@article {276, title = {Exploration of Glycosyl Dithioimidocarbonates in Photoinduced Desulfurative Cross-Coupling Reactions}, journal = {Organic Letters}, volume = {26}, year = {2024}, month = {09/2024}, chapter = {8498}, abstract = {

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.

}, doi = {https://doi.org/10.1021/acs.orglett.4c03035}, url = {https://pubs.acs.org/doi/10.1021/acs.orglett.4c03035}, author = {Ji Zhang and Qi Liu and Angus Chiang and Mark Nitz} } @article {264, title = {Oxidation-Controlled, Strain-Promoted Tellurophene-Alkyne Cycloaddition (OSTAC): A Bioorthogonal Tellurophene-Dependent Conjugation Reaction}, journal = {Journal of the American Chemical Society}, year = {2024}, month = {09/2024}, abstract = {

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{\textendash}1\ s{\textendash}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.

}, doi = {10.1021/jacs.4c07275}, url = {https://pubs.acs.org/doi/10.1021/jacs.4c07275}, author = {Yong Jia Bu and Santiago Tijaro-Bulla and Haissi Cui and Mark Nitz} } @article {270, title = {Characterization of an N-Allylglyoxylamide-Based Bioorthogonal Nitrone Trap}, journal = {Bioconjugate Chemistry}, volume = {34}, year = {2023}, month = {12/2023}, chapter = {2358{\textendash}2365}, abstract = {

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{\textendash}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.

}, doi = {10.1021/acs.bioconjchem.3c00463}, url = {https://pubs.acs.org/doi/abs/10.1021/acs.bioconjchem.3c00463$\#$}, author = {Daniel Lee and Simon Latour and Michael Emblem and Hunter J. Clark and Jobette T. Santos and Jaewan Jang and Alison P. McGuigan and Mark Nitz} } @article {269, title = {Design Parameters for a Mass Cytometry Detectable HaloTag Ligand}, journal = {Bioconjugate Chemistry}, volume = {35}, year = {2023}, month = {12/2023}, chapter = {80{\textendash}91}, abstract = {

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. {\textquotedblleft}Click{\textquotedblright}-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 (kobs\ \~{} 102\ M{\textendash}1\ s{\textendash}1), regardless of the nature of the polymer. Comparison of the polymer with a small molecule chelate having rapid HaloTag labeling kinetics (kobs\ \~{} 104\ M{\textendash}1\ s{\textendash}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\ 175Lu 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.

}, author = {Nicole Potter and Simon Latour and Edmond C. N. Wong and Mitchell A. Winnik and Hartland W. Jackson and Alison P. McGuigan and Mark Nitz} } @article {236, title = {The Metabolic Usage and Glycan Destinations of GlcNAz in E. coli}, journal = {ACS Chem. Biol.}, volume = {19}, year = {2023}, month = {12/2023}, chapter = {69{\textendash}80}, abstract = {

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 labelling in E. coli. The cytosolic production of UDP-GlcNAz was confirmed 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) towards UDP-GlcNAz in vitro and 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. Competing Interest StatementThe authors have declared no competing interest.

}, doi = {10.1101/2023.08.17.553294}, url = {https://www.biorxiv.org/content/early/2023/08/17/2023.08.17.553294}, author = {Alexander Eddenden and Manoj K. Dooda and Zachary A. Morrison and Adithya Shankara Subramanian and P. Lynne Howell and Jerry M. Troutman and Mark Nitz} } @article {268, title = {Reagents for Mass Cytometry}, journal = {Chemical Reviews}, volume = {123}, year = {2023}, month = {01/2023}, chapter = {1166{\textendash}1205}, abstract = {

Mass cytometry (cytometry by time-of-flight detection [CyTOF]) is a bioanalytical technique that enables the identification and quantification of diverse features of cellular systems with single-cell resolution. In suspension mass cytometry, cells are stained with stable heavy-atom isotope-tagged reagents, and then the cells are nebulized into an inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS) instrument. In imaging mass cytometry, a pulsed laser is used to ablate ca. 1 μm2\ spots of a tissue section. The plume is then transferred to the CyTOF, generating an image of biomarker expression. Similar measurements are possible with multiplexed ion bean imaging (MIBI). The unit mass resolution of the ICP-TOF-MS detector allows for multiparametric analysis of (in principle) up to 130 different parameters. Currently available reagents, however, allow simultaneous measurement of up to 50 biomarkers. As new reagents are developed, the scope of information that can be obtained by mass cytometry continues to increase, particularly due to the development of new small molecule reagents which enable monitoring of active biochemistry at the cellular level. This review summarizes the history and current state of mass cytometry reagent development and elaborates on areas where there is a need for new reagents. Additionally, this review provides guidelines on how new reagents should be tested and how the data should be presented to make them most meaningful to the mass cytometry user community.

}, doi = {10.1021/acs.chemrev.2c00350}, url = {https://pubs.acs.org/doi/abs/10.1021/acs.chemrev.2c00350}, author = {Loryn P. Arnett and Rahul Rana and Wilson Wai-Yip Chung and Xiaochong Li and Mahtab Abtahi and Daniel Majonis and Jay Bassan and Mark Nitz and Mitchell A. Winnik} } @article {271, title = {Small Molecule Inhibition of an Exopolysaccharide Modification Enzyme is a Viable Strategy To Block Pseudomonas aeruginosa Pel Biofilm Formation}, journal = {Microbiology Spectrum}, volume = {11}, year = {2023}, month = {04/2023}, abstract = {

Biosynthesis of the Pel exopolysaccharide in\ Pseudomonas aeruginosa\ requires all seven genes of the\ pelABCDEFG\ operon. The periplasmic modification enzyme PelA contains a C-terminal deacetylase domain that is necessary for Pel-dependent biofilm formation. Herein, we show that extracellular Pel is not produced by a\ P. aeruginosa\ PelA deacetylase mutant. This positions PelA deacetylase activity as an attractive target to prevent Pel-dependent biofilm formation. Using a high-throughput screen (n = 69,360), we identified 56 compounds that potentially inhibit PelA esterase activity, the first enzymatic step in the deacetylase reaction. A secondary biofilm inhibition assay identified methyl 2-(2-pyridinylmethylene) hydrazinecarbodithioate (SK-017154-O) as a specific Pel-dependent biofilm inhibitor. Structure-activity relationship studies identified the thiocarbazate as a necessary functional group and that the pyridyl ring could be replaced with a phenyl substituent (compound 1). Both SK-017154-O and compound 1 inhibit Pel-dependent biofilm formation in\ Bacillus cereus\ ATCC 10987, which has a predicted extracellular PelA deacetylase in its\ pel\ operon. Michaelis-Menten kinetics determined SK-017154-O to be a noncompetitive inhibitor of PelA, while compound 1 did not directly inhibit PelA esterase activity. Cytotoxicity assays using human lung fibroblast cells showed that compound 1 is less cytotoxic than SK-017154-O. This work provides proof of concept that biofilm exopolysaccharide modification enzymes are important for biofilm formation and can serve as useful antibiofilm targets.

}, doi = {10.1128/spectrum.00296-23}, url = {https://journals.asm.org/doi/10.1128/spectrum.00296-23}, author = {Erum Razvi and Benjamin R. DiFrancesco and Gregory A. Wasney and Zachary A. Morrison and John Tam and Anick Auger and Perrin Baker and Noor Alnabelseya and Jacquelyn D. Rich and Piyanka Sivarajah and Gregory B. Whitfield and Joe J. Harrison and Roman A. Melnyk and Mark Nitz and P. Lynne Howell} } @article {223, title = {Allosteric modulation of the adenosine A receptor by cholesterol.}, journal = {Elife}, volume = {11}, year = {2022}, month = {2022 Jan 05}, abstract = {

Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A receptor (AR) in reconstituted phospholipid nanodiscs. This model system conveniently excludes possible effects arising from cholesterol-induced phase separation or receptor oligomerization and focuses on the question of allostery. GTP hydrolysis assays show that cholesterol weakly enhances the basal signaling of AR while decreasing the agonist EC. Fluorine nuclear magnetic resonance (F NMR) spectroscopy shows that this enhancement arises from an increase in the receptor{\textquoteright}s active state population and a G-protein-bound precoupled state. F NMR of fluorinated cholesterol analogs reveals transient interactions with AR, indicating a lack of high-affinity binding or direct allosteric modulation. The combined results suggest that the observed allosteric effects are largely indirect and originate from cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.

}, issn = {2050-084X}, doi = {10.7554/eLife.73901}, author = {Huang, Shuya Kate and Almurad, Omar and Pejana, Reizel J and Morrison, Zachary A and Pandey, Aditya and Picard, Louis-Philippe and Nitz, Mark and Sljoka, Adnan and Prosser, R Scott} } @article {229, title = {Applications of an inactive Dispersin B probe to monitor biofilm polysaccharide production.}, journal = {Methods Enzymol}, volume = {665}, year = {2022}, month = {2022}, pages = {209-231}, abstract = {

Bacterial biofilms consist of surface-attached communities that secrete polymeric substances to form a biofilm matrix, generating a local microenvironment which helps protect from external factors. One such matrix component produced by a diverse list of microorganisms is the polysaccharide poly-β-1,6-N-acetylglucosamine (PNAG). Dispersin B is a PNAG-specific glycosyl hydrolase, which by leveraging its unique specificity, can be used to design a macromolecular fluorescent PNAG binding probe. An active site mutant of Dispersin B was fused to a fluorescent protein, to generate a probe that bound PNAG but did not hydrolyze its polysaccharide target. The ease and versatility of this strategy has made it possible to study PNAG in the context of maturing biofilms, as the probe tends to sequester in regions of high PNAG density. In this chapter, typical workflows from probe construction to cell-binding and imaging experiments are described.

}, keywords = {Acetylglucosamine, Bacterial Proteins, Biofilms, Catalytic Domain, Polysaccharides}, issn = {1557-7988}, doi = {10.1016/bs.mie.2021.11.006}, author = {Eddenden, Alexander and Nitz, Mark} } @article {225, title = {An Engineered Paper-Based 3D Coculture Model of Pancreatic Cancer to Study the Impact of Tissue Architecture and Microenvironmental Gradients on Cell Phenotype.}, journal = {Adv Healthc Mater}, year = {2022}, month = {2022 Oct 29}, pages = {e2201846}, abstract = {

The spatial configuration of cells in the tumor microenvironment (TME) affects both cancer and fibroblast cell phenotypes contributing to the clinical challenge of tumor heterogeneity and therapeutic resistance. This is a particular challenge in stroma-rich pancreatic ductal adenocarcinoma (PDAC). Here, a versatile system is described to study the impact of tissue architecture on cell phenotype using PDAC as a model system. This fully human system encompassing both primary pancreatic stellate cells and primary organoid cells using the TRACER platform to allow the creation of user-defined TME architectures that have been inferred from clinical PDAC samples and are analyzed by CyTOF to characterize cells extracted from the system. High dimensional characterization using CyTOF demonstrates that tissue architecture leads to distinct hypoxia and proliferation gradients. Furthermore, phenotypic markers for both cell types are also graded in ways that cannot be explained by either hypoxia or coculture alone. This demonstrates the importance of using complex models encompassing cancer cells, stromal cells, and allowing control over architecture to explore the impact of tissue architecture on cell phenotype. It is anticipated that this model will help decipher how tissue architecture and cell interactions regulate cell phenotype and hence cellular and tissue heterogeneity.

}, issn = {2192-2659}, doi = {10.1002/adhm.202201846}, author = {Cadavid, Jose L and Latour, Simon and Nowlan, Ferris and Co, Ileana L and Landon-Brace, Natalie and Wouters, Bradly G and Gr{\"u}nwald, Barbara T and Nitz, Mark and Jackson, Hartland Warren and McGuigan, Alison P} } @article {227, title = {Tellurophene-Tagging of Teniposide Facilitates Monitoring by Mass Cytometry.}, journal = {Chembiochem}, volume = {23}, year = {2022}, month = {2022 Oct 19}, pages = {e202200284}, abstract = {

Target engagement and the biodistribution of exogenously administered small molecules is rarely homogenous. Methods to determine the biodistribution at the cellular level are limited by the ability to detect the small molecule and simultaneously identify the cell types or tissue structures with which it is associated. The highly multiplexed nature of mass cytometry could facilitate these studies provided a heavy isotope label was available in the molecule of interest. Here we show it is possible to append a tellurophene to a known chemotherapeutic, teniposide, to follow this molecule in vivo. A semi-synthetic approach offers an efficient route to the teniposide analogue which is found to have similar characteristics when compared with the parent teniposide in vitro. Using mass cytometry we find the teniposide analogue has significant nonspecific binding to cells. In vivo the tellurium bearing teniposide produces the expected DNA damage in a PANC-1 xenograft model. The distribution of Te in the tissue is near the limits of detection and further work will be required to characterize the localization of this analogue with respect to cell type distributions.

}, keywords = {DNA Damage, Humans, Tellurium, Teniposide, Tissue Distribution}, issn = {1439-7633}, doi = {10.1002/cbic.202200284}, author = {Rana, Rahul and Vellanki, Ravi N and Wouters, Bradly G and Nitz, Mark} } @article {230, title = {Termination of Poly--acetylglucosamine (PNAG) Polymerization with -Acetylglucosamine Analogues.}, journal = {ACS Chem Biol}, volume = {17}, year = {2022}, month = {2022 Nov 18}, pages = {3036-3046}, abstract = {

Bacteria require polysaccharides for structure, survival, and virulence. Despite their central role in microbiology, few tools are available to manipulate their production. In , the glycosyltransferase complex PgaCD produces poly--acetylglucosamine (PNAG), an extracellular matrix polysaccharide required for biofilm formation. We report that C6-substituted (H, F, N, SH, NH) UDP-GlcNAc substrate analogues are inhibitors of PgaCD. , the inhibitors cause PNAG chain termination, consistent with the mechanism of PNAG polymerization from the nonreducing terminus. , expression of the GlcNAc-1-kinase NahK in provided a non-native GlcNAc salvage pathway that produced the UDP-GlcNAc analogue inhibitors . The 6-fluoro and 6-deoxy derivatives were potent inhibitors of biofilm formation in the transformed strain, providing a tool to manipulate this key exopolysaccharide. Characterization of the UDP-GlcNAc pool and quantification of PNAG generation support PNAG termination as the primary mechanism of biofilm inhibition by 6-fluoro UDP-GlcNAc.

}, keywords = {Acetylglucosamine, Biofilms, Escherichia coli, Polymerization, Uridine Diphosphate}, issn = {1554-8937}, doi = {10.1021/acschembio.1c00855}, author = {Morrison, Zachary A and Eddenden, Alexander and Subramanian, Adithya Shankara and Howell, P Lynne and Nitz, Mark} } @article {228, title = {The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing.}, journal = {PLoS Pathog}, volume = {18}, year = {2022}, month = {2022 Aug}, pages = {e1010750}, abstract = {

The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de-N-acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.

}, keywords = {Amidohydrolases, Biofilms, Periplasmic Proteins, Polymers, Tetratricopeptide Repeat}, issn = {1553-7374}, doi = {10.1371/journal.ppat.1010750}, author = {Pfoh, Roland and Subramanian, Adithya S and Huang, Jingjing and Little, Dustin J and Forman, Adam and DiFrancesco, Benjamin R and Balouchestani-Asli, Negar and Kitova, Elena N and Klassen, John S and Pom{\`e}s, R{\'e}gis and Nitz, Mark and Howell, P Lynne} } @article {226, title = {Using a high-throughput, whole-cell hydrogenase assay to identify potential small molecule inhibitors of [NiFe]-hydrogenase.}, journal = {Metallomics}, volume = {14}, year = {2022}, month = {2022 Oct 08}, abstract = {

[NiFe]-hydrogenases are used by several human pathogens to catalyze the reversible conversion between molecular hydrogen and protons and electrons. Hydrogenases provide an increased metabolic flexibility for pathogens, such as Escherichia coli and Helicobacter pylori, by allowing the use of molecular hydrogen as an energy source to promote survival in anaerobic environments. With the rise of antimicrobial resistance and the desire for novel therapeutics, the [NiFe]-hydrogenases are alluring targets. Inhibiting the nickel insertion pathway of [NiFe]-hydrogenases is attractive as this pathway is required for the generation of functional enzymes and is orthogonal to human biochemistry. In this work, nickel availability for the production and function of E. coli [NiFe]-hydrogenase was explored through immunoblot and activity assays. Whole-cell hydrogenase activities were assayed in high throughput against a small molecule library of known bioactives. Iodoquinol was identified as a potential inhibitor of the nickel biosynthetic pathway of [NiFe]-hydrogenase through a two-step screening process, but further studies with immunoblot assays showed confounding effects dependent on the cell growth phase. This study highlights the significance of considering the growth phenotype for whole-cell based assays overall and its effects on various cellular processes influenced by metal trafficking and homeostasis.

