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The enumeration of absolute cell numbers and cell proliferation in clinical samples is important for diagnostic and research purposes. Detection of cellular DNA with fluorescent dyes is the most commonly used approach for cell enumeration in cytometry. Inductively coupled plasma mass spectrometry (ICPMS) has been recently introduced to the field of protein and cell surface antigen identification via ICPMS-linked immunoassays using element-labeled affinity reagents such as gold and lanthanide-conjugated antibodies. In the present work, we describe novel methods for using metallointercalators that irreversibly bind DNA and low concentrations of rare earth metals added to cell growth media for rapid and sensitive measurement of cell numbers by mass spectrometry. We show that Ir- and Rh-containing metallointercalators are useful reagents for labeling cells and normalizing signals when studying antigen expression on different types and numbers of cells. Results are presented for solution analysis performed by conventional ICPMS and compared to measurements obtained on the novel flow cytometer mass spectrometer (FC-MS) instrument, designed to analyze multiple antigens and DNA simultaneously in single cells.

The oral pathogen Aggregatibacter actinomycetemcomitans uses pga gene locus for the production of an exopolysaccharide made up of a linear homopolymer of β-1,6-N-acetyl-d-glucosamine (PGA). An enzyme encoded by the pgaB of the pga operon in A. actinomycetemcomitans is a de-N-acetylase, which is used to alter the PGA. The full length enzyme (AaPgaB) and the N-terminal catalytic domain (residues 25-290, AaPgaBN) from A. actinomycetemcomitans were cloned, expressed and purified. The enzymatic activities of the AaPgaB enzymes were determined using 7-acetoxycoumarin-3-carboxylic acid as the substrate. The AaPgaB enzymes displayed significantly lower de-N-acetylase activity compared with the activity of the deacetylase PdaA from Bacillus subtilis, a member of the CE4 family of enzymes. To delineate the differences in the activity and the active site architecture, the structure of AaPgaBN was determined. The AaPgaBN structure has two metal ions in the active site instead of one found in other CE4 enzymes. Based on the crystal structure comparisons among the various CE4 enzymes, two residues, Q51 and R271, were identified in AaPgaB, which could potentially affect the enzyme activity. Of the two mutants generated, Q51E and R271K, the variant Q51E showed enhanced activity compared with AaPgaB, validating the requirement that an activating aspartate residue in the active site is essential for higher activity. In summary, our study provides the first structural evidence for a di-nuclear metal site at the active site of a member of the CE4 family of enzymes, evidence that AaPgaBN is catalytically active and that mutant Q51E exhibits higher de-N-acetylase activity. [ABSTRACT FROM AUTHOR]

The major residue from beer production is the brewer’s spent grain (BSG). At present, BSG is mainly used as feed for cattle. Spent grains have been considered as a potential source of protein and energy for human nutrition. For such use, BSG must be dried to reduce its mass and increase its shelf life. In this study, the drying kinetics of BSG were investigated in a tray dryer and in a pulsed fluidized bed (PFB) dryer. Five different combinations of temperature and air velocity (70°C and 1.0 m/s, 70°C and 1.7 m/s, 80°C and 1.35 m/s, 90°C and 1.0 m/s, 90°C and 1.35 m/s) were investigated for the tray dryer and four for the PFB at 900 rpm (70°C and 0.52 m/s, 70°C and 0.72 m/s, 90°C and 0.52 m/s, 90°C and 0.72 m/s). Six thin-layer drying models were fitted to the experimental data obtained for the tray dryer: Lewis, Page, Modified Page, Midilli et al., Verma et al. and Henderson-Pabis. Both temperature and air flow influenced drying rate. A moisture ratio reduction of 90% was four times faster at 90°C and 1.7 m/s than at 70°C and 1.0 m/s in the tray dryer. The drying models, except Lewis, showed good fit. Drying in the PFB was considerably faster compared to the tray dryer. A moisture reduction of 90% was twice as fast at 90°C and 0.72 m/s (PFB) than at 90°C and 1.70 m/s (tray dryer).

