IR action spectroscopy of glycosaminoglycan oligosaccharides

Glycosaminoglycans (GAGs) are a physio- and pharmacologically highly relevant class of complex saccharides, possessing a linear sequence and strongly acidic character. Their repetitive linear core makes them seem structurally simple at first glance, yet differences in sulfation and epimerization lead to an enormous structural diversity with only a few GAGs having been successfully characterized to date. Recent infrared action spectroscopic experiments on sulfated mono- and disaccharide ions show great promise. Here, we assess the potential of two types of gas-phase action spectroscopy approaches in the range from 1000 to 1800 cm−1 for the structural analysis of complex GAG oligosaccharides. Synthetic tetra- and pentasaccharides were chosen as model compounds for this benchmark study. Utilizing infrared multiple photon dissociation action spectroscopy at room temperature, diagnostic bands are largely unresolved. In contrast, cryogenic infrared action spectroscopy of ions trapped in helium nanodroplets yields resolved infrared spectra with diagnostic features for monosaccharide composition and sulfation pattern. The analysis of GAGs could therefore significantly benefit from expanding the conventional MS-based toolkit with gas-phase cryogenic IR spectroscopy

SLC7A11 Overexpression in Glioblastoma Is Associated with Increased Cancer Stem Cell-Like Properties

System x_c^− is a sodium-independent electroneutral transporter, comprising a catalytic subunit xCT (SLC7A11), which is involved in importing cystine. Certain cancers such as gliomas upregulate the expression of system x_c^−, which confers a survival advantage against the detrimental effects of reactive oxygen species (ROS) by increasing generation of the antioxidant glutathione. However, ROS have also been shown to function as targeted, intracellular second messengers in an array of physiological processes such as proliferation. Several studies have implicated ROS in important cancer features such as migration, invasion, and contribution to a cancer stem cell (CSC)-like phenotype. The role of system x_c^− in regulating these ROS-sensitive processes in glioblastoma multiforme (GBM), the most aggressive malignant primary brain tumor in adults, remains unknown. Stable SLC7A11 knockdown and overexpressing U251 glioma cells were generated and characterized to understand the role of redox and system x_c^− in glioma progression. SLC7A11 knockdown resulted in higher endogenous ROS levels and enhanced invasive properties. On the contrary, overexpression of SLC7A11 resulted in decreased endogenous ROS levels as well as decreased migration and invasion. However, SLC7A11-overexpressing cells displayed actin cytoskeleton changes reminiscent of epithelial-like cells and exhibited an increased CSC-like phenotype. The enhanced CSC-like phenotype may contribute to increased chemoresistance and suggests that overexpression of SLC7A11 in the context of GBM may contribute to tumor progression. These findings have important implications for cancer management where targeting system x_C^− in combination with other chemotherapeutics can reduce cancer resistance and recurrence and improve GBM patient survival

Increased Expression of System x_c^- in Glioblastoma Confers an Altered Metabolic State and Temozolomide Resistance

Glioblastoma multiforme is the most aggressive malignant primary brain tumor in adults. Several studies have shown that glioma cells upregulate the expression of xCT (SLC7A11), the catalytic subunit of system x_c^-, a transporter involved in cysteine import, that modulates glutathione production and glioma growth. However, the role of system x_c^- in regulating the sensitivity of glioma cells to chemotherapy is currently debated. Inhibiting system x_c^- with sulfasalazine decreased glioma growth and survival via redox modulation, and use of the chemotherapeutic agent temozolomide together with sulfasalazine had a synergistic effect on cell killing. To better understand the functional consequences of system x_c^- in glioma, stable SLC7A11-knockdown and -overexpressing U251 glioma cells were generated. Modulation of SLC7A11 did not alter cellar proliferation but overexpression did increase anchorage-independent cell growth. Knockdown of SLC7A11 increased basal reactive oxygen species (ROS) and decreased glutathione generation resulting in increased cell death under oxidative and genotoxic stress. Overexpression of SLC7A11 resulted in increased resistance to oxidative stress and decreased chemosensitivity to temozolomide. In addition, SLC7A11 overexpression was associated with altered cellular metabolism including increased mitochondrial biogenesis, oxidative phosphorylation, and ATP generation. These results suggest that expression of SLC7A11 in the context of glioma contributes to tumorigenesis, tumor progression, and resistance to standard chemotherapy

