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Initiative recently coordinated a multiinstitutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze

Objectives The goal of this study was to create and validate a new set of sentence lists that could be used to evaluate the speech perception abilities of listeners with hearing loss in cases where adult materials are inappropriate due to difficulty level or content. Our intention was to generate a large number of sentence lists with an equivalent level of difficulty for the evaluation of performance over time and across conditions. Design The original Pediatric-AzBio sentence corpus included 450 sentences recorded from one female talker. All sentences included in the corpus were successfully repeated by kindergarten and first grade students with normal hearing. The mean intelligibility of each sentence was estimated by processing each sentence through a cochlear implant simulation and calculating the mean percent correct score achieved by 15 normal-hearing listeners. After sorting sentences by mean percent correct scores, 320 sentences were assigned to 16 lists of equivalent difficulty. List equivalency was then validated by presenting all sentence lists, in a novel random order, to adults and children with hearing loss. A final-validation stage examined single-list comparisons from adult and pediatric listeners tested in research or clinical settings. Results The results of the simulation study allowed for the creation of 16 lists of 20 sentences. The average intelligibility of each list ranged from 78.4% to 78.7%. List equivalency was then validated, when the results of 16 adult cochlear implant users and 9 pediatric hearing aid and cochlear implant users revealed no significant differences across lists. The binomial distribution model was used to account for the inherent variability observed in the lists. This model was also used to generate 95% confidence intervals for one and two list comparisons. A retrospective analysis of 361 instances from 78 adult cochlear implant users and 48 instances from 36 pediatric cochlear implant users revealed that the 95% confidence intervals derived from the model captured 94% of all responses (385/409). Conclusions The cochlear implant simulation was shown to be an effective method for estimating the intelligibility of individual sentences for use in the evaluation of cochlear implant users. Further the method used for constructing equivalent sentence lists and estimating the inherent variability of the materials has also been validated. Thus, the AzBio Pediatric Sentence Lists are equivalent and appropriate for the assessment of speech understanding abilities of children with hearing loss as well as adults for whom performance on AzBio sentences is near the floor.

Objective One of the hallmarks of severe pneumonia and associated Acute Lung Injury (ALI) is neutrophil recruitment to the lung. Leptin is thought to be up-regulated in the lung following injury and to exert diverse effects on leukocytes, influencing both chemotaxis and survival. We hypothesized that pulmonary leptin contributes directly to the development of pulmonary neutrophilia during pneumonia and ALI. Design Controlled human and murine in vivo and ex vivo experimental studies. Settings Research laboratory of a university hospital. Subjects Healthy human volunteers and subjects hospitalized with bacterial and H1N1 pneumonia. C57Bl/6 and db/db mice were also used. Interventions Lung samples from patients and mice with either bacterial or H1N1 pneumonia and associated ALI were immunostained for leptin. Human bronchoalveolar-lavage (BAL) samples obtained after lipopolysaccharide (LPS)-induced lung injury were assayed for leptin. C57Bl/6 mice were examined after oropharyngeal aspiration of recombinant leptin alone or in combination with E.coli- or K.pneumonia-induced pneumonia. Leptin-resistant (db/db) mice were also examined using the E.coli model. BAL neutrophilia and cytokine levels were measured. Leptin-induced chemotaxis was examined in human blood- and murine marrow-derived neutrophils in vitro. Measurements and Main Results Injured human and murine lung tissue showed leptin induction compared to normal lung, as did human BAL following LPS instillation. BAL neutrophilia in uninjured and infected mice was increased and lung bacterial-load decreased by airway leptin administration, whereas BAL neutrophilia in infected leptin-resistant mice was decreased. In sterile lung injury by LPS, leptin also appeared to decrease airspace neutrophil apoptosis. Both human and murine neutrophils migrated towards leptin in vitro, and this required intact signaling through the JAK2/PI3K pathway. Conclusion We demonstrate that pulmonary leptin is induced in injured human and murine lungs and that this cytokine is effective in driving alveolar airspace neutrophilia. This action appears to be caused by direct effects of leptin on neutrophils.

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We have previously reported that obesity attenuates pulmonary inflammation in both patients with acute respiratory distress syndrome (ARDS) and in mouse models of the disease. We hypothesized that obesity-associated hyperleptinemia, and not body mass per se, drives attenuation of the pulmonary inflammatory response and that this e_ect could also impair the host response to pneumonia. We examined the correlation between circulating leptin levels and risk, severity, and outcome of pneumonia in 2 patient cohorts (NHANES III and ARDSNet-ALVEOLI) and in mouse models of diet-induced obesity and lean hyperleptinemia. Plasma leptin levels in ambulatory subjects (NHANES) correlated positively with annual risk of respiratory infection independent of BMI. In patients with severe pneumonia resulting in ARDS (ARDSNet-ALVEOLI), plasma leptin levels were found to correlate positively with subsequent mortality. In obese mice with pneumonia, plasma leptin levels were associated with pneumonia severity, and in obese mice with sterile lung injury, leptin levels were inversely related to bronchoalveolar lavage neutrophilia, as well as to plasma IL-6 and G-CSF levels. These results were recapitulated in lean mice with experimentally induced hyperleptinemia. Our findings suggest that the association between obesity and elevated risk of pulmonary infection may be driven by hyperleptinemia.

BackgroundRecent progress in method development for characterising the branched structures of complex carbohydrates has now enabled higher throughput technology. Automation of structure analysis then calls for software development since adding meaning to large data collections in reasonable time requires corresponding bioinformatics methods and tools. Current glycobioinformatics resources do cover information on the structure and function of glycans, their interaction with proteins or their enzymatic synthesis. However, this information is partial, scattered and often difficult to find to for non-glycobiologists.MethodsFollowing our diagnosis of the causes of the slow development of glycobioinformatics, we review the "objective" difficulties encountered in defining adequate formats for representing complex entities and developing efficient analysis software.ResultsVarious solutions already implemented and strategies defined to bridge glycobiology with different fields and integrate the heterogeneous glyco-related information are presented.ConclusionsDespite the initial stage of our integrative efforts, this paper highlights the rapid expansion of glycomics, the validity of existing resources and the bright future of glycobioinformatics.

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