Biological synthesis of gold nanoparticles by the xylotrophic basidiomycete Lentinula edodes

This is the first study to demonstrate that the medicinal basidiomycete Lentinula edodes can reduce gold (III) ions from hydrogen tetrachloaurate (chloroauric acid) H[AuCl₄] to the elementary state with the formation of spherical nanoparticles (nanospheres). When a culture was grown under submerged conditions in the presence of chloroauric acid, the appearance of an intense purple-red color of L. edodes filamentous hyphae was recorded, which indicates that gold ions were reduced to gold nanoparticles. Using transmission electron microscopy and X-ray fluorescence, we observed accumulation of colloidal gold by the fungal mycelium in the form of electron-dense nanospheres of 5 to 50 nm in diameter on the surface and inside fungal cells.     

Investigation of cell adhesion to structured surfaces using total internal reflection fluorescence and confocal laser scanning microscopy

Adhesion of adherent cells on structured surfaces is influenced by the surface pattern given. Here, we designed a structured gold relief surface based on cell adhesion patterns we had previously observed. We analysed the geometric parameters and the overall distribution of focal adhesion kinase in focal adhesions on unstructured glass surfaces using optical microscopy. The basic structural elements obtained from this analysis were arranged in regular clusters that resembled the shape of a polarised migratory cell. In time-lapse studies we observed that the cells adhere preferentially to the gold pads and adopt the shape of the clusters. Staining of the actin cytoskeleton revealed that the actin filaments are aligned to the gold pads of the elementary structure.     

Diversity of dermal denticle structure in sharks: Skin surface roughness and three‐dimensional morphology

Shark skin is covered with numerous placoid scales or dermal denticles. While previous research has used scanning electron microscopy and histology to demonstrate that denticles vary both around the body of a shark and among species, no previous study has quantified three‐dimensional (3D) denticle structure and surface roughness to provide a quantitative analysis of skin surface texture. We quantified differences in denticle shape and size on the skin of three individual smooth dogfish sharks (Mustelus canis) using micro‐CT scanning, gel‐based surface profilometry, and histology. On each smooth dogfish, we imaged between 8 and 20 distinct areas on the body and fins, and obtained further comparative skin surface data from leopard, Atlantic sharpnose, shortfin mako, spiny dogfish, gulper, angel, and white sharks. We generated 3D images of individual denticles and measured denticle volume, surface area, and crown angle from the micro‐CT scans. Surface profilometry was used to quantify metrology variables such as roughness, skew, kurtosis, and the height and spacing of surface features. These measurements confirmed that denticles on different body areas of smooth dogfish varied widely in size, shape, and spacing. Denticles near the snout are smooth, paver‐like, and large relative to denticles on the body. Body denticles on smooth dogfish generally have between one and three distinct ridges, a diamond‐like surface shape, and a dorsoventral gradient in spacing and roughness. Ridges were spaced on average 56 µm apart, and had a mean height of 6.5 µm, comparable to denticles from shortfin mako sharks, and with narrower spacing and lower heights than other species measured. We observed considerable variation in denticle structure among regions on the pectoral, dorsal, and caudal fins, including a leading‐to‐trailing edge gradient in roughness for each region. Surface roughness in smooth dogfish varied around the body from 3 to 42 microns.     

Synthesis of gold nanotriangles and silver nanoparticles using Aloe vera plant extract

Biogenic gold nanotriangles and spherical silver nanoparticles were synthesized by a simple procedure using Aloe vera leaf extract as the reducing agent. This procedure offers control over the size of the gold nanotriangle and thereby a handle to tune their optical properties, particularly the position of the longitudinal surface plasmon resonance. The kinetics of gold nanotriangle formation was followed by UV-vis-NIR absorption spectroscopy and transmission electron microscopy (TEM). The effect of reducing agent concentration in the reaction mixture on the yield and size of the gold nanotriangles was studied using transmission electron microscopy. Monitoring the formation of gold nanotriangles as a function of time using TEM reveals that multiply twinned particles (MTPs) play an important role in the formation of gold nanotriangles. It is observed that the slow rate of the reaction along with the shape directing effect of the constituents of the extract are responsible for the formation of single crystalline gold nanotriangles. Reduction of silver ions by Aloe vera extract however, led to the formation of spherical silver nanoparticles of 15.2 nm +/- 4.2 nm size.     

