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BackgroundAnkle fracture is a common injury with a strong evidence base focused on effectiveness of treatments. However, there are no reporting guidelines on distal tibia and ankle fractures. This has led to heterogeneity in outcome reporting and consequently, restricted the contribution of evidence syntheses. Over the past decade, core outcome sets have been developed to address this issue and are available for several common fractures, including those of the hip, distal radius, and open tibial fractures. This protocol describes the process to co-produce—with patient partners and other key stakeholders—a multi-stakeholder derived Core Outcome Set for distal Tibia and Ankle fractures (COSTA). The scope of COSTA will be for clinical trials.MethodsThe study will have five-stages which will include the following: (i) systematic reviews of existing qualitative studies and outcome reporting in randomised controlled trial studies to inform a developing list of potential outcome domains; (ii) qualitative interviews (including secondary data) and focus groups with patients and healthcare professionals to explore the impact of ankle fracture and the outcomes that really matter; (iii) generation of meaningful outcome statements with the study team, international advisory group and patient partners; (iv) a multi-round, international e-Delphi study to achieve consensus on the core domain set; and (v) an evidence-based consensus on a core measurement set will be achieved through a structured group consensus meeting, recommending best assessment approaches for each of the domains in the core domain set.DiscussionDevelopment of COSTA will provide internationally endorsed outcome assessment guidance for clinical trials for distal tibia and ankle fractures. This will enhance comparative reviews of interventions, potentially reducing reporting bias and research waste.

Background Major trauma is the leading cause of death in people aged Objectives To assess the deep surgical site infection rate, disability, quality of life, patient assessment of the surgical scar and resource use in patients with surgical incisions associated with fractures following major trauma to the lower limbs treated with incisional negative-pressure wound therapy versus standard dressings. Design A pragmatic, multicentre, randomised controlled trial. Setting Twenty-four specialist trauma hospitals representing the UK Major Trauma Network. Participants A total of 1548 adult patients were randomised from September 2016 to April 2018. Exclusion criteria included presentation > 72 hours after injury and inability to complete questionnaires. Interventions Incisional negative-pressure wound therapy (n = 785), in which a non-adherent absorbent dressing covered with a semipermeable membrane is connected to a pump to create a partial vacuum over the wound, versus standard dressings not involving negative pressure (n = 763). Trial participants and the treating surgeon could not be blinded to treatment allocation. Main outcome measures Deep surgical site infection at 30 days was the primary outcome measure. Secondary outcomes were deep infection at 90 days, the results of the Disability Rating Index, health-related quality of life, the results of the Patient and Observer Scar Assessment Scale and resource use collected at 3 and 6 months post surgery. Results A total of 98% of participants provided primary outcome data. There was no evidence of a difference in the rate of deep surgical site infection at 30 days. The infection rate was 6.7% (50/749) in the standard dressing group and 5.8% (45/770) in the incisional negative-pressure wound therapy group (intention-to-treat odds ratio 0.87; 95% confidence interval 0.57 to 1.33; p = 0.52). There was no difference in the deep surgical site infection rate at 90 days: 13.2% in the standard dressing group and 11.4% in the incisional negative-pressure wound therapy group (odds ratio 0.84, 95% confidence interval 0.59 to 1.19; p = 0.32). There was no difference between the two groups in disability, quality of life or scar appearance at 3 or 6 months. Incisional negative-pressure wound therapy did not reduce the cost of treatment and was associated with a low probability of cost-effectiveness. Limitations Owing to the emergency nature of the surgery, we anticipated that some patients who were randomised would subsequently be unable or unwilling to participate. However, the majority of the patients (85%) agreed to participate. Therefore, participants were representative of the population with lower-limb fractures associated with major trauma. Conclusions The findings of this study do not support the use of negative-pressure wound therapy in patients having surgery for major trauma to the lower limbs. Future work Our work suggests that the use of incisional negative-pressure wound therapy dressings in other at-risk surgical wounds requires further investigation. Future research may also investigate different approaches to reduce postoperative infections, for example the use of topical antibiotic preparations in surgical wounds and the role of orthopaedic implants with antimicrobial coatings when fixing the associated fracture. Trial registration Current Controlled Trials ISRCTN12702354 and UK Clinical Research Network Portfolio ID20416. Funding This project was funded by the National Institute for Health Research Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 24, No. 38. See the NIHR Journals Library for further project information.

