Technology transfer under China’s economic reforms:business environment and success factors

The opening up of the Chinese economy and the associated transfer of technology from abroad have been taking place at an accelerating pace. Technology is crucial to China's industrial development. It is a productive resource and has a vital role in the process of economic and social development. This article provides an overview of technology transfer into China, focusing on recent developments, and examines the macroenvironmental and microenvironmental influences which foreign enterprises must consider when making investments or technology transfer decisions. Cases of companies engaged in international technology transfer are used to illustrate the discussion on the microenvironment. To be successful, foreign investors and suppliers of technology must respond to China's industrial priorities and pursue projects that are compatible with the country's broad policy goals as well as the corporate objectives of Chinese partners. The article concludes by listing a number of points to which attention should be paid before a decision is made to transfer technology to China.

Modulation of tissue transglutaminase in tubular epithelial cells alters extracellular matrix levels: a potential mechanism of tissue scarring

The up-regulation and trafficking of tissue transglutaminase (TG2) by tubular epithelial cells (TEC) has been implicated in the development of kidney scarring. TG2 catalyses the crosslinking of proteins via the formation of highly stable e(?-glutamyl) lysine bonds. We have proposed that TG2 may contribute to kidney scarring by accelerating extracellular matrix (ECM) deposition and by stabilising the ECM against proteolytic decay. To investigate this, we have studied ECM metabolism in Opossum kidney (OK) TEC induced to over-express TG2 by stable transfection and in tubular cells isolated from TG2 knockout mice. Increasing the expression of TG2 led to increased extracellular TG2 activity (p < 0.05), elevated e(?-glutamyl) lysine crosslinking in the ECM and higher levels of ECM collagen per cell by 3H-proline labelling. Immunofluorescence demonstrated that this was attributable to increased collagen III and IV levels. Higher TG2 levels were associated with an accelerated collagen deposition rate and a reduced ECM breakdown by matrix metalloproteinases (MMPs). In contrast, a lack of TG2 was associated with reduced e(?-glutamyl) lysine crosslinking in the ECM, causing reduced ECM collagen levels and lower ECM per cell. We report that TG2 contributes to ECM accumulation primarily by accelerating collagen deposition, but also by altering the susceptibility of the tubular ECM to decay. These findings support a role for TG2 in the expansion of the ECM associated with kidney scarring.

Neuronal convergence in early contrast vision: binocular summation is followed by response nonlinearity and area summation

We assessed summation of contrast across eyes and area at detection threshold ( C t). Stimuli were sine-wave gratings (2.5 c/deg) spatially modulated by cosine- and anticosine-phase raised plaids (0.5 c/deg components oriented at ±45°). When presented dichoptically the signal regions were interdigitated across eyes but produced a smooth continuous grating following their linear binocular sum. The average summation ratio ( C t1/([ C t1+2]) for this stimulus pair was 1.64 (4.3 dB). This was only slightly less than the binocular summation found for the same patch type presented to both eyes, and the area summation found for the two different patch types presented to the same eye. We considered 192 model architectures containing each of the following four elements in all possible orders: (i) linear summation or a MAX operator across eyes, (ii) linear summation or a MAX operator across area, (iii) linear or accelerating contrast transduction, and (iv) additive Gaussian, stochastic noise. Formal equivalences reduced this to 62 different models. The most successful four-element model was: linear summation across eyes followed by nonlinear contrast transduction, linear summation across area, and late noise. Model performance was enhanced when additional nonlinearities were placed before binocular summation and after area summation. The implications for models of probability summation and uncertainty are discussed.

Controlling protein molecular dynamics:how to accelerate folding while preserving the native state

The dynamics of peptides and proteins generated by classical molecular dynamics (MD) is described by using a Markov model. The model is built by clustering the trajectory into conformational states and estimating transition probabilities between the states. Assuming that it is possible to influence the dynamics of the system by varying simulation parameters, we show how to use the Markov model to determine the parameter values that preserve the folded state of the protein and at the same time, reduce the folding time in the simulation. We investigate this by applying the method to two systems. The first system is an imaginary peptide described by given transition probabilities with a total folding time of 1 micros. We find that only small changes in the transition probabilities are needed to accelerate (or decelerate) the folding. This implies that folding times for slowly folding peptides and proteins calculated using MD cannot be meaningfully compared to experimental results. The second system is a four residue peptide valine-proline-alanine-leucine in water. We control the dynamics of the transitions by varying the temperature and the atom masses. The simulation results show that it is possible to find the combinations of parameter values that accelerate the dynamics and at the same time preserve the native state of the peptide. A method for accelerating larger systems without performing simulations for the whole folding process is outlined.

