Application of transmission electron microscopy to the clinical study of viral and bacterial infections: present and future

Transmission electron microscopy has had a profound impact on our knowledge and understanding of viruses and bacteria. The 1000-fold improvement in resolution provided by electron microscopy (EM) has allowed visualization of viruses, the existence of which had previously only been suspected as the causative agents of transmissible infectious disease. Viruses are grouped into families based on their morphology. Viruses from different families look different and these morphological variances are the basis for identification of viruses by EM. Electron microscopy initially came to prominence in diagnostic microbiology in the late 1960s when it was used in the rapid diagnosis of smallpox, by differentiating, on a morphological basis, poxviruses from the less problematic herpesviruses in skin lesions. Subsequently, the technique was employed in the diagnosis of other viral infections, such as hepatitis B and parvovirus B19. Electron microscopy has led to the discovery of many new viruses, most notably the various viruses associated with gastroenteritis, for which it remained the principal diagnostic method until fairly recent times. Development of molecular techniques, which offer greater sensitivity and often the capacity to easily process large numbers of samples, has replaced EM in many areas of diagnostic virology. Hence the role of EM in clinical virology is evolving with less emphasis on diagnosis and more on research, although this is likely only to be undertaken in specialist centres. However, EM still offers tremendous advantages to the microbiologist, both in the speed of diagnosis and the potential for detecting, by a single test, any viral pathogen or even multiple pathogens present within a sample. There is continuing use of EM for the investigation of new and emerging agents, such as SARS and human monkeypox virus. Furthermore, EM forms a vital part of the national emergency response programme of many countries and will provide a frontline diagnostic service in the event of a bioterrorism incident, particularly in the scenario of a deliberate release of smallpox virus. In the field of bacteriology, EM is of little use diagnostically, although some bacterial pathogens can be identified in biopsy material processed for EM examination. Electron microscopy has been used, however, to elucidate the structure and function of many bacterial features, such as flagellae, fimbriae and spores and in the study of bacteriophages. The combined use of EM and gold-labelled antibodies provides a powerful tool for the ultrastructural localisation of bacterial and viral antigens.     

New in situ hybridization towards single-RNA detection

Fluorescence in situ hybridization (ISH) could be a powerful tool for analyzing the dynamics of memory phases if a semi-quantitative measurement of mRNA levels could be performed with a cellular resolution. While such a method does not yet exist, we show preliminary results in that direction where direct labeling of RNA probes with fluorescent dyes is combined with single-molecule-like detection.     

Structural Changes in Lymphocytes Membrane of Chernobyl Clean-up Workers from Latvia

ABM (3-aminobenzanthrrone derivative) developed at the Riga Technical University, Riga, Latvia) has been previously shown as a potential probe for determination of the immune state of patients with different pathologies .The fist study (using probe ABM) of peripheral blood mononuclear cells (PBMC) membranes of 97 Chernobyl clean-up workers from Latvia was conducted in 1997. Now we repeatedly examine the same (n = 54) individuals in dynamics. ABM spectral parameters in PBMC suspension, fluorescence anisotropy and blood plasma albumin characteristics were recorded. In 1997 screening showed 5 different patterns of fluorescence spectra, from which in 2007 we obtained only two. These patterns of spectra had never been previously seen in healthy individuals or patients with tuberculosis, multiple sclerosis, rheumatoid arthritis, etc., examined by us. Patterns of ABM fluorescence spectra are associated with membrane anisotropy and conformational changes of blood plasma albumin. We observed that in dynamics 1997–2007 the lipid compartment of the membrane became more fluid while the lipid-protein interface became more rigid. The use of probe ANS and albumin auto-fluorescence allowed show conformational alterations in Chernobyl clean-up workers blood plasma. It is necessary to note that all investigated parameters significantly differ in observed groups of patients. These findings reinforce our understanding that that the cell membrane is a significant biological target of radiation. The role of the membrane in the expression and course of cell damage after radiation exposure must be considered. So ten years dynamic of PBMC membrane characteristics by ABM (spectral shift and anisotropy indexes) in Chernobyl clean-up workers reveal progressive trend toward certain resemblance with those of chronic B-cell lymphoid leukemia.     

