The structure of proteoglycan aggregate determined by atomic force microscopy

Proteoglycan aggregate is the major extracellular matrix component in cartilage, comprising about 18% of the dry weight of hyaline cartilage. The proteoglycan aggregate is the major substance in cartilage which resists compression in the joint. The purpose of this study was to utilize the newly developed imaging technique, Atomic force Microscopy (AFM), to visualize the ultrastructure of proteoglycan aggregates. The proteoglycan aggregate molecules were imaged in air using the tapping mode of the AFM. The images illustrated the ultrastructure of the aggregates, especially the individual proteoglycan and the core hyaluronic acid. In addition to the length and width of each molecule, the height of the proteoglycan aggregates and the individual proteoglycans could be directly measured. The images of the ultrastructures of proteoglycan aggregates visualized from the AFM are comparable with those using conventional electron microscopy approaches. Nevertheless, the sample preparation for AFM imaging does not involve fixation, staining, coating, and other routine procedures required for traditional electron microscopy imaging. Thus, this technique could be a simple alternative approach for future analysis of proteoglycan aggregate and its assembly.     

Second-order stereology and ultrastructural examination of the spatial arrangements of tissue compartments within glomeruli of normal and diabetic kidneys

The present study explores 3D spatial arrangements of compartments within the rat renal glomerulus and tests for differences after chemically-induced diabetes. In particular, the arrangements of capillaries, podocytes, mesangium and urinary space are quantified and compared between (a) kidneys within groups and (b) kidneys from streptozotocin-diabetic rats and age-matched controls. The stereological tool employed is the pair correlation function which is estimated by counting linear dipole probes of different sizes superimposed on ultrathin sections so as to be random in position and orientation. Unbiased estimates of the volume density of each glomerular component were estimated by point counting. Thereafter, estimates of the covariance and pair correlation function were determined from corresponding dipole counts. Plots of covariance and pair correlation functions against dipole length were almost identical in control and diabetic groups, indicating that diabetes did not disturb the normal spatial arrangements within glomeruli. However, differences were detected between compartments within groups. Whilst volume elements within all compartments were clustered at distances below about 8 μm (the approximate size of the basic cellular or other structural unit), the cluster size varied between compartments. The pattern was one of progressively smaller clusters in the sequence capillaries, podocytes, urinary space, mesangium. Beyond a distance of 8 μm, all glomerular components (in both control and diabetic groups) were arranged as expected for a 'random' (meaning neither clustered nor repulsed) volume process. These studies re-emphasize the relative invariance of biological organization and the value and limitations of covariance analysis for quantifying different levels of organization in different tissues and experimental groups.     

Quantitative morphology for biologists and computer scientists: I. Computer-aided tutorial for biological stereology (version 1.0).

This paper describes a computer-aided tutorial for biological stereology. Stereology, a type of quantitative morphology, includes a collection of statistical methods that quantify the structural compartments that can be viewed in sections with light and electron microscopy. These methods provide volume, surface, length, shape, and number data, and help define the quantitative relationships among the structural compartments of biological hierarchies. Hierarchies, which connect structural data ranging in size from molecules to organs, serve as a central core to which the data of biological databases can be linked. The tutorial focuses on two objectives. It provides the user primarily interested in using quantitative morphology databases with background information, and offers a set of state-of-the-art tools to researchers wishing to use these methods in the laboratory. The main topics of the tutorial include: introduction to quantitative morphology, symbols/terms, data types, sampling, hierarchies, data interpretation, and utilities. The tutorial runs under the MS-DOS operating system and requires at least an IBM PC AT (or compatible), a color monitor (EGA, VGA), 540 KB of RAM, and 3 MB of hard disk space.     

