A novel computational method for predicting the transmembrane structure of G-protein coupled receptors: application to human C5aR and C3aR

A novel algorithm was applied to the sequences of bacteriorhodopsin (BRh), of rhodopsin (Rh), and of the two human anaphylatoxin receptors, C5a-receptor (hC5aR) and C3a-receptor (hC3aR), that predicts their transmembrane domains (TMD) according to energy criteria alone, on the basis of their sequences and a template structure for each. Two consecutive criteria were applied for the predictions: the first is hydrophobicity of a sequence of residues, which determines the candidate stretches of residues that form one of the transmembrane helices. The second criterion is an energy function composed of inter residue contact energies, of hydrophobic contributions due to membrane exposure and of the interactions of a few residues with the phospholipid head groups. The sequence of candidate residues for each helix is longer than that of the template, and is finally determined by threading each of the candidate stretches on each of the template helices and evaluating the energy for all possible configurations. Contact energies between residues were taken from a database (Miyazawa S and Jernigan RL (1996) J Mol Biol 256 623-44). The algorithm predicts well the TMD structure of BRh based on its own template, and the TMD structure of Rh conforms well with the model of Baldwin et al (Baldwin JM Schertler GFX and Unger VM (1997) J Biol Chem 272 144-64). Results for the construction of the TMD of hC5aR and hC3aR were compared, employing the template structure of Rh. Most of the results for these receptors are in accord with alignments and with mutation experiments on hC5aR and hC3aR. The predictions may serve as a basis for future mutagenesis experiments of these receptors.     

Activating mutations of the lutropin choriogonadotropin receptor in precocious puberty

The human receptor for lutropin and chorionic gonadotropin (LHR) is a member of the G-protein-coupled receptor superfamily and plays a key role in normal and abnormal reproductive physiology. Naturally occurring mutations of the LHR gene that lead to hormone-independent signaling are associated with abnormal Leydig cell growth and precocious puberty in boys. These mutations affect 13 different residues in the LHR, with the majority clustered in the cytoplasmic half of transmembrane helix 6. Germline LHR mutations that produce constitutive activation of the Gs pathway are found in cases of Leydig cell hyperplasia, while a unique somatic LHR mutation capable of activating both the Gs and Gq signaling pathways has been identified in Leydig cell tumors. The newly determined crystal structure of bovine rhodopsin provides a useful framework for interpreting the effects of these disease-associated LHR mutations in the context of conserved structural motifs in helix 3 and helix 7 that are known to be involved in receptor activation. As with rhodopsin, conformational signaling appears to depend on the rearrangement of key electrostatic, hydrogen-bond, and hydrophobic interactions that normally serve to stabilize the inactive LHR conformation.     

Unmasking different constitutive activity of four chemoattractant receptors using Na+ as universal stabilizer of the inactive (R) state.

Neutrophils express receptors for the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) complement C5a, leukotriene B4 (LTB4) and platelet-activating factor (PAF). The aim of this study was to analyze the constitutive activity of chemoattractant receptors by studying binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to the G-protein Gi alpha 2 beta 1 gamma 2 expressed in Sf9 cells. We used Na+ as modulator of constitutive activity because there are no known inverse agonists for the C5a receptor (C5aR), LTB4 receptor (BLTR) and PAF receptor (PAFR). In the absence of NaCl, PAF and LTB4 exhibited larger relative stimulatory effects on GTP gamma S binding than fMLP and C5a. NaCl showed larger inhibitory effects on basal GTP gamma S binding in membranes expressing the formyl peptide receptor (FPR) and C5aR than in membranes expressing BLTR and PAFR. The order of potency of NaCl at inhibiting basal GTP gamma S binding was FPR > C5aR approximately BLTR > PAFR. As a result of the inhibitory effect of NaCl on basal GTP gamma S binding, the relative stimulatory effects of agonists were increased. By quantitatively analyzing the expression levels of chemoattractant receptors and Gi alpha 2 and the stoichiometry of receptor/G-protein coupling we obtained no evidence for structural instability of constitutively active receptors and catalytical G-protein activation. Taken together, the FPR and C5aR exhibit higher constitutive activity than the BLTR and PAFR. Na+ acts as a universal stabilizer of the inactive (R) state in chemoattractant receptors. The different potencies of NaCl at suppressing basal G-protein activity with different receptors indicate that chemoattractant receptors differ from each other in their Na(+)-affinity.     

