Sulfation Sites Research Articles

Quantitative Conversion of Methanol to Methyl Formate on Graphene-Confined Nano-Oxides

SummaryWe demonstrate the nearly quantitative conversion of methanol to methyl formate (MF) with a reliable durability on the reduced-graphene-oxide-confined VTiOx nanoparticles (rGO@VTiO). The rGO@VTiO exhibits superior low-temperature reactivity than the rGO-free VTiO, and the MF yield of 98.8% is even comparable with the noble metal catalysts. Both experiments and simulations demonstrate that the ultrathin rGO shell significantly impacts the shell/core interfacial electronic structure and the surface chemistry of the resultant catalysts, leading to remarkable reactivity in methanol to MF. rGO enhances the dispersion and loading rates of active monomeric/oligomeric VOx. In particular, the electron migration between the rGO shell and oxides core reinforces the acidity of rGO@VTiO in the absence of sulfate acidic sites. Moreover, both in situ NAP-XPS and DRIFTS investigations suggest that the lattice oxygen was involved in the oxidation of methanol and the MF was formed via the hemiacetal mechanism.

Distribution behavior of arsenate into α-calcium sulfate hemihydrate transformed from gypsum in solution

Transforming gypsum into α-calcium sulfate hemihydrate (α-HH) provides a promising utilization pathway for the abundant amount of chemical gypsum. The transformation follows the route of “dissolution-recrystallization”, during which the arsenic pollutant in gypsum is released into the solution, and hence raises the possibility of being distributed into the product of α-HH, a potential harm that has always been neglected. Investigation of the transformation process at neutral pH revealed that the arsenate ions in solution were distributed into α-HH and generated an enrichment of arsenic by 4–6 times. Arsenate ions distributed into α-HH by substitution for lattice sulfate, adsorption on α-HH facets and occupation for surface sulfate sites. While at higher concentrations, calcium arsenate coprecipitated with α-HH or even crystallized independently. Increasing temperature accelerated the phase transformation and restrained arsenate migration into α-HH due to the lag of distribution balance. The pH of solution modulated the dominant arsenate species and decreasing pH weakened arsenate substitution capacity for sulfate in α-HH. This work uncovers arsenate distribution mechanism during the transformation of gypsum into α-HH and provides a feasible method to restrain arsenate distribution into product, which helps to understand arsenate behavior in hydrothermal solution with high concentration of sulfate minerals and provides a guidance for controlling pollutants distribution into product.

Regioselective sulfation of β-glucan from Ganoderma lucidum and structure-anticoagulant activity relationship of sulfated derivatives

In the present study, regioselective sulfation of β-glucan (GLP) from Ganoderma lucidum were firstly established by using 4,4′-dimethoxytrityl chloride and hexamethyldisilazane as protecting precursor. 2,4,6-O-sulfated, 6-O-sulfated and 2,4-O-sulfated GLP derivatives were prepared and the molecular weights (Mw) of derivatives were determined to range from 0.94 × 104 to 6.27 × 104 g/mol, while the degrees of sulfation (DS) were calculated to vary from 0.83 to 1.74. The regioselective sulfation of GLP was confirmed by FT-IR, 13C NMR spectroscopy and methylation analysis. Results indicated that the sulfated substitution sites were predominantly at C-6 in 6-O-sulfated GLP (S6-OGLP) and C-4 in 2,4-O-sulfated GLP (S2,4-OGLP), respectively. Clotting assays (APTT, PT and TT) in vitro showed that sulfate groups were essential for anticoagulant activity and S6-OGLP exhibited much higher than others. Meanwhile, sulfated GLP with higher DS and Mw showed stronger anticoagulant activity in the case of the same condition.

Recombination Events and Conserved Nature of Receptor Binding Motifs in Coxsackievirus A9 Isolates.

