New urinary EPO drug testing method using two-dimensional gel electrophoresis

We present a two-dimensional electrophoresis (2DE) method for the detection of the drug, recombinant erythropoietin (rHuEPO) in urine and its separation from endogenous erythropoietin (HuEPO). This method involves a one-step acetonitrile precipitation of the proteins in urine, addition of an internal standard, two-dimensional gel electrophoresis (2D PAGE), a single Western blot and chemiluminescent immunodetection. Results: The 2DE method separates HuEPO and rHuEPO isoforms by both iso-electric point and molecular mass. We have identified several urinary proteins with which the monoclonal EPO antibody used in the current test has non-specific binding. The iso-electric points of these cross-reactive proteins overlap with HuEPO and rHuEPO however, they separate distinctly by the 2DE method. Alpha-2-HS-glycoprotein (HSGP) was identified by peptide mass fingerprinting as one of the urinary cross-reacting proteins, and commercially available purified HSGP was chosen to be added into urine samples as an internal standard prior to separation. Software (EpIQ) was specifically developed that applies four separate criteria to the detection of the migration of rHuEPO and HuEPO relative to the internal standard. Conclusion: The combination of sample preparation, two-dimensional separation, internal standard, standardized blotting procedures and image analysis software enables the 2DE test for rHuEPO in urine to be performed reproducibly and accurately. © 2005 Elsevier B.V. All rights reserved

Visualizing neuroinflammation with fluorescence and luminescent lanthanide-based in situ hybridization

© 2019 The Author(s). Background: Neurokine signaling via the release of neurally active cytokines arises from glial reactivity and is mechanistically implicated in central nervous system (CNS) pathologies such as chronic pain, trauma, neurodegenerative diseases, and complex psychiatric illnesses. Despite significant advancements in the methodologies used to conjugate, incorporate, and visualize fluorescent molecules, imaging of rare yet high potency events within the CNS is restricted by the low signal to noise ratio experienced within the CNS. The brain and spinal cord have high cellular autofluorescence, making the imaging of critical neurokine signaling and permissive transcriptional cellular events unreliable and difficult in many cases. Methods: In this manuscript, we developed a method for background-free imaging of the transcriptional events that precede neurokine signaling using targeted mRNA transcripts labeled with luminescent lanthanide chelates and imaged via time-gated microscopy. To provide examples of the usefulness this method can offer to the field, the mRNA expression of toll-like receptor 4 (TLR4) was visualized with traditional fluorescent in situ hybridization (FISH) or luminescent lanthanide chelate-based in situ hybridization (LISH) in mouse BV2 microglia or J774 macrophage phenotype cells following lipopolysaccharide stimulation. TLR4 mRNA staining using LISH- and FISH-based methods was also visualized in fixed spinal cord tissues from BALB/c mice with a chronic constriction model of neuropathic pain or a surgical sham model in order to demonstrate the application of this new methodology in CNS tissue samples. Results: Significant increases in TLR4 mRNA expression and autofluorescence were visualized over time in mouse BV2 microglia or mouse J774 macrophage phenotype cells following lipopolysaccharide (LPS) stimulation. When imaged in a background-free environment with LISH-based detection and time-gated microscopy, increased TLR4 mRNA was observed in BV2 microglia cells 4 h following LPS stimulation, which returned to near baseline levels by 24 h. Background-free imaging of mouse spinal cord tissues with LISH-based detection and time-gated microscopy demonstrated a high degree of regional TLR4 mRNA expression in BALB/c mice with a chronic constriction model of neuropathic pain compared to the surgical sham model. Conclusions: Advantages offered by adopting this novel methodology for visualizing neurokine signaling with time-gated microscopy compared to traditional fluorescent microscopy are provided

The Minimum Information Required for a Glycomics Experiment (MIRAGE) project: improving the standards for reporting glycan microarray-based data

MIRAGE (Minimum Information Required for A Glycomics Experiment) is an initiative that was created by experts in the fields of glycobiology, glycoanalytics, and glycoinformatics to produce guidelines for reporting results from the diverse types of experiments and analyses used in structural and functional studies of glycans in the scientific literature. As a sequel to the guidelines for sample preparation (Struwe et al. 2016, Glycobiology, 26, 907-910) and mass spectrometry (MS) data (Kolarich et al. 2013, Mol. Cell Proteomics. 12, 991-995), here we present the first version of guidelines intended to improve the standards for reporting data from glycan microarray analyses. For each of eight areas in the workflow of a glycan microarray experiment, we provide guidelines for the minimal information that should be provided in reporting results. We hope that the MIRAGE glycan microarray guidelines proposed here will gain broad acceptance by the community, and will facilitate interpretation and reproducibility of the glycan microarray results with implications in comparison of data from different laboratories and eventual deposition of glycan microarray data in international databases

