Development of a novel workflow method to characterize and validate clinical data quality acquired through the Veterinary Committee on Trauma registry.

OBJECTIVE: To generate and apply a novel workflow method to assess the quality of data from the Veterinary Committee on Trauma (VetCOT) registry. ANIMALS: Canine and feline trauma patient data entered by identified and verified Veterinary Trauma Centers into the VetCOT registry between April 2017-December 2018 were retrieved for analysis. METHODS: Analysis software (RVetQual) was created in the R programming language to compare 5,000 cases exported from the VetCOT registry with samples of original corresponding records from 6 veterinary trauma centers. In addition, an evaluation of the consistency and completeness of the trauma registry was conducted. RESULTS: The utilization of this analysis tool allowed an assessment of the VetCOT trauma registry. Some of the variables effecting the accuracy, consistency, and completeness of the VetCOT trauma registry were canine and feline age, weight, trauma time entered, and mismatches in blood glucose. However, the completeness of the database was minimally affected. CLINICAL RELEVANCE: RVetQual is an efficient, accessible, and adjustable tool that facilitates the assessment of the data quality of the VetCOT registry. Such an assessment can lead to improvement of the quality of information serving to guide further trauma patient care.

[Factors influencing the choice of nondonor families in a French organ-harvesting center]. / Analyse des raisons motivant le refus du don d'organes par les familles de patients en état de mort encéphalique dans un centre régional de prélèvement.

OBJECTIVES: Report the reasons that lead families to refuse organ donation during their close solicitation by hospital coordination. MATERIAL AND METHODS: A retrospective study was conducted between 2012 and 2015, including 148 (34%) refusal of organ donation among 426 patients identified in a state of brain death. A questionnaire of the family was completed for each interview. Collected data concerned patient characteristics, cause of death, description of the interview and reasons for refusal. A descriptive statistical analysis was performed. RESULTS: The median age of patients was 50 years with a sex ratio of 1.4 men to 1 woman. The most common reason for non-donor family was the desire to maintain the integrity of the body of the patient (28%) followed by a religious order pattern (11%), brutality and suddenness of death (9%), the denial of death (6%) and early age of the donor (5%). In 39% of cases, the family said that the donor had expressed a written or oral refusal in his lifetime. CONCLUSION: A better understanding of the reasons leading to the refusal of non-donor family could provide assistance to the medical team on actions to general public with the aim to reduce the refusal rate. LEVEL OF EVIDENCE: 4.

Acute insulin response to arginine in deceased donors predicts the outcome of human islet isolation.

Despite a stringent donor selection, human islet isolation remains frustratingly unpredictable. In this study, we measured acute insulin response to arginine (AIRarg), an in vivo surrogate measure of islet mass, in 29 human deceased donors before organ donation, and correlated values with the outcome of islet isolation. Thirteen isolations (45%) met the threshold for clinical islet transplantation. Among all measured donor characteristics, the only discriminating variable between successful or unsuccessful isolations was donor AIRarg (p < 0.01). Using a threshold of 55 microIU/mL (ROC curve AUC: 72%), isolation was successful in 12/19 donors with high AIRarg and in 1/10 donors with low AIRarg (p < 0.001). The negative and positive predictive values were 90 and 63%, respectively. If used to select donors in the entire cohort, AIRarg would have increased our success rate by 40% and avoided 56% of unsuccessful isolations while missing only 8% of successful preparations. Our results suggest that donor AIRarg is markedly superior to body mass index (BMI) and other criteria currently used to predict isolation outcome. If routinely performed in deceased donors, this simple test could significantly reduce the failure rate of human islet isolation.

The role of spiral CT for the assessment of the intracranial circulation in suspected brain-death.