}, keywords = {Anti-Infective Agents, Escherichia coli, Humans, Hydrogen, Hydrogenase, Iodoquinol, Nickel, Protons}, issn = {1756-591X}, doi = {10.1093/mtomcs/mfac073}, author = {Sebastiampillai, Stephanie and Lacasse, Michael J and McCusker, Susan and Campbell, Tracey and Nitz, Mark and Zamble, Deborah B} } @article {215, title = {Incorporation of TePhe into Expressed Proteins is Minimally Perturbing.}, journal = {Chembiochem}, volume = {22}, year = {2021}, month = {2021 Jul 15}, pages = {2449-2456}, abstract = {

Tellurium is a versatile heavy chalcogen with numerous applications in chemical biology, providing valuable probes in mass cytometry, fluorescence imaging and structural biology. L-Tellurienylalanine (TePhe) is an analogue of the proteinogenic amino acid L-phenylalanine (Phe) in which the phenyl side chain has been replaced by a 5-membered tellurophene moiety. High incorporation level of TePhe in expressed proteins at defined sites is expected to facilitate studies in proteomics, protein NMR spectroscopy, and structure elucidation. As a model we chose immunoglobulin-binding Protein G, B1 domain (GB1) to validate TePhe as a suitable structural analogue for Phe. We demonstrate that approximately 1 in 2 of all Phe sites within GB1 can be substituted with TePhe through expression in standard non-Phe-auxotrophic E. coli in Phe-deficient media containing glyphosate, an inhibitor of aromatic amino acid biosynthesis. The TePhe content of the GB1 sample can be further increased to 85 \% through HPLC. Using NMR and CD spectroscopy, we confirm that the Phe-to-TePhe substitution has negligible impact on the global structure and stability of GB1.

}, issn = {1439-7633}, doi = {10.1002/cbic.202100160}, author = {Jia Bu, Yong and Nitz, Mark} } @article {216, title = {An Iodinated DAPI-Based Reagent for Mass Cytometry.}, journal = {Chembiochem}, volume = {22}, year = {2021}, month = {2021 02 02}, pages = {532-538}, abstract = {

Multiparametric single-cell analysis has seen dramatic improvements with the introduction of mass cytometry (MC) and imaging mass cytometry (IMC ). These technologies expanded the number of biomarkers that can be identified simultaneously by using heavy-isotope-tagged antibody reagents. Small-molecule probes bearing heavy isotopes are emerging as additional useful functional reporters of cellular features. Realizing this, we explored the iodination of DAPI to produce a heavy-atom-substituted derivative of the commonly used fluorescent DNA stain. Although exhibiting a drastically reduced fluorescence emission profile, I-DAPI retains strong binding affinity for DNA. I-DAPI was used to detect cellular DNA in MC and IMC{\texttrademark} assays with comparable efficiency to known Ir-containing DNA intercalators. This work suggests repurposing well-known colorimetric stains through simple reactions could be an effective strategy to develop new, functional MC and IMC{\texttrademark} reagents.

}, keywords = {Animals, Cell Line, DNA, Flow Cytometry, Fluorescent Dyes, Halogenation, Humans, Indicators and Reagents, Indoles, Mice, Molecular Structure, Optical Imaging, Spectrometry, Fluorescence}, issn = {1439-7633}, doi = {10.1002/cbic.202000369}, author = {Rana, Rahul and Chang, Qing and Bassan, Jay and Chow, Sue and Hedley, David and Nitz, Mark} } @article {217, title = {Multiplexed single-cell analysis of organoid signaling networks.}, journal = {Nat Protoc}, volume = {16}, year = {2021}, month = {2021 10}, pages = {4897-4918}, abstract = {

Organoids are biomimetic tissue models comprising multiple cell types and cell states. Post-translational modification (PTM) signaling networks control cellular phenotypes and are frequently dysregulated in diseases such as cancer. Although signaling networks vary across cell types, there are limited techniques to study cell type-specific PTMs in heterocellular organoids. Here, we present a multiplexed mass cytometry (MC) protocol for single-cell analysis of PTM signaling and cell states in organoids and organoids co-cultured with fibroblasts and leukocytes. We describe how thiol-reactive organoid barcoding in situ (TOBis) enables 35-plex and 126-plex single-cell comparison of organoid cultures and provide a cytometry by time of flight (CyTOF) signaling analysis pipeline (CyGNAL) for computing cell type-specific PTM signaling networks. The TOBis MC protocol takes ~3 d from organoid fixation to data acquisition and can generate single-cell data for \>40 antibodies from millions of cells across 126 organoid cultures in a single MC run.

}, keywords = {Cell Differentiation, Fibroblasts, Humans, Organoids, Single-Cell Analysis}, issn = {1750-2799}, doi = {10.1038/s41596-021-00603-4}, author = {Sufi, Jahangir and Qin, Xiao and Rodriguez, Ferran Cardoso and Bu, Yong Jia and Vlckova, Petra and Zapatero, Mar{\'\i}a Ramos and Nitz, Mark and Tape, Christopher J} } @article {195, title = {Cell-type-specific signaling networks in heterocellular organoids}, journal = {Nature Methods}, volume = {17}, year = {2020}, pages = {335{\textendash}342}, abstract = {

Despite the widespread adoption of organoids as biomimetic tissue models, methods to comprehensively analyze cell-type-specific post-translational modification (PTM) signaling networks in organoids are absent. Here, we report multivariate single-cell analysis of such networks in organoids and organoid cocultures. Simultaneous analysis by mass cytometry of 28 PTMs in \>1 million single cells derived from small intestinal organoids reveals cell-type- and cell-state-specific signaling networks in stem, Paneth, enteroendocrine, tuft and goblet cells, as well as enterocytes. Integrating single-cell PTM analysis with thiol-reactive organoid barcoding in situ (TOBis) enables high-throughput comparison of signaling networks between organoid cultures. Cell-type-specific PTM analysis of colorectal cancer organoid cocultures reveals that shApc, KrasG12D and Trp53R172H cell-autonomously mimic signaling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type-specific signaling analysis of healthy and cancerous organoids.

}, issn = {15487105}, doi = {10.1038/s41592-020-0737-8}, url = {http://dx.doi.org/10.1038/s41592-020-0737-8}, author = {Qin, Xiao and Sufi, Jahangir and Vlckova, Petra and Kyriakidou, Pelagia and Acton, Sophie E. and Li, Vivian S.W. and Nitz, Mark and Tape, Christopher J.} } @article {144, title = {An Inactive Dispersin B Probe for Monitoring PNAG Production in Biofilm Formation.}, journal = {ACS Chem Biol}, volume = {15}, year = {2020}, month = {2020 05 15}, pages = {1204-1211}, abstract = {

The bacterial exopolysaccharide poly-β-1,6--acetylglucosamine is a major extracellular matrix component in biofilms of both Gram-positive and Gram-negative organisms. We have leveraged the specificity of the biofilm-dispersing glycoside hydrolase Dispersin B (DspB) to generate a probe (Dispersin B PNAG probe, DiPP) for monitoring PNAG production and localization during biofilm formation. Mutation of the active site of Dispersin B gave DiPP, which was an effective probe despite its low affinity for PNAG oligosaccharides ( \~{} 1-10 mM). Imaging of PNAG-dependent and -independent biofilms stained with a fluorescent-protein fusion of DiPP (GFP-DiPP) demonstrated the specificity of the probe for the structure of PNAG on both single-cell and biofilm levels, indicating a high local concentration of PNAG at the bacterial cell surface. Through quantitative bacterial cell binding assays and confocal microscopy analysis using GFP-DiPP, discrete areas of local high concentrations of PNAG were detected on the surface of early log phase cells. These distinct areas were seen to grow, slough from cells, and accumulate in interbacterial regions over the course of several cell divisions, showing the development of a PNAG-dependent biofilm. A potential helical distribution of staining was also noted, suggesting some degree of organization of PNAG production at the cell surface prior to cell aggregation. Together, these experiments shed light on the early stages of PNAG-dependent biofilm formation and demonstrate the value of a low-affinity-high-specificity probe for monitoring the production of bacterial exopolysaccharides.

}, issn = {1554-8937}, doi = {10.1021/acschembio.9b00907}, author = {Eddenden, Alexander and Kitova, Elena N and Klassen, John S and Nitz, Mark} } @article {140, title = {Intrinsic Turn-On Response of Thioflavin T in Complexes.}, journal = {Chemistry}, volume = {26}, year = {2020}, month = {2020 Mar 18}, pages = {3479-3483}, abstract = {

The fluorescence enhancement ("turn-on") response of the amyloid-sensing dye thioflavin T (ThT) is examined in vacuo, where solvent interactions are absent. Upon the complexation of ThT with a derivatized β-cyclodextrin, heptakis-[6-deoxy-6-(3-sulfanylpropanoic acid)]-β-cyclodextrin, turn-on responses in both the gas phase and solution phase were observed. In contrast, turn-on response was not detected when ThT was bound to gaseous cucurbit[7]uril or human telomeric DNA 22AG, whereas clear turn-on response occurs in solution. The observed difference in turn-on response in the gas phase emphasizes the key interplay between chromophore, host and solvent and demonstrates the utility of gas-phase spectroscopy to tease out the balance among intermolecular forces driving the behavior of important chromophores in solution.

}, issn = {1521-3765}, doi = {10.1002/chem.201905100}, author = {Kung, Jocky C K and Vurgun, Nesrin and Chen, JoAnn C and Nitz, Mark and Jockusch, Rebecca A} } @article {150, title = {A new ELISA assay demonstrates sex differences in the concentration of serum polysialic acid.}, journal = {Anal Biochem}, volume = {600}, year = {2020}, month = {2020 07 01}, pages = {113743}, abstract = {

Male and female immune systems are strikingly different and yet little is known about sex differences in immune glycans, though glycans play central roles in regulating the immune response. Polysialic acid (polySia) occurs on the majority of leukocytes and is a potent immunomodulatory glycan which enables cell migration and serves as an immune checkpoint. Due to widespread influence of polySia on the immune system, we aimed to characterize its levels in serum, its presence on specific proteins, and differences in the amounts of polySia in male and female serum. However, polySia is difficult to quantify and detect on specific proteins, which makes it challenging to elucidate the molecular details of polySia function. We developed a sandwich ELISA that allows for the quantification of polySia as well as specific polysialylated proteins in complex mixtures without any pretreatment or harsh conditions. The assay is quick, linear, and robust under a wide variety of conditions and gave a limit of detection of approximately 0.2\ ng polySia per mL of serum. We then quantified polySia and polysialylated CD56 in human and mouse serum. These studies strongly support our hypothesis of differences in glycosylation between the sexes as significantly less polySia was observed in female samples than in male samples.

}, issn = {1096-0309}, doi = {10.1016/j.ab.2020.113743}, author = {Tajik, Amanda and Phillips, Kaetlyn L and Nitz, Mark and Willis, Lisa M} } @article {145, title = {PelX is a UDP--acetylglucosamine C4-epimerase involved in Pel polysaccharide-dependent biofilm formation.}, journal = {J Biol Chem}, volume = {295}, year = {2020}, month = {2020 Aug 21}, pages = {11949-11962}, abstract = {

Pel is a GalNAc-rich bacterial polysaccharide that contributes to the structure and function of biofilms. The operon is highly conserved among diverse bacterial species, and Pel may therefore be a widespread biofilm determinant. Previous annotation of gene clusters has helped us identify an additional gene, , that is present adjacent to in \>100 different bacterial species. The gene is predicted to encode a member of the short-chain dehydrogenase/reductase (SDR) superfamily, but its potential role in Pel-dependent biofilm formation is unknown. Herein, we have used Pf-5 as a model to elucidate PelX function as lacks a homologue in its gene cluster. We found that forms Pel-dependent biofilms; however, despite expression of under these conditions, biofilm formation was unaffected in a Δ strain. This observation led us to identify a paralogue, PFL_5533, which we designate here PgnE, that appears to be functionally redundant to In line with this, a Δ Δ double mutant was substantially impaired in its ability to form Pel-dependent biofilms. To understand the molecular basis for this observation, we determined the structure of PelX to 2.1 {\r A} resolution. The structure revealed that PelX resembles UDP-GlcNAc C4-epimerases. Using H NMR analysis, we show that PelX catalyzes the epimerization between UDP-GlcNAc and UDP-GalNAc. Our results indicate that Pel-dependent biofilm formation requires a UDP-GlcNAc C4-epimerase that generates the UDP-GalNAc precursors required by the Pel synthase machinery for polymer production.

}, issn = {1083-351X}, doi = {10.1074/jbc.RA120.014555}, author = {Marmont, Lindsey S and Whitfield, Gregory B and Pfoh, Roland and Williams, Rohan J and Randall, Trevor E and Ostaszewski, Alexandra and Razvi, Erum and Groves, Ryan A and Robinson, Howard and Nitz, Mark and Parsek, Matthew R and Lewis, Ian A and Whitney, John C and Harrison, Joe J and Howell, P Lynne} } @article {153, title = {SCO-Spondin Defects and Neuroinflammation Are Conserved Mechanisms Driving Spinal Deformity across Genetic Models of Idiopathic Scoliosis.}, journal = {Curr Biol}, volume = {30}, year = {2020}, month = {2020 Jun 22}, pages = {2363-2373.e6}, abstract = {

Adolescent idiopathic scoliosis (AIS) affects 3\% to 4\% of children between the ages of 11 and 18 [1, 2]. This disorder, characterized by abnormal three-dimensional spinal curvatures that typically develop during periods of rapid growth, occurs in the absence of congenital vertebral malformations or neuromuscular defects [1]. Genetic heterogeneity [3] and a historical lack of appropriate animal models [4] have confounded basic understanding of AIS biology; thus, treatment options remain limited [5, 6]. Recently, genetic studies using zebrafish have linked idiopathic-like scoliosis to irregularities in motile cilia-mediated cerebrospinal fluid flow [7-9]. However, because loss of cilia motility in human primary ciliary dyskinesia patients is not fully associated with scoliosis [10, 11], other pathogenic mechanisms remain to be determined. Here, we demonstrate that zebrafish scospondin (sspo) mutants develop late-onset idiopathic-like spinal curvatures in the absence of obvious cilia motility defects. Sspo is a large secreted glycoprotein functionally associated with the subcommissural organ and Reissner{\textquoteright}s fiber [12]-ancient and enigmatic organs of the brain ventricular system reported to govern cerebrospinal fluid homeostasis [13, 14], neurogenesis [12, 15-18], and embryonic morphogenesis [19]. We demonstrate that irregular deposition of Sspo within brain ventricles is associated with idiopathic-like scoliosis across diverse genetic models. Furthermore, Sspo defects are sufficient to induce oxidative stress and neuroinflammatory responses implicated in AIS pathogenesis [9]. Through screening for chemical suppressors of sspo mutant phenotypes, we also identify potent agents capable of blocking severe juvenile spine deformity. Our work thus defines a new preclinical model of AIS and provides tools to realize novel therapeutic strategies.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2020.04.020}, author = {Rose, Chloe D and Pompili, David and Henke, Katrin and Van Gennip, Jenica L M and Meyer-Miner, Anne and Rana, Rahul and Gobron, St{\'e}phane and Harris, Matthew P and Nitz, Mark and Ciruna, Brian} } @article {136, title = {Synthesis of C6-substituted UDP-GlcNAc derivatives.}, journal = {Carbohydr Res}, volume = {495}, year = {2020}, month = {2020 Sep}, pages = {108071}, abstract = {

UDP-sugar analogs are useful for the study of glycosyltransferases and the production of unnatural glycans. The preparation of five UDP-GlcNAc derivatives is reported with 6-deoxy, 6-azido, 6-amino, 6-mercapto, or 6-fluoro substitutions. A concise chemoenzymatic synthesis was developed using the kinase NahK (B. longum JCM1217) and the uridyl transferase GlmU (E. coli K12).

}, issn = {1873-426X}, doi = {10.1016/j.carres.2020.108071}, author = {Morrison, Zachary A and Nitz, Mark} } @article {138, title = {TBAF Effects 3,6-Anhydro Formation from 6--Tosyl Pyranosides.}, journal = {Org Lett}, volume = {22}, year = {2020}, month = {2020 02 21}, pages = {1453-1457}, abstract = {

3,6-Anhydro sugars are common structures in algal polysaccharides and occur in the furanodictine and sauropunol natural products. We have found that treatment of 6--tosylpyranosides with tetrabutylammonium fluoride provides a mild, high-yielding synthesis of 3,6-anhydro sugars. Using -glycoside substrates, 3,6-anhydropyranosides are isolated and the use of -dimethyl hydroxylamine glycosides yields 3,6-anhydrofuranosides. Applying this approach, concise synthetic routes to several 3,6-anhydro sugar natural products are reported, including furanodictine A and sauropunols A-D.

