Tin-containing fluoride solutions as anti-erosive agents in enamel: an in vitro tin-uptake, tissue-loss, and scanning electron micrograph study

Tin-containing fluoride solutions can reduce erosive tissue loss, but the effects of the reaction between tin and enamel are still not clear. During a 10-d period, enamel specimens were cyclically demineralized (0.05 M citric acid, pH 2.3, 6 x 5 min d(-1)) and remineralized (between the demineralization cycles and overnight). In the negative-control group, no further treatment was performed. Three groups were treated (2 x 2 min d(-1)) with tin-containing fluoride solutions (400, 1,400 or 2,100 ppm Sn2+, all 1,500 ppm F-, pH 4.5). Three additional groups were treated with test solutions twice daily, but without demineralization. Tissue loss was determined profilometrically. Energy-dispersive X-ray spectroscopy was used to measure the tin content on and within three layers (10 mum each) beneath the surface. In addition, scanning electron microscopy was conducted. All test preparations significantly reduced tissue loss. Deposition of tin on surfaces was higher without erosion than with erosion, but no incorporation of tin into enamel was found without demineralization. Under erosive conditions, both highly concentrated solutions led to the incorporation of tin up to a depth of 20 mum; the less-concentrated solution led to small amounts of tin in the outer 10 mum. The efficacy of tin-containing solutions seems to depend mainly on the incorporation of tin into enamel.

Projection structure of DtpD (YbgH), a prokaryotic member of the peptide transporter family

Cellular uptake of di- and tripeptides has been characterized in numerous organisms, and various transporters have been identified. In contrast, structural information on peptide transporters is very sparse. Here, we have cloned, overexpressed, purified, and biochemically characterized DtpD (YbgH) from Escherichia coli, a prokaryotic member of the peptide transporter family. Its homologues in mammals, PEPT1 (SLC15A1) and PEPT2 (SLC15A2), not only transport peptides but also are of relevance for uptake of drugs as they accept a large spectrum of peptidomimetics such as beta-lactam antibiotics, antivirals, peptidase inhibitors, and others as substrates. Uptake experiments indicated that DtpD functions as a canonical peptide transporter and is, therefore, a valid model for structural studies of this family of proteins. Blue native polyacrylamide gel electrophoresis, gel filtration, and transmission electron microscopy of single-DtpD particles suggest that the transporter exists in a monomeric form when solubilized in detergent. Two-dimensional crystallization of DtpD yielded first tubular crystals that allowed the determination of a projection structure at better than 19 A resolution. This structure of DtpD represents the first structural view of a member of the peptide transporter family.

Sprouting and intussusceptive angiogenesis in postpneumonectomy lung growth: mechanisms of alveolar neovascularization

In most rodents and some other mammals, the removal of one lung results in compensatory growth associated with dramatic angiogenesis and complete restoration of lung capacity. One pivotal mechanism in neoalveolarization is neovascularization, because without angiogenesis new alveoli can not be formed. The aim of this study is to image and analyze three-dimensionally the different patterns of neovascularization seen following pneumonectomy in mice on a sub-micron-scale. C57/BL6 mice underwent a left-sided pneumonectomy. Lungs were harvested at various timepoints after pneumonectomy. Volume analysis by microCT revealed a striking increase of 143 percent in the cardiac lobe 14 days after pneumonectomy. Analysis of microvascular corrosion casting demonstrated spatially heterogenous vascular densitities which were in line with the perivascular and subpleural compensatory growth pattern observed in anti-PCNA-stained lung sections. Within these regions an expansion of the vascular plexus with increased pillar formations and sprouting angiogenesis, originating both from pre-existing bronchial and pulmonary vessels was observed. Also, type II pneumocytes and alveolar macrophages were seen to participate actively in alveolar neo-angiogenesis after pneumonectomy. 3D-visualizations obtained by high-resolution synchrotron radiation X-ray tomographic microscopy showed the appearance of double-layered vessels and bud-like alveolar baskets as have already been described in normal lung development. Scanning electron microscopy data of microvascular architecture also revealed a replication of perialveolar vessel networks through septum formation as already seen in developmental alveolarization. In addition, the appearance of pillar formations and duplications on alveolar entrance ring vessels in mature alveoli are indicative of vascular remodeling. These findings indicate that sprouting and intussusceptive angiogenesis are pivotal mechanisms in adult lung alveolarization after pneumonectomy. Various forms of developmental neoalveolarization may also be considered to contribute in compensatory lung regeneration.