The effect of thermal annealing on dopant site choice in conjugated polymers

Solution-processed organic electronic devices often consist of layers of polar and non-polar polymers. In addition, either of these layers could be doped with small molecular dopants. It is extremely important for device stability to understand the diffusion behavior of these molecular dopants under the thermal stress and whether the dopants have preference for the polar or the non-polar polymer layers. In this work, a widely used molecular dopant 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (F4TCNQ) was chosen to investigate dopant site preference upon thermal annealing between the polar thiophene poly(thiophene-3-[2-(2-methoxy-ethoxy)ethoxy]-2,5-diyl) (S–P3MEET) and non-polar thiophene poly(3-hexylthiophene) (P3HT). F4TCNQ is able to p-type dope both P3HT and S–P3MEET. Further doping studies of S–P3MEET using near edge X-ray absorption fine structure spectroscopy, conductivity measurements and atomic force microscopy show that the F4TCNQ additive competes for doping sites with the covalently attached dopants on the S–P3MEET. Calorimetry measurements reveal that the F4TCNQ interacts strongly with the side-chains of the S–P3MEET, increasing the melting temperature of the side-chains by 30 °C with 5 wt% dopant loading. Next, the thermal stability of doping in the polar/non-polar (S–P3MEET/P3HT) bilayer architectures was investigated. Steady-state absorbance and fluorescence results show that F4TCNQ binds much more strongly in S–P3MEET than P3HT and very little F4TCNQ is found in the P3HT layer after annealing. In combination with reflectometry measurements, we show that F4TCNQ remains in the SP3MEET layer with annealing to 210 °C even though the sublimation temperature for neat F4TCNQ is about 80 °C. In contrast, F4TCNQ slowly diffuses out of P3HT at room temperature. We attribute this difference in binding the F4TCNQ anion to the ability of the ethyl-oxy side-chains of the S–P3MEET to orient around the charged dopant molecule and thereby to stabilize its position. This study suggests that polar side-chains could be engineered to increase the thermal stability of molecular dopant position.     

纵向导电翼片屏蔽螺旋线结构的色散计算

本文采用螺旋带模型,综合考虑了介质、纵向导电翼片屏蔽筒及导丝尺寸对结构色散的影响。导出了设计公式,与实测结果相当符合。 当R/S1.1,S/1.5时,相速理论值的误差在5％以下。提供了通用设计计算曲线,可供工程设计之用。     

Disulfide bond structure and accessibility of cysteines in the ectodomain of the cholecystokinin receptor: specific mono-reactive receptor constructs examine charge-sensitivity of loop regions

Cysteine residues play a unique role in structural analysis. We examined endogenous cysteine residues in the cholecystokinin receptor to determine participation in disulfide bonds and accessibility to methanethiosulfonate (MTS) reagents. Bonds linking Cys114 to Cys196 and Cys18 to Cys29 were demonstrated, with the first functionally important and the amino-terminal bond having no apparent function. Cys94, in the second transmembrane segment, was also accessible. Mutation of this residue to serine (C94S) was key for establishing a null cysteine-reactive pseudo-wild type receptor that could act as a template for insertion of a reactive cysteine (N102C, A204C, and T341C). Modification of T341C with a negatively charged MTS reagent reduced CCK agonist binding, while this binding was enhanced by a positively charged MTS reagent. This pattern was repeated in mutants having the same residue directly replaced with a charged residue.     

参麦注射液与人血清白蛋白相互作用的多光谱研究

参麦注射液是一种中药复合物，广泛应用于癌症患者的辅助治疗中。在生理（PH7.4）环境下，通过荧光光谱与紫外吸收光谱研究了参麦注射液与人血清白蛋白的相互作用。荧光光谱和紫外吸收光谱实验结果表明，参麦注射液能够有效地引起人血清白蛋白的内荧光源淬灭，其淬灭机理为动态淬灭。利用Stern-Volmer方程对荧光光谱数据进行分析，得到不同温度（296，303，310 K）下的结合常数（KA）。通过Van't Hoff方程计算出热力学参数（△G0，△H 0，△S 0），该结果表明疏水力是人血清白蛋白与参麦注射液结合时的主要相互作用力及结合过程是一个自发过程。此外，同步荧光光谱实验结果表明，当参麦注射液与人血清白蛋白结合时，主要结合点位于酪氨酸残基，且引起了人血清白蛋白的结构变化。     

