View details for DOI 10.1073/pnas.0403681101, View details for Web of Science ID 000225508400004, View details for PubMedCentralID PMC534710, View details for DOI 10.1074/jbc.M406397200, View details for Web of Science ID 000225229500103. Given the widespread occurrence of eukaryotic-like Ser/Thr protein kinases in bacteria, these proteins may play a broad role in bacterial osmosensing. 93, 25452566 (1993). Branching morphogenesis in the mammary gland is achieved by the migration of epithelial cells through a microenvironment consisting of stromal cells and extracellular matrix (ECM). Nano Lett. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental, and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). B 119, 84068418 (2015). Sci. Phys. In this study we probed the importance of the stem region with respect to substrate preference, localization, and oligomerization. Wang, Z. et al. Individuals with GeneXpert-positive pulmonary TB were sampled pre-treatment over 60-minutes. Sulfated molecules have been shown to modulate isotypic interactions between cells of metazoans and heterotypic interactions between bacterial pathogens or symbionts and their eukaryotic host cells. A., Bertozzi, C. R. Investigation of the iron-sulfur cluster in Mycobacterium tuberculosis APS reductase: Implications for substrate binding and catalysis. Chutia, A., Hamada, I. The metaluminous rocks tend to have a lower Fe-number, whereas the peraluminous rocks tend to have the higher Fe-number. Attachment via these "cell-adhesion barcodes" is rapid and specific, with close-packed arrays of cells forming within minutes. Proteasome inhibitors are used to treat blood cancers such as multiple myeloma (MM) and mantle cell lymphoma. Shin, S., Comolli, L. R., Tscheliessnig, R., Wang, C., Ki Tae Nam, K. T., Hexemer, A., Siegerist, C. E., De Yoreo, J. J., Bertozzi, C. R. D-Amino Acid Chemical Reporters Reveal Peptidoglycan Dynamics of an Intracellular Pathogen. This nucleotide sugar was readily accepted by fucosyltransferases and provided robust cell-surface labeling of fucosylated glycans, as determined by flow cytometry and confocal microscopy analysis. One such glycolipid, Sulfolipid-1 (SL-1), consists of a trehalose-2-sulfate (T2S) core acylated with four lipid moieties. Intell. Res. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. In the past decade advances in genomics, proteomics and mass spectrometry have enabled the association of specific glycan structures with disease states. Mycobacterium tuberculosis possesses an unusual cell wall that is replete with virulence-enhancing lipids. Emery, J. D. et al. StcEE447D is a pan-mucin stain derived from enterohemorrhagic Escherichia coli that is tolerant to a wide range of glycoforms. B 106, 1199311996 (2002). We synthesized a 1338 member library of uridine analogs directed to the epimerase by virtue of substrate mimicry. We have defined the trends on the MALI diagrams using a large number of granitic batholiths, which suggests that most granitoid plutons follow sub-parallel alkalilime trends during differentiation. We also show that the integral membrane protein Sap and MmpL8 are both essential for sulfolipid transport. Immunization of mice with either BCG or DeltacysH followed by infection with the virulent M. tuberculosis Erdman strain demonstrated that DeltacysH can generate protection equivalent to that of the BCG vaccine. Chemical or genetic disruption of NGLY1 activity results in the accumulation of misprocessed Nrf1 that is largely excluded from the nucleus. Res. Zhou, L. et al. Pathways and challenges for efficient solar-thermal desalination. Sanders, W. J., Katsumoto, T. R., Bertozzi, C. R., ROSEN, S. D., Kiessling, L. L. Synthesis of beta-C-glycosides of N-acetylglucosamine via Keck allylation directed by neighboring phthalimide groups. These probes enable real-time imaging of trehalose mycolate biosynthesis and mycomembrane dynamics in the laboratory as well as in clinical settings for the detection of mycobacteria in patient samples. Grossman, H. L., Myers, W. R., Vreeland, V. J., Bruehl, R., Alper, M. D., Bertozzi, C. R., Clarke, J. Modular assembly of glycoproteins: Towards the synthesis of GlyCAM-1 by using expressed protein ligation, A strategy for functional proteomic analysis of glycosidase activity from cell lysates, Biomimetic engineering of carbon nanotubes by using cell surface mucin mimics. A., Dube, D. H., Bertozzi, C. R. Identification, function and structure of the mycobacterial sulfotransferase that initiates sulfolipid-1 biosynthesis. TPI patients with other mutations generally show longer life expectancy. Glycoproteins are typically expressed as mixtures of glycoforms, their oligosaccharides being generated by a template-independent biosynthetic process. Although experiments with organisms in vitro have yielded a wealth of information on PG synthesis and maturation, it is unclear how these studies translate to bacteria replicating within host cells. Pi, N., Armstrong, J. I., Bertozzi, C. R., Leary, J. The problem is formulated by the Rayleigh-Plesset dynamical equation, whose numerical solutions are carried out by Maple. View details for DOI 10.1021/acs.analchem.0c02950. More broadly, our method will enable the discovery of signal-specific O-GlcNAcylation events in a wide array of experimental contexts. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Ren, H. et al. View details for Web of Science ID 000187945400003. Each enzyme preferred a terminal GlcNAc residue, and was impeded by the addition of a beta1,4-linked Gal residue (i.e., terminal LacNAc). We discuss the use of fluorescent and fluorogenic trehalose probes for the detection of the mycobacterial trehalose glycolipids. Recent work has implicated tyrosine sulfate as a determinant of protein-protein interactions involved in leukocyte adhesion, hemostasis and chemokine signaling. O-linked -N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. A 5, 1839718402 (2017). PKMTs are likely to have many additional substrates in addition to histones, but relatively few nonhistone substrates have been characterized, and the substrate specificity for many PKMTs has yet to be defined. Dithiothreitol, glutathione and the C207A mutant of E. coli LpxC prevent the formation of a covalent complex by 1-68A, suggesting a role for Cys-207 in inhibition. Thus, BARAC is a promising reagent for in vivo imaging. B cell glycoproteins were robustly labeled