Investigation of sequence features of hinge-bending regions in proteins with domain movements using kernel logistic regression

Background: Hinge-bending movements in proteins comprising two or more domains form a large class of functional movements. Hinge-bending regions demarcate protein domains and collectively control the domain movement. Consequently, the ability to recognise sequence features of hinge-bending regions and to be able to predict them from sequence alone would benefit various areas of protein research. For example, an understanding of how the sequence features of these regions relate to dynamic properties in multi-domain proteins would aid in the rational design of linkers in therapeutic fusion proteins. Results: The DynDom database of protein domain movements comprises sequences annotated to indicate whether the amino acid residue is located within a hinge-bending region or within an intradomain region. Using statistical methods and Kernel Logistic Regression (KLR) models, this data was used to determine sequence features that favour or disfavour hinge-bending regions. This is a difficult classification problem as the number of negative cases (intradomain residues) is much larger than the number of positive cases (hinge residues). The statistical methods and the KLR models both show that cysteine has the lowest propensity for hinge-bending regions and proline has the highest, even though it is the most rigid amino acid. As hinge-bending regions have been previously shown to occur frequently at the terminal regions of the secondary structures, the propensity for proline at these regions is likely due to its tendency to break secondary structures. The KLR models also indicate that isoleucine may act as a domain-capping residue. We have found that a quadratic KLR model outperforms a linear KLR model and that improvement in performance occurs up to very long window lengths (eighty residues) indicating long-range correlations. Conclusion: In contrast to the only other approach that focused solely on interdomain hinge-bending regions, the method provides a modest and statistically significant improvement over a random classifier. An explanation of the KLR results is that in the prediction of hinge-bending regions a long-range correlation is at play between a small number amino acids that either favour or disfavour hinge-bending regions. The resulting sequence-based prediction tool, HingeSeek, is available to run through a webserver at hingeseek.cmp.uea.ac.uk

CRISPR/Cas9-mediated gene manipulation to create single-amino-acid-substituted and floxed mice with a cloning-free method.

Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a powerful tool to manipulate the genome with extraordinary simplicity and speed. To generate genetically modified animals, CRISPR/Cas9-mediated genome editing is typically accomplished by microinjection of a mixture of Cas9 DNA/mRNA and single-guide RNA (sgRNA) into zygotes. However, sgRNAs used for this approach require manipulation via molecular cloning as well as in vitro transcription. Beyond these complexities, most mutants obtained with this traditional approach are genetically mosaic, yielding several types of cells with different genetic mutations. Recently, a growing body of studies has utilized commercially available Cas9 protein together with sgRNA and a targeting construct to introduce desired mutations. Here, we report a cloning-free method to target the mouse genome by pronuclear injection of a commercial Cas9 protein:crRNA:tracrRNA:single-strand oligodeoxynucleotide (ssODN) complex into mouse zygotes. As illustration of this method, we report the successful generation of global gene-knockout, single-amino-acid-substituted, as well as floxed mice that can be used for conditional gene-targeting. These models were produced with high efficiency to generate non-mosaic mutant mice with a high germline transmission rate

Indosinian high-strain deformation for the Yunkaidashan tectonic belt, south China : Kinematics and 40Ar/39Ar geochronological constraints

Structural and 40Ar/39Ar data from the Yunkaidashan Belt document kinematic and tectonothermal characteristics of early Mesozoic Indosinian orogenesis in the southern part of the South China Block. The Yunkaidashan Belt is tectonically divided from east to west into the Wuchuang-Sihui shear zone, Xinyi-Gaozhou block, and the Fengshan-Qinxi shear zone. Indosinian structural elements ascribed to the Indosinian orogeny include D2 and D3 deformation. The early D2 phase is characterized by folding and thrusting with associated foliation and lineation development, related to NW-SE transpression under amphibolite- to greenschist-facies conditions. This event is heterogeneously overprinted by D3 deformation characterized by a gentle-dipping S-3 foliation, subhorizontally to shallowly plunging L3 lineation, some reactived-D2 folds and low-angle normal faults. The D3 fabrics suggest a sinistral transtensional regime under greenschist-facies metamorphism. The timing of the D2 and D3 events have been constrained to the early to middle Triassic (similar to 248-220 Ma) and late Triassic (similar to 220-200 Ma) respectively on the basis of 40Ar/39Ar geochronology and regional geological relations. The change from oblique thrusting (D2) to sinistral transtension (D3) may reflect oblique convergence and crustal thickening followed by relaxation of the overthickened crust. In combination with the regional relations from Xuefengshan to Yunkaidashan and on to Wuyishan, the early phase of the Indosinian orogeny constituted a large-scale positive flower structure and is related to the intracontinental convergence during the assembly of Pangea in which the less competent South China Orogen was squeezed between the more competent North China and Indosinian Blocks.Peer reviewe

