Chromatographic separation and C-13 NMR characterization of aromatic diester-diacids isomers

Reverse phase high performance liquid chromatography (HPLC) was used to separate and quantify aromatic diester-diacids isomers which arise from the opening selectivity of anhydride rings towards methanol. C-13 NMR spectroscopy was a supplementary tool to characterize the isomer structure. It was found that a meta-position attack is slightly preferred in pyromellitic dianhydride (PMDA), while the preferred position of an attack in bridged dianhydrides is determined by the chemical nature (donors or accepters) of the bridged group. The stronger its electron-withdrawing abilities, the lower the probability of a meta-position attack.

AN EXPERT SYSTEM FOR SOLVENT-EXTRACTION OF RARE-EARTHS

An expert system for solvent extraction of rare earths has been developed using LISP. The goal of this project was to mimic the chemists' inferential abilities to assist in the process of solvent extraction of rare earths. The system includes frequently used extractants, separation of specific rare earths, recommendation of procedures for the separation of mixtures of rare earths using (2-ethylhexyl)phosphonic acid 2-ethylhexyl monoester, selection of parameters for counter-current extraction and methods for evaluation of the technique, and the economics of the processing. The expert system runs on an IBM-PC/XT.

Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50 degrees C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.

Identification and characterization of a virulence-associated protease from a pathogenic Pseudomonas fluorescens strain

Pseudomonas fluorescens is an aquaculture pathogen that can infect a number of fish species. The virulence mechanisms of aquatic P. fluorescens remain largely unknown. Many P. fluorescens strains are able to secrete an extracellular protease called AprX, yet no AprX-like proteins have been identified in pathogenic P. fluorescens associated with aquaculture. In this study, a gene encoding an AprX homologue was cloned from TSS, a pathogenic A fluorescens strain isolated from diseased fish. In TSS, AprX is secreted into the extracellular milieu, and the production of AprX is controlled by growth phase and calcium. Mutation of aprX has multiple effects, which include impaired abilities in interaction with cultured host cells, adherence to host mucus, modulation of host immune response, and dissemination and survival in host tissues and blood. Purified recombinant AprX exhibits apparent proteolytic activity, which is optimal at pH 8.0 and 50 degrees C. The protease activity of recombinant AprX is enhanced by Ca2+ and Zn2+ and reduced by Co2+. Cytotoxicity analyses showed that purified recombinant AprX has profound toxic effect on cultured fish cells. These results demonstrate that AprX is an extracellular metalloprotease that is involved in bacterial virulence. (C) 2009 Elsevier B.V. All rights reserved.

Heterologous expression and purification of recombinant allophycocyanin in marine Streptomyces sp isolate M097

Allophycocyanin is one of the most important marine active peptides. Previous studies suggested that recombinant allophycocyanin (rAPC) could remarkably inhibit the S-180 carcinoma in mice, indicating its potential pharmaceutical uses. Based on intergeneric conjugal transfer, heterologous expression of rAPC was first achieved in marine Streptomyces sp. isolate M097 through inserting the apc gene into the thiostrepton-induced vector pIJ8600. The transformation frequency for this system was approximately 10(-4) exconjugants/recipient. In the transformed Streptomyces sp. isolate M097, the yield of purified rAPC could amount to about 38 mg/l using a simple purification protocol, and HPLC analysis showed that the purity of the protein reached about 91.5%. In vitro activity tests also revealed that the purified rAPC had effective scavenging abilities on superoxide and hydroxyl radicals. This would widen the usefulness of the marine Streptomyces as a host to express the rAPC and to offer industrial strain for the production of rAPC.

