Jeannette V. Taylor

TEM analysis of the nanostructure of normal and osteoporotic human trabecular bone

Transmission electron microscopy (TEM) was used to investigate the crystal-collagen interactions in normal and osteoporotic human trabecular bone at the nanostructural level. More specifically, two-dimensional TEM observations were used to infer the three-dimensional information on the shape, the size, the orientation, and the alignment of apatite crystals in collagen fibrils in normal and osteoporotic bone. We found that crystals were of platelet shape with irregular edges and that there was no substantial difference in crystal length or crystal thickness between normal and osteoporotic trabecular bone. The crystal arrangement in cross-sectioned fibrils did not neatly conform to the parallel arrangement of crystals seen in longitudinally-sectioned fibrils. Instead, the crystal arrangement in both normal and osteoporotic trabecular bone took on more of a random, undulated arrangement, with certain localized areas demonstrating circular oriented patterns. The TEM imaging was done using bright fields only. Thus, the results presented are within the limitations of this approach.

Long-Term Biostability of Self-Assembling Protein Polymers in the Absence of Covalent Crosslinking

Unless chemically crosslinked, matrix proteins, such as collagen or silk, display a limited lifetime in vivo with significant degradation observed over a period of weeks. Likewise, amphiphilic peptides, lipopeptides, or glycolipids that self-assemble through hydrophobic interactions to form thin films, fiber networks, or vesicles do not demonstrate in vivo biostability beyond a few days. We report herein that a self-assembling, recombinant elastin-mimetic triblock copolymer elicited minimal inflammatory response and displayed robust in vivo stability for periods exceeding 1 year, in the absence of either chemical or ionic crosslinking. Specifically, neither a significant inflammatory response nor calcification was observed upon implantation of test materials into the peritoneal cavity or subcutaneous space of a mouse model. Moreover, serial quantitative magnetic resonance imaging, evaluation of pre- and post-explant ultrastructure by cryo-high resolution scanning electron microscopy, and an examination of implant mechanical responses revealed substantial preservation of form, material architecture, and biomechanical properties, providing convincing evidence of a non-chemically or ionically crosslinked protein polymer system that exhibits long-term stability in vivo.

Peptides Organized as Bilayer Membranes

From the organizing poten-tial of two-dimensional phospholipid membranes to theinformation-rich DNA helices, from the mechanical actinand tubulin cables to the structural collagen and elastinnetworks, these self-assembling asymmetric arrays define thearchitectures of all cells and tissues. Recent covalent hybridsof traditional biological macromolecular families (e.g.,nucleic acids with proteins

Microscopic Studies on the Aspergillus Flavus Infected Kernels of Commercial Peanuts in Georgia

This article describes the use of microscopy to prove the presence of the aflatoxin producing pathogen, Aspergillus flavus Link ex Fries in commercially available edible peanuts in Georgia. Light microscopy in combination with electron microscopy has been used to describe the infection course established by the fungus. The alkali maceration technique used in the study was successful and sufficient to detect the kernel infection of A. flavus and monitor the infection percentage in edible peanuts. Percentage of infected kernel varied from one commercial outlet to another in the region. Briefly, peanut seeds from Cartersville had the highest percentage of A. flavus infection. Electron microscopy confirmed the seed-borne infection of this mold. Mycelium established inside the host tissues both intercellularly and intracellularly aided by active, continuous branching of young hyphae. Establishment of mycelium was also detected in the xylem vessels of roots indicative of systemic infection. Thus, edible peanuts can form an important source of inoculum and facilitate the spread of the fungus from one peanut to another in commercial outlets and elsewhere. Present study provides strong evidence that A. flavus can escape detection at selling points and lands in commercial outlets via edible peanuts. That these contaminated peanuts could pose public health hazards is discussed.

