bluuebunny/crossref_metadata_2025_split 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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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 (&lt;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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.