ObjectiveTo determine research priorities for the management of broken bones of the upper limb in people over 50, which represent the shared priorities of patients, their families, carers and healthcare professionals.Design/settingA national (UK) research priority setting partnership.ParticipantsPeople aged 50 and over who have experienced a fracture of the upper limb, carers involved in their care, family and friends of patients, healthcare professionals involved in the treatment of these patients.MethodsUsing a multiphase methodology in partnership with the James Lind Alliance over 15 months (September 2017 to December 2018), a national scoping survey asked respondents to submit their research uncertainties. These were amalgamated into a smaller number of research questions. The existing evidence was searched to ensure that the questions had not already been answered. A second national survey asked respondents to prioritise the research questions. A final shortlist of 25 questions was taken to a multi-stakeholder workshop where a consensus was reached on the top 10 priorities.ResultsThere were 1898 original uncertainties submitted by 328 respondents to the first survey. These original uncertainties were refined into 51 research questions of which 50 were judged to be true uncertainties following a review of the research evidence. There were 209 respondents to the second (interim prioritisation) survey. The top 10 priorities encompass a broad range of uncertainties in management and rehabilitation of upper limb fractures.ConclusionsThe top 10 UK research priorities highlight uncertainties in how we assess outcomes, provide information, achieve pain control, rationalise surgical intervention, optimise rehabilitation and provide psychological support. The breadth of these research areas highlights the value of this methodology. This work should help to steer research in this area for the next 5–10 years and the challenge for researchers now is to refine and deliver answers to these research priorities.

The transient transport of electrolytes in thermally-activated batteries is studied using electron probe micro-analysis (EPMA), demonstrating the robust capability of EPMA as a useful tool for studying and quantifying mass transport within porous materials, particularly in difficult environments where classical flow measurements are challenging. By tracking the mobility of bromine and potassium ions from the electrolyte stored within the separator into the lithium silicon anode and iron disulfide cathode, we are able to quantify the transport mechanisms and physical properties of the electrodes including permeability and tortuosity. Due to the micron to submicron scale porous structure of the initially dry anode, a fast capillary pressure driven flow is observed into the anode from which we are able to set a lower bound on the permeability of 10−1 mDarcy. The transport into the cathode is diffusion-limited because the cathode originally contained some electrolyte before activation. Using a transient one-dimensional diffusion model, we estimate the tortuosity of the cathode electrode to be 2.8 ± 0.8.

ObjectivesThe time taken for older people to recover from hip fracture can be extensive. The aim of this study was to gain an understanding of patient and informal carer experience of recovery in the early stage, while in acute care.DesignA phenomenological (lived experience) approach was used to guide the design of the study. Interviews and observation took place between March 2016 and December 2016 in acute care.SettingTrauma wards in a National Health Service Foundation Trust in the South West of England.ParticipantsA purposive sample of 25 patients were interviewed and observation taking 52 hours was undertaken with 13 patients and 12 staff. 11 patients had memory loss, 2 patients chose to take part in an interview and observation. The age range was 63–91 years (median 83), 10 were men. A purposive sample of 25 informal carers were also interviewed, the age range was 42–95 years (mean 64), 11 were men.ResultsThe results identified how participants moved forward together after injury by sharing the journey. This was conveyed through three themes: (1) sustaining relationships while experiencing strong emotions and actively helping, (2) becoming aware of uncertainty about the future and working through possible outcomes, (3) being changed, visibly looking different, not being able to walk, and enduring indignity and pain.ConclusionThis study identified the experience of patients and informal carers as they shared the journey during a challenging life transition. Strategies that support well-being and enable successful negotiation of the emotional and practical challenges of acute care may help with longer term recovery. Research should focus on developing interventions that promote well-being during this transition to help provide the foundation for patients and carers to live fulfilled lives.