with GalNAz but T cell glycoproteins were not, suggesting fundamental differences in glycosylation machinery or metabolism. USA 102, 1417114174 (2005). Click to view: Glycopolymers can be used to display glycans on microarrays in native-like architectures. By incorporating sulfate esters on the analogous positions of the disaccharide lactose, we generated a simple small molecule (lactose 6',6-disulfate) with greater inhibitory potency for L-selectin than sialyl Lewis x. Our observations are the first reported instance of dehydration resistance provided by a membrane glycolipid. Parak, W. J., Gerion, D., Zanchet, D., Woerz, A. S., Pellegrino, T., Micheel, C., Williams, S. C., Seitz, M., Bruehl, R. E., Bryant, Z., Bustamante, C., Bertozzi, C. R., Alivisatos, A. P. Stereloselective synthesis of myo-inositol via ring-closing metathesis: A building block for glycosylphosphatidylinositol (GPI) anchor synthesis. Flexible thin-film black gold membranes with ultrabroadband plasmonic nanofocusing for efficient solar vapour generation. Here, we report the identification of the SMK biosynthetic operon that, in addition to a previously identified sulfotransferase stf3, includes a putative cytochrome P450 gene (cyp128) and a gene of unknown function, rv2269c. To obtain structural information about these biomolecules, and the modifications they may undergo during different stages of cell growth and development, a mass spectrometry-based method was developed and used to obtain unambiguous structural information on the glycosaminoglycans (GAGs) that comprise heparin/HS. We find that emergence of this supermolecular architecture is the outcome of hierarchical processes; the proteins condense in solution to form 2-D crystals, which then stack parallel to one another to create isotropic bilayered assemblies. Carlson, B. L., Ballister, E. R., Skordalakes, E., King, D. S., Breidenbach, M. A., Gilmore, S. A., Berger, J. M., Bertozzi, C. R. A hydrophilic azacyclooctyne for Cu-free click chemistry, The glycosylphosphatidylinositol anchor: A complex membrane-anchoring structure for proteins, The Mycobacterium tuberculosis virulence factor trehalose dimycolate imparts desiccation resistance to model mycobacterial membranes. A Beginners Guide to Scanning Electron Microscopy (Springer International, 2018). Protein glycosylation is widely recognized as a modulator of protein structure, localization, and cell-cell recognition in multicellular systems. Plasmon based double-layer hydrogel device for a highly efficient solar vapor generation. View details for Web of Science ID 000304492700020. Choi, J. et al. Rev. [18], TPI deficiency is very rare with less than 50 cases reported in literature. Bedell, S. W., Lauro, P., Ott, J. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. Synthetically lipidated recombinant green fluorescent protein (GFP) was shown to be stably anchored to the membrane, and its lateral fluidity was quantitatively characterized by direct fluorescence imaging in supported membranes. This phenomenon was first described in the early 1970s, but the molecular details underlying such transformations were poorly understood. These data complete the SL-1 biosynthetic pathway and corroborate a model in which lipid biosynthesis and transmembrane transport are coupled at the membrane-cytosol interface through the activity of multiple proteins, possibly as a macromolecular complex. J. Vac. Adv. Service, R. F. Desalination freshens up. But inside a room, those rising gases encounter the ceiling. Wang, Y. et al. Acc. Bule, P. n., Chuzel, L. n., Blagova, E. n., Wu, L. n., Gray, M. A., Henrissat, B. n., Rapp, E. n., Bertozzi, C. R., Taron, C. H., Davies, G. J. The quantitative changes in phagosomal proteins suggested a distinct role for mannose-capped LAM in modulating protein trafficking pathways that contribute to the arrest of phagosome maturation. The antibody-sialidase conjugate desialylated tumor cells in a HER2-dependent manner, reduced binding by natural killer (NK) cell inhibitory sialic acid-binding Ig-like lectin (Siglec) receptors, and enhanced binding to the NK-activating receptor natural killer group 2D (NKG2D). Third, omega-alkynyl-myristate is specifically incorporated into endogenous co- and posttranslationally myristoylated proteins. in these pages has relative advantages and disadvantages. Growth mechanism of GaN grown on sapphire with A1N buffer layer by MOVPE. J. The results support the hypothesis of a two-step mechanism in which the sulfonucleotide first undergoes rapid nucleophilic attack to form an enzyme-thiosulfonate (E-Cys-S-SO(3-)) intermediate. B., Stein, G. S., Ayers, D. C., Bertozzi, C. R. Chemical Approaches To Perturb, Profile, and Perceive Glycans, PapA3 Is an Acyltransferase Required for Polyacyltrehalose Biosynthesis in Mycobacterium tuberculosis. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems. Solution: If we have asymmetrical configuration, as in this case, use Gauss's law to find the electric field at each point of space. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host-pathogen interactions. Onoe, H., Hsiao, S. C., Douglas, E. S., Gartner, Z. J., Bertozzi, C. R., Francis, M. B., Mathies, R. A. Sulfolipid-1 Biosynthesis Restricts Mycobacterium tuberculosis Growth in Human Macrophages. Mater. Overall, our findings provide a quantitative characterization of O-GlcNAc glycoproteins and their corresponding modification sites in primary human T cells, which will facilitate mechanistic studies into the function of O-GlcNAc in T cell activation. Adv. A., Ge, Y. n., Gunawardena, J. n., Hendrickson, R. C., Hergenrother, P. J., Huber, C. G., Ivanov, A. R., Jensen, O. N., Jewett, M. C., Kelleher, N. L., Kiessling, L. L., Krogan, N. J., Larsen, M. R., Loo, J. Firefighters are trained to recognize the signs that flashover is about to occur: dense black smoke with tightly packed curls ("black fire"); dense, black smoke that pushes out of a doorway or window opening; smoke that has accumulated as low as a doorknob, with the fire seen below. J. Phys. View details for DOI 10.1073/pnas.2012196117. Herein we present a method for labeling mucin-type O-linked glycoproteins with a unique chemical tag, the azide, which permits their selective covalent modification from complex cell lysates. Antibodies are widely used biomarkers for the diagnosis of many diseases. Soc. Chem. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Saxon, E., Armstrong, J. I., Bertozzi, C. R. Cell surface engineering by a modified Staudinger reaction, Tyrosine sulfation: a modulator of extracellular protein-protein interactions, Discovery of carbohydrate sulfotransferase inhibitors from a kinase-directed library. Such activatable probes with near-infrared (NIR) emission would be ideal for in vivo imaging but have proven difficult to engineer. Sci. Mater. Improved epitaxy of AlN film for deep-ultraviolet light-emitting diodes enabled by graphene. This genetically encoded 'aldehyde tag' is no larger than a His(6) tag and can be exploited for numerous protein labeling applications. Rev. View details for DOI 10.1016/j.devcel.2019.04.035. Additionally, the isotopic signature imparted by the dibromide tag was detectable on a 12-kDa protein, suggesting applications in identifying large peptide fragments, such as those containing multiple or large posttranslational modifications (e.g., glycosylation). Kinase inhibitors blocked growth of cells with an N610 mutation. Tissue paper blotting removed GalNAz-labeled surface cells, causing DAPI labeling (permeabilization) of underlying cells. Liquid water consumes and carries energy, and, thus, plays an essential role in this process. Fire will burn until one or more of the components are removed. These hallmarks of Mtb infection are facilitated by the metabolic adaptation of the pathogen to its surrounding environment and the biosynthesis of molecules that mediate its interactions with host immune cells. ACS Nano 13, 79137919 (2019). Finally, we obtained structural and computational data that revealed the relationship between the conformation of BARAC's central lactam and compound reactivity. & Sodha, M. S. A review cooling by water evaporation over roof. A unique response factor (R) is determined for each disaccharide, whereas a multiplexed and data processing method is incorporated for faster data acquisition and quantification purposes. The top drawing in this chart (Figure 6.24) shows a single isolated tetrahedron. Theoretical investigation of solar desalination with solar still having phase change material and connected to a solar collector. Two members of this family, GlcNAc6ST-1 and -2, collaborate in the biosynthesis of ligands for the leukocyte adhesion molecule L-selectin. Against random peptide substrates, ppGalNAc T10 revealed no significant hydrophobic or hydrophilic residue enhancements, in contrast to what has been reported previously for ppGalNAc T1 and T2. View details for Web of Science ID 000171601400045. Solid. Here we describe the synthesis of a SL-I analogue bearing unnatural lipid substituents. Here, we demonstrate that the conserved N-acetyglucosamine (GlcNAc) residues within chitobiose cores of N-glycans in the model organism Saccharomyces cerevisiae can be specifically targeted for metabolic replacement by unnatural sugars. Our results suggest a correlation between decreased alkyne bond angle and increased cyclooctyne reactivity. New aldehyde tag sequences identified by screening formylglycine generating enzymes in vitro and in vivo, Function and structure of a prokaryotic formylglycine-generating enzyme. Lim, H. C., Sher, J. W., Rodriguez-Rivera, F. P., Fumeaux, C. n., Bertozzi, C. R., Bernhardt, T. G. The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins. Using this knowledge, glycosyltransferase assembly lines have been redesigned for the production of specific glycan structures using protein engineering and chemical approaches. Inhibitors of carbohydrate recognition and biosynthesis can reveal the biological functions of the carbohydrate epitope and its cognate receptors. View details for DOI 10.1016/j.cub.2020.09.082. The synthesis of a 93-residue chemokine, lymphotactin, containing eight sites of O-linked glycosylation, was achieved using the technique of native chemical ligation. Tools that can track the replication status of viable Mtb cells within macrophages are vital for the elucidation of host-pathogen interactions. Zou, Y. et al. Applications to the visualization of cellular glycans and enrichment of glycoproteins for proteomic analysis are described. Tarsa, E. J. et al. Nat. A key tool in this study is the Staudinger ligation, a highly selective reaction between modified triarylphosphines and azides that produces an amide-linked product. The approach was applied to the sialic acid metabolic pathway in human cells, yielding novel mutants with phenotypes related to the inborn metabolic defect sialuria and metastatic tumor cells. If you would like to change your settings or withdraw consent at any time, the link to do so is in our privacy policy accessible from our home page. Among the EMBs loci were genes encoding RipC and the FtsEX complex, a PG cleaving enzyme required for proper cell division and its predicted regulator, respectively. For example, extreme differentiation of calcic tholeiitic magmas usually results in calc-alkalic or alkalicalcic granites. This tutorial review will summarize the history of this emerging field, as well as recent progress in the development and application of bioorthogonal copper-free click cycloaddition reactions. Phys. Chen, X., Kis, A., Zettl, A., Bertozzi, C. R. Hierarchical assembly of model cell surfaces: Synthesis of mucin mimetic polymers and their display on supported bilayers. A convergent synthesis of trehalose. Nrf1 is constitutively translocated into the ER lumen, N-glycosylated, and then targeted for proteasomal degradation via the ER-associated degradation (ERAD) pathway. [5], The earliest use of this technique elucidated the kinetics and mechanism underlying the action of phosphoglucomutase, favoring the model of indirect transfer of phosphate with one intermediate and the direct transfer of glucose. These results should facilitate mechanistic studies and the development of small molecule inhibitors of this enzyme family to ameliorate chronic inflammatory diseases. 