Monitoring long distance WDM communication lines using a high-loss loopback supervisory system

In this paper, we present experimental results for monitoring long distance WDM communication links using a line monitoring system suitable for legacy optically amplified long-haul undersea systems. This monitoring system is based on setting up a simple, passive, low cost high-loss optical loopback circuit at each repeater that provides a connection between the existing anti-directional undersea fibres, and can be used to define fault location. Fault location is achieved by transmitting a short pulse supervisory signal along with the WDM data signals where a portion of the overall signal is attenuated and returned to the transmit terminal by the loopback circuit. A special receiver is used at the terminal to extract the weakly returned supervisory signal where each supervisory signal is received at different times corresponding to different optical repeaters. Therefore, the degradation in any repeater appears on its corresponding supervisory signal level. We use a recirculating loop to simulate a 4600 km fibre link, on which a high-loss loopback supervisory system is implemented. Successful monitoring is accomplished through the production of an appropriate supervisory signal at the terminal that is detected and identified in a satisfactory time period after passing through up to 45 dB attenuation in the loopback circuit. © 2012 Elsevier B.V. All rights reserved.

Optimal production planning for PCB assembly

It is indisputable that printed circuit boards (PCBs) play a vital role in our daily lives. With the ever-increasing applications of PCBs, one of the crucial ways to increase a PCB manufacturer’s competitiveness in terms of operation efficiency is to minimize the production time so that the products can be introduced to the market sooner. Optimal Production Planning for PCB Assembly is the first book to focus on the optimization of the PCB assembly lines’ efficiency. This is done by: • integrating the component sequencing and the feeder arrangement problems together for both the pick-and-place machine and the chip shooter machine; • constructing mathematical models and developing an efficient and effective heuristic solution approach for the integrated problems for both types of placement machines, the line assignment problem, and the component allocation problem; and • developing a prototype of the PCB assembly planning system. The techniques proposed in Optimal Production Planning for PCB Assembly will enable process planners in the electronics manufacturing industry to improve the assembly line’s efficiency in their companies. Graduate students in operations research can familiarise themselves with the techniques and the applications of mathematical modeling after reading this advanced introduction to optimal production planning for PCB assembly.

Cost effectiveness of production control systems

This thesis deals with the problems associated with the planning and control of production, with particular reference to a small aluminium die casting company. The main problem areas were identified as: (a) A need to be able to forecast the customers demands upon the company's facilities. (b) A need to produce a manufacturing programme in which the output of the foundry (or die casting section) was balanced with the available capacity in the machine shop. (c) The need to ensure that the resultant system enabled the company's operating budget to have a reasonable chance of being achieved. At the commencement of the research work the major customers were members of the automobile industry and had their own system of forecasting, from which they issued manufacturing schedules to their component suppliers, The errors in the forecast were analysed and the distributions noted. Using these distributions the customer's forecast was capable of being modified to enable his final demand to be met with a known degree of confidence. Before a manufacturing programme could be developed the actual manufacturing system had to be reviewed and it was found that as with many small companies there was a remarkable lack of formal control and written data. Relevant data with regards to the component and the manufacturing process had therefore to be collected and analysed. The foundry process was fixed but the secondary machining operations were analysed by a technique similar to Component Flow Analysis and as a result the machines were arranged in a series of flow lines. A system of manual production control was proposed and for comparison, a local computer bureau was approached and a system proposed incorporating the production of additional management information. These systems are compared and the relative merits discussed and a proposal made for implementation.

Development of techniques to predict production line efficiency

This industrial based research project was undertaken for British Leyland and arose as a result of poor system efficiency on the Maxi and Marina vehicle body build lines. The major factors in the deterioration of system efficiency were identified as: a) The introduction of a 'Gateline' system of vehicle body build. b) The degeneration of a newly introduced measured daywork payment scheme. By relating the conclusions of past work on payment systems to the situation at Cowley, it was concluded that a combination of poor industrial relations and a lack of managerial control had caused the measured daywork scheme to degenerate into a straightforward payment for time at work. This ellminated the monetary incentive to achieve schedule with the consequence that both inefficiency and operating costs increased. To analyse further the cause of inefficiency, a study of Marina gateline stoppage logs was carried out. This revealed that poor system efficiency on the gateline was caused more by the nature of its design than poor reliability on individual items of' plant. The consideration given to system efficiency at the design stage was found to be negligible, the main obstacles being: a) A lack of understanding pertaining to the influence of certain design factors on the efficiency of a production line. b) The absence of data and techniques to predict system efficiency at the design stage. To remedy this situation, a computer simulation study of' the design factors was carried out from which relationships with system efficiency were established and empirical efficiency equations developed. Sets of tables were compiled from the equations and efficiency data relevant to vehicle body building established from the gateline stoppage logs. Computer simulation, the equations and the tables,when used in conjunction. with good efficiency data, are shown to be accurate methods of predicting production line system.efficiency.

