bluuebunny/crossref_metadata_2025 huggingface dataset:
Morphological Characterization of a Sustained-Release Drug Implant by Scanning Electron Microscopy, Polarized Light Microscopy and Image Analysis
Authors: Neilly JP , Deng JS , House JL , Fagerland JA
Published: July 2020

Septacin is a sustained-release antibiotic currently under development by the Hospital Products Division of Abbott Laboratories. The product is designed to be used as an anti-infective implant in orthopedic surgical procedures with a sustained drug release for up to six weeks in vivo. It consists of gentamicin sulfate formulated with a bioerodable polyanhydride copolymer. The polymer is biodegradable and has been approved by the FDA for human clinical trials. The final product is obtained by mixing 20% gentamicin sulfate with molten polymer and injection molding it to form cylindrical Septacin beads.

The microstructure of drug particles and polymer matrix is critical to the performance of sustained release products, thus scanning electron microscopy (SEM) and polarized light microscopy (PLM) were utilized in this study. SEM has proven useful for evaluating the microstructure of drug formulations3 and was used to examine the drug-polymer matrix structure. Average drug particle size and distribution were determined, and the drug-polymer boundary was evaluated.
Quantification of Bone Formation on Calcium Phosphate Ceramic Thin Film, in Vitro by Tetracycline Labelling
Authors: Davies JE , Krasnoshtein F , Hryhorenko L , Sindrey D
Published: July 2020

Introduction

We have previously described a quantitative in vitro method for the assessment of bone matrix deposition, employing the fluorescent antibiotic tetracycline1. In a present study we demonstrate the relationship of bone matrix formation by osteoblasts as a function of cell seeding density, and time, on calcium phosphate ceramic thin film substrates.

Materials and Methods

Substrata. Commercially available 3x1 inch quartz Osteologic™ chamber slides (Millenium Biologix Inc., Kingston, Ont.) previously shown to support bone growth, were used as substrates for osteoblastic cell growth2. These chamber slides are coated with a sintered sub-micron calcium phosphate ceramic thin film, and are fitted with a removable 16 well superstructure.

Rat Bone Marrow Cell Culture. Bone marrow cells from young adult male Wistar rat (115-120g) femora were cultured according to a previously described protocol. Each well of each Osteologic™ chamber slide (14/16 wells; 2 wells contained media, but without cells, to provide background for tetracycline fluorescence) was inoculated with a 250 μl of cell suspension.
Bone Formation by Human Oseoblasts on Implant Materials
Authors: Gronowicz G , Ahmad M
Published: July 2020

We have developed an in vitro system to study the process by which osteoblasts attach and secrete extracellular matrix (ECM) proteins that subsequently mineralize in response to implant materials. In vivo mineralization is characterized by the sequential expression by osteoblasts of specific ECM proteins that are capable of calcifying in an orderly manner. A similar sequence of events is shown to occur in this in vitro system. The extent and pattern of mineralization was visualized by fluorescence light microscopy and transmission electron microscopy (TEM), and was dependent on the substrate to which osteoblasts adhered.

Materials and Methods

Disks (22 mm diameter) composed of Tivanium (Tiv) (Ti6AI4V, ASTM F-136) and Zimaloy (Zim)(CoCrMo, ASTM F-75), prepared identically to clinical orthopaedic implants, were supplied by Zimmer, Inc. Glass disks were used as controls. Osteoblast-like cells (Saos-2), derived from a human osteosarcoma, or primary human osteoblasts from a 41 year-old male patient were added to the disks in a-MEM medium with 10% fetal bovine serum,
Characterization of Titanium Alloy Particulates to Study the in Vitro Release of Inflammatory Mediators by Human Peripheral Blood Monocytes.
Authors: Darien BJ , Sims P , Robinson T , Manley P , Albrecht R
Published: July 2020

Particulate wear debris found around joint arthroplasties has been linked to the premature failure of artificial joints. Cytokines and eicosanoids released by macrophages are known mediators of inflammation and osteolysis. Previous in vitro studies of wear debris-mediated inflammation have relied upon poorly characterized particulates and peripheral blood cell fractions of mixed cellular composition. The objective of this study was to utilize titanium particulates that closely match wear debris found around joint arthroplasties and to study their effects on the biosynthesis of inflammatory mediators by isolated monocytes.

To mimic wear debris that surrounds artificial joints, titanium alloy particulates (1-3 μm in diameter) were obtained. A submicron fraction of these particulates was isolated by differential sedimentation. The morphology, size and concentration of the resulting population was characterized utilizing scanning electron microscopy. Titanium particulate size was estimated from photomicrographs of calibrated latex microspheres mixed with titanium particles (Figure 1). A known volume of titanium particulates in suspension was applied to a carbon planchette and allowed to dry.
Nanoscale Correlation of Structure and Mechanical Properties of a Human Tooth
Authors: Fong Hanson , Sarikaya Mehmet
Published: July 2020

The human tooth is a functionally-gradient structural composite material composed of collagen and hydroxyapatite (HAP). The outer most region is a hard enamel layer consisting primarily of HAP. Underneath is dentin region, which is composed of HAP with a significant fraction of collagen fibers. It has been demonstrated that it is possible to perform nano-indentation on sub-micron regions of tooth using either a standard nanoindentor or modified cantilever type atomic force microscope tip (AFM). In this study, we report nano-mechanical properties of dentin and enamel regions of a human tooth, measured with a nanoindentor attached to an AFM using a vertical force, in correlation with the microstructures observed with TEM.

