纳米材料层状双氢氧化物（LDH）对茄二十八星瓢虫dsECR致死效应的影响

纳米材料被视为重要的RNA干扰增效物质。层状双氢氧化物（LDH）是具有生物相容性的可降解纳米材料,可以显著提高dsRNA防控黄瓜花叶病毒的效果。为明确LDH能否提高昆虫RNA干扰的效果,以期促进RNA生物农药在农业害虫防治中的应用和推广,本研究选择茄二十八星瓢虫Henosepilachna vigintioctopunctata的EcR基因进行RNA干扰介导的致死效应研究。将LDH与dsEcR组装形成复合物LDH-dsEcR,利用琼脂糖凝胶电泳确定LDH-dsEcR的最佳装载比例。通过静置法检测LDH-dsEcR的稳定性,借助透射电子显微镜验证其组装状态。采用浸叶法测定LDH-dsEcR的增效性,使用体视镜观察其对试虫形态的影响。结果表明：LDH与dsEcR可形成稳定的复合物,其最佳装载比为16∶1。LDH对茄二十八星瓢虫无毒性,但经LDH与dsEcR处理,部分试虫虫体呈黑褐色,或无法蜕皮化蛹,或维持在预蛹状态。遗憾的是LDH-dsEcR复合物的增效作用不明显,推测昆虫肠道的酸碱度可能是影响LDH-dsRNA增效的因素之一。本研究首次为利用LDH研究昆虫RNA生物农药提供了可观的理论基础。     

Mg-Al layered double hydroxide intercalated with porphyrin anions: molecular simulations and experiments

Molecular modeling in combination with powder X-ray diffraction (XRD) provided new information on the organization of the interlayer space of Mg-Al layered double hydroxide (LDH) containing intercalated porphyrin anions [5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS)]. Anion-exchange and rehydration procedures were used for the preparation of TPPS-containing LDH with an Mg/Al molar ratio of 2. Molecular modeling was carried out in the Cerius² and Materials Studio modeling environment. Three types of models were created in order to simulate the experimental XRD patterns of LDH intercalates with a TPPS loading of 70–80% with respect to the theoretical anion exchange capacity (AEC). The models represent single-phase systems with a 100% TPPS loading in the interlayer space (Type 1) and models represent the coexistence of two phases corresponding to the total exchange from 75 to 92% (Type 2). To cover other possible arrangements, models with the coexistence of both TPPS and NO₃⁻ anions in the same interlayer space were calculated (Type 3). The models are described and compared with experimental data. In all cases, guest TPPS anions are tilted with respect to the hydroxide layers, and are horizontally shifted to each other by up to one-half of the TPPS diameter. According to the energy characteristics and simulated XRD, the most probable arrangement is of Type 2, where some layers are saturated with TPPS anions and others are filled with original NO₃⁻ anions.     

Novel organo-mineral phases obtained by intercalation of maleic anhydride-allyl ether copolymers into layered calcium aluminum hydrates

The work presented here deals with the intercalation of worm- and brush-shaped polycarboxylates (PC) into calcium aluminum layered double hydroxide (Ca-Al-LDH). The nanocomposite materials were obtained from tricalcium aluminate hydration in presence of polycarboxylate copolymers with different side chain lengths. As polycarboxylate compound, amphiphilic copolymers composed of maleic anhydride and α-allyl-ω-methoxy-poly(ethylene glycol) ether with side chain lengths of n = 7, 10, 34, 70 and 90 ethylene oxide units (EOUs) were chosen. These polymers possess a high side chain density due to strictly alternating copolymerization. Powder X-ray diffraction (XRD) of the synthesized Ca-Al-PC-LDH composites revealed that basal spacings (d-values) increase with the number n of EOUs in the side chain. An extremely high d-value of 4.85 nm was obtained for the polymer with n = 34 EOUs. According to elemental analysis data, the amounts of organic material present in the different composites were found to lie between 48 and 77 wt.%, respectively. Additionally, IR spectroscopy and thermogravimetric measurements were carried out in order to characterize the intercalates. The layered structure of the organo-mineral materials was confirmed by transmission electron microscopy (TEM).     