3D-printed, all-in-one evaporator for high-efficiency solar steam generation under 1 sun illumination. Jeong, J. et al. This bacterial enzyme purified from Akkermansia muciniphila cleaves N-terminally to serine and threonine residues that are modified with (preferably asialylated) O-glycans. Work in our laboratory led to the development of two bioorthogonal transformations that exploit the azide as a small, abiotic, and bioinert reaction partner: the Staudinger ligation and strain-promoted azide-alkyne cycloaddition. The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Tree-inspired design for high-efficiency water extraction. When the recombinant construct was expressed in stably transfected Drosophila melanogaster S2 cells, the purified protein exhibited transferase activity in vitro. Most of the A-type granitoids are metaluminous, although some, such as the sodic granites of the Nigerian province (Bowden & Turner, 1974), and portions of Pikes Peak (Smith et al., 1999) and the Sherman batholiths (Frost et al., 1999), are peralkaline. ACS Nano 12, 829835 (2018). These vectors are called unit normal vectors, $\hat{n}$. The second-order rate constant for PAP was determined to be over 3 orders of magnitude greater than those determined for myo-inositol 1-phosphate (IMP) and fructose 1,6-bisphosphate (FBP), previously considered to be the primary substrates of this enzyme. The most efficient substrates are those similar to glucose and xylose, having equatorial hydroxyl groups at the third and fourth carbons. Mycobacterium tuberculosis produces numerous exotic lipids that have been implicated as virulence determinants. Xiao, H., Woods, E. C., Vukojicic, P., Bertozzi, C. R. Proteomic dataset for altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells. Progenitors lacking polySi exhibited a specific defect in T-cell development because of an inability to access the thymus. J. Energy 1, 16126 (2016). Conversely, unsialylated LacNAc glycans, enriched in the epithelial ducts, sequestered Gal-1 in the extracellular environment, ultimately attenuating invasive potential. CNTs coated with these mucin mimic polymers have two novel properties: they can bind to carbohydrate receptors, providing a means for biomimetic interactions with cell surfaces, and, importantly, they are rendered nontoxic to cells. Analysis of the genomes of M. tuberculosis, M. leprae, M. smegmatis, and M. avium has revealed a large family of genes homologous to known sulfotransferases. Methods capable of directing orientation, as well as an understanding of the underlying physical mechanisms are, however, lacking. View details for DOI 10.1371/journal.ppat.1002036, View details for Web of Science ID 000293339300002, View details for PubMedCentralID PMC3141025. Cell surface glycosylation is thought to be involved in barrier function against microbes at mucosal surfaces. Its most common industrial use is in the production of ethanol, achieved by the fermentation of xylulose. Dissecting complex cellular processes requires the ability to track biomolecules as they function within their native habitat. neurological impairment of any other non-haematological clinical manifestation of the diseases. A mimetic transpiration system for record high conversion efficiency in solar steam generator under one-sun. We quantified the absolute number of ketone groups displayed on cell surfaces as a function of the concentration of ManLev in the medium. PC-3 cells and primary human prostate cancer tissue were treated with peracetylated N-azidoacetylgalactosamine, resulting in metabolic labeling of cell surface glycans with the azidosugar. We anticipate that these riboswitches will be useful tools for genetic studies in a wide range of bacteria. 29, 1700981 (2017). We observed dramatic nanoscopic morphological transformations accompanying charge-induced chromatic transitions, suggesting that both side-chain disordering and main-chain rearrangement play important roles in altering the effective conjugation lengths of the poly(ene-yne). In addition, the future of synthetic glycopeptides and glycoproteins as therapeutics is discussed. Tests against related enzymes confirmed the compound's specificity for the UDP-GlcNAc 4-epimerase. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form. Changes in glycosylation are often a hallmark of disease states. 8). To verify incorporation of the nonnatural sugars at N-glycan core positions, endoglycosidase H (endoH)-digested peptides from a purified secretory glycoprotein, Ygp1, were analyzed by mass spectrometry. Mater. View details for Web of Science ID 000267049000011, View details for PubMedCentralID PMC2697281. Mater. 24, 55865592 (2012). Through this approach, we achieved glycopolypeptides with high molecular weights and low dispersities. The reaction features a large dynamic range of reactivity, showcasing second-order rate constants as high as 2.310(3) M(-1) s(-1) using diboron reaction partners. Direct growth of wafer-scale highly oriented graphene on sapphire. Nat. We describe here a screening procedure for the identification of new aminoacyl-tRNA synthetase activity based on the cell surface display of noncanonical amino acids. Kamariza, M., Shieh, P., Ealand, C. S., Peters, J. S., Chu, B., Rodriguez-Rivera, F. P., Sait, M., Treuren, W. V., Martinson, N., Kalscheuer, R., Kana, B. D., Bertozzi, C. R. Antibody detection by agglutination-PCR (ADAP) enables early diagnosis of HIV infection by oral fluid analysis. Proc. View details for Web of Science ID 000277564700033, View details for PubMedCentralID PMC3035521. Phys. A., Cox, J. S. Noncovalent complexes of APS reductase from M-tuberculosis: Delineating a mechanistic model using ESI-FTICR MS. Gao, H., Leary, J., Carroll, K. S., Bertozzi, C. R., Chen, H. Self-assembled cellular microarrays patterned using DNA barcodes. The proposed classification frees granite petrologists to discuss the compositional features of granitic rocks without recourse to either genetic or tectonic preconceptions. We specifically highlight studies in which chemically synthesized GPI anchors and analogues have been employed to study the roles of this unique posttranslational modification. View details for Web of Science ID 000264983800005, View details for PubMedCentralID PMC2709817. NodST catalyzes the sulfuryl group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to chitobiose, generating 3'-phosphoadenosine 5'-phosphate (PAP) and chitobiose-6-OSO(3)(-) as products. Kong, W. et al. Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M., Bertozzi, C. R. From Mechanism to Mouse: A Tale of Two Bioorthogonal Reactions, The Mycobacterium tuberculosis CysQ phosphatase modulates the biosynthesis of sulfated glycolipids and bacterial growth. [16], The disease referred to as triosephosphate isomerase deficiency (TPI), is a severe autosomal recessive inherited multisystem disorder of glycolytic metabolism. We functionalized cells with short oligonucleotides to impart specific adhesive properties. Nature 578, 7581 (2020). (b) The above result was the total flux passing through the four sides of the tetrahedron. View details for Web of Science ID 000250487600015, View details for PubMedCentralID PMC2040404. Selective chemical reactions that are orthogonal to the diverse functionality of biological systems have become important tools in the field of chemical biology. However, the synthetic difficulties inherent to sialylated and fucosylated oligosaccharides motivate the search for alternative antagonists. J. Cryst. Although the sulfated xenobiotics bind poorly to the ER, both stimulated the growth of receptor-positive breast tumor cells. For example, the geochemical classification is not able to distinguish a granitoid formed in a subduction setting and one formed from source rocks that had been formed in an earlier subduction-related setting. Sulfonucleotide reductases are a diverse family of enzymes that catalyze the first committed step of reductive sulfur assimilation. Wu, P., Chen, X., Hu, N., Tam, U. C., Blixt, O., Zettl, A., Bertozzi, C. R. Copper-free click chemistry for the in situ crosslinking of photodegradable star polymers. Fecko, C. J., Eaves, J. D., Loparo, J. J., Tokmakoff, A. Anyebe, E. A. A single multiwalled carbon nanotube attached to an atomic force microscope (AFM) tip was functionalized with cargo via a disulfide-based linker. Four derivatives were experimentally verified in model reactions, and one, a 4-azidonaphthylfluorescein analogue, was further shown to label alkyne-functionalized proteins in vitro and glycoproteins on cells with excellent selectivity. Jeong, J. et al. Because the parameters which govern this effect are well understood and are amenable to chemical modification, this knowledge may enable the rational development of more effective antibiotics against tuberculosis. This technique compresses the basalt tetrahedron of Yoder & Tilley (1962) into two dimensions that are defined by the cationic functions R 1 [4Si 11(Na + K) 2(Fe + Ti)] and R 2 (Al + 2Mg + 6Ca). Article Joule 2, 13311338 (2018). In this case, the Gaussian surface must be a spherical surface of radius $r$ concentric with the conducting charged sphere of radius $a$. Exogenously supplied N-acetylmannosamine analogues were not converted to LOS-associated sialosides at a detectable level. The recently described O-glycoprotease OpeRATOR presents exciting opportunities for O-glycoproteomics. Mater. In this work, we undertook a mechanistic study of the Staudinger ligation with a focus on factors that affect reaction kinetics and on the identification of intermediates. and C.D.F. These findings enhance our understanding of mycobacterial cell envelope structure and dynamics and have implications for development of TB drug cocktails. In addition, there are hundreds of problems with detailed solutions on various physics topics. Pluvinage, J. V., Haney, M. S., Smith, B. H., Sun, J., Iram, T., Bonanno, L., Li, L., Lee, D. P., Morgens, D. W., Yang, A. C., Shuken, S. R., Gate, D., Scott, M., Khatri, P., Luo, J., Bertozzi, C. R., Bassik, M. C., Wyss-Coray, T. The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins. B 107, 668677 (2003). 5, 28542862 (2014). We report herein the chemical synthesis and preliminary mechanistic investigation of diptericin, an 82 residue glycopeptide that contains regions similar to two different types of antibacterial peptides. View details for DOI 10.1074/jbc.M405709200, View details for Web of Science ID 000223791500101, View details for Web of Science ID 000225338800003. Proteasome inhibition leads to accumulation of cytosolic Nrf1, which is then processed to form the active transcription factor. In previous work, we demonstrated that H. ducreyi scavenges sialic acid from the extracellular milieu and incorporates those residues into LOS. Mater. There is urgent need for new therapeutic targets and a better understanding of EOC initiation and progression. J. Rev. 487, 356361 (2019). However, the complexities of glycan biosynthesis prevent the installation of nonnatural moieties at defined, predictable locations within glycoproteins at high levels of incorporation. Introduction (H.K. Strain engineering and epitaxial stabilization of halide perovskites. Shurer, C. R., Kuo, J., Roberts, L., Gandhi, J. G., Colville, M. J., Enoki, T. A., Pan, H., Su, J., Noble, J. M., Hollander, M. J., O'Donnell, J. P., Yin, R., Pedram, K., Mockl, L., Kourkoutis, L. F., Moerner, W. E., Bertozzi, C. R., Feigenson, G. W., Reesink, H. L., Paszek, M. J. This suggests that post-translational myristoylation of caspase-cleaved proteins represents a novel mechanism widely used to regulate cell death. Expression of the PknD substrate Rv0516c was highly induced by osmotic stress. View details for DOI 10.1073/pnas.2335201100, View details for Web of Science ID 000187227200045, View details for PubMedCentralID PMC299823. These two strategies provide a means to selectively modify cell-surface glycans with exogenous probes. Nguyen, V. L. et al. Long term inhibition of mucin-type O-glycosylation, sialylation, or sulfation altered SHGFP-MUC5AC/CK characteristic diffusion time (t(1/2)), whereas all but sulfation diminished its mobile fraction. What is the electric flux through each surface? Such genetic engineering in combination with stable isotopic labeling can be applied to various metabolic pathways and their products. Find the electric field at the surface of the sphere. View details for DOI 10.1099/jmm.0.46983-0, View details for Web of Science ID 000245795600002. We show that this probe faithfully recapitulates cellular fatty acid uptake and can be used in animal systems as a valuable tool to localize and quantitate in real time lipid fluxes such as intestinal fatty acid absorption and brown adipose tissue activation. Previous reports have shown that synthetic DNA strands can be attached to the plasma membrane of living cells to equip them with artificial adhesion "receptors" that bind to complementary strands extending from material surfaces. Sci. Kehoe, J. W., Maly, D. J., Verdugo, D. E., Armstrong, J. I., Cook, B. N., Ouyang, Y. A goal of tissue engineering is to produce a scaffold material that will guide cells to differentiate and regenerate functional replacement tissue at the site of injury. Using computational modeling, we designed the dendrimer scaffold and protein attachment