Hotspot DAXX, PTCH2 and CYFIP2 mutations in pancreatic neuroendocrine neoplasms.

Mutations in DAXX/ATRX, MEN1 and genes involved in the phosphoinositide-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway have been implicated in pancreatic neuroendocrine neoplasms (pNENs). However, mainly mutations present in the majority of tumor cells have been identified, while proliferation-driving mutations could be present only in small fractions of the tumor. This study aims to identify high- and low-abundance mutations in pNENs using ultra-deep targeted resequencing. Formalin-fixed paraffin-embedded matched tumor-normal tissue of 38 well-differentiated pNENs was sequenced using a HaloPlex targeted resequencing panel. Novel amplicon-based algorithms were used to identify both single nucleotide variants (SNVs) and insertion-deletions (indels) present in >10% of reads (high abundance) and in <10% of reads (low abundance). Found variants were validated by Sanger sequencing. Sequencing resulted in 416,711,794 reads with an average target base coverage of 2663 ± 1476. Across all samples, 32 high-abundance somatic, 3 germline and 30 low-abundance mutations were withheld after filtering and validation. Overall, 92% of high-abundance and 84% of low-abundance mutations were predicted to be protein damaging. Frequently, mutated genes were MEN1, DAXX, ATRX, TSC2, PI3K/Akt/mTOR and MAPK-ERK pathway-related genes. Additionally, recurrent alterations on the same genomic position, so-called hotspot mutations, were found in DAXX, PTCH2 and CYFIP2. This first ultra-deep sequencing study highlighted genetic intra-tumor heterogeneity in pNEN, by the presence of low-abundance mutations. The importance of the ATRX/DAXX pathway was confirmed by the first-ever pNEN-specific protein-damaging hotspot mutation in DAXX. In this study, both novel genes, including the pro-apoptotic CYFIP2 gene and hedgehog signaling PTCH2, and novel pathways, such as the MAPK-ERK pathway, were implicated in pNEN.

Perdeuterated and 13C-enriched myo-inositol for DNP assisted monitoring of enzymatic phosphorylation by inositol-3-kinase.

The synthesis of perdeuterated and 13C enriched myo-inositol is presented. Myo-inositol and its derivatives are of interest as substrates for enzymes producing phosphorylated species with regulatory functions in many organisms. Its utility in monitoring real-time phosphorylation by myo-inositol-3-kinase is illustrated using dynamic nuclear polarization (DNP) to enhance NMR observation.

Involvement of water in carbohydrate-protein binding.

The interactions of trimannosides 1 and 2 with Con A were studied to reveal the effects of displacement of well-ordered water molecules on the thermodynamic parameters of protein-ligand complexation. Trisaccharide 2 is a derivative of 1, in which the hydroxyl at C-2 of the central mannose unit is replaced by a hydroxyethyl moiety. Upon binding, this moiety displaces a conserved water molecule present in the Con A binding site. Structural studies by NMR spectroscopy and MD simulations showed that the two compounds have very similar solution conformational properties. MD simulations of the complexes of Con A with 1 and 2 demonstrated that the hydroxyethyl side chain of 2 can establish the same hydrogen bonds in a low energy conformation with the protein binding site as those mediated by the water molecule in the complex of 1 with Con A. Isothermal titration microcalorimetry (ITC) measurements showed that 2 has a more favorable entropy of binding compared to 1. This term, which was expected, arises from the return of the highly ordered water molecule to bulk solution. The favorable entropy term was, however, offset by a relatively large unfavorable enthalpy term. This observation was rationalized by comparing the extent of hydrogen bond and solvation changes during binding. It is proposed that an indirect interaction through a water molecule will provide a larger number of hydrogen bonds in the complex that have higher occupancies than in bulk solution, thereby stabilizing the complex.

Identification of a developmental chemoattractant in Myxococcus xanthus through metabolic engineering.