Geometric approach to segmentation and protein localization in cell culture assays

Cell‐based fluorescence imaging assays are heterogeneous and require the collection of a large number of images for detailed quantitative analysis. Complexities arise as a result of variation in spatial nonuniformity, shape, overlapping compartments and scale (size). A new technique and methodology has been developed and tested for delineating subcellular morphology and partitioning overlapping compartments at multiple scales. This system is packaged as an integrated software platform for quantifying images that are obtained through fluorescence microscopy. Proposed methods are model based, leveraging geometric shape properties of subcellular compartments and corresponding protein localization. From the morphological perspective, convexity constraint is imposed to delineate and partition nuclear compartments. From the protein localization perspective, radial symmetry is imposed to localize punctate protein events at submicron resolution. Convexity constraint is imposed against boundary information, which are extracted through a combination of zero‐crossing and gradient operator. If the convexity constraint fails for the boundary then positive curvature maxima are localized along the contour and the entire blob is partitioned into disjointed convex objects representing individual nuclear compartment, by enforcing geometric constraints. Nuclear compartments provide the context for protein localization, which may be diffuse or punctate. Punctate signal are localized through iterative voting and radial symmetries for improved reliability and robustness. The technique has been tested against 196 images that were generated to study centrosome abnormalities. Corresponding computed representations are compared against manual counts for validation.     

Relative labelling index: a novel stereological approach to test for non-random immunogold labelling of organelles and membranes on transmission electron microscopy thin sections

Simple and efficient protocols for quantifying immunogold labelling of antigens localized in different cellular compartments (organelles or membranes) and statistically evaluating resulting labelling distributions are presented. Two key questions are addressed: (a) is compartmental labelling within an experimental group (e.g. control or treated) consistent with a random distribution? and (b) do labelling patterns vary between groups (e.g. control vs. treated)? Protocols rely on random sampling of cells and compartments. Numbers of gold particles lying on specified organelle compartments provide an observed frequency distribution. By superimposing test‐point lattices on cell profiles, design‐based stereology is used to determine numbers of points lying on those same compartments. Random points hit compartments with probabilities determined by their relative sizes and so provide a convenient internal standard, namely, the expected distribution if labelling is purely random. By applying test‐line lattices, and counting sites at which these intersect membrane traces, analogous procedures provide observed and expected labelling distributions for different classes of membranes. Dividing observed golds by expected golds provides a relative labelling index (RLI) for each compartment and, for random labelling, the predicted RLI = 1. In contrast to labelling densities of organelles (golds µm^?2) or membranes (golds µm^?1), RLI values are estimated without needing to know lattice constants (area per point or length per intersection) or specimen magnification. Gold distributions within a group are compared by chi‐squared analysis to test if the observed distribution differs significantly from random and, if it is non‐random, to identify compartments which are preferentially labelled (RLI > 1). Contingency table analysis allows labelling distributions in different groups of cells to be compared. Protocols are described and illustrated using worked specimen examples and real data.     

运动链拓扑胚图的同构判断

平面并联机构基于胚图的综合方法中,拓扑胚图的同构判断是关键的环节。针对不含二元杆的运动链拓扑胚图,解决它们之间的同构判断问题。运动链的拓扑胚图与拓扑图相比有着独特的个性,由于在支链上没有二元点,所以图的顶点之间的关系主要是顶点之间的相对位置。根据拓扑胚图的特性,从关于图的邻接矩阵的经典理论出发,建立图的任意顶点之间的路径数矩阵,将路径数矩阵的元素按照一定规则排列成路径数组。论证了拓扑胚图同构的条件。在度序列和一阶路径数组相等的前提下,利用二阶路径数组来判断拓扑胚图是否同构。举实例说明判断过程及具体应用。拓扑胚图同构判断问题的解决不仅为基于胚图的型综合奠定了基础,而且对于某些运动链拓扑图的同构判断也具有普遍意义。     

Structural modification of the hamster sperm acrosome during posttesticular development in the epididymis

