Focused Ion Beam-Scanning Electron Microscopy for Investigating Plasmodesmal Densities.

Plasmodesmata (PD) facilitate the exchange of nutrients and signaling molecules between neighboring plant cells, and they are therefore essential for proper growth and development. PD have been studied extensively in efforts to elucidate the ultrastructure of individual PD nanopores and the distribution of PD in a variety of cell walls. These studies often involved the use of serial ultrathin sections and manual quantification of PD by transmission electron microscopy (TEM). In recent years, a variety of techniques that offer more amenable approaches for quantifying PD distribution have been reported. Here, we describe the quantification of PD densities using the serial scanning electron microscopy technique called focused ion beam-scanning electron microscopy (FIB-SEM). For this, resin-embedded samples prepared by standard TEM methods undergo successive rounds of imaging by SEM interspersed with milling of the sample surface by a focused beam of gallium ions to reveal a new surface. In this way, the details of the sample are sequentially revealed and imaged. Over the course of a few hours, repetitive milling and imaging facilitates the automated collection of nanometer-resolution data of several µm of sample depth. FIB-SEM can be targeted to interrogate specific cell walls and cell wall junctions, and the subsequent three-dimensional renderings of the data can be used to visualize the ultrastructural details of the sample. PD densities can then be rapidly quantified by calculating the number of PD per µm2 of cell wall observed in the renderings.

Pulmonary silicosis in 2 rock hyraxes, and literature review.

Two rock hyraxes (Procavia capensis), from the Chattanooga Zoo, were submitted separately for autopsy at the University of Tennessee Veterinary Medical Center. The first was a 4-y-old intact female that died without premonitory signs and the second was a 10-y-old intact male that was euthanized because of severe renal disease. Microscopically, the lungs of both hyraxes had multifocal-to-coalescing, <1-mm diameter aggregates of epithelioid macrophages separated by streams of fibrous tissue. Macrophages contained intracytoplasmic, clear, acicular, birefringent crystals. Transmission electron microscopy and energy-dispersive x-ray spectroscopy findings on the lung samples were consistent with silica crystal deposition. The hyraxes had been housed together on commercially sourced play sand composed of 99-99.5% quartz, a crystalline silica polymorph. The microscopic findings, transmission electron microscopy, and energy-dispersive x-ray spectroscopy of the intrahistiocytic crystals, in addition to the history of exposure to crystalline silica, were consistent with pulmonary silicosis. Pulmonary silicosis has not been reported previously in rock hyraxes, to our knowledge.

Low-Cost Transformation of Biomass-Derived Carbon to High-Performing Nano-graphite via Low-Temperature Electrochemical Graphitization.

Graphite, an essential component of energy storage devices, is traditionally synthesized via an energy-intensive thermal process (Acheson process) at ∼3300 K. However, the battery performance of such graphite is abysmal under fast-charging conditions, which is deemed essential for the propulsion of electric vehicles to the next level. Herein, a low-temperature electrochemical transformation approach has been demonstrated to afford a highly crystalline nano-graphite with the capability of tuning interlayer spacing to enhance the lithium diffusion kinetics in molten salts at 850 °C. The essence of our strategy lies in the effective electrocatalytic transformation of carbon to graphite at a lower temperature that could significantly increase the energy savings, reduce the cost, shorten the synthesis time, and replace the traditional graphite synthesis. The resulting graphite exhibits high purity, crystallinity, a high degree of graphitization, and a nanoflake architecture that all ensure fast lithium diffusion kinetics (∼2.0 × 10-8 cm2 s-1) through its nanosheet. Such unique features enable outstanding electrochemical performance (∼200 mA h g-1 at 5C for 1000 cycles, 1C = 372 mA g-1) as a fast-charging anode for lithium-ion batteries. This finding paves the way to make high energy-density fast-charging batteries that could boost electromobility.

Ionic Liquid-Directed Nanoporous TiNb2 O7 Anodes with Superior Performance for Fast-Rechargeable Lithium-Ion Batteries.