BACKGROUND AND PURPOSE: The aim of this study was to assess the role of spiral CT for the diagnosis of brain death. METHODS: Over a 12-month period, 15 patients that fulfilled the clinical criteria of brain death were referred from the intensive care unit to evaluate remaining intracranial blood flow by spiral CT. The clinical diagnosis was confirmed by an apnea test in all cases. Two phases of spiral CT were performed at 20 and 60 seconds after the start of contrast media injection. Qualitative analysis included the evaluation of vessel opacification (arteries and veins) by two radiologists in consensus. RESULTS: The cortical segments of the middle cerebral artery (MCA) were assessable in all patients, whereas the internal cerebral veins could not be evaluated in five patients due to artifacts or intracranial hemorrhage. Opacification of the major branches of the circle of Willis was observed in seven cases. Unilateral opacification of cortical branches of the MCA occurred in one. We did not observe bilateral enhancement of cortical MCA branches. The internal cerebral veins did not enhance in brain death. CONCLUSION: The absence of internal cerebral vein opacification and the absence of bilateral enhancement of cortical MCA branches constituted the best criteria of brain death by contrast enhanced spiral CT.

The nematode Caenorhabditis elegans synthesizes unusual O-linked glycans: identification of glucose-substituted mucin-type O-glycans and short chondroitin-like oligosaccharides.

The free-living nematode Caenorhabditis elegans is a relevant model for studies on the role of glycoconjugates during development of multicellular organisms. Several genes coding for glycosyltransferases involved in the synthesis of N- and O-linked glycans have already been isolated, but, apart from repetitive dimers of glycosaminoglycans, no detailed structure of either type of component has been published so far. This study aimed to establish the structures of the major O-glycans synthesized by C. elegans to give an insight into the endogenous glycosyltransferase activities expressed in this organism. By the use of NMR and MS, we have resolved the sequence of seven of these components that present very unusual features. Most of them were characterized by the type-1 core substituted on Gal and/or GalNAc by (beta1-4)Glc and (beta1-6)Glc residues. Another compound exhibited the GalNAc(beta1-4)N-acetylglucosaminitol sequence in the terminal position, to which was attached a tetramer of beta-Gal substituted by both Fuc and 2-O-methyl-fucose residues. Our experimental procedure led also to the isolation of glycosaminoglycan-like components and oligomannosyl-type N-glycans. In particular, the data confirmed that C. elegans synthesizes the ubiquitous linker sequence GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl.

Diversity of O-linked glycosylation patterns between species. Characterization of 25 carbohydrate chains from oviducal mucins of Rana ridibunda.

Amphibia egg jelly coats are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they pass along the different oviducal portions. Mucin type glycoproteins are the major constituents of the egg jelly coats. In this study, the O-linked carbohydrate chains of the jelly coats surrounding the eggs of Rana ridibunda were released by alkaline borohydride treatment. Fractionation of the mixture of O-linked oligosaccharide-alditols was achieved by a combination of chromatographic techniques including gel-permeation chromatography, ion-exchange chromatography and high-performance liquid chromatography using an amino-bonded silica column. The primary structures of these O-glycans were determined by one-dimensional and two-dimensional 1H-NMR spectroscopy and matrix-assisted laser-desorption-ionization-time-of flight mass spectrometry. 25 oligosaccharide structures, possessing a core consisting of Gal(beta1-3)GalNAc-ol with or without branching through a GlcNAc residue linked (beta1-6) to the GalNAc residue (core type 2 or core type 1, respectively) are described. The most representative antennae are: HSO3(6)[Fuc(alpha1-3)]GlcNAc; Gal(beta1-2)Gal; Gal(beta1-2)Gal(alpha1-3)[Fuc(alpha1-2)]Gal; GlcA(beta1-3)-Gal(beta1-3)[Fuc(alpha1-2)]Gal; GalNAc(alpha1-4)Gal(beta1-4)Gal; Gal(beta1-3)GalNAc(alpha1-4)Gal(beta1-4)Gal and GlcA(beta1-3)Gal(beta1-3)GalNAc. These results confirm the species-specific O-glycosylation of Amphibia oviducal mucins. The significance of this observation should be linked to a symbiotic role of carbohydrates involved in host-parasite interactions.

Recombinant human interleukins IL-1alpha, IL-1beta, IL-4, IL-6, and IL-7 show different and specific calcium-independent carbohydrate-binding properties.

