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.

Digital Collections in Regional Public Library “P. R. Slaveikov” – Veliko Tarnovo in the Context of the National Program for Preservation and Promotion of Literary Heritage

Nowadays the great public libraries in Bulgaria are gaining the appearance of digital centers which provide new informational resources and services in the digital space. The digital conversion as a way of preservation is one of the important priorities of Regional Public Library in Veliko Tarnovo. In the last few years we persistently search for possible ways of financing by national and foreign programs in this direction. In the beginning the strategy was oriented to digitalization of the funds with most urgent conversion – these of the local studies periodicals from 1878 till 1944 year. The digitalization of funds will create conditions of laying the basement of full text database of Bulgarian periodical publications. The technology that is offered gives opportunities for including other libraries in the Unified index, which can develop it into a National Unified index of periodical publications. The integrated informational environment that is created is an attractive, comfortable and useful place for work in home or at work for researchers, historians, art experts, bibliographers. The library readers use very actively all informational services of the library internet page and work competently with the on-line indexes provided there, they find the necessary title, which can be demanded later for usage in home or in the library, using electronic means again.

Compilation of downward flux observations from sediment trap deployments in the Atlantic Ocean - Contribution to EURO-BASIN's Data integration

We provide a compilation of downward fluxes (total mass, POC, PON, BSiO2, CaCO3, PIC and lithogenic/terrigenous fluxes) from over 6000 sediment trap measurements distributed in the Atlantic Ocean, from 30 degree North to 49 degree South, and covering the period 1982-2011. Data from the Mediterranean Sea are also included. Data were compiled from different sources: data repositories (BCO-DMO, PANGAEA), time series sites (BATS, CARIACO), published scientific papers and/or personal communications from PI's. All sources are specifed in the data set. Data from the World Ocean Atlas 2009 were extracted to provide each flux observation with contextual environmental data, such as temperature, salinity, oxygen (concentration, AOU and percentage saturation), nitrate, phosphate and silicate.

Spatially explicit estimates of stock size, structure and biomass of North Atlantic albacore tuna (Thunnus alalunga) in the North Atlantic for the period 1956-2010, compiled from the International Commission for the Conservation of Atlantic Tunas

The development of the ecosystem approach and models for the management of ocean marine resources requires easy access to standard validated datasets of historical catch data for the main exploited species. They are used to measure the impact of biomass removal by fisheries and to evaluate the models skills, while the use of standard dataset facilitates models inter-comparison. North Atlantic albacore tuna is exploited all year round by longline and in summer and autumn by surface fisheries and fishery statistics compiled by the International Commission for the Conservation of Atlantic Tunas (ICCAT). Catch and effort with geographical coordinates at monthly spatial resolution of 1° or 5° squares were extracted for this species with a careful definition of fisheries and data screening. In total, thirteen fisheries were defined for the period 1956-2010, with fishing gears longline, troll, mid-water trawl and bait fishing. However, the spatialized catch effort data available in ICCAT database represent a fraction of the entire total catch. Length frequencies of catch were also extracted according to the definition of fisheries above for the period 1956-2010 with a quarterly temporal resolution and spatial resolutions varying from 1°x 1° to 10°x 20°. The resolution used to measure the fish also varies with size-bins of 1, 2 or 5 cm (Fork Length). The screening of data allowed detecting inconsistencies with a relatively large number of samples larger than 150 cm while all studies on the growth of albacore suggest that fish rarely grow up over 130 cm. Therefore, a threshold value of 130 cm has been arbitrarily fixed and all length frequency data above this value removed from the original data set.

The production effect: the role of attentional resources

The production effect is the benefit in memory found for produced (i.e., read aloud) words relative to words read silently. It is proposed that the production effect occurs as a result of the enhanced distinctiveness associated with the produced items. The current research investigated whether attentional resources are required to encode and/or retrieve the distinctive information associated with the produced words. The literature suggests that the encoding of this distinctive information occurs automatically, but at test, purposeful attention is required to retrieve this distinctive information. To test this, participants read words aloud and silently, under either full or divided attention. Participants then completed either a recognition (Experiment 1) or free recall (Experiment 2) memory test under either full or divided attention. The findings show that when attention is divided at encoding, the benefit for aloud words remains for both recognition and free recall. When attention is divided at test, however, the benefit for aloud words remains for recognition but is absent for free recall. Overall, these results suggest that the distinctive information associated with produced words is encoded automatically, but it may not be accessible at test under attentionally demanding conditions.