Nanoporous TiNb2 O7 (NPTNO) material is synthesized by a sol-gel method with an ionic liquid (IL) as the nanoporous structure directing template. NPTNO exhibits a high reversible capacity of 210 mAh g-1 even at the charging rate of 50 C and an excellent cyclability of half-cell capacity retention of 74% for 1000 cycles at 5 C and LiNi0.5 Mn1.5 O4 -coupled full-cell capacity retentions of 81% and 87% for 1000 cycles at 1 C and 2 C, respectively. The studies of the 1000 cycled NPTNO electrode illustrate that the IL-directed mesoporous structure can enhance the cyclability of NPTNO cells due to the alleviation of repetitive mechanical stress and volume fluctuation induced by the repetitive Li+ insertion-extraction processes. The measured Li+ diffusion coefficients from the galvanostatic intermittent titration technique suggest that the IL-templating strategy indeed ensures the fast rechargeability of NPTNO cells based on the fast Li+ diffusion kinetics. Benefitting from the nanoporous structure, NPTNO with unhindered Li+ diffusion pathways achieves a superior rate capability in the titanium-based oxide materials and the best full-cell cyclability in the TNO materials. Therefore, the templating potential of IL is demonstrated, and the superb electrochemical performance establishes the IL-directed NPTNO as a promising anode candidate for fast-rechargeable LIBs.

Comparison of Staphylococcus aureus surface protein extraction methods and immunogenicity.

Staphylococcus aureus is the major contagious bovine mastitis pathogen and has no effective vaccine. Strain variation and limited knowledge of common immunogenic antigen/s are among major constraints for developing effective vaccines. S. aureus cell surface proteins that are exposed to the host immune system constitute good vaccine candidates. The objective of this study was to compare two novel S. aureus surface protein extraction methods with biotinylation method and evaluate immune-reactivity of extracted proteins. Surface proteins were extracted from nine genetically distinct S. aureus strains from cases of bovine mastitis. After extraction, bacterial cell integrity was examined by Gram staining and electron microscopy to determine if extraction methods caused damage to cells that may release non-surface proteins. The extracted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and evaluated for immune-reactivity using western blot. Results showed that all three extraction methods provided multiple protein bands on SDS-PAGE. Western blot result showed several immunoreactive surface proteins, in which some proteins strongly (well-resolved, thick, dark, and intense band) reacted across the nine strains tested. The three methods are valid for the extraction of surface proteins and hexadecane, and cholic acid methods are more feasible than biotinylation since both are easier, cheaper, and have minor effects on the bacterial cell. Strongly immune-reactive surface proteins may serve as potential candidates for a vaccine to control S. aureus mastitis in dairy cows.

Characterization of Fungal Colonization of Indwelling Esophagostomy Tubes.

Fungal colonization of feeding tubes occurs rapidly in people, resulting in decreased structural integrity and complications such as luminal obstruction and tube failure. Esophagostomy tubes (E-tubes) are commonly used in dogs and cats for enteral support, but data are lacking regarding colonizing fungi and the impact of colonization on tube integrity. In this study, esophagostomy tubes were collected in lieu of disposal from dogs and cats undergoing feeding tube exchange. Fungi were isolated with culture and identified using morphological characteristics. Scanning electron microscopy was used to evaluate the surface characteristics of the tubes. Two silicone and one polyurethane E-tube were evaluated. Fungi associated with the normal microbiota, including Candida sp. and Penicillium sp., as well as environmental fungi were identified. This case series represents the first documentation of fungal colonization of silicone and polyurethane E-tubes in dogs and cats. Additionally, this is the first report to document degenerative changes in a silicone E-tube.

Ultrastructure and Enzymatic Hydrolysis of Deuterated Switchgrass.

Neutron scattering of deuterated plants can provide fundamental insight into the structure of lignocellulosics in plant cell walls and its deconstruction by pretreatment and enzymes. Such plants need to be characterized for any alterations to lignocellulosic structure caused by growth in deuterated media. Here we show that glucose yields from enzymatic hydrolysis at lower enzyme loading were 35% and 30% for untreated deuterated and protiated switchgrass, respectively. Lignin content was 4% higher in deuterated switchgrass but there were no significant lignin structural differences. Transmission electron microscopy showed differences in lignin distribution and packing of fibers in the cell walls that apparently increased surface area of cellulose in deuterated switchgrass, increasing cellulose accessibility and lowering its recalcitrance. These differences in lignification were likely caused by abiotic stress due to growth in deuterated media.

Spatial distribution of organelles in leaf cells and soybean root nodules revealed by focused ion beam-scanning electron microscopy.