Droplet and Fluid Gating by Biomimetic Janus Membranes

The ability to gate (i.e., allow or block) droplet and fluid transport in a directional manner represents an important form of liquid manipulation and has tremendous application potential in fields involving intelligent liquid management. Inspired by passive transport across cell membranes which regulate permeability by transmembrane hydrophilic/hydrophobic interactions, macroscopic hydrophilic/hydrophobic Janus‐type membranes are prepared by facile vapor diffusion or plasma treatments for liquid gating. The resultant Janus membrane shows directional water droplet gating behavior in air‐water systems. Furthermore, membrane‐based directional gating of continuous water flow is demonstrated for the first time, enabling Janus membranes to act as facile fluid diodes for one‐way flow regulation. Additionally, in oil‐water systems, the Janus membranes show directional gating of droplets with integrated selectivity for either oil or water. The above remarkable gating properties of the Janus membranes could bring about novel applications in fluid rectifying, microchemical reaction manipulation, advanced separation, biomedical materials and smart textiles.     

In vitro binding characteristics and affinity for sulfatide of Escherichia coli STb enterotoxin.

It has previously been demonstrated that sulfatide (3'-sulfogalactosyl-ceramide), present at the surface of epithelial cells of the small intestine of pigs, interacts with the thermostable enterotoxin b (STb) produced by ETEC, and that this molecule is implicated in the mechanism of action of the toxin. However, few things are known about the affinity and physical characteristics of the interaction between these two macromolecules. In this study, using a microtiter plate binding assay (MPBA), we showed that STb toxin has a strong specificity for sulfatide and that this binding is dose-dependent and saturable. A very weak binding occurred with galactosyl-ceramide whereas attachment to 3'-sulfolactosyl-ceramide corresponded to 76% of the binding to sulfatide. STb toxin was shown to possess a lectin-like property; a significative binding was observed when a terminal beta-galactose was present in the glycosphingolipids tested and an increased binding was observed in presence of a sulfate group in position 3 on the galactose. These findings suggest that a sulfated galactosyl residue seems to represent the epitope recognized by the toxin. The reaction between sulfatide and STb toxin is also time and temperature dependent and is not affected by pH. The interaction was not inhibited by free sugars, sulfated polymers, glycolipids or free ions, but was partly inhibited by high concentrations of charged sugars. STb-sulfatide binding process is a low affinity interaction, as demonstrated by the determined Kd of 2-6 +/- 1.5 microM.     

S、C、X波段螺旋线慢波结构的宽频带行波管研究

为满足现代信息化战争对宽带行波管的需求,对S、C、X波段螺旋线行波管慢波结构的性能进行了研究分析.为适应宽频带要求,选用了带有T形加载翼片以及品形氮化硼夹持杆的螺旋线慢波结构;通过采用动态速度渐变技术,得到了较高的电子效率,并根据仿真计算结果成功研制出了S、C、X波段螺旋线行波管样管.     

Scanning mutagenesis studies of the M1 muscarinic acetylcholine receptor

Following the solution of the structure of bovine rhodopsin by X-ray crystallography, it has been possible to build an improved homology model of the M(1) muscarinic acetylcholine receptor. This has been used to interpret the outcome of an extensive series of scanning and point mutagenesis studies on the transmembrane domain of the receptor. Potential intramolecular interactions enhancing the stability of the protein fold have been identified. The residues contributing to the binding site for the antagonist, N-methylscopolamine, and the agonist, acetylcholine have been mapped. The positively charged headgroups of these ligands appear to bind in a charge-stabilized aromatic cage formed by amino acid side chains in transmembrane (TM) helices 3, 6, and 7, while residues in TM 4 may participate in a peripheral docking site. Closure of the cage around the headgroup of acetylcholine may help to transduce binding energy into receptor activation, possibly disrupting a set of Van der Waals interactions between a set of residues underlying the binding site which help to constrain the receptor to the inactive state, in the absence of agonist. This may trigger the reorganization of a hydrogen bonding network between highly conserved residues in the core of the receptor, whose integrity is crucial for activation.     