A noção de relação na teoria linguística de Ferdinand de Saussure

Our work aims at investigating the notion of relation in Ferdinand de Saussure’s linguistic reflections. As we observe his theoretical production, we see this notion was amply worked in the elaboration of other concepts of his, for example, language, signe, value, arbitrariness, among others. However, beyond the capacity of the notion of relation to produce other concepts, the way it was conceived and the theoretical space it was placed allowed Saussure to break through the essentially historical-comparative studies of the epoch, what made a new way of thinking language to have its starting point. Taking it into account, firstly, by reading the work of Herman Paul, the one who represents the neogrammarians in our text, we examine in which way Saussure is distant from his contemporary linguistics when he distinguishes the linguistic relations and give them a new status in his investigation. Secondly, we study how the notion of relation has it own place in Saussure’s theoretical process of thinking the fundamentals which later would be considered the milestone for Modern Linguistics. For that, we analyse one of his manuscripts, Notes pour un livre sur la linguistique générale, and the Course in General Linguistics. The first is composed by sketches which would become a book, as Saussure promised in a letter to Meillet; and the latter contains the principals for the science of Linguistics. So we aim at researching how the notion of relation is treated in this corpus in order to see the way it is conceived both in the particular production of a manuscript written by Saussure itself and the book which was published and considered the turning point for Linguistics.

Woven cultures : New insights into Pictish and Viking culture contact using the implements of textile production

This paper is based on the undergraduate dissertation of Lindsey Stirling, which was supervised by Karen Milek, and which won the Society of Medieval Archaeology's John Hurst Memorial Dissertation Prize Acknowledgements This research would not have been possible without the assistance of Dr Martin Goldberg at the National Museum of Scotland, Lynda Aiano at Tankerness House Museum, Orkney, and Beverley Ballin Smith. The authors also wish to thank the two anonymous reviewers who provided valuable comments on earlier versions of this paper.

Woven cultures : New insights into Pictish and Viking culture contact using the implements of textile production

This paper is based on the undergraduate dissertation of Lindsey Stirling, which was supervised by Karen Milek, and which won the Society of Medieval Archaeology's John Hurst Memorial Dissertation Prize Acknowledgements This research would not have been possible without the assistance of Dr Martin Goldberg at the National Museum of Scotland, Lynda Aiano at Tankerness House Museum, Orkney, and Beverley Ballin Smith. The authors also wish to thank the two anonymous reviewers who provided valuable comments on earlier versions of this paper.

Asymmetric 3D video coding based on regions of perceptual relevance

This dissertation presents a study and experimental research on asymmetric coding of stereoscopic video. A review on 3D technologies, video formats and coding is rst presented and then particular emphasis is given to asymmetric coding of 3D content and performance evaluation methods, based on subjective measures, of methods using asymmetric coding. The research objective was de ned to be an extension of the current concept of asymmetric coding for stereo video. To achieve this objective the rst step consists in de ning regions in the spatial dimension of auxiliary view with di erent perceptual relevance within the stereo pair, which are identi ed by a binary mask. Then these regions are encoded with better quality (lower quantisation) for the most relevant ones and worse quality (higher quantisation) for the those with lower perceptual relevance. The actual estimation of the relevance of a given region is based on a measure of disparity according to the absolute di erence between views. To allow encoding of a stereo sequence using this method, a reference H.264/MVC encoder (JM) has been modi ed to allow additional con guration parameters and inputs. The nal encoder is still standard compliant. In order to show the viability of the method subjective assessment tests were performed over a wide range of objective qualities of the auxiliary view. The results of these tests allow us to prove 3 main goals. First, it is shown that the proposed method can be more e cient than traditional asymmetric coding when encoding stereo video at higher qualities/rates. The method can also be used to extend the threshold at which uniform asymmetric coding methods start to have an impact on the subjective quality perceived by the observers. Finally the issue of eye dominance is addressed. Results from stereo still images displayed over a short period of time showed it has little or no impact on the proposed method.

Biomass of large protists from the Infrakingdom Rhizaria in the global ocean

The present data set is a worldwide compilation from 11 oceanographic expeditions during which an underwater vision profiler (UVP) was deployed in situ to determine the vertical distribution (biomass) of 4 taxonomic groups of plankton larger than 600 µm, belonging to the Infrakingdom Rhizaria, including Collodaria, Acantharia, Phaeodaria and other Rhizaria. Vertical distributions are binned in four layers: 0-100, 0-200, 100-500 and 0-500 m.

