SIGNATURE: a single-particle selection system for molecular electron microscopy

SIGNATURE is a particle selection system for molecular electron microscopy. It applies a hierarchical screening procedure to identify molecular particles in EM micrographs. The user interface of the program provides versatile functions to facilitate image data visualization, particle annotation and particle quality inspection. The system design emphasizes both functionality and usability. This software has been released to the EM community and has been successfully applied to macromolecular structural analyses.     

Trapping and Scattering of a Relativistic Charged Particle by Resonance in a Magnetic Field and an Electromagnetic Wave

A study is made of the dynamics of a relativistic charged particle in an electromagnetic wave (with an electrostatic component) in a constant uniform magnetic field. A system with a high-frequency wave is a Hamiltonian system with two degrees of freedom and with fast and slow variables. The trapping of a particle into resonance and its scattering on resonance in such a system is described.     

Modeling the C. elegans nematode and its environment using a particle system

A particle system, as understood in computer science, is a novel technique for modeling living organisms in their environment. Such particle systems have traditionally been used for modeling the complex dynamics of fluids and gases. In the present study, a particle system was devised to model the movement and feeding behavior of the nematode Caenorhabditis elegans in three different virtual environments: gel, liquid, and soil. The results demonstrate that distinct movements of the nematode can be attributed to its mechanical interactions with the virtual environment. These results also revealed emergent properties associated with modeling organisms within environment-based systems.     

Study of the RESS Process for Producing Beclomethasone-17,21-Dipropionate Particles Suitable for Pulmonary Delivery

The purpose of this research was to micronize beclomethasone-17,21-dipropionate (BDP), an anti-inflammatory inhaled corticosteroid commonly used to treat asthma, using the rapid expansion of supercritical solution (RESS) technique. The RESS technique was chosen for its ability to produce both micron particles of high purity for inhalation, and submicron/nano particles as a powder handling aid for use in next generation dry powder inhalers (DPIs). Particle formation experiments were carried out with a capillary RESS system to determine the effect of experimental conditions on the particle size distribution (PSD). The results indicated that the RESS process conditions strongly influenced the particle size and morphology; with the BDP mean particle size decreasing to sub-micron and nanometer dimensions. An increase in the following parameters, i.e. nozzle diameter, BDP mol fraction, system pressure, and system temperature; led to larger particle sizes. Aerodynamic diameters were estimated from the SEM data using three separate relations, which showed that the RESS technique is promising to produce particles suitable for pulmonary delivery.     

Nitrite reductase system involved in the terminal oxidation of the Streptomyces griseus respiratory particle.

A nitrite reductase system which was associated with the electron transfer system of the respiratory particle in Streptomyces griseus was studied. The electron transfer pathway consisted of the cytochrome oxidase and the nitrite reductase systems under aerobic and anaerobic conditions respectively, and these systems showed the exact opposite response to 2-n-heptyl-4-hydroxyquinoline-N-oxide and azide. Azide inhibited specifically the nitrite reductase system. It seems that cytochrome d works as the nitrite reductase and the reduced cytochrome b works as an intermediate electron donor for cytochrome d respectively. The respiratory particle also had a hydroxylamine reductase activity and ammonia was identified as the product of hydroxylamine reduction by the respiratory particle. A terminal electron transfer pathway in Streptomyces griseus was proposed.     

Calculating the local nonequilibrium configurational entropy in quasi-one-dimensional heat conduction

In a quasi-one-dimensional (QOD) system the particles remain ordered from left to right allowing the association of a volume element to the particle which on average resides there. Thus the properties of that single particle give the local densities in the volume element. With reservoirs of different temperatures connected to each end of the system a steady heat current with an anomalous thermal conductivity results. The largest contribution to the local configurational entropy density is calculated from two-particle correlation functions which vary locally within the nonequilibrium system. This contribution to the local configurational entropy is calculated for a QOD system in a nonequilibrium steady state.     

Characterization of early steps in the poliovirus infection process: receptor-decorated liposomes induce conversion of the virus to membrane-anchored entry-intermediate particles

