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Berlin Brandenburg

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  • 1
    In: American Journal of Clinical Pathology, 2012, Vol. 138(suppl1), pp.A224-A224
    Description: Carcinoma of the urethra is a rare tumor affecting females more often than males, and African Americans are affected more commonly than Caucasians. The most common histologic subtypes are squamous cell, transitional cell, and adenocarcinoma, …
    Keywords: Medicine ; Biology;
    ISSN: 0002-9173
    E-ISSN: 1943-7722
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  • 2
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 06 March 2018, Vol.115(10), pp.2353-2358
    Description: Regulation of gene expression at the level of protein synthesis is a crucial element in driving how the genetic landscape is expressed. However, we are still limited in technologies that can quantitatively capture the immediate proteomic changes that allow cells to respond to specific stimuli. Here, we present a method to capture and identify nascent proteomes in situ across different cell types without disturbing normal growth conditions, using O-propargyl-puromycin (OPP). Cell-permeable OPP rapidly labels nascent elongating polypeptides, which are subsequently conjugated to biotin-azide, using click chemistry, and captured with streptavidin beads, followed by digestion and analysis, using liquid chromatography-tandem mass spectrometry. Our technique of OPP-mediated identification (OPP-ID) allows detection of widespread proteomic changes within a short 2-hour pulse of OPP. We illustrate our technique by recapitulating alterations of proteomic networks induced by a potent mammalian target of rapamycin inhibitor, MLN128. In addition, by employing OPP-ID, we identify more than 2,100 proteins and uncover distinct protein networks underlying early erythroid progenitor and differentiation states not amenable to alternative approaches such as amino acid analog labeling. We present OPP-ID as a method to quantitatively identify nascent proteomes across an array of biological contexts while preserving the subtleties directing signaling in the native cellular environment.
    Keywords: Erythropoiesis ; Mtor ; Proteomics ; Puromycin ; Translation ; Cell Differentiation -- Physiology ; Proteome -- Analysis ; Proteomics -- Methods ; Signal Transduction -- Physiology
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 3
    Language: English
    In: The Journal of biological chemistry, 12 February 2016, Vol.291(7), pp.3224-38
    Description: The degree of phosphorylation and phosphoethanolaminylation of lipid A on neisserial lipooligosaccharide (LOS), a major cell-surface antigen, can be correlated with inflammatory potential and the ability to induce immune tolerance in vitro. On the oligosaccharide of the LOS, the presence of phosphoethanolamine and sialic acid substituents can be correlated with in vitro serum resistance. In this study, we analyzed the structure of the LOS from 40 invasive isolates and 25 isolates from carriers of Neisseria meningitidis without disease. Invasive strains were classified as groups 1-3 that caused meningitis, septicemia without meningitis, and septicemia with meningitis, respectively. Intact LOS was analyzed by high resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Prominent peaks for lipid A fragment ions with three phosphates and one phosphoethanolamine were detected in all LOS analyzed. LOS from groups 2 and 3 had less abundant ions for highly phosphorylated lipid A forms and induced less TNF-α in THP-1 monocytic cells compared with LOS from group 1. Lipid A from all invasive strains was hexaacylated, whereas lipid A of 6/25 carrier strains was pentaacylated. There were fewer O-acetyl groups and more phosphoethanolamine and sialic acid substitutions on the oligosaccharide from invasive compared with carrier isolates. Bioinformatic and genomic analysis of LOS biosynthetic genes indicated significant skewing to specific alleles, dependent on the disease outcome. Our results suggest that variable LOS structures have multifaceted effects on homeostatic innate immune responses that have critical impact on the pathophysiology of meningococcal infections.
    Keywords: Neisseria Meningitidis ; Acylation ; Bioinformatics ; Infection ; Lipid A ; Lipooligosaccharide ; Mass Spectrometry (MS) ; Phosphoethanolamine ; Phosphorylation ; Sialic Acid ; Antigens, Bacterial -- Toxicity ; Carrier State -- Microbiology ; Lipopolysaccharides -- Toxicity ; Meningitis, Meningococcal -- Microbiology ; Meningococcal Infections -- Microbiology ; Neisseria Meningitidis, Serogroup B -- Pathogenicity ; Neisseria Meningitidis, Serogroup C -- Pathogenicity
    E-ISSN: 1083-351X
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  • 4
    Language: English
    In: Proceedings of the National Academy of Sciences, 02/13/2017, p.201612927
    ISSN: 0027-8424
    E-ISSN: 1091-6490
    Source: CrossRef
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  • 5
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 28 February 2017, Vol.114(9), pp.2218-2223
    Description: Multidrug-resistant (MDR) gram-negative bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin containing lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response. One type of lipid A modification involves the addition of phosphoethanolamine to the 1 and 4' headgroup positions by phosphoethanolamine transferases. Previous structural work on a truncated form of this enzyme suggested that the full-length protein was required for correct lipid substrate binding and catalysis. We now report the crystal structure of a full-length lipid A phosphoethanolamine transferase from , determined to 2.75-Å resolution. The structure reveals a previously uncharacterized helical membrane domain and a periplasmic facing soluble domain. The domains are linked by a helix that runs along the membrane surface interacting with the phospholipid head groups. Two helices located in a periplasmic loop between two transmembrane helices contain conserved charged residues and are implicated in substrate binding. Intrinsic fluorescence, limited proteolysis, and molecular dynamics studies suggest the protein may sample different conformational states to enable the binding of two very different- sized lipid substrates. These results provide insights into the mechanism of endotoxin modification and will aid a structure-guided rational drug design approach to treating multidrug-resistant bacterial infections.
