•A straightforward enzymatic method was developed to synthesize specific lyso-phospholipids.•20:1 lyso-PS is a true agonist of the TLR2/6 hetero-dimer.•Both chainlength and the headgroup of 20:1 ...lyso-PS determine this interaction.
Toll-like receptor 2 (TLR2) is an important pattern recognition receptor on the surface of host immune cells that binds a variety of ligands that are released by microorganisms as well as by damaged or dying host cells. According to the current concept, TLR2/1 and TLR2/6 heterodimers are activated by tri- or di-acylated ligands, respectively. However, also mono-acyl phospholipid containing lipid fractions derived from parasites, were reported to be able to activate TLR2. In order to provide conclusive evidence for the TLR2 activating capacity of mono-acyl phospholipids derived from pathogens, we developed a biosynthetic method to enzymatically convert commercially available phospholipids into several mono-acyl-phospholipid variants that were examined for their TLR2 activating capacity. These investigations demonstrated that 1-(11Z-eicosenoyl)-glycero-3-phosphoserine 20:1 (20:1 lyso-PS) is a true agonist of the TLR2/6 heterodimer and that its polar headgroup as well as the length of the acyl chain are crucial for TLR2 activation. In silico modelling further confirmed 20:1 mono-acyl PS as a ligand for TLR2/6 heterodimer, as this predicted that multiple hydrogen bonds are formed between the polar headgroup of 20:1 mono-acyl PS and amino acid residues of both TLR2 and TLR6. Future studies can now be performed to further assess the functions of 20:1 lyso-PS as an immunological mediator, because this enzymatic method enables its preparation in larger quantities than is possible by isolation from the parasite that naturally produces this compound, Schistosoma mansoni, the source of the original discovery (Van der Kleij et al., 2002).
Carbapenemase-resistant bacteria are increasingly spreading worldwide causing public concern due to their ability to elude antimicrobial treatment. Early identification of these bacteria is therefore ...of high importance. Here, we describe the development of a simple and robust protocol for the detection of carbapenemase activity in clinical isolates of Enterobacteriaceae, suitable for routine and clinical applications.
The final protocol involves cellular lysis and enzyme extraction from a defined amount of bacterial cells followed by the addition of a benchmark drug (e.g. the carbapenem antibiotic imipenem or ertapenem). Carbapenem inactivation is mediated by enzymatic hydrolysis (cleavage) of the β-lactam common structural motif, which can be detected using MALDI-TOF MS.
A total of 260 strains were studied (208 carbapenemase producers and 52 non-carbapenemase producers) resulting in 100% sensitivity and 100% specificity for the KPC, NDM and OXA-48-like PCR-confirmed positive isolates using imipenem as benchmark. Differences between the benchmark (indicator) antibiotics imipenem and ertapenem, buffer constituents and sample preparation methods have been investigated. Carbapenemase activity was further characterized by performing specific inhibitor experiments. Intraday and interday reproducibility (coefficient of variation) of the observed hydrolysis results were 15% and 30%, respectively. A comparative study of our extraction method and a recently published method using whole bacterial cells is presented and differences are discussed.
Using this method, an existing carbapenemase activity can be directly read from the mass spectrum as a ratio of hydrolysed product and substrate, setting an important step towards routine application in clinical laboratories.
Collagen has a triple helix form, structured by a -Gly-Xaa-Yaa- repetition, where Xaa and Yaa are amino acids. This repeating unit can be post-translationally modified by enzymes, where proline is ...often hydroxylated into hydroxyproline (Hyp). Two Hyp isomers occur in collagen: 4-hydroxyproline (4Hyp, Gly-Xaa-Pro, substrate for 4-prolyl hydroxylase) and 3-hydroxyproline (3Hyp, Gly-Pro-4Hyp, substrate for 3-prolyl hydroxylase). If 4Hyp is lacking at the Yaa position, then Pro at the Xaa position should remain unmodified. Nevertheless, in literature 41 positions have been described where Hyp occurs at the Xaa position (?xHyp) lacking an adjacent 4Hyp. We report four additional positions in liver and colorectal liver metastasis tissue (CRLM). We studied the sequence commonalities between the 45 known positions of ?xHyp. Alanine and glutamine were frequently present adjacent to ?xHyp. We showed that proline, position 584 in COL1A2, had a lower rate of modification in CRLM than in healthy liver. The isomeric identity of ?xHyp, that is, 3- and/or 4Hyp, remains unknown. We present a proof of principle identification of ?xHyp. This identification is based on liquid chromatography retention time differences and mass spectrometry using ETD-HCD fragmentation, complemented by ab initio calculations. Both techniques identify ?xHyp at position 584 in COL1A2 as 4-hydroxyproline (4xHyp).