Immobilization and biocatalytic activity of fungal protease on gold nanoparticle-loaded zeolite microspheres

Gold nanoparticles are excellent biocompatible surfaces for the immobilization of enzymes. However, separation of the gold nanoparticle-enzyme bioconjugate material from the reaction medium is often difficult. In this study, we investigate the assembly of the gold nanoparticles on the surface of the amine-functionalized zeolite microspheres in the formation of zeolite-gold nanoparticle "core-shell" structures and, thereafter, the use of this structure in immobilization of fungal protease. The assembly of gold nanoparticles on the zeolite surface occurs through the amine groups present in 3-aminopropyltrimethoxysilane (3-APTS). The fungal proteases bound to the massive "core-shell" structures were easily separated from the reaction medium by mild centrifugation and exhibited excellent reuse characteristics. The biocatalytic activity of fungal protease in the bioconjugate was marginally enhanced relative to the free enzyme in solution. The bioconjugate material also showed significantly enhanced pH and temperature stability and a shift in the optimum temperature of operation.     

Whole-cell-mount cytochemistry by the colloidal gold labeling method. Combined transmission and scanning electron microscopic study of ConA binding sites in mouse macrophages

Binding, redistribution, and endocytosis of colloidal gold (CG)-labeled concanavalin A (ConA) were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Mouse peritoneal macrophages were cultured on Formvar-coated platinum grids. Either fixed or unfixed cells were labeled by the indirect ConA-CG labeling method. Specimens were critical-point-dried and observed by TEM and SEM in the same region. Surface-bound ConA-CG was easily seen by SEM. Stereomicroscopic observation by TEM clearly showed the three-dimensional distribution of ConA on the cell surface as well as in the cytoplasmic vesicles and vacuoles. In the prefixed cells, CG was distributed randomly on the cell surface. When unfixed cells were labeled at 0 degree C, a similar binding pattern was observed, although the density of bound CG was decreased. When cells labeled with ConA-CG at 0 degree C were further incubated at 37 degrees C, redistribution and endocytosis of the label were seen. Endocytosed CG in the cytoplasmic vesicles and vacuoles was clearly seen by TEM. In addition, three-dimensional location and relationship with other organelles were easily observed. Combined TEM and SEM observation of CG-labeled whole-cell-mount specimens is a useful method to study the dynamics of cell-bound ligands.     

Blood lysate staining, a new microscopic method for diagnosis of fungemia using peripheral blood

We developed a microscopy method for the detection of fungal cells in peripheral blood, termed blood lysate staining, using an approximately 5x5 mm dotted blood lysate. This method was able to detect the emerging fungal pathogen Trichosporon asahii in murine models of systemic fungal infection and fungemia in patients quickly and at minimal cost. Pathogenic yeasts were successfully detected in 6 of 8 blood samples which were taken from feverish immunocompromised patients who were clinically suspected of having fungal infections. Fungal cells were observed as ovoid to elongated, 3x3 to 7x10 microm, and occurred singly, budding, and in short chains and clusters in a periodic acid-Schiff-stained blood smear. The yeast cells were easily distinguished from blood-cell debris by their size, shape and smooth yet rigid outline.     

Immunolocalization of chitin synthases in the phytopathogenic dimorphic fungus Ustilago maydis

Conserved polypeptides of the chitin synthase genes UmCHS3 and UmCHS6 from the phytopathogenic fungus Ustilago maydis were utilized as immunogens to obtain polyclonal antibodies that were purified by affinity procedures. Because of their similarities at the regions encoded by either polypeptide, it was concluded that anti-Chs3 antibodies recognized both Chs3 and Chs4 chitin synthases, whereas anti-Chs6 antibodies recognized Chs6 and Chs8 polypeptides. These antibodies were used to analyze the localization of the corresponding chitin synthases in U. maydis cells, using both indirect immunofluorescence microscopy and immunoelectron microscopy with colloidal-gold-labeled secondary antibodies. It was observed that chitin synthase proteins were accumulated both in the surface and in the cytoplasm of the fungal cells. Electron microscopy images revealed the accumulation of clusters of gold particles in vesicles, providing evidence for the possible origin and destination of chitin synthases in the fungal cells.     