The mechanism by which poliovirus infects the cell has been characterized by a combination of biochemical and structural studies, leading to a working model for cell entry. Upon receptor binding at physiological temperature, native virus (160S) undergoes a conformational change to a 135S particle from which VP4 and the N terminus of VP1 are externalized. These components interact with the membrane and are proposed to form a membrane pore. An additional conformational change in the particle is accompanied by release of the infectious viral RNA genome from the particle and its delivery, presumably through the membrane pore into the cytoplasm, leaving behind an empty 80S particle. In this report, we describe the generation of a receptor-decorated liposome system, comprising nickel-chelating nitrilotriacetic acid (NTA) liposomes and His-tagged poliovirus receptor, and its use in characterizing the early events in poliovirus infection. Receptor-decorated liposomes were able to capture virus and induce a temperature-dependent virus conversion to the 135S particle. Upon conversion, 135S particles became tethered to the liposome independently of receptor by a membrane interaction with the N terminus of VP1. Converted particles had lost VP4, which partitioned with the membrane. The development of a simple model membrane system provides a novel tool for studying poliovirus entry. The liposome system bridges the gap between previous studies using either soluble receptor or whole cells and offers a flexible template which can be extrapolated to electron microscopy experiments that analyze the structural biology of nonenveloped virus entry.     

Application of europium(III) chelate-dyed nanoparticle labels in a competitive atrazine fluoroimmunoassay on an ITO waveguide

We have demonstrated the use of an optical indium tin oxide (ITO) (quartz) waveguide as a new platform for immunosensors with fluorescent europium(III) chelate nanoparticle labels (Seradyn) in a competitive atrazine immunoassay. ITO as a solid surface facilitated the successful use of particulate labels in a competitive assay format. The limit of detection in the new nanoparticle assay was similar to a conventional ELISA. The effect of particle size on bioconjugate binding kinetics was studied using three sizes of bioconjugated particle labels (107, 304, and 396nm) and a rabbit IgG/anti-IgG system in a 96-well plate. A decrease in particle size resulted in faster binding but did not increase the assay sensitivity. Flux calculations based on the particle diffusivity prove that faster binding of the small particles in this study was primarily due to diffusion kinetics and not necessarily to a higher density of antibodies on the particle surface. The results suggest that ITO could make a good platform for an optical immunosensor using fluorescent nanoparticle labels in a competitive assay format for small molecule detection. However, when used in combination with fluorescent particulate labels, a highly sensitive excitation/detection system needs to be developed to fully utilize the kinetic advantage from small particle size. Different regeneration methods tested in this study showed that repeated washings with 0.1 M glycine-HCl facilitated the reuse of the ITO waveguide.     

Particle Size, Moisture, and Fluidization Variations Described by Indirect In-line Physical Measurements of Fluid Bed Granulation

The aim of this study was to evaluate an instrumentation system for a bench scale fluid bed granulator to determine the parameters expressing the changing conditions during the spraying phase of a fluid bed process. The study focused mainly on four in-line measurements (dependent variables): fluidization parameter (calculated by inlet air flow rate and rotor speed), pressure difference over the upper filters, pressure difference over the granules (lower filter), and temperature of the fluidizing mass. In-line particle size measured by the spatial filtering technique was an essential predictor variable. Other physical process measurements of the automated granulation system, 25 direct and 12 derived parameters, were also utilized for multivariate modeling. The correlation and partial least squares analyses revealed significant relationships between various process parameters highlighting the particle size, moisture, and fluidization effect. Fluidization parameter and pressure difference over upper filters were found to correlate with in-line particle size and therefore could be used as estimates of particle size during granulation. The pressure difference over the granules and the temperature of the fluidizing mass expressed the moisture conditions of wet granulation. The instrumentation system evaluated here is an invaluable aid to gaining more control for fluid bed processing to obtain repeatable granules for further processing.     

Automated monitoring of cell concentration and viability using an image analysis system

In order to automate measurements of cell concentration and viability in a suspended animal cell culture, we have developed anin situ microscopic image analysis system with an effective cell recognition algorithm. With a small amount of sample, this system can measure the cell density rapidly and aseptically. In addition, it can measure a cell size histogram including cell debris small particle distribution. These small particles have been found to be related to the viability of the mouse-mouse hybridoma STK1 cell line. By using cell debris small particle density as an indicator of cell viability, the developed system provides non-destructive viability monitoring without trypan blue staining.     

Computer-assisted analysis of particulate axoplasmic flow in organized CNS tissue cultures

Using organized nervous system tissue cultures of embryonic mouse spinal cord, time-lapse motion pictures of intra-axonal particle movements were made using Nomarski optics. The paths of 272 particles were followed and analyzed using digital computer methods. Particles had a bidirectional saltatory motility with net anterograde and retrograde velocities which were shown to be the same (about 1 micron per second). When anterograde moving and retrograde moving particles were analyzed separately for anterograde and retrograde saltations, the velocities of saltations in the direction of net movement were found to be 1.5 times greater than saltations against the direction of net movement. These differences were statistically significant. In addition, it was shown that there were regions within the axon where variations in particle motion were similar from particle to particle as they passed, and that more disturbed motion was found to occur near intra-axonal objects which were judged to be mitochondria. No decrease of particle velocity was noted in regions away from mitochondria nor was an increase in velocity noted near them. Computer-drawn particle plots illustrate the various facets of particle motion.     

