Structure of the N-terminal oligomerization domain of DnaD reveals a unique tetramerization motif and provides insights into scaffold formation

DnaD is a primosomal protein that remodels supercoiled plasmids. It binds to supercoiled forms and converts them to open forms without nicking. During this remodeling process, all the writhe is converted to twist and the plasmids are held around the periphery of large scaffolds made up of DnaD molecules. This DNA-remodeling function is the sum of a scaffold-forming activity on the N-terminal domain and a DNA-dependent oligomerization activity on the C-terminal domain. We have determined the crystal structure of the scaffold-forming N-terminal domain, which reveals a winged-helix architecture, with additional structural elements extending from both N- and C-termini. Four monomers form dimers that join into a tetramer. The N-terminal extension mediates dimerization and tetramerization, with extensive interactions and distinct interfaces. The wings and helices of the winged-helix domains remain exposed on the surface of the tetramer. Structure-guided mutagenesis and atomic force microscopy imaging indicate that these elements, together with the C-terminal extension, are involved in scaffold formation. Based upon our data, we propose a model for the DnaD-mediated scaffold formation.

The Bacillus subtilis primosomal protein DnaD untwists supercoiled DNA

The essential Bacillus subtilis DnaD and DnaB proteins have been implicated in the initiation of DNA replication. Recently, DNA remodeling activities associated with both proteins were discovered that could provide a link between global or local nucleoid remodeling and initiation of replication. DnaD forms scaffolds and opens up supercoiled plasmids without nicking to form open circular complexes, while DnaB acts as a lateral compaction protein. Here we show that DnaD-mediated opening of supercoiled plasmids is accompanied by significant untwisting of DNA. The net result is the conversion of writhe (Wr) into negative twist (Tw), thus maintaining the linking number (Lk) constant. These changes in supercoiling will reduce the considerable energy required to open up closed circular plectonemic DNA and may be significant in the priming of DNA replication. By comparison, DnaB does not affect significantly the supercoiling of plasmids. Binding of the DnaD C-terminal domain (Cd) to DNA is not sufficient to convert Wr into negative Tw, implying that the formation of scaffolds is essential for duplex untwisting. Overall, our data suggest that the topological effects of the two proteins on supercoiled DNA are different; DnaD opens up, untwists and converts plectonemic DNA to a more paranemic form, whereas DnaB does not affect supercoiling significantly and condenses DNA only via its lateral compaction activity. The significance of these findings in the initiation of DNA replication is discussed.

Propiedades dieléctricas y térmicas de cristales líquidos: dímeros y bent-core

194 p.

Complete structure of an epithelial keratin dimer: implications for intermediate filament assembly

Keratins are cytoskeletal proteins that hierarchically arrange into filaments, starting with the dimer sub-unit. They are integral to the structural support of cells, in skin, hair and nails. In skin, keratin is thought to play a critical role in conferring the barrier properties and elasticity of skin. In general, the keratin dimer is broadly described by a tri-domain structure: a head, a central rod and a tail. As yet, no atomistic-scale picture of the entire dimer structure exists; this information is pivotal for establishing molecular-level connections between structure and function in intermediate filament proteins. The roles of the head and tail domains in facilitating keratin filament assembly and function remain as open questions. To address these, we report results of molecular dynamics simulations of the entire epithelial human K1/K10 keratin dimer. Our findings comprise: (1) the first three-dimensional structural models of the complete dimer unit, comprising of the head, rod and tail domains; (2) new insights into the chirality of the rod-domain twist gained from analysis of the full domain structure; (3) evidence for tri-subdomain partitioning in the head and tail domains; and, (4) identification of the residue characteristics that mediate non-covalent contact between the chains in the dimer. Our findings are immediately applicable to other epithelial keratins, such as K8/K18 and K5/K14, and to intermediate filament proteins in general.