Gas-Phase Structural Analysis of Sulfated Glycans

This work investigates the use of ion mobility spectrometry and gas-phase infrared spectroscopy in combination with mass spectrometry for the structural analysis of isomeric sulfated glycans. Both methods have been successfully applied for the analysis of glycans before. Ion mobility spectrometry offers a structural dimension, missing in classical mass spectrometry for the differentiation of isomeric structures, while infrared spectroscopy can provide detailed structural information of even gas-phase conformations. These methods allowed for the investigation of the interaction of glycosaminoglycan with magnetic resonance imaging probes, revealing an intricate binding motive and a surprising dissociation behaviour. They allowed for the separation and quantification of twelve partially isomeric sulfated disaccharides, which previously were analyzed by tedious and time-consuming liquid chromatography methods. And finally, they revealed two unknown sulfated glycan-specific gas-phase rearrangement processes, which further consolidate the unique challenges faced in the structural analysis of sulfated glycans

A 3D-Printed Offline Nano-ESI Source for Bruker MS Instruments

Nanoelectrospray ionization (nano-ESI) is a highly efficient and a widely used technique for the ionization of minute amounts of analyte. Offline nano-ESI sources are convenient for the direct infusion of complex mixtures that suffer from high matrix content and are crucial for the native mass spectrometric analysis of proteins. For Bruker instruments, no such source is readily available. Here we close this gap and present a 3D-printable nano-ESI source for Bruker instruments, which can be assembled by anyone with access to 3D printers. The source can be fitted to any Bruker mass spectrometer with an ionBooster ESI source and only requires minor, reversible changes to the original Bruker hardware. The general utility was demonstrated by recording high-resolution MS spectra of small molecules, intact proteins, as well as complex biological samples in negative and positive ion mode on two different Bruker instruments

Investigation of Glycosaminoglycans with Ion Mobility-Mass Spectrometry and Gas-Phase IR Spectroscopy

Glycosaminoglycans (GAGs) are a family of polydisperse polysaccharides, which are widely distributed at cell surfaces and in the extracellular matrix. Although structurally simple at first glance, with a repeating backbone of alternating hexuronic acid and hexosamine dimers, they display a highly complex structure, which results from their heterogeneous sulfation pattern. Even though structurally poorly understood, there has been increasing evidence that GAGs can transchelate gadolinium-based magnetic resonance imaging (MRI) contrast agents. This unintended release of gadolinium is the leading cause of nephrogenic systemic fibrosis. To date, however, the lack of structurally well-defined GAG samples has hindered a detailed elucidation of the underlying mechanism and only soft evidence of a GAG-induced gadolinium release has been reported. In this work we demonstrate the purification and isolation of GAG fragments from low- molecular-weight-heparin (LMWH) enoxaparin using size-exclusion chromatography. Additionally, we provide the first direct evidence for GAG-gadolinium binding using the synthetic model substance fondaparinux and LMWH enoxaparin. Gas-phase IR spectroscopy and CID-MS/MS experiments revealed possible binding sites of gadolinium on fondaparinux, with surprisingly strong binding contribution from hexuronic acid site chains. This data represent the first direct insights into this complex interaction and will in the future help to unravel the molecular details of GAG-induced gadolinium transchelation from MRI contrast agents

A 3D-Printed Offline Nano-ESI Source for Bruker MS Instruments

Nanoelectrospray ionization (nano-ESI) is a highly efficient and a widely used technique for the ionization of minute amounts of analyte. Offline nano-ESI sources are convenient for the direct infusion of complex mixtures that suffer from high matrix content and are crucial for the native mass spectrometric analysis of proteins. For Bruker instruments, no such source is readily available. Here we close this gap and present a 3D-printable nano-ESI source for Bruker instruments, which can be assembled by anyone with access to 3D printers. The source can be fitted to any Bruker mass spectrometer with an ionBooster ESI source and only requires minor, reversible changes to the original Bruker hardware. The general utility was demonstrated by recording high-resolution MS spectra of small molecules, intact proteins, as well as complex biological samples in negative and positive ion mode on two different Bruker instruments

Improving the interface of an e-commerce website by applying universal design principles

This paper presents an analysis of two websites in terms of accessibility and usability. An authorial e-commerce website with improvements for people with disabilities was implemented. The website was compared with a popular commercial service. The study was conducted on a group of students and used the eye tracking method, a questionnaire developed for the purpose of the study and the LUT checklist. Additionally, an accessibility study was performed using the WAVE web accessibility evaluation tool. In the eye tracking study five measures were selected to evaluate the websites: the task completion time, the mean fixation time, the mean number of fixations, the mean saccade duration, and the mean number of saccades. On the basis of the obtained results and after their initial processing basic statistics and box plots were created to facilitate interpretation of the results

Theory of scattering by an array of lossy dielectric, ferrite and conducting cylinders, Journal of Telecommunications and Information Technology, 2003, nr 1

Theory of scattering by lossy dielectric, ferrite and/or conducting cylinders is investigated using a combination of an iterative scattering procedure and the orthogonal expansion method. The addition theorems for vector cylindrical harmonics, which transform harmonics from one coordinate system to another, are presented