Studies on the transmission of WSSV (white spot syndrome virus) in juvenile Marsupenaeus japonicus via marine microalgae

We studied the possible role that marine microalgae may play during the outbreaks of WSS (white spot syndrome). In order to elucidate the possibility of marine microalgae carrying WSSV (white spot syndrome virus), six marine microallgae (Isochr.vsis galbana, Skeletonema costatum, Chlorella sp., Heterosigma akashiwo, Scrippsiella trochoidea, Dunaliella salina) were co-cultured with adult Marsupenaeus japollicus infected with WSSV and were assayed daily by nested-PCR to study whether they could carry WSSV. Further experiments were conducted to investigate whether the virus carried by microalgae could re-infect juvenile M. japonicus. Results showed that all of the experimental microalgae, except H. akashiwo could carry WSSV, and among them, Chlorella sp. and S. trochoidea had the strongest WSSV-carrying ability. Unlike other invertebrate carriers of WSSV, the WSSV detections in microalgae, which were positive after I and 3 days, were negative after 10 days of incubation. WSSV detection results in juvenile M. japonicus showed that the juvenile shrimp were re-infected by co-cultured Chlorella sp., although the juvenile M. japonicus carried so small an amount of WSSV that it could only be detected by nested-PCR. The results of this experiment suggest that microalgae might be one possible horizontal transmission pathway for WSSV. Further research, however, is required to better understand the factors behind the different carrying abilities and virus-carrying mechanisms of different microalgae. (c) 2007 Elsevier Inc. All rights reserved.

In vitro determination of antioxidant activity of proteins from jellyfish Rhopilema esculentum

In this study, the antioxidant activity of proteins isolated from jellyfish, Rhopilema esculentum Kishinouye (R. esculentum), was determined by various antioxidant assays, including superoxide anion radical-scavenging, hydroxyl radical-scavenging, total antioxidant activity, reducing power and metal chelating activity. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), alpha-tocopherol, vitamin C and mannitol were used as standards in those various antioxidant activities. The crude protein (CP) and the protein fractions isolated by Sephadex chromatography, first peak (FP) and second peak (SP), had very low reductive power and metal chelating abilities compared to EDTA, but they showed strong scavenging effects on the superoxide anion radical, hydroxyl radical and varying total antioxidant activity. FP and SP exhibited stronger scavenging effects on the superoxide anion radical than BHA, BHT or a-tocopherol. The EC50 values of FP and SP were 6.12 and 0.88 mu g/ml, respectively, while values EC50 of BHA, BHT and alpha-tocopherol were 31, 61 and 88 mu g/ml, respectively. CP, FP and SP showed far higher hydroxyl radical-scavenging activities than did vitamin C or mannitol. The EC50 values of CP, FP and SP were 48.76, 45.42 and 1.52 mu g/ml, but EC50 values of vitamin C and mannitol were 1907 and 4536 mu g/ml, respectively. In a beta-carotene-linoleate system, SP and CP showed antioxidant activity, but lower than BHA. Of the three samples, SP had the strongest antioxidant activity. So, SP may have a use as a possible supplement in the food and pharmaceutical industries. (c) 2005 Elsevier Ltd. All rights reserved.

Studies on two coordination polymers [M(mu(4)-pz25dc)](n) (M = Cd or Zn, pz25dc=pyrazine-2,5-dicarboxylato) with three-dimensional pillared-layer three-nodal framework: Synthesis, structural characterization, strong optical non-linearities and optical limiting properties

Two isomorphous new candidates [M(mu(4)-pz25dc)](n) (M = Cd, 1; Zn, 2; pz25dc = pyrazine-2,5-dicarboxylato)for nonlinear optical (NLO) materials have been synthesized hydrothermally and characterized crystallographically as pillared-layer three-nodal frameworks with one four-connected metal nodes and two crystallographically different four-connected ligand nodes. Their optical non-linearities are measured by the Z-Scan technique with an 8 ns pulsed laser at 532 nm. These two coordination polymers both exhibit strong NLO absorptive abilities [alpha(2) = (63 +/- 6) x 10 (12) mW (1) 1, ( 46 +/- 6) x 10 (11) mW (1) 2] and effective self-focusing performance [n(2) = (67 +/- 5) x 10 (18) 1, (13 +/- 3) x 10 (18) m(2) W (1) 2] in 1.02 x 10 (4) 1 and 1.05 x 10 (4) mol dm (3) 2 DMF solution separately. The values of the limiting threshold are also measured from the optical limiting experimental data. The heavy atom effect plays important role in the enhancement of optical non-linearities and optical limiting properties. (C) 2009 Elsevier B. V. All rights reserved.

