Normal Neurodevelopment and Fertility in Juvenile Male Rats Exposed to Polyethylene Glycol Following Dosing With PEGylated rFIX (Nonacog Beta Pegol, N9-GP): Evidence from a 10-Week Repeat-Dose Toxicity Study.

N9-GP/Rebinyn/Refixia is an approved PEGylated (polyethylene glycol-conjugated) recombinant human factor IX intended for prophylactic and/or on-demand treatment in adults and children with haemophilia B. A juvenile neurotoxicity study was conducted in male rats to evaluate effects on neurodevelopment, sexual maturation, and fertility following repeat-dosing of N9-GP. Male rats were dosed twice weekly from Day 21 of age with N9-GP or vehicle for 10 weeks, followed by a dosing-free recovery period for 13 weeks and terminated throughout the dosing and recovery periods.

Higher-Order Structure Characterization of Pharmaceutical Proteins by 2D Nuclear Magnetic Resonance Methyl Fingerprinting.

A key challenge in the analytical assessment of therapeutic proteins is the comprehensive characterization of their higher-order structure (HOS). To directly assess HOS, a new type of assay is warranted. The most sensitive and detailed method for characterizing HOS is unquestionably nuclear magnetic resonance (NMR) spectroscopy.

An efficient arabinoxylan-debranching α-L-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site.

An α-L-arabinofuranosidase of GH62 from Aspergillus nidulans FGSC A4 (AnAbf62A-m2,3) has an unusually high activity towards wheat arabinoxylan (WAX) (67 U/mg; k cat = 178/s, K m = 4.90 mg/ml) and arabinoxylooligosaccharides (AXOS) with degrees of polymerisation (DP) 3-5 (37-80 U/mg), but about 50 times lower activity for sugar beet arabinan and 4-nitrophenyl-α-L-arabinofuranoside. α-1,2- and α-1,3-linked arabinofuranoses are released from monosubstituted, but not from disubstituted, xylose in WAX and different AXOS as demonstrated by NMR and polysaccharide analysis by carbohydrate gel electrophoresis (PACE).

Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin.

A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior.

Season is an unreliable predictor of Lyme neuroborreliosis.

Introduction: Lyme neuroborreliosis (LNB) is a tick-borne infection of the nervous system caused by the spirochete Borrelia burgdorferi sensu lato. The primary symptoms are usually painful radiculitis, facial palsy and lymphocytic meningitis. The aim of this study was to provide data on the seasonal variation, anamnesis, symptoms, laboratory data and course of the disease in adults (≥ 16 years).

Frequency, risk factors, and outcomes of vancomycin-resistant Enterococcus colonization and infection in patients with newly diagnosed acute leukemia: different patterns in patients with acute myelogenous and acute lymphoblastic leukemia.

OBJECTIVE To determine the frequency, risk factors, and outcomes for vancomycin-resistant Enterococcus (VRE) colonization and infection in patients with newly diagnosed acute leukemia. DESIGN Retrospective clinical study with VRE molecular strain typing. SETTING A regional referral center for acute leukemia.

A diverse range of bacterial and eukaryotic chitinases hydrolyzes the LacNAc (Galβ1-4GlcNAc) and LacdiNAc (GalNAcβ1-4GlcNAc) motifs found on vertebrate and insect cells.

There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galβ1-4GlcNAcβ-tetramethylrhodamine (LacNAc-TMR (Galβ1-4GlcNAcβ(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures.

Structural determination of Streptococcus pneumoniae repeat units in serotype 41A and 41F capsular polysaccharides to probe gene functions in the corresponding capsular biosynthetic loci.

We report the repeating unit structures of the native capsular polysaccharides of Streptococcus pneumoniae serotypes 41A and 41F. Structural determinations yielded six carbohydrate units in the doubly branched repeating unit to give the following structure for serotype 41A: The structure determinations were motivated (1) by an ambition to help close the remaining gaps in S. pneumoniae capsular polysaccharide structures, and (2) by the attempt to derive functional annotations of carbohydrate active enzymes in the biosynthesis of bacterial polysaccharides from the determined structures.

Photoperiodic variations induce shifts in the leaf metabolic profile of Chrysanthemum morifolium.

