废弃蛋壳源高性能钙基吸收剂的制备及其碳捕集性能

基于食品工业废弃蛋壳,本文利用不同有机酸反应制取乙酸钙、柠檬酸钙及葡萄糖酸钙共三种蛋壳源有机钙。在高温固定床反应器及热重分析仪上研究了不同前体所制成钙基吸收剂的碳循环捕集性能及碳酸化特性。进一步通过XRD分析了不同钙基吸收剂的物相组成,通过N₂吸附仪及SEM分析了循环前后钙基吸收剂结构特性及微观形貌的变化。结果表明,在三种蛋壳源有机钙中,葡萄糖酸钙所制成的钙基吸收剂具有较高的反应活性和相对最佳的碳捕集性能,首次碳酸化转化率高达85.33%,其钙基吸收剂相比其他吸收剂晶粒更小,20~100nm孔径范围内的孔隙较为发达,具有相对较强的抗烧结能力。经过20次循环实验发现,随着循环次数的增加,几种钙基吸收剂小颗粒均团聚烧结成大颗粒,造成孔隙结构缺失,孔隙率降低,影响其后续碳捕集性能。     

Studies of the structure of the N-terminal domain from the Y4 receptor - a G protein-coupled receptor - and its interaction with hormones from the NPY family

Binding of peptide hormones to G protein-coupled receptors is believed to be mediated through formation of contacts of the ligands with residues of the extracellular loops of family 1 GPCRs. Here we have investigated whether additional binding sites exist within the N-terminal domain, as studied in the form of binding of peptides from the neuropeptide Y (NPY) family to the N terminus of the Y4 receptor (N-Y4). The N-terminal domain of the Y4 receptor has been expressed in isotopically enriched form and studied by solution NMR spectroscopy. The peptide is unstructured in solution, whereas a micelle-associated helical segment is formed in the presence of dodecylphosphocholine (DPC) or sodium dodecylsulfate (SDS). As measured by surface plasmon resonance (SPR) spectroscopy, N-Y4 binds with approximately 50 microM affinity to the pancreatic polypeptide (PP), a high-affinity ligand to the Y4 receptor, whereas binding to neuropeptide Y (NPY) and peptide YY (PYY) is much weaker. Residues critical for binding in PP and in N-Y4 have been identified by site-directed mutagenesis. The data indicate that electrostatic interactions dominate and that this interaction is mediated by acidic ligand and basic receptor residues. Residues of N-Y4 are likely to contribute to the binding of PP, and in addition might possibly also help to transfer the hormone from the membrane-bound state into the receptor binding pocket.     

Characterization of Structure and Dynamics of the Guanidine-II Riboswitch from Escherichia coli by NMR Spectroscopy and Small-Angle X-ray Scattering (SAXS)

Riboswitches are regulatory RNA elements that undergo functionally important allosteric conformational switching upon binding of specific ligands. The here investigated guanidine-II riboswitch binds the small cation, guanidinium, and forms a kissing loop-loop interaction between its P1 and P2 hairpins. We investigated the structural changes to support previous studies regarding the binding mechanism. Using NMR spectroscopy, we confirmed the structure as observed in crystal structures and we characterized the kissing loop interaction upon addition of Mg²⁺ and ligand for the riboswitch aptamer from Escherichia coli. We further investigated closely related mutant constructs providing further insight into functional differences between the two (different) hairpins P1 and P2. Formation of intermolecular interactions were probed by small-angle X-ray scattering (SAXS) and NMR DOSY data. All data are consistent and show the formation of oligomeric states of the riboswitch induced by Mg²⁺ and ligand binding.     