一种7DOF冗余手臂构形的运动学与动力学特性研究

冗余度机构具有高度的灵活性和避障能力,利用冗余自由度可以改善仿人机器人手臂的运动性能。以实现人体手臂的运动特性和操作灵活性为目标,研制了一种7DOF冗余仿人机器人手臂,根据冗余手臂的机构学特点,研究了手臂的运动学和动力学性能,并在工作空间、肘部关节的自运动能力以及手臂自重引起的关节重力矩等方面与其它两种典型的冗余手臂构形进行了比较分析。

小型自治水下机器人运动控制系统研究

以主推加舵控制的小型自治水下机器人为研究对象,建立了水下机器人的数学模型并进行了分析。根据机器人结构的特点,对模型进行了必要的简化。设计了机器人的运动控制系统。通过湖试验正控制器的性能。

远程自治水下机器人三维实时避障方法研究

海洋环境通常是不确定、非结构化和未知的 ,而远程AUV(LAUV)的避障声纳对环境感知有一定的局限 ,因此很难建立起精确、完整和统一的三维环境模型 .LAUV实时避障是一个实时性很强的动态过程 ,它不但与环境有关 ,而且还与LAUV的运动学约束、动力学特性和操纵性有关 .针对上述问题本文提出了一种基于复合控制的三维实时模糊避障方法

Fourier有限差分法深度偏移与正演模拟

With the development of seismic exploration, the target becomes more and more complex, which leads to a higher demand for the accuracy and efficiency in 3D exploration. Fourier finite-difference (FFD) method is one of the most valuable methods in complex structure exploration, which keeps the ability of finite-differenc method in dealing with laterally varing media and inherits the predominance of the phase-screen method in stablility and efficiency. In this thesis, the accuracy of the FFD operator is highly improved by using simulated annealing algorithm. This method takes the extrapolation step and band width into account, which is more suitable to various band width and discrete scale than the commonely-used optimized method based on velocity contrast alone. In this thesis, the FFD method is extended to viscoacoustic modeling. Based on one-way wave equation, the presented method is implemented in frequency domain; thus, it is more efficient than two-way methods, and is more convenient than time domain methods in handling attenuation and dispersion effects. The proposed method can handle large velocity contrast and has a high efficiency, which is helpful to further research on earth absorption and seismic resolution. Starting from the frequency dispersion of the acoustic VTI wave equation, this thesis extends the FFD migration method to the acoustic VTI media. Compared with the convetional FFD method, the presented method has a similar computational efficiency, and keeps the abilities of dealing with large velocity contrasts and steep dips. The numerical experiments based on the SEG salt model show that the presented method is a practical migration method for complex acoustical VTI media, because it can handle both large velocity contrasts and large anisotropy variations, and its accuracy is relatively high even in strong anisotropic media. In 3D case, the two-way splitting technique of FFD operator causes artificial azimuthal anisotropy. These artifacts become apparent with increasing dip angles and velocity contrasts, which prevent the application of the FFD method in 3D complex media. The current methods proposed to reduce the azimuthal anisotropy significantly increase the computational cost. In this thesis, the alternating-direction-implicit plus interpolation scheme is incorporated into the 3D FFD method to reduce the azimuthal anisotropy. By subtly utilizing the Fourier based scheme of the FFD method, the improved fast algorithm takes approximately no extra computation time. The resulting operator keeps both the accuracy and the efficiency of the FFD method, which is helpful to the inhancements of both the accuracy and the efficiency for prestack depth migration. The general comparison is presented between the FFD operator and the generalized-screen operator, which is valuable to choose the suitable method in practice. The percentage relative error curves and migration impulse responses show that the generalized-screen operator is much sensiutive to the velocity contrasts than the FFD operator. The FFD operator can handle various velocity contrasts, while the generalized-screen operator can only handle some range of the velocity contrasts. Both in large and weak velocity contrasts, the higher order term of the generalized-screen operator has little effect on improving accuracy. The FFD operator is more suitable to large velocity contrasts, while the generalized-screen operator is more suitable to middle velocity contrasts. Both the one-way implicit finite-difference migration and the two-way explicit finite-differenc modeling have been implemented, and then they are compared with the corresponding FFD methods respectively. This work gives a reference to the choosen of proper method. The FFD migration is illustrated to be more attractive in accuracy, efficiency and frequency dispertion than the widely-used implicit finite-difference migration. The FFD modeling can handle relatively coarse grids than the commonly-used explicit finite-differenc modeling, thus it is much faster in 3D modeling, especially for large-scale complex media.