ObjectiveTo determine research priorities in fragility fractures of the lower limb and pelvis which represent the shared priorities of patients, their friends and families, carers and healthcare professionals.Design/settingA national (UK) research priority setting partnership.ParticipantsPatients over 60 years of age who have experienced a fragility fracture of the lower limb or pelvis; carers involved in their care (both in and out of hospital); family and friends of patients; healthcare professionals involved in the treatment of these patients including but not limited to surgeons, anaesthetists, paramedics, nurses, general practitioners, physicians, physiotherapists and occupational therapists.MethodsUsing a multiphase methodology in partnership with the James Lind Alliance over 18 months (August 2016–January 2018), a national scoping survey asked respondents to submit their research uncertainties. These were amalgamated into a smaller number of research questions. The existing evidence was searched to ensure that the questions had not been answered. A second national survey asked respondents to prioritise the research questions. A final shortlist of 25 questions was taken to a multistakeholder workshop where a consensus was reached on the top 10 priorities.ResultsThere were 963 original uncertainties submitted by 365 respondents to the first survey. These original uncertainties were refined into 88 research questions of which 76 were judged to be true uncertainties following a review of the research evidence. Healthcare professionals and other stakeholders (patients, carers, friends and families) were represented equally in the responses. The top 10 represent uncertainties in rehabilitation, pain management, anaesthesia and surgery.ConclusionsWe report the top 10 UK research priorities in patients with fragility fractures of the lower limb and pelvis. The priorities highlight uncertainties in rehabilitation, postoperative physiotherapy, pain, weight-bearing, infection and thromboprophylaxis. The challenge now is to refine and deliver answers to these research priorities.

A study was performed to validate a first-principles model for whisker and hillock formation based on the cyclic dynamic recrystallization (DRX) mechanism in conjunction with long-range diffusion. The test specimens were evaporated Sn films on Si having thicknesses of 0.25 μm, 0.50 μm, 1.0 μm, 2.0 μm, and 4.9 μm. Air annealing was performed at 35°C, 60°C, 100°C, 120°C, or 150°C over a time duration of 9 days. The stresses, anelastic strains, and strain rates in the Sn films were predicted by a computational model based upon the constitutive properties of 95.5Sn-3.9Ag-0.6Cu (wt.%) as a surrogate for pure Sn. The cyclic DRX mechanism and, in particular, whether long whiskers or hillocks were formed, was validated by comparing the empirical data against the three hierarchal requirements: (1) DRX to occur at all: ec = A D o Z n , (2) DRX to be cyclic: D o

Many challenges must be overcome in order to create reliable electrochemical energy storage devices with not only high energy but also high power densities. Gaps exist in both battery and supercapacitor technologies, with neither one satisfying the need for both large power and energy densities in a single device. To begin addressing these challenges (and others), we report a process to create a self-assembled array of electrochemically active nanoparticles bound directly to a current collector using extremely short (2 nm or less) conductive tethers. The tethered array of nanoparticles, MnO in this case, bound directly to a gold current collector via short conducting linkages eliminates the need for fillers, resulting in a material which achieves 99.9% active material by mass (excluding the current collector). This strategy is expected to be both scalable as well as effective for alternative tethers and metal oxide nanoparticles.

Time-lapse SEM studies of Sn whiskers were conducted to estimate growth kinetics and document whisker morphologies. For straight whiskers, growth rates of 3 to 4 microns per day were measured at room temperature. Two types of kinked whiskers were observed. For Type A kinks, the original growth segment spatial orientation remains unchanged, there are no other changes in morphology or diameter, and growth continues. For Type B kinks, the spatial orientation of the original segment changes and it appears that the whisker bends over. Whiskers with Type B kinks show changes in morphology and diameter at the base, indicating grain boundary motion in the film, which eliminates the conditions suitable for long-term whisker growth. To estimate the errors in the whisker growth measurements, a technique is presented to correct for SEM projection effects. With this technique, the actual growth angles and lengths of a large number of whiskers were collected. It was found that most whiskers grow at moderate or shallow angles with respect to the surface; few straight whiskers grow nearly normal to the surface. In addition, there is no simple correlation between growth angles and lengths for whiskers observed over an approximate 2-year period.

The recognition of single-stranded RNA (ssRNA) is an important aspect of gene regulation, and a number of different classes of protein domains that recognize ssRNA in a sequence-specific manner have been identified. Recently, we demonstrated that the RanBP2-type zinc finger (ZnF) domains from the human splicing factor ZnF Ran binding domain-containing protein 2 (ZRANB2) can bind to a sequence containing the consensus AGGUAA. Six other human proteins, namely, Ewing's sarcoma (EWS), translocated in liposarcoma (TLS)/FUS, RNA-binding protein 56 (RBP56), RNA-binding motif 5 (RBM5), RNA-binding motif 10 (RBM10) and testis-expressed sequence 13A (TEX13A), each contains a single ZnF with homology to the ZRANB2 ZnFs, and several of these proteins have been implicated in the regulation of mRNA processing. Here, we show that all of these ZnFs are able to bind with micromolar affinities to ssRNA containing a GGU motif. NMR titration data reveal that binding is mediated by the corresponding surfaces on each ZnF, and we also show that sequence selectivity is largely limited to the GGU core motif and that substitution of the three flanking adenines that were selected in our original selection experiment has a minimal effect on binding affinity. These data establish a subset of RanBP2-type ZnFs as a new family of ssRNA-binding motifs.

