Functional split and crosslinking of the membrane domain of the β subunit of proton-translocating transhydrogenase from Escherichia coli

Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α subunit with the NAD(H)-binding domain I and a β subunit with the NADP(H)-binding domain III. The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic parts of the α and β subunits. The interface in domain II between the α and the β subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the α subunit and loops connecting the nine transmembrane helices in the β subunit. However, to investigate the organization of the nine transmembrane helices in the β subunit, a split was introduced by creating a stop codon in the loop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existing downstream start codon. The resulting enzyme was composed of the wild-type α subunit and the two new peptides β1 and β2. As compared to other split membrane proteins, the new transhydrogenase was remarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD + by NADPH, the cyclic reduction of 3-acetylpyridine-NAD + by NADH (mediated by bound NADP(H)), and proton pumping, amounting to about 50-107% of the corresponding wild-type activities. These high activities suggest that the α subunit was normally folded, followed by a concerted folding of β1 + β2. Cross-linking of a βS105C-βS237C double cysteine mutant in the functional split cysteine-free background, followed by SDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity. This is the first time that cross-linking between helices in the same β subunit has been demonstrated.

Distributed routing algorithms for interconnected FDDI LANS

The Fibre Distributed Data Interface (FDDI) represents the new generation of local area networks (LANs). These high speed LANs are capable of supporting up to 500 users over a 100 km distance. User traffic is expected to be as diverse as file transfers, packet voice and video. As the proliferation of FDDI LANs continues, the need to interconnect these LANs arises. FDDI LAN interconnection can be achieved in a variety of different ways. Some of the most commonly used today are public data networks, dial up lines and private circuits. For applications that can potentially generate large quantities of traffic, such as an FDDI LAN, it is cost effective to use a private circuit leased from the public carrier. In order to send traffic from one LAN to another across the leased line, a routing algorithm is required. Much research has been done on the Bellman-Ford algorithm and many implementations of it exist in computer networks. However, due to its instability and problems with routing table loops it is an unsatisfactory algorithm for interconnected FDDI LANs. A new algorithm, termed ISIS which is being standardized by the ISO provides a far better solution. ISIS will be implemented in many manufacturers routing devices. In order to make the work as practical as possible, this algorithm will be used as the basis for all the new algorithms presented. The ISIS algorithm can be improved by exploiting information that is dropped by that algorithm during the calculation process. A new algorithm, called Down Stream Path Splits (DSPS), uses this information and requires only minor modification to some of the ISIS routing procedures. DSPS provides a higher network performance, with very little additional processing and storage requirements. A second algorithm, also based on the ISIS algorithm, generates a massive increase in network performance. This is achieved by selecting alternative paths through the network in times of heavy congestion. This algorithm may select the alternative path at either the originating node, or any node along the path. It requires more processing and memory storage than DSPS, but generates a higher network power. The final algorithm combines the DSPS algorithm with the alternative path algorithm. This is the most flexible and powerful of the algorithms developed. However, it is somewhat complex and requires a fairly large storage area at each node. The performance of the new routing algorithms is tested in a comprehensive model of interconnected LANs. This model incorporates the transport through physical layers and generates random topologies for routing algorithm performance comparisons. Using this model it is possible to determine which algorithm provides the best performance without introducing significant complexity and storage requirements.

A naturally shaped silicone ventricle evaluated in a mock circulation loop: a preliminary study

Mock circulation loops are used to evaluate the performance of cardiac assist devices prior to animal and clinical testing. A compressible, translucent silicone ventricle chamber that mimics the exact size, shape and motion of a failing heart is desired to assist in flow visualization studies around inflow cannulae during VAD support. The aim of this study was therefore to design and construct a naturally shaped flexible left ventricle and evaluate its performance in a mock circulation loop. The ventricle shape was constructed by the use of CT images taken from a patient experiencing cardiomyopathic heart failure and used to create a 3D image and subsequent mould to produce a silicone ventricle. Different cardiac conditions were successfully simulated to validate the ventricle performance, including rest, left heart failure and VAD support.