}, issn = {1523-7052}, doi = {10.1021/acs.orglett.0c00045}, author = {Morrison, Zachary A and Nitz, Mark} } @article {127, title = {Validation of l-Tellurienylalanine as a Phenylalanine Isostere.}, journal = {Chembiochem}, volume = {21}, year = {2020}, month = {2020 Apr 17}, pages = {1136-1139}, abstract = {

Mass cytometry (MC) and imaging mass cytometry (IMC ) have emerged as important tools for the study of biological heterogeneity. We recently demonstrated the use of l-2-tellurienylalanine (TePhe), a mimic of phenylalanine (Phe), as an MC- and IMC-compatible protein synthesis reporter. In this work, the biochemical similarity of TePhe and its cognate analogue, Phe, are examined in the context of the RNase S complex. Isothermal titration calorimetry studies show that incorporation of TePhe preserves the interaction of S-peptide with S-protein, and the dissociation constants for the interaction of the Phe and TePhe peptides are within a factor of two. The resulting RNase S complex is catalytically active without significant alterations in the enzyme{\textquoteright}s kinetic parameters. Furthermore, circular dichroism spectroscopy does not reveal any changes to the secondary structure of TePhe-substituted RNase S. These findings provide strong evidence that TePhe functions as a Phe isostere in the context of a folded protein. It is anticipated that incorporation of TePhe into peptides or peptidomimetic scaffolds will enable facile generation of MC and IMC probes.

}, issn = {1439-7633}, doi = {10.1002/cbic.201900635}, author = {Vurgun, Nesrin and Nitz, Mark} } @article {128, title = {DNA directed damage using a brominated DAPI derivative.}, journal = {Chem Commun (Camb)}, volume = {55}, year = {2019}, month = {2019 Aug 15}, pages = {9971-9974}, abstract = {

Photodynamic therapy (PDT) is a clinically approved cancer treatment that uses light, oxygen and a photosensitizer to produce localized reactive oxygen species (ROS). Due to the short lifetime of ROS, the location of the photosensitizer in the cell is believed to be the key determinant governing the outcome of PDT. To explore the effect of direct association between a photosensitizer and DNA a well know DNA-binding dye, DAPI, was converted into a photosensitizer. Br-DAPI - unlike native DAPI - upon irradiation produces ROS. We demonstrate that the ROS are only effective in inducing dsDNA breaks when Br-DAPI is bound to DNA. In cancer cells (A549) Br-DAPI causes rapid light dependent cell death. This work supports the design of photosensitizers which bind with high affinity to the DNA of target cells for potentially more effective PDT.

}, keywords = {A549 Cells, Apoptosis, Bromine, Cell Survival, DNA, DNA Damage, Fluorescent Dyes, Humans, Indoles, Light, Photochemotherapy, Photosensitizing Agents, Proof of Concept Study, Reactive Oxygen Species}, issn = {1364-548X}, doi = {10.1039/c9cc03942b}, author = {Digby, Elyse M and Rana, Rahul and Nitz, Mark and Beharry, Andrew A} } @article {132, title = {Efficacy of ruthenium coordination complex-based Rutherrin in a preclinical rat glioblastoma model.}, journal = {Neurooncol Adv}, volume = {1}, year = {2019}, month = {2019 May-Dec}, pages = {vdz006}, abstract = {

Background: Glioblastoma is an aggressive brain cancer in adults with a grave prognosis, aggressive radio and chemotherapy provide only a 15 months median survival.

Methods: We evaluated the tolerability and efficacy of the Ruthenium-based photosensitizer TLD-1433 with apo-Transferrin (Rutherrin) in the rat glioma 2 (RG-2) model. The specific tumor uptake ratio and photodynamic therapy (PDT) threshold of the rat glioblastoma and normal brain were determined, survival and CD8T-cell infiltration post-therapy were analyzed. Results were compared with those obtained for 5-aminolevulinic acid (ALA)-induced Protoporphyrin IX (PpIX)-mediated photodynamic therapy in the same animal model. As both photosensitizers have different photophysical properties, the number of absorbed photons required to achieve an equal cell kill was determined for in vitro and in vivo studies.

Results: A significantly lower absorbed energy was sufficient to achieve LD with Rutherrin versus PpIX-mediated PDT. Rutherrin provides a higher specific uptake ratio (SUR) \>20 in tumors versus normal brain, whereas the SUR for ALA-induced PpIX was 10.6. To evaluate the short-term tissue response in vivo, enhanced T2-weighted magnetic resonance imaging (MRI) provided the spatial extent of edema, post PpIX-PDT at twice the cross-section versus Rutherrin-PDT suggesting reduced nonspecific damage, typically associated with a secondary wave of neuronal damage. Following a single therapy, a significant survival increase was observed in rats bearing glioma for PDT mediated by Rutherrin versus PpIX for the selected treatment conditions. Rutherrin-PDT also demonstrated an increased CD8+T-cell infiltration in the tumors.

Conclusion: Rutherrin-PDT was well tolerated providing a safe and effective treatment of RG-2 glioma.

}, issn = {2632-2498}, doi = {10.1093/noajnl/vdz006}, author = {Munegowda, Manjunatha Akathatti and Fisher, Carl and Molehuis, Daniel and Foltz, Warren and Roufaiel, Mark and Bassan, Jay and Nitz, Mark and Mandel, Arkady and Lilge, Lothar} } @article {135, title = {Ega3 from the fungal pathogen is an endo-α-1,4-galactosaminidase that disrupts microbial biofilms.}, journal = {J Biol Chem}, volume = {294}, year = {2019}, month = {2019 09 13}, pages = {13833-13849}, abstract = {

is an opportunistic fungal pathogen that causes both chronic and acute invasive infections. Galactosaminogalactan (GAG) is an integral component of the biofilm matrix and a key virulence factor. GAG is a heterogeneous linear α-1,4-linked exopolysaccharide of galactose and GalNAc that is partially deacetylated after secretion. A cluster of five co-expressed genes has been linked to GAG biosynthesis and modification. One gene in this cluster, ega3, is annotated as encoding a putative α-1,4-galactosaminidase belonging to glycoside hydrolase family 114 (GH114). Herein, we show that recombinant Ega3 is an active glycoside hydrolase that disrupts GAG-dependent and Pel polysaccharide-dependent biofilms at nanomolar concentrations. Using MS and functional assays, we demonstrate that Ega3 is an endo-acting α-1,4-galactosaminidase whose activity depends on the conserved acidic residues, Asp-189 and Glu-247. X-ray crystallographic structural analysis of the apo Ega3 and an Ega3-galactosamine complex, at 1.76 and 2.09 {\r A} resolutions, revealed a modified (β/α)-fold with a deep electronegative cleft, which upon ligand binding is capped to form a tunnel. Our structural analysis coupled with docking studies also uncovered the molecular determinants for galactosamine specificity and substrate binding at the -2 to +1 binding subsites. The findings in this study increase the structural and mechanistic understanding of the GH114 family, which has \>600 members encoded by plant and opportunistic human pathogens, as well as in industrially used bacteria and fungi.

}, keywords = {Aspergillus fumigatus, Biofilms, Crystallography, X-Ray, Fungal Proteins, Fungi, Glycoside Hydrolases, Hexosaminidases, Polysaccharides, Virulence}, issn = {1083-351X}, doi = {10.1074/jbc.RA119.009910}, author = {Bamford, Natalie C and Le Mauff, Fran{\c c}ois and Subramanian, Adithya S and Yip, Patrick and Mill{\'a}n, Claudia and Zhang, Yongzhen and Zacharias, Caitlin and Forman, Adam and Nitz, Mark and Cod{\'e}e, Jeroen D C and Us{\'o}n, Isabel and Sheppard, Donald C and Howell, P Lynne} } @article {130, title = {Lanthanide nanoparticles for high sensitivity multiparameter single cell analysis.}, journal = {Chem Sci}, volume = {10}, year = {2019}, month = {2019 Mar 14}, pages = {2965-2974}, abstract = {

Mass cytometry (MC) is a high throughput multiparameter analytical technique for determining biomarker expression in cells. In MC, antibodies (Abs) are tagged with heavy metal isotopes conjugation to metal chelating polymers (MCPs). To improve the sensitivity of MC towards low abundance biomarkers, we are developing nanoparticle (NP)-based reagents as mass tags for Abs. We examine the use of silica-coated NaHoF NPs ( \~{} 12 nm) decorated with PEG5k conjugated to thiol-modified primary or secondary Abs for MC assays. We compare the sensitivity of NP-Ab conjugates to MCP-Ab conjugates towards seven biomarkers with varying expression levels across six cell lines. We also perform a multi-parameter assay using a cocktail of both NP- and MCP-based reagents to detect seven cellular markers in peripheral blood mononuclear cells (PBMCs). In the case of highly abundant markers, signal enhancements from NP-Ab conjugates offer minimal advantages over MCP-Ab conjugates, which already give strong signals. In the case of biomarkers with lower abundance, the level of signal enhancements depended on the nature of the biomarker being detected, or on the type of detection method used. When comparing the indirect detection of CD14 on THP-1 cells using NPs or MCPs conjugated to secondary Abs, the NP reagents offered little signal enhancements compared to the MCP reagents. However, in the case of direct CD14 detection on THP-1 or U937 cells using NPs or MCPs conjugated to primary Abs, a 30- or 450-fold signal enhancement was seen from the NP-based reagent. In the experiments where both NP-Ab and MCP-Ab conjugates were used together to stain PBMCs, we found that the presence of the NP-Ab conjugates did not affect the function of MCP-Ab conjugates, and the NP-Ab conjugates showed minimal non-specific interaction with cells without the target biomarker (CD14). Furthermore, these NP-Ab conjugates could be used to identify rare CD14 monocytes from the PBMC mixture with a 20-fold signal increase when compared to the use of only MCP-Ab conjugates. Collectively, the strong signal amplification obtained from NP reagents demonstrate the potential of these reagents to be used in conjunction with MCP-reagents to detect rare cellular markers or cell types that may otherwise be overlooked when using MCP-reagents alone.

}, issn = {2041-6520}, doi = {10.1039/c8sc04407d}, author = {Pichaandi, Jothirmayanantham and Zhao, Guangyao and Bouzekri, Alexandre and Lu, Elsa and Ornatsky, Olga and Baranov, Vladimir and Nitz, Mark and Winnik, Mitchell A} } @article {129, title = {Methods for analyzing tellurium imaging mass cytometry data.}, journal = {PLoS One}, volume = {14}, year = {2019}, month = {2019}, pages = {e0221714}, abstract = {

Imaging mass cytometry (IMC) is a technique allowing visualization and quantification of over 40 biological parameters in a single experiment with subcellular spatial resolution, however most IMC experiments are limited to endpoint analysis with antibodies and DNA stains. Small molecules containing tellurium are promising probes for IMC due to their cell permeability, synthetic versatility, and most importantly their application to sequential labelling with isotopologous probes (SLIP) experiments. SLIP experiments with tellurium-containing probes allow quantification of intracellular biology at multiple timepoints with IMC. Despite the promise of tellurium in IMC, there are unique challenges in image processing associated with tellurium IMC data. Here, we address some of these issues by demonstrating the removal of xenon background signal, combining channels to improve signal-to-noise ratio, and calculating isotope transmission efficiency biases. These developments add accuracy to the unique temporal resolution afforded by tellurium IMC probes.

}, keywords = {Animals, Humans, Image Cytometry, Image Processing, Computer-Assisted, Isotopes, Jejunum, Mice, Molecular Probe Techniques, Molecular Probes, Signal-To-Noise Ratio, Subtraction Technique, Tellurium, Xenon}, issn = {1932-6203}, doi = {10.1371/journal.pone.0221714}, author = {Bassan, Jay and Nitz, Mark} } @article {126, title = {Signal Amplification for Imaging Mass Cytometry.}, journal = {Bioconjug Chem}, volume = {30}, year = {2019}, month = {2019 11 20}, pages = {2805-2810}, abstract = {

An enzyme-catalyzed reporter deposition stain has been developed for Imaging Mass Cytometry (IMC). The reagent consists of an alkaline phosphatase substrate tethered to a tellurophene which serves as reporter group for mass cytometry. Upon phosphate hydrolysis, a quinone methide is released which covalently labels local nucleophiles. This strategy is a useful complement to heavy isotope antibody conjugates as it facilitates signal amplification for low-abundance biomarker detection. The workflow is conveniently integrated with standard IMC antibody staining to allow multiparametric antigen detection.

}, keywords = {Alkaline Phosphatase, Animals, Carbonic Anhydrase IX, Colonic Neoplasms, Humans, Image Cytometry, Indolequinones, Mice, Tumor Cells, Cultured, Xenograft Model Antitumor Assays}, issn = {1520-4812}, doi = {10.1021/acs.bioconjchem.9b00559}, author = {Rana, Rahul and G{\'o}mez-Biagi, Rodolfo F and Bassan, Jay and Nitz, Mark} } @article {131, title = {Synthesis of defined mono-de-N-acetylated β-(1{\textrightarrow}6)-N-acetyl-d-glucosamine oligosaccharides to characterize PgaB hydrolase activity.}, journal = {Org Biomol Chem}, volume = {17}, year = {2019}, month = {2019 11 06}, pages = {9456-9466}, abstract = {

Many clinically-relevant biofilm-forming bacterial strains produce partially de-N-acetylated poly-β-(1{\textrightarrow}6)-N-acetyl-d-glucosamine (dPNAG) as an exopolysaccharide. In Gram-negative bacteria, the periplasmic protein PgaB is responsible for partial de-N-acetylation of PNAG prior to its export to the extracellular space. In addition to de-N-acetylase activity found in the N-terminal domain, PgaB contains a C-terminal hydrolase domain that can disrupt dPNAG-dependent biofilms and hydrolyzes dPNAG but not fully acetylated PNAG. The role of this C-terminal domain in biofilm formation has yet to be determined in vivo. Further characterization of the enzyme{\textquoteright}s hydrolase activity has been hampered by a lack of specific dPNAG oligosaccharides. Here, we report the synthesis of a defined mono de-N-acetylated dPNAG penta- and hepta-saccharide. Using mass spectrometry analysis and a fluorescence-based thin-layer chromatography (TLC) assay, we found that our defined dPNAG oligosaccharides are hydrolase substrates. In addition to the expected cleavage site, two residues to the reducing side of the de-N-acetylated residue, minor cleavage products on the non-reducing side of the de-N-acetylation site were observed. These findings provide quantitative data to support how PNAG is processed in Gram-negative bacteria.

}, keywords = {Acetylation, Acetylglucosamine, Amidohydrolases, Biofilms, Escherichia coli Proteins, Hydrolysis, Molecular Conformation, Oligosaccharides}, issn = {1477-0539}, doi = {10.1039/c9ob02079a}, author = {Forman, Adam and Pfoh, Roland and Eddenden, Alexander and Howell, P Lynne and Nitz, Mark} } @article {90, title = {TePhe, a tellurium-containing phenylalanine mimic, allows monitoring of protein synthesis in vivo with mass cytometry.}, journal = {Proc Natl Acad Sci U S A}, volume = {116}, year = {2019}, month = {2019 Apr 23}, pages = {8155-8160}, abstract = {

Protein synthesis is central to maintaining cellular homeostasis and its study is critical to understanding the function and dysfunction of eukaryotic systems. Here we report L-2-tellurienylalanine (TePhe) as a noncanonical amino acid for direct measurement of protein synthesis. TePhe is synthetically accessible, nontoxic, stable under biological conditions, and the tellurium atom allows its direct detection with mass cytometry, without postexperiment labeling. TePhe labeling is competitive with phenylalanine but not other large and aromatic amino acids, demonstrating its molecular specificity as a phenylalanine mimic; labeling is also abrogated in vitro and in vivo by the protein synthesis inhibitor cycloheximide, validating TePhe as a translation reporter. In vivo, imaging mass cytometry with TePhe visualizes translation dynamics in the mouse gut, brain, and tumor. The strong performance of TePhe as a probe for protein synthesis, coupled with the operational simplicity of its use, suggests TePhe could become a broadly applied molecule for measuring translation in vitro and in vivo.

