An intranasal cationic liposomal polysaccharide vaccine elicits humoral immune responses against Streptococcus pneumoniae.

Diseases caused by S. pneumoniae are the leading cause of child mortality. As antibiotic resistance of S. pneumoniae is rising, vaccination remains the most recommended solution. However, the existing pneumococcal polysaccharides vaccine (Pneumovax® 23) proved only to induce T-independent immunity, and strict cold chain dependence of the protein conjugate vaccine impedes its promotion in developing countries, where infections are most problematic. Affordable and efficient vaccines against pneumococcus are therefore in high demand. Here, we present an intranasal vaccine Lipo+CPS12F&αGC, containing the capsular polysaccharides of S. pneumoniae 12F and the iNKT agonist α-galactosylceramide in cationic liposomes. In BALB/cJRj mice, the vaccine effectively activates iNKT cells and promotes B cells maturation, stimulates affinity-matured IgA and IgG production in both the respiratory tract and systemic blood, and displays sufficient protection both in vivo and in vitro. The designed vaccine is a promising, cost-effective solution against pneumococcus, which can be expanded to cover more serotypes and pathogens.

Chemical synthesis and immunological evaluation of cancer vaccines based on ganglioside antigens and α-galactosylceramide.

iNKT cells - often referred as the "Swiss Army knife" of the immune system - have emerged as central players in cancer vaccine therapies. Glycolipids activating iNKT cells, such as α-galactosylceramide (αGalCer), can enhance the immune response against co-delivered cancer antigens and have been applied in the design of self-adjuvanting anti-tumor vaccines. In this context, this work focuses on the chemical synthesis of ganglioside tumor-associated carbohydrate antigens (TACAs), namely GM3 and (Neu5Gc)GM3 antigens, their conjugation to αGalCer, and their formulation into liposomes as an efficient platform for their in vivo delivery. Liposomes containing GM3-αGalCer, (Neu5Gc)GM3-αGalCer, and equimolar amounts of the two conjugates have been fully characterized and their ability to activate iNKT cell has been confirmed ex vivo in mouse and human cell assays. The candidates were tested in in vivo immunization studies, demonstrating an ability to induce both TH1 and TH2 cytokines leading to the production of all subclasses of IgG antibodies. Notably, the study also demonstrated that serum antibodies raised against the two TACAs, alone and in combination, were cross-reactive. This finding has consequences for future vaccine designs - even if a highly tumor-selective antigen is chosen, the resulting antibody response may be broader than anticipated.

Chemical synthesis of natural and azido-modified UDP-rhamnose and -arabinofuranose for glycan array-based characterization of plant glycosyltransferases.

Chemical syntheses of UDP-rhamnose and UDP-arabinofuranose and respective azido-modified analogues are reported. The prepared substrates are useful for the glycan array-based analysis of glycosyltransferases, as exemplified with the plant cell wall-biosynthetic enzymes PvXAT3, AtRRT4 and PtRRT5.

Synthesis of fluoro- and seleno-containing D-lactose and D-galactose analogues.

Synthetic deoxy-fluoro-carbohydrate derivatives and seleno-sugars are useful tools in protein-carbohydrate interaction studies using nuclear magnetic resonance spectroscopy because of the presence of the 19F and 77Se reporter nuclei. Seven saccharides containing both these atoms have been synthesized, three monosaccharides, methyl 6-deoxy-6-fluoro-1-seleno-ß-D-galactopyranoside (1) and methyl 2-deoxy-2-fluoro-1-seleno-α/ß-D-galactopyranoside (2α and 2ß), and four disaccharides, methyl 4-O-(ß-D-galactopyranosyl)-2-deoxy-2-fluoro-1-seleno-ß-D-glucopyranoside (3), methyl 4-Se-(ß-D-galactopyranosyl)-2-deoxy-2-fluoro-4-seleno-ß-D-glucopyranoside (4), and methyl 4-Se-(2-deoxy-2-fluoro-α/ß-D-galactopyranosyl)-4-seleno-ß-D-glucopyranoside (5α and 5ß), the three latter compounds with an interglycosidic selenium atom. Selenoglycosides 1 and 3 were obtained from the corresponding bromo sugar by treatment with dimethyl selenide and a reducing agent, while compounds 2α/2ß, 4, and 5α/5ß were synthesized by the coupling of a D-galactosyl selenolate, obtained in situ from the corresponding isoselenouronium salt, with either methyl iodide or a 4-O-trifluoromethanesulfonyl D-galactosyl moiety. While benzyl ether protecting groups were found to be incompatible with the selenide linkage during deprotection, a change to acetyl esters afforded 4 in a 17% overall yield and over 9 steps from peracetylated D-galactosyl bromide. The synthesis of 5 was performed similarly, but the 2-fluoro substituent led to reduced stereoselectivity in the formation of the isoselenouronium salt (α/ß âˆ¼ 1 : 2.3). However, the ß-anomer of the uronium salt could be obtained almost pure (∼98%) by precipitation from the reaction mixture. The following displacement reaction occurred without anomerisation, affording, after deacetylation, pure 5ß.

