The Aspergillus fumigatus sialidase is a 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid hydrolase (KDNase): structural and mechanistic insights

Aspergillus fumigatus is a filamentous fungus that can cause severe respiratory disease in immunocompromised individuals. A putative sialidase from A. fumigatus was recently cloned and shown to be relatively poor in cleaving N-acetylneuraminic acid (Neu5Ac) in comparison with bacterial sialidases. Here we present the first crystal structure of a fungal sialidase. When the apo structure was compared with bacterial sialidase structures, the active site of the Aspergillus enzyme suggested that Neu5Ac would be a poor substrate because of a smaller pocket that normally accommodates the acetamido group of Neu5Ac in sialidases. A sialic acid with a hydroxyl in place of an acetamido group is 2-keto-3-deoxynononic acid (KDN). We show that KDN is the preferred substrate for the A. fumigatus sialidase and that A. fumigatus can utilize KDN as a sole carbon source. A 1.45-Å resolution crystal structure of the enzyme in complex with KDN reveals KDN in the active site in a boat conformation and nearby a second binding site occupied by KDN in a chair conformation, suggesting that polyKDN may be a natural substrate. The enzyme is not inhibited by the sialidase transition state analog 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) but is inhibited by the related 2,3-didehydro-2,3-dideoxy-d-glycero-d-galacto-nonulosonic acid that we show bound to the enzyme in a 1.84-Å resolution crystal structure. Using a fluorinated KDN substrate, we present a 1.5-Å resolution structure of a covalently bound catalytic intermediate. The A. fumigatus sialidase is therefore a KDNase with a similar catalytic mechanism to Neu5Ac exosialidases, and this study represents the first structure of a KDNase.     

LOCALIZATION OF IRON WITHIN CENTRIC DIATOMS OF THE GENUS THALASSIOSIRA1

The cellular iron (Fe) quota of centric diatoms has been shown to vary in response to the ambient dissolved Fe concentration; however, it is not known how centric diatoms store excess intracellular Fe. Here, we use synchrotron X‐ray fluorescence (SXRF) element mapping to identify Fe storage features in cells of Thalassiosira pseudonana Hasle et Heimdal and Thalassiosira weissflogii G. A. Fryxell et Hasle grown at low and high Fe concentrations. Localized intracellular Fe storage features, defined as anomalously high Fe concentrations in regions of relatively low phosphorus (P), sulfur (S), silicon (Si), and zinc (Zn), were twice as common in T. weissflogii cells grown at high Fe compared to low‐Fe cells. Cellular Fe quotas of this strain increased 2.9‐fold, the spatial extent of the features increased 4.6‐fold, and the Fe content of the features increased 14‐fold under high‐Fe conditions, consistent with a vacuole storage mechanism. The element stoichiometry of the Fe features is consistent with polyphosphate‐bound Fe as a potential vacuolar Fe storage pool. Iron quotas increased 2.5‐fold in T. pseudonana grown at high Fe, but storage features contained only 2‐fold more Fe and did not increase in size compared to low‐Fe cells. The differences in Fe storage observed between T. pseudonana and T. weissflogii may have been due to differences in the growth states of the cultures.     

Molecular interaction of delta-conotoxins with voltage-gated sodium channels

Various neurotoxic peptides modulate voltage-gated sodium (Na(V)) channels and thereby affect cellular excitability. Delta-conotoxins from predatory cone snails slow down inactivation of Na(V) channels, but their interaction site and mechanism of channel modulation are unknown. Here, we show that delta-conotoxin SVIE from Conus striatus interacts with a conserved hydrophobic triad (YFV) in the domain-4 voltage sensor of Na(V) channels. This site overlaps with that of the scorpion alpha-toxin Lqh-2, but not with the alpha-like toxin Lqh-3 site. Delta-SVIE functionally competes with Lqh-2, but exhibits strong cooperativity with Lqh-3, presumably by synergistically trapping the voltage sensor in its "on" position.     

A1M Ameliorates Preeclampsia-Like Symptoms in Placenta and Kidney Induced by Cell-Free Fetal Hemoglobin in Rabbit