Cross-strand disulfides in the non-hydrogen bonding site of antiparallel β-sheet (aCSDns): poised for biological switching

Forbidden disulfides are stressed disulfides found in recognisable protein contexts previously defined as structurally forbidden. The torsional strain of forbidden disulfides is typically higher than for structural disulfides, but not so high as to render them immediately susceptible to reduction under physionormal conditions. The meta-stability of forbidden disulfides makes them likely candidates as redox switches. Here we mined the Protein Data Bank for examples of the most common forbidden disulfide, the aCSDn. This is a canonical motif in which disulfide-bonded cysteine residues are positioned directly opposite each other on adjacent anti-parallel β-strands such that the backbone hydrogen bonded moieties are directed away from each other. We grouped these aCSDns into homologous clusters and performed an extensive physicochemical and informatic analysis of the examples found. We estimated their torsional energies using quantum chemical calculations and studied differences between the preferred conformations of the computational model and disulfides found in solved protein structures to understand the interaction between the forces imposed by the disulfide linkage and typical constraints of the surrounding β-sheet. In particular, we assessed the twisting, shearing and buckling of aCSDn-containing β-sheets, as well as the structural and energetic relaxation when hydrogen bonds in the motif are broken. We show the strong preference of aCSDns for the right-handed staple conformation likely arises from its compatibility with the twist, shear and Cα separation of canonical β-sheet. The disulfide can be accommodated with minimal distortion of the sheet, with almost all the strain present as torsional strain within the disulfide itself. For each aCSDn cluster, we summarise the structural and strain data, taxonomic conservation and any evidence of redox activity. aCSDns are known substrates of thioredoxin-like enzymes. This, together with their meta-stability, means they are ideally suited to biological switching roles and are likely to play important roles in the molecular pathways of oxidative stress.

Optimising technical skills and physical loading in small-sided basketball games

Differences in physiological, physical, and technical demands of small-sided basketball games related to the number of players, court size, and work-to-rest ratios are not well characterised. A controlled trial was conducted to compare the influence of number of players (2v2/4v4), court size (half/full court) and work-to-rest ratios (4x2.5 min/2x5 min) on the demands of small-sided games. Sixteen elite male and female junior players (aged 15-19 years) completed eight variations of a small-sided game in randomised order over a six-week period. Heart rate responses and rating of perceived exertion (RPE) were measured to assess the physiological load. Movement patterns and technical elements were assessed by video analysis. There were ∼60% more technical elements in 2v2 and ∼20% more in half court games. Heart rate (86 ± 4% & 83 ± 5% of maximum; mean ± SD) and RPE (8 ± 2 & 6 ± 2; scale 1-10) were moderately higher in 2v2 than 4v4 small-sided games, respectively. The 2v2 format elicited substantially more sprints (36 ±12%; mean ±90% confidence limits) and high intensity shuffling (75 ±17%) than 4v4. Full court games required substantially more jogging (9 ±6%) compared to half court games. Fewer players in small-sided basketball games substantially increases the technical, physiological and physical demands.

Crystallographic analysis of intervariant boundaries in a lath martensite

In the current study, the crystallographic of intervariant boundary planes distribution in the lath martensite has been measured as a function of lattice misorientation and boundary plane orientation using five macroscopic parameters approach. The distribution revealed a relatively high anisotropy with a tendency for the lath interfaces to terminate on (110) planes. This results from the crystallographic constraints associated with the shear transformation rather than a low energy interface configuration. The lath martensite habit plane was determined to be mostly (110) or near (110). The relative populations of boundaries with [111] and [110] misorientations were greater than other high index misorientations, mostly characterised as (110) symmetric tilt and (110) twist boundary types, respectively.

Online stretching of directly electrospun nanofiber yarns

Nanofiber yarns are important building blocks for making three-dimensional nanostructures, e.g. through a knitting or weaving process, with better mechanical properties than nanofiber nonwovens and well-controlled fibrous construction. However, it still remains challenging to produce quality nanofiber yarns in a sufficient rate. In this study, we have proven that online stretching during electrospinning of nanofiber yarns can considerably improve fiber alignment and molecular orientation within the yarn and increase yarn tensile strength, but reduce fiber/yarn diameters. By compensating twist during online stretching, the device can prepare nanofiber yarns with different stretch levels, but maintaining the same twist multiplier. This allows us to examine the effect of stretching on fiber and yarn morphology. It was interesting to find that on increasing the stretching ratio from 0% to 95%, the yarn diameter reduced from 135.1 ± 20.3 μm to 46.2 ± 10.2 μm, and the fiber diameter reduced from 998 ± 141 nm to 631 ± 98 nm, whereas the yarn tensile strength increased from 48.2 ± 5.6 MPa to 127.7 ± 5.4 MPa. Such an advanced yarn electrospinning technique can produce nanofiber yarn with an overall yarn production rate as high as 10 m min−1. This may be useful for production of nanofiber yarns for various applications.

