Search

• NMR spectroscopy is used to characterize reactivity in bromoacetyl-dPRP. • Reaction with 2-mercapto imidazole provides proton NMR assignments in BrAc-dPRP. • Bromine loss from BrAc-dPRP before conjugation can be monitored using NMR. PedvaxHIB® is a highly purified capsular polysaccharide (polyribosylribitol phosphate or PRP) of Haemophilus influenzae type b (Hib) that is covalently linked to an outer membrane protein complex (OMPC) of Neisseria meningitidis pediatric vaccine. This Hib protein conjugate vaccine is very effective in protecting infants from invasive gram-negative bacterium Hib. Before conjugation of PRP to OMPC, PRP is first derivatized with butadiamine and bromoacetyl group which reacts with thiolated OMPC. This report describes an NMR method to analyze the reactivity of bromoacetyl group in derivatized PRP (BrAc-derivatized PRP) toward small molecule thiol group, 2-mercapto imidazole (MI) which is used as a surrogate for thiolated OMPC. Reaction with MI helped identify observable amide protons in BrAc-derivatized PRP according to presence or absence of bromine. Furthermore, the new method can be used to monitor the percent of bromoacetylation during manufacturing and potentially a stability indicating assay for loss of bromine in BrAc-derivatized PRP. [ABSTRACT FROM AUTHOR]

Conjugation with polyethylene glycol (PEG) is a strategy for improving the pharmaceutical properties of therapeutic proteins. In nonclinical studies of PEGylated compounds, microscopic tissue vacuolation is often observed, characterized ultrastructurally in this report by lysosomal distension. Although PEGylation-associated vacuolation appears to be of limited toxicologic concern when alternative therapies are limited, the risk-benefit considerations may be impacted by uncertainty about reversibility, lack of methods for monitoring PEG accumulation in vivo without biopsy, and the variability in tissues affected depending on species studied. We demonstrate the use of magnetic resonance spectroscopy (MRS) to measure PEG concentrations at multiple time points in vivo in the kidney with comparison to PEG concentrations ex vivo in body fluids and tissue extracts using nuclear magnetic resonance (NMR) spectroscopy. Furthermore, we demonstrate the use of these techniques to study distribution and elimination of PEG in a dog model of PEGylation-associated vacuolation. This report suggests that MRS could be further investigated as a feasible imaging-based method for monitoring PEG accumulation in a clinical setting in conjunction with NMR quantitation of PEG in plasma and urine. [ABSTRACT FROM AUTHOR]

Abstract: PedvaxHIB is a pediatric vaccine that protects children from severe disease caused by the gram-negative bacterium Haemophilus influenzae type b (Hib). The vaccine is made by chemically conjugating Hib capsular polysaccharide to the outer membrane protein complex of Neisseria meningitidis. The protein-conjugated vaccine has proven to be extremely effective in preventing invasive Hib disease in infants and young children. This paper presents the nuclear magnetic resonance (NMR) methodology for the quantitative characterization of derivatized polysaccharide and its validation closely following ICH guidelines. The assay has been shown to be precise and accurate (relative standard deviation [RSD] ⩽1%), specific (no observable matrix interference), rugged (RSD <3% for day-to-day and operator-to-operator variations), and robust (to changes in temperature and polysaccharide concentration). Therefore, the quantitative NMR assay can be used as a reliable product release or process monitoring method and can replace several labor-intensive chromatographic and colorimetric methods. [Copyright &y& Elsevier]

Abstract: Purified capsular polysaccharide preparations from Streptococcus pneumoniae that are used for vaccine production typically contain residual levels of C-polysaccharide (C-Ps). Residual C-Ps is typically found in one of two forms, either chemically linked to the capsular polysaccharide (bound) or present by itself (free). Two analytical methods have been developed and applied to determine the relative percentages of the two C-Ps forms present in various capsular polysaccharide preparations. Both methods differentiate the two forms of C-Ps according to the difference of their hydrodynamic sizes. One method is based on labeling C-Ps with a fluorescent tag and separating the two forms of C-Ps by high-performance size exclusion chromatography with on-line refractive index and fluorescence detection, and the other method is based on measuring self-diffusion rates of the two forms of C-Ps by nuclear magnetic resonance (NMR) and quantifying each form with deconvolution. Both methods were evaluated for relative accuracy, precision, and ease of application, and they were found to provide comparable results for a large number of pneumococcal polysaccharide preparations. These analyses, combined with other quantitative NMR measurement of total C-Ps in the polysaccharide powder, provide a more refined means of evaluating the amount of each form of C-Ps in polysaccharide preparations targeted for vaccine production. [Copyright &y& Elsevier]