Microscopy techniques in flavivirus research

The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells. Fluorescence microscopy contributes greatly in understanding the fundamental viral protein localizations and virus–host protein interactions during infection. Electron microscopy remains the gold standard for visualizing ultra-structural features of virus particles and infected cells. New imaging techniques and combinatory applications are continuously being developed to push the limit of resolution and extract more quantitative data. Currently, correlative live cell imaging and high resolution three-dimensional imaging have already been achieved through the tandem use of optical and electron microscopy in analyzing biological specimens. Microscopy techniques are also used to measure protein binding affinities and determine the mobility pattern of proteins in cells. This chapter will consolidate on the applications of various well-established microscopy techniques in flavivirus research, and discuss how recently developed microscopy techniques can potentially help advance our understanding in these membrane viruses.     

Formation of two-dimensional crystals of icosahedral RNA viruses

The formation of 2D arrays of three small icosahedral RNA viruses with known 3D structures (tomato bushy stunt virus, turnip yellow mosaic virus and bromegrass mosaic virus) has been investigated to determine the role of each component of a negative staining solution containing ammonium molybdate and polyethylene glycol. Virion association was monitored by dynamic light scattering (DLS) and virus array formation was visualised by conventional transmission electron microscopy and cryo-electron microscopy after negative staining. The structural properties of viral arrays prepared in vitro were compared to those of microcrystals found in the leaves of infected plants. A novel form of macroscopic 3D crystals of turnip yellow mosaic virus has been grown in the negative staining solution. On the basis of the experimental results, the hypothesis is advanced that microscopic arrays might be planar crystallisation nuclei. The formation of 2D crystals and the enhancing effect of polyethylene glycol on the self-organisation of virions at the air/water interface are discussed. SYNOPSIS: The formation of 2D arrays of icosahedral viruses was investigated by spectroscopic and transmission electron microscopic methods.     

Statistical properties of thermodynamically predicted RNA secondary structures in viral genomes

By performing a comprehensive study on 1832 segments of 1212 complete genomes of viruses, we show that in viral genomes the hairpin structures of thermodynamically predicted RNA secondary structures are more abundant than expected under a simple random null hypothesis. The detected hairpin structures of RNA secondary structures are present both in coding and in noncoding regions for the four groups of viruses categorized as dsDNA, dsRNA, ssDNA and ssRNA. For all groups, hairpin structures of RNA secondary structures are detected more frequently than expected for a random null hypothesis in noncoding rather than in coding regions. However, potential RNA secondary structures are also present in coding regions of dsDNA group. In fact, we detect evolutionary conserved RNA secondary structures in conserved coding and noncoding regions of a large set of complete genomes of dsDNA herpesviruses.     

Single-strand DNA detection using a planar photonic-crystal-waveguide-based sensor

We report an experimental demonstration of single-strand DNA (ssDNA) detection at room temperature using a photonic-crystal-waveguide-based optical sensor. The sensor surface was previously biofunctionalized with ssDNA probes to be used as specific target receptors. Our experiments showed that it is possible to detect these hybridization events using planar photonic-crystal structures, reaching an estimated detection limit as low as 19.8 nM for the detection of the complementary DNA strand.     

Clustering gene expression data analysis using an improved EM algorithm based on multivariate elliptical contoured mixture models

Clustering gene expression data is an important research topic in bioinformatics because knowing which genes act similarly can lead to the discovery of important biological information. Many clustering algorithms have been used in the field of gene clustering. The multivariate Gaussian mixture distribution function was frequently used as the component of the finite mixture model for clustering, however the clustering cannot be restricted to the normal distribution in the real dataset. In order to make the cluster algorithm strong adaptability, this paper proposes a new scheme for clustering gene expression data based on the multivariate elliptical contoured mixture models (MECMMs). To solve the problem of over-reliance on the initialization, we propose an improved expectation maximization (EM) algorithm by adding and deleting initial value for the classical EM algorithm, and the number of clusters can be treated as a known parameter and inferred with the QAIC criterion. The improved EM algorithm based on the MECMMs is tested and compared with some other clustering algorithms, the performance of our clustering algorithm has been extensively compared over several simulated and real gene expression datasets. Our results indicated that improved EM clustering algorithm is superior to the classical EM algorithm and the support vector machines (SVMs) algorithm, and can be widely used for gene clustering.     