Microstructure characteristics of the (111) oriented grains in a Fe-30Ni-Nb model austenitic steel deformed in hot uniaxial compression

The work presents a detailed investigation of the microstructure characteristics of the (111) oriented grains in a Fe-30Ni-Nb austenitic model steel subjected to hot uniaxial compression at 925 °C at a strain rate of 1 s- 1. The above grains exhibited a tendency to split into deformation bands having alternating orientations and largely separated by transition regions comprising arrays of closely spaced, extended sub-boundaries collectively accommodating large misorientations across very small distances. On a fine scale, the (111) oriented grains typically contained a mix of "microbands" (MBs) closely aligned with {111} slip planes and those significantly deviated from these planes. The above deformation substructure thus markedly differed from the microstructure type, comprising strictly non-{111} aligned MBs, expected within such grains on the basis of the uniaxial compression experiments performed using aluminium. Both the crystallographic MBs and their non-crystallographic counterparts typically displayed similar misorientations and formed self-screening arrays characterized by systematically alternating misorientations. The crystallographic MBs were exclusively aligned with {111} slip planes containing slip systems whose sum of Schmid factors was the largest among the four available slip planes. The corresponding boundaries appeared to mainly display either a large twist or a large tilt component.

A comparison of the yaw constraining performance of SCARA-tau parallel manipulator variants via screw theory

The SCARA-Tau parallel manipulator was derived with the objective to overcome the limited workspace-to-footprint ratio of the DELTA parallel manipulator while maintaining its many benefits. The SCARA-Tau family has later been extended and a large number of variants have been proposed. In this paper, we analyse four of these variants, which together encompass the main differences between all the proposed SCARA-Tau manipulators. The analysed manipulator variants utilise an identical arrangement of five of the six linkages connecting the actuated arms and the manipulated platform and exhibit the same input-output Jacobian. The normalised reciprocal product between the wrench of the sixth linkage and the twist of the platform occurring without this linkage provides a measure on how effectively the sixth linkage constrains the manipulated platform. A comparison of the manipulator variants with respect to this measure demonstrates each variants suitability for specific applications.

Human rights in Papua New Guinea: is this where we should be settling refugees?

Australia has had a long, and at times tumultuous, relationship with our nearest neighbour, Papua New Guinea. This relationship took a twist in late 2012, with the re-opening of the off-shore processing centre on Manus Island, and again in February 2014, when Iranian asylum seeker Reza Berati was murdered by locals during a violent disturbance at the centre. The latest test of the strength and endurance of the relationship between PNG and Australia came in April 2016, when the PNG Supreme Court ruled that the detention of asylum seekers on Manus Island breached the right to personal liberty in the PNG constitution. This article provides much-needed insight into the human rights situation in PNG, and makes recommendations regarding the prospect of resettling refugees in that country.

Efficient algorithms for beacon routing in polygons

In a paper by Biro et al. [7], a novel twist on guarding in art galleries is introduced. A beacon is a fixed point with an attraction pull that can move points within the polygon. Points move greedily to monotonically decrease their Euclidean distance to the beacon by moving straight towards the beacon or sliding on the edges of the polygon. The beacon attracts a point if the point eventually reaches the beacon. Unlike most variations of the art gallery problem, the beacon attraction has the intriguing property of being asymmetric, leading to separate definitions of attraction region and inverse attraction region. The attraction region of a beacon is the set of points that it attracts. For a given point in the polygon, the inverse attraction region is the set of beacon locations that can attract the point. We first study the characteristics of beacon attraction. We consider the quality of a "successful" beacon attraction and provide an upper bound of $\sqrt{2}$ on the ratio between the length of the beacon trajectory and the length of the geodesic distance in a simple polygon. In addition, we provide an example of a polygon with holes in which this ratio is unbounded. Next we consider the problem of computing the shortest beacon watchtower in a polygonal terrain and present an $O(n \log n)$ time algorithm to solve this problem. In doing this, we introduce $O(n \log n)$ time algorithms to compute the beacon kernel and the inverse beacon kernel in a monotone polygon. We also prove that $\Omega(n \log n)$ time is a lower bound for computing the beacon kernel of a monotone polygon. Finally, we study the inverse attraction region of a point in a simple polygon. We present algorithms to efficiently compute the inverse attraction region of a point for simple, monotone, and terrain polygons with respective time complexities $O(n^2)$, $O(n \log n)$ and $O(n)$. We show that the inverse attraction region of a point in a simple polygon has linear complexity and the problem of computing the inverse attraction region has a lower bound of $\Omega(n \log n)$ in monotone polygons and consequently in simple polygons.