基于匹配追踪方法的地震信号时频分析研究

Seismic signal is a typical non-stationary signal, whose frequency is continuously changing with time and is determined by the bandwidth of seismic source and the absorption characteristic of the media underground. The most interesting target of seismic signal’s processing and explaining is to know about the local frequency’s abrupt changing with the time, since this kind of abrupt changing is indicating the changing of the physical attributes of the media underground. As to the seismic signal’s instantaneous attributes taken from time-frequency domain, the key target is to search a effective, non-negative and fast algorithm time-frequency distribution, and transform the seismic signal into this time-frequency domain to get its instantaneous power spectrum density, and then use the process of weighted adding and average etc. to get the instantaneous attributes of seismic signal. Time-frequency analysis as a powerful tool to deal with time variant non-stationary signal is becoming a hot researching spot of modern signal processing, and also is an important method to make seismic signal’s attributes analysis. This kind of method provides joint distribution message about time domain and frequency domain, and it clearly plots the correlation of signal’s frequency changing with the time. The spectrum decomposition technique makes seismic signal’s resolving rate reach its theoretical level, and by the method of all frequency scanning and imaging the three dimensional seismic data in frequency domain, it improves and promotes the resolving abilities of seismic signal vs. geological abnormal objects. Matching pursuits method is an important way to realize signal’s self-adaptive decomposition. Its main thought is that any signal can be expressed by a series of time-frequency atoms’ linear composition. By decomposition the signal within an over completed library, the time-frequency atoms which stand for the signal itself are selected neatly and self-adaptively according to the signal’s characteristics. This method has excellent sparse decomposition characteristics, and is widely used in signal de-noising, signal coding and pattern recognizing processing and is also adaptive to seismic signal’s decomposition and attributes analysis. This paper takes matching pursuits method as the key research object. As introducing the principle and implementation techniques of matching pursuits method systematically, it researches deeply the pivotal problems of atom type’s selection, the atom dictionary’s discrete, and the most matching atom’s searching algorithm, and at the same time, applying this matching pursuits method into seismic signal’s processing by picking-up correlative instantaneous messages from time-frequency analysis and spectrum decomposition to the seismic signal. Based on the research of the theory and its correlative model examination of the adaptively signal decomposition with matching pursuit method, this paper proposes a fast optimal matching time-frequency atom’s searching algorithm aimed at seismic signal’s decomposition by frequency-dominated pursuit method and this makes the MP method pertinence to seismic signal’s processing. Upon the research of optimal Gabor atom’s fast searching and matching algorithm, this paper proposes global optimal searching method using Simulated Annealing Algorithm, Genetic Algorithm and composed Simulated Annealing and Genetic Algorithm, so as to provide another way to implement fast matching pursuit method. At the same time, aimed at the characteristics of seismic signal, this paper proposes a fast matching atom’s searching algorithm by means of designating the max energy points of complex seismic signal, searching for the most optimal atom in the neighbor area of these points according to its instantaneous frequency and instantaneous phase, and this promotes the calculating efficiency of seismic signal’s matching pursuit algorithm. According to these methods proposed above, this paper implements them by programmed calculation, compares them with some open algorithm and proves this paper’s conclusions. It also testifies the active results of various methods by the processing of actual signals. The problems need to be solved further and the aftertime researching targets are as follows: continuously seeking for more efficient fast matching pursuit algorithm and expanding its application range, and also study the actual usage of matching pursuit method.