A new tool based on two micromanipulators facilitates the handling of ultrathin cryosection ribbons

A close to native structure of bulk biological specimens can be imaged by cryo-electron microscopy of vitreous sections (CEMOVIS). In some cases structural information can be combined with X-ray data leading to atomic resolution in situ. However, CEMOVIS is not routinely used. The two critical steps consist of producing a frozen section ribbon of a few millimeters in length and transferring the ribbon onto an electron microscopy grid. During these steps, the first sections of the ribbon are wrapped around an eyelash (unwrapping is frequent). When a ribbon is sufficiently attached to the eyelash, the operator must guide the nascent ribbon. Steady hands are required. Shaking or overstretching may break the ribbon. In turn, the ribbon immediately wraps around itself or flies away and thereby becomes unusable. Micromanipulators for eyelashes and grids as well as ionizers to attach section ribbons to grids were proposed. The rate of successful ribbon collection, however, remained low for most operators. Here we present a setup composed of two micromanipulators. One of the micromanipulators guides an electrically conductive fiber to which the ribbon sticks with unprecedented efficiency in comparison to a not conductive eyelash. The second micromanipulator positions the grid beneath the newly formed section ribbon and with the help of an ionizer the ribbon is attached to the grid. Although manipulations are greatly facilitated, sectioning artifacts remain but the likelihood to investigate high quality sections is significantly increased due to the large number of sections that can be produced with the reported tool.

Three-dimensional structure of tropism-switching Bordetella bacteriophage.

Bacteriophage BPP-1, which infects Bordetella species, can switch its specificity by mutations to the ligand-binding surface of its major tropism-determinant protein, Mtd. This targeted mutagenesis results from the activity of a phage-encoded diversity-generating retroelement. Purified Mtd binds its receptor with low affinity, yet BPP-1 binding and infection of Bordettella cells are efficient because of high-avidity binding between phage-associated Mtd and its receptor. Here, using an integrative approach of three-dimensional (3D) structural analyses of the entire phage by cryo-electron tomography and single-prticle cryo-electron microscopy, we provide direct localization of Mtd in the phage and the structural basis of the high-avidity binding of the BPP-1 phage. Our structure shows that each BPP-1 particle has a T = 7 icosahedral head and an unusual tail apparatus consisting of a short central tail "hub," six short tail spikes, and six extended tail fibers. Subtomographic averaging of the tail fiber maps revealed a two-lobed globular structure at the distal end of each long tail fiber. Tomographic reconstructions of immuno-gold-labeled BPP-1 directly localized Mtd to these globular structures. Finally, our icosahedral reconstruction of the BPP-1 head at 7A resolution reveals an HK97-like major capsid protein stabilized by a smaller cementing protein. Our structure represents a unique bacteriophage reconstruction with its tail fibers and ligand-binding domains shown in relation to its tail apparatus. The localization of Mtd at the distal ends of the six tail fibers explains the high avidity binding of Mtd molecules to cell surfaces for initiation of infection.

Crystal structure of the alpha(6)beta(6) holoenzyme of propionyl-coenzyme A carboxylase.

Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants. The holoenzyme of PCC is an alpha(6)beta(6) dodecamer, with a molecular mass of 750 kDa. The alpha-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the beta-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-A resolution of a bacterial PCC alpha(6)beta(6) holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-A resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the alpha-subunits are arranged as monomers in the holoenzyme, decorating a central beta(6) hexamer. A hitherto unrecognized domain in the alpha-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the beta-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 A, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the beta-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) and eukaryotic acetyl-CoA carboxylase (ACC).

Cryo-electron tomography of Kaposi's sarcoma-associated herpesvirus capsids reveals dynamic scaffolding structures essential to capsid assembly and maturation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a recently discovered DNA tumor virus that belongs to the gamma-herpesvirus subfamily. Though numerous studies on KSHV and other herpesviruses, in general, have revealed much about their multilayered organization and capsid structure, the herpesvirus capsid assembly and maturation pathway remains poorly understood. Structural variability or irregularity of the capsid internal scaffolding core and the lack of adequate tools to study such structures have presented major hurdles to earlier investigations employing more traditional cryo-electron microscopy (cryoEM) single particle reconstruction. In this study, we used cryo-electron tomography (cryoET) to obtain 3D reconstructions of individual KSHV capsids, allowing direct visualization of the capsid internal structures and systematic comparison of the scaffolding cores for the first time. We show that B-capsids are not a structurally homogenous group; rather, they represent an ensemble of "B-capsid-like" particles whose inner scaffolding is highly variable, possibly representing different intermediates existing during the KSHV capsid assembly and maturation. This information, taken together with previous observations, has allowed us to propose a detailed pathway of herpesvirus capsid assembly and maturation.