Atherosclerosis is a lipid and inflammation-driven disease of the arteries that is characterized by gradual buildup of plaques in the vascular wall. A so-called vulnerable plaque, consisting of a ...lipid-rich necrotic core contained by a thin fibrous cap, may rupture and trigger thrombus formation, which can lead to ischemia in the heart (heart attack) or in the brain (stroke). In this study, we present a protocol to investigate the lipid composition of advanced human carotid plaques using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI), providing a framework that should enable the discrimination of vulnerable from stable plaques based on lipid composition. We optimized the tissue preparation and imaging methods by systematically analyzing data from three specimens: two human carotid endarterectomy samples (advanced plaque) and one autopsy sample (early stage plaque). We show a robust data reduction method and evaluate the variability of the endarterectomy samples. We found diacylglycerols to be more abundant in a thrombotic area compared to other plaque areas and could distinguish advanced plaque from early stage plaque based on cholesteryl ester composition. We plan to use this systematic approach to analyze a larger dataset of carotid atherosclerotic plaques.
A total of 164 cerebrospinal fluid (CSF) samples taken from neurological patients were classed into four groups according to the clinical diagnosis: multiple sclerosis (MScl, n = 44), clinically ...isolated syndrome of demyelination (CIS, n = 40), other inflammatory neurological disease (OIND, n = 26) and other neurological disease (OND, n = 54). After tryptic digestion, the samples were measured by MALDI-TOF MS. Spectra were analyzed using the R-project software package, in which a peak detection algorithm was developed. Subsequently, the peak lists were compared based on ranked data (non-parametric). Significant differences were observed in the comparisons of MScl vs. OND and CIS vs. OND. The comparisons of MScl vs. OIND, and CIS vs. OIND showed fewer significant differences. No significant differences were found in comparisons MScl vs. CIS and OIND vs. OND. MScl and CIS had strikingly similar profiles, probably a reflection of common pathological mechanisms. Three differentially expressed proteins in the comparison of MScl vs. OND were identified: chromogranin A, a potential marker for neurodegeneration; and two important factors in complement-mediated inflammatory reaction, clusterin and complement C3. CSF chromogranin A levels were confirmed to be significantly elevated in the MScl group using an ELISA.
Proton affinities and ion enthalpies Holmes, John L; van Huizen, Nick A; Burgers, Peter C
European journal of mass spectrometry (Chichester, England),
12/2017, Letnik:
23, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Proton affinities of a number of alkyl acetates (CH3–C(=O)–OR) and of methyl alkanoates (R–C(=O)–OCH3, R=H, alkyl) have been assembled from the literature or measured using the kinetic method. It was ...observed that the proton affinities for the isomeric species CH3–C(=O)–OR and R–C(=O)–OCH3 are almost identical, an unexpected result as the charge in these protonated ester molecules is largely at the keto carbon atom and so this site should be more sensitive to alkyl substitution. Analysis of the data, including those from lone pair ionisation and core-electron ionisation experiments available from the literature, indicate that after protonation, extensive charge relaxation (or polarisation) takes place (as is also the case, according to the literature, after core-electron ionisation). By contrast, after lone pair ionisation, which results in radical cations, such relaxation processes are relatively less extensive. As a consequence, changes in ion enthalpies of these protonated molecules follow more closely the changes in neutral enthalpies, compared with changes in enthalpies of the corresponding radical cations, formed by electron detachment. Preliminary analyses of published energetic data indicate that the above finding for organic esters may well be another example of a more general phenomenon.