Bioengineered Dual-Targeting Protein Nanocage for Stereoscopical Loading of Synergistic Hydrophilic/Hydrophobic Drugs to Enhance Anticancer Efficacy

A biocompatible and modifiable protein nanocarrier is a promising candidate for tumor targeted drug delivery. However, it is challenging to effectively load hydrophobic drugs, not to mention to upload both hydrophilic and hydrophobic drugs on one protein nanocarrier. Here, an amphiphilic multi-drug loading protein nanocage (Am-PNCage) is presented which is generated by replacing the fifth helix of human H-ferritin (HFn) subunit with a functional motif composed of hydrophobic–hydrophilic-RGD peptides. The Am-PNCage possesses a dual targeting property resulting from the intrinsic CD71 targeting ability of HFn and the integrin α vβ3 targeting ability of displayed RGD peptides. Through the hydrophilic drug entry channel in the protein nanocage and hydrophobic peptides displayed on the outer surface, amphiphilic epirubicin (132)/camptothecin (50) are stereoscopically loaded into the inner cavity/outer protein shell, respectively, for one Am-PNCage, exhibiting cascade drug release pattern. The dual-targeted Am-PNCage promotes the loaded drugs penetrating various 3D tumor models in vitro, as well as traversing the brain blood barrier and accumulating in brain tumors in vivo. Moreover, the drug loaded Am-PNCage shows reduced side effects and significantly enhances synergistic efficacy against brain tumor, metastatic liver cancers, and drug resistant breast tumor. Thus, the Am-PNCage represents a novel promising protein nanocarrier for targeted combination chemotherapy.     

Limitations on the noise figure of microwave amplifiers of the beam type

A general model is developed for microwave amplifiers of the beam type based on the usual assumptions of a small signal, one-dimensional, single velocity flow of the electrons in the beam. The model includes arbitrary noise smoothing schemes and all amplifiers whose input and output structures have no direct rf connection; the rf coupling is provided solely by means of the modulated electron beam. A klystron, a space-charge wave amplifier, and a traveling-wave tube with a severed helix (or a helix with strong localized attenuation) are all special cases of this model. It is shown that the minimum noise figure of such an amplifier is of the form Fmin=1+2Π/kT|K| [S(z0,w)- K/|K| Π(z0,w) where S(z0,w) and Π(z0,w) are parameters of the beam noise expressed at a cross section z0slightly beyond the potential minimum in the electron gun, k is Boltzmann's constant, T is the temperature of the circuit, and K is a constant dependent upon the particular structures used. The dependence of K upon the microwave structures employed is discussed under restrictive assumptions about the loss in the rf structure of the amplifier. Two cases are possible. In the first case, the minimum possible value of K is unity. In the second, K is less than unity. It is shown, however, that in the latter case the available gain, G0, of the amplifier is given by G0=1/1-K     

NIR‐Triggered Synergic Photo‐chemothermal Therapy Delivered by Reduced Graphene Oxide/Carbon/Mesoporous Silica Nanocookies

A novel photo‐responsive drug carrier that doubles as a photothermal agent with a nanocookie‐like structure is constructed by coating amorphous carbon on a mesoporous silica support self‐assembled on a sheet of reduced graphene oxide. With a large payload (0.88 mmolg^?1) of a hydrophobic anticancer drug, (S)‐(+)‐camptothecin (CPT), nanocookies simultaneously provide a burst‐like drug release and intense heat upon near‐infrared exposure. Being biocompatible yet with a high efficiency for cell uptake, nanocookies have successfully eradicated subcutaneous tumors in 14 days following a single 5 min NIR irradiation without distal damage. These results demonstrate that the nanocookie is an excellent new delivery platform for local, on‐demand, NIR‐responsive, combined chemotherapy/hyperthermia for tumor treatment and other biomedical applications.     

Analysis of Helix Slow Wave Structure for High Efficiency Space TWT

This paper describes the analysis of helix slow-wave structure (SWS) for a high efficiency space traveling wave tube that is carried out using Ansoft HFSS and CST microwave studio, which is a 3D electromagnetic field simulators. Two approaches of simulating the dispersion and impedance characteristics of the helix slow wave structure have been discussed and compared with measured results. The dispersion characteristic gives the information about axial propagation constant (Beta). Which in turn yields the phase velocity at a particular frequency. The dispersion and impedance characteristics can be used in finding the pertinent design parameters of the helix slow-wave structure. Therefore a new trend has been initiated at CEERI to use Ansoft HFSS code to analysis of the helix slow wave structure in its real environment. The analysis of the helix SWS for Ku-band 140W space TWT has been carried out and compared with experimental results, and found is close agreement.     

Bacteriorhodopsin in a periodic boundary water-vacuum-water box as an example towards stable molecular dynamics simulations of G-protein coupled receptors.