Analysis of cellular ultrastructure has been dominated by transmission electron microscopy (TEM), so images collected by this technique have shaped our current understanding of cellular structure. More recently, three-dimensional (3D) analysis of organelle structures has typically been conducted using TEM tomography. However, TEM tomography application is limited by sample thickness. Focused ion beam-scanning electron microscopy (FIB-SEM) uses a dual beam system to perform serial sectioning and imaging of a sample. Thus FIB-SEM is an excellent alternative to TEM tomography and serial section TEM tomography. Animal tissue samples have been more intensively investigated by this technique than plant tissues. Here, we show that FIB-SEM can be used to study the 3D ultrastructure of plant tissues in samples previously prepared for TEM via commonly used fixation and embedding protocols. Reconstruction of FIB-SEM sections revealed ultra-structural details of the plant tissues examined. We observed that organelles packed tightly together in Nicotiana benthamiana Domin leaf cells may form membrane contacts. 3D models of soybean nodule cells suggest that the bacteroids in infected cells are contained within one large membrane-bound structure and not the many individual symbiosomes that TEM thin-sections suggest. We consider the implications of these organelle arrangements for intercellular signalling.

The protein and neutral lipid composition of lipid droplets isolated from the fission yeast, Schizosaccharomyces pombe.

Lipid droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer with bound proteins. Much of the information on lipid droplet function comes from proteomic and lipodomic studies that identify the components of droplets isolated from organisms throughout the phylogenetic tree. Here, we add to that important inventory by reporting lipid droplet factors from the fission yeast, Schizosaccharomyces pombe. Unique to this study was the fact that cells were cultured in three different environments: 1) late log growth phase in glucose-based media, 2) stationary phase in glucosebased media, and 3) late log growth phase in media containing oleic acid. We confirmed colocalization of major factors with lipid droplets using live-cell fluorescent microscopy. We also analyzed droplets from each of the three conditions for sterol ester (SE) and triacylglycerol (TAG) content, along with their respective fatty acid compositions. We identified a previously undiscovered lipid droplet protein, Vip1p, which affects droplet size distribution. The results provide further insight into the workings of these ubiquitous organelles.

Tandem high-pressure freezing and quick freeze substitution of plant tissues for transmission electron microscopy.

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Translational control of Arabidopsis meristem stability and organogenesis by the eukaryotic translation factor eIF3h.

Essentially all aboveground plant tissues develop from the stem cells in the primary shoot apical meristem. Proliferation of the stem cell population in the Arabidopsis shoot apical meristem is tightly controlled by a feedback loop formed primarily by the homeodomain transcription factor WUSCHEL (WUS) and the CLAVATA ligand-receptor system. In this study, it is shown that mutation of a translation initiation factor, eIF3h, causes a tendency to develop a strikingly enlarged shoot apical meristem with elevated and ectopic expression of WUS and CLAVATA3 (CLV3). Many of the mRNAs that function in apical meristem maintenance possess upstream open reading frames (uORFs), translational attenuators that render translation partially dependent on eIF3h. Specifically, the mRNA for the receptor kinase, CLV1, is undertranslated in the eif3h mutant as shown by transient and transgenic expression assays. Concordant phenotypic observations include defects in organ polarity and in translation of another uORF-containing mRNA, ASYMMETRIC LEAVES 1 (AS1), in eif3h. In summary, the expression of developmental regulatory mRNAs is attenuated by uORFs, and this attenuation is balanced in part by the translation initiation factor, eIF3h. Thus, translational control plays a key role in Arabidopsis stem cell regulation and organogenesis.

Synthesis of millimeter-size hexagon-shaped graphene single crystals on resolidified copper.

We present a facile method to grow millimeter-size, hexagon-shaped, monolayer, single-crystal graphene domains on commercial metal foils. After a brief in situ treatment, namely, melting and subsequent resolidification of copper at atmospheric pressure, a smooth surface is obtained, resulting in the low nucleation density necessary for the growth of large-size single-crystal graphene domains. Comparison with other pretreatment methods reveals the importance of copper surface morphology and the critical role of the melting-resolidification pretreatment. The effect of important growth process parameters is also studied to determine their roles in achieving low nucleation density. Insight into the growth mechanism has thus been gained. Raman spectroscopy and selected area electron diffraction confirm that the synthesized millimeter-size graphene domains are high-quality monolayer single crystals with zigzag edge terminations.

Very bright orange fluorescent plants: endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants.

