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The EUROCarbDB project is a design study for a technical framework, which provides sophisticated, freely accessible, open-source informatics tools and databases to support glycobiology and glycomic research. EUROCarbDB is a relational database containing glycan structures, their biological context and, when available, primary and interpreted analytical data from high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance experiments. Database content can be accessed via a web-based user interface. The database is complemented by a suite of glycoinformatics tools, specifically designed to assist the elucidation and submission of glycan structure and experimental data when used in conjunction with contemporary carbohydrate research workflows. All software tools and source code are licensed under the terms of the Lesser General Public License, and publicly contributed structures and data are freely accessible. The public test version of the web interface to the EUROCarbDB can be found at http://www.ebi.ac.uk/eurocarb.

The bioinformatic tool GlycosidIQTM was developed for computerized interpretation of oligosaccharide mass spectrometric fragmentation based on matching experimental data with theoretically fragmented oligosaccharides generated from the database GlycoSuiteDBTM. This use of the software for glycofragment mass fingerprinting obviates a large part of the manual, labor intensive, and technically challenging interpretation of oligosaccharide fragmentation. Using 130 negative ion electrospray ionization-tandem mass spectrometry fragment spectra from identified oligosaccharide structures, it was shown that the GlycosidIQ scoring algorithms were able to correctly identify oligosaccharides in the great majority of cases (correct structure top ranked in 78% of the cases and an additional 17% were ranked second highest in the sample set).

Background. A history of loss of consciousness (LOC) is frequently used as an indication for cranial computed tomography (CT) in the emergency department (ED) evaluation of children with blunt head trauma.Objective. We sought to determine whether an isolated LOC and/or amnesia is predictive of traumatic brain injury (TBI) in children with blunt head trauma.Methods. We prospectively enrolled children 1 week, persistent neurologic deficits, or hospitalization for ≥2 nights. We then investigated the association of LOC and/or amnesia with TBI in those patients without other symptoms or signs of TBI (“isolated” LOC and/or amnesia).Results. Of eligible children, 2043 (77%) were enrolled, 1271 (62%) of whom underwent CT; 1159 (91%) of these 1271 had their LOC and/or amnesia status known. A total of 801 (39%) of the 2043 enrolled children had a documented history of LOC and/or amnesia. Of the 745 with documented LOC and/or amnesia who underwent CT, 70 (9.4%; 95% confidence interval [CI]: 7.4%, 11.7%) had TBI identified on CT versus 11 of 414 (2.7%; 95% CI: 1.3%, 4.7%) without LOC and/or amnesia (difference: 6.7%; 95% CI: 4.1%, 9.3%). Of the 801 children known to have had LOC and/or amnesia (regardless of whether they underwent CT), 77 (9.6%; 95% CI: 7.7%, 11.9%) had TBI requiring acute intervention versus 11 of 1115 (1%; 95% CI: 0.5%, 1.8%) of those without LOC and/or amnesia (difference: 8.6%; 95% CI: 6.5%, 10.7%). For those with an isolated LOC and/or amnesia without other signs or symptoms of TBI, however, 0 of 142 (95% CI: 0%, 2.1%) had TBI identified on CT, and 0 of 164 (95% CI: 0%,1.8%) had TBI requiring acute intervention.Conclusions. Isolated LOC and/or amnesia, defined by the absence of other clinical findings suggestive of TBI, are not predictive of either TBI on CT or TBI requiring acute intervention. Elimination of an isolated LOC and/or amnesia as an indication for CT may decrease unnecessary CT use in those patients without an appreciable risk of TBI.

Computed tomography (CT) is frequently used in evaluating children with blunt head trauma. Routine use of CT, however, has disadvantages. Therefore, we sought to derive a decision rule for identifying children at low risk for traumatic brain injuries.We enrolled children with blunt head trauma at a pediatric trauma center in an observational cohort study between July 1998 and September 2001. We evaluated clinical predictors of traumatic brain injury on CT scan and traumatic brain injury requiring acute intervention, defined by a neurosurgical procedure, antiepileptic medications for more than 1 week, persistent neurologic deficits, or hospitalization for at least 2 nights. We performed recursive partitioning to create clinical decision rules.Two thousand forty-three children were enrolled, 1,271 (62%) underwent CT, 98 (7.7%; 95% confidence interval [CI] 6.3% to 9.3%) had traumatic brain injuries on CT scan, and 105 (5.1%; 95% CI 4.2% to 6.2%) had traumatic brain injuries requiring acute intervention. Abnormal mental status, clinical signs of skull fracture, history of vomiting, scalp hematoma (in childrenor =2 years of age), or headache identified 97/98 (99%; 95% CI 94% to 100%) of those with traumatic brain injuries on CT scan and 105/105 (100%; 95% CI 97% to 100%) of those with traumatic brain injuries requiring acute intervention. Of the 304 (24%) children undergoing CT who had none of these predictors, only 1 (0.3%; 95% CI 0% to 1.8%) had traumatic brain injury on CT, and that patient was discharged from the ED without complications.Important factors for identifying children at low risk for traumatic brain injuries after blunt head trauma included the absence of: abnormal mental status, clinical signs of skull fracture, a history of vomiting, scalp hematoma (in childrenor =2 years of age), and headache.