A method was developed for the determination of putative lectin activities of cytokines. It involved the immunoblotting measurement of the quantity of these cytokines unbound to a series of different immobilized glycoconjugates and displacement of the bound cytokines with oligosaccharides of known structures. This method allows demonstrating that the following interleukins specifically recognize different oligosaccharide structures in a calcium-independent mechanism: interleukin-1alpha binds to the biantennary disialylated N-glycan completed with two Neu5Acalpha2-3 residues; interleukin-1beta to a GM4 sialylated glycolipid Neu5Acalpha2-3Galbeta1-Cer having very long and unusual long-chain bases; interleukin-4 to the 1,7 intramolecular lactone of N-acetyl-neuraminic acid; interleukin-6 to compounds having N-linked and O-linked HNK-1-like epitopes; and interleukin-7 to the sialyl-Tn antigen. Because the glycan ligands are rare structures in human circulating cells, it is suggested that such activities could be essential for providing specific signaling systems to cells having both the receptors and the oligosaccharide ligands of the interleukin at their cell surface.

O-glycan variability of egg-jelly mucins from Xenopus laevis: characterization of four phenotypes that differ by the terminal glycosylation of their mucins.

Eggs from Xenopus laevis are surrounded by several layers of jelly that are needed for proper fertilization. Jelly coat is composed of high-molecular-mass glycoconjugates to which are bound many globular proteins. O-glycans released from the jelly coat of X. laevis have been partially described in previous studies. In this study, we compared the glycosylation pattern of the egg jelly coat isolated from six specimens of X. laevis. The O-glycans were released from jelly coats by alkali/borohydride treatment. Structural characterization was performed through a combination of one- and two-dimensional (1)H-NMR and methylation analysis. This allowed the description of a new family of sulphated O-glycans present in jelly coats of all X. laevis. However, the jelly O-glycans showed a low extent of polymorphism between specimens. This intra-specific variability was restricted to the terminal substitution of O-linked oligosaccharides. The differential expression of two glycosyltransferase [an alpha-(1-->4) galactosyltransferase and an alpha-(1-->3) fucosyltransferase] activities resulted in the characterization of four phenotypes of X. laevis. Furthermore, electrophoretic analysis suggested that the high-molecular-mass fraction of jelly coat was mostly composed of mucin-type glycoproteins. Blot analysis with lectins confirmed that the glycan variability was borne by these mucin-type components. However, fertilization assays suggested that the glycan polymorphism had no repercussion on egg fertilizability.

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin. Characterization of the sequence Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2)Man.

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21. 90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and 1H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N'-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (alpha 2-6) or (alpha 2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (alpha 1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2 )Man(alpha 1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(alpha 1-6). In fraction mTf-V, which was found to be very heterogeneous by (1)H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri'-antennary glycans sialylated by Neu5Gc alpha-2,6- and alpha-2, 3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(alpha 2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (alpha 2-6)GlcNAc sialyltransferase.

Capsular polysaccharide produced by the thermophilic cyanobacterium Mastigocladus laminosus. Structural study of an undecasaccharide obtained by lithium degradation.

The capsular polysaccharide produced by the thermophilic cyanobacterium Mastigocladus laminosus has been subjected to a specific degradation with lithium in ethylenediamine. The released undecasaccharide attached to one unit of tetrahydroxycyclopentanecarboxylic acid has been characterized by a combination of 2D nuclear magnetic resonance spectroscopy, mass spectrometry, monosaccharidic composition and linkage analyses. From the overlap of the structure of this oligosaccharide with previously identified di-, tri- and pentasaccharides released by mild acid hydrolysis, the capsular polysaccharide was deduced to have a pentadecasaccharide repeating unit with the following structure:

Structural analysis of three sulfated oligosaccharides isolated from human milk.

The structures of two sulfated octasaccharides and one sulfated nonasaccharide isolated from human milk have been investigated. Using 13C and 1H NMR spectroscopy and ESMS, the following structures 1-3 were established: [formula: see text].

Acquisition of species-specific O-linked carbohydrate chains from oviducal mucins in Rana arvalis. A case study.