}, issn = {1091-6490}, doi = {10.1073/pnas.1821151116}, author = {Bassan, Jay and Willis, Lisa M and Vellanki, Ravi N and Nguyen, Alan and Edgar, Landon J and Wouters, Bradly G and Nitz, Mark} } @article {93, title = {Highly Functionalized β-Cyclodextrins by Solid-Supported Synthesis.}, journal = {Chemistry}, volume = {24}, year = {2018}, month = {2018 Mar 20}, pages = {4459-4467}, abstract = {

Using covalent capture, a high yielding selective mono-functionalization of heptakis-[6-deoxy-6-(2-aminoethylsulfanyl)]-β-CD with a 5-mercaptopentyl functional group has been achieved. Here, we demonstrate the immobilization of the mono-thiol functionalized β-CD on PEGA resin via a disulfide bond, enabling solid-phase elaboration of the remaining six primary amines. To showcase the potential of this method, the amines were elaborated to tripeptides through standard Fmoc-peptide chemistry. A small library of CD-tripeptide conjugates was generated which, when reduced from the solid support, could be tagged at the released thiol with an environmentally sensitive fluorophore. The resulting library of sensors showed potential for the differential sensing of various bile salts. The described methodology provides a rapid and versatile route to synthesize highly functionalized libraries of CD derivatives that may be tailored towards applications in sensing, catalysis, and multivalent displays.

}, keywords = {Acrylic Resins, Amines, beta-Cyclodextrins, Fluorescent Dyes, Molecular Structure, Oligopeptides, Polyethylene Glycols, Solid-Phase Synthesis Techniques, Spectrometry, Fluorescence, Sulfhydryl Compounds}, issn = {1521-3765}, doi = {10.1002/chem.201800028}, author = {Vurgun, Nesrin and Nitz, Mark} } @article {91, title = {Monosaccharide inhibitors targeting carbohydrate esterase family 4 de-N-acetylases.}, journal = {Bioorg Med Chem}, volume = {26}, year = {2018}, month = {2018 11 15}, pages = {5631-5643}, abstract = {

The Carbohydrate Esterase family 4 contains virulence factors which modify peptidoglycan and biofilm-related exopolysaccharides. Despite the importance of this family of enzymes, a potent mechanism-based inhibition strategy has yet to emerge. Based on the postulated tridentate binding mode of the tetrahedral de-N-acetylation intermediate, GlcNAc derivatives bearing metal chelating groups at the 2 and 3 positions were synthesized. These scaffolds include 2-C phosphonate, 2-C sulfonamide, 2-thionoacetamide warheads as well as derivatives bearing thiol, amine and azide substitutions at the 3-position. The inhibitors were assayed against a representative peptidoglycan deacetylase, Pgda from Streptococcus pneumonia, and a representative biofilm-related exopolysaccharide deacetylase, PgaB from Escherichia coli. Of the inhibitors evaluated, the 3-thio derivatives showed weak to moderate inhibition of Pgda. The strongest inhibitor was benzyl 2,3-dideoxy-2-thionoacetamide-3-thio-β-d-glucoside, whose inhibitory potency showed an unexpected dependence on metal concentration and was found to have a partial mixed inhibition mode (K = 2.9 {\textpm} 0.6 μM).

}, issn = {1464-3391}, doi = {10.1016/j.bmc.2018.10.008}, author = {DiFrancesco, Benjamin R and Morrison, Zachary A and Nitz, Mark} } @article {137, title = {PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms.}, journal = {PLoS Pathog}, volume = {14}, year = {2018}, month = {2018 04}, pages = {e1006998}, abstract = {

Poly-β(1,6)-N-acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in Escherichia coli and Bordetella species require the protein products encoded by the pgaABCD operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show that the C-terminal domains of Bordetella bronchiseptica PgaB (PgaBBb) and E. coli PgaB (PgaBEc) function as glycoside hydrolases. These enzymes hydrolyze purified deacetylated PNAG (dPNAG) from Staphylococcus aureus, disrupt PNAG-dependent biofilms formed by Bordetella pertussis, Staphylococcus carnosus, Staphylococcus epidermidis, and E. coli, and potentiate bacterial killing by gentamicin. Furthermore, we found that PgaBBb was only able to hydrolyze PNAG produced in situ by the E. coli PgaCD synthase complex when an active deacetylase domain was present. Mass spectrometry analysis of the PgaB-hydrolyzed dPNAG substrate showed a GlcN-GlcNAc-GlcNAc motif at the new reducing end of detected fragments. Our 1.76 {\r A} structure of the C-terminal domain of PgaBBb reveals a central cavity within an elongated surface groove that appears ideally suited to recognize the GlcN-GlcNAc-GlcNAc motif. The structure, in conjunction with molecular modeling and site directed mutagenesis led to the identification of the dPNAG binding subsites and D474 as the probable catalytic acid. This work expands the role of PgaB within the PNAG biosynthesis machinery, defines a new glycoside hydrolase family GH153, and identifies PgaB as a possible therapeutic agent for treating PNAG-dependent biofilm infections.

}, keywords = {Acetylation, Amidohydrolases, beta-Glucans, Biofilms, Bordetella, Crystallography, X-Ray, Escherichia coli, Escherichia coli Proteins, Glycoside Hydrolases, Operon, Protein Conformation}, issn = {1553-7374}, doi = {10.1371/journal.ppat.1006998}, author = {Little, Dustin J and Pfoh, Roland and Le Mauff, Fran{\c c}ois and Bamford, Natalie C and Notte, Christina and Baker, Perrin and Guragain, Manita and Robinson, Howard and Pier, Gerald B and Nitz, Mark and Deora, Rajendar and Sheppard, Donald C and Howell, P Lynne} } @article {92, title = {Tellurium-based mass cytometry barcode for live and fixed cells.}, journal = {Cytometry A}, volume = {93}, year = {2018}, month = {2018 07}, pages = {685-694}, abstract = {

Mass cytometry is a revolutionary technology that allows for the simultaneous quantification of \>40 different biomarkers with cellular resolution. The biomarkers are detected using metal-labeled antibodies as well as small-molecule probes of cell size, viability, and biochemical status. Barcoding is an important component of sample preparation because it reduces processing time, eliminates sample-to-sample variation, discriminates cell doublets, reduces the amount of antibody needed, and conserves sample. We developed a thiol-reactive tellurium-based barcode, TeMal. TeMal is nontoxic at working concentrations, compatible with metal-labeled antibodies, and can readily be applied to live or fixed cells, making it advantageous and complementary compared to existing barcoding reagents. We have demonstrated the utility of TeMal by barcoding microscale samples in situ to facilitate analysis of cells from an automated cell culture system using mass cytometry.

}, issn = {1552-4930}, doi = {10.1002/cyto.a.23495}, author = {Willis, Lisa M and Park, Hanuel and Watson, Michael W L and Majonis, Daniel and Watson, Jessica L and Nitz, Mark} } @article {95, title = {Access to Versatile β-Cyclodextrin Scaffolds through Guest-Mediated Monoacylation.}, journal = {Chemistry}, volume = {22}, year = {2016}, month = {2016 Jan 18}, pages = {1062-9}, abstract = {

Herein, we report the selective mono-derivatization of heptakis[6-deoxy-6-(2-aminoethylsulfanyl)]-β-CD (1) through a guest-mediated covalent capture strategy. The use of guests functionalized with cleavable linkers enables the installation of an amine-orthogonal thiol group on the primary rim of 1 as a handle for further transformations to the β-CD scaffold. Applying this methodology, two novel monoderivatized β-CDs were obtained in good yield and high purity. Both of these monoacylated CDs were amenable to facile linker cleavage and further modification at the resulting thiol group. This methodology can be applied towards the synthesis heterofunctionalized β-CD constructs for analyte sensing, drug delivery, and other applications.

}, keywords = {Acylation, beta-Cyclodextrins, Cyclodextrins, Drug Delivery Systems, Molecular Structure}, issn = {1521-3765}, doi = {10.1002/chem.201503131}, author = {Vurgun, Nesrin and G{\'o}mez-Biagi, Rodolfo F and Nitz, Mark} } @article {94, title = {Isotopologous Organotellurium Probes Reveal Dynamic Hypoxia In Vivo with Cellular Resolution.}, journal = {Angew Chem Int Ed Engl}, volume = {55}, year = {2016}, month = {2016 10 10}, pages = {13159-13163}, abstract = {

Changes in the oxygenation state of microenvironments within solid tumors are associated with the development of aggressive cancer phenotypes. Factors that influence cellular hypoxia have been characterized; however, methods for measuring the dynamics of oxygenation at a cellular level in vivo have been elusive. We report a series of tellurium-containing isotopologous probes for cellular hypoxia compatible with mass cytometry (MC)-technology that allows for highly parametric interrogation of single cells based on atomic mass spectrometry. Sequential labeling with the isotopologous probes (SLIP) in pancreatic tumor xenograft models revealed changes in cellular oxygenation over time which correlated with the distance from vasculature, the proliferation of cell populations, and proximity to necrosis. SLIP allows for capture of spatial and temporal dynamics in vivo using enzyme activated probes.

}, keywords = {Animals, Cell Hypoxia, Cell Line, Tumor, Humans, Mice, Molecular Probes, Neoplasms, Experimental, Organometallic Compounds, Tellurium, Tissue Distribution}, issn = {1521-3773}, doi = {10.1002/anie.201607483}, author = {Edgar, Landon J and Vellanki, Ravi N and McKee, Trevor D and Hedley, David and Wouters, Bradly G and Nitz, Mark} } @article {98, title = {Direct Staudinger-Phosphonite Reaction Provides Methylphosphonamidates as Inhibitors of CE4 De-N-acetylases.}, journal = {Chembiochem}, volume = {16}, year = {2015}, month = {2015 Jun 15}, pages = {1350-6}, abstract = {

De-N-acetylases of β-(1{\textrightarrow}6)-D-N-acetylglucosamine polymers (PNAG) and β-(1{\textrightarrow}4)-D-N-acetylglucosamine residues in peptidoglycan are attractive targets for antimicrobial agents. PNAG de-N-acetylases are necessary for biofilm formation in numerous pathogenic bacteria. Peptidoglycan de-N-acetylation facilitates bacterial evasion of innate immune defenses. To target these enzymes, transition-state analogue inhibitors containing a methylphosphonamidate have been synthesized through a direct Staudinger-phosphonite reaction. The inhibitors were tested on purified PgaB, a PNAG de-N-acetylase from Escherichia coli, and PgdA, a peptidoglycan de-N-acetylase from Streptococcus pneumonia. Herein, we describe the most potent inhibitor of peptidoglycan de-N-acetylases reported to date (Ki =80 μM). The minimal inhibition of PgaB observed provides insight into key structural and functional differences in these enzymes that will need to be considered during the development of future inhibitors.

}, keywords = {Acetylglucosamine, Amidohydrolases, Bacterial Proteins, Enzyme Inhibitors, Escherichia coli, Escherichia coli Infections, Escherichia coli Proteins, Humans, Methylation, Organophosphorus Compounds, Peptidoglycan, Pneumococcal Infections, Streptococcus pneumoniae}, issn = {1439-7633}, doi = {10.1002/cbic.201500091}, author = {Ariyakumaran, Rishikesh and Pokrovskaya, Varvara and Little, Dustin J and Howell, P Lynne and Nitz, Mark} } @article {97, title = {Organotellurium scaffolds for mass cytometry reagent development.}, journal = {Org Biomol Chem}, volume = {13}, year = {2015}, month = {2015 Jul 07}, pages = {7027-33}, abstract = {

Mass cytometry (MC) is a powerful tool for studying heterogeneous cell populations. In previous work, our laboratory has developed an MC probe for hypoxia bearing a methyl telluride mass tag. The methyl telluride was unoptimized, displaying stability and toxicity limitations. Here, we investigate three classes of organotelluriums as MC mass tags: methyl tellurides, trifluoromethyl tellurides and 2-alkyl-tellurophenes. NMR was used to compare the stability of these compounds in aqueous and organic solutions and the compounds were analysed for toxicity in Jurkat cells. The methyl tellurides were moderately stable to aerobic oxidation in organic solution under dry ambient conditions. The trifluoromethyl tellurides were stable to aerobic oxidation in organic solution but decomposed in aqueous solution. The 2-alkyl-tellurophenes proved to be stable in both organic and aqueous solutions under ambient conditions and showed limited toxicity (IC50 \> 200 μM) in cell based assays. The synthetic feasibility, chemically stability, and limited toxicity of tellurophenes suggests these groups will be good choices for MC reagent development.

}, issn = {1477-0539}, doi = {10.1039/c5ob00593k}, author = {Park, Hanuel and Edgar, Landon J and Lumba, Matthew A and Willis, Lisa M and Nitz, Mark} } @article {96, title = {The protein BpsB is a poly-β-1,6-N-acetyl-D-glucosamine deacetylase required for biofilm formation in Bordetella bronchiseptica.}, journal = {J Biol Chem}, volume = {290}, year = {2015}, month = {2015 Sep 11}, pages = {22827-40}, abstract = {

Bordetella pertussis and Bordetella bronchiseptica are the causative agents of whooping cough in humans and a variety of respiratory diseases in animals, respectively. Bordetella species produce an exopolysaccharide, known as the Bordetella polysaccharide (Bps), which is encoded by the bpsABCD operon. Bps is required for Bordetella biofilm formation, colonization of the respiratory tract, and confers protection from complement-mediated killing. In this report, we have investigated the role of BpsB in the biosynthesis of Bps and biofilm formation by B. bronchiseptica. BpsB is a two-domain protein that localizes to the periplasm and outer membrane. BpsB displays metal- and length-dependent deacetylation on poly-β-1,6-N-acetyl-d-glucosamine (PNAG) oligomers, supporting previous immunogenic data that suggests Bps is a PNAG polymer. BpsB can use a variety of divalent metal cations for deacetylase activity and showed highest activity in the presence of Ni(2+) and Co(2+). The structure of the BpsB deacetylase domain is similar to the PNAG deacetylases PgaB and IcaB and contains the same circularly permuted family four carbohydrate esterase motifs. Unlike PgaB from Escherichia coli, BpsB is not required for polymer export and has unique structural differences that allow the N-terminal deacetylase domain to be active when purified in isolation from the C-terminal domain. Our enzymatic characterizations highlight the importance of conserved active site residues in PNAG deacetylation and demonstrate that the C-terminal domain is required for maximal deacetylation of longer PNAG oligomers. Furthermore, we show that BpsB is critical for the formation and complex architecture of B. bronchiseptica biofilms.

}, keywords = {Amidohydrolases, Amino Acid Motifs, Bacterial Proteins, beta-Glucans, Biofilms, Bordetella bronchiseptica, Cobalt, Nickel, Protein Structure, Tertiary}, issn = {1083-351X}, doi = {10.1074/jbc.M115.672469}, author = {Little, Dustin J and Milek, Sonja and Bamford, Natalie C and Ganguly, Tridib and DiFrancesco, Benjamin R and Nitz, Mark and Deora, Rajendar and Howell, P Lynne} } @article {151, title = {Functional PEG-PAMAM-tetraphosphonate capped NaLnF$_{4}$ nanoparticles and their colloidal stability in phosphate buffer.}, journal = {Langmuir}, volume = {30}, year = {2014}, month = {2014 Jun 17}, pages = {6980-9}, abstract = {

Developing surface coatings for NaLnF4 nanoparticles (NPs) that provide long-term stability in solutions containing competitive ions such as phosphate remains challenging. An amine-functional polyamidoamine tetraphosphonate (NH2-PAMAM-4P) as a multidentate ligand for these NPs has been synthesized and characterized as a ligand for the surface of NaGdF4 and NaTbF4 nanoparticles. A two-step ligand exchange protocol was developed for introduction of the NH2-PAMAM-4P ligand on oleate-capped NaLnF4 NPs. The NPs were first treated with methoxy-poly(ethylene glycol)-monophosphoric acid (M(n) = 750) in tetrahydrofuran. The mPEG750-OPO3-capped NPs were stable colloidal solutions in water, where they could be ligand-exchanged with NH2-PAMAM-4P. The surface amine groups on the NPs were available for derivatization to attach methoxy-PEG (M(n) = 2000) and biotin-terminated PEG (M(n) = 2000) chains. The surface coverage of ligands on the NPs was examined by thermal gravimetric analysis, and by a HABA analysis for biotin-containing NPs. Colloidal stability of the NPs was examined by dynamic light scattering. NaGdF4 and NaTbF4 NPs capped with mPEG2000-PAMAM-4P showed colloidal stability in DI water and in phosphate buffer (10 mM, pH 7.4). A direct comparison with NaTbF4 NPs capped with a mPEG2000-lysine-based tetradentate ligand that we reported previously (Langmuir 2012, 28, 12861-12870) showed that both ligands provided long-term stability in phosphate buffer, but that the lysine-based ligand provided better stability in phosphate-buffered saline.

