particles and also fibrous material. These complexes lacked RNA and contained mainly VP1 and VP3. We interpret the results as indicating that the polypeptide VP2 may have a specific location at the apical region of the capsid.

Electron Microscopic Observation on Dolichos Enation Mosaic Virus

SUMMARY Sap from plants infected with a strain (NDEMV) of dolichos enation mosaic virus (DEMV) contains many ringlike particles, but few particles of about 3000 Å, the normal length for viruses of the tobacco mosaic virus group. In purified preparations the mean length of normal particles of NDEMV was 3000 Å and that of DEMV, 2860 Å. Staining with uranyl formate clearly revealed the fine structure of these particles and in the ringlike particles it penetrated to the expected position of the ribonucleic acid.

The Quality of Virus as Affected by the Ambient Temperature

SUMMARY Dolichos enation mosaic virus is a serotype of tobacco mosaic virus infecting leguminous plants; it produces a large proportion of particles shorter than 3000 Å, the accepted length of infective tobacco mosaic virus. A new strain of dolichos enation mosaic virus isolated from a single necrotic lesion on French bean caused local and systemic necroses in French bean plants. It produced many more defective particles than the parent strain when infected plants were kept at 20° but not at temperatures above 32°. Both strains multiplied faster at 32° than at 20°, but the effect of increasing temperature was greater with the new strain. The effect on the new strain appeared greater when virus content was assayed hy infectivity tests than when it was assayed serologically, suggesting that infectivity per unit weight of virus was also greater at 32° than at 20°. No evidence was found that leaves infected with the new strain contained free infective RNA. The ultraviolet absorption spectra of purified preparations of the new strain produced at 20° had a greater optical density at 280 mαt than at 260 mα, whereas the parent strain had greater density at 260 mα, showing that some of the particles of the new strain were without RNA. The type of particles produced by the two strains differed when purified preparations in 0·06 m- phosphate buffer at pH 8 were subjected to analytical ultracentrifugation in sucrose gradient columns or fractionation through agar columns. When produced in plants at 20°, the new strain consisted mainly of ring-like particles, virus protein, some free RNA and very few infective particles. By contrast, the parent strain consisted mainly of infective virus and broken particles of various lengths, of which a particle 400 Å long was plentiful enough to give a peak in the analytical centrifuge and a zone in sucrose gradient columns. The new strain produced in plants at 32 to 36° did not differ in appearance or infectivity from the parent strain. The distribution of particle lengths in sprayed droplets, measured in the electron microscope, confirmed the difference found by other means between the type of particle in the two strains. Changing the pH value of a purified preparation of the new strain (produced at 20°) from 5·2 to 8 released some RNA in amounts suggesting that about a third of the particles in the preparation released their RNA.