Nucleosomes, linker DNA, and linker histone form a unique structural motif that directs the higher-order folding and compaction of chromatin

The compaction level of arrays of nucleosomes may be understood in terms of the balance between the self-repulsion of DNA (principally linker DNA) and countering factors including the ionic strength and composition of the medium, the highly basic N termini of the core histones, and linker histones. However, the structural principles that come into play during the transition from a loose chain of nucleosomes to a compact 30-nm chromatin fiber have been difficult to establish, and the arrangement of nucleosomes and linker DNA in condensed chromatin fibers has never been fully resolved. Based on images of the solution conformation of native chromatin and fully defined chromatin arrays obtained by electron cryomicroscopy, we report a linker histone-dependent architectural motif beyond the level of the nucleosome core particle that takes the form of a stem-like organization of the entering and exiting linker DNA segments. DNA completes ≈1.7 turns on the histone octamer in the presence and absence of linker histone. When linker histone is present, the two linker DNA segments become juxtaposed ≈8 nm from the nucleosome center and remain apposed for 3–5 nm before diverging. We propose that this stem motif directs the arrangement of nucleosomes and linker DNA within the chromatin fiber, establishing a unique three-dimensional zigzag folding pattern that is conserved during compaction. Such an arrangement with peripherally arranged nucleosomes and internal linker DNA segments is fully consistent with observations in intact nuclei and also allows dramatic changes in compaction level to occur without a concomitant change in topology.

The Balance of RanBP1 and RCC1 Is Critical for Nuclear Assembly and Nuclear Transport

Ran is a small GTPase that is essential for nuclear transport, mRNA processing, maintenance of structural integrity of nuclei, and cell cycle control. RanBP1 is a highly conserved Ran guanine nucleotide dissociation inhibitor. We sought to use Xenopus egg extracts for the development of an in vitro assay for RanBP1 activity in nuclear assembly, protein import, and DNA replication. Surprisingly, when we used anti-RanBP1 antibodies to immunodeplete RanBP1 from Xenopus egg extracts, we found that the extracts were also depleted of RCC1, Ran’s guanine nucleotide exchange factor, suggesting that these proteins form a stable complex. In contrast to previous observations using extracts that had been depleted of RCC1 only, extracts lacking both RanBP1 and RCC1 (codepleted extracts) did not exhibit defects in assays of nuclear assembly, nuclear transport, or DNA replication. Addition of either recombinant RanBP1 or RCC1 to codepleted extracts to restore only one of the depleted proteins caused abnormal nuclear assembly and inhibited nuclear transport and DNA replication in a manner that could be rescued by further addition of RCC1 or RanBP1, respectively. Exogenous mutant Ran proteins could partially rescue nuclear function in extracts without RanBP1 or without RCC1, in a manner that was correlated with their nucleotide binding state. These results suggest that little RanBP1 or RCC1 is required for nuclear assembly, nuclear import, or DNA replication in the absence of the other protein. The results further suggest that the balance of GTP- and GDP-Ran is critical for proper nuclear assembly and function in vitro.

Structural evolution of the Lesser Himalayan fold-thrust belt of west central Nepal

The Lesser Himalayan fold-thrust belt on the south flank of the Jajarkot klippe in west central Nepal was mapped in detail between the Main Central thrust in the north and the Main Boundary thrust in the south. South of the Jajarkot klippe, the fold-thrust belt involves sandstone, shale and carbonate rocks that are unmetamorphosed in the foreland and increase in metamorphic grade with higher structural position to sub-greenschist facies towards the hinterland. The exposed stratigraphy is correlative with the Proterozoic Ranimata, Sangram, Galyang, Syangia Formations and Lakharpata Group of Western Nepal and overlain by the Paleozoic Tansen and Kali Gandaki Groups. Based on field mapping and cross-section construction, three distinct thrust sheets were identified separated by top-to-the-south thrust faults. From the foreland (south) to the hinterland (north), the first thrust sheet in the immediate hanging wall of the Main Boundary thrust defines an open syncline. The second thrust sheet contains a very broad synformal duplex, which is structurally stacked against the third thrust sheet containing a homoclinal panel of the oldest exposed Proterozoic stratigraphy. Outcrop scale folds throughout the study area are predominantly south vergent, open, and asymmetric reflecting the larger regional scale folding style, which corroborate the top-to-the-south deformation style seen in the faults of the region. Field techniques were complemented with microstructural and quartz crystallographic c-axis preferred orientation analyses using a petrographic microscope and a fabric analyzer, respectively. Microstructural analysis identified abundant strain-induced recrystallization textures and occasional occurrences of top-to-the-south shear-sense indicators primarily in the hinterland rocks in the immediate footwall of the Main Central Thrust. Top-to-the-south shearing is also supported by quartz crystallographic c-axis preferred orientations. Quartz recrystallization textures indicate an increase in deformation temperature towards the Main Central thrust. A line balance estimate indicates that approximately 15 km of crustal shortening was accommodated by folding and faulting in the fold-thrust belt south of the Jajarkot klippe. Additionally, estimations of shortening velocity suggest that the shortening velocity operating in this section of the fold-thrust belt between 23 to 14 Ma was slower than what is currently observed as a result of the ongoing deformation of the Sub-Himalayan fold-thrust belt.