Fruiting body formation of Myxococcus xanthus requires the ordered migration of tens of thousands of cells by using a form of surface motility known as gliding and chemical signal(s) that have yet to be elucidated. Directed movement is regulated by phosphatidylethanolamine (PE) purified from M. xanthus cell membranes. Because the purified PE preparation contains a remarkably diverse mixture of fatty acids, metabolic engineering was used to elucidate the biologically active fatty acid component. The mutational block in an esg mutant, which renders it defective in producing primers for branched-chain fatty acid biosynthesis, was bypassed with one of a series of primers that enriches for a particular family of branched-chain fatty acids. Each PE enrichment was observed for chemotactic activity by using an excitation assay and for fatty acid content. The excitation activity of a PE preparation was generally proportional with the concentration of the fatty acid 16:1 omega 5c. 1,2-O-Bis[11-(Z)-hexadecenoyl]-sn-glycero-3-phosphoethanolamine (PE-16:1 omega 5c/16:1 omega 5c) was synthesized and elicited an excitation peak at 2 ng. This peak activity occurred at a 1,000-fold lower concentration than dilauroyl PE (PE-12:0/12:0) and the peak magnitude was 2-fold higher. PE containing 16:1 omega 5c is likely to play a role in development because it is active at physiological concentrations and only under developmental conditions.

A stereoselective approach for the synthesis of alpha-sialosides.

A highly efficient synthesis of the human melanoma associated antigen GD(3) derivative has been described. A key feature of the synthetic approach was the use of sialyl donors that were protected with a C-5 trifluoroacetamide moiety. These sialyl donors gave high yields and excellent alpha-anomeric selectivities in direct glycosylations with a wide variety of glycosyl acceptors ranging from C-8 hydroxyls of sialic acids and C-3 hydroxyls of galactosides to reactive primary alcohols.

A highly efficient synthetic strategy for polymeric support synthesis of Le(x), Le(y), and H-type 2 oligosaccharides.

The O-protecting groups Levulinoyl (Lev) and 9-fluroenylmethoxycarbonyl (Fmoc) offer an attractive set of orthogonal protecting groups which are compatible with base sensitive N-trichloroethoxylcarbonyl (Troc) group. B exploiting these orthogonal protecting groups and a novel phenolic ester linker, a series of oligosaccharide of biological importance, Le(x), H-type 2, and Le(y), were synthesized on the polytheylene glycol resin MPEG (Mw 5000). The products bearing a p-hydroxybenzyl group could be easily converted into glycosyl donors for further synthesis. Using this strategy, a spacer containing tumor antigen Le(y)-Lac hexasaccharide was described. The artificial spacer at the reducing end provides an opportunity for selective conjugation to an appropriate carrier protein for immunlogical studies.

A highly convergent synthesis of a complex oligosaccharide derived from group B type III Streptococcus.

An efficient synthesis of a heptasaccharide derived from group B type III Streptococcus carrying an artificial spacer (1) is described. Rapid assembly of a protected heptasaccharide (16a) is accomplished from readily available building blocks 2-5 without a single protecting group manipulation between glycosylation steps. The synthetic strategy may be applied to the assembly of other branched complex oligosaccharides. The deprotected heptasaccharide 1 was coupled to a poly[N-(acryloyloxy)succinimide, and the resulting material will be used for the development of an ELISA assay to detect antibodies against GBS, type III in pregnant women.

Thioglycosides protected as trans-2,3-cyclic carbonates in chemoselective glycosylations.

Thioglycosides protected as trans-2,3-cyclic carbonates have significantly lower anomeric reactivities than fully acylated and N-acyl-protected thioglycosides and can be used as acceptors in chemoselective glycosylations with a wide range of thioglycosyl donors. The resulting thioglycosides can be further activated to give 1,2-cis-linked glycosides. [reaction: see text]

Intermolecular aglycon transfer of ethyl thioglycosides can be prevented by judicious choice of protecting groups.

Unexpected intermolecular aglycon transfer occurred in chemoseletive glycosylations using glycosyl fluorides or trichloroacetimidates as glycosyl donors and partially protected thioglycosides as glycosyl acceptors. It is shown that this problem can be addressed by fine-tuning of the reactivity of the anomeric thioalkyl moiety and hydroxyl of the acceptor.

Synthesis and supramolecular characterization of a novel class of glycopyranosyl-containing amphiphiles.

A novel class of glycopeptidolipids is described, which potentially can be used as a novel antigen-delivery system. The compounds have been prepared by a combination of solid-supported and solution-based methods. The use of the orthogonally protected FmocLysDde derivative provided an opportunity to incorporate two different types lipids. It was found that the model compound 1 forms aggregates in aqueous media which can be described as rod or tubelike structures. The aggregates can be stabilized by topotactic photopolymerization. Studies on the structural analogues 2-5 revealed the effect of the carbohydrate, peptide, and lipid moiety on the aggregation properties. It is concluded that none of the structure elements can lay claim to be exclusively important for the formation of highly organized aggregates such as tubes, fibers, or helical ribbons from 1, but the presence of all of these structural elements afforded the most uniformly shaped extended structures.