TEM and nano-indentation samples were prepared from a juvenile human incisor tooth. The tooth was first sectioned into 1 mm thick slices along its length, each containing both dentin and enamel regions. A slice was taken to prepare a TEM sample and an adjacent slice was used for nano-indentation measurements.
Effect of “Stress-Absorbing” Dentin Adhesives on the Interaction of Composites with Human Dentin an SEM Study.
Authors: Francci Carlos , Ritter Andre V , Perdigao Jorge , Rosa Bruno T
Published: July 2020

Current dental restorative materials include tooth-colored materials called composite resins, which are made of a resin matrix and an inorganic filler coated with a silane coupling agent. Composite resins can shrink considerably upon setting. This shrinkage stress can generate gaps along the composite/dentin interface. Low-viscosity fluid resins and primers have been used to bond composite resins to dentin. The adhesive resin layer may be able to act as an elastic medium and compensate for the polymerization stresses that develop within the composite resin. The hypothesis tested in this study was that the use of a “stress-absorbing” intermediary dentin adhesive would result in lower incidence of gaps at the restoration/cavity interface and in a thicker resin/dentin interdifusion layer or hybrid layer.

Dentin disks with a thickness of 900 um±100 μm were obtained from extracted human molars and assigned to two adhesive systems (One-Step [OS] and Single Bond [SB]), three degrees of intermediary “stress-absorbing” treatments (none, Scotchbond Multi-Purpose [SBMP], and OptiBond FL [OB]) and two composite resins (Ælite-Flo [ÆF] and Z100
Cryo-Electron Microscopy of Aura Viruses
Authors: Zhang W , Olson N H , McKinney B R , Kuhn R J , Baker T S
Published: July 2020

Alphaviruses are a group of enveloped viruses in the Togaviridae family. Studies of several alphaviruses, including Ross River, Sindbis and Semliki Forest viruses, by cryo-electron microscopy (cryo-EM), three-dimensional (3D) image resconstruction and other techniques have illustrated that these spherical viruses have a T=4, multi-layered structure.

Aura virus, which is closely related to Sindbis, was first isolated in South America. Unlike the other alphaviruses, both genomic RNA (12kb, 49S) and subgenomic RNA(4.2kb, 26S) are encapsidated efficiently and form mature virions. Studies on negatively-stained virus particles demonstrated that there are two major size classes. The first contains particles of ∼72nm diameter, which are most similar to wild type virus, whereas the second class includes particles of ∼62nm in diameter. The 72nm particles are believed to have one copy of genomic RNA or one to three copies of subgenomic RNA, and a T=4 structure. The 62nm particles probably only have a single copy of subgenomic RNA and are presumed to be T=3 structures.
Nuclear Pore Transport: Insight in Situ
Authors: Aebi U , Stoffler D , Feja B , Goldie K
Published: July 2020

The nuclear envelope (NE) physically separates the genetic machinery residing in the nucleus from protein synthesis taking place in the cytoplasm. Bidirectional transport of proteins, RNAs and RNP particles between these two compartments is mediated by the nuclear pore complexes (NPCs), large (-120 MDa) supramolecular assemblies embedded in the NE and being built of -100 different polypeptides, called nucleoporins. Extensive structural studies have revealed the 3D architecture of NPCs, and epitopes of several nucleoporins have been localized within their 3D structure.

In an attempt to go beyond the current consensus model of the NPC (Fig. lc, inset), we have embarked on a more systematic structural analysis of the molecular architecture of native NPCs. This is achieved by field-emission (FETEM; Fig. la) or energy-filtering (EFTEM; Fig. lb) TEM of completely unfixed and unstained Xenopus oocyte NEs embedded in thick (i.e. -200 nm) amorphous ice so that the 3D organization of the cytoplasmic and nuclear periphery of the NPCs (i.e. the cytoplasmic fibrils and nuclear baskets) is fully preserved (Fig. lb).
High Resolution Epitope Mapping of the Hepatitis B Virus Capsid by Cryo-Electron Microscopy
Authors: Conway JF , Cheng N , Zlotnick A , Stahl SJ , Wingfield PT , Belnap DM , Steven AC
Published: July 2020

The capsid structure of the Hepatitis B virus (HBV) has been studied to resolutions below 10Å by cryo-electron microscopy, revealing much of its a-helical substructure and an apparently novel fold for a capsid protein. Although this represents a significant improvement in resolution for such studies, it is nonetheless still too low for complete tracing of the polypeptide chain. With the aim of establishing fiducial markers to aid in the process of chain-tracing, we have used cryo-microscopy to definitively localize specific peptides on the surface of the capsid. In one such study a gold cluster label was attached to a single cysteine residue engineered on to the C-terminus of the HBcAg assembly domain. The reconstructed density reveals a single gold cluster under each of the icosahedral 5-fold and 2-fold axes and connected to sites at either ends of the undersides of the dimers, thus pin-pointing the location of the C-terminus.
The Ryanodine Receptor/Calcium Release Channel and its Interaction Partners
Authors: Wagenknecht Terry , Samso Montserrat
Published: July 2020

Ryanodine receptors (RyRs) function as the major intracellular calcium release channels in striated muscle, where they also play a central role in excitation-contraction (e-c) coupling, the signal transduction process by which neuron-induced depolarization of the muscle plasma membrane leads to release of Ca from the sarcoplasmic reticulum. Structurally, RyRs are the largest ion channels known, being composed of 4 identical large subunits (565 kDa). In situ, RyRs interact with numerous proteins that are essential for e-c coupling or regulation thereof. Some of these ligands include calmodulin, a 12-kDa FK506-binding protein (FKBP, an immunophi1 in), calsequestrin, triadin, and the dihydropyridine receptor (DHPR).