O-Glycosylation of snails

The glycosylation abilities of snails deserve attention, because snail species serve as intermediate hosts in the developmental cycles of some human and cattle parasites. In analogy to many other host-pathogen relations, the glycosylation of snail proteins may likewise contribute to these host-parasite interactions. Here we present an overview on the O-glycan structures of 8 different snails (land and water snails, with or without shell): Arion lusitanicus, Achatina fulica, Biomphalaria glabrata, Cepaea hortensis, Clea helena, Helix pomatia, Limax maximus and Planorbarius corneus. The O-glycans were released from the purified snail proteins by β-elimination. Further analysis was carried out by liquid chromatography coupled to electrospray ionization mass spectrometry and – for the main structures – by gas chromatography/mass spectrometry. Snail O-glycans are built from the four monosaccharide constituents: N-acetylgalactosamine, galactose, mannose and fucose. An additional modification is a methylation of the hexoses. The common trisaccharide core structure was determined in Arion lusitanicus to be N-acetylgalactosamine linked to the protein elongated by two 4-O-methylated galactose residues. Further elongations by methylated and unmethylated galactose and mannose residues and/or fucose are present. The typical snail O-glycan structures are different to those so far described. Similar to snail N-glycan structures they display methylated hexose residues

An Improved Protocol for N-Glycosylation Analysis of Gel-Separated Sialylated Glycoproteins by MALDI-TOF/TOF

Different glycoforms of some proteins have been identified as differential spots for certain diseases in 2-DE, indicating disease-related glycosylation changes. It is routine to determine the site-specific glycosylation of nonsialylated N-glycoproteins from a single gel spot, but some obstacles still exist in analyzing sialylated glycoproteins due to the lability and higher detection limit of acid glycans in MALDI-TOF/TOF analysis. Thus, we present an improved protocol here. Tryptic glycopeptides were separated and subjected to MALDI-TOF/TOF analysis, resulting in the identification of site-specific glycosylation of high-intensity glycopeptides. Sequential deglycosylation and desialylation were used to improve the identification of glycosylation sites and desialylated glycans. The site-specific glycosylation of large glycopeptides and low-intensity glycopeptides was deduced based on the masses of glycopeptides, deglycosylated peptides and desialylated glycans. By applying it to 2-DE separated human serum, the difference of N-glycosylation was successfully determined for α1-antitrypsin between different gel spots

Soil fungal networks maintain local dominance of ectomycorrhizal trees

The mechanisms regulating community composition and local dominance of trees in species-rich forests are poorly resolved, but the importance of interactions with soil microbes is increasingly acknowledged. Here, we show that tree seedlings that interact via root-associated fungal hyphae with soils beneath neighbouring adult trees grow faster and have greater survival than seedlings that are isolated from external fungal mycelia, but these effects are observed for species possessing ectomycorrhizas (ECM) and not arbuscular mycorrhizal (AM) fungi. Moreover, survival of naturally-regenerating AM seedlings over ten years is negatively related to the density of surrounding conspecific plants, while survival of ECM tree seedlings displays positive density dependence over this interval, and AM seedling roots contain greater abundance of pathogenic fungi than roots of ECM seedlings. Our findings show that neighbourhood interactions mediated by beneficial and pathogenic soil fungi regulate plant demography and community structure in hyperdiverse forests

Structure of the dimeric N-glycosylated form of fungal β-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies

<p>Abstract</p> <p>Background</p> <p>Fungal β-<it>N</it>-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal β-<it>N</it>-acetylhexosaminidase. The fungal β-<it>N</it>-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from <it>Aspergillus oryzae </it>was purified and its sequence was determined.</p> <p>Results</p> <p>The complete primary structure of the fungal β-<it>N</it>-acetylhexosaminidase from <it>Aspergillus oryzae </it>CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the <it>N</it>-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate – chitobiose with a stable value of binding energy during the molecular dynamics simulation.</p> <p>Conclusion</p> <p>Whereas the intracellular bacterial β-<it>N</it>-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal β-<it>N</it>-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and <it>N</it>-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys⁴⁴⁸with Cys⁴⁸³stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.</p

Analysis of urinary oligosaccharides in lysosomal storage disorders by capillary high-performance anion-exchange chromatography–mass spectrometry

Many lysosomal storage diseases are characterized by an increased urinary excretion of glycoconjugates and oligosaccharides that are characteristic for the underlying enzymatic defect. Here, we have used capillary high-performance anion-exchange chromatography (HPAEC) hyphenated to mass spectrometry to analyze free oligosaccharides from urine samples of patients suffering from the lysosomal storage disorders fucosidosis, α-mannosidosis, GM1-gangliosidosis, GM2-gangliosidosis, and sialidosis. Glycan fingerprints were registered, and the patterns of accumulated oligosaccharides were found to reflect the specific blockages of the catabolic pathway. Our analytical approach allowed structural analysis of the excreted oligosaccharides and revealed several previously unpublished oligosaccharides. In conclusion, using online coupling of HPAEC with mass spectrometric detection, our study provides characteristic urinary oligosaccharide fingerprints with diagnostic potential for lysosomal storage disorders