Biosynthesis of extracellular and intracellular gold nanoparticles by Aspergillus fumigatus and A. flavus

Green chemistry is a boon for the development of safe, stable and ecofriendly nanostructures using biological tools. The present study was carried out to explore the potential of selected fungal strains for biosynthesis of intra- and extracellular gold nanostructures. Out of the seven cultures, two fungal strains (SBS-3 and SBS-7) were selected on the basis of development of dark pink colour in cell free supernatant and fungal beads, respectively indicative of extra- and intracellular gold nanoparticles production. Both biomass associated and cell free gold nanoparticles were characterized using X-ray diffractogram (XRD) analysis and transmission electron microscopy (TEM). XRD analysis confirmed crystalline, face-centered cubic lattice of metallic gold nanoparticles along with average crystallite size. A marginal difference in average crystallite size of extracellular (17.76 nm) and intracellular (26 and 22 nm) Au-nanostructures was observed using Scherrer equation. In TEM, a variety of shapes (triangles, spherical, hexagonal) were observed in both extra- and intracellular nanoparticles. 18S rRNA gene sequence analysis by multiple sequence alignment (BLAST) indicated 99 % homology of SBS-3 to Aspergillus fumigatus with 99 % alignment coverage and 98 % homology of SBS-7 to Aspergillus flavus with 98 % alignment coverage respectively. Native-PAGE and activity staining further confirmed enzyme linked synthesis of gold nanoparticles.     

An easy method to discover cell membrane antigen with atomic force microscopy

Atomic force microscopy (AFM) obtains a high resolution at nanometer. In the current study, we found that, before and after adding CD34, CD44 or CD29 antibodies, the AFM images of mouse adipose tissue derived mesenchymal stem cells (ADMSCs) had significant changes in both cell morphous (shape) and the average roughness of cell membrane. However, there was no significant difference in the cell shape and the average roughness of cell membrane, after adding CD45 or CD144 antibodies. Therefore, we could use the AFM to scan cell shape or to calculate the average roughness changes of cell membrane to analyze the existence of cell membrane antigen qualitatively.     

Static magnetic fields affect cell size, shape, orientation, and membrane surface of human glioblastoma cells, as demonstrated by electron, optic, and atomic force microscopy.

BACKGROUND: It is common knowledge that static magnetic fields (SMF) do not interact with living cells; thus, fewer studies of SMF compared with variable magnetic fields are carried out. However, evidence demonstrated that SMF affect cellular structures. To investigate the effect of exposure to increasing doses of SMF on cell morphology, human glioblastoma cells were exposed to SMF ranging between 80 and 3,000 G (8 and 300 mT). METHODS: Cell morphology of human glioblastoma cells, derived from a primary culture, was studied by electron and optic microscopy. FITC-phalloidin staining of actin filaments was also investigated. Finally, cell surface structure changes were detected by atomic force microscopy. RESULTS: Scanning electron microscopy demonstrated a dose-dependent cell shape modification, progressive cell detachment, loss of the long villi, and appearance of membrane roughness and blebs. FITC-phalloidin staining confirmed the villi retention and cell dimension decrease. At 3,000 G, the appearance of apoptotic morphology was also observed by transmission electron microscopy. Cell exposed to SMF showed different orientation and alignment when compared with nonexposed cells. The atomic force microscopy of the exposed cells' membrane surfaces demonstrated the disappearance of the ordered surface ripples and furrows typical of the unexposed cells, and the occurrence of surface membrane corrugation at increasing dose exposure CONCLUSIONS: Our experimental procedures demonstrated that exposure to SMF affects not only cell size, shape, and orientation but also human glioblastoma cells' membrane surfaces.     