Detergent-solubilized, purified RyRs appear to retain their native structure as assessed by electron cryo-microscopy, and are amenable to three-dimensional reconstruction by single-particle image processing techniques. In Fig. 1, a solid-body representation of the reconstructed skeletal muscle RyR shows the structural complexity that is revealed at moderate resolutions (3-4 nm).
3D Reconstruction of Mannosidase II from Single Particle Distributions: Noise Reduction Approaches for Higher Resolution
Authors: Ottensmeyer FP , Fernandes AB , Timmer M , Kroft J , Varga K , Moremen KW
Published: July 2020

3D electron microscopy of macromolecules is now moving beyond the very successful tomographic approach of using images of a tilt-series of 2D crystals at known angles to single randomly oriented particles with less and less internal symmetry. The success of 3D reconstruction of symmetrical objects was predicated primarily on the redundancy of information due to lateral symmetry in 2D crystals, or to internal rotational symmetry in the icosahedral, helical or cylindrically symmetric particles. Such a spatially predictable redundancy permitted both a reduction in irradiation dose, to reduce radiationinduced structural alterations of the molecules, and an averaging of image noise due to the low number of electrons used and from the specimen support.

For 3D reconstruction from single particles the noise level in the images is crucial for the accurate determination of the relative orientation of the particles. In principle the central line theorem was recognized to be as applicable to asymmetric particles as it was to symmetrical viruses.
Structure of Wild Type Yeast RNA Polymerase II and Location of RPB4 and RPB7
Authors: Jensen Grant J , Meredith Gavin , Bushnell David A , Kornberg Roger D
Published: July 2020

Nucleic acid polymerase structure has been studied by both X-ray and electron crystallography. To date, only the smaller, single subunit polymerases have been subjected to X-ray analysis, including the bacteriophage T7 RNA polymerase, which is the only RNA polymerase whose structure is known to atomic resolution. Lower resolution structures of several multisubunit polymerases have been determined by electron crystallography, including a mutant form of yeast RNA polymerase II which lacks subunits Rpb4 and Rpb7 (denoted A4/7 polymerase). All polymerase structures obtained by both X-ray and electron crystallography show a large cleft appropriate in size for binding duplex DNA, and further appear to contain a mobile arm allowing open and closed conformations of the cleft, presumably permitting entry and retention of DNA. Subunits Rpb4 and Rpb7 of RNA polymerase II form a dissociable subcomplex that has been implicated in the stress response and in the initiation of transcription. Human homologs of Rpb4 and Rpb7 have been identified.
Strategies Insuring the Optimal use of IgG or FAB* Fragments Covalently Bound to 1.4 NM Nanogold ™ in Immunogold Labeling Procedures
Authors: Ackerley CA , Tilups A , Becker LE
Published: July 2020

Gold particles as markers in immunocytochemical procedures have undergone tremendous changes since their introduction by Faulk and Taylor (1). With the development of both small colloidal gold (<3 nm) and 1.4 nm gold clusters (Nanogold™) for immunocytochemical labeling, steric hindrance has been significantly reduced. In a comparative study using colloidal gold-anti CD3 complexes and particle sizes of 5 nm, 3 nm and less than 3 nm on surface labeling of lymphocytes there was a 1.5 fold increase in labeling density when 3 nm particles were used. There was no increase when smaller particles were used (unpublished data). In another unpublished experiment, labeling densities on an astrocytoma cell line were compared between 3nm colloidal gold-CD44 Fab’ fragments and 1.4 nm gold-CD44 Fab’ fragments. Again there was no detectable differences between particle sizes. The limiting factors remain the size of the IgG or Fab’ fragment, the ability to bind it whether by absorption or by covalent bonding without rendering it inactive and also the accessibility of the target molecule in the tissue.
Molecular Cloning and Expression of 6HIS - GFP Reporter Gene for Fluorescent and Electron Spectroscopic Imaging.
Authors: Malecki M , Sanchez S , Skowron P , Case G
Published: July 2020

Molecular cloning offers an opportunity for designing constructs for expressing chimeric proteins containing incorporated reporter molecules. In this approach, molecules are identified based upon reporter molecules which are expressed within cells as a result of transfection, instead of marking them through immunolabeling, in situ hybridization, or derivative incorporation. This approach is particularly useful for studies involving integrated microscopy. Integrated microscopy allows us to assemble images of the same cell obtained with different microscopes into one comprehensive message concerning cellular functions. Integration of fluorescence and electron spectroscopic imaging is particularly promising. The main advantage of this approach relies in overcoming limitations of each type of microscopy alone i.e. in studies on living cells limitations in spatial resolution of light microscopy and in analysis of supramolecular organization, limitation of electron microscopy to frozen or fixed cells. For this purpose, special reporter molecules suitable for selected modes of microscopy have to be used.
New Advances in Super-Sensitive DNA-, RNA- and Antigen Detection: Combination of Labeled Tyramides with Nanogold-Silver Staining (NGSS)
Authors: Hacker GW , Hauser-Kronberger C , Zehbe I , Su H , Tubbs R
Published: July 2020