Development of novel NMR pulse sequences

To elucidate the structures of orgamc molecules in solution using pulse FT NMR, heteronuclear pulse sequence experiments to probe carbon-13 (13C) and proton (1H) spin systems are invaluable. The one-dimensional insensitive nucleus detected PENDANT experiment finds popular use for structure determination via one-bond 13C-1H scalar couplings. PENDANT facilitates the desired increase in 13C signal-to-noise ratio, and unlike many other pulse sequence experiments (e.g., refocused INEPT and DEPT), allows the simultaneous detection of 13C quaternary nuclei. The tlrst chapter herein details the characterisation of PENDANT and the successful rectification of spectral anomalies that occur when it is used without proton broadband decoupling. Multiple-bond (long-range) l3C-1H scalar coupling correlations can yield important bonding information. When the molecule under scrutiny is devoid of proton spectral crowding, and more sensitive 'inverse' pulse sequence experiments are not available, one may use insensitive nucleus detected long-range selective one-dimensional correlation methods, rather than more time consuming and insensitive multidimensional analogues. To this end a novel long-range selective one-dimensional correlation pulse sequence experiment has been invented. Based on PENDANT, the new experiment is shown to rival the popular selective INEPT technique because it can determine the same correlations while simultaneously detecting isolated 13C quaternary nuclei. INEPT cannot facilitate this, potentially leaving other important quaternary nuclei undetected. The novel sequence has been modified further to yield a second novel experiment that simultaneously yields selective 13C transient nOe data. Consequently, the need to perform the two experiments back-to-back is conveniently removed, and the experimental time reduced. Finally, the SNARE pulse sequence was further developed. SNARE facilitates the reduction of experimental time by accelerating the relaxation of protons upon which pulse sequences, to which SNARE is appended, relies. It is shown, contrary to the original publication, that reiaxation time savings can be derived from negative nOes.

NMR spectroscopy in the presence of ultrasound and other perturbations

The work described in this thesis has been concerned with exploring the potential uses of ultrasound in Nuclear Magnetic Resonance (NMR) spectroscopy, The NMR spectra of liquids provide detailed structural information that may be deduced from the chemical shifts and spin-spin coupling, that are evident in the narrow resonances, arising from some of the nuclear broadening interactions being reduced to zero. In the solid state, all of the nuclear broadening interactions are present and broad lines in the NMR spectrum are observed. Current techniques employed to reduce the line widths in solids are based on coherent averaging techniques such as MAS NMR1,2 which can remove first order interactions. Recently DOR3 and DAS4 have become available to remove higher order interactions. SINNMR (Sonically Induced Narrowing of the NMR spectra of solids) has been reported by Homer et al5 and developed by Homer and Howard6 to reduce the line widths of solids. The basis of their work is the proposal that a colloidal suspension of solid particles can be made to move like large molecules by using ultrasonic agitation. The advantage of the technique is that the particles move incoherently removing all of the nuclear interactions responsible for broad lines. This thesis describes work on the extension of SINNMR by showing that the line width of 27AI and 11B for the glass Na20/B203/AI203 can be reduced by placing solid particles in a colloidal suspension. Further line width reduction is possible by applying ultrasound, at 2 MHz, of sufficient intensity. It is proposed that a cavitation field is responsible for imparting sufficient rotational motion to the solid particles to partially average the nuclear interactions responsible for broad lines. Rapid stirring of the colloidal suspension generates turbulent flow, however, the motion is insufficient to narrow the line widths for 27AI in the glass. Investigations of sonochemical reactions for in situ rate measurements by NMR have been made. 8y using the Weissler reaction7, it has been shown that ultrasonic cavitation is possible up to 10MHz. Preliminary studies have been carried out into the rate of ultrasonic polymerisation of methylmethacrylate by NMR. Long range order in liquid crystals can imposed when they are aligned in the presence a magnetic field. The degree of alignment can be monitored by NMR using, for example a deuterated solute added to the liquid crystal8. Ultrasonic streaming can then be employed to deflect the directors of the liquid crystal from their equilibrium position, resulting in a change In the NMR spectrum. The angle of deflection has been found for the thermotropic liquid crystal (I35) to be ca, 35° and for the lyotropic (ZLI-1167) to be ca, 20°, Mechanical stirring can used to re- orientate the liquid crystal but was found to give a smaller deflection, In a separate study, that did not use ultrasound, it has been found that the signal to noise ratio of 13C NMR signals can be enhanced by rapidly stirring a Iiquid. Accelerating the diffusion of nuclei out of the coil region enables M0 to be re-established more rapidly than the normal relaxation process. This allows the pulse repetition rate to be reduced without saturating the spin system. The influence of varying the relaxation delay, acquisition time and inter-pulse delay have been studied and parameters optimised. By studying cholesterol the technique was found to be most effective for nuclei with long relaxation times, such as quaternary carbon sites. Key Worde: NMR, Ulf.rasciund, 1,.lqi.fi!:l cryllltalt!h SCll1C1otlemlstryl I!r1hano~d algnflllf