Biosynthesis of nanoparticles of metals and metalloids by basidiomycetes. Preparation of gold nanoparticles by using purified fungal phenol oxidases

The work shows the ability of cultured Basidiomycetes of different taxonomic groups—Lentinus edodes, Pleurotus ostreatus, Ganoderma lucidum, and Grifola frondosa—to recover gold, silver, selenium, and silicon, to elemental state with nanoparticles formation. It examines the effect of these metal and metalloid compounds on the parameters of growth and accumulation of biomass; the optimal cultivation conditions and concentrations of the studied ion-containing compounds for recovery of nanoparticles have been identified. Using the techniques of transmission electron microscopy, dynamic light scattering, X-ray fluorescence and X-ray phase analysis, the degrees of oxidation of the bioreduced elements, the ζ-potential of colloidal solutions uniformity, size, shape, and location of the nanoparticles in the culture fluid, as well as on the surface and the inside of filamentous hyphae have been determined. The study has found the part played by homogeneous chromatographically pure fungal phenol-oxidizing enzymes (laccases, tyrosinases, and Mn-peroxidases) in the recovery mechanism with formation of electrostatically stabilized colloidal solutions. A hypothetical mechanism of gold(III) reduction from HAuCl₄ to gold(0) by phenol oxidases with gold nanoparticles formation of different shapes and sizes has been introduced.

     

Biosynthesis of Stable Polyshaped Gold Nanoparticles from Microwave-Exposed Aqueous Extracellular Anti-malignant Guava (Psidium guajava) Leaf Extract

Addition of microwave-exposed aqueous extracellular anti-malignant guava (Psidium guajava) leaf extract to the aqueous gold chloride solution yielded stable polyshaped gold nanoparticles of high composition. Microwave-assisted route selected for the preparation of aqueous guava leaf extract was to suppress the enzymatic action. The formation of nanoparticles was understood from the UV–visible and X-ray diffraction studies. The size and shape analysis was done using field emission scanning electron microscopy, transmission electron microscopy, and atomic force microscopy. Zeta potential experiment shows that the bio-functionalized gold nanoparticles colloidal solution obtained as above will maintain its stability even after 30 weeks of storage. It is observed that the flavonoids which are separated during microwave heating of extracellular solution of the guava leaves are responsible for the biosynthesis of gold nanoparticles.     

Whole-cell-mount cytochemistry by the colloidal gold labeling method

Summary Binding, redistribution, and endocytosis of colloidal gold (CG)-labeled concanavalin A (ConA) were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Mouse peritoneal macrophages were cultured on Formvar-coated platinum grids. Either fixed or unfixed cells were labeled by the indirect ConA-CG labeling method. Specimens were critical-pointdried and observed by TEM and SEM in the same region. Surface-bound ConA-CG was easily seen by SEM. Stereomicroscopic observation by TEM clearly showed the threedimensional distribution of ConA on the cell surface as well as in the cytoplasmic vesicles and vacuoles. In the prefixed cells, CG was distributed randomly on the cell surface. When unfixed cells were labeled at 0° C, a similar binding pattern was observed, although the density of bound CG was decreased. When cells labeled with ConA-CG at 0° C were further incubated at 37° C, redistribution and endocytosis of the label were seen. Endocytosed CG in the cytoplasmic vesicles and vacuoles was clearly seen by TEM. In addition, three-dimensional location and relationship with other organelles were easily observed. Combined TEM and SEM observation of CG-labeled whole-cell-mount specimens is a useful method to study the dynamics of cellbound ligands.This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan, and the Japan Private School Promotion Foundation     

A multiple-staining ultrastructural procedure simultaneously detecting three membrane antigens on suspended cells by monoclonal antibodies and preembedding immunogold labelling

Summary A triple ultrastructural immunogold staining method for the simultaneous demonstration of three surface antigens of peripheral blood mononuclear cells at the electron microscope level is described. A six-step pre-embedding immunoelectron microscopy procedure was developed, using commercially available reagents. The CD11b antigen was first detected, through a two-step (indirect) method with 40 nm-sized gold particles; after a blocking step, the HLA-DR surface antigen was subsequently detected, through a two-step (biotin-streptavidin) method with 20 nm-sized gold particles; the CD4 antigen was finally detected, through a one-step (direct) method, using 5 nm-sized gold particles. Electron microscopic examination revealed firstly the presence of a triple-labelled cell subpopulation, which showed gold granules of the three sizes simultaneously decorating the cell membrane. Thus, the cells of such a subset simultaneously expressed the three antigens investigated. In contrast, either gold particles of only one size or no gold particles were observed on the cell surface of other subpopulations. This technique is a model demonstrating the importance of varying the size of particles in pre-embedding gold immunoelectron microscopy for a better analysis of the expression of surface antigens in isolated cells.     