The extracellular matrix surrounding amphibian eggs is composed of mucin-type glycoproteins, highly O-glycosylated and plays an important role in the fertilization process. Oligosaccharide-alditols were released from the oviducal mucins of the anuran Rana arvalis by alkali-borohydride treatment in reduced conditions. Neutral and acidic oligosaccharides were fractionated by ion-exchange chromatographies and purified by HPLC. Each compound was identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) spectrometry, NMR spectroscopy, electrospray ionization-tandem mass spectroscopy (ESI-MS/MS) and permethylation analyses. This paper reports on the structures of 19 oligosaccharide-alditols, 12 of which have novel structures. These structures range in size from disaccharide to octasaccharide. Some of them are acidic, containing either a glucuronic acid or, more frequently, a sulfate group, located either at the 6 position of GlcNAc or the 3 or 4 positions of Gal. This latter sulfation is novel and has only been characterized in the species R. arvalis. This structural analysis led to the establishment of several novel carbohydrate structures, demonstrating the structural diversity and species-specificity of amphibian glycoconjugates.

Structural analysis of 13 neutral oligosaccharide-alditols released by reductive beta-elimination from oviducal mucins of Rana temporaria.

Amphibian eggs are always surrounded by an extracellular matrix, named the jelly coat. This is mainly composed of a highly O-glycosylated, mucin-type glycoprotein. This work has consisted of isolating O-linked neutral oligosaccharides from oviducal mucin of Rana temporaria, with a view to determining their primary structure. Hence, these carbohydrate chains have been released by alkaline borohydride treatment leading to stable glycans. The oligosaccharide-alditols have been purified by ion-exchange chromatography and separated by HPLC. The primary structure of 13 of these carbohydrate chains have been obtained by 1D/2D 1H-NMR spectroscopy and methylation analyses, in combination with MALDI-TOF mass spectroscopy. The results confirm what has been observed for six other amphibians about the species-specificity of the carbohydrate moieties and their likely involvement in the species-specific gamete recognition.

The Candida albicans phospholipomannan is a family of glycolipids presenting phosphoinositolmannosides with long linear chains of beta-1,2-linked mannose residues.

In a series of studies, we have shown that Candida albicans synthesizes a glycolipid, phospholipomannan (PLM), which reacted with antibodies specific for beta-1,2-oligomannosides and was biosynthetically labeled by [(3)H]mannose, [(3)H]palmitic acid, and [(32)P]phosphorus. PLM has also been shown to be released from the C. albicans cell wall and to bind to and stimulate macrophage cells. In this study, we show by thin layer chromatography scanning of metabolically radiolabeled extracts that the C. albicans PLM corresponds to a family of mannose and inositol co-labeled glycolipids. We describe the purification process of the molecule and the release of its glycan fraction through alkaline hydrolysis. Analysis of this glycan fraction by radiolabeling and methylation-methanolysis confirmed the presence of inositol and of 1, 2-linked mannose units. NMR studies evidenced linear chains of beta-1,2-oligomannose as the major PLM components. Mass spectrometry analysis revealed that these chains were present in phosphoinositolmannosides with degrees of polymerization varying from 8 to 18 sugar residues. The PLM appears as a new type of eukaryotic inositol-tagged glycolipid in relationship to both the absence of glucosamine and the organization of its glycan chains. This first structural evidence for the presence of beta-1, 2-oligomannosides in a glycoconjugate other than the C. albicans phosphopeptidomannan may have some pathophysiological relevance to the adhesive, protective epitope, and signaling properties thus far established for these residues.

Structural analysis of 20 oligosaccharide-alditols released from the jelly coat of Rana palustris eggs by reductive beta-elimination characterization of the polymerized sequence [Gal(beta1, 3)GalNAc(alpha1-4)]n.