Coxsackievirus A9 (CVA9) is an enterically transmitted enterovirus and one of the most pathogenic type among human enteroviruses. CVA9 isolates use a distinctive RGD (Arg-Gly-Asp) motif within VP1 capsid protein that defines its ability to bind to integrin receptor(s) for cellular entry. To investigate CVA9 evolution and pathogenicity, genetic relationships and recombination events were analyzed between 54 novel clinical isolates of CVA9, as well as 21 previously published full length CVA9 sequences from GenBank. Samples were investigated by partial sequencing of the novel VP1 and 3Dpol genes, as well as including the corresponding areas from GenBank sequences. Phylogenetic analyses were combined with clinical data in a further attempt to analyze whether sequence evolution reflects CVA9 pathogenicity in the phylogenies. Furthermore, VP1 gene was also analyzed for receptor binding sites including the RGD motif and the putative heparan sulfate (HS) site. Analysis of the 559-nucleotide-long VP1 sequences identified six clades. Although most of the strains within each clade showed geographical clustering, the grouping pattern of the isolates in the analysis of the VP1 gene was strikingly different from grouping of 3Dpol, which suggests that recombination events may have occurred in the region encoding the nonstructural proteins. Inclusion of clinical data did not provide any evidence of symptom based phylogenetic clustering of CVA9 isolates. Amino acid sequence analysis of the VP1 polypeptide demonstrated that the RGD motif was fully conserved among the isolates while the putative HS binding site was only found in one isolate. These data suggest that integrin binding is essential for virus tropism, but do not explain the symptom repertoire.

Ion-specific interactions at calcite-brine interfaces: a nano-scale study of the surface charge development and preferential binding of polar hydrocarbons.

This research provides an atomic-level insight into the synergic contribution of mono- and divalent ions to interfacial characteristics of calcite surfaces exposed to electrolyte solution containing organic compounds. The emphasis was placed on the ionic interactions responsible for charge developing mechanisms of calcite surfaces and also the capacity for adsorption of polar hydrocarbons, represented by benzoic acid (BA), at different brine compositions. For this purpose, molecular dynamics (MD) simulations were employed to explore the interplay of the main constituent ions of natural brines (Na+, Cl-, Mg2+, and SO42-) and BA at the interface of CaCO3. It was observed that surface accumulation of Na+ cations produces a positively charged layer immediate to the basal plane of calcite, validating the typical positive surface charge of carbonates reported by laboratory experiences. Meanwhile, a negatively charged layer appears beyond the sodium layer as a result of direct and solvent-mediated pairing of anions with Na+ cations lodged on the calcite substrate. In this process, sulfate adsorption severely diminishes surface charge to even a negative value in the case of a SO42--rich solution, providing an interpretation for the measurements reported in the literature. Our results revealed the inhibition of direct binding of BA molecules onto the calcite surface through complexation with protruding oxygen atoms of basal carbonates by the residing Na+ cations. Further, we noticed the sulfate-mediated pairing of BA molecules to the Na+ layer, which in effect intensifies surface adsorption of BA. However, BA-SO42- interaction is considerably reduced by magnesium cations shielding sulfate sites in the Mg2+-augmented brine. The findings presented in this study are of fundamental importance to advance our microscopic understanding of interfacial interactions in brine/oil/carbonate systems; with broad scientific and applied implications in the context of mobilizing organic contaminants trapped in aquifer sediments and enhancement of hydrophilicity of subsurface oil-bearing carbonate reservoirs by injecting ion-modified brine solutions.

Unraveling Heparan Sulfate Proteoglycan Binding Motif for Cancer Cell Selectivity.

Membrane heparan sulfate proteoglycans (HSPG) regulate cell proliferation, migration, and differentiation and are therefore considered key players in cancer cell development processes. Here, we used the NT4 peptide to investigate how the sulfation pattern of HSPG on cells drives binding specificity. NT4 is a branched peptide that binds the glycosaminoglycan (GAG) chains of HSPG. It has already been shown to inhibit growth factor-induced migration and invasiveness of cancer cells, implying antagonist binding of HSPG. The binding affinity of NT4 with recombinant HSPG showed that NT4 bound glypican-3 and -4 and, with lower affinity, syndecan-4. NT4 binding to the cancer cell membrane was inversely correlated with sulfatase expression. NT4 binding was higher in cell lines with lower expression of SULF-1 and SULF-2, which confirms the determinant role of sulfate groups for recognition by NT4. Using 8-mer and 9-mer heparan sulfate (HS) oligosaccharides with analog disaccharide composition and different sulfation sites, a possible recognition motif was identified that includes repeated 6-O-sulfates alternating with N- and/or 2-O-sulfates. Molecular modeling provided a fully descriptive picture of binding architecture, showing that sulfate groups on opposite sides of the oligosaccharide can interact with positive residues on two peptide sequences of the branched structure, thus favoring multivalent binding and explaining the high affinity and selectivity of NT4 for highly sulfated GAGs. NT4 and possibly newly selected branched peptides will be essential probes for reconstructing and unraveling binding sites for cancer-involved ligands on GAGs and will pave the way for new cancer detection and treatment options.