Asprich: A novel aspartic acid-rich protein family from the prismatic shell matrix of the bivalve Atrina rigida

Almost all mineralized tissues contain proteins that are unusually acidic. As they are also often intimately associated with the mineral phase, they are thought to fulfill important functions in controlling mineral formation. Relatively little is known about these important proteins, because their acidic nature causes technical difficulties during purification and characterization procedures. Much effort has been made to overcome these problems, particularly in the study of mollusk-shell formation. To date about 16 proteins from mollusk-shell organic matrices have been sequenced, but only two are unusually rich in aspartic and glutamic acids. Here we screened a cDNA library made from the mRNA of the shell-forming cells of a bivalve, Atrina rigida, using probes for short Asp-containing repeat sequences, and identified ten different proteins. Using more specific probes designed from one subgroup of conserved sequences, we obtained the full sequences of a family of seven aspartic acid-rich proteins, which we named "Asprich"; a subfamily of the unusually acidic shell-matrix proteins. Polyclonal antibodies raised against a synthetic peptide of the conserved acidic1 domain of these proteins reacted specifically with the matrix components of the calcitic prismatic layer, but not with those of the aragonitic nacreous layer. Thus the Asprich proteins are constituents of the prismatic layer shell matrix. We can identify different domains within these sequences, including a signal peptide characteristic of proteins destined for extracellular secretion, a conserved domain rich in aspartic acid that contains a sequence very similar to the calcium-binding domain of Calsequestrin, and another domain rich in aspartic acid, that varies between the seven sequences. We also identified a domain with DEAD repeats that may have Mg-binding capabilities. Although we do not know, as yet, the function of these proteins, their generally conserved sequences do indicate that they might well fulfill basic functions in shell formation.     

Solution‐NMR Characterization of Outer‐Membrane Protein A from E. coli in Lipid Bilayer Nanodiscs and Detergent Micelles

X‐ray crystallography and solution NMR of detergent‐reconstituted OmpA (outer membrane protein A from E. coli) had shown that this protein forms an eight‐stranded transmembrane β‐barrel, but only limited information was obtained for the extracellular loops. In NMR studies of OmpA in two different detergent micelles, “NMR‐invisible” amino acid residues in‐between the extracellular loops and the β‐barrel prevented complete structural characterization. Here, we show that this NMR‐invisible ring around the β‐barrel of OmpA is also present in lipid bilayer nanodiscs and in mixed micelles with a third detergent, thus suggesting that the implicated rate processes have a functional role rather than representing an artifact of the protein reconstitution. In addition to sequence‐specific NMR assignments for OmpA in the nanodiscs, the present results are based on a protocol of micro‐coil TROSY‐ and CRINEPT‐type NMR diffusion measurements for studying the hydrodynamic properties and the foldedness of [²H,¹⁵N]‐labeled membrane proteins in nanodiscs. This protocol can be applied under conditions closely similar to those used for NMR structure determinations or crystallization trials.     

The Importance of Therapeutically Targeting the Binary Toxin from Clostridioides difficile

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell’s cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.     

Chromatographic characterisation of aptamer-modified poly(EDMA-co-GMA) monolithic disk format for protein binding and separation

The introduction of aptameric ligands onto disk-monolithic adsorbent, representing a unique strategy for convective isolation of target molecules with high specificity and selectivity, is investigated for the first time. Experimental results showed that the disk monolith possessed a good permeability of 1.67 ± 0.05 × 10^–14 m² (RSD = 3.2%). The aptameric ligand density for the aptamer-modified disk monolith was 480 pmol/uL. Chromatographic analysis of the aptamer disk-monolith efficiency showed an optimum linear velocity of 126 cm/min (≈0.25 mL/min) at room temperatures 25 ± 2°C. The theoretical number of plates corresponding to the optimum linear velocity was 128.2 with an height equivalent to the theoretical plate of 0.022 mm. The disk aptamer-immobilised monolithic system demonstrated good selectivity and isolation of thrombin from non-targets.     

