David Gene Morgan

Electron Microscopic Analysis of a Spherical Mitochondrial Structure

Background: Mitochondria are dynamic organelles with variable morphological features under different functional status. Results: Mitochondria treated with an uncoupler presented a spherical structure with an internal lumen containing cytosolic materials as defined by serial sections and electron tomography. Conclusion: Mitochondria were able to undergo a three-dimensional structural transformation under oxidative stress. Significance: Mitochondrial spheroid formation represents a novel mitochondrial dynamics. Mitochondria undergo dynamic structural alterations to meet changing needs and to maintain homeostasis. We report here a novel mitochondrial structure. Conventional transmission electron microscopic examination of murine embryonic fibroblasts treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, found that more than half of the mitochondria presented a ring-shaped or C-shaped morphology. Many of these mitochondria seemed to have engulfed various cytosolic components. Serial sections through individual mitochondria indicated that they formed a ball-like structure with an internal lumen surrounded by the membranes and containing cytosolic materials. Notably, the lumen was connected to the external cytoplasm through a small opening. Electron tomographic reconstruction of the mitochondrial spheroids demonstrated the membrane topology and confirmed the vesicular configuration of this mitochondrial structure. The outside periphery and the lumen were defined by the outer membranes, which were lined with the inner membranes. Matrix and cristae were retained but distributed unevenly with less being kept near the luminal opening. Mitochondrial spheroids seem to form in response to oxidative mitochondrial damage independently of mitophagy. The structural features of the mitochondrial spheroids thus represent a novel mitochondrial dynamics.

Functionalization of monodisperse iron oxide NPs and their properties as magnetically recoverable catalysts.

Here we report the functionalization of monodisperse iron oxide nanoparticles (NPs) with commercially available functional acids containing multiple double bonds such as linolenic (LLA) and linoleic (LEA) acids or pyridine moieties such as 6-methylpyridine-2-carboxylic acid, isonicotinic acid, 3-hydroxypicolinic acid, and 6-(1-piperidinyl)pyridine-3-carboxlic acid (PPCA). Both double bonds and pyridine groups can be reacted with noble metal compounds to form catalytically active species in the exterior of magnetic NPs, thus making them promising magnetically recoverable catalysts. We determined that both LLA and LEA stabilize magnetic iron oxide NPs, allowing the formation of π-complexes with bis(acetonitrile)dichloropalladium(II) in the NP shells. In both cases, this leads to the formation of NP aggregates because of interparticle complexation. In the case of pyridine-containing ligands, only PPCA with two N-containing rings is able to provide NP stabilization and functionalization whereas other pyridine-containing acids did now allow sufficient steric stabilization. The interaction of PPCA-based particles with Pd acetate also leads to aggregation because of interparticle interactions, but the aggregates that are formed are much smaller. Nevertheless, the catalytic properties in the selective hydrogenation of dimethylethynylcarbinol (DMEC) to dimethylvinylcarbinol were the best for the catalyst based on LLA, demonstrating that the NP aggregates in all cases are penetrable for DMEC. Easy magnetic separation of this catalyst from the reaction solution makes it promising as a magnetically recoverable catalyst.

The Spirochete FlaA Periplasmic Flagellar Sheath Protein Impacts Flagellar Helicity

Spirochete periplasmic flagella (PFs), including those from Brachyspira (Serpulina), Spirochaeta, Treponema, and Leptospira spp., have a unique structure. In most spirochete species, the periplasmic flagellar filaments consist of a core of at least three proteins (FlaB1, FlaB2, and FlaB3) and a sheath protein (FlaA). Each of these proteins is encoded by a separate gene. Using Brachyspira hyodysenteriae as a model system for analyzing PF function by allelic exchange mutagenesis, we analyzed purified PFs from previously constructed flaA::cat, flaA::kan, and flaB1::kan mutants and newly constructed flaB2::cat and flaB3::cat mutants. We investigated whether any of these mutants had a loss of motility and altered PF structure. As formerly found with flaA::cat, flaA::kan, and flaB1::kan mutants, flaB2::cat and flaB3::cat mutants were still motile, but all were less motile than the wild-type strain, using a swarm-plate assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis indicated that each mutation resulted in the specific loss of the cognate gene product in the assembled purified PFs. Consistent with these results, Northern blot analysis indicated that each flagellar filament gene was monocistronic. In contrast to previous results that analyzed PFs attached to disrupted cells, purified PFs from a flaA::cat mutant were significantly thinner (19.6 nm) than those of the wild-type strain and flaB1::kan, flaB2::cat, and flaB3::cat mutants (24 to 25 nm). These results provide supportive genetic evidence that FlaA forms a sheath around the FlaB core. Using high-magnification dark-field microscopy, we also found that flaA::cat and flaA::kan mutants produced PFs with a smaller helix pitch and helix diameter compared to the wild-type strain and flaB mutants. These results indicate that the interaction of FlaA with the FlaB core impacts periplasmic flagellar helical morphology.