BACKGROUND: The expression of fluorescent protein (FP) genes as real-time visual markers, both transiently and stably, has revolutionized plant biotechnology. A palette of colors of FPs is now available for use, but the diversity has generally been underutilized in plant biotechnology. Because of the green and far-red autofluorescent properties of many plant tissues and the FPs themselves, red and orange FPs (RFPs, and OFPs, respectfully) appear to be the colors with maximum utility in plant biotechnology. Within the color palette OFPs have emerged as the brightest FP markers in the visible spectra. This study compares several native, near-native and modified OFPs for their "brightness" and fluorescence, therefore, their usability as marker genes in transgenic plant tissues. RESULTS: The OFPs DsRed2, tdTomato, mOrange and pporRFP were all expressed under the control of the CaMV 35S promoter in agroinfiltration-mediated transient assays in Nicotiana benthamiana. Each of these, as well as endoplasmic reticulum (ER)-targeted versions, were stably expressed in transgenic Nicotiana tabacum and Arabidopsis thaliana. Congruent results were observed between transient and stable assays. Our results demonstrated that there are several adequate OFP genes available for plant transformation, including the new pporRFP, an unaltered tetramer from the hard coral Porites porites. When the tandem dimer tdTomato and the monomeric mOrange were targeted to the ER, dramatic, ca. 3-fold, increase in plant fluorescence was observed. CONCLUSIONS: From our empirical data, and a search of the literature, it appears that tdTomato-ER and mOrange-ER are the two highest fluorescing FPs available as reporters for transgenic plants. The pporRFP is a brightly fluorescing tetramer, but all tetramer FPs are far less bright than the ER-targeted monomers we report here.

Lipid droplet de novo formation and fission are linked to the cell cycle in fission yeast.

Cells sequester neutral lipids in bodies called lipid droplets. Thus, the formation and breakdown of the droplets are important for cellular metabolism; unfortunately, these processes are difficult to quantify. Here, we used time-lapse confocal microscopy to track the formation, movement and size changes of lipid droplets throughout the cell cycle in fission yeast Schizosaccharomyces pombe. In theory, the number of lipid droplets in these cells must increase for daughter cells to have the same number of droplets as the parent at a reference point in the cell cycle. We observed stable droplet formation events in G2 phase that were divided evenly between de novo formation of nascent droplets and fission of preexisting droplets. The observations that lipid droplet number is linked to the cell cycle and that droplets can form via fission were both new discoveries. Thus, we scrutinized each fission event for multiple signatures to eliminate possible artifacts from our microscopy. We augmented our time-lapse confocal microscopy with electron microscopy, which showed lipid droplet 'intermediates': droplets shaped like dumbbells that are potentially in transition states between two spherical droplets. Using these complementary microscopy techniques and also dynamic simulations, we show that lipid droplets can form by fission.

Marivita roseacus sp. nov., of the family Rhodobacteraceae, isolated from a temperate estuary and an emended description of the genus Marivita.

A gram-negative, non-motile, pigmented, rod-shaped and strictly aerobic bacterium (CB1052(T)) was isolated from a temperate estuary. On the basis of 16S rRNA gene sequence similarity, strain CB1052(T) belongs to the α-3 subclass of the Proteobacteria, within the family Rhodobacteraceae, having the highest similarity to members of the genus Marivita (97.8%) of the Roseobacter lineage. Pylogenetic analysis showed CB1052(T) to be a distinct sister clade to M. litorea and M. cryptomonadis and DNA-DNA relatedness was quite low amongst the strains (< 35%). Strain CB1052(T) cells are non-motile and display a needle-like filamentous form, where individual cells can become quite elongated (up to 15 µm). Similar to M. litorea and M. cryptomonadis, CB1052(T) harbors aerobic anoxygenic photosynthesis genes. However, in contrast to other described Marivita species, strain CB1052(T) actively produces bacteriochlorophyll a. Further physiological features, including antibiotic sensitivities, differentiate strain CB1052(T) from the other members of the genus. Therefore, strain CB1052(T) is considered to represent a novel species of the genus Marivita, for which the name Marivita roseacus sp. nov. is proposed, with the type strain CB1052(T) (=DSM 23118(T) =ATCC BAA 1914(T)).

May-Hegglin anomaly in a dog.

An 8-year-old female spayed Pug dog was presented for evaluation of cutaneous lesions occurring secondary to immunosuppressive treatment of presumed immune-mediated thrombocytopenia. Abnormal hematologic findings included persistent thrombocytopenia, macrothrombocytes, and variably shaped, often fusiform, blue cytoplasmic inclusions in neutrophils. May-Hegglin anomaly (MHA) was suspected based on the morphologic appearance of platelets and neutrophils. Examination of cells by transmission electron microscopy revealed normal platelet ultrastructure; neutrophil inclusions had features similar to those reported for inclusions in human MHA. Neutrophil function was within normal limits based on flow cytometric analysis. Thrombelastography indicated a prolonged clotting time (r), and PlateletMapping showed a lack of response to 2 µM ADP compared with a moderate response in the control dog. Immunocytochemical staining of blood smears using 2 commercially available antibodies against MYH9 protein (nonmuscle myosin heavy chain II) yielded negative results. However, genomic DNA sequencing analysis of the dog's MYH9 gene identified a single point mutation, resulting in substitution of lysine for glutamine at the 1841 amino acid position; this mutation is identical to one identified in people with MHA. To our knowledge, this is the first report of an MYH9 mutation in the dog. MHA-associated macrothrombocytopenia may be mistaken for immune-mediated thrombocytopenia.