In situ detection of specific antigens and nucleic acid sequences is possible with a variety of immunohistochemical (IHC) and in situ hybridization (ISH) methods. The drawback of conventional methodologies is their relatively low detection sensitivity. Improved detection efficiency is required in preparations containing only minute amounts of detectable antigens or only few copies of specific DNA and/or RNA sequences, which is often the case in routine paraffin cytoand histopathologic material. The use of gold as the label, followed by silver amplification (autometallography) avoids hazardous reagents and also gives an improved resolution compared to „standard” methods. Sensitivity problems may now be overcome by our specific combinations of recent technologies, utilitizing reporter molecule amplification with labeled tyramides (CARD, catalyzed reporter deposition, or tyramide signal amplification), followed by streptavidin bound to a new gold label, Nanogold™ (Nanoprobes, Stony Brook, NY), and silver acetate autometallography.
Use of Green Fluorescent Protein (GFT)-Chimeras to Study Cytoskeletal Assembly and Dynamics
Authors: Gundersen G G , Mikhailov A , Martys J L , Ho L , Liem R K H , Smelinov L , Marcantonio EE
Published: July 2020

The cytoskeleton plays an important role in cell structure, polarity, locomotion and division. Individual elements of the cytoskeleton are composed of subunit proteins which assemble and disassemble in specific places and times within the cell. Knowledge of the temporal and spatial regulation of subunit assembly and disassembly is essential to understanding how the cytoskeleton contributes to cellular activities. The assembly and dynamics of two cytoskeletal structures, namely adhesion plaques (APs) and intermediate filaments (IFs), have been difficult to study by traditional methods. We have generated GFP-chimeras to label these structures and to study their dynamics in motile fibroblasts.

To study the dynamics of APs, we prepared stable 3T3 cell lines expressing a GFP-β1 integrin chimera. The chimera was prepared by fusing GFP to the transmembrane and cytoplasmic portions of β1 intergrin, since previous studies had shown that the cytoplasmic tail of β integrins is sufficient to direct integrins to APs.
Microscopic Investigations of the Infection Process of Choristoneura Fumiferana Nucleopolyhedrovirus in the Spruce Budworm
Authors: Brownwright A J , Barrett J W , Ladd T R , Primavera M , Sohi S S , Arif B M , Retnakaran A , Palli S R
Published: July 2020

Choristoneura fumiferana nucleopolyhedrovirus(CfMNPV) is a baculovirus that infects the spruce budworm, Choristoneura fumiferana(Cf), and a few other related species. To study the infection process of this virus within the spruce budworm, we have constructed a recombinant virus (CfMNPV-GFP) expressing the green fluorescence protein(GFP) under the control of the polyhedrin promoter of Autographa californica nucleopolyhedrovirus. Each larva was fed 10,000 CfMNPV-GFP occlusion bodies(OB) applied to diet plugs and various tissues were dissected from the larvae at 24 hr intervals and examined for the presence of the OB and/or GFP using a fluorescence microscope. Larvae were also fixed and embedded for light and electron microscopy.

GFP-specific fluorescence was first detected as a few spots at the posterior end of the midgut at 72 hr post feeding(PF). By 96 hr PF, the fluorescence had expanded to include adjacent cells and some hemocytes.
Light Microscopy Beyond the Wavelength Limit: Methods for Characterizing Cell Surface Membranes
Authors: Edidin Michael , Kenworthy Anne K , Gheber Levi
Published: July 2020

The cell surface mediates the flow of information and metabolites between a cell and its environment. We are interested in understanding the lateral organization of the surface, as part of our program in understanding its function. The model of lateral organization of cell surface membranes is evolving from one which emphasizes mobility and autonomy of membrane constituent molecules, to another which emphasizes the lateral concentration of membrane proteins and lipids into patches and membrane microdomains. Indirect evidence suggests that diameters of these patches and microdomains are often ≤ a wavelength of visible light, and so cannot be readily resolved by conventional light microscopy.

We have developed two complementary techniques for detecting patches and microdomains on the nm scale: image fluorescence resonance energy transfer, FRET, and near-field scanning optical microscopy, NSOM. The first of these techniques detects proximity of membrane proteins and lipids on a scale of a few nm in terms of the interactions of donor and acceptor fluorophores.
Immunoperoxidase Labeling Demonstrates an Early Divergence of Chlamydia Trachomatis from the Endosomal-Lysosomal Pathway
Authors: Fischer Elizabeth R , Scidmore-Carlson Marci A , Hackstadt Ted
Published: July 2020

Chlamydia trachomatis is responsible for several significant human diseases including trachoma, the primary source of preventable blindness in developing countries, and is the most common cause of sexually transmitted disease. C. trachomatis is an obligate intracellular prokaryote (ICP) relying on eukaryotic host cells for growth and replication. Typically, microorganisms engulfed by host cells, are trafficked through maturing endosomes to the lysosomal pathway and ultimately destroyed. Survival in a host cell requires the invading organism to either adapt or modify their host environment to avoid fusion with lysosomal vesicles. Organisms such as Mycobacterium tuberculosis have evolved mechanisms to arrest maturation of the endosomes, such that they avoid lysosomal fusion.3 C trachomatis has developed alternative strategies for successful intracellular survival and growth.