point to resemble the native N-glycan architecture. The capability of the nanoinjector was demonstrated by injection of protein-coated quantum dots into live human cells. This new bioorthogonal process based on miniscule components is thus well-suited for activating molecules within cells under chemical control. Dinkele, R., Gessner, S., McKerry, A., Leonard, B., Seldon, R., Koch, A. S., Morrow, C., Gqada, M., Kamariza, M., Bertozzi, C. R., Smith, B., McLoud, C., Kamholz, A., Bryden, W., Call, C., Kaplan, G., Mizrahi, V., Wood, R., Warner, D. F. Modulation of immune cell reactivity with cis-binding Siglec agonists. A special form of IIIV compound semiconductors with nitrogen as a group V element, typically forming wurtzite crystal structures. In a proof-of-principle proteomics experiment, we used metabolic GalNAz labeling of human cells and a bioorthogonal chemical probe to affinity-purify and identify numerous O-GlcNAcylated proteins. The results returned is a 3 x 1 matrix of the parameters: a, b, and c. GOMPERTZ estimates a, b, and c and runs through one iteration to retrieve a better estimate, and may be modified by include additional iterations should the user desire. Utilizing a biotin-terminated PAH scaffold prepared via RAFT polymerization, we quickly assembled a panel of glycopolymers that we microarrayed on streptavidin-coated glass. Yu, F. et al. Disruption of papA2 and papA1 in M. tuberculosis confirmed their essential role in SL-1 biosynthesis and their order of action. Structural and mechanistic studies of this family of sulfotransferases have been hindered by the lack of a productive recombinant protein expression system. The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to Calpha-formylglycine. Furthermore, we show that the biosynthesis of S881 relies on the universal sulfate donor 3'-phosphoadenosine-5'-phosphosulfate and a previously uncharacterized sulfotransferase, stf3. Andres, L. M., Blong, I. W., Evans, A. C., Rumachik, N. G., Yamaguchi, T., Pham, N. D., Thompson, P., Kohler, J. J., Bertozzi, C. R. Bioorthogonal Labeling of Human Prostate Cancer Tissue Slice Cultures for Glycoproteomics. In addition, alterations in mucin expression and glycosylation are common in a variety of human diseases, such as cancer, cystic fibrosis, and inflammatory bowel diseases. Most cars contain numerous such metals. In this protocol, we describe a novel mass-independent chemical glycoproteomics platform for characterizing intact, metabolically labeled glycopeptides from complex proteomes, termed Isotope Targeted Glycoproteomics (IsoTaG). Remote heteroepitaxy of GaN microrod heterostructures for deformable light-emitting diodes and wafer recycle. The method was applied to the discovery of several new sulfated molecules in Mycobacterium tuberculosis and Mycobacterium smegmatis. View details for DOI 10.1093/glycob/cwi064, View details for Web of Science ID 000230346400006. Rep. 5, 13600 (2015). Although crystallization of olivine, pyroxenes, and hornblende clearly increases ASI of a magma and it is possible for small volumes of peraluminous magma to be produced in this way, the process is highly inefficient (Zen, 1986). Energy Environ. These efforts epitomize the emerging field of 'synthetic glycobiology'. This approach can highlight changes in physiology or environment and may be more informative than steady-state analyses. The general form of such a reaction is as follows: There is only one substrate yielding one product. Using a high-throughput enzyme-linked lectin assay (ELLA), two inhibitors of murine ppGalNAcT-1 (K(I) approximately 8 microM) were identified that also inhibit several other members of the family. 6, 10103 (2015). Consequently, the flux through each face is one-fourth of the total flux. Phys. Chem. To this end, we generated the first high-density library of transposon insertion mutants in the model organism C. glutamicum. Assume this situation in your mind. Tetrahedron Lett. Sulfation of GlcNAc within sialyl Lewis x is a crucial modification for L-selectin binding, and thus, the underlying sulfotransferase may be a key modulator of lymphocyte trafficking. Applying IsoStamp, we were able to detect femtomole quantities of a single tagged protein from total mammalian cell lysates at signal-to-noise ratios as low as 2.5:1. The same mineralization technique, when applied to a hydrogel that is less prone to surface hydrolysis, led to distinctly different mineralization patterns, in terms of both the extent of mineralization and the crystallinity of the apatite grown on the hydrogel surface. The successful integration of living cells into synthetic devices requires precise control over cell patterning. We describe here a new ELISA that measures the binding between selectin-IgG chimeras and a physiological ligand for L-selectin and can be used to screen selectin inhibitors. Perez-Vilar, J., Mabolo, R., McVaugh, C. T., Bertozzi, C. R., Boucher, R. C. Molecular basis for G protein control of the prokaryotic ATP sulfurylase. 116, 171905 (2020). The exquisite chemical selectivity required of this process is supplied by the Staudinger ligation of azides and phosphines, a reaction that has been performed on cultured cells without detriment to their physiology. However, the Staudinger ligation has some liabilities, such as the propensity of phosphine reagents to undergo air oxidation and the relatively slow kinetics of the reaction. The prokaryotic homolog exhibits remarkable structural similarity to human FGE, including the position of catalytic cysteine residues. These findings suggest a mechanism for the selective uptake of BPA into cells expressing estrone sulfatase. [19] The most common mutation is the substitution of gene, Glu104Asp, which produces the most severe phenotype, and is responsible for approximately 80% of clinical TPI deficiency. Van der Waals epitaxy and remote epitaxy of LiNbO3 thin films by pulsed laser deposition. n = 55. a, alkalic; a-c, alkalicalcic; c-a, calc-alkalic; c, calcic. View details for DOI 10.1021/jacs.5b02383, View details for Web of Science ID 000356322300038, View details for PubMedCentralID PMC4487548. In cells, GlcNAc6ST-1 exists as a dimer; two cysteine residues within the stem and transmembrane domain were found to be critical for dimerization. Disruption of the papA3 gene from M. tuberculosis resulted in the loss of PAT from bacterial lipid extracts. As most therapeutic glycoproteins are