Scanning electron microscope study of the developing microvasculature in the postnatal rat lung.

During postnatal growth the parenchymal septa of rat lung undergo an impressive restructuring. While immature septa are thick and contain two capillary layers, mature septa are slender and contain a single microvascular network. Using the Mercox casting technique and scanning electron microscopy, we investigated the mode and the timing of the transformation of the pulmonary capillary bed. During the third postnatal week the parenchymal septa rapidly mature to match adult morphology. Even in adult lungs, however, remnants of the immature status are present: A capillary bilayer is regularly found at the base and the tip of the septa. Our observations support the concept that reduction of intervening tissue, partial fusion of the two capillary networks, and preferential growth lead to the mature vascular arrangement. The fact that true mature interalveolar septa show a denser capillary network than alveolar walls abutting onto pleura, bronchi, or larger vessels is consonant with the fusion theory. Towards the nonparenchyma, the capillary network surrounding every airspace had no counterpart to fuse with. From quantitative data it can be calculated that owing to lung growth, mesh size should increase more than four times between birth and adult age. The adult lung network, however, is denser than the one in young animals. This means that new meshes must be added during growth. We propose that small holes observed in sheet-like regions of the microvasculature enlarge to form new capillary meshes. With this mechanism of in-itself or intussusceptional growth, sprouting of individual capillary segments to increase network size is no longer needed.

Anti-PSMA antibody-coupled gold nanorods detection by optical and electron microscopies

While cancer is one of the greatest challenges to public health care, prostate cancer was chosen as cancer model to develop a more accurate imaging assessment than those currently available. Indeed, an efficient imaging technique which considerably improves the sensitivity and specificity of the diagnostic and predicting the cancer behavior would be extremely valuable. The concept of optoacoustic imaging using home-made functionalized gold nanoparticles coupled to an antibody targeting PSMA (prostate specific membrane antigen) was evaluated on different cancer cell lines to demonstrate the specificity of the designed platform. Two commonly used microscopy techniques (indirect fluorescence and scanning electron microscopy) showed their straightforwardness and versatility for the nanoparticle binding investigations regardless the composition of the investigated nanoobjects. Moreover most of the research laboratories and centers are equipped with fluorescence microscopes, so indirect fluorescence using Quantum dots can be used for any active targeting nanocarriers (polymers, ceramics, metals, etc.). The second technique based on backscattered electron is not only limited to gold nanoparticles but also suits for any study of metallic nanoparticles as the electronic density difference between the nanoparticles and binding surface stays high enough. Optoacoustic imaging was finally performed on a 3D cellular model to assess and prove the concept of the developed platform.

Capture of activity-induced ultrastructural changes at synapses by high-pressure freezing of brain tissue.

Electron microscopy (EM) allows for the simultaneous visualization of all tissue components at high resolution. However, the extent to which conventional aldehyde fixation and ethanol dehydration of the tissue alter the fine structure of cells and organelles, thereby preventing detection of subtle structural changes induced by an experiment, has remained an issue. Attempts have been made to rapidly freeze tissue to preserve native ultrastructure. Shock-freezing of living tissue under high pressure (high-pressure freezing, HPF) followed by cryosubstitution of the tissue water avoids aldehyde fixation and dehydration in ethanol; the tissue water is immobilized in ∼50 ms, and a close-to-native fine structure of cells, organelles and molecules is preserved. Here we describe a protocol for HPF that is useful to monitor ultrastructural changes associated with functional changes at synapses in the brain but can be applied to many other tissues as well. The procedure requires a high-pressure freezer and takes a minimum of 7 d but can be paused at several points.

Direct observation of liquid crystals using cryo-TEM: Specimen preparation and low-dose imaging

Liquid crystals (LCs) represent a challenging group of materials for direct transmission electron microscopy (TEM) studies due to the complications in specimen preparation and the severe radiation damage. In this paper, we summarize a series of specimen preparation methods, including thin film and cryo-sectioning approaches, as a comprehensive toolset enabling high-resolution direct cryo-TEM observation of a broad range of LCs. We also present comparative analysis using cryo-TEM and replica freeze-fracture TEM on both thermotropic and lyotropic LCs. In addition to the revisits of previous practices, some new concepts are introduced, e.g., suspended thermotropic LC thin films, combined high-pressure freezing and cryo-sectioning of lyotropic LCs, and the complementary applications of direct TEM and indirect replica TEM techniques. The significance of subnanometer resolution cryo-TEM observation is demonstrated in a few important issues in LC studies, including providing direct evidences for the existence of nanoscale smectic domains in nematic bent-core thermotropic LCs, comprehensive understanding of the twist-bend nematic phase, and probing the packing of columnar aggregates in lyotropic chromonic LCs. Direct TEM observation opens ways to a variety of TEM techniques, suggesting that TEM (replica, cryo, and in situ techniques), in general, may be a promising part of the solution to the lack of effective structural probe at the molecular scale in LC studies. Microsc. Res. Tech. 77:754-772, 2014. © 2014 Wiley Periodicals, Inc.