Low temperature, Sn-based Pb-free solders were developed by making alloy additions to the starting material, 96.5Sn-3.5Ag (mass%). The melting behavior was determined using Differential Scanning Calorimetry (DSC). The solder microstructure was evaluated by optical microscopy and electron probe microanalysis (EPMA). Shear strength measurements, hardness tests, intermetallic compound (IMC) layer growth measurements, and solderability tests were performed on selected alloys. Three promising ternary alloy compositions and respective solidus temperatures were: 91.84Sn-3.33Ag-4.83Bi, 212 � C; 87.5Sn-7.5Au-5.0Bi, 200 � C; and 86.4Sn-5.1Ag-8.5Au, 205 � C. A quaternary alloy had the composition 86.8Sn-3.2Ag-5.0Bi-5.0Au and solidus temperature of 194 � C. The shear strength of this quaternary alloy was nearly twice that of the eutectic Sn-Pb solder. The 66Sn-5.0Ag-10Bi-5.0Au-10In-4.0Cu alloy had a solidus temperature of 178 � C and good solderability on Cu. The lowest solidus temperature of 159 � C was realized with the alloy 62Sn-5.0Ag-10Bi-4.0Au-10In-4.0Cu-5.0Ga. The contributing factor towards the melting point depression was the composition of the solid solution, Sn-based matrix phase of each solder.

As scientists, we are comfortable communicating our work to our peers. However, communicating with equal passion to those outside our field is something we as a community often shy away from. This reticence has often been exploited by those wishing to present their own view of the science we practice. Herein, we urge the scientific community to actively re-engage with the general public, openly and creatively sharing our endeavors beyond these pages, in order to reconnect with the real consumers of the work we produce.

The ninth and tenth type III domains of fibronectin each contain specific cell binding sequences, RGD in FIII10 and PHSRN in FIII9, that act synergistically in mediating cell adhesion. We investigated the relationship between domain-domain orientation and synergistic adhesive activity of the FIII9 and FIII10 pair of domains. The interdomain interaction of the FIII9-10 pair was perturbed by introduction of short flexible linkers between the FIII9 and FIII10 domains. Incremental extensions of the interdomain link between FIII9 and FIII10 reduced the initial cell attachment, but had a much more pronounced effect on the downstream cell adhesion events of spreading and phosphorylation of focal adhesion kinase. The extent of disruption of cell adhesion depended upon the length of the interdomain linker. Nuclear magnetic resonance spectroscopy of the wild type and mutant FIII9-10 proteins demonstrated that the structure of the RGD-containing loop is unaffected by domain-domain interactions. We conclude that integrin-mediated cell adhesion to the central cell binding domain of fibronectin depends not only upon specific interaction sites, but also on the relative orientation of these sites. These data have implications for the molecular mechanisms by which integrin-ligand interactions are achieved.

The structure of mosaic proteins depends on the nature and strength of interactions between individual modules. Here we investigated the structural significance of module-module interactions in the RGD-depen dent cell binding region of human fibronectin, comprising the ninth and tenth fibronectin type III. A combination of protein engineering, thermodynamics and nuclear magnetic resonance methods was employed to establish a relationship between intermodular protein - protein interactions and the structural properties of the module pair. A poly(glycine) peptide link connecting the C terminus of the ninth and the N terminus of the tenth module was introduced to probe the range of the interaction. NMR studies (Chemical shifts and 15N relaxation) together with equilibrium and kinetic unfolding experiments were carried out on five different single and double module constructs. The results show that non-specific protein - protein interactions provide the bulk of the thermodynamic stabilization and the motional constraint of the two modules. Specific interactions between the two modules are restricted to the wild-type module pair and decline very rapidly with the insertion of additional linker residues. This low level of specificity is nonetheless sufficient to fine-tune the precise module-module orientation and to provide the full biological activity of the wild-type pair. This suggests that individual modules in mosaic proteins can achieve a high degree of motional constraint and mutual stabilization without the requirement for intricate and specific interactions in the module-module interfaces.