Advancing MSn spatial resolution and documentation for glycosaminoglycans by sulfate-isotope exchange.

Glycosaminoglycans (GAGs) are carbohydrate polyionic polymers that participate in a host of critically important biological processes. A significant difficulty in the comprehensive structural characterization of GAGs is the determination of specific sulfation position isomers. We chose to circumvent sulfate lability by its liberation followed by specific isotope exchange that makes it amenable to methylation, collisional induced dissociation, and MSn disassembly for a detailed structural characterization. A set of chemistries that include sulfate release, isotopic (CD3- and CD3-CO-) replacement, and methylation have been modified to yield a stable product ideal for sequencing by MSn. Disassembly of these samples provides a detailed read-out of sequence inclusive of all sulfation sites. As documenting steps, we applied these chemical modifications to a series of disaccharides and a synthetic GAG pentamer, Arixtra®. Upon disassembly, glycosidic and cross-ring cleavages define the monomer composition including individual sulfation positions. The N- and O-sulfates are differentiated by deuterium-containing mass compositions. The uronic methylesters do not significantly alter the fragmentation patterns. A fragment library of these products is being assembled as an adjunct to our larger fragment library, some 15years in the making.

ISulfoTyr-PseAAC: Identify Tyrosine Sulfation Sites by Incorporating Statistical Moments via Chou's 5-steps Rule and Pseudo Components.

Background:The amino acid residues, in protein, undergo post-translation modification (PTM) during protein synthesis, a process of chemical and physical change in an amino acid that in turn alters behavioral properties of proteins. Tyrosine sulfation is a ubiquitous posttranslational modification which is known to be associated with regulation of various biological functions and pathological pro-cesses. Thus its identification is necessary to understand its mechanism. Experimental determination through site-directed mutagenesis and high throughput mass spectrometry is a costly and time taking process, thus, the reliable computational model is required for identification of sulfotyrosine sites.Methodology:In this paper, we present a computational model for the prediction of the sulfotyrosine sites named iSulfoTyr-PseAAC in which feature vectors are constructed using statistical moments of protein amino acid sequences and various position/composition relative features. These features are in-corporated into PseAAC. The model is validated by jackknife, cross-validation, self-consistency and in-dependent testing.Results:Accuracy determined through validation was 93.93% for jackknife test, 95.16% for cross-validation, 94.3% for self-consistency and 94.3% for independent testing.Conclusion:The proposed model has better performance as compared to the existing predictors, how-ever, the accuracy can be improved further, in future, due to increasing number of sulfotyrosine sites in proteins.

Genesis of placental sequestration in malaria and possible targets for drugs for placental malaria.