断陷湖盆缓坡带成藏模式研究

Halfgraben-like depressions have multiple layers of subtle traps, multiple coverings of oil-bearing series and multiple types of reservoirs. But these reservoirs have features of strong concealment and are difficult to explore. For this reason, many scholars contribute efforts to study the pool-forming mechanism for this kind of basins, and establish the basis for reservoir exploration and development. However, further study is needed. This paper takes HuiMin depression as an example to study the pool-forming model for the gentle slope belts of fault-depression lake basins. Applying multi-discipline theory, methods and technologies including sedimentary geology, structural geology, log geology, seismic geology, rock mechanics and fluid mechanics, and furthermore applying the dynamo-static data of oil reservoir and computer means in maximum limitation, this paper, qualitatively and quantitatively studies the depositional system, structural framework, structural evolution, structural lithofacies and tectonic stress field, as well as fluid potential field, sealing and opening properties of controlling-oil faults and reservoir prediction, finally presents a pool-forming model, and develops a series of methods and technologies suited to the reservoir prediction of the gentle slope belt. The results obtained in this paper richen the pool-forming theory of a complex oil-gas accumulative area in the gentle slope belt of a continental fault-depression basin. The research work begins with the study of geometric shape of fracture system, then the structural form, activity stages and time-space juxtaposition of faults with different level and different quality are investigated. On the basis of study of the burial history, subsidence history and structural evolution history, this paper synthesizes the studied results of deposition system, analyses the structural lithofacies of the gentle slope belt in the HuiMing Depression and its controlling roles to oil reservoir in the different structural lithofacies belts in time-space, and presents their evolution patterns. The study of structural stress field and fluid potential field indicates that the stress field has a great change from the Dong Ying stages to nowadays. One marked point among them is that the Dong Ying double peak- shaped nose structures usually were the favorable directional area for oil and gas migration, while the QuDi horst became favorable directional area since the GuanTao stage. Based on the active regular of fractures and the information of crude oil saturation pressure, this paper firstly demonstrates that the pool-forming stages of the LingNan field were prior to the stages of the QuDi field, whici provides new eyereach and thinking for hydrocarbon exploration in the gentle slope belt. The BeiQiao-RenFeng buried hill belt is a high value area with the maximum stress values from beginning to end, thus it is a favorable directional area for oil and gas migration. The opening and sealing properties of fractures are studied. The results obtained demonstrate their difference in the hydrocarbon pool formation. The seal abilities relate not only with the quality, direction and scale of normal stress, with the interface between the rocks of two sides of a fault and with the shale smear factor (SSF), but they relate also with the juxtaposition of fault motion stage and hydrocarbon migration. In the HuiMin gentle slope belt, the fault seal has difference both in different stages, and in different location and depth in the same stage. The seal extent also displays much difference. Therefore, the fault seal has time-space difference. On the basis of study of fault seal history, together with the obtained achievement of structural stress field and fluid potential field, it is discovered that for the pool-forming process of oil and gas in the studied area the fault seal of nowadays is better than that of the Ed and Ng stages, it plays an important role to determine the oil column height and hydrocarbon preservation. However, the fault seal of the Ed and Ng stages has an important influence for the distribution state of oil and gas. Because the influential parameters are complicated and undefined, we adopt SSF in the research work. It well reflects synthetic effect of each parameter which influences fault seal. On the basis of the above studies, three systems of hydrocarbon migration and accumulation, as well as a pool-forming model are established for the gentle slope belt of the HuiMin depression, which can be applied for the prediction of regular patterns of oil-gas migration. Under guidance of the pool-forming geological model for the HuiMin slope belt, and taking seismic facies technology, log constraint evolution technology, pattern recognition of multiple parameter reservoir and discrimination technology of oil-bearing ability, this paper develops a set of methods and technologies suited to oil reservoir prediction of the gentle slope belt. Good economic benefit has been obtained.