Fine structure of synapses on dendritic spines

Camillo Golgi's "Reazione Nera" led to the discovery of dendritic spines, small appendages originating from dendritic shafts. With the advent of electron microscopy (EM) they were identified as sites of synaptic contact. Later it was found that changes in synaptic strength were associated with changes in the shape of dendritic spines. While live-cell imaging was advantageous in monitoring the time course of such changes in spine structure, EM is still the best method for the simultaneous visualization of all cellular components, including actual synaptic contacts, at high resolution. Immunogold labeling for EM reveals the precise localization of molecules in relation to synaptic structures. Previous EM studies of spines and synapses were performed in tissue subjected to aldehyde fixation and dehydration in ethanol, which is associated with protein denaturation and tissue shrinkage. It has remained an issue to what extent fine structural details are preserved when subjecting the tissue to these procedures. In the present review, we report recent studies on the fine structure of spines and synapses using high-pressure freezing (HPF), which avoids protein denaturation by aldehydes and results in an excellent preservation of ultrastructural detail. In these studies, HPF was used to monitor subtle fine-structural changes in spine shape associated with chemically induced long-term potentiation (cLTP) at identified hippocampal mossy fiber synapses. Changes in spine shape result from reorganization of the actin cytoskeleton. We report that cLTP was associated with decreased immunogold labeling for phosphorylated cofilin (p-cofilin), an actin-depolymerizing protein. Phosphorylation of cofilin renders it unable to depolymerize F-actin, which stabilizes the actin cytoskeleton. Decreased levels of p-cofilin, in turn, suggest increased actin turnover, possibly underlying the changes in spine shape associated with cLTP. The findings reviewed here establish HPF as an appropriate method for studying the fine structure and molecular composition of synapses on dendritic spines.

In vitro characterization of a synthetic calcium phosphate bone graft on periodontal ligament cell and osteoblast behavior and its combination with an enamel matrix derivative.

OBJECTIVES Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials. AIMS This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. MATERIALS AND METHODS EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining. RESULTS Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points. CONCLUSION The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings. CLINICAL RELEVANCE The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.

Detergent-induced stabilization and improved 3D map of the human heteromeric amino acid transporter 4F2hc-LAT2.

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.

Placental Uric Acid Transport System: Glucose Transporter 9 (SLC2A9)

Placental Uric Acid Transport System: Glucose Transporter 9 (SLC2A9). INTRODUCTION: Pre-eclampsia, a pregnancy-specific disease, contributes substantially to perinatal morbidity and mortality of both the mother and her child. Pre-eclampsia is often associated with high maternal urate serum levels, which in turn has been shown to play a role in the pathogenesis of this disease. The aim of this study was to investigate the glucose transporter GLUT9-mediated placental uric acid transport system. METHODS: In this study western blot, immunofluorescence techniques as well as a transepithelial transport (Transwell) model were used to assess GLUT9 protein expression and, respectively, uric acid transport activity. Electrophysiological techniques and transmission electron microscopy (TEM) were used to characterize the properties and the structure of GLUT9. RESULTS: Uric acid is transported across a BeWo choriocarcinoma cell monolayer with 530 pmol/min. We could successfully overexpress and for the first time purify the GLUT9b isoform using the Xenopus laevis oocytes expression system. Chloride seems to modulate the urate transport system. TEM revealed that GLUT9b isoform is present as monomer and dimmer in the Xenopus laevis overexpression model. A class average of all the particles allowed us to develop a first model of human GLUT9b structure, which was derived from the published crystal structure of the bacterial homologue of GLUT1-4. CONCLUSIONS: In vitro the “materno-fetal” transport of uric acid is slow indicating that in vivo the fetus might be protected from short-term fluctuations of maternal urate serum levels. The low-resolution structure obtained from TEM validates the proposed homology model regarding the structure of human GLUT9b. In ongoing studies this model is used to perform virtual screening to identify novel modulators of the urate transport system enabling the development of novel therapies in pregnancy complications.