}, keywords = {Colloids, Dendrimers, Nanoparticles, Phosphates, Polyethylene Glycols}, issn = {1520-5827}, doi = {10.1021/la501142v}, author = {Zhao, Guangyao and Tong, Lemuel and Cao, Pengpeng and Nitz, Mark and Winnik, Mitchell A} } @article {171, title = {A high-sensitivity lanthanide nanoparticle reporter for mass cytometry: tests on microgels as a proxy for cells.}, journal = {Langmuir}, volume = {30}, year = {2014}, month = {2014 Mar 25}, pages = {3142-53}, abstract = {

This paper addresses the question of whether one can use lanthanide nanoparticles (e.g., NaHoF4) to detect surface biomarkers expressed at low levels by mass cytometry. To avoid many of the complications of experiments on live or fixed cells, we carried out proof-of-concept experiments using aqueous microgels with a diameter on the order of 700 nm as a proxy for cells. These microgels were used to test whether nanoparticle (NP) reagents would allow the detection of as few as 100 proteins per "cell" in cell-by-cell assays. Streptavidin (SAv), which served as the model biomarker, was attached to the microgel in two different ways. Covalent coupling to surface carboxyls of the microgel led to large numbers (\>10(4)) of proteins per microgel, whereas biotinylation of the microgel followed by exposure to SAv led to much smaller numbers of SAv per microgel. Using mass cytometry, we compared two biotin-containing reagents, which recognized and bound to the SAvs on the microgel. One was a metal chelating polymer (MCP), a biotin end-capped polyaspartamide containing 50 Tb(3+) ions per probe. The other was a biotinylated NaHoF4 NP containing 15 000 Ho atoms per probe. Nonspecific binding was determined with bovine serum albumin (BSA) conjugated microgels. The MCP was effective at detecting and quantifying SAvs on the microgel with covalently bound SAv (20 000 SAvs per microgel) but was unable to give a meaningful signal above that of the BSA-coated microgel for the samples with low levels of SAv. Here the NP reagent gave a signal 2 orders of magnitude stronger than that of the MCP and allowed detection of NPs ranging from 100 to 500 per microgel. Sensitivity was limited by the level of nonspecific adsorption. This proof of concept experiment demonstrates the enhanced sensitivity possible with NP reagents in cell-by-cell assays by mass cytometry.

}, keywords = {Animals, Biomimetics, Biotin, Cattle, Cytological Techniques, Gels, Lanthanoid Series Elements, Nanoparticles, Polymers, Serum Albumin, Bovine, Streptavidin}, issn = {1520-5827}, doi = {10.1021/la403627p}, author = {Lin, Wanjuan and Hou, Yi and Lu, Yijie and Abdelrahman, Ahmed I and Cao, Pengpeng and Zhao, Guangyao and Tong, Lemuel and Qian, Jieshu and Baranov, Vladimir and Nitz, Mark and Winnik, Mitchell A} } @article {99, title = {Identification of hypoxic cells using an organotellurium tag compatible with mass cytometry.}, journal = {Angew Chem Int Ed Engl}, volume = {53}, year = {2014}, month = {2014 Oct 20}, pages = {11473-7}, abstract = {

Mass cytometry (MC) offers unparalleled potential for the development of highly parameterized assays for characterization of single cells within heterogeneous populations. Current reagents compatible with MC analysis employ antibody-metal-chelating polymer conjugates to report on the presence of biomarkers. Here, we expand the utility of MC by developing the first activity-based probe designed specifically for use with the technology. A compact MC-detectable telluroether is linked to a bioreductively sensitive 2-nitroimidazole scaffold, thereby generating a probe sensitive to cellular hypoxia. The probe exhibits low toxicity and is able to selectively label O2-deprived cells. A proof-of-concept experiment employing metal-bound DNA intercalators demonstrates that a heterogeneous mixture of cells with differential exposure to O2 can be effectively discriminated by the quantity of tellurium-labeling. The organotellurium reagents described herein provide a general approach to the development of a large toolkit of MC-compatible probes for activity-based profiling of single cells.

}, keywords = {Cell Hypoxia, Cell Separation, Molecular Probes, Organic Chemicals, Tellurium}, issn = {1521-3773}, doi = {10.1002/anie.201405233}, author = {Edgar, Landon J and Vellanki, Ravi N and Halupa, Adrienne and Hedley, David and Wouters, Bradly G and Nitz, Mark} } @article {143, title = {Modification and periplasmic translocation of the biofilm exopolysaccharide poly-β-1,6-N-acetyl-D-glucosamine.}, journal = {Proc Natl Acad Sci U S A}, volume = {111}, year = {2014}, month = {2014 Jul 29}, pages = {11013-8}, abstract = {

Poly-β-1,6-N-acetyl-D-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of medically important bacteria. Polyglucosamine subunit B (PgaB) is responsible for the de-N-acetylation of PNAG, a process required for polymer export and biofilm formation. PgaB is located in the periplasm and likely bridges the inner membrane synthesis and outer membrane export machinery. Here, we present structural, functional, and molecular simulation data that suggest PgaB associates with PNAG continuously during periplasmic transport. We show that the association of PgaB{\textquoteright}s N- and C-terminal domains forms a cleft required for the binding and de-N-acetylation of PNAG. Molecular dynamics (MD) simulations of PgaB show a binding preference for N-acetylglucosamine (GlcNAc) to the N-terminal domain and glucosammonium to the C-terminal domain. Continuous ligand binding density is observed that extends around PgaB from the N-terminal domain active site to an electronegative groove on the C-terminal domain that would allow for a processive mechanism. PgaB{\textquoteright}s C-terminal domain (PgaB310-672) directly binds PNAG oligomers with dissociation constants of \~{}1-3 mM, and the structures of PgaB310-672 in complex with β-1,6-(GlcNAc)6, GlcNAc, and glucosamine reveal a unique binding mode suitable for interaction with de-N-acetylated PNAG (dPNAG). Furthermore, PgaB310-672 contains a β-hairpin loop (βHL) important for binding PNAG that was disordered in previous PgaB42-655 structures and is highly dynamic in the MD simulations. We propose that conformational changes in PgaB310-672 mediated by the βHL on binding of PNAG/dPNAG play an important role in the targeting of the polymer for export and its release.

}, keywords = {Amidohydrolases, beta-Glucans, Biofilms, Biological Transport, Active, Escherichia coli, Escherichia coli Proteins, Molecular Docking Simulation, Periplasm, Polysaccharides, Bacterial, Protein Structure, Secondary, Protein Structure, Tertiary}, issn = {1091-6490}, doi = {10.1073/pnas.1406388111}, author = {Little, Dustin J and Li, Grace and Ing, Christopher and DiFrancesco, Benjamin R and Bamford, Natalie C and Robinson, Howard and Nitz, Mark and Pom{\`e}s, R{\'e}gis and Howell, P Lynne} } @article {100, title = {Protecting-group-free O-glysosidation using p-toluenesulfonohydrazide and glycosyl chloride donors.}, journal = {Carbohydr Res}, volume = {386}, year = {2014}, month = {2014 Mar 11}, pages = {73-7}, abstract = {

A range of N{\textquoteright}-glycosylsulfonohydrazides (GSHs) display good reactivity but poor stereoselectivity in protecting-group-free O-glycosidations when a moderate excess of the model acceptor n-decanol is employed. This stable, readily-accessed class of donor may be more tractable for the glycosylation of non-volatile acceptors than Fischer{\textquoteright}s glycosidation conditions. It is possible to generate unprotected glycosyl chlorides from GSHs in situ. In an effort to find conditions to improve glycosidation stereoselectivity, methanolysis of unprotected glucosyl chloride under halide-ion exchange conditions was examined. Relative to its tetra-O-benzyl analogue, this donor displays moderate, inverted stereoselectivity and a significantly faster reaction rate.

}, keywords = {Chlorides, Glucose, Glycosides, Glycosylation, Ion Exchange, Kinetics, Stereoisomerism, Tosyl Compounds}, issn = {1873-426X}, doi = {10.1016/j.carres.2013.08.019}, author = {Williams, Rohan J and Paul, Caroline E and Nitz, Mark} } @article {142, title = {Structural basis for the De-N-acetylation of Poly-β-1,6-N-acetyl-D-glucosamine in Gram-positive bacteria.}, journal = {J Biol Chem}, volume = {289}, year = {2014}, month = {2014 Dec 26}, pages = {35907-17}, abstract = {

Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In staphylococci, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by the extracellular protein IcaB is required for biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of IcaB from Ammonifex degensii (IcaBAd) has been determined to 1.7 {\r A} resolution. The structure of IcaBAd reveals a (β/α)7 barrel common to the family four carbohydrate esterases (CE4s) with the canonical motifs circularly permuted. The metal dependence of IcaBAd is similar to most CE4s showing the maximum rates of de-N-acetylation with Ni(2+), Co(2+), and Zn(2+). From docking studies with β-1,6-GlcNAc oligomers and structural comparison to PgaB from Escherichia coli, the Gram-negative homologue of IcaB, we identify Arg-45, Tyr-67, and Trp-180 as key residues for PNAG binding during catalysis. The absence of these residues in PgaB provides a rationale for the requirement of a C-terminal domain for efficient deacetylation of PNAG in Gram-negative species. Mutational analysis of conserved active site residues suggests that IcaB uses an altered catalytic mechanism in comparison to other characterized CE4 members. Furthermore, we identified a conserved surface-exposed hydrophobic loop found only in Gram-positive homologues of IcaB. Our data suggest that this loop is required for membrane association and likely anchors IcaB to the membrane during polysaccharide biosynthesis. The work presented herein will help guide the design of IcaB inhibitors to combat biofilm formation by staphylococci.

}, keywords = {Acetylation, Amidohydrolases, Amino Acid Sequence, Bacterial Proteins, beta-Glucans, Catalytic Domain, Conserved Sequence, Crystallography, X-Ray, Gram-Positive Endospore-Forming Rods, Hydrophobic and Hydrophilic Interactions, Molecular Docking Simulation, Molecular Sequence Data, Protein Binding, Protein Structure, Secondary, Zinc}, issn = {1083-351X}, doi = {10.1074/jbc.M114.611400}, author = {Little, Dustin J and Bamford, Natalie C and Pokrovskaya, Varvara and Robinson, Howard and Nitz, Mark and Howell, P Lynne} } @article {101, title = {Functional characterization of Staphylococcus epidermidis IcaB, a de-N-acetylase important for biofilm formation.}, journal = {Biochemistry}, volume = {52}, year = {2013}, month = {2013 Aug 13}, pages = {5463-71}, abstract = {

A polymer of partially de-N-acetylated β-1,6-linked N-acetylglucosamine (dPNAG), also known as the polysaccharide intercellular adhesin (PIA), is an important component of many bacterial biofilm matrices. In Staphyloccocus epidermidis, the poly-N-acetylglucosamine polymer is partially de-N-acetylated by the extracellular protein IcaB. To understand the mechanism of action of IcaB, the enzyme was overexpressed and purified. IcaB demonstrates metal-dependent de-N-acetylase activity on β-1,6-linked N-acetylglucosamine oligomers with a broad preference for divalent metals. Steady-state kinetic analysis reveals the low catalytic efficiency (pentasaccharide kcat/KM 0.03 M(-1) s(-1)) of the enzyme toward the oligomeric substrates. While IcaB displays similar rates of de-N-acetylation with tri- through hexasaccharide PNAG oligomers, position specific de-N-acetylation was only observed with penta- and hexasaccharides. The enzyme preferentially de-N-acetylates the second residue from the reducing terminus in the pentasaccharide and second and third residues from the reducing terminus in the hexasaccharide. The data described here represent an important step toward a detailed understanding of dPNAG biosynthesis in S. epidermidis, an important nosocomial pathogen, as well as in other Gram-positive bacteria. The low catalytic activity of IcaB is consistent with reports of other enzymes which act on biofilm-related polysaccharides, and this emerging trend may indicate a common feature among this group of polysaccharide processing enzymes.

}, keywords = {Acetylesterase, Acetylglucosamine, Amidohydrolases, Biofilms, Carbohydrate Sequence, Extracellular Matrix, Kinetics, Periplasm, Polysaccharides, Polysaccharides, Bacterial, Staphylococcus epidermidis}, issn = {1520-4995}, doi = {10.1021/bi400836g}, author = {Pokrovskaya, Varvara and Poloczek, Joanna and Little, Dustin J and Griffiths, Heather and Howell, P Lynne and Nitz, Mark} } @article {154, title = {Combining in situ proteolysis and mass spectrometry to crystallize Escherichia coli PgaB.}, journal = {Acta Crystallogr Sect F Struct Biol Cryst Commun}, volume = {68}, year = {2012}, month = {2012 Jul 01}, pages = {842-5}, abstract = {

The periplasmic poly-β-1,6-N-acetyl-D-glucosamine (PNAG) de-N-acetylase PgaB from Escherichia coli was overexpressed and purified, but was recalcitrant to crystallization. Use of the in situ proteolysis technique produced crystals of PgaB, but these crystals could not be optimized for diffraction studies. By analyzing the initial crystal hits using SDS-PAGE and mass spectrometry, the boundaries of the protein species that crystallized were determined. The re-engineered protein target crystallized reproducibly without the addition of protease and with significantly increased crystal quality. Crystals of the selenomethionine-incorporated protein exhibited the symmetry of space group P2(1)2(1)2(1) and diffracted to 2.1 {\r A} resolution.

}, keywords = {Amidohydrolases, Crystallization, Escherichia coli, Escherichia coli Proteins, Mass Spectrometry, Proteolysis}, issn = {1744-3091}, doi = {10.1107/S1744309112022075}, author = {Little, Dustin J and Whitney, John C and Robinson, Howard and Yip, Patrick and Nitz, Mark and Howell, P Lynne} } @article {102, title = {Improving lanthanide nanocrystal colloidal stability in competitive aqueous buffer solutions using multivalent PEG-phosphonate ligands.}, journal = {Langmuir}, volume = {28}, year = {2012}, month = {2012 Sep 04}, pages = {12861-70}, abstract = {

The range of properties available in the lanthanide series has inspired research into the use of lanthanide nanoparticles for numerous applications. We aim to use NaLnF(4) nanoparticles for isotopic tags in mass cytometry. This application requires nanoparticles of narrow size distribution, diameters preferably less than 15 nm, and robust surface chemistry to avoid nonspecific interactions and to facilitate bioconjugation. Nanoparticles (NaHoF(4), NaEuF(4), NaGdF(4), and NaTbF(4)) were synthesized with diameters from 9 to 11 nm with oleic acid surface stabilization. The surface ligands were replaced by a series of mono-, di-, and tetraphosphonate PEG ligands, whose synthesis is reported here. The colloidal stability of the resulting particles was monitored over a range of pH values and in phosphate containing solutions. All of the PEG-phosphonate ligands were found to produce non-aggregated colloidally stable suspensions of the nanoparticles in water as judged by DLS and TEM measurements. However, in more aggressive solutions, at high pH and in phosphate buffers, the mono- and diphosphonate PEG ligands did not stabilize the particles and aggregation as well as flocculation was observed. However, the tetraphosphonate ligand was able to stabilize the particles at high pH and in phosphate buffers for extended periods of time.

}, keywords = {Buffers, Colloids, Hydrogen-Ion Concentration, Lanthanoid Series Elements, Ligands, Nanoparticles, Organophosphonates, Polyethylene Glycols, Solutions, Water}, issn = {1520-5827}, doi = {10.1021/la302690h}, author = {Cao, Pengpeng and Tong, Lemuel and Hou, Yi and Zhao, Guangyao and Guerin, Gerald and Winnik, Mitchell A and Nitz, Mark} } @article {149, title = {In vivo uptake of β-amyloid by non-plaque associated microglia.}, journal = {Curr Alzheimer Res}, volume = {9}, year = {2012}, month = {2012 Oct}, pages = {890-901}, abstract = {

The role of microglia in β-amyloid (Aβ) deposition or clearance in the Alzheimer{\textquoteright}s disease (AD) brain remains unclear. Previous in vivo studies have focused primarily on the association of microglia with Aβ-positive parenchymal plaques, but have given little consideration to the possible interaction between Aβ and non-plaque associated microglia. Further, it is not known if microglia play a direct role in mediating Aβ uptake following anti-aggregant treatment. We report here the identification of Aβ-positive processes throughout the cortex and hippocampus of TgCRND8 mice expressing the human Swedish (KM670/671NL) and Indiana (V717F) amyloid precursor protein mutations, which localized to ionized calcium binding protein-1-positive resident microglia that were not associated with extracellular plaques. Oral administration of 1-deoxy-1-fluoro-scyllo-inositol, a scyllo-inositol analogue, to TgCRND8 mice improved spatial memory impairments and suppressed amyloid pathology in a dose-dependent manner. Further, treatment with 1-deoxy-1- fluoro-scyllo-inositol significantly increased hippocampal intra-microglial Aβ levels without stimulating microglial proliferation or peripheral macrophage recruitment. These results reveal a novel, beneficial role for non-plaque associated microglia in the regulation of cerebral Aβ levels in a mouse model of AD.