The acrosome of the mature spermatozoon functions as a regulated secretory vesicle which performs several critical functions in mammalian fertilization. Acrosome assembly occurs throughout spermiogenesis and continues during posttesticular sperm maturation in the epididymis, resulting in a structurally polarized membrane-bounded organelle that contains an assortment of hydrolases and a stable infrastructure termed the acrosomal matrix. The role of stable acrosomal matrix assemblies in acrosomal biogenesis and function are poorly understood. This article presents ultrastructural, immunocytochemical, and biochemical data on the remodeling of the hamster acrosomal matrix during spermiogenesis and posttesticular sperm maturation in the epididymis. Specific posttranslational modifications of the major acrosomal matrix protein are evident in late, step 16, spermatids and matrix protein processing continues within specific acrosomal subdomains of caput epididymal spermatozoa. At the completion of sperm maturation, the acrosomal matrix consists of two structurally distinct domains which are adherent to the outer acrosomal membrane and exhibit a localized distribution pattern. Coincident with acrosomal matrix differentiation, a paracrystalline cytoskeletal complex is assembled onto the outer acrosomal membrane of epididymal spermatozoa. This cytoskeletal network appears to establish transmembrane structural interactions with the acrosomal matrix and may maintain attachment of the acrosomal cap to the sperm head during the early steps of the acrosome reaction.     

Influence of proteoglycan contents and of tissue hydration on the frictional characteristics of articular cartilage

In this work, the hypothesis that water content and substances present on the articular surface play an important role in lubrication through the formation of a layer with a high content of water on the articular surface is analysed. The hydrophilic properties of proteoglycans exposed at the articular surface and hydration of tissue are the main responsible factors for the formation of this layer. The role of the articular surface in the frictional characteristics of articular cartilage was examined using specimens (femoral condyles of pigs) with intact and wiped surfaces tested in intermittent friction tests. Results indicated that the intact condition presented low friction in comparison with the wiped condition. The measured water loss of the articular cartilage after sliding and loading indicated a gradual decrease in the water content as the time evolved, and rehydration was observed after the submersion of unloaded specimens in the saline bath solution. Micrographic analyses indicated the presence of a layer covering the articular surface, and histological analyses indicated the presence of proteoglycans in this superficial layer. The hydration of the cartilage surface layer and proteoglycan in this layer influence lubrication.     

Molecular self-recognition and adhesion via proteoglycan to proteoglycan interactions as a pathway to multicellularity: atomic force microscopy and color coded bead measurements in sponges

During the emergence of multicellular organisms, molecular mechanisms evolved to allow maintenance of anatomical integrity and self-recognition. We propose that carbohydrates from proteoglycans, as the most peripheral cell surface, and matrix molecules might have provided these key adhesion and recognition functions. If so, the Porifera as the simplest metazoans alive today should retain, at least in part, proteoglycan adhesion and recognition mechanisms. Early work on cell adhesion of dissociated marine sponge cells provided important phenomenological evidence for cell sorting. Here is reviewed recent work on molecular mechanisms of cell recognition and adhesion mediated by cell surface proteoglycans purified from three marine sponge species, Microciona prolifera, Halichondria panicea, and Cliona celata. Biochemical characterization of isolated proteoglycans showed that each species expressed a unique type of primordial molecule named glyconectins. These proteoglycans displayed species-specific self-recognition and adhesion in color-coded bead, cell, and blotting assays. The specificity of homophilic proteoglycan to proteoglycan interactions in the Porifera approaches the binding selectivity of the evolutionarily advanced immunoglobulin superfamily system. Such xeno-selectivity may be a new paradigm for the molecular self-recognition, which was a fundamental requirement in the self/non-self discrimination during the emergence of multicellularity and further divergence of species. We have used atomic force microscopy (AFM) technology to directly measure intermolecular binding strength between individual pairs of ligand and receptor molecules in physiological solution. Homophilic glyconectin interactions were investigated by AFM after covalent attachment of the protein core to the sensor tip and to a flat surface, leaving the carbohydrates unmodified. AFM measurements of the binding strength between glyconectins indicated that one pair of molecules could theoretically hold the weight of 1,600 cells in physiological solution. These results provided the first essential and quantitative evidence that proteoglycan-proteoglycan binding can perform the adhesion function that we have assigned to it. Our investigations with purified proteoglycans from the marine sponge M. prolifera (glyconectin 1) using bead and cell adhesion assays have provided evidence that a new molecular mechanism of polyvalent and specific glycan-glycan binding between proteoglycans can mediate cell recognition and adhesion. Partial sequencing of the glycans has revealed two new cell adhesion carbohydrate structures: (3)GlcNAc(3OSO3)beta1-3Fuc and Pyr4,6Galbeta1-4GlcNAcbeta1-3Fuc.     