Malaria during pregnancy results in intrauterine growth restriction, fetal anemia, and infant mortality. Women are more susceptible to malaria during pregnancy due to malaria‐induced inflammation and the sequestration of infected red blood cells in the placenta, which bind to the chondroitin sulfate portion of syndecan‐1 on the syncytiotrophoblast and in the intervillous space. Syndecan‐1 is a dimeric proteoglycan with an extracellular ectodomain that is cleaved from the transmembrane domain (referred to as “shedding”) by matrix metalloproteinases (MMPs), likely the secreted MMP‐9. The ectodomain includes four binding sites for chondroitin sulfate, which are proximal to the transmembrane domain, and six distal binding sites primarily for heparan sulfate. This “shedding” of syndecan‐1 is inhibited by the presence of the heparan sulfate chains, which can be removed by heparanase. The intervillous space contains fibrin strands and syndecan‐1 ectodomains free of heparan sulfate. The following is proposed as the sequence of events that leads to and is primarily responsible for sequestration in the intervillous space of the placenta. Inflammation associated with malaria triggers increased heparanase activity that degrades the heparan sulfate on the membrane‐bound syndecan‐1. Inflammation also upregulates MMP‐9 and the removal of heparan sulfate gives MMP‐9 access to cleave syndecan‐1, thereby releasing dimeric syndecan‐1 ectodomains with at least four chondroitin sulfate chains attached. These multivalent ectodomains bind infected RBCs together leading to their aggregation and entrapment in intervillous fibrin. This mechanism suggests possible new targets for anti‐placental malaria drugs such as the inhibition of MMP‐9. Doxycycline is an antimalarial drug which inhibits MMP‐9.

Evaluation and extension of the two-site, two-step model for binding and activation of the chemokine receptor CCR1

Interactions between secreted immune proteins called chemokines and their cognate G protein-coupled receptors regulate the trafficking of leukocytes in inflammatory responses. The two-site, two-step model describes these interactions. It involves initial binding of the chemokine N-loop/β3 region to the receptor's N-terminal region and subsequent insertion of the chemokine N-terminal region into the transmembrane helical bundle of the receptor concurrent with receptor activation. Here, we test aspects of this model with C-C motif chemokine receptor 1 (CCR1) and several chemokine ligands. First, we compared the chemokine-binding affinities of CCR1 with those of peptides corresponding to the CCR1 N-terminal region. Relatively low affinities of the peptides and poor correlations between CCR1 and peptide affinities indicated that other regions of the receptor may contribute to binding affinity. Second, we evaluated the contributions of the two CCR1-interacting regions of the cognate chemokine ligand CCL7 (formerly monocyte chemoattractant protein-3 (MCP-3)) using chimeras between CCL7 and the non-cognate ligand CCL2 (formerly MCP-1). The results revealed that the chemokine N-terminal region contributes significantly to binding affinity but that differences in binding affinity do not completely account for differences in receptor activation. On the basis of these observations, we propose an elaboration of the two-site, two-step model-the "three-step" model-in which initial interactions of the first site result in low-affinity, nonspecific binding; rate-limiting engagement of the second site enables high-affinity, specific binding; and subsequent conformational rearrangement gives rise to receptor activation.

Spectro-thermal characterization of the nature of sulfate groups immobilized on tetragonal zirconium oxide: Consequences of doping the oxide with Al or Mg cations

The present investigation was designed to characterize influence of doping with Al or Mg ions on nature and acidity of sulfate species dispersed on tetragonal-zirconia. Pure and doped zirconias were prepared by the citrate method. Sulfating of the thus produced zirconias was carried out by impregnation with aqueous solutions of (NH4)2SO4. The material bulk structure was elucidated by X-ray powder diffractometry and Laser Raman spectroscopy, whereas the surface sulfate was characterized via thermal and mass spectrometric analyses, and Fourier-transform infrared spectroscopy. The sulfate acidity was probed by IR νCCN spectra of adsorbed pyridine, and TPD of adsorbed n-propylamine. Results obtained showed the Al-doping to improve sulfate monolayer at the expense of overlayer structure, as well as its acidity. In contrast, Mg-doping influenced favorable bonding sites for polydentate sulfate of high thermal stability but of insignificant acidity.

Thermodynamic mixing properties of alunite supergroup minerals: Quantum-mechanical modeling and thermodynamic analysis of sulfate, chromate, selenate, phosphate, and arsenate solid solutions, as well as uranyl incorporation