The eggs of amphibians are surrounded by three to eight layers of jelly coats. This extracellular matrix is mainly composed of hydrated mucin-type glycoproteins. These highly glycosylated molecules are synthesized by oviduct and play an important role in the fertilization process. Recent structural analyses have shown the strict species-specificity of the O-linked oligosaccharides which constitute 60-70% of these oviducal mucins. Consequently, these carbohydrate chains represent new phenotypic markers, and from a biological point of view, can influence parasite tropism or can be involved in species-specific interaction of gametes. The primary structure of 20 oligosaccharide-alditols, released by alkali/borohydride treatment from the mucin of Rana palustris egg jelly coats, was established by 1H and 13C-NMR analysis. Thirteen of these components possess new structures and the polymerization of the sequence Gal(beta1-3)GalNAc(alpha1-4) characterizes the species-specificity of R. palustris.

Activity of enzymes catalyzing formation of beta-L-fucosyl phosphate and GDP-beta-L-fucose in amphibian tissues and their application in chemo-enzymic synthesis of GDP-beta-L-fucose.

Amphibian oviduct and liver were shown to contain enzymes that catalyze the formation of GDP-beta-L-fucose from GDP-alpha-D-mannose or L-fucose. The conversion of L-fucose into beta-L-fucopyranosyl phosphate was achieved on a preparative scale using high-activity fucokinase in toad liver extracts. For chemo-enzymic preparation of GDP-beta-L-fucose, a convenient modification for pyrophosphate synthesis through phosphomorpholidate which does not require anhydrous conditions is suggested.

A reexamination of the role of GABA in the mammalian suprachiasmatic nucleus.

Three independent electrophysiological approaches in hypothalamic slices were used to test the hypothesis that gamma-amino butyric acid (GABA)A receptor activation excites suprachiasmatic nucleus (SCN) neurons during the subjective day, consistent with a recent report. First, multiple-unit recordings during either the subjective day or night showed that GABA or muscimol inhibited firing activity of the SCN population in a dose-dependent manner. Second, cell-attached recordings during the subjective day demonstrated an inhibitory effect of bath- or microapplied GABA on action currents of single SCN neurons. Third, gramicidin perforated-patch recordings showed that bicuculline increased the spontaneous firing rate during the subjective day. Therefore, electrophysiological data obtained by three different experimental methods provide evidence that GABA is inhibitory rather than excitatory during the subjective day.

Carbohydrate binding, quaternary structure and a novel hydrophobic binding site in two legume lectin oligomers from Dolichos biflorus.

The seed lectin (DBL) from the leguminous plant Dolichos biflorus has a unique specificity among the members of the legume lectin family because of its high preference for GalNAc over Gal. In addition, precipitation of blood group A+H substance by DBL is slightly better inhibited by a blood group A trisaccharide (GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal) containing pentasaccharide, and about 40 times better by the Forssman disaccharide (GalNAc(alpha1-3)GalNAc) than by GalNAc. We report the crystal structures of the DBL-blood group A trisaccharide complex and the DBL-Forssman disaccharide complex.A comparison with the binding sites of Gal-binding legume lectins indicates that the low affinity of DBL for Gal is due to the substitution of a conserved aromatic residue by an aliphatic residue (Leu127). Binding studies with a Leu127Phe mutant corroborate these conclusions. DBL has a higher affinity for GalNAc because the N-acetyl group compensates for the loss of aromatic stacking in DBL by making a hydrogen bond with the backbone amide group of Gly103 and a hydrophobic contact with the side-chains of Trp132 and Tyr104. Some legume lectins possess a hydrophobic binding site that binds adenine and adenine-derived plant hormones, i.e. cytokinins. The exact function of this binding site is unknown, but adenine/cytokinin-binding legume lectins might be involved in storage of plant hormones or plant growth regulation. The structures of DBL in complex with adenine and of the dimeric stem and leaf lectin (DB58) from the same plant provide the first structural data on these binding sites. Both oligomers possess an unusual architecture, featuring an alpha-helix sandwiched between two monomers. In both oligomers, this alpha-helix is directly involved in the formation of the hydrophobic binding site. DB58 adopts a novel quaternary structure, related to the quaternary structure of the DBL heterotetramer, and brings the number of know legume lectin dimer types to four.