C. trachomatis exists in two morphologically and functionally distinct forms which multiply in vacuoles termed inclusions. A small dense form known as the elementary body (EB), is the stable extracellular stage of the life cycle capable of attachment and entry into host cells.
Apoptosis And Resistance To Anti-Microtubule Agents
Authors: Willingham M C
Published: July 2020

Several clinically important anti-cancer agents exert their effects on tumor cells through interference with the function of microtubules. In addition to the Vinca alkaloids, such as vinblastine and vincristine, the taxanes, such as paclitaxel (Trade Name: Taxol), kill tumor cells through a microtubular target. Treatment with taxol leads to the inability of microtubules to depolymerize, leading to the formation of large intracellular microtubular bundles. In tumor cells that progress through the cell cycle, this leads to the inability of these cells to disassembly interphase microtubule networks and a failure to form functional mitotic spindles. These cells arrest in M phase, from which they eventually progress, either by the induction of apoptotic cell death, or by micronucleation and the formation of tetraploid cells. There is also the possibility that taxol has other effects on the regulation of genes or other systems to enhance cell killing, perhaps through lowering the threshold of cells to the induction of apoptotic cell death.
Applications of Correlative Microscopy in Diagnostic and Investigative Pathology
Authors: Howell D N , Miller S E
Published: July 2020

Correlative microscopy is employed in a great variety of settings by both diagnostic and investigative pathologists. Combinations of conventional light microscopy (LM), immunohistology, and electron microscopy (EM) are used in a wide range of diagnostic settings, including the analysis of tumors, autoimmune disorders, and infectious diseases. Valuable diagnostic information is also frequently obtained by simultaneous or sequential examination of exfoliated or aspirated cell suspensions (cytopathology) and tissue sections (histopathology) from the same lesion. An even wider range of correlative microscopic methods is employed by pathologists in basic and clinical research. The rationales for using correlative techniques are many and varied, but in most cases fall within a limited number of categories.

Pathologists frequently use a second microscopic or preparative technique to improve on the resolution afforded by an initial technique. Electron microscopy is often used to refine the analysis of features initially detected by routine LM or immunohistology.
Immune System Activation After Cisplatin or “Poly-Plat” Treatment: An in Vivo Study
Authors: Telgen D J , Aggarwal S K
Published: July 2020

Cisplatin (cw-diamminedichloroplatinum II; CDDP) is a broad spectrum anti-cancer agent with severe toxic side effects. New platinum based chemotherapeutic agents with similar properties are in constant development. Poly-plat (Poly-[(teans-l,2-diaminocyclohexane) platinum]-carboxyamylose), a novel second generation platinum chemotherapeutic agent, has been shown to be less toxic compared to CDDP. The purpose of this paper was to examine the morphological changes in the Kupffer cells in the liver after poly-plat treatment in rats and compare the results obtained after CDDP. Peripheral blood was also examined for levels of circulating leukocytes.

Wistar rats (120-160 g) were given intraperitoneal injections of either CDDP (9 mg/kg in 0.9% sodium chloride) or poly-plat (10 mg/kg in 0.9% sodium chloride) over a five day period. Controls were treated with equal amounts of the vehicle of injection. Peripheral blood was collected via tail vein beginning 1 day prior to treatment and continuing 10 days after treatment.
Negative Stain Electron Microscopy…a Valuable Diagnostic Method in a Molecular Probe Diagnostic World
Authors: Humphrey C D , Monroe S S , Glass R I
Published: July 2020

Endemic and epidemic viral gastroenteritis are major causes of morbidity worldwide and are frequently a major cause of death in the developing world. Rotavirus, adenovirus, calicivirus, astrovirus, and the Norwalk virus family are the viruses most often associated with gastroenteritis. Of these, the major cause of epidemic viral gastroenteritis in the US is Norwalk-like viruses. Gastroenteritis caused by this virus group is responsible for such outbreaks in settings such as cruise ships, schools, camps, hospitals, day-care and extended care facilities, football games and festivals.

As early as the 1950s and 1960s electron microscopy (EM), including negative stain EM, was established as a diagnostic tool for identifying viruses. Negative stain EM is especially useful for identifying viruses that are difficult or impossible to culture (eg. viruses in the Norwalk family). The first Norwalk virus was identified in the 1969 by immune-negative stain EM. Many antigenic types were identified soon afterward that did not cross-react with the prototype Norwalk virus.
Microvascular Architecture of the Lingual Papillae in Rabbits.
Authors: Masuko T S , König B , Schmidt BR
Published: July 2020

The comparative morphology of the tongue microvasculature has been studied in different animals, such as in man (Okada et al, 1993, Matsukawa and Okada, 1994), rats (Nagato et al., 1989, Ohshima, et al, 1990, Selliseth and Slvig, 1993), dogs (Kishi et al, 1986, Kishi et al 1988), goats(Scala et al, 1992), monkeys (Patterson, et al, 1992, Okada et al, 1993, Matsukawa and Okada, 1994), marmosets (Okada et al, 1993, Matsukawa and Okada, 1994) and shrews (Okada et al, 1993, Matsukawa and Okada, 1994), but apparently none in rabbits. The disposition of the blood vessels of the rabbit's lingual papillae were studied by injecting them with Mercox resin and posterior corrosion, thus obtained microcorrosion casts were observed in the scanning electron microscope.