Rhamnogalacturonan II: Chemical Synthesis of a Substructure Including α-2,3-Linked Kdo*.

The synthesis of a fully deprotected Kdo-containing rhamnogalacturonan II pentasaccharide is described. The strategy relies on the preparation of a suitably protected homogalacturonan tetrasaccharide backbone, through a post-glycosylation oxidation approach, and its stereoselective glycosylation with a Kdo fluoride donor.

Fluorinated Carbohydrates as Lectin Ligands: Simultaneous Screening of a Monosaccharide Library and Chemical Mapping by 19F NMR Spectroscopy.

Molecular recognition of carbohydrates is a key step in essential biological processes. Carbohydrate receptors can distinguish monosaccharides even if they only differ in a single aspect of the orientation of the hydroxyl groups or harbor subtle chemical modifications. Hydroxyl-by-fluorine substitution has proven its merits for chemically mapping the importance of hydroxyl groups in carbohydrate-receptor interactions. 19F NMR spectroscopy could thus be adapted to allow contact mapping together with screening in compound mixtures. Using a library of fluorinated glucose (Glc), mannose (Man), and galactose (Gal) derived by systematically exchanging every hydroxyl group by a fluorine atom, we developed a strategy combining chemical mapping and 19F NMR T2 filtering-based screening. By testing this strategy on the proof-of-principle level with a library of 13 fluorinated monosaccharides to a set of three carbohydrate receptors of diverse origin, i.e. the human macrophage galactose-type lectin, a plant lectin, Pisum sativum agglutinin, and the bacterial Gal-/Glc-binding protein from Escherichia coli, it became possible to simultaneously define their monosaccharide selectivity and identify the essential hydroxyls for interaction.

S-Glycosides: synthesis of S-linked arabinoxylan oligosaccharides.

S-Glycosides are important tools for the elucidation of specific protein-carbohydrate interactions and can significantly aid structural and functional studies of carbohydrate-active enzymes, as they are often inert or act as enzyme inhibitors. In this context, this work focuses on the introduction of an S-linkage into arabinoxylan oligosaccharides (AXs) in order to obtain a small collection of synthetic tools for the study of AXs degrading enzymes. The key step for the introduction of the S-glycosidic linkage involved anomeric thiol S-alkylation of an orthogonally protected l-arabinopyranoside triflate. The resulting S-linked disaccharide was subsequently employed in a series of glycosylation reactions to obtain a selectively protected tetrasaccharide. This could be further elaborated through chemoselective deprotection and glycosylation reactions to introduce branching l-arabinofuranosides.

Unraveling Sugar Binding Modes to DC-SIGN by Employing Fluorinated Carbohydrates.

A fluorine nuclear magnetic resonance (19F-NMR)-based method is employed to assess the binding preferences and interaction details of a library of synthetic fluorinated monosaccharides towards dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN), a lectin of biomedical interest, which is involved in different viral infections, including HIV and Ebola, and is able to recognize a variety of self- and non-self-glycans. The strategy employed allows not only screening of a mixture of compounds, but also obtaining valuable information on the specific sugar-protein interactions. The analysis of the data demonstrates that monosaccharides Fuc, Man, Glc, and Gal are able to bind DC-SIGN, although with decreasing affinity. Moreover, a new binding mode between Man moieties and DC-SIGN, which might have biological implications, is also detected for the first time. The combination of the 19F with standard proton saturation transfer difference (1H-STD-NMR) data, assisted by molecular dynamics (MD) simulations, permits us to successfully define this new binding epitope, where Man coordinates a Ca2+ ion of the lectin carbohydrate recognition domain (CRD) through the axial OH-2 and equatorial OH-3 groups, thus mimicking the Fuc/DC-SIGN binding architecture.

Synthesis of lactosamine-based building blocks on a practical scale and investigations of their assembly for the preparation of 19F-labelled LacNAc oligomers.