Abstract In various geological settings, alunite supergroup minerals, including the end members alunite [KAl3(SO4)2(OH)6] and jarosite [KFe3(SO4)2(OH)6], occur in extensive solid solution on the cation and anion sites, enabling them to accommodate a variety of elements. Jarosite is known for scavenging heavy metals on the K site (e.g., Pb in solid solution between jarosite and plumbojarosite) in acid mine drainage settings, while both jarosite and alunite can be important in controlling toxic anions such as selenate, chromate, or arsenate. However, there is no thermodynamic information on the solubility of these important cations and anions as a function of temperature, in part because this information is difficult to obtain experimentally. In this study, thermodynamic mixing properties of sulfate-chromate (S-Cr), sulfate-selenate (S-Se), and sulfate-phosphate-arsenate (S-P-As) solid solutions in alunite supergroup minerals are investigated based on quantum-mechanical modeling and statistical thermodynamic analysis. S-Cr and S-Se solid solutions in alunite and jarosite are due to the mixing of anions with equivalent charges on the sulfate site. The enthalpy of mixing ( Δ H mix ) is lowest at 0 K (−273 °C) and increases with increasing temperature; it also depends on the arrangement of atoms on the sulfate site (i.e., atomic ordering). Δ H mix is almost constant at −70 °C and higher temperatures. These findings imply that S-Cr and S-Se solid solutions tend to be complete at room temperature and no ordering is acquired at or above ambient conditions. The Gibbs free energy of mixing ( Δ G mix ) indicates that jarosite is more flexible to accommodate chromate and selenate at the sulfate site than the alunite structure and that complete solid solution can be formed between jarosite and both Se- and Cr-analogues at temperatures above −80 °C. Our modeling results of solid solutions in jarosite and alunite demonstrate the critical role of alunite supergroup minerals in controlling toxic elements for long-term immobilization including the relatively favorable incorporation of uranyl (UO22+) into plumbojarosite. The S-P-As solid solution is also explored between alunite family minerals [DAl3(TO4)2(OH, H2O)6] (D is K+, Na+ or Ca2+; TO4 is SO42−, PO43− or AsO43−). The energetic barrier for mixing (reflected by the peak enthalpy of mixing) between ions with different charges (i.e., S-P and As-S solid solution; ∼2–3 kJ/mol of exchangeable atoms) is higher by a factor of two or three compared to anions with the equal charge (P-As solid solution; 1–1.5 kJ/mol of exchangeable atoms). Below 700 °C, ternary S-P-As mixing over a range of compositions in ternary space having alunite, crandallite [CaAl3(PO4)6(OH)5(H2O)], and arsenocrandallite [CaAl3(AsO4)6(OH)5(H2O)] as end members shows large miscibility gaps at compositions close to the ratio of S:P:As = 4:1:1 and 1:1:1 at the TO4 site. Ternary S-P-As mixing between woodhouseite, arsenowoodhouseite, and either alunite or natroalunite shows that arsenate is more compatible with sulfate in natroalunite than sulfate in alunite, whereas substitution of sulfate with phosphate is energetically more favorable in alunite than natroalunite. Our computed phase diagrams of S-P-As mixing suggest that binary solid solutions between pairs of sulfate, phosphate and arsenate in alunite family minerals scarcely occur below 100 °C, is limited at temperatures from 100 to 300 °C and become extensive or complete above 300 °C.

UNESCO WORLD KARST NATURAL HERITAGE SITES: GEOGRAPHICAL AND GEOLOGICAL REVIEW

The paper is devoted to the review of the World Karst Natural Heritage and the perspectives of such new properties in the Russia. Presently there are 37 World Karst Natural Heritage sites on the globe; 34 of them have the natural heritage and 3 sites have the mixed, natural-cultural significance. The World Karst Heritage distribution by the countries and by parts of the world was analyzed. A brief description of outstanding universal value of the “Lena Pillars Nature Park”, being currently the only Russian karst area nominated at the List of World Natural Heritage by a specific type of the karst – ground frozen karst, was given. The necessity of the new World Karst Heritage properties characterizing by the karst development in sulfate and salt rock sites are considered. The karst areas, located in extreme (cold or arid) climate conditions, are in the focus of separate attention.