NMR spectroscopy can be used to characterize bacterial polysaccharides such as that of Streptococcus pneumoniae type 1 which is a component of the 23-valent pneumococcal vaccine in clinical use. This particular polysaccharide gives NMR spectra with wide lines apparently due to restricted molecular mobility and chain flexibility which leads to rapid dipolar T2 relaxation limiting the possibility of detailed spectral analysis. Removal of O-acetyl groups found on approximately two thirds of the repeating subunits of pneumococcal type 1 capsule leads to narrower NMR lines facilitating a complete assignment of the 1H and 13C NMR spectra. Degradation of the polysaccharide by periodate oxidation followed by base treatment leads to an oligosaccharide fragment of approximately three repeating trisaccharide units. This oligosaccharide has narrow NMR lines and 1H and 13C assignments very similar to those of the O-deacetylated polysaccharide. In the native polysaccharide, O-acetyl groups are located on the 2- and 3-positions of the 4-linked galacturonic acid residue providing protection against periodate oxidation. Analysis of NOESY spectra combined with molecular modeling of the oligosaccharide shows that flexibility occurs in certain of the saccharide linkages. [Copyright &y& Elsevier]

Describes a method of constructing conformational models of flexible complex polysaccharides on the basis of nuclear magnetic resonance data and molecular modeling. Application of the method to a polysaccharide which is a lectin-biding receptor important in coaggregation of oral bacteria; Measurements of coupling constants for a 13C-enriched bacterial polysaccharide.

Studies the dynamics of the motion of a complex polysaccharide having seven sugar residues in the repeating subunit and which is a receptor for lectin interaction in the coaggregation of oral bacteria. Measurements of the longitudinal and the rotating frame relaxation rates; Dynamics of a flexible polysaccharide.

Investigates the involvement of univalent cations in stabilizing G-quartet structures of the oligonucleotide d(T2G4T). Synthesizing of oligonucleotide; Nuclear magnetic resonance measurements; Smear of electron density; Types of binding environments for univalent cations; NMR behaviors of 23Na+ and 39K+; Potassium; Resonances of amino protons.

Examines assumptions about duplex DNA structure. Sensitivity to ionic conditions; Binding of simple cations to DNA in a nonspecific fashion; Experimental procedures; Oligonucleotides; Quadrupole coupling constants.

Focuses on the structural features of Vibrio cholerae O139 Bengal strain. Nuclear magnetic resonance frequency (NMR) spectra; Chemical shifts; Remarks on structural polysaccharide; Effect of transposon insertion.

Indoleamine-2,3-dioxygenase 1 (IDO1) and tryptophan-2,3-dioxygenase 2 (TDO2) degrade tryptophan (Trp) to kynurenine (Kyn), and these enzymes have promise as therapeutic targets. A comprehensive characterization of potential safety liabilities of IDO1 and TDO2 inhibitors using knockout (KO) mice has not been assessed, nor has the dual Ido1/Tdo2 KO been reported. Here we characterized male and female mice with KOs for Ido1 , Tdo2 , and Ido1/Tdo2 and compared findings to the wild type (WT) mouse strain, evaluated for 14 days, using metabolomics, transcriptional profiling, behavioral analysis, spleen immunophenotyping, comprehensive histopathological analysis, and serum clinical chemistry. Multiple metabolomic changes were seen in KO mice. For catabolism of Trp to Kyn and anthranilic acid, both substrates were decreased in liver of Tdo2 and dual KO mice. Metabolism of Trp to serotonin and its metabolites resulted in an increase in 5-Hydroxyindole-3-acetic acid in the Tdo2 and dual KO mice. Ido1 and dual KO mice displayed a Kyn reduction in plasma but not in liver. Nicotinamide synthesis and conversion of glucose to lactic acid were not impacted. A slight decrease in serum alkaline phosphatase was seen in all KOs, and small changes in liver gene expression of genes unrelated to tryptophan metabolism were observed. Regarding other parameters, no genotype-specific changes were observed. In summary, this work shows metabolomic pathway changes for metabolites downstream of tryptophan in these KO mice, and suggests that inhibition of the IDO1 and TDO2 enzymes would be well tolerated whether inhibited individually or in combination since no safety liabilities were uncovered. • IDO1 & TDO2 degrade tryptophan to kynurenine, & these enzymes have promise as therapeutic targets. • To understand target liabilities, single & dual Ido1 & Tdo2 KO mice were thoroughly evaluated. • Tryptophan metabolite changes were seen, & other subtle findings had no toxicologic consequence. • This implies inhibition of these enzymes would be well tolerated. [ABSTRACT FROM AUTHOR]

Polar codes, as the coding scheme for the control channel in fifth-generation mobile communication technology (5G), have attracted widespread attention since their proposal. As a mainstream decoding algorithm for polar codes, the successive cancellation list (SCL) decoder usually improves the error correction performance by increasing the list size, but this method suffers from the problems of high decoding complexity. To address this problem, this paper proposes a layered-search bit-flipping (LS-SCLF) decoding algorithm based on SCL decoding. Firstly, a new flip-bit metric is proposed, which derives a formula to approximate the probability of an error occurring in an information bit. This formula introduces a perturbation parameter to improve the calculation accuracy. Secondly, a compromise scheme for determining the perturbation parameter is proposed. The scheme uses Monte Carlo simulation to determine an optimized parameter for the precise positioning of the first erroneous decoded bit under different decoding conditions. Finally, a layered search strategy is adopted to sequentially search the erroneous decoded bits from the low order to high order, which can correct up to multiple bits at the same time. Simulation results show that the proposed algorithm achieves improved error correction performance with a slight increase in decoding complexity compared to the generalized SCL-Flip (GSCLF) decoding algorithm. This algorithm also achieves a good balance between the error correction performance and decoding complexity. [ABSTRACT FROM AUTHOR]