The ubiquitous disaccharide N-acetyllactosamine (LacNAc type 2, Galß1,4GlcNAc) is often over-expressed on the surface of cancer cells where it is bound by tumour secreted galectins contributing to cancer-related processes such as metastasis, adhesion, tumour survival, and immune escape. To facilitate NMR investigations into the binding interactions between oligo-LacNAc structures and galectins, which can show both exo- and endo-binding behaviour, a library of regioselectively 19F-labelled oligo-LacNAc structures was required. Herein, the synthesis on a practical scale of various N-protected (Troc, Phth, TFAc) lactosamine donors is reported starting from commercially available lactosamine hydrochloride. Investigations into their glycosylations with lactosamine acceptors to form 19F-containing LacNAc oligomers showed that benzylated acceptors significantly improved the yields over acetylated ones, and that, gratifyingly, the almost untried N-trifluoroacetamide (NTFAc) protected donors, already containing the desired 19F-label, were found to be optimal, both considering reaction yields and purification of the glycosylation reactions. The NTFAc group of reducing end acceptors was introduced through N-amide transacylation of linker-equipped LacNAc structures. A [2 + 2] synthetic approach was optimized for the preparation of tetrasaccharide LacNAc/TFAc-dimers and also further expanded to the synthesis of hexasaccharide LacNAc/TFAc-trimer structures.

A Tomato Tocopherol-Binding Protein Sheds Light on Intracellular α-Tocopherol Metabolism in Plants.

Tocopherols are non-polar compounds synthesized in the plastids, which function as major antioxidants of the plant cells and are essential in the human diet. Both the intermediates and final products of the tocopherol biosynthetic pathway must cross plastid membranes to reach their sites of action. So far, no protein with tocopherol binding activity has been reported in plants. Here, we demonstrated that the tomato SlTBP protein is targeted to chloroplasts and able to bind α-tocopherol. SlTBP-knockdown tomato plants exhibited reduced levels of tocopherol in both leaves and fruits. Several tocopherol deficiency phenotypes were apparent in the transgenic lines, such as alterations in photosynthetic parameters, dramatic distortion of thylakoid membranes and significant variations in the lipid profile. These results, along with the altered expression of genes related to photosynthesis, and tetrapyrrole, lipid, isoprenoid, inositol/phosphoinositide and redox metabolism, suggest that SlTBP may act in conducting tocopherol (or its biosynthetic intermediates) between the plastid compartments and/or at the interface between chloroplast and endoplasmic reticulum membranes, affecting interorganellar lipid metabolism.

Exploring functional pairing between surface glycoconjugates and human galectins using programmable glycodendrimersomes.

Precise translation of glycan-encoded information into cellular activity depends critically on highly specific functional pairing between glycans and their human lectin counter receptors. Sulfoglycolipids, such as sulfatides, are important glycolipid components of the biological membranes found in the nervous and immune systems. The optimal molecular and spatial design aspects of sulfated and nonsulfated glycans with high specificity for lectin-mediated bridging are unknown. To elucidate how different molecular and spatial aspects combine to ensure the high specificity of lectin-mediated bridging, a bottom-up toolbox is devised. To this end, negatively surface-charged glycodendrimersomes (GDSs), of different nanoscale dimensions, containing sulfo-lactose groups are self-assembled in buffer from a synthetic sulfatide mimic: Janus glycodendrimer (JGD) containing a 3'-O-sulfo-lactose headgroup. Also prepared for comparative analysis are GDSs with nonsulfated lactose, a common epitope of human membranes. These self-assembled GDSs are employed in aggregation assays with 15 galectins, comprising disease-related human galectins, and other natural and engineered variants from four families, having homodimeric, heterodimeric, and chimera architectures. There are pronounced differences in aggregation capacity between human homodimeric and heterodimeric galectins, and also with respect to their responsiveness to the charge of carbohydrate-derived ligand. Assays reveal strong differential impact of ligand surface charge and density, as well as lectin concentration and structure, on the extent of surface cross-linking. These findings demonstrate how synthetic JGD-headgroup tailoring teamed with protein engineering and network assays can help explain how molecular matchmaking operates in the cellular context of glycan and lectin complexity.

Effects of different rest-stop durations at control posts during a long journey on the welfare of sheep.