Confocal microscopy of whole mount embryonic cartilage: Intracellular localization of F-actin, chick prolyl hydroxylase and type II collagen mRNA

We show that whole mount preparations of embryonic chick sterna can be analyzed with confocal laser scanning microscopy (CLSM). This technique replaces the traditional sectioning of cartilage or culturing of chondrocytes. Whole ‘chunks’ of cartilage can be stained with dyes, used for immunohistochemistry or in situ hybridization. Although other stains have been used, the stains presented include phalloidin and propidium iodide which stain filamentous actin (F-actin) and the DNA and RNA of cells, respectively. Collagen secreting endoplasmic reticulum (ER) was localized with a primary antibody to chick prolyl hydroxylase (CPH) that was detected with a secondary antibody conjugated to FITC. The intracellular localization of type II collagen mRNA was analyzed using in situ hybridization. The cDNA probe specific for the C-propeptide region of the 1 type (II) collagen mRNA was nick translated and labeled with biotin-16-dUTP. Biotin labeled probes were visualized with avidin-FITC. Depending on the intensity of the stain, we were able to analyze approximately 3–10 layers of chondrocytes. Stains penetrated into the cartilage better than antibodies and biotin-avidin labeled cDNA probes. The F-actin was located as bands of filaments in the superficial layers of the cartilage and was associated with the membranes that marked cell boundaries as deep as 10 layers of chondrocytes. The ER stained with anti-chick prolyl hydroxylase was prominent in perinuclear regions of the cells, but the antibody was only able to penetrate 4–5 cell layers. Single label in situ hybridization studies show that chondrocytes are positive for type II collagen mRNA. Similar to the immunohistochemistry, in situ hybridization probes were only able to penetrate 4–5 cell layers. The type II collagen mRNA appeared perinuclear in the chondrocytes, similar to the ER staining pattern.     

On the filamentation instability in degenerate relativistic plasmas

The filamentation instability of the electromagnetic (EM) beam in an underdense plasma with high level of degeneracy is examined by means of the momentum equation, continuity equation and Maxwell's equations. It has been demonstrated that the instability develops for weakly as well as strongly relativistic degenerate plasma and arbitrary strong amplitude of EM beams.     

New diphenylhexatriene derivatives as fluorescent membrane probes: Partitioning properties

Three new diphenylhexatriene derivatives, two phospholipids and one single-chain amphiphilic molecule, have been synthesized and considered as probes for measuring membrane fluidity by fluorescence anisotropy. The possibility of using these probes to determine specifically fluidity of inner leaflets of cellular plasma membranes was inferred from their partitioning properties between gel and liquid crystalline phases of phospholipid vesicles of binary composition.     

Electron plasma waves generation in suddenly created isotropicplasma

Electron plasma waves excitation in suddenly created isotropic plasma as a result of weak nonlinear interaction of linearly polarized plane electromagnetic (EM) wave and electrons has been considered. By the use of standard perturbation method the problem is solved in closed form for the case of a simple harmonic source EM wave. The appearance of the second harmonic and time independent modes have been demonstrated. The efficiency of excitation of these modes is possible to control by varying the frequency of the source wave     

等离子体覆盖立方散射体目标雷达散射截面的时域有限差分法分析

采用分段线性电流密度递归卷积时域有限差分（PLJERC-FDTD）算法计算了均匀非磁化等离子体覆盖三维立方体目标的散射特性.分析了等离子体厚度、密度和碰撞频率对雷达散射截面（RCS）的影响.计算结果表明：等离子体包层能有效地减小雷达目标的RCS，当等离子体频率比入射电磁波频率小得多时，主要靠增大等离子体的厚度使立方散射体目标的RCS值减小，增大等离子体碰撞频率对立方散射体目标的RCS值影响不大；当等离子体频率约为入射电磁波频率的一半时，增大等离子体厚度和碰撞频率都对立方散射体目标的RCS值减小有影响；当等 关键词： FDTD算法 电磁波 等离子体隐身 雷达散射截面     

The effect of infected external computers on the spread of viruses: A compartment modeling study

Inevitably, there exist infected computers outside of the Internet. This paper aims to understand how infected external computers affect the spread of computer viruses. For that purpose, a new virus–antivirus spreading model, which takes into account the effect of infected/immune external computers, is established. A systematic study shows that, unlike most previous models, the proposed model admits no virus-free equilibrium and admits a globally asymptotically stable viral equilibrium. This result implies that it would be practically impossible to eradicate viruses on the Internet. As a result, inhibiting the virus prevalence to below an acceptable level would be the next best thing. A theoretical study reveals the effect of different parameters on the steady virus prevalence. On this basis, a number of suggestions are made so as to contain virus spreading.     