The Early Modern Colonial State in Asia: Private Agency and Family Networks in the English East India Company

This thesis studies the formation of the early modern colonial state in Asia. Through an exploration of the English East India Company, it examines the dynamics which shaped political authority, colonial governance and the performance of state power in the seventeenth and eighteenth centuries. Specifically, the following research argues that a process of political decentralisation took place within the Company. This was driven by the pursuit of ‘private interests’ on behalf of the Company’s servants in Asia, who, as a result neglected, resisted or subverted the ‘public interests’ of their masters in London. Key to this reconfiguration of power were the family networks established by Company servants between Europe and Asia, and across Asia itself in this period. As constructs of exchange, circulation and movement, family networks allowed Company servants to exercise considerable political agency, distinct from metropolitan authorities. In so doing, they transformed the political landscape around them, laying the foundations of the early modern colonial state through a process of private state formation from the turn of the eighteenth century onwards

Flood magnitude-frequency and lithologic control on bedrock river incision in post-orogenic terrain

Mixed bedrock-alluvial rivers - bedrock channels lined with a discontinuous alluvial cover - are key agents in the shaping of mountain belt topography by bedrock fluvial incision. Whereas much research focuses upon the erosional dynamics of such rivers in the context of rapidly uplifting orogenic landscapes, the present study investigates river incision processes in a post-orogenic (cratonic) landscape undergoing extremely low rates of incision (> 5 m/Ma). River incision processes are examined as a function of substrate lithology and the magnitude and frequency of formative flows along Sandy Creek gorge, a mixed bedrock-alluvial stream in arid SE-central Australia. Incision is focused along a bedrock channel with a partial alluvial cover arranged into riffle-pool macrobedforms that reflect interactions between rock structure and large-flood hydraulics. Variations in channel width and gradient determine longitudinal trends in mean shear stress (τb) and therefore also patterns of sediment transport and deposition. A steep and narrow, non-propagating knickzone (with 5% alluvial cover) coincides with a resistant quartzite unit that subdivides the gorge into three reaches according to different rock erodibility and channel morphology. The three reaches also separate distinct erosional styles: bedrock plucking (i.e. detachment-limited erosion) prevails along the knickzone, whereas along the upper and lower gorge rock incision is dependent upon large formative floods exceeding critical erosion thresholds (τc) for coarse boulder deposits that line 70% of the channel thalweg (i.e. transport-limited erosion). The mobility of coarse bed materials (up to 2 m diameter) during late Holocene palaeofloods of known magnitude and age is evaluated using step-backwater flow modelling in conjunction with two selective entrainment equations. A new approach for quantifying the formative flood magnitude in mixed bedrock-alluvial rivers is described here based on the mobility of a key coarse fraction of the bed materials; in this case the d84 size fraction. A 350 m3/s formative flood fully mobilises the coarse alluvial cover with τb200-300 N/m2 across the upper and lower gorge riffles, peaking over 500 N/m2 in the knickzone. Such floods have an annual exceedance probability much less than 10- 2 and possibly as low as 10- 3. The role of coarse alluvial cover in the gorge is discussed at two scales: (1) modulation of bedrock exposure at the reach-scale, coupled with adjustment to channel width and gradient, accommodates uniform incision across rocks of different erodibility in steady-state fashion; and (2) at the sub-reach scale where coarse boulder deposits (corresponding to <i>τ</i><sub>b</sub> minima) cap topographic convexities in the rock floor, thereby restricting bedrock incision to rare large floods. While recent studies postulate that decreasing uplift rates during post-orogenic topographic decay might drive a shift to transport-limited conditions in river networks, observations here and elsewhere in post-orogenic settings suggest, to the contrary, that extremely low erosion rates are maintained with substantial bedrock channel exposure. Although bed material mobility is known to be rate-limiting for bedrock river incision under low sediment flux conditions, exactly how a partial alluvial cover might be spatially distributed to either optimise or impede the rate of bedrock incision is open to speculation. Observations here suggest that the small volume of very stable bed materials lining Sandy Creek gorge is distributed so as to minimise the rate of bedrock fluvial incision over time