Mathematical modelling and control of agitated extraction columns

Liquid-liquid extraction has long been known as a unit operation that plays an important role in industry. This process is well known for its complexity and sensitivity to operation conditions. This thesis presents an attempt to explore the dynamics and control of this process using a systematic approach and state of the art control system design techniques. The process was studied first experimentally under carefully selected. operation conditions, which resembles the ranges employed practically under stable and efficient conditions. Data were collected at steady state conditions using adequate sampling techniques for the dispersed and continuous phases as well as during the transients of the column with the aid of a computer-based online data logging system and online concentration analysis. A stagewise single stage backflow model was improved to mimic the dynamic operation of the column. The developed model accounts for the variation in hydrodynamics, mass transfer, and physical properties throughout the length of the column. End effects were treated by addition of stages at the column entrances. Two parameters were incorporated in the model namely; mass transfer weight factor to correct for the assumption of no mass transfer in the. settling zones at each stage and the backmixing coefficients to handle the axial dispersion phenomena encountered in the course of column operation. The parameters were estimated by minimizing the differences between the experimental and the model predicted concentration profiles at steady state conditions using non-linear optimisation technique. The estimated values were then correlated as functions of operating parameters and were incorporated in·the model equations. The model equations comprise a stiff differential~algebraic system. This system was solved using the GEAR ODE solver. The calculated concentration profiles were compared to those experimentally measured. A very good agreement of the two profiles was achieved within a percent relative error of ±2.S%. The developed rigorous dynamic model of the extraction column was used to derive linear time-invariant reduced-order models that relate the input variables (agitator speed, solvent feed flowrate and concentration, feed concentration and flowrate) to the output variables (raffinate concentration and extract concentration) using the asymptotic method of system identification. The reduced-order models were shown to be accurate in capturing the dynamic behaviour of the process with a maximum modelling prediction error of I %. The simplicity and accuracy of the derived reduced-order models allow for control system design and analysis of such complicated processes. The extraction column is a typical multivariable process with agitator speed and solvent feed flowrate considered as manipulative variables; raffinate concentration and extract concentration as controlled variables and the feeds concentration and feed flowrate as disturbance variables. The control system design of the extraction process was tackled as multi-loop decentralised SISO (Single Input Single Output) as well as centralised MIMO (Multi-Input Multi-Output) system using both conventional and model-based control techniques such as IMC (Internal Model Control) and MPC (Model Predictive Control). Control performance of each control scheme was. studied in terms of stability, speed of response, sensitivity to modelling errors (robustness), setpoint tracking capabilities and load rejection. For decentralised control, multiple loops were assigned to pair.each manipulated variable with each controlled variable according to the interaction analysis and other pairing criteria such as relative gain array (RGA), singular value analysis (SVD). Loops namely Rotor speed-Raffinate concentration and Solvent flowrate Extract concentration showed weak interaction. Multivariable MPC has shown more effective performance compared to other conventional techniques since it accounts for loops interaction, time delays, and input-output variables constraints.

Expert systems for fault tree synthesis

Hazard and operability (HAZOP) studies on chemical process plants are very time consuming, and often tedious, tasks. The requirement for HAZOP studies is that a team of experts systematically analyse every conceivable process deviation, identifying possible causes and any hazards that may result. The systematic nature of the task, and the fact that some team members may be unoccupied for much of the time, can lead to tedium, which in turn may lead to serious errors or omissions. An aid to HAZOP are fault trees, which present the system failure logic graphically such that the study team can readily assimilate their findings. Fault trees are also useful to the identification of design weaknesses, and may additionally be used to estimate the likelihood of hazardous events occurring. The one drawback of fault trees is that they are difficult to generate by hand. This is because of the sheer size and complexity of modern process plants. The work in this thesis proposed a computer-based method to aid the development of fault trees for chemical process plants. The aim is to produce concise, structured fault trees that are easy for analysts to understand. Standard plant input-output equation models for major process units are modified such that they include ancillary units and pipework. This results in a reduction in the nodes required to represent a plant. Control loops and protective systems are modelled as operators which act on process variables. This modelling maintains the functionality of loops, making fault tree generation easier and improving the structure of the fault trees produced. A method, called event ordering, is proposed which allows the magnitude of deviations of controlled or measured variables to be defined in terms of the control loops and protective systems with which they are associated.

Lead time reduction for new products

The research, which was given the terms of reference, "To cut the lead time for getting new products into volume production", was sponsored by a company which develops and manufactures telecommunications equipment. The research described was based on studies made of the development of two processors which were designed to control telephone exchanges in the public network. It was shown that for each of these products, which were large electronic systems containing both hardware and software, most of their lead time was taken up with development. About half of this time was consumed by activities associated with redesign resulting from changes found to be necessary after the original design had been built. Analysing the causes of design changes showed the most significant to be Design Faults. The reasons why these predominated were investigated by seeking the collective opinion from design staff and their management using a questionnaire. Using the results from these studies to build upon the works of other authors, a model of the development process of large hierarchical systems is derived. An important feature of this model is its representation of iterative loops due to design changes. In order to reduce the development time, two closely related philosophies are proposed: By spending more time at the early stages of development (detecting and remedying faults in the design) even greater savings can be made later on, The collective performance of the development organisation would be improved by increasing the amount and speed of feedback about that performance. A trial was performed to test these philosophies using readily available techniques for design verification. It showed that about an 11 per cent saving would be made on the development time and that the philosophies might be equally successfully applied to other products and techniques.