Plants have a high ability to adjust their metabolism, growth and development to changes in the light environment and to photoperiodic variation, but the current knowledge on how changes in metabolite contents are associated with growth and development is limited. We investigated the effect of three different photoperiodic treatments with similar daily light integral (DLI) on the growth responses and diurnal patterns in detected leaf metabolites in the short day plant Chrysanthemum×morifolium Ramat. Treatments were long day (LD, 18h light/6h dark), short day (SD, 12h light/12h dark) and short day with irregular night interruptions (NI-SD,12h light/12h dark, applied in a weekly pattern, shifting from day-to-day).

Probing the structural details of xylan degradation by real-time NMR spectroscopy.

The biodegradation of abundantly available cell wall polysaccharides has recently received much attention, not least because cell wall polysaccharides are substrates for the human gut microbiota and for environmentally sustainable processes of biomass conversion to value-added compounds. A major fraction of cereal cell wall polysaccharides consists of arabinoxylans. Arabinoxylan and its degradation products are therefore present in a variety of agro-industrial residues and products.

Determination of native capsular polysaccharide structures of Streptococcus pneumoniae serotypes 39, 42, and 47F and comparison to genetically or serologically related strains.

The diversity of capsular polysaccharides of the bacterial pathogen Streptococcus pneumoniae leads to at least 91 different serotypes. While the genetic loci for capsular biosynthesis have been characterized for all serotypes, the determination of resultant polysaccharide structures remains incomplete. Here, we report the chemical structures of the capsular polysaccharides of serotypes 39, 42, and 47F from the genetic cluster 4, and discuss the structures in the context of structures from serologically and genetically related serotypes.

NMR characterization of chemically synthesized branched α-dextrin model compounds.

1H and 13C NMR chemical shifts were accurately determined by consistent referencing for an extensive set of chemically synthesized branched α-glucan model compounds. The model compounds include anomerically fixed and reducing oligosaccharides ranging in size from isomaltose to a doubly branched decasaccharide. Both the 13C1 chemical shift and the 13C6 chemical shifts in α-(1→6) glycosidic bonds are strongly dependent on the chemical structure in the vicinity of the branch point, especially on the addition of glucopyranosyl units towards the non-reducing end of the backbone chain.

Structural elucidation of the capsular polysaccharide from Streptococcus pneumoniae serotype 47A by NMR spectroscopy.

The structure of the serotype 47A (Danish nomenclature system) capsular polysaccharide from Streptococcus pneumoniae was elucidated by NMR spectroscopy. The following structure of the repeating heptasaccharide was deduced: [structure: see text]. The serotype 47A capsular polysaccharide is one of 91 structurally and serologically distinct capsular polysaccharides that have been recognized in S.

(1)H NMR spectroscopy for profiling complex carbohydrate mixtures in non-fractionated beer.

A plethora of biological and biotechnological processes involve the enzymatic remodelling of carbohydrates in complex mixtures whose compositions affect both the processes and products. In the current study, we employed high-resolution (1)H NMR spectroscopy for the analysis of cereal-derived carbohydrate mixtures as exemplified on six beer samples of different styles. Structural assignments of more than 50 carbohydrate moieties were obtained using (1)H1-(1)H2 groups as structural reporters.

Profiling of carbohydrate mixtures at unprecedented resolution using high-precision 1H-13C chemical shift measurements and a reference library.

Complex mixtures of carbohydrates pose distinct challenges in routine and high-throughput analysis, so that only a few carbohydrate components are routinely resolved and identified in biofluids, extracts, foods and other complex mixtures. Here, we conduct precise measurements of (1)H and (13)C anomeric chemical shifts to construct a reference library of specific carbohydrate signals with high-resolution two-dimensional (1)H-(13)C NMR spectra. High-resolution multidimensional NMR spectra largely abolish resolution problems in carbohydrate analysis with state-of-the-art instrumentation.

Time-resolved in-situ observation of starch polysaccharide degradation pathways.

Analytical challenges in the direct time-resolved observation of starch metabolism have been addressed by using optimized multidimensional NMR experiments. Starch provides the main source of human dietary energy intake and is a raw material for beverage and renewable fuel production. Use of direct in situ observations of starch remodeling pathways could facilitate our understanding and control of processes of biotechnological, medical, and environmental relevance.