The use of organotellurium heterocycles as precursors for novel organometallic compounds

The reactions of group 16 heterocycles with organometallic reagents are described. Thiophenes have been used as models for organic sulfur in coal and their reactivity towards triiron dodecacarbonyl has been investigated. Reaction of unsubstituted thiophene with Fe3(CO)12 results in desulfurisation of the heterocycle, with the organic fragment being recovered in the form of the ferrole, C4H4.Fe2(CO)6. In addition a novel organometallic compound of iron is isolated, the formula of which is shown to be C4H4.Fe3(CO)8. Bezothiophene reacts with Fe3(CO)12 to yield benzothiaferrole, C8H6S.Fe2(CO)6, in which the sulfur is retained in the heterocycle. Dibenzothiophene, a more accurate model for organic sulfur in coal, displays no reactivity towards the iron carbonyl, suggesting that the more condensed systems will desulfurise less readily. Microwave methodology has been successful in accelerating the reactions of thiophenes with Fe3(CO)12. However, reaction of benzothiophene does not proceed to the desulfurisation stage while dibenzothiophene is unreactive even under microwave conditions. Tellurophenes (Te analogues of thiophenes) are shown to mimic the behaviour of thiophenes towards certain organometallic reagents with the advantage that their greater reactivity enables recovery of products in higher yields. Hence, reaction of tellurophene with Fe3(CO)12 again affords the ferrole but with an almost ten-fold increase in yield over thiophene. More significantly, dibenzotellurophene is also detellurated by the iron carbonyl affording the previously inaccessible dibenzoferrole, C12H8.Fe2(CO)6, thereby demonstrating the mechanistic feasibility of dechalcogenation of the more condensed aromatic molecules. The potential of tellurium heterocycles to act as precursors for novel organometallics is also recognised owing to the relatively facile elimination of the heteroatom from these systems. Thus, 2-telluraindane reacts with Fe3(CO)12 to yield a novel organometallic compound of formula C16H16.Fe(CO)3, arising from the unsymmetric dimerisation of two organic fragments.

Photochemical degradation of polystyrene

In this work the oxidative degradation of pure polystyrene, polybutadiene and butadiene-modified polystyrene (normally called high impact polystyrene or HIPS) have been studied using a variety of physical and chemical techniques. The changes in dynamic-mechanical properties occurring during the ultra-violet light accelerated weathering of these polymers were followed by a visco-elastometric technique (Rheovibron) in the solid phase over a wide temperature range. Selective cross-linking of the polybutadiene in high-impact polystyrene caused the depression of the low temperature damping peak (tan d) with a corresponding sharp peak in tan d at ambient temperature accompanied by an integral rise in complex modulus. During the same period of photoxidation, the hydroperoxide concentration and gel content increased rapidly, reaching a maximum before decomposing photolytically with the destruction of unsaturation and with the formation of stable oxidation products. Infra-red spectroscopy showed the formation of carbonyl and hydroxyl groups. a,ß-unsaturated carbonyl was also identified and was formed by decomposition of both allylic hydroperoxide and initial peroxidic gel by ß-scission of the graft between polybutadiene and polystyrene. With further photoxidation a more stable ether gel was formed involving the destruction of the conjugating double bond of a,ß-unsaturated carbonyl. Addition of saturated and unsaturated ketones which are potential sensitisers of photoxidation to high-impact polystyrene and polybutadiene failed to photo-initiate the oxygen absorption of the polymers. A prior thermal oxidative treatment on the other hand eliminated the auto- accelerating stage leading to linear kinetics as the concentration of thermally-produced hydroperoxide approached a maximum. Antioxidants which act by destroying hydroperoxide lengthened the induction period to rapid oxygen absorption, whilst a phenolic antioxidant behaved as a weak photo-activator initially and a retarder later. Prior photolysis of high-impact polystyrene photo-activated the unsaturated component and caused similar changes in dynamic-mechanical properties to those found during photoxidation although at a much lower rate. Polybutadiene behaves as a photo-pro-oxidant for the destruction of polystyrene in high-impact polystyrene.