Multiple roles of charged amino acids in cytoplasmic loop 7 for expression and function of the multidrug and organic anion transporter MRP1 (ABCC1)

Multidrug resistance protein MRP1 mediates the ATP-dependent efflux of many chemotherapeutic agents and organic anions. MRP1 has two nucleotide binding sites (NBSs) and three membrane spanning domains (MSDs) containing 17 transmembrane helices linked by extracellular and cytoplasmic loops (CL). Homology models suggest that CL7 (amino acids 1141-1195) is in a position where it could participate in signaling between the MSDs and NBSs during the transport process. We have individually replaced eight charged residues in CL7 with Ala, and in some cases, an amino acid with the same charge, and then investigated the effects on MRP1 expression, transport activity, and nucleotide and substrate interactions. A triple mutant in which Glu(1169), Glu(1170), and Glu(1172) were all replaced with Ala was also examined. The properties of R1173A and E1184A were comparable with those of wild-type MRP1, whereas the remaining mutants were either poorly expressed (R1166A, D1183A) or exhibited reduced transport of one or more organic anions (E1144A, D1179A, K1181A, (1169)AAQA). Same charge mutant D1183E was also not expressed, whereas expression and activity of R1166K were similar to wild-type MRP1. The moderate substrate-selective changes in transport activity displayed by mutants E1144A, D1179A, K1181A, and (1169)AAQA were accompanied by changes in orthovanadate-induced trapping of [alpha-(32)P]azidoADP by NBS2 indicating changes in ATP hydrolysis or release of ADP. In the case of E1144A, estradiol glucuronide no longer inhibited trapping of azidoADP. Together, our results demonstrate the extreme sensitivity of CL7 to mutation, consistent with its critical and complex dual role in both the proper folding and transport activity of MRP1.

Semantic linking through spaces for cyber-physical-socio intelligence:a methodology

Humans consciously and subconsciously establish various links, emerge semantic images and reason in mind, learn linking effect and rules, select linked individuals to interact, and form closed loops through links while co-experiencing in multiple spaces in lifetime. Machines are limited in these abilities although various graph-based models have been used to link resources in the cyber space. The following are fundamental limitations of machine intelligence: (1) machines know few links and rules in the physical space, physiological space, psychological space, socio space and mental space, so it is not realistic to expect machines to discover laws and solve problems in these spaces; and, (2) machines can only process pre-designed algorithms and data structures in the cyber space. They are limited in ability to go beyond the cyber space, to learn linking rules, to know the effect of linking, and to explain computing results according to physical, physiological, psychological and socio laws. Linking various spaces will create a complex space — the Cyber-Physical-Physiological-Psychological-Socio-Mental Environment CP3SME. Diverse spaces will emerge, evolve, compete and cooperate with each other to extend machine intelligence and human intelligence. From multi-disciplinary perspective, this paper reviews previous ideas on various links, introduces the concept of cyber-physical society, proposes the ideal of the CP3SME including its definition, characteristics, and multi-disciplinary revolution, and explores the methodology of linking through spaces for cyber-physical-socio intelligence. The methodology includes new models, principles, mechanisms, scientific issues, and philosophical explanation. The CP3SME aims at an ideal environment for humans to live and work. Exploration will go beyond previous ideals on intelligence and computing.

Bistable regimes in an optically injected mode-locked laser

We study experimentally the dynamics of quantum-dot (QD) passively mode-locked semiconductor lasers under external optical injection. The lasers demonstrated multiple dynamical states, with bifurcation boundaries that depended upon the sign of detuning variation. The area of the hysteresis loops grew monotonically at small powers of optical injection and saturated at moderate powers. At high injection levels the hysteresis decreased and eventually disappeared.