All livestock transport within the European Union must comply with the EC Regulation 1÷2005. For sheep, this law prescribes a maximum journey of 29 hours after which animals must rest in control posts (CP) for 24 hours before further transportation. However, there is no scientific evidence de ning the effects of di erent stop duration on sheep recovery during long journeys. The aim of this study was to assess if a shorter stop could be provided without impairing ewes' welfare. Ninety-six adult ewes were divided into 4 homogenous groups. One group stayed at the farm (control) and the other 3 were transported for 29 hours (long-transport, LT), stopped at CP for di erent times (8 hours (S8 group); 16 hours (S16 group); 24 hours (S24 group)) and were re-transported for 6 hours (short-transport, ST). Blood and saliva were collected to assess dehydration, muscular damage, and adrenocortical stress before departure, after LT, after the stop, and after ST. The LT a ected the hydration of all transported groups (i.e. higher BUN÷creatinine levels than controls, p<0.001), but basal values were restored after the ST, regardless of the stop duration. After the ST, S8 group had higher muscular damage than the other groups (p<0.05). No di erences in stress level were observed. These results suggest that, in this trial, ewe's welfare was not impaired by a stop reduction from 24 hours to 16 hours.

Intra- and intermolecular interactions of human galectin-3: assessment by full-assignment-based NMR.

Galectin-3 is an adhesion/growth-regulatory protein with a modular design comprising an N-terminal tail (NT, residues 1-111) and the conserved carbohydrate recognition domain (CRD, residues 112-250). The chimera-type galectin interacts with both glycan and peptide motifs. Complete (13)C/(15)N-assignment of the human protein makes NMR-based analysis of its structure beyond the CRD possible. Using two synthetic NT polypeptides covering residues 1-50 and 51-107, evidence for transient secondary structure was found with helical conformation from residues 5 to 15 as well as proline-mediated, multi-turn structure from residues 18 to 32 and around PGAYP repeats. Intramolecular interactions occur between the CRD F-face (the 5-stranded ß-sheet behind the canonical carbohydrate-binding 6-stranded ß-sheet of the S-face) and NT in full-length galectin-3, with the sequence P(23)GAW(26)…P(37)GASYPGAY(45) defining the primary binding epitope within the NT. Work with designed peptides indicates that the PGAX motif is crucial for self-interactions between NT/CRD. Phosphorylation at position Ser6 (and Ser12) (a physiological modification) and the influence of ligand binding have minimal effect on this interaction. Finally, galectin-3 molecules can interact weakly with each other via the F-faces of their CRDs, an interaction that appears to be assisted by their NTs. Overall, our results add insight to defining binding sites on galectin-3 beyond the canonical contact area for ß-galactosides.

Effect of perphenazine enanthate on open-field test behaviour and stress-induced hyperthermia in domestic sheep.

The open-field test (OFT) and stress-induced hyperthermia (SIH) have been used to measure individual differences in fear. The present study has been designed as a pharmacological validation of OFT and SIH as indicators of fear in sheep using perphenazine enanthate (PPZ), a long-acting neuroleptic. Twenty four ewes of two breeds, Lacaune and Ripollesa, were tested in an arena measuring 5mx2.5m. Treatment group received one dose of 1.5mg/kg of PPZ and control group received sterile sesame oil. All animals were tested for 10min and behaviours were recorded. Rectal temperature was measured at the beginning (T1) and at the end (T2) of the test. SIH was defined as the difference between T2 and T1. Sheep were tested on days 1, 2, 3, 4, 7 and 9 after PPZ injection. Variables were analysed using a mixed model. PPZ decreased bleats on days 2, 3, 4 and the SIH response on days 2 and 3. Breed differences were observed. Treated animals showed positive correlations between SIH and bleats; squares entered; attempts to escape and negative correlation between SIH and visits to the food bucket. Our results suggest that behaviour and SIH on the OFT are useful measures of fear in sheep.

Signos de alarma en el recien nacido, nacido pretermino / Signs of alarm in infant, newborn

Se pretende dejar claro cuales son los signos de alarma que comunmente se evidencian en estos pacientes, de manera que la deteccion temprana de ellos sin lugar a dudas colaborará significativamente en la evolución favorable, esto depende de la habilidad y destreza en la observación de todo el equipo de enfermería de la Ucin, para valorar a cada uno de los recien nacidos

Signos de alarma en el recien nacido, nacido pretermino / Signs of alarm in infant, newborn

Se pretende dejar claro cuales son los signos de alarma que comunmente se evidencian en estos pacientes, de manera que la deteccion temprana de ellos sin lugar a dudas colaborará significativamente en la evolución favorable, esto depende de la habilidad y destreza en la observación de todo el equipo de enfermería de la Ucin, para valorar a cada uno de los recien nacidos (AU)