Preeclampsia is one of the most serious pregnancy-related diseases and clinically manifests as hypertension and proteinuria after 20 gestational weeks. The worldwide prevalence is 3-8% of pregnancies, making it the most common cause of maternal and fetal morbidity and mortality. Preeclampsia lacks an effective therapy, and the only “cure” is delivery. We have previously shown that increased synthesis and accumulation of cell-free fetal hemoglobin (HbF) in the placenta is important in the pathophysiology of preeclampsia. Extracellular hemoglobin (Hb) and its metabolites induce oxidative stress, which may lead to acute renal failure and vascular dysfunction seen in preeclampsia. The human endogenous protein, α₁-microglobulin (A1M), removes cell-free heme-groups and induces natural tissue repair mechanisms. Exogenously administered A1M has been shown to alleviate the effects of Hb-induced oxidative stress in rat kidneys. Here we attempted to establish an animal model mimicking the human symptoms at stage two of preeclampsia by administering species-specific cell-free HbF starting mid-gestation until term, and evaluated the therapeutic effect of A1M on the induced symptoms. Female pregnant rabbits received HbF infusions i.v. with or without A1M every second day from gestational day 20. The HbF-infused animals developed proteinuria and a significantly increased glomerular sieving coefficient in kidney that was ameliorated by co-administration of A1M. Transmission electron microscopy analysis of kidney and placenta showed both intracellular and extracellular tissue damages after HbF-treatment, while A1M co-administration resulted in a significant reduction of the structural and cellular changes. Neither of the HbF-treated animals displayed any changes in blood pressure during pregnancy. In conclusion, infusion of cell-free HbF in the pregnant rabbits induced tissue damage and organ failure similar to those seen in preeclampsia, and was restored by co-administration of A1M. This study provides preclinical evidence supporting further examination of A1M as a potential new therapy for preeclampsia.     

EUROCarbDB: An open-access platform for glycoinformatics

The EUROCarbDB project is a design study for a technical framework, which provides sophisticated, freely accessible, open-source informatics tools and databases to support glycobiology and glycomic research. EUROCarbDB is a relational database containing glycan structures, their biological context and, when available, primary and interpreted analytical data from high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance experiments. Database content can be accessed via a web-based user interface. The database is complemented by a suite of glycoinformatics tools, specifically designed to assist the elucidation and submission of glycan structure and experimental data when used in conjunction with contemporary carbohydrate research workflows. All software tools and source code are licensed under the terms of the Lesser General Public License, and publicly contributed structures and data are freely accessible. The public test version of the web interface to the EUROCarbDB can be found at http://www.ebi.ac.uk/eurocarb.     

Stability of targeted metabolite profiles of urine samples under different storage conditions

Introduction

Few studies have investigated the influence of storage conditions on urine samples and none of them used targeted mass spectrometry (MS).

Objectives

We investigated the stability of metabolite profiles in urine samples under different storage conditions using targeted metabolomics.

Methods

Pooled, fasting urine samples were collected and stored at ?80 °C (biobank standard), ?20 °C (freezer), 4 °C (fridge), ~9 °C (cool pack), and ~20 °C (room temperature) for 0, 2, 8 and 24 h. Metabolite concentrations were quantified with MS using the AbsoluteIDQ? p150 assay. We used the Welch-Satterthwaite-test to compare the concentrations of each metabolite. Mixed effects linear regression was used to assess the influence of the interaction of storage time and temperature.

Results

The concentrations of 63 investigated metabolites were stable at ?20 and 4 °C for up to 24 h when compared to samples immediately stored at ?80 °C. When stored at ~9 °C for 24 h, few amino acids (Arg, Val and Leu/Ile) significantly decreased by 40% in concentration (P < 7.9E?04); for an additional three metabolites (Ser, Met, Hexose H1) when stored at ~20 °C reduced up to 60% in concentrations. The concentrations of four more metabolites (Glu, Phe, Pro, and Thr) were found to be significantly influenced when considering the interaction between exposure time and temperature.

Conclusion

Our findings indicate that 78% of quantified metabolites were stable for all examined storage conditions. Particularly, some amino acid concentrations were sensitive to changes after prolonged storage at room temperature. Shipping or storing urine samples on cool packs or at room temperature for more than 8 h and multiple numbers of freeze and thaw cycles should be avoided.

     

Engineered <Emphasis Type="Italic">E. coli</Emphasis> W enables efficient 2,3-butanediol production from glucose and sugar beet molasses using defined minimal medium as economic basis

Background

Efficient microbial production of chemicals is often hindered by the cytotoxicity of the products or by the pathogenicity of the host strains. Hence 2,3-butanediol, an important drop-in chemical, is an interesting alternative target molecule for microbial synthesis since it is non-cytotoxic. Metabolic engineering of non-pathogenic and industrially relevant microorganisms, such as Escherichia coli, have already yielded in promising 2,3-butanediol titers showing the potential of microbial synthesis of 2,3-butanediol. However, current microbial 2,3-butanediol production processes often rely on yeast extract as expensive additive, rendering these processes infeasible for industrial production.

Results

The aim of this study was to develop an efficient 2,3-butanediol production process with E. coli operating on the premise of using cost-effective medium without complex supplements, considering second generation feedstocks. Different gene donors and promoter fine-tuning allowed for construction of a potent E. coli strain for the production of 2,3-butanediol as important drop-in chemical. Pulsed fed-batch cultivations of E. coli W using microaerobic conditions showed high diol productivity of 4.5 g l^?1 h^?1. Optimizing oxygen supply and elimination of acetoin and by-product formation improved the 2,3-butanediol titer to 68 g l^?1, 76% of the theoretical maximum yield, however, at the expense of productivity. Sugar beet molasses was tested as a potential substrate for industrial production of chemicals. Pulsed fed-batch cultivations produced 56 g l^?1 2,3-butanediol, underlining the great potential of E. coli W as production organism for high value-added chemicals.