Structural investigation of porcine stomach mucin by X-ray fiber diffraction and homology modeling

The basic understanding of the three dimensional structure of mucin is essential to understand its physiological function. Technology has been developed to achieve orientated porcine stomach mucin molecules. X-ray fiber diffraction of partially orientated porcine stomach mucin molecules show d-spacing signals at 2.99, 4.06, 4.22, 4.7, 5.37 and 6.5 Å. The high intense d-spacing signal at 4.22 Å is attributed to the antiparallel β-sheet structure identified in the fraction of the homology modeled mucin molecule (amino acid residues 800–980) using Nidogen–Laminin complex structure as a template. The X-ray fiber diffraction signal at 6.5 Å reveals partial organization of oligosaccharides in porcine stomach mucin. This partial structure of mucin will be helpful in establishing a three dimensional structure for the whole mucin molecule.     

Layered double hydroxide nanoparticles as cellular delivery vectors of supercoiled plasmid DNA

We prepared stable homogeneous suspensions with layered double hydroxide (LDH) nanoparticles for in vitro gene delivery tests. The viability of HEK 293T cells in the presence of LDH nanoparticles at different concentrations was investigated. This revealed 50% cell viability at 500 microg/mL of LDH nanoparticles that is much higher than 50-100 microg/mL used for the delivery tests. The supercoiled pEF-eGFP plasmid (ca. 6100 base pairs) was mixed with LDH nanoparticle suspensions for anion exchange at a weight ratio of DNA/LDH between 1:25 and 1:100. In vitro experiments show that GFP expression in HEK 293T cells starts in the first day, reaches the maximum levels by the second day and continues in the third day. The GFP expression generally increases with the increase in DNA loading in DNA-LDH nanohybrids. However, the delivery efficiency with LDH nanoparticles as the agent is low. For example, the relative efficiency is 7%-15% of that of the commercial agent FuGENE 6. Three to 6% of total cells expressed GFP in an amount detectable by the FACS cytometry 2 days after transfection at 1 microg/mL of plasmid DNA with 25 microg/mL of LDH nanomaterial. The lower delivery efficiency could be attributed to the aggregation of LDH nanoparticles caused by the long-chain plasmid DNA.     

Direct electrochemistry and electrocatalysis based on a film of horseradish peroxidase intercalated into Ni-Al layered double hydroxide nanosheets

Positively charged Ni-Al layered double hydroxide nanosheets (Ni-Al LDHNS) have been used for the first time as matrices for immobilization of horseradish peroxidase (HRP) in order to fabricate enzyme electrodes for the purpose of studying direct electron transfer between the redox centers of proteins and underlying electrodes. X-ray diffraction (XRD) and high-resolution transmission electron microscopy (HRTEM) revealed that the HRP-Ni-Al LDHNS film had an ordered structure and that HRP was intercalated into Ni-Al LDHNS with a monolayer arrangement. Field emission scanning electron microscopy (FESEM) showed that the HRP-Ni-Al LDHNS film had a uniform, porous morphology. UV-vis spectroscopy indicated that the intercalated HRP retained its native structure after incorporation in the Ni-Al LDHNS film. The immobilized HRP in Ni-Al LDHNS on the surface of a glassy carbon electrode (GCE) exhibited good direct electrochemical and electrocatalytic responses to the reduction of hydrogen peroxide (H(2)O(2)) and trichloroacetic acid (TCA). The resulting H(2)O(2) biosensor showed a wide linear range from 6.00x10(-7)M to 1.92x10(-4)M, low detection limit (4.00x10(-7)M) and good stability. The results show that Ni-Al LDHNS provide a novel and efficient platform for the immobilization of enzymes and realizing direct electrochemistry and that the materials have potential applications in the fabrication of third-generation biosensors.     

Acetan:glucomannan interactions--a molecular modeling study

X-ray fiber diffraction patterns from deacylated acetan and glucomannan (konjac mannan) blends are diagnostic of good orientation and modest polycrystallinity. The meridional reflection on the sixth layer line suggests that the binary complex is a 6-fold helix of pitch 55.4 A. A molecular modeling study incorporating this information reveals that a double helix in which one strand is acetan and the other glucomannan is stereochemically feasible. While the backbone and side groups are sufficiently flexible to allow the chains to associate with the same or opposite polarity, the parallel model is superior in terms of unit cell packing. The results are compatible with the observed synergy; namely the weak gelation behavior of the complex. The molecular model can be generalized for the binary system when acetan is replaced by xanthan or glucomannan by galactomannan.     