Modification of form, material and modularity of threaded acetabulum cups]

The fixation principle of threaded cups ensures high primary stability. Inadequate results with first-generation threaded cups led to modifications of surface machining. 10 threaded cups of the first generation, and 27 of the second and third generations were systematically analysed and their shapes measured using a no-touch light section technique. In addition, measurements of surface roughness were performed. Implants of the first generation made of polyethylene, ceramic or cobalt-chrome have an average surface roughness (Ra) of 1.5 microns. Approximately one-half of these implants have a conical shape, and one-third a height that is greater than the radius. Threaded cups of the second generation are made either of CP-titanium or titanium alloy. The average corundum-blasted surface roughness is 4.5 microns. Hydroxyapatite-coated (HA) implants have a surface roughness of 5.0 microns. Approximately 45% of the implants have a conical, biconical or flattened-conical shape, while one-third are of hemispherical shape. Approximately 90% of the cups have a height that is up to 23% smaller than the radius. A few cups have a height that approximates the radius. Implants of the third generation with identical surface structure can be supplied with crosslinked-polyethylene inlays or, optionally, with metal/metal or ceramic/ceramic contact surfaces. Primary stability, biocompatible materials and a structured surface are essential for ensuring osseointegration over the long-term. Corundum-blasted pure titanium or titanium alloys with corundum-blasted or HA-coated implants can be considered standard for these cups.     

A Review of the Potential and Versatility of Colloidal Gold Cytochemical Labeling for Molecular Morphology

In the present article we review several postembedding cytochemical techniques using the colloidal gold marker. Owing to the high atomic number of gold, the colloidal gold particles are electron dense. They are spherical in shape and can be prepared in sizes from 1 to 25 nm, which renders this marker among the best for electron microscopy. In addition, because it can be bound to several molecules, this marker has the advantage of being extremely versatile. Combined to immunoglobulins or immunoglobulin-binding proteins (protein A), it has been applied successfully in immunocytochemistry. Colloidal gold particles 5–15 nm in size are excellent for postembedding cytochemistry. Particles of smaller size, such as 1 nm, must be silver enhanced to be visualized by transmission electron microscopy. We have elected to review the superiority of indirect immunocytochemical approaches using IgG-gold or protein A-gold (protein G-gold and protein AG-gold). Lectins or enzymes can be tagged with colloidal gold particles, and the corresponding lectin-gold and enzyme-gold techniques have specific advantages and great potential. Using an indirect digoxigenin-tagged nucleotide and an antidigoxigenin probe, colloidal gold technology can also be used for in situ hybridization at the electron microscope level. Affinity characteristics lie behind all cytochemical techniques and several molecules displaying high affinity properties can also be beneficial for colloidal gold electron microscopy cytochemistry. All of these techniques can be combined in various ways to produce multiple labelings of several binding sites on the same tissue section. Colloidal gold is particulate and can easily be counted; thus the cytochemical signal can be evaluated quantitatively, introducing further advantages to the use of the colloidal gold marker. Finally, several combinations and multiple step procedures have been designed to amplify the final signal which renders the techniques more sensitive. The approaches reviewed here have been applied successfully in different fields of cell and molecular biology, cell pathology, plant biology and pathology, microbiology and virology. The potential of the approaches is emphasized in addition to different ways to assess specificity, sensitivity and accuracy of results.     

A review of the potential and versatility of colloidal gold cytochemical labeling for molecular morphology.