    Keywords: Neisseria ; Lipid Modification ; Membrane Protein Structure ; Molecular Dynamics ; Multidrug Resistance ; Bacterial Proteins -- Chemistry ; Ethanolaminephosphotransferase -- Chemistry ; Lipid A -- Chemistry ; Neisseria Meningitidis -- Chemistry ; Periplasm -- Chemistry
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 6
    Language: English
    In: PLoS ONE, 2012, Vol.7(6), p.e38303
    Description: Neisseria gonorrhoeae , the causative agent of gonorrhea, can form biofilms in vitro and in vivo . In biofilms, the organism is more resistant to antibiotic treatment and can serve as a reservoir for chronic infection. We have used stable isotope labeling by amino acids in cell culture (SILAC) to compare protein expression in biofilm and planktonic organisms. Two parallel populations of N. gonorrhoeae strain 1291, which is an arginine auxotroph, were grown for 48 h in continuous-flow chambers over glass, one supplemented with 13 C 6 -arginine for planktonic organisms and the other with unlabeled arginine for biofilm growth. The biofilm and planktonic cells were harvested and lysed separately, and fractionated into three sequential protein extracts. Corresponding heavy (H) planktonic and light (L) biofilm protein extracts were mixed and separated by 1D SDS-PAGE gels, and samples were extensively analyzed by liquid chromatography-mass spectrometry. Overall, 757 proteins were identified, and 152 unique proteins met a 1.5-fold cutoff threshold for differential expression with p-values 〈0.05. Comparing biofilm to planktonic organisms, this set included 73 upregulated and 54 downregulated proteins. Nearly a third of the upregulated proteins were involved in energy metabolism, with cell envelope proteins making up the next largest group. Of the downregulated proteins, the largest groups were involved in protein synthesis and energy metabolism. These proteomics results were compared with our previously reported results from transcriptional profiling of gonococcal biofilms using microarrays. Nitrite reductase and cytochrome c peroxidase, key enzymes required for anaerobic growth, were detected as highly upregulated in both the proteomic and transcriptomic datasets. These and other protein expression changes observed in the present study were consistent with a shift to anaerobic respiration in gonococcal biofilms, although changes in membrane proteins not explicitly related to this shift may have other functions.
    Keywords: Research Article ; Biology ; Microbiology ; Biochemistry
    E-ISSN: 1932-6203
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  • 7
    Language: English
    In: Journal of The American Society for Mass Spectrometry, 2016, Vol.27(7), pp.1263-1276
    Description: Lipooligosaccharides (LOS) are major microbial virulence factors displayed on the outer membrane of rough-type Gram-negative bacteria. These amphipathic glycolipids are comprised of two domains, a core oligosaccharide linked to a lipid A moiety. Isolated LOS samples are generally heterogeneous mixtures of glycoforms, with structural variability in both domains. Traditionally, the oligosaccharide and lipid A components of LOS have been analyzed separately following mild acid hydrolysis, although important acid-labile moieties can be cleaved. Recently, an improved method was introduced for analysis of intact LOS by MALDI-TOF MS using a thin layer matrix composed of 2,4,6-trihydroxyacetophenone (THAP) and nitrocellulose. In addition to molecular ions, the spectra show in-source “prompt” fragments arising from regiospecific cleavage between the lipid A and oligosaccharide domains. Here, we demonstrate the use of traveling wave ion mobility spectrometry (TWIMS) for IMS-MS and IMS-MS/MS analyses of intact LOS from Neisseria spp. ionized by MALDI. Using IMS, the singly charged prompt fragments for the oligosaccharide and lipid A domains of LOS were readily separated into resolved ion plumes, permitting the extraction of specific subspectra, which led to increased confidence in assigning compositions and improved detection of less abundant ions. Moreover, IMS separation of precursor ions prior to collision-induced dissociation (CID) generated time-aligned, clean MS/MS spectra devoid of fragments from interfering species. Incorporating IMS into the profiling of intact LOS by MALDI-TOF MS exploits the unique domain structure of the molecule and offers a new means of extracting more detailed information from the analysis. Graphical Abstract ᅟ
    Keywords: Ion mobility spectrometry ; Traveling wave ion mobility ; IMS ; TWIMS ; IMS-MS ; IMS-MS/MS ; Lipooligosaccharides ; Lipopolysaccharides ; LOS ; LPS
    ISSN: 1044-0305
    E-ISSN: 1879-1123
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  • 8