}, keywords = {Alzheimer Disease, Amyloid beta-Peptides, Animals, Brain, Disease Models, Animal, Enzyme-Linked Immunosorbent Assay, Female, Humans, Immunohistochemistry, Inositol, Male, Maze Learning, Mice, Mice, Transgenic, Microglia, Neuroprotective Agents}, issn = {1875-5828}, doi = {10.2174/156720512803251084}, author = {Hawkes, Cheryl A and Deng, LeHua and Fenili, Daniela and Nitz, Mark and McLaurin, JoAnne} } @article {141, title = {Protecting-group-free synthesis of glycosyl 1-phosphates.}, journal = {Org Lett}, volume = {14}, year = {2012}, month = {2012 Aug 17}, pages = {4226-9}, abstract = {

Glycosyl 1-phosphates enriched in the α-anomer are obtained without the use of protecting groups in two steps starting from the free hemiacetal. Condensation of free hemiacetals with toluenesulfonylhydrazide yields a range of glycosylsulfonohydrazide donors which can be oxidized using cupric chloride in the presence of phosphoric acid and the coordinating additive 2-methyl-2-oxazoline to give useful yields of the fully deprotected glycosyl 1-phosphates.

}, keywords = {Combinatorial Chemistry Techniques, Copper, Glycosides, Glycosylation, Molecular Structure, Organophosphates, Phosphoric Acids, Stereoisomerism}, issn = {1523-7052}, doi = {10.1021/ol3019083}, author = {Edgar, Landon John G and Dasgupta, Somnath and Nitz, Mark} } @article {148, title = {Site-selective affinity labelling of maltose binding protein in bacterial cells.}, journal = {Chembiochem}, volume = {13}, year = {2012}, month = {2012 Apr 16}, pages = {783-7}, keywords = {Affinity Labels, Bacterial Proteins, Maltose, Maltose-Binding Proteins, Models, Molecular, Spectrophotometry, Ultraviolet, Substrate Specificity}, issn = {1439-7633}, doi = {10.1002/cbic.201200046}, author = {Rullo, Anthony and Beharry, Andrew A and G{\'o}mez-Biagi, Rodolfo F and Zhao, Xiong and Nitz, Mark} } @article {161, title = {The structure- and metal-dependent activity of Escherichia coli PgaB provides insight into the partial de-N-acetylation of poly-β-1,6-N-acetyl-D-glucosamine.}, journal = {J Biol Chem}, volume = {287}, year = {2012}, month = {2012 Sep 07}, pages = {31126-37}, abstract = {

Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In Escherichia coli, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-D-glucosamine (PNAG) by the periplasmic protein PgaB is required for polysaccharide intercellular adhesin-dependent biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of PgaB in complex with Ni(2+) and Fe(3+) have been determined to 1.9 and 2.1 {\r A} resolution, respectively, and its activity on β-1,6-GlcNAc oligomers has been characterized. The structure of PgaB reveals two (β/α)(x) barrel domains: a metal-binding de-N-acetylase that is a member of the family 4 carbohydrate esterases (CE4s) and a domain structurally similar to glycoside hydrolases. PgaB displays de-N-acetylase activity on β-1,6-GlcNAc oligomers but not on the β-1,4-(GlcNAc)(4) oligomer chitotetraose and is the first CE4 member to exhibit this substrate specificity. De-N-acetylation occurs in a length-dependent manor, and specificity is observed for the position of de-N-acetylation. A key aspartic acid involved in de-N-acetylation, normally seen in other CE4s, is missing in PgaB, suggesting that the activity of PgaB is attenuated to maintain the low levels of de-N-acetylation of PNAG observed in vivo. The metal dependence of PgaB is different from most CE4s, because PgaB shows increased rates of de-N-acetylation with Co(2+) and Ni(2+) under aerobic conditions, and Co(2+), Ni(2+) and Fe(2+) under anaerobic conditions, but decreased activity with Zn(2+). The work presented herein will guide inhibitor design to combat biofilm formation by E. coli and potentially a wide range of medically relevant bacteria producing polysaccharide intercellular adhesin-dependent biofilms.

}, keywords = {Acetylation, Amidohydrolases, beta-Glucans, Biofilms, Crystallography, X-Ray, Escherichia coli, Escherichia coli Proteins, Iron, Nickel, Protein Structure, Tertiary}, issn = {1083-351X}, doi = {10.1074/jbc.M112.390005}, author = {Little, Dustin J and Poloczek, Joanna and Whitney, John C and Robinson, Howard and Nitz, Mark and Howell, P Lynne} } @article {174, title = {Syntheses of carbocyclic analogues of α-D-glucosamine, α-D-mannose, α-D-mannuronic acid, β-L-idosamine, and β-L-gulose.}, journal = {J Org Chem}, volume = {77}, year = {2012}, month = {2012 Sep 07}, pages = {7401-10}, abstract = {

A versatile synthesis of orthogonally protected derivatives of carba-α-D-glucosamine, carba-α-D-mannose, carba-α-D-mannuronic acid, carba-β-L-idosamine, and carba-β-L-gulose from methyl α-D-mannoside is described. Our synthetic strategy utilizes the palladium-promoted Ferrier carbocyclization and persistent butane-2,3-diacetal protection to produce a key chiral cyclohexanone intermediate, from which all five carbasugar derivatives can readily be obtained.

}, keywords = {Carbohydrate Conformation, Glucosamine, Hexoses, Hexuronic Acids, Mannose, Stereoisomerism}, issn = {1520-6904}, doi = {10.1021/jo301240j}, author = {Sun, Yunshan and Nitz, Mark} } @article {103, title = {Synthesis and evaluation of inhibitors of E. coli PgaB, a polysaccharide de-N-acetylase involved in biofilm formation.}, journal = {Org Biomol Chem}, volume = {10}, year = {2012}, month = {2012 Sep 21}, pages = {7103-7}, abstract = {

Many medically important biofilm forming bacteria produce similar polysaccharide intercellular adhesins (PIA) consisting of partially de-N-acetylated β-(1 {\textrightarrow} 6)-N-acetylglucosamine polymers (dPNAG). In Escherichia coli, de-N-acetylation of the β-(1 {\textrightarrow} 6)-N-acetylglucosamine polymer (PNAG) is catalysed by the carbohydrate esterase family 4 deacetylase PgaB. The de-N-acetylation of PNAG is essential for productive PNAG-dependent biofilm formation. Here, we describe the development of a fluorogenic assay to monitor PgaB activity in vitro and the synthesis of a series of PgaB inhibitors. The synthesized inhibitors consist of a metal chelating functional group on a glucosamine scaffold to target the active site metal ion of PgaB. Optimal inhibition was observed with N-thioglycolyl amide (K(i) = 480 μM) and N-methyl-N-glycolyl amide (K(i) = 320 μM) glucosamine derivatives. A chemoenzymatic synthesis of an N-thioglycolyl amide PNAG pentasaccharide led to an inhibitor with an improved K(i) of 280 μM.

}, keywords = {Acetylglucosamine, Amidohydrolases, Biofilms, Catalytic Domain, Enzyme Inhibitors, Escherichia coli, Escherichia coli Infections, Escherichia coli Proteins, Glucosamine, Humans}, issn = {1477-0539}, doi = {10.1039/c2ob26105g}, author = {Chibba, Anthony and Poloczek, Joanna and Little, Dustin J and Howell, P Lynne and Nitz, Mark} } @article {157, title = {Characterization of the poly-β-1,6-N-acetylglucosamine polysaccharide component of Burkholderia biofilms.}, journal = {Appl Environ Microbiol}, volume = {77}, year = {2011}, month = {2011 Dec}, pages = {8303-9}, abstract = {

We demonstrated the production of poly-β-1,6-N-acetylglucosamine (PNAG) polysaccharide in the biofilms of Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia cepacia, and Burkholderia cenocepacia using an immunoblot assay for PNAG. These results were confirmed by further studies, which showed that the PNAG hydrolase, dispersin B, eliminated immunoreactivity of extracts from the species that were tested (B. cenocepacia and B. multivorans). Dispersin B also inhibited biofilm formation and dispersed preformed biofilms of Burkholderia species. These results imply a role for PNAG in the maintenance of Burkholderia biofilm integrity. While PNAG was present in biofilms of all of the wild-type test organisms, a ΔpgaBC mutant of B. multivorans (Mu5) produced no detectable PNAG, indicating that these genes are needed for Burkholderia PNAG formation. Furthermore, restoration of PNAG production in PNAG negative E. coli TRXWMGΔC (ΔpgaC) by complementation with B. multivorans pgaBCD confirmed the involvement of these genes in Burkholderia PNAG production. While the confocal scanning laser microscopy of untreated wild-type B. multivorans showed thick, multilayered biofilm, Mu5 and dispersin B-treated wild-type biofilms were thin, poorly developed, and disrupted, confirming the involvement of PNAG in B. multivorans biofilm formation. Thus, PNAG appears to be an important component of Burkholderia biofilms, potentially contributing to its resistance to multiple antibiotics and persistence during chronic infections, including cystic fibrosis-associated infection.

}, keywords = {Bacterial Proteins, beta-Glucans, Biofilms, Burkholderia, Escherichia coli, Gene Deletion, Genetic Complementation Test, Glycoside Hydrolases, Immunoblotting, Microscopy, Confocal}, issn = {1098-5336}, doi = {10.1128/AEM.05814-11}, author = {Yakandawala, Nandadeva and Gawande, Purushottam V and LoVetri, Karen and Cardona, Silvia T and Romeo, Tony and Nitz, Mark and Madhyastha, Srinivasa} } @article {166, title = {Comparisons of [18F]-1-deoxy-1-fluoro-scyllo-inositol with [18F]-FDG for PET imaging of inflammation, breast and brain cancer xenografts in athymic mice.}, journal = {Nucl Med Biol}, volume = {38}, year = {2011}, month = {2011 Oct}, pages = {953-9}, abstract = {

INTRODUCTION: The aim of the study was to evaluate the uptake of [(18)F]-1-deoxy-1-fluoro-scyllo-inositol ([(18)F]-scyllo-inositol) in human breast cancer (BC) and glioma xenografts, as well as in inflammatory tissue, in immunocompromised mice. Studies of [(18)F]-2-fluoro-2-deoxy-d-glucose ([(18)F]-FDG) under the same conditions were also performed.

METHODS: Radiosynthesis of [(18)F]-scyllo-inositol was automated using a commercial synthesis module. Tumour, inflammation and normal tissue uptakes were evaluated by biodistribution studies and positron emission tomography (PET) imaging using [(18)F]-scyllo-inositol and [(18)F]-FDG in mice bearing subcutaneous MDA-MB-231, MCF-7 and MDA-MB-361 human BC xenografts, intracranial U-87 MG glioma xenografts and turpentine-induced inflammation.

RESULTS: The radiosynthesis of [(18)F]-scyllo-inositol was automated with good radiochemical yields (24.6\%{\textpm}3.3\%, uncorrected for decay, 65{\textpm}2 min, n=5) and high specific activities (>=195 GBq/μmol at end of synthesis). Uptake of [(18)F]-scyllo-inositol was greatest in MDA-MB-231 BC tumours and was comparable to that of [(18)F]-FDG (4.6{\textpm}0.5 vs. 5.5{\textpm}2.1 \%ID/g, respectively; P=.40), but was marginally lower in MDA-MB-361 and MCF-7 xenografts. Uptake of [(18)F]-scyllo-inositol in inflammation was lower than [(18)F]-FDG. While uptake of [(18)F]-scyllo-inositol in intracranial U-87 MG xenografts was significantly lower than [(18)F]-FDG, the tumour-to-brain ratio was significantly higher (10.6{\textpm}2.5 vs. 2.1{\textpm}0.6; P=.001).

CONCLUSIONS: Consistent with biodistribution studies, uptake of [(18)F]-scyllo-inositol was successfully visualized by PET imaging in human BC and glioma xenografts, with lower accumulation in inflammatory tissue than [(18)F]-FDG. The tumour-to-brain ratio of [(18)F]-scyllo-inositol was also significantly higher than that of [(18)F]-FDG for visualizing intracranial glioma xenografts in NOD SCID mice, giving a better contrast.

}, keywords = {Animals, Biological Transport, Brain Neoplasms, Breast Neoplasms, Cell Line, Tumor, Female, Fluorodeoxyglucose F18, Humans, Inflammation, Inositol, Male, Mice, Mice, Inbred NOD, Mice, Nude, Mice, SCID, Neoplasm Transplantation, Positron-Emission Tomography, Radiochemistry, Transplantation, Heterologous}, issn = {1872-9614}, doi = {10.1016/j.nucmedbio.2011.02.017}, author = {McLarty, Kristin and Moran, Matthew D and Scollard, Deborah A and Chan, Conrad and Sabha, Nesrin and Mukherjee, Joydeep and Guha, Abhijit and McLaurin, JoAnne and Nitz, Mark and Houle, Sylvain and Wilson, Alan A and Reilly, Raymond M and Vasdev, Neil} } @article {155, title = {Development of mass cytometry methods for bacterial discrimination.}, journal = {Anal Biochem}, volume = {419}, year = {2011}, month = {2011 Dec 01}, pages = {1-8}, abstract = {

Fluorescent flow cytometry has become the method of choice for interrogation of bacterial populations at the single-cell level. However, limitations of this technique include issues of dynamic range, spectral overlap, photobleaching, and overall low signal intensity due to the small size of bacteria. The recent development of mass cytometry allows single-cell analysis with the resolution of inductively coupled plasma mass spectrometry, facilitating multiparametric analysis. Using a combination of a metal-based membrane stain and lectins conjugated to lanthanide-chelating polymers, we demonstrate that individual Escherichia coli cells can be differentiated based on their cell surface polysaccharides using mass cytometry. The model E. coli system involves evaluation of three different surface polysaccharides using element-tagged concanavalin A and wheat germ agglutinin lectins. Finally, this technique enabled experiments designed to follow the export of O-antigen substituted lipopolysaccharide in a conditional mutant. These studies revealed that the culture responds as a uniform population and that lipopolysaccharide export is approximately 10 times faster than the logarithmic bacterial doubling time.

}, keywords = {Cell Membrane, Cell Separation, Chelating Agents, Cytophotometry, Escherichia coli, Lanthanoid Series Elements, Lectins, Lipopolysaccharides, Mass Spectrometry, Mutation, Pentetic Acid, Single-Cell Analysis, Staining and Labeling}, issn = {1096-0309}, doi = {10.1016/j.ab.2011.07.035}, author = {Leipold, Michael D and Ornatsky, Olga and Baranov, Vladimir and Whitfield, Chris and Nitz, Mark} } @article {181, title = {Mono-acylation of a polyamine-β-cyclodextrin based on guest mediated acyl migration.}, journal = {Chem Commun (Camb)}, volume = {47}, year = {2011}, month = {2011 Aug 14}, pages = {8614-6}, abstract = {

The S {\textrightarrow} N acylation rates of thioester functionalized coumarins on heptakis-[6-deoxy-6-(2-aminoethylsulfanyl)]-β-cyclodextrin were measured. A high yield of mono-acylation was achieved with products that form self-inclusion compounds. The differential fluorescence response of the functionalized cyclodextrins upon binding biomacromolecules shows the potential of the constructs as probes.

}, keywords = {Acylation, Coumarins, Cyclodextrins, Polyamines}, issn = {1364-548X}, doi = {10.1039/c1cc12646f}, author = {G{\'o}mez-Biagi, Rodolfo F and Nitz, Mark} } @article {170, title = {Multiplexed protease assays using element-tagged substrates.}, journal = {Anal Biochem}, volume = {408}, year = {2011}, month = {2011 Jan 01}, pages = {157-9}, abstract = {

Inductively coupled plasma-mass spectrometry (ICP-MS)-based assays lend themselves to multiplexing due to the high resolution between mass channels, the sensitivity, and the reliability of the technique. Here the potential of ICP-MS-based protease assays is demonstrated with a quadruplex assay of cysteine proteases and metalloproteases. Four orthogonal peptide substrates were synthesized for the proteases calpain-1, caspase-3, matrix metalloprotease-9 (MMP-9), and a disintegrin and metalloprotease-10 (ADAM10). Each substrate carries a biotin tag at the C terminus and a diethylenetriaminepentaacetic acid (DTPA)-based lanthanide complex at the N terminus. The results demonstrate that this is a simple and reproducible analysis technique with excellent correlation between the single and multiplex assay formats.