The Opportunistic Pathogen Vibrio vulnificus Produces Outer Membrane Vesicles in a Spatially Distinct Manner Related to Capsular Polysaccharide

Vibrio vulnificus, a bacterial species that inhabits brackish waters, is an opportunistic pathogen of humans. V. vulnificus infections can cause acute gastroenteritis, invasive septicemia, tissue necrosis, and potentially death. Virulence factors associated with V. vulnificus include the capsular polysaccharide (CPS), lipopolysaccharide, flagellum, pili, and outer membrane vesicles (OMVs). The aims of this study were to characterize the morphology of V. vulnificus cells and the formation and arrangement of OMVs using cryo-electron microscopy (cryo-EM). cryo-EM and cryo-electron tomography imaging of V. vulnificus strains grown in liquid cultures revealed the presence of OMVs (diameters of ∼45 nm for wild-type, ∼30 nm for the unencapsulated mutant, and ∼50 nm for the non-motile mutant) in log-phase growth. Production of OMVs in the stationary growth phase was limited and irregular. The spacing of the OMVs around the wild-type cells was in regular, concentric rings. In wild-type cells and a non-motile mutant, the spacing between the cell envelope and the first ring of OMVs was ∼200 nm; this spacing was maintained between subsequent OMV layers. The size, arrangement, and spacing of OMVs in an unencapsulated mutant was irregular and indicated that the polysaccharide chains of the capsule regulate aspects of OMV production and order. Together, our results revealed the distinctive organization of V. vulnificus OMVs that is affected by expression of the CPS.

Pleomorphic Structures in Human Blood Are Red Blood Cell-Derived Microparticles, Not Bacteria

Background Red blood cell (RBC) transfusions are a common, life-saving therapy for many patients, but they have also been associated with poor clinical outcomes. We identified unusual, pleomorphic structures in human RBC transfusion units by negative-stain electron microscopy that appeared identical to those previously reported to be bacteria in healthy human blood samples. The presence of viable, replicating bacteria in stored blood could explain poor outcomes in transfusion recipients and have major implications for transfusion medicine. Here, we investigated the possibility that these structures were bacteria. Results Flow cytometry, miRNA analysis, protein analysis, and additional electron microscopy studies strongly indicated that the pleomorphic structures in the supernatant of stored RBCs were RBC-derived microparticles (RMPs). Bacterial 16S rDNA PCR amplified from these samples were sequenced and was found to be highly similar to species that are known to commonly contaminate laboratory reagents. Conclusions These studies suggest that pleomorphic structures identified in human blood are RMPs and not bacteria, and they provide an example in which laboratory contaminants may can mislead investigators.

The Morphology and Assembly of Respiratory Syncytial Virus Revealed by Cryo-Electron Tomography

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. With repeat infections throughout life, it can also cause substantial disease in the elderly and in adults with compromised cardiac, pulmonary and immune systems. RSV is a pleomorphic enveloped RNA virus in the Pneumoviridae family. Recently, the three-dimensional (3D) structure of purified RSV particles has been elucidated, revealing three distinct morphological categories: spherical, asymmetric, and filamentous. However, the native 3D structure of RSV particles associated with or released from infected cells has yet to be investigated. In this study, we have established an optimized system for studying RSV structure by imaging RSV-infected cells on transmission electron microscopy (TEM) grids by cryo-electron tomography (cryo-ET). Our results demonstrate that RSV is filamentous across several virus strains and cell lines by cryo-ET, cryo-immuno EM, and thin section TEM techniques. The viral filament length varies from 0.5 to 12 μm and the average filament diameter is approximately 130 nm. Taking advantage of the whole cell tomography technique, we have resolved various stages of RSV assembly. Collectively, our results can facilitate the understanding of viral morphogenesis in RSV and other pleomorphic enveloped viruses.

Correlating Cryo-Electron Microscopy Methods for Structural Studies of Bacteria

Vibrio vulnificus, a halotrophic Gram-negative bacterium, which lives in coastal waters, is associated with shellfish in natural environments and is an opportunistic pathogen of humans. A number of studies have identified many of the virulence factors associated with V. vulnificus [1]. Some of the virulence factors identified include external structures of the bacteria such as the capsular polysaccharide (CPS), flagellum, and pili. However, limited efforts have been aimed at determining the ultrastructure of these factors and the arrangement of their individual components. In order to address how ultrastructure of the bacteria influences pathogenesis, we probed the architecture of the cells, the flagella, the pili, and the CPS with high-resolution scanning electron microscopy (HRSEM), conventional transmission electron microscopy (TEM) of cryo-preserved specimens, and cryo-electron tomography (cryo-ET).