Colonization of implanted medical devices by coagulase-negative staphylococci such as Staphylococcus epidermidis is mediated by the bacterial polysaccharide intercellular adhesin (PIA), a polymer of β-(1→6)-linked glucosamine substituted with N-acetyl and O-succinyl constituents. The icaADBC locus containing the biosynthetic genes for production of PIA has been identified in both S. epidermidis and S. aureus. Whereas it is clear that PIA is a constituent that contributes to the virulence of S. epidermidis, it is less clear what role PIA plays in infection with S. aureus. Recently, identification of a novel polysaccharide antigen from S. aureus termed poly N-succinyl β-(1→6)-glucosamine (PNSG) has been reported. This polymer was composed of the same glycan backbone as PIA but was reported to contain a high proportion of N-succinylation rather than acetylation. We have isolated a glucosamine-containing exopolysaccharide from the constitutive over-producing MN8m strain of S. aureus in order to prepare polysaccharide–protein conjugate vaccines. In this report we demonstrate that MN8m produced a high-molecular-weight (>300,000 Da) polymer of β-(1→6)-linked glucosamine containing 45–60% N-acetyl, and a small amount of O-succinyl (approx 10% mole ratio to monosaccharide units). By detailed NMR analyses of polysaccharide preparations, we show that the previous identification of N-succinyl was an analytical artifact. The exopolysaccharide we have isolated is active in in vitro hemagglutination assays and is immunogenic in mice when coupled to a protein carrier. We therefore conclude that S. aureus strain MN8m produces a polymer that is chemically and biologically closely related to the PIA produced by S. epidermidis. [Copyright &y& Elsevier]

Examines the structure of capsular polysaccharide from Vibrio vulnificus bacterium in strain 6353. Derivation of proton homonuclear coupling constants; Differences in the detailed structure of the polysaccharide; Composition of sugar residues.

Mutations in either the mitochondrial or nuclear genomes can give rise to respiratory chain disease (RCD), a large class of devastating metabolic disorders. Their clinical management is challenging, in part because we lack facile and accurate biomarkers to aid in diagnosis and in the monitoring of disease progression. Here we introduce a sequential strategy that combines biochemical analysis of spent media from cell culture with analysis of patient plasma to identify disease biomarkers. First, we applied global metabolic profiling to spotlight 32 metabolites whose uptake or secretion kinetics were altered by chemical inhibition of the respiratory chain in cultured muscle . These metabolites span a wide range of pathways and include lactate and alanine, which are used clinically as biomarkers of RCD. We next measured the cell culture-defined metabolites in human plasma to discover that creatine is reproducibly elevated in two independent cohorts of RCD patients, exceeding lactate and alanine in magnitude of elevation and statistical significance. In cell culture extracellular creatine was inversely related to the intracellular phosphocreatine:creatine ratio suggesting that the elevation of plasma creatine in RCD patients signals a low energetic state of tissues using the phosphocreatine shuttle. Our study identifies plasma creatine as a potential biomarker of human mitochondrial dysfunction that could be clinically useful. More generally, we illustrate how spent media from cellular models of disease may provide a window into the biochemical derangements in human plasma, an approach that could, in principle, be extended to a range of complex diseases. [ABSTRACT FROM AUTHOR]

Bile acid binding to sevelamer HCl. Background. Clinical studies have shown sevelamer HCl (Renagel®) to be effective for the reduction of serum phosphate in hemodialysis patients. These studies also consistently have demonstrated a significant reduction of low-density lipoprotein (LDL) cholesterol following treatment with sevelamer. Methods. Equilibrium binding of bile acids and oleic acid was determined by incubating sevelamer with ligand containing buffer. Aliquots of the solution were filtered and the free ligand concentrations quantitated by high-pressure liquid chromatography (HPLC). Flow kinetics were determined using a cylindrical flow cell containing trapped sevelamer. Bile acid and oleic acid were pumped through the stirred cell in a manner designed to mimic the in vivo situation. Binding was monitored by HPLC. Results. Sevelamer binds bile acids cooperatively and with high capacity. At low binding densities, the presence of the more hydrophobic bile acids enhances the binding of the less hydrophobic bile acids, and the presence of oleic acid enhances the binding of all bile acids. At saturating oleic acid concentrations, the bile acid binding capacity of sevelamer is reduced by only a factor of two. Moreover, the presence of oleic acid dramatically diminishes the release rate of bile acids from sevelamer. Conclusions. The favorable bile acid binding characteristics of sevelamer provide a compelling explanation for its ability to lower LDL cholesterol in hemodialysis patients and in healthy volunteers. [ABSTRACT FROM AUTHOR]