Copurification of the Lac repressor with polyhistidine-tagged proteins in immobilized metal affinity chromatography.

One of the commonly used resins for immobilized metal affinity purification of polyhistidine-tagged recombinant proteins is TALON resin, a cobalt (II)--carboxymethylaspartate-based matrix linked to Sepharose CL-6B. Here, we show that TALON resin efficiently purifies the native form of Lac repressor, which represents the major contaminant when (His)(6)-tagged proteins are isolated from Escherichia coli host cells carrying the lacI(q) gene. Inspection of the crystal structure of the repressor suggests that three His residues (residues 163, 173, and 202) in each subunit of the tetramer are optimally spaced on an exposed face of the protein to allow interaction with Co(II). In addition to establishing a more efficient procedure for purification of the Lac repressor, these studies indicate that non-lacI(q)-based expression systems yield significantly purer preparations of recombinant polyhistidine-tagged proteins.

Plastic limit solution for the response of plate anchors under combined translational and torsional undrained loading

Plate anchors are increasingly being used to moor large floating offshore structures in deep and ultradeep water. These facilities impart substantial vertical uplift loading to plate anchors. However, extreme operating conditions such as hurricane loading often result in partial system failures, with significant change in the orientation of the remaining intact mooring lines. The purpose of this study is to investigate the undrained pure translational (parallel to plate) and torsional bearing capacity of anchor plates idealized as square and rectangular shaped plates. Moreover, the interaction response of plate anchors under combined translational and torsional loading is studied using a modified plastic limit analysis (PLA) approach. The previous PLA formulation which did not account for shear-normal force interaction on the vertical end faces of the plate provides an exact solution to the idealized problem of an infinitely thin plate but only an approximate solution to the problem of a plate of finite thickness. This is also confirmed by the three-dimensional finite element (FE) results, since the PLA values exceed FE results as the thickness of the plate increases. By incorporating the shear-normal interaction relationship in the modified solution, the torsional bearing capacity factors, as well as the plate interaction responses are enhanced as they show satisfactory agreement with the FE results. The interaction relationship is then obtained for square and rectangular plates of different aspect ratios and thicknesses. The new interaction relationships could also be used as an associated plastic failure locus for combined shear and torsional loading to predict plastic displacements and rotations in translational and torsional loading modes as well. Copyright © 2011 by the International Society of Offshore and Polar Engineers (ISOPE).

Industry perceptions of barriers to commercialization of regenerative medicine products in the UK.

AIMS: Regenerative medicine is an emerging field with the potential to provide widespread improvement in healthcare and patient wellbeing via the delivery of therapies that can restore, regenerate or repair damaged tissue. As an industry, it could significantly contribute to economic growth if products are successfully commercialized. However, to date, relatively few products have reached the market owing to a variety of barriers, including a lack of funding and regulatory hurdles. The present study analyzes industry perceptions of the barriers to commercialization that currently impede the success of the regenerative medicine industry in the UK. MATERIALS & METHODS: The analysis is based on 20 interviews with leading industrialists in the field. RESULTS: The study revealed that scientific research in regenerative medicine is thriving in the UK. Unfortunately, lack of access to capital, regulatory hurdles, lack of clinical evidence leading to problems with reimbursement, as well as the culture of the NHS do not provide a good environment for the commercialization of regenerative medicine products. CONCLUSION: Policy interventions, including increased translational government funding, a change in NHS and NICE organization and policies, and regulatory clarity, would likely improve the general outcomes for the regenerative medicine industry in the UK.

A modular planar robotic manipulandum with end-point torque control.

Robotic manipulanda are extensively used in investigation of the motor control of human arm movements. They permit the application of translational forces to the arm based on its state and can be used to probe issues ranging from mechanisms of neural control to biomechanics. However, most current designs are optimized for studying either motor learning or stiffness. Even fewer include end-point torque control which is important for the simulation of objects and the study of tool use. Here we describe a modular, general purpose, two-dimensional planar manipulandum (vBOT) primarily optimized for dynamic learning paradigms. It employs a carbon fibre arm arranged as a parallelogram which is driven by motors via timing pulleys. The design minimizes the intrinsic dynamics of the manipulandum without active compensation. A novel variant of the design (WristBOT) can apply torques at the handle using an add-on cable drive mechanism. In a second variant (StiffBOT) a more rigid arm can be substituted and zero backlash belts can be used, making the StiffBOT more suitable for the study of stiffness. The three variants can be used with custom built display rigs, mounting, and air tables. We investigated the performance of the vBOT and its variants in terms of effective end-point mass, viscosity and stiffness. Finally we present an object manipulation task using the WristBOT. This demonstrates that subjects can perceive the orientation of the principal axis of an object based on haptic feedback arising from its rotational dynamics.

Three-dimensional effects on sliding and waving wings

Like large insects, micro air vehicles operate at low Reynolds numbers O(1; 000 - 10; 000) in a regime characterized by separated flow and strong vortices. The leading-edge vortex has been identified as a significant source of high lift on insect wings, but the conditions required for the formation of a stably attached leading-edge vortex are not yet known. The waving wing is designed to model the translational phase of an insect wing stroke by preserving the unsteady starting and stopping motion as well as three-dimensionality in both wing geometry (via a finite-span wing) and kinematics (via wing rotation). The current study examines the effect of the spanwise velocity gradient on the development of the leading-edge vortex along the wing as well as the effects of increasing threedimensionalityby decreasing wing aspect ratio from four to two. Dye flow visualization and particle image velocimetry reveal that the leading-edge vortices that form on a sliding or waving wing have a very high aspect ratio. The structure of the flow is largely two-dimensional on both sliding and waving wings and there is minimal interaction between the leading-edge vortices and the tip vortex. Significant spanwise flow was observed on the waving wing but not on the sliding wing. Despite the increased three-dimensionality on the aspect ratio 2 waving wing, there is no evidence of an attached leading-edge vortex and the structure of the flow is very similar to that on the higher-aspect-ratio wing and sliding wing. © Copyright 2010.