The motivation of junior high school pupils to learn English in provincial Indonesia

The purpose of this work is to explore the motivation of young Indonesians to learn English over the first two years of formal study in a provincial junior high school. The national education system has always struggled to produce competent users of the language, yet the country's need for such graduates is never greater than at the beginning of the 21" century as it responds to the social, economic and political challenges of globalization. Meanwhile motivation has always been recognised as an important factor in language learning success, but recent work has stressed its complexity and changeability over time and in particular contexts, encouraging the possibility of new discoveries in this academically unexplored territory. Defining motivation as a dynamic constellation of contextually sensitive cognitions and affects stimulating individuals to learn, the study adopted a mixed method strategy, using questionnaires at beginning and end of the 20-month research period to track motivational trends across the whole school year group (n = 195) and developing indepth portraits of 12 individuals through interview and classroom observation at three points. The eight school English teachers were also interviewed at the beginning. Results showed a very high level of motivation to learn English, reflected in much autonomous learning of the language outside of school. Although there was evidence of dissatisfaction with aspects of school English lessons, this motivation was largely sustained throughout the period under study and appeared to contribute to significant gains in competence in the language among some learners. It is argued that this motivation derives its strength from identification processes, nurtured and developed through social interaction at home and in the community, which encouraged many young Indonesians in this context to view English as integral to their future lives. 'Me study strongly suggests that understanding differences in the way learners identify with the language is an important direction for future research into L2 motivation in general. Understanding how schools and teachers promote or challenge pupils' L2 identities could lead to improvements in language pedagog

Methodological improvements for the analysis of domain movements in large biomolecular complexes

Domain movements play a prominent role in the function of many biomolecules such as the ribosome and F0F1-ATP synthase. As more structures of large biomolecules in different functional states become available as experimental techniques for structure determination advance, there is a need to develop methods to understand the conformational changes that occur. DynDom and DynDom3D were developed to analyse two structures of a biomolecule for domain movements. They both used an original method for domain recognition based on clustering of “rotation vectors”. Here we introduce significant improvements in both the methodology and implementation of a tool for the analysis of domain movements in large multimeric biomolecules. The main improvement is in the recognition of domains by using all six degrees of freedom required to describe the movement of a rigid body. This is achieved by way of Chasles’ theorem in which a rigid-body movement can be described as a screw movement about a unique axis. Thus clustering now includes, in addition to rotation vector data, screw-axis location data and axial climb data. This improves both the sensitivity of domain recognition and performance. A further improvement is the recognition and annotation of interdomain bending regions, something not done for multimeric biomolecules in DynDom3D. This is significant as it is these regions that collectively control the domain movement. The new stand-alone, platform-independent implementation, DynDom6D, can analyse biomolecules comprising protein, DNA and RNA, and employs an alignment method to automatically achieve the required equivalence of atoms in the two structures

Improved K-mer Based Prediction of Protein-Protein Interactions With Chaos Game Representation, Deep Learning and Reduced Representation Bias

Protein-protein interactions drive many biological processes, including the detection of phytopathogens by plants' R-Proteins and cell surface receptors. Many machine learning studies have attempted to predict protein-protein interactions but performance is highly dependent on training data; models have been shown to accurately predict interactions when the proteins involved are included in the training data, but achieve consistently poorer results when applied to previously unseen proteins. In addition, models that are trained using proteins that take part in multiple interactions can suffer from representation bias, where predictions are driven not by learned biological features but by learning of the structure of the interaction dataset. We present a method for extracting unique pairs from an interaction dataset, generating non-redundant paired data for unbiased machine learning. After applying the method to datasets containing _Arabidopsis thaliana_ and pathogen effector interations, we developed a convolutional neural network model capable of learning and predicting interactions from Chaos Game Representations of proteins' coding genes