The effect of barium substitution on the ferroelectric properties of Sr2 Nb2 O7 Ceramics

This work revealed that the solid solution compounds of Sr 2-xBaxNb2O7 are promising lead-free materials for high-temperature piezoelectric sensor application. These compounds were confirmed as ferroelectric materials with high Curie points (> 900°C) by their piezoelectric activity after poling, ferroelectric domain switching in their P-E hysteresis loops and thermal depoling behavior. The effect of Ba substitution on the structure and properties of Sr 2-xBaxNb2O7 (x < 1.0) was investigated. The solid solution limit of Sr2-xBaxNb 2O7 was determined by XRD as x < 0.6. The a-, b-, c- axes, and cell volume increase with Ba addition. The textured ceramics of Sr2-xBaxNb2O7 were prepared for the first time. The highest d33 was measured as 3.6 ± 0.1 pC/N for Sr1.8Ba0.2Nb2O7. © 2012 The American Ceramic Society.

On the structural repertoire of pools of short, random RNA sequences

A detailed knowledge of the mapping between sequence and structure spaces in populations of RNA molecules is essential to better understand their present-day functional properties, to envisage a plausible early evolution of RNA in a prebiotic chemical environment and to improve the design of in vitro evolution experiments, among others. Analysis of natural RNAs, as well as in vitro and computational studies, show that certain RNA structural motifs are much more abundant than others, pointing out a complex relation between sequence and structure. Within this framework, we have investigated computationally the structural properties of a large pool (10 molecules) of single-stranded, 35 nt-long, random RNA sequences. The secondary structures obtained are ranked and classified into structure families. The number of structures in main families is analytically calculated and compared with the numerical results. This permits a quantification of the fraction of structure space covered by a large pool of sequences. We further show that the number of structural motifs and their frequency is highly unbalanced with respect to the nucleotide composition: simple structures such as stem-loops and hairpins arise from sequences depleted in G, while more complex structures require an enrichment of G. In general, we observe a strong correlation between subfamilies-characterized by a fixed number of paired nucleotides-and nucleotide composition. Our results are compared to the structural repertoire obtained in a second pool where isolated base pairs are prohibited. © 2008 Elsevier Ltd. All rights reserved.

Bistable regimes in an optically injected mode-locked laser

We study experimentally the dynamics of quantum-dot (QD) passively mode-locked semiconductor lasers under external optical injection. The lasers demonstrated multiple dynamical states, with bifurcation boundaries that depended upon the sign of detuning variation. The area of the hysteresis loops grew monotonically at small powers of optical injection and saturated at moderate powers. At high injection levels the hysteresis decreased and eventually disappeared.

A short regulatory domain restricts glycerol transport through yeast Fps1p

The controlled export of solutes is crucial for cellular adaptation to hypotonic conditions. In the yeast Saccharomyces cerevisiae glycerol export is mediated by Fpslp, a member of the major intrinsic protein (MIP) family ]of channel proteins. Here we describe a short regulatory domain that restricts glycerol transport through Fpslp. This domain is required for retention of cellular glycerol under hypertonic stress and hence acquisition of osmotolerance. It is located in the N-terminal cytoplasmic extension close to the first transmembrane domain. Several residues within that domain and its precise position are critical for channel control while the proximal residues 13-215 of the N-terminal extension are not required. The sequence of the regulatory domain and its position are perfectly conserved in orthologs from other yeast species. The regulatory domain has an amphiphilic character, and structural predictions indicate that it could fold back into the membrane bilayer. Remarkably, this domain has structural similarity to the channel forming loops B and E of Fpslp and other glycerol facilitators. Intragenic second-site suppressor mutations of the sensitivity to high osmolarity conferred by truncation of the regulatory domain caused diminished glycerol transport, confirming that elevated channel activity is the cause of the osmosensitive phenotype.

Near-constant time delay response of fibre grating Sagnac loop filters

A theoretical and experimental investigation of the time delay characteristics of fiber Bragg grating-based Sagnac loops (FBGSLs) is presented. Analytic expressions for the phase and time delay of the FBGSL have been derived and excellent agreement is found between their predictions and experimental results for configurations incorporating uniform-period and chirped-period gratings. For symmetrical grating structures, it is found that the FBGSL time delay response is similar to that of the incorporated grating; with asymmetrical gratings, the FBGSL response is quite different. It is shown that wavelength-division-multiplexing filters exhibiting near-zero dispersion characteristics can be implemented using FBGSLs.

Anderson localization in synthetic photonic lattices

We experimentally demonstrate Anderson localization for optical pulses in time domain, using a photonic mesh lattice implemented with coupled optical fiber loops. We also discuss interplay of photonic band-gaps and disorder in such lattices. © OSA 2015.