In the present article we review several postembedding cytochemical techniques using the colloidal gold marker. Owing to the high atomic number of gold, the colloidal gold particles are electron dense. They are spherical in shape and can be prepared in sizes from 1 to 25 nm, which renders this marker among the best for electron microscopy. In addition, because it can be bound to several molecules, this marker has the advantage of being extremely versatile. Combined to immunoglobulins or immunoglobulin-binding proteins (protein A), it has been applied successfully in immunocytochemistry. Colloidal gold particles 5-15 nm in size are excellent for postembedding cytochemistry. Particles of smaller size, such as 1 nm, must be silver enhanced to be visualized by transmission electron microscopy. We have elected to review the superiority of indirect immunocytochemical approaches using IgG-gold or protein A-gold (protein G-gold and protein AG-gold). Lectins or enzymes can be tagged with colloidal gold particles, and the corresponding lectin-gold and enzyme-gold techniques have specific advantages and great potential. Using an indirect digoxigenin-tagged nucleotide and an antidigoxigenin probe, colloidal gold technology can also be used for in situ hybridization at the electron microscope level. Affinity characteristics lie behind all cytochemical techniques and several molecules displaying high affinity properties can also be beneficial for colloidal gold electron microscopy cytochemistry. All of these techniques can be combined in various ways to produce multiple labelings of several binding sites on the same tissue section. Colloidal gold is particulate and can easily be counted; thus the cytochemical signal can be evaluated quantitatively, introducing further advantages to the use of the colloidal gold marker. Finally, several combinations and multiple step procedures have been designed to amplify the final signal which renders the techniques more sensitive. The approaches reviewed here have been applied successfully in different fields of cell and molecular biology, cell pathology, plant biology and pathology, microbiology and virology. The potential of the approaches is emphasized in addition to different ways to assess specificity, sensitivity and accuracy of results.     

Differential mechanisms for calcium-dependent protein/membrane association as evidenced from SPR-binding studies on supported biomimetic membranes

In this work, two different types of supported biomimetic membranes were designed to study the membrane binding properties of two different proteins that both interact with cellular membranes in a calcium-dependent manner. The first one, neurocalcin, is a member of a subfamilly of EF-hand calcium-binding proteins that exhibit a calcium-myristoyl switch. The second protein is a bacterial toxin, the adenylate cyclase produced by Bordetella pertussis, the causative agent of whooping cough. The biomimetic membranes constructed in this study were either hybrid bilayer membranes or polymer-tethered membranes. Hemimembrane formation was obtained in two steps: a monolayer of 1-octadecanethiol or octadecyltrichlorosilane was self-assembled on top of the gold or glass surface, respectively, and then the egg-phosphatidyl choline (PC) vesicle fused on the hydrophobic alkyl layer. Polymer-tethered membranes on solid support were obtained using N-hydroxysuccinimide (NHS)-terminated-poly(ethyleneglycol) (PEG)-phospholipids as anchoring molecules. Egg-PC/1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-poly(ethyleneglycol)-N-hydroxy-succinimide (DSPE-PEG-NHS) mixture liposomes were injected on the top of an amine grafted surface (cysteamine-coated gold or silanized glass); vesicles were linked to the surface and disrupted, leading to the formation of a bilayer. The biomimetic membrane constructions were followed by surface plasmon spectroscopy, while membrane fluidity and continuity were observed by fluorescence microscopy. Protein/membrane binding properties were determined by resonance surface plasmon measurements. The tethered bilayer, designed here, is very versatile as it can be adapted easily to different types of support. The results demonstrate the potentialities of such polymer-tethered artificial membranes for the study of proteins that insert into biological membranes such as toxins and/or integral membrane proteins.     

Fabrication of Gold Nanostructures and Studies of Their Morphological and Surface Plasmonic Properties

We report fabrication of gold nanostructures on glass and indium tin oxide (ITO)-coated glass substrates using high fluence and highly energetic gold ions generated by hot, dense, and strongly non-equilibrium plasma. Nanodots and nanorods are observed in scanning electron microscopy (SEM) of nanostructures grown on glass substrate with single and double shots of gold ions which is in conformity with the transmission electron microscopy image. SEM images for single and double shots of gold ions on ITO-coated glass substrate show only nanodots. The mean diameter of nanodots obtained on both glass and ITO-coated glass is found to increase with increase in the number of gold ions shot from one to two. The gold nanostructures exhibit red shift in surface plasmon resonance with increased interaction which is in agreement with other reported work.