Male and adult rabbits were used to obtain our models. After perfusion with heparin 0,1M, pH 7.4 and glutaraldehyde, both in the same buffer, the vessels were filled as reported above.
Prevention of changes in Endothelial Cells Cultured in High Glucose
Authors: Taylor CA , Imdad Z , Puchalski J , Lemley-Gillespie S , Mandal AK
Published: July 2020

The vascular endothelium is the interface with hyperglycemia in uncontrolled diabetes mellitus and its complications, including nephropathy, myocardial infarction, blindness, and gangrene. To mimic the vascular endothelium in a controlled experimental situation, we use the endothelial cell line (CRL 1998) obtained from The American Type Culture Collection. Cells are grown to confluency in Media 199 containing 10% fetal calf serum and gentamicin (50mg/ml). The growth media is replaced with experimental media containing high glucose (30mM), insulin (20 Units/ml), or heparin (500 Units/ml) either separately or in combinations. Cells are briefly trypsinized, pelleted, embedded, and sectioned with a Diatome diamond knife for TEM or for SEM, grown on 0.45um filters, followed by fixation and air-drying in situ: this approach to culture allows cells to obtain a polar, more natural phenotype than monolayers on glass or plastic.

Incubation in high glucose results in loss of confluency, production of intercellular gaps, and fewer mitotic figures.
Pericyte Ultrastructural Pathology in Autoimmune Inflammatory Myopathies
Authors: Finol HJ , Marquez A , Pulido-Mendez M , Muller B , Montes de Oca I , Tonino P
Published: July 2020

Ultrastructural Pathology of capillary endothelial cells in autoimmune inflammatory myopathies has been intensively investigated in the last two decades, but much less is known about the alterations of capillary pericytes. In other diseases as diabetes and arterial hypertension perycites abnormalities have been reported. In this work we report the pericyte ultrastructural pathology in autoimmune inflammatory myopathies.

Patients admitted to the study were attending the rheumatology clinics at the Caracas University Hospital. Diagnoses were systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, and muscle paraneoplastic phenomenon.

Needle muscle biopsies were processed with routine techniques for transmission electron microscopy and observed in a Hitachi H-500 electron microscope.

Two different processes were observed, pericyte hypertrophy and cell degeneration. In the first one cytoplasmic proliferation is evident (Fig. 1). Some capillaries embraced by the pericyte were found (Fig. 2). In the second one cytoplasmic vacuolation and capillary necrosis (Fig. 3) were evident. Usually, pericyte and endothelial degeneration were concomitant.
Ultrastructural Localization of CD34 in Soft Tissue Tumors
Authors: Minda J M , Lawton T J , Rijn M van de
Published: July 2020

The CD34 antigen is a single chain, heavily glycosylated transmembrane protein originally identified in hematopoietic stem cells. Subsequently it was found to be expressed in cells of normal endothelium and a variety of soft tissue tumors, including: epithelioid sarcoma, solitary fibrous tumor,’ gastrointestinal stromal tumor and dermatofibrosarcoma protuberans. Using paraffin immunohistochemistry, membranous and cytoplasmic staining for CD34 can be seen in these lesions, with most showing a combination of both. The findings of cytoplasmic staining is unexpected, as CD34 is a transmembrane cell surface protein. We analyzed the ultrastructural localization of CD34 on two soft tissue tumors with membranous and combined membranous and cytoplasmic staining.

Biopsy specimens were fixed with a mixture of 4% paraformaldehyde and 1% glutaraldehyde in 0.1M sodium cacodylate buffer, pH = 7.4 for 1-2 hours, and embedded in Poly/Bed 812 with osmium tetroxide post-fixation, or in LR White without osmication. The immunogold method was performed on ultrathin sections placed on Nickel grids,
Tissue Response to Bone Substitute Materials Supporting Dental Implants
Authors: Krizan K E , Lew D , Rubey T , Keller J C
Published: July 2020

The purpose of this study was to evaluate the biological response of a bone substitute material placed around Ti dental implants surgically positioned in tooth extraction sites of canine mandible. Under general anesthesia six adult mongrel canine had bilateral extractions of the fourth premolarbicuspid. Each extraction site was modeled into a 4 wall defect with box-like dimensions (1.0 mm vertically x 1.2 mm horizontally x 0.75 mm laterally). Utilizing the Branemark technique, screwtype implants with healing screws (10 mm x 4 mm), were placed in the prepared sites. A synthetic hydroxyapatite bone cement (HAC) [BoneSource®, Howmedica Laboratories, Inc.] was prepared and filled in around each implant. As controls, three canine had HAC placed in prepared sites without implants. Prior to periodontal flap suture closure a n-butyl-2-cyanoacrylate biodegradable tissue adhesive [Histoacryl®], was placed over implants and HAC. One canine each was euthanized at one month (m), two m, and three m postop.
Ultrastructural Distribution of Basic Fibroblast Growth Factor-Like Molecules in Peripheral Nerves
Authors: Kayton Robert J , Aktas Ranan Gulhan
Published: July 2020

Basic Fibroblast Growth Factor (bFGF) is a multifunctional polypeptide which has been shown to play a pivotal role in the survival and differentiation of nerve cells. Several trophic and non-trophic functions of this protein have been suggested in peripheral nerves. In spite of ample information about the distribution and effects of bFGF in central nervous system, few data are available concerning the localization of this protein in peripheral nerves. In view of the role of bFGF in regulation of trophic and non-trophic functions, we particularly focused on the presence and precise location of bFGF in peripheral nerves at the electron microscope level.