The durability of glass fibre reinforced cements made with new cementitious matrices

Widespread use of glass fibre reinforced cement (GRC) has been impeded by concerns over its durability. Three degradation mechanisms are proposed - fibre corrosion, Ca(OHh precipitation and matrix densification - although their relative importance is debated. Matrices with reduced alkalinities and Ca(OH)2 contents are being developed; the aim of this study was to investigate their hydration and interaction with alkali-resistant fibres to determine the factors controlling their long-term durability, and assess the relevancy of accelerated ageing. The matrices studied were: OPC/calcium-sulphoaluminate cement plus metakaolin (C); OPC plus metakaolin (M); blast-furnace slag cement plus a micro-silica based additive (D); and OPC (O). Accelerated ageing included hot water and cyclic regimes prior to tensile testing. Investigations included pore solution expression, XRD, DTA/TG, SEM and optical petrography. Bond strength was determined from crack spacings using microstructural parameters obtained from a unique image analysis technique. It was found that, for the new matrices - pore solution alkalinities were lower; Ca(OH)2 was absent or quickly consumed; different hydrates were formed at higher immersion temperatures; degradation under 65°C immersion was an order of magnitude slower, and no interfilamental Ca(OH)2 was observed .It was concluded that: fibre weakening caused by flaw growth was the primary degradation mechanism and was successfully modelled on stress corrosion/static fatigue principles. OPC inferiority was attributed partly to its higher alkalinity but chiefly to the growth of Ca(OH)2 aggravating the degradation; and hot water ageing although useful in model formulation and contrasting the matrices, changed the intrinsic nature of the composites rather than simply accelerating the degradation mechanisms.

The influence of Arabian Gulf environment on mechanisms of reinforcement corrosion

The reduction in the useful-service life of reinforced concrete construction in the Arabian Gulf is attributed to reinforcement corrosion. While this phenomenon is primarily related to chloride ions, the concomitant pressure of sulfate salts may accelerate the deterioration process. Another factor which might influence reinforcement corrosion is the elevated ambient temperature. While few studies have been conducted to evaluate the individual effect of sulfate contamination and temperature on chloride binding and reinforcement corrosion, the synergistic effect of these factors on concrete durability, viz.-a-viz., reinforcement corrosion, needs to be evaluated. Further, the environmental conditions of the Arabian Gulf are also conducive for accelerated carbonation. However, no data are available on the concomitant effect of chloride-sulfate contamination and elevated temperature on the carbonation behaviour of plain and blended cements.This study was conducted to evaluate the conjoint effect of chloride-sulfate contamination and temperature on the pore solution chemistry and reinforcement corrosion. The effect of chloride-sulfate contamination and elevated temperature on carbonation in plain and blended cements was also investigated. Pore solution extraction and analysis, X-ray diffraction, differential thermal analysis, scanning electron microscopy, DC linear polarization resistance and AC impedance spectroscopy techniques were utilized to study the effect of experimental parameters on chloride binding, reinforcement corrosion and carbonation.The results indicated that the concomitant presence of chloride and sulfate salts and temperature significantly influences the durability performance of concrete by: (i) decreasing the chloride binding, (ii) increasing reinforcement corrosion, and (iii) accelerating the carbonation process. To avoid such deterioration, it is advisable to minimize both chloride and sulfate contamination contributed by the mixture ingredients. Due to the known harmful role of sulfate ions in decreasing the chloride binding and increasing reinforcement corrosion, limits on allowable sulfate contamination in concrete should also be established.