Identification,SAR Studies,and X‐ray Co‐crystallographic Analysis of a Novel Furanopyrimidine Aurora Kinase A Inhibitor

Herein we reveal a simple method for the identification of novel Aurora kinase A inhibitors through substructure searching of an in‐house compound library to select compounds for testing. A hydrazone fragment conferring Aurora kinase activity and heterocyclic rings most frequently reported in kinase inhibitors were used as substructure queries to filter the in‐house compound library collection prior to testing. Five new series of Aurora kinase inhibitors were identified through this strategy, with IC₅₀ values ranging from ～300 nM to ～15 μM , by testing only 133 compounds from a database of ～125 000 compounds. Structure–activity relationship studies and X‐ray co‐crystallographic analysis of the most potent compound, a furanopyrimidine derivative with an IC₅₀ value of 309 nM toward Aurora kinase A, were carried out. The knowledge gained through these studies could help in the future design of potent Aurora kinase inhibitors.     

Unique Crystal Structure of a Novel Surfactant Protein from the Foam Nest of the Frog Leptodactylus vastus

Breeding by releasing eggs into stable biofoams (“foam nests”) is a peculiar reproduction mode within anurans, fish, and tunicates; not much is known regarding the biochemistry or molecular mechanisms involved. Lv‐ranaspumin (Lv‐RSN‐1) is the predominant protein from the foam nest of the frog Leptodactylus vastus. This protein shows natural surfactant activity, which is assumed to be crucial for stabilizing foam nests. We elucidated the amino acid sequence of Lv‐RSN‐1 by de novo sequencing with mass‐spectrometry and determined the high‐resolution X‐ray structure of the protein. It has a unique fold mainly composed of a bundle of 11 α‐helices and two small antiparallel β‐strands. Lv‐RSN‐1 has a surface rich in hydrophilic residues and a lipophilic cavity in the region of the antiparallel β‐sheet. It possesses intrinsic surface‐active properties, reducing the surface tension of water from 73 to 61 mN m^?1 (15 μg mL^?1). Lv‐RSN‐1 belongs to a new class of surfactants proteins for which little has been reported regarding structure or function.     

Biosynthesis and spectroscopic characterization of 2-TM fragments encompassing the sequence of a human GPCR, the Y4 receptor

This paper presents a divide‐and‐conquer approach towards obtaining solution structures of G protein‐coupled receptors. The human Y4 receptor was dissected into two to three transmembrane helix fragments, which were individually studied by solution NMR. We systematically compared various biosynthetic routes for the expression of the fragments in Escherichia coli and discuss purification strategies. In particular, we have compared the production of transmembrane (TM) fragments as inclusion bodies by using the ΔTrp leader sequence, with membrane‐directed expression by using Mistic as the fusion partner, and developed methods for enzymatic cleavage. In addition, direct expression of two‐TM fragments into inclusion bodies is a successful route in some cases. With the exception of TM13, we could produce all fragments in isotope‐labeled form in quantities sufficient for NMR studies. Almost complete backbone resonance assignment was obtained for the first two helices, as well as for helices 5 and 7, and a high degree was obtained for TM6, while conformational exchange processes resulted in the disappearance of many signals from TM4. In addition, complete assignments were obtained for all residues of the N‐terminal domain, as well as the extracellular and cytosolic loops (with the exception of an undecapeptide segment in the second extracellular loop, EC2) and for the complete cytosolic C‐terminal tail. In total, backbone resonances of 78 % of all residues were assigned for the Y4 receptor. Predictions of secondary structure based on backbone chemical shifts indicate that most residues from the TM regions adopt helical conformations, with exception of those around polar residues or prolines. However, the domain boundaries differ slightly from those predicted for homology models. We suggest that the obtained chemical shifts might be useful in assigning the full‐length receptor.     

