John R. Dunlap

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.

The dual specificity phosphatase Cdc14B bundles and stabilizes microtubules

ABSTRACT The Cdc14 dual-specificity phosphatases regulate key events in the eukaryotic cell cycle. However, little is known about the function of mammalian CDC14B family members. Here, we demonstrate that subcellular localization of CDC14B protein is cell cycle regulated. CDC14B can bind, bundle, and stabilize microtubules in vitro independently of its catalytic activity. Basic amino acid residues within the nucleolar targeting domain are important for both retaining CDC14B in the nucleolus and preventing microtubule bundling. Overexpression of CDC14B resulted in the formation of cytoplasmic CDC14B and microtubule bundles in interphase cells. These microtubule bundles were resistant to microtubule depolymerization reagents and enriched in acetylated α-tubulin. Expression of cytoplasmic forms of CDC14B impaired microtubule nucleation from the microtubule organization center. CDC14B is thus a novel microtubule-bundling and -stabilizing protein, whose regulated subcellular localization may help modulate spindle and microtubule dynamics in mitosis.

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.

Cdc14B depletion leads to centriole amplification, and its overexpression prevents unscheduled centriole duplication

Centrosome duplication is tightly controlled in coordination with DNA replication. The molecular mechanism of centrosome duplication remains unclear. Previous studies found that a fraction of human proline-directed phosphatase Cdc14B associates with centrosomes. However, Cdc14Bs involvement in centrosome cycle control has never been explored. Here, we show that depletion of Cdc14B by RNA interference leads to centriole amplification in both HeLa and normal human fibroblast BJ and MRC-5 cells. Induction of Cdc14B expression through a regulatable promoter significantly attenuates centriole amplification in prolonged S phase–arrested cells and proteasome inhibitor Z-L3VS–treated cells. This inhibitory function requires centriole-associated Cdc14B catalytic activity. Together, these results suggest a potential function for Cdc14B phosphatase in maintaining the fidelity of centrosome duplication cycle.

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

Abstract 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.

Bovine lactoferrin serves as a molecular bridge for internalization of Streptococcus uberis into bovine mammary epithelial cells.

Streptococcus uberis, an environmental mastitis pathogen, is an important causative agent of mastitis in dairy cattle throughout the world. Research from our laboratory demonstrated that bovine lactoferrin (LF), a whey protein present in milk and nonlactating cow mammary secretions, significantly enhanced adherence of S. uberis to mammary epithelial cells in culture. Subsequent research from our laboratory identified S. uberis adhesion molecule (SUAM) showing an affinity for LF. The objective of the present investigation was to test the hypothesis that the interaction between SUAM, bovine LF, and a putative LF receptor on the bovine mammary epithelial cell surface could serve as a bridging molecule for internalization of S. uberis into mammary epithelial cells. When internalization assays were conducted using cell growth medium containing bovine LF, a significant increase in internalization of S. uberis into mammary epithelial cells was observed. However, this effect was reversed when assays were conducted in the presence of antibodies to bovine LF suggesting that internalization of S. uberis into mammary epithelial cells, at least in part, was mediated by LF ligands. When S. uberis was pretreated with antibodies to SUAM, internalization in the presence of LF was reduced in the same manner as observed with antibodies to LF. Transmission and scanning electron microscopy results demonstrated that streptavidin-coated gold particles specifically localized on biotinylated LF receptors on S. uberis and mammary epithelial cell surfaces supporting the availability of LF receptors. Collectively, these results suggest that LF serves as a bridging molecule between SUAM located on the surface of S. uberis and LF receptors located on the surface of mammary epithelial cells thus enhancing internalization of S. uberis into host cells. Exploitation of LF as a molecular bridge for internalization of S. uberis into mammary epithelial cells may confer a significant advantage allowing mammary gland infection.

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

BackgroundThe 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.ResultsThe 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.ConclusionsFrom 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.

Cytological and taxonomic studies of the Euglenales. II: Comparative microarchitecture and cytochemistry of envelopes of Strombomonas and trachelomonas

Envelopes of Strombomonas conspersa, like those of Trachelomonas, may range in colour from hyaline to dark brown. Cytochemical staining with Alcian Blue reveals differences between light and dark envelopes of both genera: acidic and sulphated glycosaminoglycans (mucopolysaccharides) are present in all the organic matrices; however, in dark envelopes the sulphate groups are masked by the Mn and are detectable only when the metal is removed. The TEM and EM-EDS comparative studies of the microarchitecture and elemental composition of envelopes of Strombomonas conspersa and several species of Trachelomonas show similar results: hyaline envelopes are characterized by unmineralized strands that anastomose to form a complex reticulum, whereas dark envelopes, heavily mineralized with Mn, are composed of dense needle-like components enmeshed in a mucilaginous matrix. In the light of these findings, we propose that Strombomonas conspersa be removed to its original taxonomic position in the genus Trachelomonas, as T...

Microarchitecture and elemental spatial segregation of envelopes ofTrachelomonas lefevrei (Euglenophyceae)

SummaryEnvelopes of the euglenoid flagellate,Trachelomonas lefevrei may range in color from hyaline to golden to dark brown and may be variously ornamented with spines, papillae and puncta. Elemental composition affects both envelope color and microarchitecture, which may be granular and/or microcrystalline. TEM-EDS microanalyses of granular envelopes or regions show Fe as the predominant element, whereas Mn is the predominant element in envelopes or regions with high concentrations of needle-like microcrystallites. In envelopes composed of both structural components, there is a spatial segregation of Fe and Mn that corresponds to the distribution of the granular and/or microcrystalline entities. Extraction of the needle-like components with leucoberbelin blue indicates that they are Mn oxides. Quantitative elemental analyses show shifting ratios of Mn: Fe that correlate with envelope color and microarchitecture. This is the first direct demonstration both of such a spatial segregation of elements and of the presence of Mn oxides in envelopes ofTrachelomonas.

Cytological and taxonomic studies of the Euglenales. I. Ultrastructure and envelope elemental composition in Trachelomonas

The ultrastructural cytology, envelope microarchitecture and elemental composition are described for species from three of Pringsheims subgeneric categories of the euglenoid flagellate, Trachelomonas. Except for differences in chloroplast number and pyrenoid morphology, cellular organization in T. bulla, T. zorensis and T. lefevrei is similar; however, based on our finding that T. lefevrei has inner and outer paramylon caps over the pyrenoid, we propose that this species be moved from its present position in the subgroup with inner pyrenoids, to the subgroup with double-sheathed pyrenoids. Envelope microarchitecture, elemental composition and colour are correlated in each of the species. Elemental analyses indicate that envelopes or regions of envelopes with high iron content have a granular substructure, whereas those rich in manganese have a needle-like microcrystalline substructure. Envelopes of T. bulla never appear dark in the light microscope (L.M.) and have a granular microarchitecture with iron a...