Conclusion

A potent 2,3-butanediol producing E. coli strain was generated by considering promoter fine-tuning to balance cell fitness and production capacity. For the first time, 2,3-butanediol production was achieved with promising titer, rate and yield and no acetoin formation from glucose in pulsed fed-batch cultivations using chemically defined medium without complex hydrolysates. Furthermore, versatility of E. coli W as production host was demonstrated by efficiently converting sucrose from sugar beet molasses into 2,3-butanediol.

     

Phylogeography of island canary (Serinus canaria) populations

Island canaries (Serinus canaria) are characterised as a species living exclusively on North Atlantic islands, mainly on the Azores, Madeira and Canary Islands. Although they are very common in their habitats, their behaviour and breeding system has only recently been studied systematically. To advance the understanding of their ecology and to see if the rather isolated archipelagos are already promoting a genetic differentiation, we investigated their phylogeographic relationship as revealed by mtDNA sequences of the cytochrome b gene and investigated whether this measure corresponds to morphological characteristics within the islands. Genetic distances were very low throughout the distribution range of the species. Although the variation of genetic distances within the population of Pico (Azores) was larger than that on Madeira and Canary Islands, the genetic distances between island populations were very low throughout which prevented a clear phylogeographic differentiation. Moreover, morphological measurements did not reveal a consistent pattern to reliably separate the populations, although the measures of beak length and body weight revealed a clear island-specific differentiation. These data lead to the assumption that the colonisation of the Atlantic islands by the canaries occurred very recently, while there is no persisting gene flow between the populations.     

Arabidopsis genes encoding mitochondrial type II NAD(P)H dehydrogenases have different evolutionary origin and show distinct responses to light

In addition to proton-pumping complex I, plant mitochondria contain several type II NAD(P)H dehydrogenases in the electron transport chain. The extra enzymes allow the nonenergy-conserving electron transfer from cytoplasmic and matrix NAD(P)H to ubiquinone. We have investigated the type II NAD(P)H dehydrogenase gene families in Arabidopsis. This model plant contains two and four genes closely related to potato (Solanum tuberosum) genes nda1 and ndb1, respectively. A novel homolog, termed ndc1, with a lower but significant similarity to potato nda1 and ndb1, is also present. All genes are expressed in several organs of the plant. Among the nda genes, expression of nda1, but not nda2, is dependent on light and circadian regulation, suggesting separate roles in photosynthesis-associated and other respiratory NADH oxidation. Genes from all three gene families encode proteins exclusively targeted to mitochondria, as revealed by expression of green fluorescent fusion proteins and by western blotting of fractionated cells. Phylogenetic analysis indicates that ndc1 affiliates with cyanobacterial type II NADH dehydrogenase genes, suggesting that this gene entered the eukaryotic cell via the chloroplast progenitor. The ndc1 should then have been transferred to the nucleus and acquired a signal for mitochondrial targeting of the protein product. Although they are of different origin, the nda, ndb, and ndc genes carry an identical intron position.     

Diversity of human insulin-like growth factor (IGF) binding protein-2 fragments in plasma: primary structure, IGF-binding properties, and disulfide bonding pattern

The insulin-like growth factor binding proteins (IGFBPs) play a major role in the regulation of the effects and the bioavailability of the insulin-like growth factors (IGFs). IGFs are released from IGFBP-IGF complexes by proteolysis of IGFBPs generating fragments with reduced ligand-binding properties. To identify naturally occurring fragments of IGFBP-2, a peptide library generated from human hemofiltrate was immunologically screened. Purification of immunoreactive IGFBP-2 fragments was performed by consecutive chromatographic steps. A total of 18 different IGFBP-2 fragments was isolated and characterized. The peptides exhibited different N-terminal amino acid residues that were located in the variable midregion of IGFBP-2. Four major cleavage sites were determined to be between Tyr103 and Gly104, Leu152 and Ala153, Arg156 and Glu157, and Gln165 and Met166. The resulting fragments were further processed by amino and/or carboxy peptidases and comprised 37-185 amino acid residues. Ligand blotting, solution binding assays, and BIAcore analyses revealed that all tested fragments retained low IGF-binding capacity. The most abundant fragment IGFBP-2 (167-279) showed 10% of IGF-II binding compared to recombinant human (rh)IGFBP-2. Furthermore, the disulfide bonding pattern of the C-terminal domain of rhIGFBP-2 was defined, indicating linkages between cysteine residues 191-225, 236-247, and 249-270. This study provides the most comprehensive molecular characterization of human IGFBP-2 fragments formed in vivo, exhibiting both residual IGF-binding capacities and the integrin-binding sequence.