东营凹陷成藏组合体研究与勘探实践

With the development of petroleum exploration, subtle reservoir has become the main exploration object in Dongying Depression, which requires some new technologies and methods to further reveal the geological characteristics in step with the mature exploration stage. In this paper, on the references to the studies of petroleum system and multiple oil-gas accumulation belt with flexible maneuverability, and the application of systematic theory, the concept of reservoir assemblage is initially defined as "the association of active source rock(s) and hydrocarbon reservoir(s) that are genetically related, with the bridge of pathway system in an oil and gas bearing basin". Compared with the theories of petroleum system and multiple oil-gas accumulation belts, it emphasizes on the processes of petroleum migration and accumulation and the correlation among active source rock, trapped hydrocarbon and migration pathway, and has been confirmed to be more suitably applied to high maturely explored basin. In the first study of this paper, sequence stratigraphy and subtle analytical technology of source rock have been employed to find that two categories of source rock with their characteristic types of organic matter and substantial states occurred in Dongying Depression. The first category, consisting of the oil shales within the third-order sequences of lacustrine expanding system tracts in the upper interval of the fourth Member of Shahejie Formation and both in the middle and lower intervals of the third Member of Shahejie Formation, is featured with the highest abundance of total organic matter (TOC) and the strongest abilities of hydrocarbon generation and expulsion, which is classified into the standard of good hydrocarbon source rock. Exploration assessment confirmed that about 70-80% of hydrocarbon in Dongying Depression came from this set of source rock for which the low sedimentary rate and strong oxygen-free environment would play the key role during its generation. The second category, composed of organic matter of dark mudstone in high stand system tracts in the upper and middle intervals of the third Member of Shahejie Formation, has been characterized by low content of total organic matter which mostly dispersedly distributes, and formed in the pre-delta to delta front environments. In classification, it belongs to the ordinary standard of source rocks. In the second research part, through the studies of high frequency sequence stratigraphy, fault geometry and active history combining with geochemistry of fluid inclusion and nitrogen compound and simulation test of hydrocarbon migration and accumulation, the faults have been thought to be the principal conduits, and the sandy bodies and unconformities might played the complementary pathways for hydrocarbon migration and accumulation in Dongying Depression of the continental faulted basin. Therefore, the fault activities may mainly constrain on the development of hydrocarbon pathways in space and time. Even more, using homogenization temperatures of fluid inclusion in digenetic minerals, three critical moments for hydrocarbon accumulation have been determined as well in Dongying Depression, which happened during the late stage of Dongying Formation (Ed), the early stage of Guantao Formation (Nig) and the early stage of Minghuazhen Formation (Nim), respectively. Comparatively, the last stage is looked as the main forming-reservoir period, which has also been supported by the results of geochemical analysis and simulation experiments of hydrocarbon generation and expulsion. Clearly, the times of hydrocarbon migration and accumulation are consistent with those of the fault activities in Dongying Depression, which indicate that tectonic activities would control the forming-reservoir. A conceptual model of faulting-episodic expulsion coupled with episodic forming-reservoir has then been established in this study. In the third part of this paper, some focusing areas were selected for the fine descriptions of pathway distribution and forming-reservoir, which has given four types of reservoir assemblage in terms of the main pathway and its correlation with the reservoir and trap: (1) mainly consisted of sandy bodies; (2) mainly consisted of faults; (3) mainly consisted of unconformities; and (4) their complex with two or three types of pathways. This classified criteria has also been applied to access the risk of some prospected traps in Dongying Depression. Finally, through the application of reservoir assemblage integrated with pathway distribution to all the prospective targets in Dongying Depression, the new favorably hydrocarbon accumulated belts have been figured out, and more subtle reservoirs have also been found. For examples, during 2000 and 2002, in the mature exploration areas, such as Liangjialou and Shengtuo structural closures etc., newly proved reserves were 2274 * 104t, and forecasted oil reserves 5660-5860xl04t; and in the predicted favorable areas, newly additional controlled oil reserves was 3355xl04t. Besides those, many other favorable exploration areas need to be further appraised.