Técnicas e sistemas de desobturação canalar no retratamento endodôntico não cirúrgico

Na prática clínica, a diversidade de instrumentos manuais, rotatórios ou reciprocantes, dificulta a seleção do sistema a aplicar no retratamento dentário não cirúrgico. O presente trabalho teve como objetivo comparar diferentes instrumentos quanto a diferentes parâmetros: capacidade de remoção de Gutta-Percha (GP), extrusão apical de detritos, fratura de instrumentos, e ocorrência de iatrogenias. Neste trabalho foram utilizadas 111 publicações posteriores a 2011, obtidas via PubMed e Science Direct. A análise da bibliografia indica que, independentemente do sistema, não é possível remover todo o material obturador das paredes radiculares, sendo esta tarefa dificultada em canais curvos e na área apical. Verifica-se que a remoção de GP melhora no sentido: limas H, ProTaper, e Mtwo. O sistema Reciproc foi associado a melhores desempenhos e a menores tempo de trabalho, do que os sistemas de rotação contínua. Nenhum dos instrumentos analisados é capaz de evitar a extrusão apical de detritos na totalidade. Apesar de resultados dispares, a maioria dos estudos assume que o sistema Reciproc provoca menor extrusão apical de detritos. Em Endodontia, as duas principais causas da fratura de instrumentos são a fadiga cíclica e a torsão. A maioria dos estudos concordam que o movimento reciprocante, como o do Reciproc, aumenta a resistência à fractura e a resistência à torsão, mantendo a anatomia original do canal. Relativamente à produção de perfurações e fracturas radiculares, a superioridade dos instrumentos NiTi relativamente às limas manuais não foi clara. De acordo com a literatura, o sistema Reciproc, constituído por liga de NiTi M-Wire, está associado a menos eventos iatrogénicos. Finalmente, conclui-se que futuros estudos seriam benéficos para esclarecer o potencial dos diferentes sistemas estudados.