This study presents an optimised set-up for molecular dynamics (MD) simulations of G-protein coupled receptors (GPCR). Such simulations are complicated because (1) the experimental template structure for GPCRs (bovine rhodopsin) is of low resolution, (2) the receptor surroundings are irregular (water exposed loops vs. lipid exposed transmembrane regions) and (3) the protonation and solvation states of the inner core receptor residues are unknown. We compared various simulations of the experimentally derived and refined electron density structure of the seven helical transmembrane protein bacteriorhodopsin (bR) under different MD conditions using AMBER 4.1. Our results demonstrate that the optimal MD set-up with minimal computational effort is a periodic boundary (PB) box containing two water shells solvating the extra- and intracellular loops separated by a vacuum layer surrounding the helical transmembrane (TM) regions. It was found that the vacuum layer and water layers are stable under periodic boundary conditions during at least 1 ns of MD simulation. In this set-up the bR structure is stable without any restraints. The average bR structure during the last 500 ps of the MD run has an excellent RMSD value relative to the original bR structure (RMSD = 1.66 A for the C alpha atoms within the TM domains) and shows a very high helical stability within the TM regions (88.8% helix). The use of this MD set-up for simulations of GPCRs is discussed.     

毫米波螺旋线行波管慢波系统高频损耗

基于夹持杆分层螺旋带模型和三维电磁场分析研究了毫米波螺旋线行波管慢波系统的导体和介质损耗。螺旋带模型中介质损耗考虑为纵向传播常数的虚部,给出螺旋带中电磁场的解析解,导体损耗由螺旋线和管壳表面的面电流不连续性获得。三维电磁场分析通过本征模法,求解单周期结构的品质因数和周期储能获得有限导电率导体和夹持杆陶瓷损耗角带来的慢波系统高频损耗。结果表明,毫米波段螺旋线的导体损耗和夹持杆的介质损耗远大于管壳导体损耗,介质损耗与陶瓷损耗角呈线性关系,对高频损耗的影响不可忽略。     

Optical Gating of Photosensitive Synthetic Ion Channels

4‐oxo‐4‐(pyren‐4‐ylmethoxy) butanoic acid is used as a photolabile protecting group to show the optical gating of nanofluidic devices based on synthetic ion channels. The inner surface of the channels is decorated with monolayers of photolabile hydrophobic molecules that can be removed by irradiation, which leads to the generation of hydrophilic groups. This process can be exploited in the UV‐light‐triggered permselective transport of ionic species in aqueous solution through the channels. The optical gating of a single conical nanochannel and multichannel polymeric membranes is characterised experimentally and theoretically by means of current–voltage and selective permeation measurements, respectively. It is anticipated that the integration of nanostructures into multifunctional devices is feasible and can readily find applications in light‐induced controlled release, sensing, and information processing.     

Conducting Helical Structures from Celery Decorated with a Metallic Conjugated Polymer Give Resonances in the Terahertz Range

A method to decorate cellulose‐based helices retrieved from the plant celery with a conductive polymer is proposed. Using a layer‐by‐layer method, the decoration of the polyanionic conducting polymer poly(4‐(2,3‐dihydrothieno [3,4‐b]‐[1,4]dioxin‐2‐yl‐methoxy)‐1‐butanesulfonic acid (PEDOT‐S) is enhanced after coating the negatively charged cellulose helix with a polycationic polyethyleneimine. Microscopy techniques and two‐point probe are used to image the structure and measure the conductivity of the helix. Analysis of the optical and electrical properties of the coated helix in the terahertz (THz) frequency range shows a resonance close to 1 THz and a broad shoulder that extends to 3.5 THz, consistent with electromagnetic models. Moreover, as helical antennas, it is shown that both axial and normal modes are present, which are correlated to the orientation and antenna electrical lengths of the coated helices. This work opens the possibility of designing tunable terahertz antennas through simple control of their dimensions and orientation.     

Effects of Sulfur Passivation on Germanium MOS Capacitors With HfON Gate Dielectric

In this letter, we study the effects of sulfur (S) passivation, using aqueous ammonium sulfide ((NH₄)₂S), on germanium (Ge) MOS capacitors with sputtered HfON as gate dielectric and TaN as metal-gate electrode. Compared with control samples, the S passivation can effectively reduce both equivalent oxide thickness and interface-state density. X-ray-photoelectron-spectroscopy analysis shows that (NH₄)₂S treatment can reduce the Ge-O bonds on Ge surface. The thermal stability of the S passivation under different postmetal-annealing temperatures was also examined, and it was found that samples with (NH₄)₂S treatment exhibit stable Ge/high-fc interface upon 550-deg C postmetal-deposition annealing, whereas interface quality degrades for those samples without S passivation.