Molecular modeling of flavonoids that inhibits xanthine oxidase

The inhibition of xanthine oxidase activity by various flavonoids was assessed. All of the tested flavonoids were competitive inhibitors, and from the kinetic analysis suggested that flavonoids bind to the reactive site. To further understand the stereochemistry between these flavonoids and xanthine oxidase, structure-based molecular modeling was performed. Apigenin was the most potent inhibitor which showed the most favorable interaction in the reactive site. The bicyclic benzopyranone ring of apigenin stacked with phenyl of Phe 914, and the phenolic group stretched to the space surrounding with several hydrophobic residues. Quercetin and myricetin composed a 3-hydroxyl group on benzopyranone which resulting in reduction of binding affinity. The phenolic group of genistein positioned in opposite orientation comparison with apigenin, and resulted in a weaker interaction with xanthine oxidase. Isovitexin showed the weakest inhibitory effect among the compounds tested. The bulky group of sugar in isovitexin may hamper its interaction with xanthine oxidase.     

Molecular modeling of SWR-0342SA, a beta3-selective agonist, with beta1- and beta3-adrenoceptor

The molecular dynamics (MD) simulations study in the formation of the complex between compound SWR-0342SA and beta-ARs suggested that upon binding SWR-0342SA stimulates receptor activation through residues network (Asp104, Leu335 in beta(1)-AR; Asp117, Ser209, Leu303, Ser191 in beta(3)-AR) in an active conformation state. The models suggest that the structural origin of the selectivity of SWR-0342SA to beta(3)-AR vs. beta(1)-AR comes from the following results: (a) the tight interaction between the agonist and the TMs 3, 5, 6 and 2 nd EC loop. Asp117 interacts with the cationic amino group of the agonist molecule. (b) Additional contacts are done with Ser209, Leu303 and Ser191. These results are in good agreement with the binding affinities (pKi values) of SWR-0342SA to beta-AR family expressed in recombinant mammalian cells.     

Structure analysis of montmorillonite intercalated with rhodamine B: modeling and experiment

The intercalation process and the structure of montmorillonite intercalated with [rhodamine B]+ cations have been investigated using molecular modeling (molecular mechanics and molecular dynamics simulations), X-ray powder diffraction and IR spectroscopy. The structure of the intercalate depends strongly on the concentration of rhodamine B in the intercalation solution. The presence of two phases in the intercalated structure was revealed by modeling and X-ray powder diffraction: (i) phase with basal spacing 18 A and with bilayer arrangement of guests and (ii) phase with average basal spacing 23 A and with monolayer arrangement of guests. In both phases the monomeric and dimeric arrangement can coexist in the interlayer space. Three types of dimers in the interlayer structure have been found by modeling: (i) H-dimer (head-to-head arrangement) present in the 18 A phase, (ii) sandwich type of the head-to-tail arrangement (present in the 23 A phase) and (iii) J-dimer (head-to-tail arrangement) present in the 23 A phase. Figure Montmorillonite intercalated with rhodamine B cations. On the left: phase 18 A, bilayer dimeric arrangement of guests (H-dimers). On the right: phase 23 A, monolayer arrangement of guests prepared using intercalation solution with a low concentration of rhodamine B     

Molecular modeling and dynamics simulations of PNP from Streptococcus agalactiae

This work describes for the first time a structural model of purine nucleoside phosphorylase from Streptococcus agalactiae (SaPNP). PNP catalyzes the cleavage of N-ribosidic bonds of the purine ribonucleosides and 2-deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate. This enzyme is a potential target for the development of antibacterial drugs. We modeled the complexes of SaPNP with 15 different ligands in order to determine the structural basis for the specificity of these ligands against SaPNP. The application of a novel empirical scoring function to estimate the affinity of a ligand for a protein was able to identify the ligands with high affinity for PNPs. The analysis of molecular dynamics trajectory for SaPNP indicates that the functionally important motifs have a very stable structure. This new structural model together with a novel empirical scoring function opens the possibility to explorer larger library of compounds in order to identify the new inhibitors for PNPs in virtual screening projects.     