The nature of the N- and O- linked glycosylation of acetylxylan esterase (AXE) of the Trichoderma reesei strain Rut-C30 has been characterized using different enzymatic, chromatographic, and mass spectrometric techniques. The combined data showed that the AXE N-glycan is phosphorylated and highly mannosylated. The predominant N-glycans on the single glycosylation site on AXE can be represented as GlcNAc2Man(1–6)P. The linker–substrate binding domain peptide separated from the core by papain digestion is heavily O-glycosylated and consists of mannose, galactose, and possibly glucose as monosaccharide and disaccharide substituents. In addition to glycosylation, sulfation was observed in the linker region. Both N- and O- linked glycans show remarkable heterogeneity. Three isoforms of AXE, separated by 2D SDS–PAGE, are described with pI values of 5.0, 5.3, and 5.9. The three isoforms can be explained by posttranslational modification of the enzyme by glycans, phosphate, and sulfate. Advancing the knowledge on the nature of the glycans produced by T. reesei is elementary for its use as a host for the expression of heterologous glycoproteins of industrial and pharmaceutical importance.

Proteomics is a data-rich discipline that makes extensive use of separation tools, mass spectrometry and bioinformatics to analyze and interpret the features and dynamics of the proteome. A major challenge for the field is how proteomics data can be stored and managed, such that data become permanent and can be mined with current and future tools. This article details our experience in the development of a commercial proteomic information management system. We identify the challenges faced in data acquisition, workflow management, data permanence, security, data interpretation and analysis, as well as the solutions implemented to address these issues. We finally provide a perspective on data management in proteomics and the implications for academic and industry-based researchers working in this field.

GlycoSuiteDB, a database of glycan structures, has been constructed with an emphasis on quality, consistency and data integrity. Importance has been placed on making the database a reliable and useful resource for all researchers. This database can help researchers to identify what glycan structures are known to be attached to certain glycoproteins, as well as more generally identifying what types of glycan structures are associated with different states, for example, different species, tissues and diseases. To achieve this, a major effort has gone into data standardisation. Many rules and standards have been adopted, especially for representing glycan structure and biological source information. This paper describes some of the challenges faced during the continuous development of GlycoSuiteDB.

Cellobiohydrolase I is an industrially important exocellulase secreted in high yields by the filamentous fungus Trichoderma reesei. The nature and effect of glycosylation of CBHI and other cellulolytic enzymes is largely unknown, although many other structural and mechanistic aspects of cellulolytic enzymes are well characterised. Using a combination of liquid chromatography, electrospray mass spectrometry, solid-phase Edman degradation, and monosaccharide analysis we have identified every site of glycosylation of CBHI from a high cellulase-producing mutant strain of T. reesei, ALKO2877, and characterised each site in terms of its modifying carbohydrate and site-specific heterogeneity. The catalytic core domain comprises three N-linked glycans which each consist of a single N-acetylglucosamine residue. Within the glycopeptide linker domain, all eight threonines are variably glycosylated with between at least one, and up to three, mannose residues per site. All serines in this domain are at least partially glycosylated with a single mannose residue. This linker region has also been shown to be sulfated by a combination of ion chromatography and collision-induced dissociation electrospray mass spectrometry. The sulfate is probably mannose-linked. The biological significance of N-linked single N-acetylglucosamine in the catalytic core, and mannose sulfation in the linker region, is not known.

One characteristic of glycoproteins is that they are separated by two-dimensional electrophoresis (2-D PAGE) into typical ‘trains’ of protein spots which separate on the basis of different isoelectric point (pI) and/or molecular mass. The pattern of these trains often varies in development and disease. While the isoforms differ both in the number of sites of glycosylation and the types of carbohydrate attached to the protein, classical methods of glycan analysis are insensitive at the levels typically separated by 2-D PAGE. Developments in mass spectrometry technologies have enabled the characterization of most of the oligosaccharide attributes to be determined on picomole amounts of protein. These techniques are beginning to allow the glycoform heterogeneity on 2-D separated glycoproteins to be analyzed.