Influenza virus variation in susceptibility to inactivation by pomegranate polyphenols is determined by envelope glycoproteins.

Pomegranates have high levels of polyphenols (PPs) and may be a rich source of compounds with antiviral activity. We evaluated the direct anti-influenza activity of three commercially available pomegranate extracts: pomegranate juice (PJ), a concentrated liquid extract (POMxl), and a 93% PP powder extract (POMxp). The acidity of PJ and POMxl solutions contributed to rapid anti-influenza activity, but this was not a factor with POMxp. Studies using POMxp showed that 5min treatment at room temperature with 800µg/ml PPs resulted in at least a 3log reduction in the titers of influenza viruses PR8 (H1N1), X31 (H3N2), and a reassortant H5N1 virus derived from a human isolate. However, the antiviral activity was less against a coronavirus and reassortant H5N1 influenza viruses derived from avian isolates. The loss of influenza infectivity was frequently accompanied by loss of hemagglutinating activity. PP treatment decreased Ab binding to viral surface molecules, suggesting some coating of particles, but this did not always correlate with loss of infectivity. Electron microscopic analysis indicated that viral inactivation by PPs was primarily a consequence of virion structural damage. Our findings demonstrate that the direct anti-influenza activity of pomegranate PPs is substantially modulated by small changes in envelope glycoproteins.

Binding of Host Factors Influences Internalization and Intracellular Trafficking of Streptococcus uberis in Bovine Mammary Epithelial Cells.

We showed that internalization of Streptococcus uberis into bovine mammary epithelial cells occurred through receptor- (RME) and caveolae-mediated endocytosis (CME). We reported also that treatment of S. uberis with host proteins including lactoferrin (LF) enhanced its internalization into host cells. Since the underlying mechanism(s) involved in such enhancement was unknown we investigated if preincubation of S. uberis with host proteins drives internalization of this pathogen into host cells through CME. Thus, experiments involving coculture of collagen-, fibronectin-, and LF-pretreated S. uberis with bovine mammary epithelial cells treated with RME and CME inhibitors were conducted. Results showed that internalization of host proteins-pretreated S. uberis into mammary epithelial cells treated with RME inhibitors was higher than that of untreated controls. These results suggest that pretreatment with selected host proteins commits S. uberis to CME, thus avoiding intracellular bactericidal mechanisms and allowing its persistence into bovine mammary epithelial cells.

Phosphatidylserine synthase and phosphatidylserine decarboxylase are essential for cell wall integrity and virulence in Candida albicans.

Phospholipid biosynthetic pathways play crucial roles in the virulence of several pathogens; however, little is known about how phospholipid synthesis affects pathogenesis in fungi such as Candida albicans. A C. albicans phosphatidylserine (PS) synthase mutant, cho1 Delta/Delta, lacks PS, has decreased phosphatidylethanolamine (PE), and is avirulent in a mouse model of systemic candidiasis. The cho1 Delta/Delta mutant exhibits defects in cell wall integrity, mitochondrial function, filamentous growth, and is auxotrophic for ethanolamine. PS is a precursor for de novo PE biosynthesis. A psd1 Delta/Delta psd2 Delta/Delta double mutant, which lacks the PS decarboxylase enzymes that convert PS to PE in the de novo pathway, has diminished PE levels like those of the cho1 Delta/Delta mutant. The psd1 Delta/Delta psd2 Delta/Delta mutant exhibits phenotypes similar to those of the cho1 Delta/Delta mutant; however, it is slightly more virulent and has less of a cell wall defect. The virulence losses exhibited by the cho1 Delta/Delta and psd1 Delta/Delta psd2 Delta/Delta mutants appear to be related to their cell wall defects which are due to loss of de novo PE biosynthesis, but are exacerbated by loss of PS itself. Cho1p is conserved in fungi, but not mammals, so fungal PS synthase is a potential novel antifungal drug target.