Molecular modeling of the three-dimensional structure of GLP-1R and its interactions with several agonists

Glucagon-like peptide-1 receptor (GLP-1R) is a promising molecular target for developing drugs treating type 2 diabetes. We have predicted the complete three-dimensional structure of GLP-1R and the binding modes of several GLP-1R agonists, including GLP-1, Boc5, and Cpd1, through a combination of homology modeling, molecular docking, and long-time molecular dynamics simulation on a lipid bilayer. Our model can reasonably interpret the results of a number of mutation experiments regarding GLP-1R as well as the successful modification to GLP-1 by Liraglutide. Our model is also validated by a recently revealed crystal structure of the extracellular domain of GLP-1R. An activation mechanism of GLP-1R agonists is proposed based on the principal component analysis and normal mode analysis on our predicted GLP-1R structure. Before the complete structure of GLP-1R is determined through experimental means, our model may serve as a valuable reference for characterizing the interactions between GLP-1R and its agonists. Figure Comparison of our predicted model of rGLP-1R (left) with the recently revealed crystal structure of hGLP-1R (right)     

Development of a high analytical performance amperometric glucose biosensor based on glucose oxidase immobilized in a composite matrix: layered double hydroxides/chitosan

This paper aimed at showing the interest of the composite material based on layered double hydroxides (LDHs) and chitosan (CHT) as suitable host matrix likely to immobilize enzyme onto electrode surface for amperometric biosensing application. This hybrid material combined the advantages of inorganic LDHs and organic biopolymer, CHT. Glucose oxidase (GOD) immobilized in the composite material maintained its activity well as the usage of glutaraldehyde was avoided. The process parameters for the fabrication of the enzyme electrode and various experimental variables such as pH, applied potential and temperature, were explored for optimum analytical performance of the enzyme electrode. The enzyme electrode provided a linear response to glucose over a concentration range of 1 x 10(-6) to 3 x 10(-3) M with a high sensitivity of 62.6 mA M(-1) cm(-2) and a detection limit of 0.1 muM based on the signal-to-noise ratio of 3.     

Molecular cloning, functional expression, and molecular modeling of bothrostatin, a new highly active disintegrin from Bothrops jararaca venom

Disintegrins are among the most potent antagonists of several integrins. A cDNA encoding a novel disintegrin, bothrostatin, was cloned from a Bothrops jararaca cDNA library. The precursor of bothrostatin contains a pro, a metalloproteinase, and an RGD-disintegrin domain. The disintegrin domain expressed in Escherichia coli showed high inhibitory activity on collagen-induced platelet aggregation (IC(50) of 12nM), and thus it can be used as a useful tool for studies of integrin-ligand interaction. Furthermore, we used the comparative modeling approach to obtain a model of the 3D structure of bothrostatin. Our results suggest that bothrostatin adopts a globular, closed structure in solution. The RGD motif is exposed to the solution by the loop formed by residues 45-59 and is very close to the C-terminal domain forming a finger-like structure. The proximity of the RGD loop and the C-terminal residues, which is maintained by the Cys47-Cys66 bond, suggests that the C-terminal residues are involved in the ability of bothrostatin to interact with its ligands.     

Molecular modeling of cobalt(II) hyaluronate

Structural data for complexes of hyaluronic acid and 3d metals(II) of the fourth group of the periodic table are lacking. A combined QM/MM method was used to solve the structure of the first coordination sphere around the cobalt(II) ion. Some available experimental data were compared with the results obtained via computation and were found to be in good agreement. Our results open the way for using molecular modeling to solve the structure of other metal(II) hyaluronates.     