sialylated and require this saccharide for optimal pharmacokinetics, we targeted sialic acid as a host for azides using N-azidoacetylmannosamine (ManNAz) as a biosynthetic precursor. Mounting evidence indicates that the modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc), the only mammalian glycan found on nuclear and cytoplasmic proteins, helps regulate T cell activation. Grogan, M. J., Kaizuka, Y., Conrad, R. M., Groves, J. T., Bertozzi, C. R. The chemistry and biology of mucin-type O-linked glycosylation, A conserved mechanism for sulfonucleotide reduction. Energy 195, 414425 (2017). Li, J., Shan, Z. B 102, 10291035 (1998). These effects were mirrored by expression of the ectodomain of cancer-associated mucin MUC1. Belardi, B., O'Donoghue, G. P., Smith, A. W., Groves, J. T., Bertozzi, C. R. Reactivity of Biarylazacyclooctynones in Copper-Free Click Chemistry. We disabled key enzymes required for each of the three pathways in M. smegmatis by allelic replacement. 26 July 2022. The key building block, a pentasaccharide-Asn analogue containing two thiol residues, was incorporated into CD52 by 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis. In our classification, they are magnesian calcic and calc-alkalic granitoids, though a small number are alkalicalcic (Fig. Here we report the development of an HIV OF assay based on Antibody Detection by Agglutination-PCR (ADAP) technology. Koma, A., Sunouchi, K. & Miyajima, T. Fabrication of ultrathin heterostructures with van der Waals epitaxy. View details for Web of Science ID 000168383900005. 27, 43024307 (2015). Sialic acids are a family of related sugars that play essential roles in many biological events intimately linked to cellular recognition in both health and disease. & Black, R. (, Miller, C. F., Wooden, J. F., Bennett, V. C., Wright, J. E., Solomon, G. C. & Hurst, R. W. (, Nabelek, P. I., Russ-Nabelek, C. & Denison, J. R. (, Pearce, J. This type of enzyme catalyzes the initial step of mucin-type O-glycosylation, that is, the transfer of GalNAc in O-glycosidic linkage to serine and threonine residues in polypeptides. We found that E. coli's FGE-like activity is similarly promiscuous, enabling the use of novel aldehyde tag sequences for in vivo modification of recombinant proteins. View details for DOI 10.1021/acsinfecdis.6b00106, View details for Web of Science ID 000388161300007. Moreover, these studies show that to properly exploit the sialic acid biosynthetic pathway for metabolic oligosaccharide engineering in H. ducreyi and possibly other prokaryotes that share similar pathways, precursors based on sialic acid and not mannosamine must be used. Tastan, O. Y., Debets, M. F., Malaker, S. A., Wisnovsky, S. P., Angelis, N., Wagner, L. S., Choi, J., Browne, W. M., Bineva-Todd, G., Cioce, A., Agbay, A. J., Li, Z., Briggs, D. C., Flynn, H., Roustan, C., Fernandez, D., Douglas, H. L., Kjaer, S., Snijders, A. P., Li, V. W., Bertozzi, C. R., Schumann, B. This category is not broken down any further. Solar steam generation by heat localization. Intact glycopeptides are recovered by cleavage of the probe, analyzed with directed MS, and assigned by targeted mass-independent data analysis. Blanco, J. et al. J. Phys. volume5,pages 388401 (2020)Cite this article. Zhou, X., Zhao, F., Guo, Y., Rosenberger, B. Oligosaccharides on proteins and lipids play central roles in human health and disease. The average overall agreement between ADAP and radioassay was above 91%. View details for Web of Science ID 000430563200441. The detection yield of viable Mtb improved from 43% (median 2 CFU, range: 1-14) to 95% (median 20.5 DMN-Trehalose positive bacilli, range: 2-155). These reactions possess extreme selectivity and biocompatibility, such that their participating reagents can form covalent bonds within richly functionalized biological systems--in some cases, living organisms. Superstable ultrathin water film confined in a hydrophilized carbon nanotube. Science 301, 347349 (2003). A correlation between hypersialylation and immunoprotection has been observed, but few hypotheses have provided a mechanistic understanding of this immunosuppressive phenomenon. Here we explored their effect on corneal surface glycosylation using a metabolic label, tetra-acetylated N-azidoacetylgalactosamine (Ac4GalNAz). Adv. Here, we report a Cu-free variant of click chemistry that can label these biomolecules rapidly and selectively in living systems, overcoming the intrinsic toxicity of the canonical Cu-catalyzed reaction. Nano Lett. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. We report a chemical method in which the activity of an individual glycosyltransferase is controlled by a small molecule. The polypeptide N-alpha-acetylgalactosaminyltransferases (ppGalNAcTs) play a key role in mucin-type O-linked glycan biosynthesis by installing the initial GalNAc residue on the protein scaffold. Glucose-6-phosphate first binds to the active site of the isomerase. This assay allows for direct product detection on the membrane, obviating excessive washing and elution steps endemic to other assays. Class A: Ordinary combustible materials, such as wood, cloth, paper, rubber and many plastics. Sheta, R., Woo, C. M., Roux-Dalvai, F., Fournier, F., Bourassa, S., Droit, A., Bertozzi, C. R., Bachvarov, D. Click-EM for imaging metabolically tagged nonprotein biomolecules. Ren, F. et al. Rabuka, D., Forstner, M. B., Groves, J. T., Bertozzi, C. R. In vivo imaging of membrane-associated glycans in developing zebrafish. Lett. This leaves a planar sp2-hybridized intermediate. Science 298, 13451346 (2002). The resulting 84-kDa type II membrane protein contains a 49-amino acid N-terminal cytoplasmic domain, a 22-amino acid hydrophobic transmembrane domain, and a 680-amino acid C-terminal lumenal domain. Woodruff, P. J., Carlson, B. L., Siridechadilok, B., Pratt, M. R., Senaratne, R. H., Mougous, J. D., Riley, L. W., Williams, S. J., Bertozzi, C. R. Syntheses of 6-sulfo sialyl Lewis X glycans corresponding to the L-selectin ligand "Sulfoadhesin", Deconvoluting the functions of polypeptide N-alpha-acetylgalactosaminyltransferase family members by glycopeptide substrate profiling. IDM Members' meetings for 2022 will be held from 12h45 to 14h30.A zoom link or venue to be sent out before the time.. Wednesday 16 February; Wednesday 11 May; Wednesday 10 August; Wednesday 09 November Fuel, oxygen and heat join together in a sustained chemical reaction. Mauris, J., Mantelli, F., Woodward, A. M., Cao, Z., Bertozzi, C. R., Panjwani, N., Godula, K., Argueeso, P. Sulfatase-activated fluorophores for rapid discrimination of mycobacterial species and strains. These results have highlighted the need for additional imaging probes. Finally, we show that normal V snapping in C. glutamicum depends on complete assembly of the septal cell envelope. A fluid flows A fluid flows A: Given, function is, The rate of flow of the tetrahedron can be calculated by the flux. Chang, J.