    Language: English
    In: Infection and immunity, November 2012, Vol.80(11), pp.4014-26
    Description: The interaction of the immune system with Neisseria commensals remains poorly understood. We have previously shown that phosphoethanolamine on the lipid A portion of lipooligosaccharide (LOS) plays an important role in Toll-like receptor 4 (TLR4) signaling. For pathogenic Neisseria, phosphoethanolamine is added to lipid A by the phosphoethanolamine transferase specific for lipid A, which is encoded by lptA. Here, we report that Southern hybridizations and bioinformatics analyses of genomic sequences from all eight commensal Neisseria species confirmed that lptA was absent in 15 of 17 strains examined but was present in N. lactamica. Mass spectrometry of lipid A and intact LOS revealed the lack of both pyrophosphorylation and phosphoethanolaminylation in lipid A of commensal species lacking lptA. Inflammatory signaling in human THP-1 monocytic cells was much greater with pathogenic than with commensal Neisseria strains that lacked lptA, and greater sensitivity to polymyxin B was consistent with the absence of phosphoethanolamine. Unlike the other commensals, whole bacteria of two N. lactamica commensal strains had low inflammatory potential, whereas their lipid A had high-level pyrophosphorylation and phosphoethanolaminylation and induced high-level inflammatory signaling, supporting previous studies indicating that this species uses mechanisms other than altering lipid A to support commensalism. A meningococcal lptA deletion mutant had reduced inflammatory potential, further illustrating the importance of lipid A pyrophosphorylation and phosphoethanolaminylation in the bioactivity of LOS. Overall, our results indicate that lack of pyrophosphorylation and phosphoethanolaminylation of lipid A contributes to the immune privilege of most commensal Neisseria strains by reducing the inflammatory potential of LOS.
    Keywords: Inflammation -- Immunology ; Lipid A -- Metabolism ; Neisseria -- Immunology
    ISSN: 00199567
    E-ISSN: 1098-5522
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  • 9
    In: American Journal of Clinical Pathology, 2012, Vol. 138(suppl1), pp.A101-A101
    Description: Gallbladder cancer is an infrequent malignancy but is the sixth most common and ninth most lethal gastrointestinal malignancy in the United States. The lack of specific signs and symptoms results in an advanced tumor stage at diagnosis, resulting in a dismal prognosis. Papillary adenocarcinoma of the gallbladder has been reported to have a better prognosis, but few studies address the correlation …
    Keywords: Medicine ; Biology;
    ISSN: 0002-9173
    E-ISSN: 1943-7722
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  • 10
    In: Pathogens and Disease, 2017, Vol. 75(3)
    Description: Infections due to Neisseria meningitidis afflict more than one million people worldwide annually and cause death or disability in many survivors. The clinical course of invasive infections has been well studied, but our understanding of the cause of differences in patient outcomes has been limited because these are dependent on multiple factors including the response of the host, characteristics of the bacteria and interactions between the host and the bacteria. The meningococcus is a highly inflammatory organism, and the lipooligosaccharide (LOS) on the outer membrane is the most potent inflammatory molecule it expresses due to the interactions of the lipid A moiety of LOS with receptors of the innate immune system. We previously reported that increased phosphorylation of hexaacylated neisserial lipid A is correlated with greater inflammatory potential. Here we postulate that variability in lipid A phosphorylation can tip the balance of innate immune responses towards homeostatic tolerance or proinflammatory signaling that affects adaptive immune responses, causing disease with meningitis only, or septicemia with or without meningitis, respectively. Furthermore, we propose that studies of the relationship between bacterial virulence and gene expression should consider whether genetic variation could affect properties of biosynthetic enzymes resulting in LOS structural differences that alter disease pathobiology. This review discusses how variability in lipooligosaccharide phosphorylation can tip the balance of innate immune responses towards homeostatic tolerance or proinflammatory signaling that affect adaptive immune responses to meningococcal infection.
    Keywords: 〈Kwd〉 〈Italic Toggle="Yes"〉Neisseria Meningitidis〈/Italic〉 〈/Kwd〉 ; Lipooligosaccharide ; Innate Immunity ; Adaptive Immunity ; Proinflammatory ; Endotoxin Tolerance
    E-ISSN: 2049-632X
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