Genetic Analysis of Spirochete Flagellin Proteins and Their Involvement in Motility, Filament Assembly, and Flagellar Morphology

The filaments of spirochete periplasmic flagella (PFs) have a unique structure and protein composition. In most spirochetes, the PFs consist of a core of at least three related proteins (FlaB1, FlaB2, and FlaB3) and a sheath of FlaA protein. The functions of these filament proteins remain unknown. In this study, we used a multidisciplinary approach to examine the role of these proteins in determining the composition, shape, and stiffness of the PFs and how these proteins impact motility by using the spirochete Brachyspira (formerly Treponema, Serpulina) hyodysenteriae as a genetic model. A series of double mutants lacking combinations of these PF proteins was constructed and analyzed. The results show the following. First, the diameters of PFs are primarily determined by the sheath protein FlaA, and that FlaA can form a sheath in the absence of an intact PF core. Although the sheath is important to the PF structure and motility, it is not essential. Second, the three core proteins play unequal roles in determining PF structure and swimming speed. The functions of the core proteins FlaB1 and FlaB2 overlap such that either one of these proteins is essential for the spirochete to maintain the intact PF structure and for cell motility. Finally, linear elasticity theory indicates that flagellar stiffness directly affects the spirochetes swimming speed.

Aerosol synthesis of porous particles using simple salts as a pore template.

Ultrasonic spray pyrolysis has been used as a facile aerosol route to macroporous SiO(2) particles using simple inorganic salts as a pore template and colloidal SiO(2) as building blocks to the larger porous particles. As we found, the use of template salts with low melting points was vital to macroporous particle synthesis, as a dynamic droplet phase can be maintained throughout product formation. Otherwise, relatively compact particles are produced. Significantly, this approach avoids the need for post-isolation elimination of the template, instead permitting aqueous removal during product collection. Produced particles were characterized by SEM, TEM, and surface area and pore size measurements, while the influence of the selected salts on the reactivity between the building block SiO(2) colloids is discussed.

Design of ruthenium/iron oxide nanoparticle mixtures for hydrogenation of nitrobenzene

Here we report novel catalysts for nitrobenzene hydrogenation based on Ru/RuO2 nanoparticles (NPs) and including iron oxide NPs, allowing magnetic recovery. The solvent type, reaction temperature, and the size and composition of initial iron oxide NPs are demonstrated to be the control factors determining synthesis outcomes including the degree of NP aggregation and catalytic properties. A complete characterization of the catalysts using transmission electron microscopy (TEM), X-ray powder diffraction (XRD), x-ray photoelectron spectroscopy (XPS), and energy dispersive x-ray spectroscopy (EDS) allowed assessment of the structure–property relationships. It is revealed that coexistence of the Ru/RuO2 and iron oxide NPs in the catalyst as well as the proximity of two different NP types lead to significantly higher aniline yields and reaction rates. The catalytic properties are also influenced by the type of iron oxide NPs present in the catalytic samples.

Fabrication of magnetically recoverable catalysts based on mixtures of Pd and iron oxide nanoparticles for hydrogenation of alkyne alcohols.