Structure determination and structure refinement of Al2CuMg precipitates by quantitative high-resolution electron microscopy

The structure of the S-phase (Al2CuMg) precipitate in an Al matrix has been determined by using a combination of image processing, quantitative image comparison, and automatic refinement of imaging and structural parameters. A method for comparing images with unknown origin relationship while quickly estimating a possible translation of the origin is outlined. The optimization algorithm used in the structure determination utilizes space group symmetry, which is deduced from the crystal-zone axis and reduces the number of free parameters.     

Application of design‐based stereology for estimation of absolute volume and surface area of the articular and calcified cartilage compartments of undecalcified human femoral heads

Design‐based stereological methods using systematic uniform random sampling, the Cavalieri estimator and vertical sections are used to investigate undecalcified human femoral heads. Ten entire human femoral heads, obtained from normal women and normal men, were systematically sampled and thin undecalcified vertical sections were obtained. Absolute volumes and surface areas of the entire femoral head, the articular cartilage and the calcified cartilage compartments were estimated. In addition, the average thickness of the articular cartilage and the calcified cartilage were calculated. The stereological procedures applied to the human femoral heads resulted in average coefficient of errors, which were 0.03–0.06 for the volume estimates and 0.03–0.04 for the surface area estimates. We conclude that design‐based stereology using the Cavalieri estimator and vertical sections can successfully be used in large undecalcified tissue specimens, like the human femoral head, to estimate the absolute volume and surface area of macroscopic as well as of microscopic tissue compartments. The application of well‐known design‐based stereological methods carries potential advantage for investigating the pathology in inflammatory and degenerative joint diseases.     

Localization of acid and alkaline phosphatase

Ultrathin frozen sections of glutaraldehyde fixed yeast cells have been successfully used for the demonstration of acid and alkaline phosphatase. Acid phosphatase was localized over the cell wall of both the mother cell and the bud as well as over the newly forming cross wall (septum). Cytoplasmic vesicles (vacuoles, lysosomes?) located close to the cell wall showed a positive reaction for acid phosphatase as well. After 3 h glutaraldehyde fixation an activation of the nuclear acid phosphatase was observed. Lead precipitates were predominantly found over the nucleolar material of ‘resting’ and budding cells. Alkaline phosphatase could be demonstrated in the ‘yeast-mesosome’ and within the plasmalemma invaginations. After separation of the bud, small vesicles, probably derived from the endoplasmic reticulum showed a strong positive reaction for alkaline phosphatase. In frozen sections incubated for alkaline phosphatase, non-specific lead precipitates were found in the nucleus and along the plasmalemma invaginations.     

The structure of (100) defects in carbonated apatite crystallites: a high resolution electron microscope study

Planar defects parallel to (100) with an approximate [1/400] displacement vector have been identified by high resolution transmission electron microscopy and by micro-electron diffraction in the center of synthetic carbonated apatite crystallites. Similar intergrowths, 0.8-1.5 nm in width, have been observed in dental enamel, dentin and bone apatite crystallites. Four possible structural models of the defect core are proposed to explain these experimental features, and computer-simulated lattice images of the models are compared with the experimental images. Typical defects were consistent with a two-dimensional octacalcium phosphate inclusion, one unit cell thick, embedded in an apatite matrix.     