Computer-assisted analysis of particulate axoplasmic flow in organized CNS tissue cultures.

Using organized nervous system tissue cultures of embryonic mouse spinal cord, time-lapse motion pictures of intra-axonal particle movements were made using Nomarski optics. The paths of 272 particles were followed and analyzed using digital computer methods. Particles had a bidirectional saltatory motility with net anterograde and retrograde velocities which were shown to be the same (about 1 micron per second). When anterograde moving and retrograde moving particles were analyzed separately for anterograde and retrograde saltations, the velocities of saltation in the direction of net movement were found to be 1.5 times greater than saltations against the direction of net movement. These differences were statistically significant. In addition, it was shown that there were regions within the axon where variations in particle motion were similar from particle to particle as they passed, and that more disturbed motion was found to occur near intra-axonal objects which were judged to be mitochondria. No decrease of particle velocity was noted in regions away from mitochondria nor was in increase in velocity noted near them. Computer-drawn particle plots illustrate the various facets of particle motion.     

The role of particle size distribution on bioavailability of cyclosporine: novel drug delivery system

During the long period of cyclosporine-containing dosage forms development a lot of significant findings have been done especially in the field of drug delivery systems. Currently available drugs are based, from technological point of view, on self-dispersible drug delivery systems, which contain cyclosporine solved in pharmaceutically acceptable vehicle. One can find difference among particular systems a) at particle size distribution after dispergation, b) at composition and c) at bioavailability of cyclosporine. As far as improvement of bioavailability between original brand leader formulation Sandimmune and recent brand leader formulation Neoral was followed by significant improvement in particle size distribution it was generally assumed that the reason for this improvement have been found. Several other formulations e.g. Consupren or SangCyA--self-dispersible systems, more or less correspond with above mentioned theory that smaller is better and by this principle bridged liquid based dosage forms with solid dosage forms. Bioavailability of novel drug delivery system which gives coarse dispersion with average particle size between 1-150 microns when dispersed have been tested on healthy volunteers. No difference among pharmacokinetic parameters of novel drug delivery system and microemulsion system have been observed in spite of fact that average particle size of novel system is almost 1000x bigger.     

驱动蛋白定向运动的动力学分析

将布朗粒子的定向运动,看作是系统受到外部非平衡涨落作用的结果,并建立相应的扩散模型。通过蒙特卡罗模拟方法,得到布郎粒子定向梯跳运动曲线。结果表明：非对称锯齿势,外部含时闪烁力及加性色噪声协调与联合作用,可使布朗粒子做梯跳或锯齿运动。作为一种可能的解释,将驱动蛋白Ｋｉｎｅｓｉｎｓ沿微管定向梯跳运动看作是上述协调作用的结果。     

Viral Protein U (Vpu)-Mediated Enhancement of Human Immunodeficiency Virus Type 1 Particle Release Depends on the Rate of Cellular Proliferation

Viral protein U (Vpu) is a 17-kDa phosphoprotein that enhances the release of viral particles from human immunodeficiency virus type 1-infected cells. This study shows that the effect of Vpu on efficient particle release depends on the rate of cell proliferation. Cells arrested by contact inhibition, chemical arresting agents, or terminal differentiation (i.e., macrophages) all exhibited a striking dependence on Vpu for efficient particle release, as shown by examination of particle production from transfections with full-length clones, infections, and the vaccinia virus expression system. In contrast, actively proliferating cells did not exhibit enhanced particle release with Vpu expression. This study demonstrates the necessity of Vpu for efficient viral particle release from quiescent cells.     

Monoclonal antibodies conjugated with superparamagnetic iron oxide particles allow magnetic resonance imaging detection of lymphocytes in the mouse brain

We investigated the potential of antibody-vectorialized superparamagnetic iron oxide (SPIO) particles as cellular specific magnetic resonance contrast agents to image lymphocyte populations within the central nervous system (CNS), with the final goal of obtaining a reliable tool for noninvasively detecting and tracking specific cellular populations in vivo. We used superparamagnetic particles bound to a monoclonal antibody. The particle is the contrast agent, by means of its T?* relaxation properties; the antibody is the targeting vector, responsible for homing the particle to target a surface antigen. To investigate the efficiency of particle vectorialization by these antibodies, we compared two types of antibody-vectorialized CD3-specific particles in vivo. We successfully employed vectorialized SPIO particles to image B220? cells in a murine model of B-cell lymphoma. Likewise, we were able to identify CD3? infiltrates in a murine model of multiple sclerosis. The specificity of the technique was confirmed by immunohistochemistry and electron microscopy of corresponding sections. Our findings suggest that indirect binding of the antibody to a streptavidinated particle allows for enhanced particle vectorialization compared to covalent binding of the antibody to the particle.     