Molecular modeling and dynamics studies of purine nucleoside phosphorylase from Bacteroides fragilis

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides, except adenosine, to generate ribose 1-phosphate and the purine base. This work describes for the first time a structural model of PNP from Bacteroides fragilis (Bf). We modeled the complexes of BfPNP with six different ligands in order to determine the structural basis for specificity of these ligands against BfPNP. Comparative analysis of the model of BfPNP and the structure of HsPNP allowed identification of structural features responsible for differences in the computationally determined ligand affinities. The molecular dynamics (MD) simulation was assessed to evaluate the overall stability of the BfPNP model. The superposition of the final onto the initial minimized structure shows that there are no major conformational changes from the initial model, which is consistent with the relatively low root mean square deviation (RMSD). The results indicate that the structure of the model was stable during MD, and does not exhibit loosely structured loop regions or domain terminals.     

Molecular approaches for structural characterization of Bothrops L-amino acid oxidases with antiprotozoal activity: cDNA cloning, comparative sequence analysis, and molecular modeling

Two l-amino acid oxidases (LAAOs) were identified by random sequencing of cDNA libraries from the venom glands of Bothrops moojeni(BmooLAAO) and Bothrops jararacussu(Bjussu LAAO). Phylogenetic analysis involving other SV-LAAOs showed sequence identities within the range 83-87% being closely related to those from Agkistrodon and Trimeresurus. Molecular modeling experiments indicated the FAD-binding, substrate-binding, and helical domains of Bmoo and Bjussu LAAOs. The RMS deviations obtained by the superposition of those domains and that from Calloselasma rhodostoma LAAO crystal structure confirm the high degree of structural similarity between these enzymes. Purified BjussuLAAO-I and BmooLAAO-I exhibited antiprotozoal activities which were demonstrated to be hydrogen-peroxide mediated. This is the first report on the isolation and identification of cDNAs encoding LAAOs from Bothrops venom. The findings here reported contribute to the overall structural elucidation of SV-LAAOs and will advance the understanding on their mode of action.     

Surface and interlayer structure of vermiculite intercalated with methyl viologen

Molecular modeling using empirical force field revealed the differences between the surface and interlayer arrangement of the dye guest molecules in vermiculite intercalated with the divalent methyl viologen cation (MV²⁺). Conformation and anchoring of MV²⁺ cations on the silicate layer in the interlayer space of vermiculite host structure is different from that on the crystal surface. A preferential position has been found for the anchoring of guests on the silicate layer. Anyway the arrangement of guests in the interlayer space as well as on the crystal surface exhibits a high degree of disorder due to a certain flexibility in guest molecules arrangement and first of all due to the presence of water molecules in the interlayer space. The presence of water disturbs not only the regularity in guest positions and orientations but also in conformation of guest molecules in the interlayer space of the host structure.     

Synthesis, characterization, structure, molecular modeling studies and biological activity of sterically crowded Pt(II) complexes containing bis(imidazole) ligands

The syntheses, structures and biological evaluation of a series of cisplatin-like complexes containing bis(imidazole) derivatives - the so-called Joseph ligands - are described. Their cytotoxicity is discussed in terms of their polar surface area, rate of aquation, and lipophilicity. The X-ray crystal structure of the platinum diiodido derivative of dimethyl 2-(di(1H-imidazol-2-yl)methyl)malonate) is reported and compared to those of related systems. Molecular modeling studies are focused on the hydrogen bonding properties of such systems, and their relevance to antitumor activity.     

Molecular modeling of the bacterial outer membrane receptor energizer, ExbBD/TonB, based on homology with the flagellar motor, MotAB

The MotA/MotB proteins serve as the motor that drives bacterial flagellar rotation in response to the proton motive force (pmf). They have been shown to comprise a transmembrane proton pathway. The ExbB/ExbD/TonB protein complex serves to energize transport of iron siderophores and vitamin B₁₂ across the outer membrane of the Gram-negative bacterial cell using the pmf. These two protein complexes have the same topology and are homologous. Based on molecular data for the MotA/MotB proteins, we propose simple three-dimensional channel structures for both MotA/MotB and ExbB/ExbD/TonB using modeling methods. Features of the derived channels are discussed, and two possible proton transfer pathways for the ExbBD/TonB system are proposed. These analyses provide a guide for molecular studies aimed at elucidating the mechanism by which chemiosmotic energy can be transferred either between two adjacent membranes to energize outer membrane transport or to the bacterial flagellum to generate torque.