Adamantane Derivatives Functionalized Gold Nanoparticles for Colorimetric Detection of MiRNA

MiRNAs are riveting RNA molecules due to their close relevance to the regulation of gene expression and certain physiological or pathological processes. Rapid and sensitive methods for miRNA assay are essential for biological researches and clinical diagnosis of many diseases. In this work, gold nanoparticles (AuNPs) have been modified with adamantane derivatives and then Dwith NA probes for colorimetric detection of miRNA. Target miRNA induces a change in DNA conformation and initiates strand displacement amplification, eventually leading to massive aggregations of AuNPs. By comparing the UV–vis absorption spectra, miRNA concentration can be determined. This developed method can detect miRNA as low as 3.7 × 10^?15m with remarkable specificity. Moreover, it is successfully used to inspect the expression of miRNA in biological samples. Therefore, adamantane derivatives functionalized AuNPs are demonstrated to offer a novel platform for biosensing and the miRNA sensing strategy may find a broad spectrum of practical applications.     

Membranes,peptides, and disease: Unraveling the mechanisms of viral proteins with solid state nuclear magnetic resonance spectroscopy

The interplay between peptides and lipid bilayers drives crucial biological processes. For example, a critical step in the replication cycle of enveloped viruses is the fusion of the viral membrane and host cell endosomal membrane, and these fusion events are controlled by viral fusion peptides. Thus such membrane-interacting peptides are of considerable interest as potential pharmacological targets. Deeper insight is needed into the mechanisms by which fusion peptides and other viral peptides modulate their surrounding membrane environment, and also how the particular membrane environment modulates the structure and activity of these peptides. An important step toward understanding these processes is to characterize the structure of viral peptides in environments that are as biologically relevant as possible. Solid state nuclear magnetic resonance (ssNMR) is uniquely well suited to provide atomic level information on the structure and dynamics of both membrane-associated peptides as well as the lipid bilayer itself; further ssNMR can delineate the contribution of specific membrane components, such as cholesterol, or changing cellular conditions, such as a decrease in pH on membrane-associating peptides. This paper highlights recent advances in the study of three types of membrane associated viral peptides by ssNMR to illustrate the more general power of ssNMR in addressing important biological questions involving membrane proteins.     

FDTD Simulation of Electromagnetic Reflection of Conductive Plane Covered with Imhomogeneous Time-Varying Plasma

A finite-difference time-domain (FDTD) algorithm is applied to study the electromagnetic reflection of conduction plane covered with inhomogeneous, collision, warm, time-varying plasma. The collision frequency of plasma is a function of electron density and plasma temperature. Under the one-dimensional case, transient electromagnetic propagation through various plasmas have been obtained, and the reflection coefficient of EM wave through inhomogeneous time-varying plasma (ITVP), homogeneous time-varying plasma (HTVP) and inhomogeneous plasma (IP) are calculated under different conditions. The results illustrate that a plasma cloaking system can successfully absorb the incident EM wave.     

Inactivation of bovine immunodeficiency virus by photodynamic therapy with HMME

To investigate the effect of photodynamic therapy (PDT) with hematoporphrin monomethyl ether (HMME) on bovine immunodeficiency virus (BIV) can provide the basis theory for photoinactivation of human immunodeficiency virus (HIV). To assess the protection of HMME-PDT on the cell line Cf2Th infected with BIVR29 by 3-(4,5)-dimethylthiahiazol-2-yl-3,5-di-phenytetrazolium bromide (MTT) with power density of 5 and 25 mW/cm2 and energy density from 0.6 to 3 J/cm<'2>. To observe the inhibition of membrane fusion using a new reporter cell line BIVE by fluorescence microscope. HMME-PDT has significant protectant effects on Cf2Th-BIVR29 with both power densities, especially in the group of high power density. Fluorescent microscope shows that there is no significant difference between the group of PDT and control, which means PDT could not inhibit the BIV-mediated membrane fusion.     

Analysis of plasma sheath propagation attenuation based on ray tracing

Based on the complex Snell's law of lossy media, a ray tracing method is proposed to study the propagation attenuation characteristics of electromagnetic (EM) waves in plasma sheaths. The plasma sheath is modelled as layered media. This method considers the complex ray characteristics of inhomogeneous plane EM waves, tracks the propagation rays of EM waves in each layer of media, and calculates the propagation attenuation of EM waves in each layer of media according to the propagation direction of the complex rays. The attenuation during numerical cumulative propagation is the total attenuation of EM waves through the plasma sheath. By comparing the results with that obtained from the WKB method, the accuracy of the ray tracing algorithm is proved. The results of the propagation attenuation of a blunt cone model are calculated by the proposed method, and the effects of different parameters on the EM wave propagation attenuation in the plasma sheath are analysed at different heights, velocities, incident angles, and incident positions. Studying the propagation characteristics of EM waves in the plasma sheath is of importance in application for radar target tracking, blackout communication, and other issues.