Comparative study by computed radiography,histology, and scanning electron microscopy of the articular cartilage of normal goats and in chronic infection with caprine arthritis‐encephalitis virus

In the northeast of Brazil, caprine arthritis‐encephalitis (CAE) is one of the key reasons for herd productivity decreasing that result in considerable economic losses. A comparative study was carried out using computed radiography (CR), histological analysis (HA), and scanning electronic microscopy (SEM) of the joints of CAE infected and normal goats. Humerus head surface of positive animals presented reduced joint space, increased bone density, and signs of degenerative joint disease (DJD). The carpal joint presented no morphological alterations in CR in any of the animals studied. Tarsus joint was the most affected, characterized by severe DJD, absence of joint space, increased periarticular soft tissue density, edema, and bone sclerosis. Histological analysis showed chronic tissue lesions, complete loss of the surface zone, absence of proteoglycans in the transition and radial zones and destruction of the cartilage surface in the CAE positive animals. Analysis by SEM showed ulcerated lesions with irregular and folded patterns on the joint surface that distinguished the limits between areas of normal and affected cartilage. The morphological study of the joints of normal and CAE positive goats deepened understanding of the alteration in the tissue bioarchitecture of the most affected joints. The SEM finding sustained previous histological reports, similar to those found for rheumatoid arthritis, suggesting that the goat infected with CAE can be considered as a potential model for research in this area. Microsc. Res. Tech. 77:11–16, 2014. © 2013 Wiley Periodicals, Inc.     

Histochemical and ultrastructural studies of the mosquito Aedes aegypti fat body: effects of aging and diet type

Aedes aegypti is the principal vector of dengue world wide and a major vector of urban yellow fever. Despite its epidemiological importance, not much is known regarding cellular and structural changes in the fat body in this mosquito. Here, we applied light and transmission electron microscopies to investigate structural changes in the fat body of three groups of A. aegypti females: newly emerged, 18-day-old sugar-fed, and 18-day-old blood-fed. The fat body consists of a layer of cells attached to the abdomen integument, formed by trophocytes and oenocytes. Trophocytes are strongly positive for carbohydrates, while oenocytes are strongly positive for proteins and lipids. Ultrastructural analyses of trophocytes from newly emerged and 18-day-old blood-fed indicate that these cells are rich in glycogen and free ribosomes. Many lipid droplets and protein granules, which are broken down after the blood meal, are also detected. In 18-day-old sugar-fed, trophocytes display a disorganized cytoplasm filled with lipid droplets, and reduced numbers of free ribosomes, glycogen, rough endoplasmic reticulum (RER) and mitochondria. Following a blood meal, the RER and mitochondria display enlarged sizes, suggestive of increased activity. With regard to oenocytes, these cells display an electron-dense cytoplasm and plasma membrane infoldings facing the hemolymph. As the A. aegypti female ages, trophocyte and oenocyte cell nuclei become larger but decrease in diameter after blood feeding. Our findings suggest that the trophocytes and oenocytes remodeling is likely involved in functional changes of fat body that take place during aging and following a blood meal in A. aegypti females.     

Hapten-tagged plasma proteins as immunocytochemical probes for the study of vascular permeability.