The drying behaviour of monoammonium phosphate fertilizer was examined in a static tray dryer, establishing kinetic parameters that may be used to improve the design of dryers. Drying temperatures ranged from 80 to 95°C, and surface air velocity was 0.50 m/s. The material was previously separated into two particle sizes, between 1.40 and 2.38 mm mean diameter. Drying curves were analysed qualitatively and seven empirical kinetic thin-layer models were fitted. The drying kinetic showed to be temperature-dependent: a moisture ratio reduction of 90% was achieved twice as fast at 95°C than at 80°C. On the other hand, particle size did not exert significant influence on the drying rate. The results showed a decreasing drying rate throughout the whole process. All studied models adjusted well to the experimental data: R2 values were all above 0.999. The two-term model generated the lowest RMSE and χ2 values, between 0.00621 and 0.010743 and between 0.000044 and 0.000115, respectively.

The article presents a study which explores the habitat preferences of Sheppardia gunningi based on analyses of four years of transect surveys from two different habitats within Arabuko-Sokoke Forest (ASF) in Kenya conducted between 2005 and 2010. It notes that such species relied on ground cover and organic matter for nesting materials as well as habitat for its prey items. It also mentions that Sheppardia was more abundant in Cynometra forest and thicket than in mixed forest.

The U.S. Presidential election of 2004 was an exciting reprise of the 2000 election and was closely watched by numerous observers across the world. The election held significant ramifications for world issues such as the war in Iraq and the war on terror. Norwegian media in particular followed the election with great interest. The strong social and familial bond between Norwegians and Americans was a foundation for an interest in the role that social issues such as abortion, gay marriage, and religion played in the campaign. This article was an exploratory case study based on data from three major Norwegian newspapers. The article used framing theory as a tool to examine the way in which these newspapers covered the 2004 U.S. Presidential election. A key focus was the importance and influence of culture in this framing process. Results are presented and implications for the role of framing theory in international contexts are discussed.

Readily synthesized bench-stable glycosyl dithioimidocarbonates are useful C -glycoside precursors. Under mild photochemical conditions, these glycosides undergo desulfurative glycosyl radical generation in the presence of weak acid, 4CzIPN, and Hantzsch ester. These radicals perform well in Geise-like reactions to yield C -glycosides with high stereoselectivity.

Tellurophene-bearing small molecules have emerged as valuable tools for localizing cellular activities in vivo using mass cytometry. To broaden the utility of tellurophenes in chemical biology, we have developed a bioorthogonal reaction to facilitate tagging of tellurophene-bearing conjugates for downstream applications. Using TePhe, a tellurophene-based phenylalanine analogue, labeled recombinant proteins were generated for reaction development. Using these proteins, we demonstrate an oxidation-controlled, strain-promoted tellurophene-alkyne cycloaddition (OSTAC) reaction. Mild oxidation of the tellurophene ring with N -chlorosuccinimide produces a reactive Te(IV) species which undergoes rapid ( k > 100 M -1 s -1 ) cycloaddition with bicyclo[6.1.0]nonyne (BCN) yielding a benzo-fused cyclooctane. Selective labeling of TePhe-containing proteins can be achieved in complex protein mixtures and on fixed cells. OSTAC reactions can be combined with strain-promoted azide alkyne cycloaddition (SPAAC) and copper-catalyzed azide alkyne click (CuAAC) reactions. Demonstrating the versatility of this approach, we observe the expected staining patterns for 5-ethynyl-2'-deoxyuridine (DNA synthesis-CuAAC) and immunohistochemistry targets in combination with TePhe (protein synthesis-OSTAC) in fixed cells. The favorable properties of the OSTAC reaction suggest its broad applicability in chemical biology.