Improved de novo sequencing of heparin/heparan sulfate oligosaccharides by propionylation of sites of sulfation

The structure of heparin and heparan sulfate (Hep/HS) oligosaccharides, as determined by the length and the pattern of sulfation, acetylation, and uronic acid epimerization, dictates their biological function through modulating interactions with protein targets. But fine structural determination is a very challenging task due to the lability of the sulfate modifications and difficulties in separating isomeric HS chains. Previously, we reported a strategy for chemical derivatization involving permethylation, desulfation, and trideuteroperacetylation, combined with standard reverse phase LC-MS/MS that enables the structural sequencing for heparin/HS oligosaccharides of sizes up to dodecasaccharide by positionally replacing all sulfates with more stable trideuteroacetyl groups, allowing for robust MS/MS sequencing. However, isomeric oligosaccharides that contain both N-sulfation and N-acetylation become isotopomers after labeling, differing only in the sites of deuteration. This prevents chromatographic separation of these different mixed domain sequences post-derivatization, and makes sequencing by MS/MS difficult due to co-fragmentation of the isotopomers leading to chimeric product ion spectra. In order to improve chromatographic separation of mixed domain oligosaccharides, we have introduced a propionylation step in place of trideuteroacetylation for labeling of sites of sulfation. HS standard disaccharides have been used to evaluate the efficiency of this improved chemical derivatization. The results show that we can quantitatively replace sulfation with propionyl groups with the same high efficiency as the previously reported trideuteroacetylation. After derivatization, we demonstrate the ability to chromatographically separate two mixed domain tetrasaccharide isomers differing solely by the order of N-sulfation and N-acetylation, allowing for full sequencing of each by MS/MS. These results represent a marked improvement in the ability of our previously reported derivatization strategy to analyze complex mixtures of Hep/HS oligosaccharides without a decrease in sensitivity.

Вплив модуляторів NMDA-рецепторів на біохімічні змінив сітківці при ішемічному та травматичному ураженні зорового аналізатора

Introduction. Glutamate exitotoxicity and hyperactivation of NMDA receptors play an important role in affecting the visual analyzer in cases of ischemia and trauma. It remains unexplored the influence of modulators of various sites of NMDA receptors on metabolic processes in the retina in these pathological conditions.The aim of the work – to determine the effect of NMDA receptor modulators on metabolic changes (oxidative stress, energy deficit, state of nitrogen oxide system, glutamate content) in the retina with the ischemic injury of the visual analyzer in rats and traumatic damage to the visual analyzer in rabbits.Research Methods. Experiments were conducted on 56 white male rats and 24 rabbits. Ischemic lesions of the visual analyzer in rats was caused by the creation of a one-sided ischemia-reperfusion in the a. ophthalmica.Traumatic damage of the visual analyzer in rabbits was caused by the action of a carbon dioxide stream under pressure on the cornea of the eye. NMDA receptor modulators were administered within 7 days before and 30 minutes after pathology simulation. Polyamino site blocker amantadine sulfate (5 mg/kg i/v) and phencyclidine site blocker memantine (20 mg/kg i/g) were used. The content of AT P, markers of oxidative-nitrosative stress, glutamate levels in retina, and the level of neuronspecific enolase (NSE) in serum were determined.Results and Discussion. The use of NMDA receptor inhibitors causes de-escalation of NSE levels in the blood, reduces energy deficit, oxidative-nitrosative stress, glutamate levels in the retina of ischemia-reperfusion of the eye in rats and contusion of the eye in rabbits. The polyamino site inhibitor amantadine sulfate exceeds (in 1.2-1.5 times, p<0.05) the fencyclidine site inhibitor memantine by the ability to adjust the levels of ATP, MDA, oxide-modified proteins, to restore the activity of glutathione peroxidase in the retina on ischemic and traumatic lesions of visual analyzer.Conclusions. Thus, the modulators of the polyamino- and phencyclidine sites of the NMDA receptors showed a protective effect on neuroretina for both ischemic and traumatic damage of the visual analyzer in animals. The study of the effects of amantadine sulfate on reparative processes in the retina is a promising direction for further research.

Neuropeptidomics of the Rat Habenular Nuclei.