Polypropylene as a reductive agent for dehalogenation of brominated organic compounds

A new method for debromination of organics by a reductive medium like polypropylene is investigated. The reaction is carried out in inert atmosphere to avoid rapid oxidation of the polymer. Through this detoxification procedure, hydrogen bromide and small brominated alkanes are formed. Experiments in closed ampoules are carried out with tetrabromobisphenol A, dibromophenol, pentabromodiphenyl ether, dichlorophenol and an oil formed by pyrolysis of printed circuit boards in the Haloclean® process. The reaction is examined under isothermal conditions in a temperature range between 300 and 400°C and a residence time between 10 and 30 min. Optimal conditions were found at 350°C and at a residence time of 20 min. As chlorinated phenols are not destroyed under these conditions, the process may be a valuable procedure to gain hydrogen bromide out of mixtures of halogenated feed materials. Also, under atmospheric pressure, a reaction between polypropylene and brominated compounds takes place as could be proved by thermogravimetric analysis. Bromobenzene has an accelerating effect on the rate of weight loss of the polymer, but at higher concentrations, it can also be slowed down. © 2003 Elsevier Ltd. All rights reserved.

Advances in characterisation, calibration and data processing speed of optical coherence tomography systems

This thesis describes advances in the characterisation, calibration and data processing of optical coherence tomography (OCT) systems. Femtosecond (fs) laser inscription was used for producing OCT-phantoms. Transparent materials are generally inert to infra-red radiations, but with fs lasers material modification occurs via non-linear processes when the highly focused light source interacts with the materials. This modification is confined to the focal volume and is highly reproducible. In order to select the best inscription parameters, combination of different inscription parameters were tested, using three fs laser systems, with different operating properties, on a variety of materials. This facilitated the understanding of the key characteristics of the produced structures with the aim of producing viable OCT-phantoms. Finally, OCT-phantoms were successfully designed and fabricated in fused silica. The use of these phantoms to characterise many properties (resolution, distortion, sensitivity decay, scan linearity) of an OCT system was demonstrated. Quantitative methods were developed to support the characterisation of an OCT system collecting images from phantoms and also to improve the quality of the OCT images. Characterisation methods include the measurement of the spatially variant resolution (point spread function (PSF) and modulation transfer function (MTF)), sensitivity and distortion. Processing of OCT data is a computer intensive process. Standard central processing unit (CPU) based processing might take several minutes to a few hours to process acquired data, thus data processing is a significant bottleneck. An alternative choice is to use expensive hardware-based processing such as field programmable gate arrays (FPGAs). However, recently graphics processing unit (GPU) based data processing methods have been developed to minimize this data processing and rendering time. These processing techniques include standard-processing methods which includes a set of algorithms to process the raw data (interference) obtained by the detector and generate A-scans. The work presented here describes accelerated data processing and post processing techniques for OCT systems. The GPU based processing developed, during the PhD, was later implemented into a custom built Fourier domain optical coherence tomography (FD-OCT) system. This system currently processes and renders data in real time. Processing throughput of this system is currently limited by the camera capture rate. OCTphantoms have been heavily used for the qualitative characterization and adjustment/ fine tuning of the operating conditions of OCT system. Currently, investigations are under way to characterize OCT systems using our phantoms. The work presented in this thesis demonstrate several novel techniques of fabricating OCT-phantoms and accelerating OCT data processing using GPUs. In the process of developing phantoms and quantitative methods, a thorough understanding and practical knowledge of OCT and fs laser processing systems was developed. This understanding leads to several novel pieces of research that are not only relevant to OCT but have broader importance. For example, extensive understanding of the properties of fs inscribed structures will be useful in other photonic application such as making of phase mask, wave guides and microfluidic channels. Acceleration of data processing with GPUs is also useful in other fields.

On the Coherence Between Probability and Possibility Measures

* This paper is supported by CICYT (Spain) under Project TIN 2005-08943-C02-01.

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.

Designing immunogenic peptides

Peptides fulfill many roles in immunology, yet none are more important than their role as immunogenic epitopes driving the adaptive immune response, our ultimate bulwark against infectious disease. Peptide epitopes are mediated primarily by their interaction with major histocompatibility complexes (T-cell epitopes) and antibodies (B-cell epitopes). As pathogen genomes continue to be revealed, both experimental and computational epitope mapping are becoming crucial tools in vaccine discovery1,2. Immunoinformatics offers many tools, techniques and approaches for in silico epitope characterization, which is capable of greatly accelerating epitope design. © 2013 Nature America, Inc. All rights reserved.