}, keywords = {ADAM Proteins, Biotin, Calpain, Caspase 3, Cysteine Proteases, Disintegrins, Mass Spectrometry, Matrix Metalloproteinase 9, Metalloproteases, Streptavidin, Substrate Specificity}, issn = {1096-0309}, doi = {10.1016/j.ab.2010.09.008}, author = {Lathia, Urja S and Ornatsky, Olga and Baranov, Vladimir and Nitz, Mark} } @article {147, title = {Peptide-glycosaminoglycan cluster formation involving cell penetrating peptides.}, journal = {Biopolymers}, volume = {95}, year = {2011}, month = {2011 Oct}, pages = {722-31}, abstract = {

Glycosaminoglycans (GAGs) affect the efficiency of cellular uptake of a wide range of cell penetrating peptides (CPPs). GAGs have been proposed to cluster with CPPs at the cell surface before uptake but little is known about the formation or stability of CPP-GAG clusters. Here we apply a combination of heparin affinity chromatography, dynamic light scattering, and fluorescence spectroscopy to characterize the formation, stability, and size of the clusters formed between CPPs and heparin. Under conditions similar to those used in cell uptake experiments the CPP, penetratin (Antp), was observed to form significantly more stable clusters with heparin than the CPP TAT, despite TAT showing a comparable affinity for heparin. This difference in cluster stability may explain the origins of the preferred cell uptake pathways followed by Antp and TAT, and may be an important parameter for optimizing the efficiency of designed CPP delivery vectors.

}, keywords = {Amino Acid Sequence, Carrier Proteins, Cell-Penetrating Peptides, Gene Products, tat, Glycosaminoglycans, Heparin, Light, Macromolecular Substances, Molecular Sequence Data, Peptides, Protein Stability, Scattering, Radiation, Spectrometry, Fluorescence}, issn = {0006-3525}, doi = {10.1002/bip.21641}, author = {Rullo, Anthony and Qian, Jieshu and Nitz, Mark} } @article {163, title = {Sodium/myo-Inositol transporters: substrate transport requirements and regional brain expression in the TgCRND8 mouse model of amyloid pathology.}, journal = {PLoS One}, volume = {6}, year = {2011}, month = {2011}, pages = {e24032}, abstract = {

Inositol stereoisomers, myo- and scyllo-inositol, are known to enter the brain and are significantly elevated following oral administration. Elevations in brain inositol levels occur across a concentration gradient as a result of active transport from the periphery. There are two sodium/myo-inositol transporters (SMIT1, SMIT2) that may be responsible for regulating brain inositol levels. The goals of this study were to determine the effects of aging and Alzheimer{\textquoteright}s disease (AD)-like amyloid pathology on transporter expression, to compare regional expression and to analyze substrate requirements of the inositol transporters. QPCR was used to examine expression of the two transporters in the cortex, hippocampus and cerebellum of TgCRND8 mice, a mouse model of amyloid pathology, in comparison to non-transgenic littermates. In addition, we examined the structural features of inositol required for active transport, utilizing a cell-based competitive uptake assay. Disease pathology did not alter transporter expression in the cortex or hippocampus (p\>0.005), with only minimal effects of aging observed in the cerebellum (SMIT1: F(2,26) = 12.62; p = 0.0002; SMIT2: F(2,26) = 8.71; p = 0.0015). Overall, brain SMIT1 levels were higher than SMIT2, however, regional differences were observed. For SMIT1, at 4 and 6 months cerebellar SMIT1 levels were significantly higher than cortical and hippocampal levels (p\<0.05). For SMIT2, at all three ages both cortical and cerebellar SMIT2 levels were significantly higher than hippocampal levels (p\<0.05) and at 4 and 6 months of age, cerebellar SMIT2 levels were also significantly higher than cortical levels (p\<0.05). Inositol transporter levels are stably expressed as a function of age, and expression is unaltered with disease pathology in the TgCRND8 mouse. Given the fact that scyllo-inositol is currently in clinical trials for the treatment of AD, the stable expression of inositol transporters regardless of disease pathology is an important finding.

}, keywords = {Aging, Alzheimer Disease, Amyloid, Animals, Biological Transport, Brain, Gene Expression Regulation, Enzymologic, Inositol, Mice, Models, Animal, Sodium, Substrate Specificity, Symporters}, issn = {1932-6203}, doi = {10.1371/journal.pone.0024032}, author = {Fenili, Daniela and Weng, Ying-Qi and Aubert, Isabelle and Nitz, Mark and McLaurin, JoAnne} } @article {175, title = {Use of N,O-dimethylhydroxylamine as an anomeric protecting group in carbohydrate synthesis.}, journal = {J Org Chem}, volume = {76}, year = {2011}, month = {2011 Mar 18}, pages = {1918-21}, abstract = {

The N,O-dimethyloxyamine-N-glycosides are introduced as anomerically protected building blocks for carbohydrate synthesis. These N-glycosides are stable to a variety of protecting group manipulations including acylation, alkylation, silylation, and acetal formation. The alkoxyamine-N-glycosides can be cleaved selectively with N-chlorosuccinimide to give the desired hemiacetals in excellent yield. Furthermore, these N-glycosides are stable to the activation conditions required for glycosylation using thioglycoside and trichloroacetimidate glycosyl donors suggesting N,O-dialkoxyamine-N-glycosides will be useful for complex oligosaccharide synthesis.

}, keywords = {Dimethylamines, Glycosylation, Oligosaccharides}, issn = {1520-6904}, doi = {10.1021/jo102372m}, author = {Dasgupta, Somnath and Nitz, Mark} } @article {176, title = {Development of inductively coupled plasma-mass spectrometry-based protease assays.}, journal = {Anal Biochem}, volume = {398}, year = {2010}, month = {2010 Mar 01}, pages = {93-8}, abstract = {

Rapid, sensitive, and quantitative assays for proteases are important for drug development and in the diagnosis of disease. Here an assay for protease activity that uses inductively coupled plasma-mass spectrometry (ICP-MS) detection is described. Peptidic alpha-chymotrypsin substrates were synthesized containing a lanthanide ion chelate at the N terminus to provide a distinct elemental tag. A biotin label was appended to the C terminus of the peptide, allowing separation of uncleaved peptide from the enzymatic digestion. The enzyme activity was determined by quantifying the lanthanide ion signal of the peptide cleavage products by ICP-MS. Biotinylated substrates synthesized include Lu-DTPA-Asp-Leu-Leu-Val-Tyr approximately Asp-Lys(biotin) and Lu-DTPA-betaAla-betaAla-betaAla-betaAla-Gly-Ser-Ala-Tyr approximately Gly-Lys-Arg-Lys(biotin)-amide. Parallel assays with a commercially available fluorogenic substrate (Suc-AAPF-AMC) for alpha-chymotrypsin were performed for comparison. Using the ICP-MS assay, enzyme concentrations as low as 2pM could be readily detected, superior to the detection limit of an assay using the alpha-chymotrypsin fluorogenic substrate (Suc-AAPF-AMC). Furthermore, we demonstrated the use of this approach to detect chymotrypsin activity in HeLa cell lysates.

}, keywords = {Amino Acid Sequence, Biotin, Chymotrypsin, Enzyme Assays, Fluorescent Dyes, HeLa Cells, Humans, Lanthanoid Series Elements, Mass Spectrometry, Substrate Specificity}, issn = {1096-0309}, doi = {10.1016/j.ab.2009.11.010}, author = {Lathia, Urja S and Ornatsky, Olga and Baranov, Vladimir and Nitz, Mark} } @article {134, title = {Highly multiparametric analysis by mass cytometry.}, journal = {J Immunol Methods}, volume = {361}, year = {2010}, month = {2010 Sep 30}, pages = {1-20}, abstract = {

This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not require compensation, allowing the application of statistical techniques; this has been impossible given the constraints of fluorescence noise with traditional cytometry instruments. Instead of "colors" the mass cytometer "reads" the stable isotope tags attached to antibodies using metal-chelating labeling reagents. Because there are many available stable isotopes, and the mass spectrometer provides exquisite resolution between detection channels, many parameters can be measured as easily as one. For example, in a single tube the technique allows for the ready detection and characterization of the major cell subsets in blood or bone marrow. Here we describe mass cytometric immunophenotyping of human leukemia cell lines and leukemia patient samples, differential cell analysis of normal peripheral and umbilical cord blood; intracellular protein identification and metal-encoded bead arrays.

}, keywords = {Bone Marrow Cells, Chelating Agents, Fetal Blood, HL-60 Cells, Humans, Immunophenotyping, Jurkat Cells, Lanthanoid Series Elements, Leukemia, Mass Spectrometry, Microspheres}, issn = {1872-7905}, doi = {10.1016/j.jim.2010.07.002}, author = {Ornatsky, Olga and Bandura, Dmitry and Baranov, Vladimir and Nitz, Mark and Winnik, Mitchell A and Tanner, Scott} } @article {179, title = {Importance of the spatial display of charged residues in heparin-peptide interactions.}, journal = {Biopolymers}, volume = {93}, year = {2010}, month = {2010 Mar}, pages = {290-8}, abstract = {

Many studies have examined consensus sequences required for protein-glycosaminoglycan interactions. Through the synthesis of helical heparin binding peptides, this study probes the relationship between spatial arrangement of positive charge and heparin binding affinity. Peptides with a linear distribution of positive charge along one face of the alpha-helix had the highest affinity for heparin. Moving the basic residues away from a single face resulted in drastic changes in heparin binding affinity of up to three orders of magnitude. These findings demonstrate that amino acid sequences, different from the known heparin binding consensus sequences, will form high affinity protein-heparin binding interactions when the charged residues are aligned linearly.

}, keywords = {Amino Acid Sequence, Amino Acids, Heparin, Models, Molecular, Molecular Sequence Data, Peptides, Protein Structure, Secondary}, issn = {0006-3525}, doi = {10.1002/bip.21339}, author = {Rullo, Anthony and Nitz, Mark} } @article {168, title = {Small molecule beta-amyloid inhibitors that stabilize protofibrillar structures in vitro improve cognition and pathology in a mouse model of Alzheimer{\textquoteright}s disease.}, journal = {Eur J Neurosci}, volume = {31}, year = {2010}, month = {2010 Jan}, pages = {203-13}, abstract = {

Beta-amyloid (Abeta) peptides are thought to play a major role in the pathogenesis of Alzheimer{\textquoteright}s disease. Compounds that disrupt the kinetic pathways of Abeta aggregation may be useful in elucidating the role of oligomeric, protofibrillar and fibrillar Abeta in the etiology of the disease. We have previously reported that scyllo-inositol inhibits Abeta(42) fibril formation but the mechanism(s) by which this occurs has not been investigated in detail. Using a series of scyllo-inositol derivatives in which one or two hydroxyl groups were replaced with hydrogen, chlorine or methoxy substituents, we examined the role of hydrogen bonding and hydrophobicity in the structure-function relationship of scyllo-inositol-Abeta binding. We report here that all scyllo-inositol derivatives demonstrated reduced effectiveness in preventing Abeta(42) fibrillization compared with scyllo-inositol, suggesting that scyllo-inositol interacts with Abeta(42) via key hydrogen bonds that are formed by all hydroxyl groups. Increasing the hydrophobicity of scyllo-inositol by the addition of two methoxy groups (1,4-di-O-methyl-scyllo-inositol) produced a derivative that stabilized Abeta(42) protofibrils in vitro. Prophylactic administration of 1,4-di-O-methyl-scyllo-inositol to TgCRND8 mice attenuated spatial memory impairments and significantly decreased cerebral amyloid pathology. These results suggest that Abeta aggregation can be targeted at multiple points along the kinetic pathway for the improvement of Alzheimer{\textquoteright}s disease-like pathology.

}, keywords = {Alzheimer Disease, Amyloid, Amyloid beta-Peptides, Animals, Cognition, Female, Hydrogen Bonding, Inositol, Male, Memory, Mice, Mice, Transgenic, Molecular Structure, Neuropsychological Tests, Peptide Fragments, Protein Conformation}, issn = {1460-9568}, doi = {10.1111/j.1460-9568.2009.07052.x}, author = {Hawkes, Cheryl A and Deng, Le-Hua and Shaw, James E and Nitz, Mark and McLaurin, JoAnne} } @article {182, title = {Synthesis of a core disaccharide from the Streptococcus pneumoniae type 23F capsular polysaccharide antigen.}, journal = {Carbohydr Res}, volume = {345}, year = {2010}, month = {2010 Oct 13}, pages = {2282-6}, abstract = {

The synthesis of methyl α-l-rhamnopyranosyl-(1{\textrightarrow}2)-β-d-galactopyranoside and methyl α-l-rhamnopyranosyl-(1{\textrightarrow}2)-3-(glycer-2-yl-phosphate)-β-d-galactopyranoside disaccharides from the Streptococcuspneumoniae type 23F capsular polysaccharide is reported. A simple protecting group strategy was followed using commercially available monosaccharides and phosphorylating reagents. H-Phosphonate and phosphoramidite coupling chemistries were explored for introducing the phosphodiester. Hydrazine hydrate was found to be a mild and efficient deacetylating agent, which was required to avoid phosphate migration during the deprotection of the phosphodiester functionalized disaccharide.

}, keywords = {Bacterial Capsules, Carbohydrate Sequence, Disaccharides, Polysaccharides, Bacterial, Streptococcus pneumoniae}, issn = {1873-426X}, doi = {10.1016/j.carres.2010.08.002}, author = {Dasgupta, Somnath and Nitz, Mark} } @article {162, title = {Synthesis of a functional metal-chelating polymer and steps toward quantitative mass cytometry bioassays.}, journal = {Anal Chem}, volume = {82}, year = {2010}, month = {2010 Nov 01}, pages = {8961-9}, abstract = {

We describe the synthesis and characterization of metal-chelating polymers with a degree of polymerization of 67 and 79, high diethylenetriaminepentaacetic acid (DTPA) functionality, M(w)/M(n) <= 1.17, and a maleimide as an orthogonal functional group for conjugation to antibodies. The polymeric disulfide form of the DP(n) = 79 DTPA polymer was analyzed by thermogravimetric analysis to determine moisture and sodium-ion content and by isothermal titration calorimetry (ITC) to determine the Gd(3+) binding capacity. These results showed each chain binds 68 {\textpm} 7 Gd(3+) per chain. Secondary goat antimouse IgG was covalently labeled with the maleimide form of the DTPA polymer (DP(n) = 79) carrying (159)Tb. Conventional ICPMS analysis of this conjugate showed each antibody carried an average of 161 {\textpm} 4 (159)Tb atoms. This result was combined with the ITC result to show there are an average of 2.4 {\textpm} 0.3 polymer chains attached to each antibody. Eleven monoclonal primary antibodies were labeled with different lanthanide isotopes using the same labeling methodology. Single cell analysis of whole umbilical cord blood stained with a mixture of 11 metal-tagged antibodies was performed by mass cytometry.

}, keywords = {Animals, Antibodies, Monoclonal, Calorimetry, Chelating Agents, Fetal Blood, Flow Cytometry, Humans, Lanthanoid Series Elements, Metals, Pentetic Acid, Polymers, Staining and Labeling}, issn = {1520-6882}, doi = {10.1021/ac101901x}, author = {Majonis, Daniel and Herrera, Isaac and Ornatsky, Olga and Schulze, Maren and Lou, Xudong and Soleimani, Mohsen and Nitz, Mark and Winnik, Mitchell A} } @article {164, title = {Efficient synthesis and protein conjugation of beta-(1-->6)-D-N-acetylglucosamine oligosaccharides from the polysaccharide intercellular adhesin.}, journal = {Carbohydr Res}, volume = {344}, year = {2009}, month = {2009 Mar 31}, pages = {570-5}, abstract = {

A wide variety of medically important biofilm forming bacteria produce similar polysaccharide intracellular adhesins (PIAs). The PIA structures consist of partially de-N-acetylated beta-(1--\>6)-N-acetylglucosamine polymers. These exopolysaccharides are key components of the bacterial biofilm matrix. Here, we describe the efficient synthesis of PIA oligosaccharides using an acid reversion reaction of N-acetylglucosamine in HF.pyridine. The PIA oligosaccharides produced by this reaction can be purified to homogeneity by size exclusion chromatography. Chemistry was developed to conjugate the PIA oligosaccharides to bovine serum albumin using a new heterobifunctional linker containing a thiol and an N-methylhydroxylamine functional group. These glycoconjugates may serve as useful precursors for the development of defined conjugate vaccines against PIA producing bacterial strains.