Enigmas monetarios en el derecho local. Los mencales y maravedís en los fueros castellanos

La historia de la moneda en la Castilla medieval ha estado siempre mediatizada por la convivencia no siempre armónica entre dos sistemas monetarios muy diferentes. Uno basado en la plata, de origen europeo, otro centrado en el oro, de raíces árabes. La necesidad de conectar y de establecer unas equivalencias entre ellos se convirtió pronto en una necesidad, máxime cuando las monedas de oro incrementaron sus variantes. En esta compleja situación aparecen mencales y maravedís citados conjuntamente en muchos fueros: Zorita, Uclés, Cuenca,... sin que su naturaleza quede del todo clara. Este artículo compara estas referencias y analiza las equivalencias que los unen. Como colofón al trabajo podemos afirmar que los maravedís citados en cada texto corresponden a monedas áureas de distinto peso y valor mientras el mencal es identificado con un ponderal de oro que los relaciona.

La gárgola en la cultura visual actual

La gárgola ha poblado desde antiguo las cornisas de los edificios religiosos y civiles, sirviendo de evacuación para el agua y para diferentes misiones según las distintas interpretaciones. Su gran componente simbólico hace de ella un reclamo muy interesante para el mundo audiovisual actual. De entre las manifestaciones encontradas, estudiaremos la visión de la gárgola en estos medios y valoraremos su adecuación o no a la visión medieval. Mediante la aparición de la gárgola medieval en el mundo actual en forma de personajes y ornamentos, y gracias a la aplicación de imágenes actuales a las nuevas gárgolas, podemos realizar un recorrido visual y simbólico alrededor de las distintas visiones que la cultura actual realiza de ellas. Pero no podemos quedarnos ahí, es necesario relacionar estas nuevas visiones y significaciones con las originales, para establecer si se ha hecho un uso correcto de la imagen, o se ha roto la estructura del símbolo primigenio.

Fronteras en construcción: Diferenciación social y desterritorialización en el Sur ibérico (siglos IX-VI a.C.)

Durante las tres últimas décadas, numerosos/as arqueólogos/as han discutido extensamente sobre el ritual funerario original de las poblaciones del sur ibérico entre los siglos ix y vi a.c., esto es, cremación o inhumación. Este debate está además conectado con la existencia o no de complejidad social antes de la llegada fenicia, con la aparición de una élite “orientalizada” y con la adopción de nuevos objetos y prácticas por las poblaciones locales. En este artículo hago uso del concepto deleuziano de “desterritorialización” y lo asocio con el de “frontera” desarrollado por anzaldúa para interpretar la sociedad del sur ibérico. Para ello, analizo la evidencia funeraria indígena y cuestiono la división estricta entre cremación e inhumación en la región; así como examino la profundidad y significado de los cambios funerarios en las comunidades locales.

Ruxia i Ruxianos w romansach rycerskich autorstwa Feliciano de Silva

W niniejszym artykule przeanalizowany został sposób wykorzystania przez Feliciano de Silva autentycznego toponimu Ruxia (Ruś) przy konstruowaniu świata przedstawionego w romansie rycerskim. Jak zostaje wykazane, Feliciano de Silva jedynie częściowo wpisuje się w tendencję zaobserwowaną wcześniej m.in. u Garci Rodrigueza de Montalvo, który w swoich dziełach nakreśla jasną opozycję pomiędzy chrześcijańskim światem błędnego rycerstwa a złym i nikczemnym pogaństwem. Autor z Ciudad Rodrigo, chcąc nawiązać do chwały średniowiecznego Konstantynopola, powraca do czasów konfliktu bizantyńsko-ruskiego, który twórczo wykorzystuje w aż trzech księgach Florisel de Niquea. Ruxia, jeden z głównych przeciwników greckiego rodu, przedstawiona zostaje jako pogańska potęga militarna, której przewodzą władcy odważni, lecz zarazem owładnięci żądzą, niemoralni i okrutni.

El color en los códices prehispánicos del México Central: identificación material, cualidad plástica y valor estético

Este artículo investiga algunos de los valores plásticos y estéticos que presidieron la selección y la preparación de las materias colorantes empleadas para iluminar los códices creados por los nahuas del México Central durante el Posclásico Tardío. Estos códices son interesantes porque análisis arqueométricos y exámenes codicológicos recientes han permitido conocer la materialidad de su capa pictórica, así como las características formales y el comportamiento de los colores en estas obras. Uno de los aportes trascendentales de estos estudios ha sido averiguar que la paleta cromática que sirvió para pintar los códices del México Central era principalmente de origen orgánico, lo que contrasta con la naturaleza de los pigmentos detectados en restos de pintura mural y en esculturas creadas por los nahuas que son sobre todo minerales. El objetivo de este artículo es reflexionar sobre las razones de esas diferencias y demostrar que el uso de los colorantes orgánicos en los códices respondía a un fin plástico específico que concordaba con el canon estético imperante en la sociedad náhuatl.