Risk model for suspected acute coronary syndrome is of limited value in an emergency department.

INTRODUCTION: Among patients with acute chest pain, acute coronary syndrome (ACS) is seen only in a minority of the patients, which raises the question, whether it is possible to separate a group with a high risk of ACS for admission to a cardiac care unit (CCU) from those with a low risk who would be treated at an emergency department (ED). The aim of this study was to describe a risk stratification model for a Danish context. METHODS: This was a historic prospective cohort study of patients with suspicion of ACS. The patient was defined as a low-risk patient and admitted to the ED if: 1) electrocardiogram (ECG) was normal, 2) the patient did not have persisting chest pain and 3) there was no history of ischaemic heart disease, heart failure or cardioverter defibrillator. Otherwise, patients were admitted to the CCU. The primary outcome was whether the ACS diagnosis was confirmed or rejected. RESULTS: We included a total of 488 patients with suspicion of ACS, 50% of whom were low-risk patients. 17% had a verified ACS; 10% of those in the low-risk group and 24% of those in the high-risk group (p = 0.0001). Among the verified ACS cases, 71% went primarily to the CCU. The odds ratio for an ACS if assigned to the high-risk group was 3.0. Allocation to the high-risk group, male gender and age above 60 years was associated with a higher risk of ACS. For patients fulfilling the high-risk definition, sensitivity was 71%, specificity 55%, negative predictive value 90% and positive predictive value 24% for an ACS. CONCLUSIONS: The model for stratification separated patients into two equal groups, allocated 71% of all ACS directly to the CCU and could not be improved by any of the additional factors examined. Further development of referral strategies for chest pain patients is required. FUNDING: none. TRIAL REGISTRATION: not relevant.

Cell surface protein glycosylation in cancer.

Glycosylation of proteins is one of the most important PTMs, with more than half of all human proteins estimated to be glycosylated. It is widely known that aberrant glycosylation has been implicated in many different diseases due to changes associated with biological function and protein folding. In cancer, there is increasing evidence pertaining to the role of glycosylation in tumour formation and metastasis. Alterations in cell surface glycosylation, particularly terminal motifs, can promote invasive behaviour of tumour cells that ultimately lead to the progression of cancer. While a majority of studies have investigated protein glycosylation changes in cancer cell lines and tumour tissue for individual cancers, the review presented here represents a comprehensive, in-depth overview of literature on the structural changes of glycosylation and their associated synthetic enzymes in five different cancer types originating from the breast, colon, liver, skin and ovary. More importantly, this review focuses on key similarities and differences between these cancers that reflect the importance of structural changes of cell surface N- and O-glycans, such as sialylation, fucosylation, degree of branching and the expression of specific glycosyltransferases for each cancer. It is envisioned that the understanding of these biologically relevant glycan alterations on cellular proteins will facilitate the discovery of novel glycan-based biomarkers which could potentially serve as diagnostic and prognostic indicators of cancer.

Challenges of determining O-glycopeptide heterogeneity: a fungal glucanase model system.

O-Linked glycosylation often occurs in mucin-type domains that are heavily and heterogeneously glycosylated and are challenging to analyze. The analysis of these domains is often overlooked because of these difficulties, but changes in mucinlike domain glycosylation are implicated in many diseases. Here we have explored several strategies to determine the heterogeneity of mucinlike O-glycosylated domains. Four glucanases secreted in large quantities from Trichoderma reesei, all containing heavily O-glycosylated mucinlike linker regions, were used as a model system. The strategies involved monosaccharide compositional analysis and identification of the released glycans by HPAEC-PAD and carbon-LC ESI-MS/MS. Glycosylated peptides were generated by different protease digestions (trypsin, papain, Asp-N, PreTAQ) and enriched by HILIC microcolumns, to determine the glycopeptide heterogeneity and glycosylation sites. The complex O-glycan heterogeneity on the intact glycoproteins and the enriched mucin-type domains was determined by MALDI-MS and ESI-MS, but the dense O-glycosylation in the mucin-type domains conferred high resistance to protease cleavage. ETD-MS/MS of the glycopeptide-enriched protease digests was unsuccessful for the de novo assignment of O-glycosylation at individual sites within the mucin-type domains but allowed several previously unknown O-linked sites outside the defined linker region to be found on two of the four glucanases. The protease digests produced many glycopeptides as determined by CID-MS/MS, but ETD fragmentation of these resulted in only a few interpretable spectra, suggesting that the use of ETD for determining the heterogeneous O-glycosylation at specific sites in regions of multiple occupancy is still in its infancy.

The glycosylation and characterization of the candidate Gc macrophage activating factor.

The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF candidate was tested for its effect on cytokine release from macrophages in human whole blood.

Protein chemical characterization of Gc globulin (vitamin D-binding protein) isoforms; Gc-1f, Gc-1s and Gc-2.

Gc globulin, also called vitamin D-binding protein, is a plasma protein involved in the extracellular actin-scavenger system, vitamin D transport and possibly also other biological activities. Low levels of Gc globulin have been found to correlate with multiple organ failure and non-survival of patients with fulminant hepatic failure and trauma. Here, we characterize the dominant isoforms of plasma-derived Gc globulin from Cohn fraction IV paste with respect to amino acid sequence and posttranslational modifications. Gc globulin was purified in large scale and the isoforms separated by ion exchange chromatography. The separated isoforms and several commercial preparations of individual isoforms were characterized by mass spectrometry. This revealed that the major isoforms were non-glycosylated. Compared to the Gc-1f isoform the other dominating isoforms represented an Asp/Glu substitution (Gc-1s) and a Thr/Lys substitution (Gc-2) in agreement with DNA sequencing studies. The commercial preparations were found to represent mainly one or two isoforms. An O-linked glycan with a mass of 656 Da and terminating with a sialic acid residue was detected on a minor proportion of Gc globulin molecules.

Large-scale purification and characterization of non-glycosylated Gc globulin (vitamin D-binding protein) from plasma fraction IV.

Gc globulin, also called vitamin D-binding protein, is a plasma protein involved in the extracellular actin-scavenger system. Low levels of Gc globulin have been found to correlate with multiple organ failure and nonsurvival of patients with fulminant hepatic failure and trauma. Therefore substitution therapy with Gc globulin might be beneficial for such patients, increasing their chance of survival. In the present study, we describe a large-scale purification process for the production of a virus-safe human plasma-derived Gc globulin from Cohn fraction IV paste. The process includes three ion-exchange-chromatography steps, followed by a gel filtration, and two virus-reduction steps are implemented. The Gc globulin product was characterized with respect to purity, functional activity, glycosylation and, finally, with respect to content of endotoxin. From the results, it can be concluded that human Gc globulin purified from Cohn fraction IV is non-glycosylated. The purified Gc globulin is able to mask the presence of endotoxin by 20%.