The need for winter in the switch to flowering.

Vernalization is the process whereby the floral transition is promoted through exposure of plants to long periods of cold temperature or winter. A requirement for vernalization aligns flowering with the seasons to ensure that their reproductive phase occurs in favorable conditions. The mitotic stability of vernalization, suggestive of an epigenetic mechanism, has intrigued researchers for many years. Genetic analysis of the vernalization requirement in Arabidopsis has identified key floral repressor genes, FRI and FLC. The action of these floral repressors is antagonized by vernalization and the activity of a set of genes grouped into the autonomous floral pathway. Analysis of the vernalization pathway has defined a series of epigenetic regulators crucial for "cellular-memory" of the cold signal, whereas the autonomous pathway appears to function in part through posttranscriptional mechanisms. The mechanism of the vernalization requirement, which is now being explored in a range of plant species, should uncover the evolutionary origins of this key agronomic trait.

RNA processing and Arabidopsis flowering time control.

Plants control their flowering time in order to ensure that they reproduce under favourable conditions. The components involved in this complex process have been identified using a molecular genetic approach in Arabidopsis and classified into genetically separable pathways. The autonomous pathway controls the level of mRNA encoding a floral repressor, FLC, and comprises three RNA-binding proteins, FCA, FPA and FLK. FCA interacts with the 3'-end RNA-processing factor FY to autoregulate its own expression post-transcriptionally and to control FLC. Other components of the autonomous pathway, FVE and FLD, regulate FLC epigenetically. This combination of epigenetic and post-transcriptional control gives precision to the control of FLC expression and flowering time.

Epigenetic inheritance in plants.

The function of plant genomes depends on chromatin marks such as the methylation of DNA and the post-translational modification of histones. Techniques for studying model plants such as Arabidopsis thaliana have enabled researchers to begin to uncover the pathways that establish and maintain chromatin modifications, and genomic studies are allowing the mapping of modifications such as DNA methylation on a genome-wide scale. Small RNAs seem to be important in determining the distribution of chromatin modifications, and RNA might also underlie the complex epigenetic interactions that occur between homologous sequences. Plants use these epigenetic silencing mechanisms extensively to control development and parent-of-origin imprinted gene expression.

FY is an RNA 3' end-processing factor that interacts with FCA to control the Arabidopsis floral transition

The nuclear RNA binding protein, FCA, promotes Arabidopsis reproductive development. FCA contains a WW protein interaction domain that is essential for FCA function. We have identified FY as a protein partner for this domain. FY belongs to a highly conserved group of eukaryotic proteins represented in Saccharomyces cerevisiae by the RNA 3' end-processing factor, Pfs2p. FY regulates RNA 3' end processing in Arabidopsis as evidenced through its role in FCA regulation. FCA expression is autoregulated through the use of different polyadenylation sites within the FCA pre-mRNA, and the FCA/FY interaction is required for efficient selection of the promoter-proximal polyadenylation site. The FCA/FY interaction is also required for the downregulation of the floral repressor FLC. We propose that FCA controls 3' end formation of specific transcripts and that in higher eukaryotes, proteins homologous to FY may have evolved as sites of association for regulators of RNA 3' end processing.

An allelic series reveals essential roles for FY in plant development in addition to flowering-time control

The autonomous pathway functions to promote flowering in Arabidopsis by limiting the accumulation of the floral repressor FLOWERING LOCUS C (FLC). Within this pathway FCA is a plant-specific, nuclear RNA-binding protein, which interacts with FY, a highly conserved eukaryotic polyadenylation factor. FCA and FY function to control polyadenylation site choice during processing of the FCA transcript. Null mutations in the yeast FY homologue Pfs2p are lethal. This raises the question as to whether these essential RNA processing functions are conserved in plants. Characterisation of an allelic series of fy mutations reveals that null alleles are embryo lethal. Furthermore, silencing of FY, but not FCA, is deleterious to growth in Nicotiana. The late-flowering fy alleles are hypomorphic and indicate a requirement for both intact FY WD repeats and the C-terminal domain in repression of FLC. The FY C-terminal domain binds FCA and in vitro assays demonstrate a requirement for both C-terminal FY-PPLPP repeats during this interaction. The expression domain of FY supports its roles in essential and flowering-time functions. Hence, FY may mediate both regulated and constitutive RNA 3'-end processing.

Policy entrepreneurship in the development of public sector strategy: the case of London health reform

The development of health policy is recognized as complex; however, there has been little development of the role of agency in this process. Kingdon developed the concept of policy entrepreneur (PE) within his ‘windows’ model. He argued inter-related ‘policy streams' must coincide for important issues to become addressed. The conjoining of these streams may be aided by a policy entrepreneur. We contribute by clarifying the role of the policy entrepreneur and highlighting the translational processes of key actors in creating and aligning policy windows. We analyse the work in London of Professor Sir Ara Darzi as a policy entrepreneur. An important aspect of Darzi's approach was to align a number of important institutional networks to conjoin related problems. Our findings highlight how a policy entrepreneur not only opens policy windows but also yokes together a network to make policy agendas happen. Our contribution reveals the role of clinical leadership in health reform.