Lactic acid is the factor in blood cell extracts which enhances the ability of CMP-NANA to sialylate gonococcal lipopolysaccharide and induce serum resistance.

In previous work, a factor which enhances the ability of cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) to sialylate gonococcal lipopolysaccharide (LPS) was liberated at 4 degrees C in diffusates from high M(r) fractions of blood cell sonicates. The diffusates also contained CMP-NANA and converted serum susceptible gonococci to resistance. The enhancer has now been separated from CMP-NANA and material absorbing at 260 nm by HPLC on mu Bondapak-10 NH2. Resistance inducing activity was found only in fractions containing CMP-NANA and recovery was poor (about 25%). However, addition of enhancer fractions to CMP-NANA substantially increased its resistance inducing activity. Blood cell sonicates dialysed at 18-20 degrees C released enhancer in diffusates. These were ultrafiltered (nominal cut off 3000 Da) and fractionated on Biogel P2 which removed saccharides and most material absorbing at 260 nm. Over 90% of a fraction which was enhancer-active in nanogram quantities was identified by nuclear magnetic resonance (NMR) spectroscopy and gas chromatography/mass spectometry (GC/MS) as lactic acid. A fraction with similar properties was obtained from a different batch of diffusate by fractionation on Dowex 1. Authentic lithium L-lactate in nanogram quantities enhanced LPS sialyation by CMP-NANA and increased its serum resistance inducing activity. These results have important implications for gonococcal pathogenicity.

Minimal oligosaccharide structures required for induction of immune responses against meningococcal immunotype L1, L2, and L3,7,9 lipopolysaccharides determined by using synthetic oligosaccharide-protein conjugates.

The 12 types of meningococcal lipopolysaccharide (LPS) (immunotypes) contain immunotype-specific and cross-reactive epitopes situated on the oligosaccharide part of the LPS molecules. To identify useful cross-reactive epitopes and to determine minimal oligosaccharide structures required for the induction of an immune response against the most prevalent immunotypes, L1, L2, and L3,7,9, synthetic as well as native LPS-derived oligosaccharides were conjugated with tetanus toxoid. L3,7,9 phosphoethanolamine (PEA) group-containing oligosaccharide-tetanus toxoid conjugates evoked high immunoglobulin G (IgG) antibody levels in rabbits which were detected by an L2-, L3,7,9-, and, depending on the antiserum, L1-specific enzyme-linked immunosorbent assay (ELISA). Inhibition studies revealed that an identical antibody population was detected by L1 and L3,7,9 ELISA, indicating a similar tertiary structure of the inner core oligosaccharide of these two immunotypes. These antibodies recognize PEA group-containing epitopes present on the L1 and L3,7,9 LPS. An L2 PEA group-containing oligosaccharide-tetanus toxoid conjugate elicited L2- and L3,7,9-specific IgG antibodies, but in contrast with the L3,7,9 conjugates, no L1-specific IgG antibodies were evoked. These results indicate that L1 and L2 LPS do not contain cross-reactive epitopes, whereas both L2 and L3,7,9 LPS and L1 and L3,7,9 LPS possess common determinants. Three linear oligosaccharides and one branched oligosaccharide, representing partial structures of the inner core oligosacchardes of meningococcal LPS, were synthesized. Only the branched synthetic oligosaccharide-containing conjugate was able to induce and L1- and L3,7,9-specific immune response, whereas the linear oligosaccharide-protein conjugates evoked L2-specific immune responses. The branched oligosaccharide (beta-D-Glcp(1----4)-[L-alpha-D-Hepp(1----3)]-L-alpha-D-Hepp ) is therefore considered a minimal structure required for the induction of an immune response against L1 and L3,7,9 LPS and part of a cross-reactive epitope between these two immunotypes. For L2-specific immune responses, oligosaccharide structures terminating in beta-D-Glcp(1----4), alpha-D-GlcNAcp(1----2), or L-alpha-D-Hepp(1----5) are needed. The results suggest that it is possible to prepare an oligosaccharide structure with the ability to evoke an immune response against L1, L2, and L3,7,9 LPS. A feasible structure for such a "hybrid" oligosaccharide is discussed.