Bacteria use a diverse range of carbohydrates to generate a profusion of glycans, with amino sugars, such as N -acetylglucosamine (GlcNAc), being prevalent in the cell wall and in many exopolysaccharides. The primary substrate for GlcNAc-containing glycans, UDP-GlcNAc, is the product of the bacterial hexosamine pathway and a key target for bacterial metabolic glycan engineering. Using the strategy of expressing NahK, to circumvent the hexosamine pathway, it is possible to directly feed the analogue of GlcNAc, N -azidoacetylglucosamine (GlcNAz), for metabolic labeling in Escherichia coli . The cytosolic production of UDP-GlcNAz was confirmed by using fluorescence-assisted polyacrylamide gel electrophoresis. The key question of where GlcNAz is incorporated was interrogated by analyzing potential sites including peptidoglycan (PGN), the biofilm-related exopolysaccharide poly-β-1,6- N -acetylglucosamine (PNAG), lipopolysaccharide (LPS), and the enterobacterial common antigen (ECA). The highest levels of incorporation were observed in PGN with lower levels in PNAG and no observable incorporation in LPS or ECA. The promiscuity of the PNAG synthase (PgaCD) toward UDP-GlcNAz in vitro and the lack of undecaprenyl-pyrophosphoryl-GlcNAz intermediates generated in vivo confirmed the incorporation preferences. The results of this work will guide the future development of carbohydrate-based probes and metabolic engineering strategies.

Mass cytometry permits the high dimensional analysis of complex biological samples; however, some techniques are not yet integrated into the mass cytometry workflow due to reagent availability. The use of self-labeling protein systems, such as HaloTag, are one such application. Here, we describe the design and implementation of the first mass cytometry ligands for use with HaloTag. "Click"-amenable HaloTag warheads were first conjugated onto poly(l-lysine) or poly(acrylic acid) polymers that were then functionalized with diethylenetriaminepentaacetic acid (DTPA) lutetium metal chelates. Kinetic analysis of the HaloTag labeling rates demonstrated that the structure appended to the 1-chlorohexyl warhead was key to success. A construct with a diethylene glycol spacer appended to a benzamide gave similar rates ( k obs M 2 M -1 s -1 ), regardless of the nature of the polymer. Comparison of the polymer with a small molecule chelate having rapid HaloTag labeling kinetics ( k M obs ∼ 10 4 M -1 s -1 ) suggests the polymers significantly reduced the HaloTag labeling rate. HEK293T cells expressing surface-exposed GFP-HaloTag fusions were labeled with the polymeric constructs and 175 Lu content measured by cytometry by time-of-flight (CyTOF). Robust labeling was observed; however, significant nonspecific binding of the constructs to cells was also present. Heavily pegylated polymers demonstrated that nonspecific binding could be reduced to allow cells bearing the HaloTag protein to be distinguished from nonexpressing cells.

Aldehydes are attractive bioorthogonal coupling partners. The ease of manipulation of aldehydes and their orthogonality to other classes of bioorthogonal reactions have inspired the exploration of chemistries, which generate irreversible conjugates. Similarly, nitrones have been shown to be potent 1,3-dipoles in bioorthogonal reactions when paired with strained alkynes. Here, we combine the reactivity of nitrones with the simplicity of aldehydes using an N -allylglyoxylamide, in a cascade reaction with an N -alkylhydroxylamine to produce a bicyclic isoxazolidine. The reaction is found to be catalyzed by 5-methoxyanthranilic acid and proceeds at pH 7 with favorable kinetics. Using the HaloTag7 protein bearing an N -alkylhydroxylamine, we show the reaction to be bioorthogonal in a complex cell lysate and to proceed well at the surface of a HEK293 cell. Furthermore, the reaction is compatible with a typical strain-promoted alkyne-azide click reaction. The characteristics of this reaction suggest it will be a useful addition to the pallet of bioorthogonal reactions that have revolutionized chemical biology.