}, keywords = {Acetylglucosamine, Carbohydrate Sequence, Glycoconjugates, Models, Chemical, Molecular Sequence Data, Molecular Structure, Oligosaccharides, Polysaccharides, Bacterial, Serum Albumin, Bovine}, issn = {1873-426X}, doi = {10.1016/j.carres.2008.12.021}, author = {Leung, Carmen and Chibba, Anthony and G{\'o}mez-Biagi, Rodolfo F and Nitz, Mark} } @article {158, title = {ICP-MS-based multiplex profiling of glycoproteins using lectins conjugated to lanthanide-chelating polymers.}, journal = {J Proteome Res}, volume = {8}, year = {2009}, month = {2009 Feb}, pages = {443-9}, abstract = {

Lectins have been increasingly important in the study of glycoproteins. Here, we report a glycoprofiling method based on the covalent attachment of metal-chelating polymers to lectins for use in an ICP-MS-based assay. The labeled lectins are able to distinguish between glycoproteins covalently attached to a microtiter plate and their binding can be directly quantified by ICP-MS. Since each conjugate contains a different lanthanide, the assays can be conducted in a single or multiplex fashion, and may be readily elaborated to many different assay formats.

}, keywords = {Animals, Chelating Agents, Glycoproteins, Lanthanoid Series Elements, Lectins, Mass Spectrometry, Models, Molecular, Molecular Structure, Polymers, Protein Array Analysis}, issn = {1535-3893}, doi = {10.1021/pr800645r}, author = {Leipold, Michael D and Herrera, Isaac and Ornatsky, Olga and Baranov, Vladimir and Nitz, Mark} } @article {180, title = {Pattern-based recognition of heparin contaminants by an array of self-assembling fluorescent receptors.}, journal = {Angew Chem Int Ed Engl}, volume = {48}, year = {2009}, month = {2009}, pages = {1995-7}, abstract = {

Tracking down potential killers: Strong host-guest interactions enable the facile combination of polycationic cyclodextrin binding motifs (blue) with fluorescent reporters (orange) tethered to a hydrophobic guest molecule (dark green). An array of supramolecular fluorescent receptors prepared by this modular approach was used for the pattern-based recognition of negatively charged contaminants in the anticoagulant drug heparin.

}, keywords = {Anticoagulants, Cyclodextrins, Drug Contamination, Fluorescent Dyes, Heparin, Molecular Conformation, Polymers}, issn = {1521-3773}, doi = {10.1002/anie.200805238}, author = {Jagt, Richard B C and G{\'o}mez-Biagi, Rodolfo F and Nitz, Mark} } @article {169, title = {Preparative size-exclusion chromatography for purification and characterization of colloidal quantum dots bound by chromophore-labeled polymers and low-molecular-weight chromophores.}, journal = {J Chromatogr A}, volume = {1216}, year = {2009}, month = {2009 Jun 19}, pages = {5011-9}, abstract = {

We explore the use of preparative size-exclusion chromatography (SEC) and high-performance liquid chromatography (HPLC) to purify quantum dots (QDs) after surface modification. In one example, in which Bio-Beads (S-X1) were used as the packing material for the preparative SEC column, CdSe QDs treated with a functional coumarin dye could be separated from the excess free dye by using tetrahydrofuran (THF) as the mobile phase. This column was unable to separate polymer-coated QDs from free polymer (M approximately 8000) because of the relatively low cutoff mass of the column. Here a preparative HPLC column packed with TOYOPEARL gel allowed the effective separation of polymer-bound QDs from the excess free polymer by using N-methyl-2-pyrrolidinone (NMP) as the mobile phase. When other solvents such as absolute ethanol, acetonitrile, THF, and THF-triethylamine mixtures were used as the eluent, QDs stuck to the column. While NMP was an effective medium to remove excess free polymer from the QDs, it was difficult to transfer the purified QDs to more volatile solvents and maintain colloidal stability.

}, keywords = {Chromatography, Gel, Chromatography, High Pressure Liquid, Colloids, Coloring Agents, Furans, Molecular Weight, Organophosphorus Compounds, Polymers, Pyrrolidinones, Quantum Dots, Solvents}, issn = {1873-3778}, doi = {10.1016/j.chroma.2009.04.060}, author = {Wang, Mingfeng and Bardajee, Ghasem Rezanejade and Kumar, Sandeep and Nitz, Mark and Scholes, Gregory D and Winnik, Mitchell A} } @article {159, title = {Stability studies of hydrazide and hydroxylamine-based glycoconjugates in aqueous solution.}, journal = {Carbohydr Res}, volume = {344}, year = {2009}, month = {2009 Feb 17}, pages = {278-84}, abstract = {

Glycoconjugates can be readily formed by the condensation of a free-reducing terminus and a strong alpha-effect nucleophile, such as a hydrazide or a hydroxylamine. Further characterization of a series of glycoconjugates formed from xylose, glucose and N-acetylglucosamine, and either p-toluenesulfonyl hydrazide or an N-methylhydroxylamine, was carried out to gain insight into the optimal conditions for the formation of these useful conjugates, and their stability. Their apparent association constants (9-74 M(-1)) at pH 4.5; as well, as rate constants for hydrolysis, at pH 4.0, 5.0 and 6.0 (37 degrees C), were determined. The half-lives of the conjugates varied between 3h and 300 days. All the compounds were increasingly stable as the pH approached neutrality. Conjugate hydrolysis rates mirrored those found for O-glycoside hydrolysis where conjugates formed from electron-rich monosaccharides hydrolyzed more rapidly.

}, keywords = {Carbohydrate Sequence, Drug Stability, Glycoconjugates, Hydrazines, Hydrogen-Ion Concentration, Hydrolysis, Hydroxylamines, Molecular Structure, Solutions, Water}, issn = {1873-426X}, doi = {10.1016/j.carres.2008.11.007}, author = {Gudmundsdottir, Anna V and Paul, Caroline E and Nitz, Mark} } @article {152, title = {Synthesis and preliminary biological evaluations of [18F]-1-deoxy-1-fluoro-scyllo-inositol.}, journal = {Chem Commun (Camb)}, year = {2009}, month = {2009 Oct 07}, pages = {5527-9}, abstract = {

A novel PET radiotracer, [18F]-1-deoxy-1-fluoro-scyllo-inositol, was synthesized via a one-pot reaction in 16 +/- 3\% uncorrected radiochemical yield within 80 minutes; although this compound revealed low brain penetration it shows promise in rodent tumour models for breast cancer imaging.

}, keywords = {Animals, Cell Line, Tumor, Fluorine Radioisotopes, Humans, Inositol, Male, Mice, Positron-Emission Tomography, Radiopharmaceuticals, Rats, Rats, Sprague-Dawley, Stereoisomerism, Transplantation, Heterologous}, issn = {1364-548X}, doi = {10.1039/b913317h}, author = {Vasdev, Neil and Chio, Jason and van Oosten, Erik M and Nitz, Mark and McLaurin, JoAnne and Vines, Douglass C and Houle, Sylvain and Reilly, Raymond M and Wilson, Alan A} } @article {167, title = {Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry.}, journal = {J Anal At Spectrom}, volume = {23}, year = {2008}, month = {2008}, pages = {463-469}, abstract = {

Advances in the development of highly multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are discussed. Use of novel reagents specifically designed for immunological methods utilizing elemental analysis is presented. The major steps of method development, including selection of elements for tags, validation of tagged reagents, and examples of multiplexed assays, are considered in detail. The paper further describes experimental protocols for elemental tagging of antibodies, immunostaining of live and fixed human leukemia cells, and preparation of samples for ICP-MS analysis. Quantitative analysis of surface antigens on model cell lines using a cocktail of seven lanthanide labeled antibodies demonstrated high specificity and concordance with conventional immunophenotyping.

}, issn = {0267-9477}, doi = {10.1039/b710510j}, author = {Ornatsky, Olga I and Kinach, Robert and Bandura, Dmitry R and Lou, Xudong and Tanner, Scott D and Baranov, Vladimir I and Nitz, Mark and Winnik, Mitchell A} } @article {133, title = {Heparin dependent coiled-coil formation.}, journal = {Chembiochem}, volume = {9}, year = {2008}, month = {2008 Jul 02}, pages = {1545-8}, keywords = {Circular Dichroism, Disulfides, Heparin, Peptides, Protein Conformation, Proteins}, issn = {1439-7633}, doi = {10.1002/cbic.200800056}, author = {Nitz, Mark and Rullo, Anthony and Ding, Matias Xiao Yue} } @article {178, title = {Modulation of amyloid-beta aggregation and toxicity by inosose stereoisomers.}, journal = {FEBS J}, volume = {275}, year = {2008}, month = {2008 Apr}, pages = {1663-74}, abstract = {

Amyloid-beta (Abeta) aggregation and amyloid formation are key pathological features of Alzheimer{\textquoteright}s disease, and are considered to be two of the major contributing factors to neurodegeneration and dementia. Identification of small molecule inhibitors that are orally available, have low toxicity and high central nervous system bioavailability is one approach to the potential development of a disease-modifying treatment for Alzheimer{\textquoteright}s disease. We have previously identified inositol stereoisomers as exhibiting stereospecific inhibition of Abeta aggregation and toxicity in vitro and in vivo. We report here the effects of inosose versus inositol stereoisomers on Abeta fibrillogenesis as determined using CD and fluorescence spectroscopy and negative-stain electron microscopy. The inososes differ from inositols by the oxidation of one of the hydroxyl groups to a ketone. These molecules help in the further elucidation of the structure-activity relationships of inositol-Abeta interactions and identify both allo-inositol and epi-2-inosose as in vitro inhibitors of Abeta aggregation.

}, keywords = {Amyloid beta-Peptides, Animals, Biological Transport, Cell Survival, Circular Dichroism, Inositol, Microscopy, Electron, Molecular Structure, PC12 Cells, Rats, Stereoisomerism, Symporters}, issn = {1742-464X}, doi = {10.1111/j.1742-4658.2008.06321.x}, author = {Nitz, Mark and Fenili, Daniela and Darabie, Audrey A and Wu, Ling and Cousins, Julian E and McLaurin, JoAnne} } @article {156, title = {Protecting group free glycosidations using p-toluenesulfonohydrazide donors.}, journal = {Org Lett}, volume = {10}, year = {2008}, month = {2008 Aug 21}, pages = {3461-3}, abstract = {

N{\textquoteright}-Glycopyranosylsulfonohydrazides are introduced as glycosyl donors for protecting group free synthesis of O-glycosides, glycosyl azides, and oxazolines. Mono- and disaccharides containing a reducing terminal N-acetylglucosamine residue were condensed with p-toluenesulfonylhydrazide to give the desired beta- d-pyranose donors. These donors can be activated with NBS and then glycosidated with the desired alcohol or transformed to the oxazoline or glycosyl azide.

}, keywords = {Azides, Glycosides, Glycosylation, Molecular Structure, Oxazoles, Stereoisomerism, Tosyl Compounds}, issn = {1523-7052}, doi = {10.1021/ol801232f}, author = {Gudmundsdottir, Anna V and Nitz, Mark} } @article {172, title = {Remarkably stable inclusion complexes with heptakis-[6-deoxy-6-(2-aminoethylsulfanyl)]-beta-cyclodextrin.}, journal = {Org Biomol Chem}, volume = {6}, year = {2008}, month = {2008 Dec 21}, pages = {4622-6}, abstract = {

Complexes of heptakis-[6-deoxy-6-(2-aminoethylsulfanyl)]-beta-cyclodextrin (1) and a series of common cyclodextrin guests were studied by NMR, fluorescence spectroscopy, and ITC experiments. NMR conformational analysis shows that the thioethers of 1 are positioned over the hydrophobic cavity of the cyclodextrin, increasing potential hydrophobic interactions with guest molecules. The combination of the increased hydrophobic character, the electrostatic complementarity and a hypothesized conformational change in 1 lead to a complex with the dye 2,6-ANS (5) that is over 2000 times more stable than with the native beta-cyclodextrin. One of the most stable host-guest complexes between a cyclodextrin and a small molecule measured to date was revealed between 1 and lithocholic acid (4) with an association constant of 5.5 x 10(7) M(-1).

}, issn = {1477-0539}, doi = {10.1039/b813999g}, author = {G{\'o}mez-Biagi, Rodolfo F and Jagt, Richard B C and Nitz, Mark} } @article {165, title = {Synthesis of scyllo-inositol derivatives and their effects on amyloid beta peptide aggregation.}, journal = {Bioorg Med Chem}, volume = {16}, year = {2008}, month = {2008 Aug 01}, pages = {7177-84}, abstract = {

scyllo-Inositol has shown promise as a potential therapeutic for Alzheimer{\textquoteright}s disease, by directly interacting with the amyloid beta (Abeta) peptide to inhibit Abeta42 fiber formation. To explore the molecular details of the inositol-Abeta42 interaction, a series of scyllo-inositol derivatives have been synthesized which contain deoxy, fluoro, chloro, and methoxy substitutions. The effects of these compounds on the aggregation cascade of Abeta42 have been investigated using electron microscopy (EM). EM analyses revealed that the 1-deoxy-1-fluoro- and 1,4-dimethyl-scyllo-inositols significantly inhibit the formation of Abeta42 fibers. The other derivatives showed some alterations in the morphology of the Abeta42 fibers produced. These findings indicate the importance of all of the hydroxyl groups of scyllo-inositol for complete inhibition of Abeta aggregation.

}, keywords = {Alzheimer Disease, Amyloid beta-Peptides, Carbohydrate Conformation, Inositol}, issn = {1464-3391}, doi = {10.1016/j.bmc.2008.06.045}, author = {Sun, Yedi and Zhang, Guohua and Hawkes, Cheryl A and Shaw, James E and McLaurin, JoAnne and Nitz, Mark} } @article {173, title = {Designing fluorescent sensors of heparin.}, journal = {Chembiochem}, volume = {8}, year = {2007}, month = {2007 Mar 05}, pages = {391-4}, keywords = {Anticoagulants, Biosensing Techniques, Drug Design, Fluorescent Dyes, Heparin, Laminin, Peptide Fragments, Quinolinium Compounds}, issn = {1439-4227}, doi = {10.1002/cbic.200600361}, author = {Sauceda, Jimena C and Duke, Rebecca M and Nitz, Mark} } @article {177, title = {Hydrolysis rates of 1-glucosyl-2-benzoylhydrazines in aqueous solution.}, journal = {Carbohydr Res}, volume = {342}, year = {2007}, month = {2007 Apr 09}, pages = {749-52}, abstract = {

A series of substituted 1-glucosyl-2-benzoylhydrazines were synthesized and their pseudo-first-order rate constants for hydrolysis were determined at pH 4, 5, 6 at 50 degrees C. All the compounds hydrolyzed quickly (t(1/2)\<3h) at pH 4.0, but were increasingly stable as the pH approached neutrality.

}, keywords = {Glycoconjugates, Hydrazines, Hydrogen-Ion Concentration, Hydrolysis, Kinetics, Temperature}, issn = {0008-6215}, doi = {10.1016/j.carres.2006.12.015}, author = {Gudmundsdottir, Anna V and Nitz, Mark} } @article {146, title = {Polymer-based elemental tags for sensitive bioassays.}, journal = {Angew Chem Int Ed Engl}, volume = {46}, year = {2007}, month = {2007}, pages = {6111-4}, keywords = {Antibodies, Antigens, Surface, Chelating Agents, Immunoassay, Indicators and Reagents, Polymers}, issn = {1433-7851}, doi = {10.1002/anie.200700796}, author = {Lou, Xudong and Zhang, Guohua and Herrera, Isaac and Kinach, Robert and Ornatsky, Olga and Baranov, Vladimir and Nitz, Mark and Winnik, Mitchell A} } @article {160, title = {Lanthanide-binding tags as versatile protein coexpression probes.}, journal = {Chembiochem}, volume = {4}, year = {2003}, month = {2003 Apr 04}, pages = {265-71}, abstract = {

Comprehensive proteomic analyses require new methodologies to accelerate the correlation of gene sequence with protein function. Key tools for such efforts include biophysical probes that integrate into the covalent architecture of proteins. Lanthanide-binding tags (LBTs) are expressible, multitasking fusion partners that are optimized to bind lanthanide ions and have several desirable attributes, which include long-lived luminescence, excellent X-ray scattering power for phase determination, and magnetic properties to facilitate NMR spectroscopic structure elucidation. Herein, we present peptide sequences with a 40-fold higher affinity for Tb(3+) ions and significantly brighter luminescence intensity compared with existing peptides. Incorporation of an LBT onto ubiquitin as a prototype fusion protein allows the use of powerful protein-visualization techniques, which include rapid luminescence detection of LBT-tagged proteins in SDS-PAGE gels, as well as determination of protein concentrations in complex mixtures. The LBT strategy is a new alternative for expressing fluorescent fusion proteins by routine molecular biological techniques.

}, keywords = {Amino Acid Sequence, Binding Sites, Combinatorial Chemistry Techniques, Electrophoresis, Polyacrylamide Gel, Gels, Luminescent Measurements, Molecular Probes, Peptides, Protein Binding, Recombinant Fusion Proteins, Terbium, Ubiquitin}, issn = {1439-4227}, doi = {10.1002/cbic.200390046}, author = {Franz, Katherine J and Nitz, Mark and Imperiali, Barbara} }