Bovine serum albumin and transferrin were covalently coupled with fluorescein isothiocyanate and digoxigenin, respectively, and intravenously co-injected in equal amounts in mouse. The derivation of the two proteins induces minor alterations of their physicochemical properties as well as of their physiological functions. The two tracers were revealed within vascular and extravascular compartments of diaphragm by quantitative postembedding immunocytochemistry, using antibodies against each of the haptens in conjunction with the protein AG-gold complexes. The influence of different fixatives and embedding protocols on the immunodetectability of the hapten-tagged proteins was assessed. Both resist reasonably well to osmication and embedding in Epon. None of the haptens reacted with the heterologous antibody. At 30 minutes after injection, the tracers were detected in blood plasma, interstitium, and endothelial plasmalemmal vesicles. The presence of both proteins within the interendothelial clefts was inconspicuous. The ratios between the labeling densities found over endothelium, interstitial space, and vascular lumen were similar for both tracers. This suggests that the endothelium of mouse diaphragm capillaries might exhibit comparable permeabilities towards serum albumin and transferrin which are similar in size and charge. The study shows that hapten-tagged polypeptides are close to the corresponding native macromolecules, and represent interesting tools for the morphological study of dynamic processes such as transcytosis.     

Ultrastructural features of the AIDS virus (HIV) and its morphogenesis

HIV particles were usually seen on the surface of established lymphoid cells derived from AIDS patients or on CEM cells infected with HIV, and sometimes in cytoplasmic vacuoles. The virus particles were formed by a budding process from the plasma membrane of an infected cell. The budding particles were of a doughnut form. Various profiles of virus particles were seen extracellularly: type 1 had a bar-shaped, electron-dense core, type 2 had a central and type 3 an eccentric electron-dense round core, type 4 was doughnut-shaped, and type 5 had a layered core. However, projection patterns of the AIDS virus model suggested that type 1, 2 and 3 particles are similar. Therefore, the AIDS virus may be one of three main types: with or without a dense core, and with a layered core. It is thought that a particle with a layered core and a doughnut-type particle may be immature viruses.     

Plastic continuum models for truss lattice materials with cubic symmetry

Analytical and numerical studies on continuum models for the elastic-plastic behavior of uniformly periodic lattice materials under multi-axial loading are presented in this paper. This study firstly investigates the basic topology of unit cell structures for three different lattice materials with cubic symmetry. By homogenizing the mechanical properties of these materials within the unit volume space, the equivalent continuum models are obtained with the internal variables which result in the mechanical and geometrical characteristics of discrete truss members at the micro-scale such as structural packing, axial stiffness, and material density. Therefore, in this study, the strain hardening was applied to the material model of individual truss members in a valuable effort to explain the plastic behavior of the homogenized lattice material. The expansion of pressure-dependent stress surface at the macro-scale level is estimated by analytical predictions, which are derived from the equivalent continuum models. Analytical predictions show good agreements with existing results obtained by finite element (FE) analyses.     

6650型工业机器人运动学分析

以6650型六轴工业机器人作为研究目标,利用D-H表示法建立该机器人的连杆模型与连杆坐标系。得到机器人杆件几何参数和关节变量,进而推导出该机器人的运动学方程,并由此计算出机器人的工作空间,为机器人的轨迹规划及运动提供一定的数学基础。     

Data correlation analysis for optimal sensor placement using a bond energy algorithm

Optimal sensor placement is one of the crucial and fundamental factors for constructing a cost-effective structural health monitoring system and is related to the effective evaluation of the state of the structure. Structural responses are correlated to some extent, as the structural behavior is continuous. Based on the above two considerations, the question arises of how to obtain the maximum amount of information for understanding the structure using measurements from limited sensors and not be limited to direct monitoring at the placements where the limited sensors are located. Data correlation analysis for optimal sensor placement is proposed using a bond energy algorithm, in which the objectives, such as structural response evaluation covering the maximum structural responses using measurements from sensors located at the optimal placements, are taken into account. The data correlation analysis is conducted for the structural responses, and the correlation matrix is established. Furthermore, the optimal sensor placements and the correlation of the responses at element locations can be determined using the bond energy algorithm. A Schwedler single-layer spherical lattice dome-like structure, which is a common large space steel structure, is used to simulate the structural responses and verify the effectiveness of the proposed method by discussion of different scenarios of parameter selection.