We report a novel method for development of magnetically recoverable catalysts prepared by thermal decomposition of palladium acetylacetonate in the presence of iron oxide nanoparticles (NPs). Depending on conditions, the reaction results either in a dispersed mixture of Pd and iron oxide NPs or in their aggregates. It was demonstrated that the Pd loading, reaction temperature, solvent, and iron oxide NP size and composition are crucial to control the reaction product including the degree of aggregation of Pd and iron oxide NPs, and the catalyst properties. The aggregation controlled by polarization and magnetic forces allows faster magnetic separation, yet the aggregate sizes do not exceed a few hundred nanometers, making them suitable for various catalytic applications. These NP mixtures were studied in a selective hydrogenation of 2-methyl-3-butyn-2-ol to 2-methyl-3-buten-2-ol, demonstrating clear differences in catalytic behavior depending on the catalyst structure. In addition, one of the catalysts was also tested in hydrogenation of 3-methyl-1-pentyn-3-ol and 3-methyl-1-nonyn-3-ol, indicating some specificity of the catalyst toward different alkyne alcohols.

Magnetically Recoverable Catalysts Based on Polyphenylenepyridyl Dendrons and Dendrimers

Here, a systematic study of magnetite nanoparticle (NP) formation in the presence of functional polyphenylenepyridyl dendrons and dendrimers of different generations and structures (such as focal groups, periphery and a combination of phenylene and pyridyl moieties) has been reported. For certain dendron/dendrimer concentrations and structures, well-dispersible, multi-core, flower-like crystals are formed which display ferrimagnetic-like behavior. It is noteworthy that the least complex second generation polyphenylenepyridyl dendrons with a carboxyl focal group already allow formation of flower-like crystals. Magnetically recoverable catalysts were obtained via Pd NP formation in the dendron/dendrimer shells of magnetite NP and tested in selective hydrogenation of dimethylethynylcarbinol to dimethylvinylcarbinol. Dependences of catalytic activity and selectivity on the dendron/dendrimer generation and structure, type of Pd species, and Pd NP size have been demonstrated. High selectivity and activity of these catalysts along with easy catalyst recovery and successful repeated use make them promising in catalytic hydrogenation.

Ru-Containing Magnetically Recoverable Catalysts: A Sustainable Pathway from Cellulose to Ethylene and Propylene Glycols.

Biomass processing to value-added chemicals and biofuels received considerable attention due to the renewable nature of the precursors. Here, we report the development of Ru-containing magnetically recoverable catalysts for cellulose hydrogenolysis to low alcohols, ethylene glycol (EG) and propylene glycol (PG). The catalysts are synthesized by incorporation of magnetite nanoparticles (NPs) in mesoporous silica pores followed by formation of 2 nm Ru NPs. The latter are obtained by thermal decomposition of ruthenium acetylacetonate in the pores. The catalysts showed excellent activities and selectivities at 100% cellulose conversion, exceeding those for the commercial Ru/C. High selectivities as well as activities are attributed to the influence of Fe3O4 on the Ru(0)/Ru(4+) NPs. A facile synthetic protocol, easy magnetic separation, and stability of the catalyst performance after magnetic recovery make these catalysts promising for industrial applications.

Pseudoinfectious Venezuelan Equine Encephalitis Virus: a New Means of Alphavirus Attenuation

ABSTRACT Venezuelan equine encephalitis virus (VEEV) is a reemerging virus that causes a severe and often fatal disease in equids and humans. In spite of a continuous public health threat, to date, no vaccines or antiviral drugs have been developed for human use. Experimental vaccines demonstrate either poor efficiency or severe adverse effects. In this study, we developed a new strategy of alphavirus modification aimed at making these viruses capable of replication and efficient induction of the immune response without causing a progressive infection, which might lead to disease development. To achieve this, we developed a pseudoinfectious virus (PIV) version of VEEV. VEE PIV mimics natural viral infection in that it efficiently replicates its genome, expresses all of the viral structural proteins, and releases viral particles at levels similar to those found in wild-type VEEV-infected cells. However, the mutations introduced into the capsid protein make this protein almost incapable of packaging the PIV genome, and most of the released virions lack genetic material and do not produce a spreading infection. Thus, VEE PIV mimics viral infection in terms of antigen production but is safer due to its inability to incorporate the viral genome into released virions. These genome-free virions are referred to as virus-like particles (VLPs). Importantly, the capsid-specific mutations introduced make the PIV a very strong inducer of the innate immune response and add self-adjuvant characteristics to the designed virus. This unique strategy of virus modification can be applied for vaccine development against other alphaviruses.