Human metapneumovirus (HMPV) encodes a small hydrophobic (SH) protein of unknown function. HMPV from which the SH open reading frame was deleted (HMPVΔSH) was viable and displayed similar replication ...kinetics, cytopathic effect and plaque size compared with wild type HMPV in several cell-lines. In addition, no differences were observed in infection efficiency or cell-to-cell spreading in human primary bronchial epithelial cells (HPBEC) cultured at an air-liquid interphase. Host gene expression was analyzed in A549 cells infected with HMPV or HMPVΔSH using microarrays and mass spectrometry (MS) based techniques at multiple time points post infection. Only minor differences were observed in mRNA or protein expression levels. A possible function of HMPV SH as apoptosis blocker, as proposed for several members of the family Paramyxoviridae, was rejected based on this analysis. So far, a clear phenotype of HMPV SH deletion mutants in vitro at the virus and host levels is absent.
We introduce a matrix-assisted laser desorption ionization−Fourier transform ion cyclotron resonance (MALDI−FT-ICR) method for quantitative peptide profiling, using peak height as a measure for ...abundance. Relative standard deviations in peak height of peptides spiked over 3 orders of magnitude in concentration were below 10% and allowed for accurate comparisons between multiple sclerosis and controls. Application on a set of 163 cerebrospinal fluid (CSF) samples showed significantly differential abundant peptides, which were subsequently identified into proteins (e.g., chromogranin A, clusterin, and complement C3).
In Guillain-Barré syndrome (GBS), ganglioside mimicry of Campylobacter jejuni lipo-oligosaccharide (LOS) drives the production of cross-reactive Abs to peripheral nerve gangliosides. We determined ...whether sialic acid residues in C. jejuni LOS modulate dendritic cell (DC) activation and subsequent B cell proliferation as a possible mechanism for the aberrant humoral immune response in GBS. Highly purified sialylated LOS of C. jejuni isolates from three GBS patients induced human DC maturation and secretion of inflammatory cytokines that were inhibited by anti-TLR4 neutralizing Abs. The extent of TLR4 signaling and DC activation was greater with LOS of the wild type isolates than with nonsialylated LOS of the corresponding sialyltransferase gene knockout (cst-II mutant) strains, indicating that sialylation boosts the DC response to C. jejuni LOS. Supernatants of LOS-activated DCs induced B cell proliferation after cross-linking of surface Igs in the absence of T cells. Lower B cell proliferation indices were found with DC supernatants after DC stimulation with cst-II mutant or neuraminidase desialylated LOS. This study showed that sialylation of C. jejuni LOS enhances human DC activation and subsequent B cell proliferation, which may contribute to the development of cross-reactive anti-ganglioside Abs found in GBS patients following C. jejuni infection.
Although many patients are cured from prostate cancer (PCa) by surgery only, there are still patients who will experience rising prostate-specific antigen (PSA) levels after surgery, a condition ...known as biochemical recurrence (BCR). Novel protein prognostic markers in PCa tissue might enable finding better treatment for those patients experiencing BCR with a high chance of metastasis. In this study, we aimed to identify altered proteins in prostate cancer tissue, and to evaluate their potential role as prognostic markers. We used two proteomics strategies to analyse 34 prostate tumours (PCa) and 33 normal adjacent prostate (NAP) tissues. An independent cohort of 481 samples was used to evaluate the expression of three proteins: AGR2, FASN and LOX5 as prognostic markers of the disease. Tissue microarray immunohistochemical staining indicated that a low percentage of positive tumour cells for AGR2 (HR (95% CI) = 0.61 (0.43-0.93)), and a low percentage of positive tumour cells for LOX5 expression (HR (95% CI) = 2.53 (1.23-5.22)) are predictors of BCR after RP. In contrast, FASN expression had no prognostic value for PCa.
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