Conserved across vertebrates, the habenular nuclei are a pair of small symmetrical structures in the epithalamus. The nuclei functionally link the forebrain and midbrain by receiving input from and projecting to several brain regions. Each habenular nucleus comprises two major asymmetrical subnuclei, the medial and lateral habenula. These subnuclei are associated with different physiological processes and disorders, such as depression, nicotine addiction, and encoding aversive stimuli or omitting expected rewarding stimuli. Elucidating the functions of the habenular nuclei at the molecular level requires knowledge of their neuropeptide complement. In this work, three mass spectrometry (MS) techniques-liquid chromatography (LC) coupled to Orbitrap tandem MS (MS/MS), LC coupled to Fourier transform (FT)-ion cyclotron resonance (ICR) MS/MS, and matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS-were used to uncover the neuropeptide profiles of the rodent medial and lateral habenula. With the assistance of tissue stabilization and bioinformatics, a total of 262 and 177 neuropeptides produced from 27 and 20 prohormones were detected and identified from the medial and lateral habenula regions, respectively. Among these neuropeptides, 136 were exclusively found in the medial habenula, and 51 were exclusively expressed in the lateral habenula. Additionally, novel sites of sulfation, a rare post-translational modification, on the secretogranin I prohormone are identified. The results demonstrate that these two small brain nuclei have a rich and differentiated peptide repertoire, with this information enabling a range of follow-up studies.

Ticks from diverse genera encode chemokine-inhibitory evasin proteins

To prolong residence on their hosts, ticks secrete many salivary factors that target host defense molecules. In particular, the tick Rhipicephalus sanguineus has been shown to produce three salivary glycoproteins named "evasins," which bind to host chemokines, thereby inhibiting the recruitment of leukocytes to the location of the tick bite. Using sequence similarity searches, we have identified 257 new putative evasin sequences encoded by the genomes or salivary or visceral transcriptomes of numerous hard ticks, spanning the genera Rhipicephalus, Amblyomma, and Ixodes of the Ixodidae family. Nine representative sequences were successfully expressed in Escherichia coli, and eight of the nine candidates exhibited high-affinity binding to human chemokines. Sequence alignments enabled classification of the evasins into two subfamilies: C8 evasins share a conserved set of eight Cys residues (four disulfide bonds), whereas C6 evasins have only three of these disulfide bonds. Most of the identified sequences contain predicted secretion leader sequences, N-linked glycosylation sites, and a putative site of tyrosine sulfation. We conclude that chemokine-binding evasin proteins are widely expressed among tick species of the Ixodidae family, are likely to play important roles in subverting host defenses, and constitute a valuable pool of anti-inflammatory proteins for potential future therapeutic applications.

High-Throughput Screening of Sulfated Proteins by Using a Genome-Wide Proteome Microarray and Protein Tyrosine Sulfation System.

Protein tyrosine sulfation (PTS) is a widespread posttranslational modification that induces intercellular and extracellular responses by regulating protein-protein interactions and enzymatic activity. Although PTS affects numerous physiological and pathological processes, only a small fraction of the total predicted sulfated proteins has been identified to date. Here, we localized the potential sulfation sites of Escherichia coli proteins on a proteome microarray by using a 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase-coupled tyrosylprotein sulfotransferase (TPST) catalysis system that involves in situ PAPS generation and TPST catalysis. Among the 4256 E. coli K12 proteins, 875 sulfated proteins were identified using antisulfotyrosine primary and Cy3-labeled antimouse secondary antibodies. Our findings add considerably to the list of potential proteins subjected to tyrosine sulfation. Similar procedures can be applied to identify sulfated proteins in yeast and human proteome microarrays, and we expect such approaches to contribute substantially to the understanding of important human diseases.

Aeshnid dragonfly larvae as bioindicators of methylmercury contamination in aquatic systems impacted by elevated sulfate loading.