Assessments of training load in elite youth soccer

One of the most popular sports globally, soccer has seen a rise in the demands of the game over recent years. An increase in intensity and playing demands, coupled with growing social and economic pressures on soccer players means that optimal preparation is of paramount importance. Recent research has found the modern game, depending on positional role, to consist of approximately 60% more sprint distance in the English Premier League, which was also found to be the case for frequency and success of discrete technical actions (Bush et al., 2015). As a result, the focus on soccer training and player preparedness is becoming more prevalent in scientific research. By designing the appropriate training load, and thus periodization strategies, the aim is to achieve peak fitness in the most efficient way, whilst minimising the risk of injury and illness. Traditionally, training intensity has been based on heart rate responses, however, the emergence of tracking microtechnology such as global positioning system (GPS) and inertial sensors are now able to further quantify biomechanical load as well as physiological stress. Detailed pictures of internal and external loading indices such as these then combine to produce a more holistic view of training load experience by the player during typical drills and phases of training in soccer. The premise of this research is to gain greater understanding of the physical demands of common training methodologies in elite soccer to support optimal match performance. The coaching process may then benefit from being able to prescribe the most effective training to support these. The first experimental chapter in this thesis began by quantify gross training loads of the pre-season and in-season phases in soccer. A broader picture of the training loads inherent in these distinct phases brought more detail as to the type and extent of external loading experienced by soccer players at these times, and how the inclusion of match play influences weekly training rhythms. Training volume (total distance) was found to be high at the start compared to the end of pre-season (37 kilometres and 28 kilometres), where high cardiovascular loads were attained as part of the conditioning focus. This progressed transiently, however, to involve higher-speed, acceleration and change-of-direction stimuli at the end of pre-season compared to the start and to that in-season (1.18 kilometres, 0.70 kilometres and 0.42 kilometres high-intensity running; with 37, 25 and 23 accelerations >3m/s2 respectively) . The decrease in volume and increase in maximal anaerobic activity was evident in the training focus as friendly matches were introduced before the competitive season. The influence of match-play as being a large physical dose in the training week may then determine the change in weekly periodisation and how resulting training loads applied and tapered, if necessary. The focus of research was then directed more specifically to the most common mode of training in soccer, that also featured regularly in the pre-season period in the present study, small-sided games (SSG). The subsequent studies examined numerous manipulations of this specific form of soccer conditioning, such as player numbers as well as absolute and relative playing space available. In contrast to some previous literature, changing the number of players did not seem to influence training responses significantly, although playing format in the possession style brought about larger effects for heart rate (89.9%HRmax) and average velocity (7.6km/h-1). However, the following studies (Chapters 5, 6 and 7) revealed a greater influence of relative playing space available to players in SSG. The larger area at their disposal brought about greater aerobic responses (~90%HRmax), by allowing higher average and peak velocities (>25km/h-1), as well as greater distance acceleration behaviour at greater thresholds (>2.8m/s2). Furthermore, the data points towards space as being a large determinant in strategy of the player in small-sided games (SSG), subsequently shaping their movement behaviour and resulting physical responses. For example, higher average velocities in a possession format (8km/h-1) reflects higher work rate and heart rate load but makes achieving significant neuromuscular accelerations at a high level difficult given higher starting velocities prior to the most intense accelerations (4.2km/h-1). By altering space available and even through intentional numerical imbalances in team numbers, it may be easier for coaches to achieve the desired stimulus for the session or individual player, whether that is for aerobic and neuromuscular conditioning. Large effects were found for heart rate being higher in the underloaded team (85-90%HRmax) compared to the team with more players (80-85%HRmax) as well as for RPE (5AU versus 7AU). This was also apparent for meterage and therefore average velocity. It would also seem neuromuscular load through high acceleration and deceleration efforts were more pronounced with less numbers (given the need to press and close down opponents, and in a larger area relative to the number of players on the underloaded team. The peak accelerations and deceleration achieved was also higher when playing with less players (3-6.2m/s2 and 3-6.1m/s2) Having detailed ways in which to reach desired physical loading responses in common small training formats, Chapter 8 compared SSG to larger 9v9 formats with full-size 11v11 friendly matches. This enabled absolute and relative comparisons to be made and to understand the extent to which smaller training formats are able to replicate the required movements to be successful in competition. In relative terms, it was revealed that relative acceleration distance and Player Load were higher in smaller 4v4 games than match-play (1.1m.min-1 and 0.3m.min-1 >3m/s2; 16.9AU versus 12AU). Although the smallest format did not replicate the high-velocity demands of matches, the results confirmed their efficacy in providing significant neuromuscular load during the training week, which may then be supplemented by high-intensity interval running in order to gain exposure to more maximal speed work. In summary, the data presented provide valuable information from GPS and inertial sensor microtechnology which may then be used to understand training better to manipulate types of load according to physical conditioning objectives. For example, a library of resources to direct planning of drills of varying cardiovascular, neuromuscular and perceptual load can be created to give more confidence in session outcomes. Combining external and internal load data of common soccer training drills, and their application across different phases and training objectives may give coaches a powerful tool to plan and periodize training.

Hall Effect and transient surface photovoltage (SPV) study of Cu3BiS3 thin films

Se presentan las propiedades eléctricas del compuesto Cu3BiS3 depositado por co-evaporación. Este es un nuevo compuesto que puede tener propiedades adecuadas para ser utilizado como capa absorbente en celdas solares. Las muestras fueron caracterizadas a través de medidas de efecto Hall y fotovoltaje superficial transiente (SPV). A través de medidas de efecto Hall se encontró que la concentración de portadores de carga n es del orden de 1016 cm-3 independiente de la relación de masas de Cu/Bi. También se encontró que la movilidad de este compuesto (μ del orden de 4 cm2V -1s-1) varía de acuerdo con los mecanismos de transporte que la gobiernan en dependencia con la temperatura. A partir de las medidas de SPV se encontró alta densidad de defectos superficiales, defectos que son pasivados al superponer una capa buffer sobre el compuesto Cu3BiS3.