Identification of a potent inhibitor of human dual-specific phosphatase, VHR, from computer-aided and NMR-based screening to cellular effects

Human vaccinia H1-related phosphatase (VHR) is a dual-specific phosphatase (DSPs) that plays an important role in the mitogen-activated protein (MAP) kinase cascade regulation. It is also a potential drug target for diseases that are related to immune response. By combining a virtual and NMR-based ligand-screening strategy, we successfully identified four VHR inhibitors, of which GATPT ((glucosamine-aminoethoxy)triphenyltin) can bind to VHR with a K(i) value of 2.54 muM. The putative binding mode of GATPT was constructed by a molecular docking simulation to provide structural insights into the ligand-binding mechanism. Furthermore, we found that this compound can significantly inhibit the dephosphorylation of the extracellular regulated kinases (ERKs), and c-Jun N-terminal kinases (JNKs) and block the G(1)-S phase transition in the cell cycle. Therefore, GATPT is a promising lead structure for designing more effective inhibitors of VHR.     

Insights into the Genetic Variations of Human Cytochrome P450 2C9: Structural Analysis,Characterization and Comparison

Cytochromes P450 (CYP) are one of the major xenobiotic metabolizing enzymes with increasing importance in pharmacogenetics. The CYP2C9 enzyme is responsible for the metabolism of a wide range of clinical drugs. More than sixty genetic variations have been identified in CYP2C9 with many demonstrating reduced activity compared to the wild-type (WT) enzyme. The CYP2C9*8 allele is predominantly found in persons of African ancestry and results in altered clearance of several drug substrates of CYP2C9. The X-ray crystal structure of CYP2C9*8, which represents an amino acid variation from arginine to histidine at position 150 (R150H), was solved in complex with losartan. The overall conformation of the CYP2C9*8-losartan complex was similar to the previously solved complex with wild type (WT) protein, but it differs in the occupancy of losartan. One molecule of losartan was bound in the active site and another on the surface in an identical orientation to that observed in the WT complex. However, unlike the WT structure, the losartan in the access channel was not observed in the *8 complex. Furthermore, isothermal titration calorimetry studies illustrated weaker binding of losartan to *8 compared to WT. Interestingly, the CYP2C9*8 interaction with losartan was not as weak as the CYP2C9*3 variant, which showed up to three-fold weaker average dissociation constant compared to the WT. Taken together, the structural and solution characterization yields insights into the similarities and differences of losartan binding to CYP2C9 variants and provides a useful framework for probing the role of amino acid substitution and substrate dependent activity.     

Fabrication of red, green, and blue organic light-emitting diodes using m-MTDATA as a common hole-injection layer

Organic light-emitting diodes (OLEDs) of metal-semiconductor-metal (MSM) structure have been fabricated by using m-MTDATA [4,4′,4′’-tris (3-methylphenylphenylamino) triphenylamine] as a hole-injection layer (HIL). The m-MTDATA is shown to be an effective hole-injecting material for the OLED, in that the insertion of m-MTDATA greatly reduces the roughness of anode surface, lowers the turn-on voltage, and increases the luminous efficiency. Red, green and blue OLEDs were fabricated, and their color coordinates in CIE chromaticity were found to be (0.600, 0.389), (0.240, 0.525) and (0.171, 0.171), respectively. The luminous efficiencies of the fabricated OLEDs were 1.4 lm/W at 106 cd/m² for red, 1.4 lm/W at 100 cd/m² for green, and 2.0 lm/W at 104 cd/m² for blue.     

生物发酵骨化二醇相关杂质的制备及结构鉴定

目的 对生物发酵的骨化二醇相关杂质进行分离纯化及结构鉴定。方法 生物发酵维生素D3的发酵液经过粗提后,采用高压制备色谱多次分离得到骨化二醇的相关杂质,使其达到一定纯度后,再通过DAD扫描、质谱和核磁鉴定化合物的结构。结果 纯化后得到纯度97.5%的杂质单品,结构鉴定此杂质化学结构式为(5Z,7E)-9,10-开环胆甾-5,7,10(19)-三烯-3b,24-二醇,也即24-羟基维生素D3,为骨化二醇的同分异构体。结论 本研究结果为生物发酵的骨化二醇原料药的质量标准的建立提供依据,同时为后续实验工作提供杂质对照品.