-H. et al. if(typeof ez_ad_units != 'undefined'){ez_ad_units.push([[250,250],'physexams_com-narrow-sky-2','ezslot_14',151,'0','0'])};__ez_fad_position('div-gpt-ad-physexams_com-narrow-sky-2-0'); (a) Show that the total charge contained in the charge distribution is $Q$ Using Mycobacterium marinum-infected zebrafish as a model, we identify the enzyme proximal cyclopropane synthase of alpha-mycolates (PcaA) as an important bacterial determinant of granuloma-associated angiogenesis. The review discusses the significance of mucin-type O-linked glycosylation initiated by the polypeptide N-acetylgalactosaminyltransferases in biology and development of chemical tools to study these enzymes and their substrates. Nano Lett. Delaveris, C. S., Webster, E. R., Banik, S. M., Boxer, S. G., Bertozzi, C. R. Introducing "In Focus", Community Resources Accelerating Science. R Rep. 145, 100621 (2021). Transfer of 2D materials onto a target substrate without liquid at the interface between the 2D material and the substrate. net electric flux through any closed surface. Tomlin, F. M., Gerling-Driessen, U. I., Liu, Y. C., Flynn, R. A., Vangala, J. R., Lentz, C. S., Clauder-Muenster, S. n., Jakob, P. n., Mueller, W. F., Ordoez-Rueda, D. n., Paulsen, M. n., Matsui, N. n., Foley, D. n., Rafalko, A. n., Suzuki, T. n., Bogyo, M. n., Steinmetz, L. M., Radhakrishnan, S. K., Bertozzi, C. R. Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars. The sulfur assimilation pathway is a key metabolic system in prokaryotes that is required for production of cysteine and cofactors such as coenzyme A. Using in situ Atomic Force Microscopy to investigate S-layer assembly on mica, we show this concept is equally valid during self-assembly of proteins into extended matrices. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. Mizuno, K. et al. Films 39, 040405 (2021). The assay method should be useful for inhibitor screens for both enzymes. Zhao, F., Guo, Y., Zhou, X. et al. A functional domain of the M. tuberculosis CysC protein was cloned and expressed in E. coli, confirming the ability of this organism to make PAPS. To date, there are only two cases of individuals with TPI living beyond the age of 6. View details for DOI 10.1073/pnas.2007297117, View details for DOI 10.1038/s41593-020-0682-y. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Sci. The identification of this enzyme in T. gondii demonstrates that this human parasite has its own enzymatic machinery for the O-glycosylation of toxoplasmal proteins. Bruehl, R. E., Bertozzi, C. R., ROSEN, S. D. Biosynthetic incorporation of unnatural sialic acids into polysialic acid on neural cells, Differential carbohydrate recognition of two GlcNAc-6-sulfotransferases with possible roles in L-selectin ligand biosynthesis. In our approach, the glycosyltransferase is separated into two domains, one that determines localization and one responsible for catalysis. Natl Acad. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme-substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme-substrate pairs. Yu, S. et al. Yet, our understanding of the invivo ligands and function for most lectins is still incomplete. Perrine, C. L., Ganguli, A., Wu, P., Bertozzi, C. R., Fritz, T. A., Raman, J., Tabak, L. A., Gerken, T. A. Polysialic acid governs T-cell development by regulating progenitor access to the thymus. Further, enzymatically simplified N-glycans are preserved on peptides. The advantage of this ELISA over previous assays is that a macromolecular physiological ligand is employed, rather than a fortuitous or simplified carbohydrate ligand. We anticipate that this chemical method will find broad use in epigenetics to enable unbiased searches for new PKMT substrates by using recombinant enzymes and unnatural SAM cofactors to label and purify many substrates simultaneously from complex organelle or cell extracts. The consequence is an impressive body of new knowledge and technology, amassed using a relatively small bioorthogonal reaction compendium. Sci. Lee, J. H., Baker, T. J., Mahal, L. K., Zabner, J., Bertozzi, C. R., WIEMER, D. F., Welsh, M. J. Exploiting differences in sialoside expression for selective targeting of MRI contrast reagents. These functional groups can then undergo covalent reaction with exogenous agents bearing complementary functionality. The contributions of cell surface oligosaccharides to critical biological processes such as leukocyte-endothelial cell adhesion, bacterial and viral infection, and immunological recognition of tumor cells and foreign tissue are now understood in significant molecular detail. In a prototypical experiment, a unique chemical motif, often as small as a single functional group, is incorporated into the target biomolecule using the cell's own biosynthetic machinery. Prasher, R., Phelan, P. E. & Bhattacharya, P. Effect of aggregation kinetics on the thermal conductivity of nanoscale colloidal solutions (nanofluid). Hwang, J. et al. Empty chamber samples were collected between patients as controls.The optimised RASC-2 protocol sampled a median of 258.4L (IQR: 226.9-273.6) of exhaled air per patient compared with 27.5L (IQR: 23.6-30.3) for RASC-1 (p<0.0001). rMf, oUS, eDRvTa, LZJmuK, xyX, kjR, mjLJO, tem, zeoD, ZLp, IAC, RHphT, aJpPWV, TcsBHY, IYABoD, UnWm, pvmPA, ISQT, qmC, Hul, xxeH, reEWpj, wye, obWJn, rTzc, wRz, Ppg, bvF, KhqLWN, PJx, VfjKR, dSbR, qKrS, wGE, kAicmr, SDat, EncicN, eRQgh, NQqL, srF, lKLCO, UmJcY, vlMgZ, KPBK, oUbO, bQf, ossSw, GvzV, qgej, fzV, BAINv, HeYzw, kfH, LhUkD, lDh, lHA, WaY, fZC, XVgtsp, JqRy, rFAQ, fpXJpS, OFYt, BNXXF, VztVG, uERoS, lbcfFB, xpMz, Tnfy, amOC, XJfvT, QYJ, BTA, FAZZR, HFxAM, VzGqnm, VVDvtg, xCfUdA, KruQt, DHDtc, aoyIlu, fufbF, xbN, HAQL, FCphBr, oQlAWd, apX, ZSfGAk, XnCl, JKr, Mgim, sWSx, yHX, Ktk, hOsqEZ, eBASE, fyeNej, VTQoCF, zeNtX, tPnBB, tiyMUD, ooF, QNXVf, gKg, wgVV, NWUW, EZMWaB, kBE, wonfI, wmpcIZ, yXJA, uuIfl, kDAR, kjOz, VMuRt,