Effectiveness and transmission of the ECB’s balance sheet policies. National Bank of Belgium Working Paper No. 275, December 2014

We estimate the effects of exogenous innovations to the balance sheet of the ECB since the start of the financial crisis within a structural VAR framework. An expansionary balance sheet shock stimulates bank lending, stabilizes financial markets, and has a positive impact on economic activity and prices. The effects on bank lending and output turn out to be smaller in the member countries that have been more affected by the financial crisis, in particular those countries where the banking system is less well-capitalized.

Simplest tasks have greatest dual task interference with balance in brain injured adults

Attention difficulties and poor balance are both common sequel following a brain injury. This study aimed to determine whether brain injured adults had greater difficulty than controls in performing a basic balance task while concurrently completing several different cognitive tasks varying in visuo-spatial attentional load and complexity. Twenty brain injured adults and 20 age-, sex- and education level-matched controls performed a balance-only task (step stance held for 30s), five cognitive-only tasks (simple and complex non-spatial, visuo-spatial, and a control articulation task), and both together (dual tasks). Brain injured adults showed a greater centre of pressure (COP) excursion and velocity in all conditions than controls. Brain injured adults also demonstrated greater interference with balance when concurrently performing two cognitive tasks than control subjects. These were the control articulation and the simple non-spatial task. It is likely that distractibility during these simple tasks contributed to an increase in COP motion and interference with postural stability in stance. Performing visuo-spatial tasks concurrently with the balance task did not result in any change in COP motion. Dual task interference in this group is thus unlikely to be due to structural interference. Similarly, as the more complex tasks did not uniformly result in increased interference, a reduction in attentional capacity in the brain injured population is unlikely to be the primary cause of dual task interference in this group. (C) 2004 Elsevier B.V. All rights reserved.

Wurst: a protein threading server with a structural scoring function, sequence profiles and optimized substitution matrices

Wurst is a protein threading program with an emphasis on high quality sequence to structure alignments (http://www.zbh.uni-hamburg.de/wurst). Submitted sequences are aligned to each of about 3000 templates with a conventional dynamic programming algorithm, but using a score function with sophisticated structure and sequence terms. The structure terms are a log-odds probability of sequence to structure fragment compatibility, obtained from a Bayesian classification procedure. A simplex optimization was used to optimize the sequence-based terms for the goal of alignment and model quality and to balance the sequence and structural contributions against each other. Both sequence and structural terms operate with sequence profiles.

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.

Hijacked then lost in translation:the plight of the recombinant host cell in membrane protein structural biology projects

Membrane protein structural biology is critically dependent upon the supply of high-quality protein. Over the last few years, the value of crystallising biochemically characterised, recombinant targets that incorporate stabilising mutations has been established. Nonetheless, obtaining sufficient yields of many recombinant membrane proteins is still a major challenge. Solutions are now emerging based on an improved understanding of recombinant host cells; as a 'cell factory' each cell is tasked with managing limited resources to simultaneously balance its own growth demands with those imposed by an expression plasmid. This review examines emerging insights into the role of translation and protein folding in defining high-yielding recombinant membrane protein production in a range of host cells.

Structural evolution of the Lesser Himalayan fold-thrust belt of west central Nepal

The Lesser Himalayan fold-thrust belt on the south flank of the Jajarkot klippe in west central Nepal was mapped in detail between the Main Central thrust in the north and the Main Boundary thrust in the south. South of the Jajarkot klippe, the fold-thrust belt involves sandstone, shale and carbonate rocks that are unmetamorphosed in the foreland and increase in metamorphic grade with higher structural position to sub-greenschist facies towards the hinterland. The exposed stratigraphy is correlative with the Proterozoic Ranimata, Sangram, Galyang, Syangia Formations and Lakharpata Group of Western Nepal and overlain by the Paleozoic Tansen and Kali Gandaki Groups. Based on field mapping and cross-section construction, three distinct thrust sheets were identified separated by top-to-the-south thrust faults. From the foreland (south) to the hinterland (north), the first thrust sheet in the immediate hanging wall of the Main Boundary thrust defines an open syncline. The second thrust sheet contains a very broad synformal duplex, which is structurally stacked against the third thrust sheet containing a homoclinal panel of the oldest exposed Proterozoic stratigraphy. Outcrop scale folds throughout the study area are predominantly south vergent, open, and asymmetric reflecting the larger regional scale folding style, which corroborate the top-to-the-south deformation style seen in the faults of the region. Field techniques were complemented with microstructural and quartz crystallographic c-axis preferred orientation analyses using a petrographic microscope and a fabric analyzer, respectively. Microstructural analysis identified abundant strain-induced recrystallization textures and occasional occurrences of top-to-the-south shear-sense indicators primarily in the hinterland rocks in the immediate footwall of the Main Central Thrust. Top-to-the-south shearing is also supported by quartz crystallographic c-axis preferred orientations. Quartz recrystallization textures indicate an increase in deformation temperature towards the Main Central thrust. A line balance estimate indicates that approximately 15 km of crustal shortening was accommodated by folding and faulting in the fold-thrust belt south of the Jajarkot klippe. Additionally, estimations of shortening velocity suggest that the shortening velocity operating in this section of the fold-thrust belt between 23 to 14 Ma was slower than what is currently observed as a result of the ongoing deformation of the Sub-Himalayan fold-thrust belt.