Methylmercury (MeHg) levels in dragonfly larvae and water were measured over two years in aquatic systems impacted to varying degrees by sulfate releases related to iron mining activity. This study examined the impact of elevated sulfate loads on MeHg concentrations and tested the use of MeHg in dragonfly larvae as an indicator of MeHg levels in a range of aquatic systems including 16 river/stream sites and two lakes. MeHg concentrations in aeshnid dragonfly larvae were positively correlated (R(2) = 0.46, p < 0.01) to peak MeHg concentrations in the dissolved phase for the combined years of 2012 and 2013. This relation was strong in 2012 (R(2) = 0.85, p < 0.01), but showed no correlation in 2013 (R(2) = 0.02, p > 0.05). MeHg in dragonfly larvae were not elevated at the highest sulfate sites, but rather the reverse was generally observed. Record rainfall events in 2012 and above average rainfall in 2013 likely delivered the majority of Hg and MeHg to these systems via interflow and activated groundwater flow through reduced sediments. As a result, the impacts of elevated sulfate releases due to mining activities were not apparent in these systems where little of the sulfate is reduced. Lower bioaccumulation factors for MeHg in aeshnid dragonfly larvae were observed with increasing dissolved organic carbon (DOC) concentrations. This finding is consistent with previous studies showing that MeHg in high DOC systems is less bioavailable; an equilibrium model shows that more MeHg being associated with DOC rather than algae at the base of the food chain readily explains the lower bioaccumulation factors.

Modulation of GAG-sulfation pattern in biohybrid hydrogels to tune growth factor release

Event Abstract Back to Event Modulation of GAG-sulfation pattern in biohybrid hydrogels to tune growth factor release Passant Atallah1, Andrea Zieris1, Uwe Freudenberg1 and Carsten Werner1, 2 1 Max Bergmann Center of Biomaterials, Leibniz Institute of Polymer Research Dresden, Germany 2 Center for Regenerative Therapies Dresden, Technische Universität Dresden, Germany Introduction: GAG based hydrogels that modulate key parameters of the extracellular matrix were previously developed showing long term delivery of signaling molecules[1]. In here, the sulfation patterns of the GAG building blocks govern the electrostatic complexation of biomolecules and thus gradual desulfation should allow for modulating the release profiles of cytokines from the hydrogels. To explore options related to this, selectively desulfated heparin derivatives were prepared, thoroughly characterized, and covalently converted with star-shaped poly (ethylene glycol) into binary polymer networks and analyzed for cytokine release and cellular response in vitro and in vivo. Methods: Different solvolytic desulfation procedures yielded N-desulfated, 6O- N- heparin, 6O-desulfated and 2O-6O-N- (completely) desulfated heparin. All the heparin derivatives were thoroughly analyzed for regioselectivity of desulfation by 1H NMR, particle size analysis by asymetrric field flow fractionation, residual sulfate content by elemental analysis and anticoagulant activity by factor Xa based heparin assay. Hydrogels with graded sulfation pattern were subsequently prepared and the impact of the variation of the net negative charge on network properties was evaluated by rheometry and swelling measurements. Vascular endothelial growth factor (VEGF 165) and fibroblast growth factor 2 (FGF-2) were non-covalently conjugated to the heparin component of the hydrogel and the initial binding affinity of cytokines and their subsequent release was analyzed by ELISA[2]. The influence of the different functionalized matrices to migration and morphology of human umbilical vein endothelial cells (HUVEC) and the modulation of wound healing in diabetic mice has been analyzed. Results and discussion: Desulfated heparin chains were systematically characterized to confirm the chemical properties of the precursors complexed with the hydrogels. Analysis of physical network properties showed the influence of the charge density as well as the sulfation site specific effect on the elasticity and network defects of the formed hydrogels. Hydrogels with variable sulfation pattern show widely tunable release profiles for FGF-2 and VEGF165. In-vitro studies showed that the increased VEGF release from desulfated heparin hydrogels resulted in more pronounced proangiogenic response of HUVECs by enhanced cell migration and tubular formation. Similarly, hydrogels transplanted in db/db mice promoted wound angiogenesis, a critical stage in the wound healing process. Conclusion: Tailoring the sulfation pattern of GAG based hydrogels enables control over cytokine delivery and therefore cellular response in vitro and in vivo. The resulting new class of cell-instructive polymer matrices with tunable GAG sulfation will be instrumental for multiple applications in biotechnology and medicine. Figure 1: Schematic design of biohybrid GAG- star PEG hydrogels with regioselective desulfation resulting in modulation of cytokine release. This work was supported by the Deutsche Forschungsgemeinschaft through grant nos. SFB-TR 67/A10