Playing catch-up with Escherichia coli:using yeast to increase success rates in recombinant protein production experiments

Several host systems are available for the production of recombinant proteins, ranging from Escherichia coli to mammalian cell-lines. This article highlights the benefits of using yeast, especially for more challenging targets such as membrane proteins. On account of the wide range of molecular, genetic, and microbiological tools available, use of the well-studied model organism, Saccharomyces cerevisiae, provides many opportunities to optimize the functional yields of a target protein. Despite this wealth of resources, it is surprisingly under-used. In contrast, Pichia pastoris, a relative new-comer as a host organism, is already becoming a popular choice, particularly because of the ease with which high biomass (and hence recombinant protein) yields can be achieved. In the last few years, advances have been made in understanding how a yeast cell responds to the stress of producing a recombinant protein and how this information can be used to identify improved host strains in order to increase functional yields. Given these advantages, and their industrial importance in the production of biopharmaceuticals, I argue that S. cerevisiae and P. pastoris should be considered at an early stage in any serious strategy to produce proteins.

Monitoring long distance WDM communication lines using a high-loss loopback supervisory system

In this paper, we present experimental results for monitoring long distance WDM communication links using a line monitoring system suitable for legacy optically amplified long-haul undersea systems. This monitoring system is based on setting up a simple, passive, low cost high-loss optical loopback circuit at each repeater that provides a connection between the existing anti-directional undersea fibres, and can be used to define fault location. Fault location is achieved by transmitting a short pulse supervisory signal along with the WDM data signals where a portion of the overall signal is attenuated and returned to the transmit terminal by the loopback circuit. A special receiver is used at the terminal to extract the weakly returned supervisory signal where each supervisory signal is received at different times corresponding to different optical repeaters. Therefore, the degradation in any repeater appears on its corresponding supervisory signal level. We use a recirculating loop to simulate a 4600 km fibre link, on which a high-loss loopback supervisory system is implemented. Successful monitoring is accomplished through the production of an appropriate supervisory signal at the terminal that is detected and identified in a satisfactory time period after passing through up to 45 dB attenuation in the loopback circuit. © 2012 Elsevier B.V. All rights reserved.

Monitoring long distance WDM communication lines using a high-loss loopback supervisory system

In this paper, we present experimental results for monitoring long distance WDM communication links using a line monitoring system suitable for legacy optically amplified long-haul undersea systems. This monitoring system is based on setting up a simple, passive, low cost high-loss optical loopback circuit at each repeater that provides a connection between the existing anti-directional undersea fibres, and can be used to define fault location. Fault location is achieved by transmitting a short pulse supervisory signal along with the WDM data signals where a portion of the overall signal is attenuated and returned to the transmit terminal by the loopback circuit. A special receiver is used at the terminal to extract the weakly returned supervisory signal where each supervisory signal is received at different times corresponding to different optical repeaters. Therefore, the degradation in any repeater appears on its corresponding supervisory signal level. We use a recirculating loop to simulate a 4600 km fibre link, on which a high-loss loopback supervisory system is implemented. Successful monitoring is accomplished through the production of an appropriate supervisory signal at the terminal that is detected and identified in a satisfactory time period after passing through up to 45 dB attenuation in the loopback circuit. © 2012 Elsevier B.V. All rights reserved.

Transglutaminase overexpression sensitizes neuronal cell lines to apoptosis by increasing mitochondrial membrane potential and cellular oxidative stress

'Tissue' transglutaminase (tTG) selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Considering the central role played by mitochondria in apoptosis, we investigated the relationships existing amongst tTG expression, apoptosis and mitochondrial function. To this aim we studied the mechanisms of apoptosis in a neuronal cell line (SK-N-BE (2)) in which the tTG-expression was driven by a constitutive promoter. Furthermore, a tet-off inducible promoter was also used in 3T3 fibroblastic cells used as control. Both cell lines, when expressing tTG, appeared 'sensitized' to apoptosis. Strikingly, we found major differences in the morphological features of mitochondria among cell lines in the absence of apoptotic stimuli. In addition, these ultrastructural characteristics were associated with specific functional features: (i) constitutively hyperpolarized mitochondria and (ii) increased reactive oxygen intermediates production. Importantly, after mitochondrial-mediated apoptosis by staurosporine, a rapid loss of mitochondrial membrane potential was found in tTG cells only. Taken together, these results seem to suggest that, via hyperpolarization, tTG might act as a 'sensitizer' towards apoptotic stimuli specifically targeted to mitochondria. These results could also be of pathogenetic relevance for those diseases that are characterized by increased tTG and apoptotic rate together with impaired mitochondrial function, e.g. in some neurodegenerative disease.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

Limits to tDCS effects in language:failures to modulate word production in healthy participants with frontal or temporal tDCS

Transcranial direct current stimulation (tDCS) is a method of non-invasive brain stimulation widely used to modulate cognitive functions. Recent studies, however, suggests that effects are unreliable, small and often non-significant at least when stimulation is applied in a single session to healthy individuals. We examined the effects of frontal and temporal lobe anodal tDCS on naming and reading tasks and considered possible interactions with linguistic activation and selection mechanisms as well possible interactions with item difficulty and participant individual variability. Across four separate experiments (N, Exp 1A = 18; 1B = 20; 1C = 18; 2 = 17), we failed to find any difference between real and sham stimulation. Moreover, we found no evidence of significant effects limited to particular conditions (i.e., those requiring suppression of semantic interference), to a subset of participants or to longer RTs. Our findings sound a cautionary note on using tDCS as a means to modulate cognitive performance. Consistent effects of tDCS may be difficult to demonstrate in healthy participants in reading and naming tasks, and be limited to cases of pathological neurophysiology and/or to the use of learning paradigms.