Optical tweezers based force measurement system for quantitating binding interactions: system design and application for the study of bacterial adhesion

An optical force measurement system for quantitating forces in the pN range between micrometer-sized objects has been developed. The system was based upon optical tweezers in combination with a sensitive position detection system and constructed around an inverted microscope. A trapped particle in the focus of the high numerical aperture microscope-objective behaves like an omnidirectional mechanical spring in response to an external force. The particle's displacement from the equilibrium position is therefore a direct measure of the exerted force. A weak probe laser beam, focused directly below the trapping focus, was used for position detection of the trapped particle (a polystyrene bead). The bead and the condenser focus the light to a distinct spot in the far field, monitored by a position sensitive detector. Various calibration procedures were implemented in order to provide absolute force measurements. The system has been used to measure the binding forces between Escherichia coli bacterial adhesins and galabiose-functionalized beads.     

Formulation and evaluation of water-in-oil amoxicillin-loaded nanoemulsions using for Helicobacter pylori eradication

Antibiotics have a short residence time and have low concentrations when absorbed through the basolateral membrane in the stomach; this causes a failure to enhance drug concentrations at Helicobacter pylori infection sites. This study developed a nanocarrier system with the ability to carry amoxicillin to increase its efficacy against H. pylori. We used a water-in-oil emulsification system to prepare a positively charged nanoemulsion particle composed of amoxicillin, chitosan, and heparin. The particle size of the prepared nanoemulsion particle was controlled by the constituted compositions. The morphology of the nanoemulsion particles was spherical. In vitro analysis of amoxicillin released indicated that the nanocarrier system controlled amoxicillin release in the gastrointestinal dissolution medium and amoxicillin-loaded nanoemulsion particles localized to the site of H. pylori infection. Meanwhile, results of in vivo clearance assays indicated that the prepared amoxicillin-loaded nanoemulsion particles had a significantly greater H. pylori clearance effect in the gastric infection mouse model than the amoxicillin solution alone.     

Boundary of the adiabatic motion of a charged particle in a dipole magnetic field

The motion of a charged particle in a dipole magnetic field is considered using a quasi-adiabatic model in which the particle guiding center trajectory is approximated by the central trajectory, i.e., a trajectory that passes through the center of the dipole. A study is made of the breakdown of adiabaticity in the particle motion as the adiabaticity parameter χ (the ratio of the Larmor radius to the radius of the magnetic field line curvature in the equatorial plane) increases. Initially, for χ?0.01, the magnetic moment μ of a charged particle undergoes reversible fluctuations, which can be eliminated by subtracting the particle drift velocity. For χ?0.1, the magnetic moment μ undergoes irreversible fluctuations, which grow exponentially with χ. Numerical integration of the equations of motion shows that, during the motion of a particle from the equatorial plane to the mirror point and back to the equator in a coordinate system related to the central trajectory, the analogue of the magnetic moment μ is conserved. In the equatorial plane, this analogue undergoes a jump. The long-term particle dynamics is described in a discrete manner, by approximating the Poincaré mapping. The existence of the regions of steady and stochastic particle motion is established, and the boundary between these regions is determined. The position of this boundary depends not only on the adiabaticity parameter χ but also on the pitch angle. The calculated boundary is found to agree well with that obtained previously by using the model of a resonant interaction between particle oscillations associated with different degrees of freedom.     

A derivative of lipid A is involved in signal recognition particle/SecYEG-dependent and -independent membrane integrations

A cell-free system was developed that allows the correct integration of single and multispanning membrane proteins of Escherichia coli into proteoliposomes. We found that physiological levels of diacylglycerol were required to prevent spontaneous integration into liposomes even of the polytopic mannitol permease. Using diacylglycerol-containing proteoliposomes, we identified a novel integration-stimulating factor. Integration of mannitol permease was dependent on both the SecYEG translocon and this factor and was mediated by signal recognition particle and signal recognition particle receptor. Integration of M13 procoat, which is independent of both signal recognition particle/signal recognition particle receptor and SecYEG, was also promoted by this factor. Furthermore, the factor stimulated the post-translational translocation of presecretory proteins, suggesting that it also mediates integration of a signal sequence. This factor was found to be a lipid A-derived membrane component possessing a peptide moiety.