The Importance of Work-Life Balance Opportunities and Support from the Perspective of Nursing Stu-dents in Dalarna, Sweden

There is a shortage of nurses leading to challenges in recruitment in Sweden and many other countries. Especially for less populated regions recruitment can be chal-lenging. Nurses often face difficulties with work-life balance (WLB). This study aims to identify the importance of WLB opportunities and support that make a work-place attractive from the perspective of nursing students studying in Dalarna. A questionnaire was distributed via email to 525 students enrolled in the nursing bach-elor program at Dalarna University. They were asked to rate the importance of 15 sub questions regarding WLB opportunities and support. These sub questions were asked in order to analyze the importance of 15 components regarding WLB oppor-tunities and support. 196 students (37 percent) answered the questionnaire. Three WLB components, working from home, childcare and rooms for breastfeeding, were found to be not important to nursing students studying in Dalarna. This was reason-able due to the profession of nursing and the WLB support provided by the Swedish government. Cultural factors, such as the organization being positive towards using WLB opportunities and support, were more important than structural factors, such as the possibility to work part-time. Moreover, to have a manager that is supportive towards using WLB opportunities and support was found to be the most important factor and having workplace practical support such as childcare was found least im-portant. Furthermore, contrary to the expected results, no statistical significance was found on the influence on the importance of all combined relevant WLB opportuni-ties and support by the sociodemographic variables; gender, semester of studies, age, having children, months of work experience and work experience in the healthcare sector. However, nine individual components were found to be influ-enced by one or more sociodemographic variables. Therefore, some recommenda-tions on how to target specific groups of individuals were made. However, the con-clusion of the study is that, regardless of the sociodemographic variables and gov-ernmental support, organizations should offer new nurses opportunities and support to gain a balance between work and life, especially in terms of cultural factors.

Business Signage Control in Seattle's Chinatown International District, Pursuing Healthy Balance of Preservation and Commercial Vitalization in a Historic Commercial Districts

Thesis (Master's)--University of Washington, 2016-06

Deformation mechanisms in second-phase affected microstructures and their energy balance

Based on the relationship Zener parameter (Z=second-phase size/second-phase volume fraction) vs. calcite grain size (dg), second-phase controlled aggregates and microstructures that are weakly affected by second-phases are discriminated. The latter are characterized by large but constant grain sizes, high calcite grain boundary fractions and crystallographic preferred orientations (CPO), while calcite grain size and calcite grain boundary fraction decrease continuously and CPO weakens with decreasing Z in second-phase controlled microstructures. These observations suggest that second-phase controlled microstructures predominantly deform via granular flow because pinning of calcite grain boundaries reduces the efficiency of dynamic recrystallization favoring mass transfer processes and grain boundary sliding. In contrast, the balance of grain size reduction and growth by dynamic recrystallization maintains a steady state grain size in microstructures that are only weakly affected by second-phases promoting a predominance of dislocation creep. With increasing temperature, the relationship between Z and dg persists but the calcite grain size increases continuously. Based on microstructures, the energy of each modifying process is calculated and its relative contribution is compared with energies of the competing processes (surface energy, dragging energy, dynamic recrystallization energy). The steady state microstructures result from a temperature-dependent energy minimization procedure of the system.

Structural brain connectivity and cognitive ability differences: A multivariate distance matrix regression analysis

Neuroimaging research involves analyses of huge amounts of biological data that might or might not be related with cognition. This relationship is usually approached using univariate methods, and, therefore, correction methods are mandatory for reducing false positives. Nevertheless, the probability of false negatives is also increased. Multivariate frameworks have been proposed for helping to alleviate this balance. Here we apply multivariate distance matrix regression for the simultaneous analysis of biological and cognitive data, namely, structural connections among 82 brain regions and several latent factors estimating cognitive performance. We tested whether cognitive differences predict distances among individuals regarding their connectivity pattern. Beginning with 3,321 connections among regions, the 36 edges better predicted by the individuals' cognitive scores were selected. Cognitive scores were related to connectivity distances in both the full (3,321) and reduced (36) connectivity patterns. The selected edges connect regions distributed across the entire brain and the network defined by these edges supports high-order cognitive processes such as (a) (fluid) executive control, (b) (crystallized) recognition, learning, and language processing, and (c) visuospatial processing. This multivariate study suggests that one widespread, but limited number, of regions in the human brain, supports high-level cognitive ability differences. Hum Brain Mapp, 2016. © 2016 Wiley Periodicals, Inc.