Fast and accurate quantitation of glucans in complex mixtures by optimized heteronuclear NMR spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy is a widely used technique for mixture analysis, but it has shortcomings in resolving carbohydrate mixtures due to the narrow chemical shift range of glycans in general and fragments of homopolymers in particular. Here, we suggest a protocol toward fast spectroscopic glycan mixture analysis. We show that a plethora of oligosaccharides comprising only α-glucopyranosyl residues can be resolved into distinct quantifiable signals with NMR experiments that are substantially faster than chromatographic runs.

In vitro growth of four individual human gut bacteria on oligosaccharides produced by chemoenzymatic synthesis.

The present study aimed at examining oligosaccharides (OS) for potential stimulation of probiotic bacteria. Nineteen structurally well-defined candidate OS covering groups of β-glucosides, α-glucosides and α-galactosides with degree of polymerization 2-4 were prepared in >100 mg amounts by chemoenzymatic synthesis (i.e.

Monitoring pathways of β-glucan degradation by enzyme mixtures in situ.

The degradation of β-glucans from cereal cell walls is related to health benefits of whole grain foodstuffs and is a prominent cost in the production of bioethanol and in improving the filterability of malt-based beverages. Detailed assays of β-glucan degradation pathways by enzyme mixtures therefore promise to support the analysis of physiological and the optimization of technological processes. Physiological and biotechnological processes tend to occur in complex mixtures of catalysts and substrates and the development of advanced methodologies for mixture analysis has been attracting a great deal of attention.

Structural characterisation of a complex heteroglycan from the cyanobacterium Nostoc commune.

An alkali-extractable O-methylated ribofuranose-containing heteroglycan, Nc-5-s, was isolated from wild-growing field colonies of the cyanobacterium Nostoc commune collected in Iceland, using ethanol fractionation and anion-exchange chromatography. The average molecular weight was estimated to be 1500 kDa. Structural characterisation of the heteroglycan was performed by high-field NMR spectroscopy (1D proton, 2D-COSY, 2D-NOESY, 2D-TOCSY, (1)H (13)C-HSQC, HMBC, H2BC and HSQC-NOESY) as well as monosaccharide analysis after methanolysis by GC and supported by linkage analysis by GC-MS.

NMR assignment of structural motifs in intact β-limit dextrin and its α-amylase degradation products in situ.

An increasingly detailed and realistic view of biological processes often hinges on atomic-level characterization of biomacromolecules and of the processes they are involved in, preferably under near-physiological conditions. Structure, degradation, and synthesis of glucose storage polymers have been studied for decades with a range of analytical tools, but the detailed in situ analysis has remained an analytical challenge. Here, we report the NMR assignment of different structural motifs in the β-limit dextrin from lintnerized maize starch as a branched α-glucan model system for starch, which is depleted of repetitive α-(1→4) glycosidic bonds at non-reducing ends but has the α-(1→6) branch points intact.

Chemical and biological characterization of pectin-like polysaccharides from the bark of the Malian medicinal tree Cola cordifolia.

The bark of Cola cordifolia used in Malian traditional medicine contains unusual types of polysaccharides with immunomodulating activities. We report for the first time on the structure of a polymer designated CC1P1 having the repeating structure [2→)[α-D-Gal(1→3)]α-L-Rha(1→4)α-d-GalA(1→] as determined by NMR and GC/MS. α-Linked Gal is unusual in pectins.

Atlantinone A, a Meroterpenoid Produced by Penicillium ribeum and Several Cheese Associated Penicillium Species.

Atlantinone A has been isolated from the psychrotolerant fungus Penicillium ribeum. The exact structure of the compound was confirmed by mass spectrometric and 1- and 2D NMR experiments. Atlantinone A was originally only produced upon chemical epigenetic manipulation of P.

Characterization of a novel Salmonella Typhimurium chitinase which hydrolyzes chitin, chitooligosaccharides and an N-acetyllactosamine conjugate.

Salmonella contain genes annotated as chitinases; however, their chitinolytic activities have never been verified. We now demonstrate such an activity for a chitinase assigned to glycoside hydrolase family 18 encoded by the SL0018 (chiA) gene in Salmonella enterica Typhimurium SL1344. A C-terminal truncated form of chiA lacking a putative chitin-binding domain was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3) with an N-terminal (His)(6) tag.