Spurr's resin embedded ultrathin sections from adult rats’ sural nerves were labeled with either polyclonal (F3393-Sigma) or monoclonal antibodies (F6162-Sigma, C3316-Zymogenetics) specific for bFGF using two-step immunogold labeling method. Control samples were treated with either an equivalent volume of blocking solution (omitting the primary antibody) or an irrelevant antibody (Factor VIII, VGF, anti-histamine, anti-fibroblast 5B5).
Ciliogenesis and Axonemal Dynein Expression in Human Airway Epithelium Cultured in an Air/Liquid Interface Environment
Authors: Carson J L , Reed W , Brighton Luisa , Gambling Todd M , Collier Albert M
Published: July 2020

Axonemal dynein is a high molecular weight Mg2+-ATPase which occurs across the eukaryotic phylogenetic spectrum and interacts with the microtubular doublets of ciliary axonemes to effect ciliary beating. Unlike cytoplasmic dyneins which serve intracellular functions and are relatively ubiquitous in all cells, axonemal dynein is expressed exclusively in mature and nascent ciliated cells. Ciliated cells figure prominently in mucociliary clearance, a primary defense mechanism of the mammalian respiratory airways. Acting in concert with mucus secreting cells, the mucociliary escalator maintains virtual sterility in the airways and lung. Although ciliated cells are the predominant cell type in the airways and serve a critical function in respiratory health, they also are particularly vulnerable to injury. The strategic functional importance of the ciliated cell as well as its susceptibility to injury by infectious agents and irritants has led to development of epithelial cell culture systems which facilitate epithelium-specific studies of injury.
Microwave-Assisted Immunoelectron Microscopy of Skin
Authors: Petrali John P , Mills Kenneth R
Published: July 2020

Microwave energy (MWE) as a method of rapid tissue processing is gaining increasing support as an alternative to routine chemical processing in diagnostic laboratory environments. Chemical fixatives and conventional fixation times used for standardized preservation of tissues can result in serious alterations in morphology as a consequence of solubilization and conformational changes of proteins and lipids. These untoward changes typically result in compromised antigenicity of many tissue proteins. With MWE processing, tissue antigens can be distinctly better preserved, antigen retrieval made more replicate and histochemical and immunochemical reactions made ultrafast. The present study had two objectives: to compare MWE processed skin to conventionally processed skin for ultrastructural integrity; to compare MWE expression of antigenicity of selected skin proteins which are usually sensitive to conventional chemical processing. Skin proteins selected were bullous pemphigoid antigen (BPA) and laminin.
Confocal Laser Scanning Microscopy of Hamster Cerebellum using FM4-64 as Intracellular Staining
Authors: Castejón O , Sims P
Published: July 2020

Confocal laser scanning microscopy is an excellent method to study nerve cell morphology and the three-dimensional distribution and interrelationship of dentrites and axons in the central nervous system. The cerebellum has been taking as a model of a gray center.

The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to evaluate its properties as an intracellular stain and intracortical tracer. Slabs of hamster cerebellum,5 mm thick, were incubated in 10,30 and 100 μm solutions of FM4-64 in sodium phosphate buffer and observed in a slow scan confocal laser scanning microscope. Mossy and climbing fibers were traced in the cerebellar white and gray substances.They exhibited high fluorescence signal at the level of myelin sheath.

Mossy fibers were identified in the granular layer by their typical rosette formation and dichotomous bifurcation pattern. Climbing fibers bundles were observed crossing the granular layer and giving collateral branches around Golgi cell bodies.
Integrated Microscopy of Extracellular Adhesives of the Marine Biofouling Alga Achnanthes Longipes (Bacillariophyceae)
Authors: Wang Y , Wustman B A , Chen Y , Lavin C , Gretz M R
Published: July 2020

An integrated approach including TEM, SEM and LM was utilized to investigate adhesives produced by the marine fouling diatom Achnanthes longipes. Extracellular adhesive secretion during sequential attachment in living diatom cells was examined using video microscopy. A suite of cryotechniques including high pressure freezing (HPF), freeze-substitution (FS) and high resolution cryo-field-emission SEM (cryo-FESEM) were required for preservation and structural investigation of the hydrophilic polymers synthesized by A. longipes that promote attachment to ship hulls, oil rigs and other submerged structures.

Living cells and associated extracellular biocomposites were rapidly cryo-immoblized and observed using a Hitachi S-900 FESEM equipped with a cryo-stage. This technique allowed resolution of details of polymer organization within the pad, shaft and collar portions of the major adhesive structure termed a stalk (Fig. 1,2). This stalk has been demonstrated to consist mainly of polysaccharide cross-linked by O-linked polypeptides. In addition to permanently adhering the diatom to the substratum, the stalk elevates A. longipes, possibly enhancing the ability to successfully compete with other biofilm dwellers.
EM Study of Isopod Hemocytes
Authors: Vernon G M , Rappa E J , Murray W C , Witkus R
Published: July 2020

Crustacean hemocytes have been characterized on the basis of cell size and nature of cytoplasmic granules. Based on light microscopic morphological analysis and cytochemistry, investigators variously named the hemocyte types (agranular, small-granule, large granule, undifferentiated, hyaline cells, non-explosive, explosive granulocytes, etc.). In his study of the isopod Armadillidium vulgare Faso adopted the terminology of Benjamin and James and referred to the hemocytes as hyaline cells, semi-granulocytes and granulocytes.

In the present investigation we have studied the hemocytes of two isopods, Oniscus asellus and Armadillidium nasatum, using transmission electron microscopy. Hemolymph was collected by penetrating the posterior dorsal exoskeleton of 20 animals of each genus with a microcapillary pipette and drawing 3-5μL per isopod. The samples were processed following a standard technique. Thin sections were collected on 300 mesh copper grids, counterstained with 2% aqueous uranyl acetate and lead citrate, and viewed with a JEOL 1010 electron microscope.
Ultrastructure of Haustoria of Plant Pathogenic Fungi
Authors: Mims C W , Richardson E A
Published: July 2020

Most plant pathogenic fungi that are obligate parasites produce haustoria which are thought to be involved in nutrient absorption. A haustorium is a specialized hyphal branch that penetrates the host cell wall and invaginates the host cell plasma membrane. The host plasma membrane ensheathing the haustorium is termed the extrahaustorial membrane. This presentation provides examples of different types of haustoria produced by plant pathogenic fungi. Species considered here are 1) Cronartium quercuum f. sp.fusiforme, the cause of fusiform gall rust of pine, 2) Puccinia arachidis, the cause of peanut rust1, 3) Uncinuliella australiana, the cause of powdery mildew of crape myrtle, 4) Exobasidium camelliae, a pathogen of Camellia sasanqua2, and 5) Cercosporidium personatum, the cause of late leaf spot of peanut.

Rust fungi typically require two different host species to complete their life cycles. The dikaryotic phase of the rust life cycle consists of intercellular hyphae that give rise to specialized haustoria known as D-haustoria which are remarkably similar from one species to the next.
Identification of Papilloma-Like Virus Particles in Cell Lines Derived from Green Sea Turtle (Chelonia Mydas) With Fibropapilloma
Authors: Lu Yuanan , Nerurkar Vivek R , Weatherby Tina M , Yanagihara Richard
Published: July 2020

The near epidemic occurrence of fibropapilloma in green sea turtle (Chelonia my das) (Figure 1) significantly threatens the survival of this species which is protected under the U.S. Endangered Species Act. Although collective evidence suggests a viral etiology, the causative virus of green sea turtle fibropapilloma has not been isolated. To facilitate the isolation and characterization of the causative virus(es), we established 13 cell lines from multiple organs/tissues (tumor, kidney, lung, heart, gall bladder, testis, and skin) of green sea turtles with fibropapilloma. Serial subcultivation of cell lines derived from lungs, testis, eye soft tissues and tumors resulted in the formation of tumor-like aggregates, which attained sizes of 1-2 mm in diameter within two weeks (Figure 2). Media from such cultures, when inoculated onto cells derived from healthy turtle embryos, produced similar tumor-like aggregates, suggesting the presence of a transmissible agent.
High Resolution Immuno-Electron Microscopy Reveals That Fetal Skin Contains Microfibrils which are Heteropolymers of Fibrillin-1 and Fibrillin-2
Authors: Keene DR , Charbonneau NL , Dzamba BJ , Reinhardt DP , Ridgway CC , Ono RN , Sakai LY
Published: July 2020

Immunolocalization studies of neonate and older human tissues have previously demonstrated that fibrillin-1 is a component of elastin and non-elastin associated microfibrils. In sections taken from fixed, dehydrated and embedded tissue, microfibrils appear in the transmission electron microscope as hollow rods, 6-8 nm in diameter. When isolated from tissue by homogenization and observed following negative staining or rotary shadowing, microfibrils appear as beaded strings with a degree of extendibility. Recently, a closely related glycoprotein, fibrillin-2, has been described, which is expressed in early fetal development, prior to fibrillin-1, but then disappears in most tissues just prior to birth. We demonstrate here the characterization monoclonal antibodies specific for fibrillin-2. The antibodies are shown by ELISA and immunoblots to be fibrillin-2 specific. The matrix from a variety of tissues, including aorta, tendon, and eye are shown to contain fibrillin-2 by immunofluorescence in a pattern similar to that of fibrillin-1.
2G Alters the Utricular Macula of Chick Embryos
Authors: Hara Hirotaka , Fermin César D
Published: July 2020

Chick embryos (Gallus domesticus) exposed continuously from day one until 18 days of incubation to a 2G acceleration (hyperG chicks) force hatched with altered macular epithelia. Fertile eggs were placed in an incubator and exposed to 2G on a centrifuge by the original apparatus until 18 days of incubation. Effective 2G loads were obtained by rotating the apparatus at a constant velocity of 327.5 degree/sec with the incubator mounted at 55 cm from the center axis [1]. The vestibules of the newly hatch chicks were dissected and fixed in 2.5% glutaraldehyde. The utricles were rinsed in phosphate buffer, postfixed with 2% osmic acid and dehydrated in graded ethanol and embedded in plastic. Semithin sections were examined under Light microscopy with computerized video imaging system [2] and ultrathin sections were examined under TEM.
Unusual Mitochondrial Aggregation with Virus in Infected Transgenic Plants
Authors: Zhang L , Langenberg W G
Published: July 2020

Viruses grouped in the family Potyviridaeare long flexious rod-shaped viruses and cause the most damage of all plant vims groups. They possess a single stranded positive sense RNA genome that encodes a single polyprotein product which yields eight or more proteins by secondary processing. Considerable efforts have been made to transform plants with DNA or RNA sequences of these proteins in hopes of obtaining pathogen-derived resistant plants. Hull has suggested that a close assessment of the risks involved in field release of transformed plants would be desirable. However, to our knowledge, there have been no published reports regarding properties of viruses that do infect some transformed plants. We describe here an ultrastructural appearance of transgenic plant cells infected by potato virus Y (PVY) (ATCC PV No. 50; PVY-50) and unusual mitochondrial aggregation with the virus.