Solid state growth of intermetallic compound layers that form between hot dipped indium coatings and copper was investigated in diffusion couples aged at temperatures of 70, 100 and 135 °C and time periods of up to 300 days. At an annealing temperature of 70 °C, the metastable composition, Cu36In64, was observed at the interface. Ageing at 100 °C caused a dual layer structure with the Cu36In64 layer joined by a copper-rich intermetallic compound, Cu11In9, that is noted in the equilibrium phase diagram. An annealing temperature of 135 °C caused the eventual development of a single copper-rich intermetallic layer, Cu57In43, at the interface. Total intermetallic layer thickness was documented as a function of ageing time and temperature, exhibiting at 1/2 dependence with an apparent activation energy of 20 kJ mol−1.

Solid state intermetallic compound layer growth was examined following ther-mal aging of the 58Bi-42Sn/Cu couple for a temperature range of 55 to 120°C and time periods of from 1 to 400 days. The intermetallic compound layer was comprised of sublayers that included the traditional Cu6Sn5 stoichiometry as well as one or more complex Cu-Sn-Bi chemistries. The number of sublayers increased with aging temperature and time. Time-dependent layer thickness computations based upon the empirical expression, Atn + B, revealed a time exponent, n, that decreased with increasing temperature from a maximum of 0.551 at 70°C to 0.417 at 120°C. The apparent activation energy for growth (at 100 days) was 55± 7 kJ/mol. The Bi-Sn/Cu data, together with that from the other solder/copper systems, suggested that at a given homologous temperature, the quantity of Sn in the solder field determines the intermetallic compound layer thickness as a function of time.

There are many different techniques available to biologists and biochemists that can be used to detect and characterize the self-association of proteins. Each technique has strengths and weaknesses and it is often useful to combine several approaches to maximize the former and minimize the latter. Here we review a range of methodologies that identify protein self-association and/or allow the stoichiometry and affinity of the interaction to be determined, placing an emphasis on what type of information can be obtained and outlining the advantages and disadvantages involved. In general, in vitro biophysical techniques, such as size exclusion chromatography, analytical ultracentrifugation, scattering techniques, NMR spectroscopy, isothermal titration calorimetry, fluorescence anisotropy and mass spectrometry, provide information on stoichiometry and/or binding affinities. Other approaches such as cross-linking, fluorescence methods (e.g., fluorescence correlation spectroscopy, FCS; Förster resonance energy transfer, FRET; fluorescence recovery after photobleaching, FRAP; and proximity imaging, PRIM) and complementation approaches (e.g., yeast two hybrid assays and bimolecular fluorescence complementation, BiFC) can be used to detect protein self-association in a cellular context.

There are many different techniques available to biologists and biochemists that can be used to detect and characterize the self-association of proteins. Each technique has strengths and weaknesses and it is often useful to combine several approaches to maximize the former and minimize the latter. Here we review a range of methodologies that identify protein self-association and/or allow the stoichiometry and affinity of the interaction to be determined, placing an emphasis on what type of information can be obtained and outlining the advantages and disadvantages involved. In general, in vitro biophysical techniques, such as size exclusion chromatography, analytical ultracentrifugation, scattering techniques, NMR spectroscopy, isothermal titration calorimetry, fluorescence anisotropy and mass spectrometry, provide information on stoichiometry and/or binding affinities. Other approaches such as cross-linking, fluorescence methods (e.g., fluorescence correlation spectroscopy, FCS; Forster resonance energy transfer, FRET; fluorescence recovery after photobleaching, FRAP; and proximity imaging, PRIM) and complementation approaches (e.g., yeast two hybrid assays and bimolecular fluorescence complementation, BiFC) can be used to detect protein self-association in a cellular context.

Tin (Sn) whiskers are conductive Sn filaments that grow from Sn-plated surfaces, such as surface finishes on electronic packages. The phenomenon of Sn whiskering has become a concern in recent years due to requirements for lead (Pb)-free soldering and surface finishes in commercial electronics. Pure Sn finishes are more prone to whisker growth than their Sn-Pb counterparts and high profile failures due to whisker formation (causing short circuits) in space applications have been documented. At Sandia, Sn whiskers are of interest due to increased use of Pb-free commercial off-the-shelf (COTS) parts and possible future requirements for Pb-free solders and surface finishes in high-reliability microelectronics. Lead-free solders and surface finishes are currently being used or considered for several Sandia applications. Despite the long history of Sn whisker research and the recently renewed interest in this topic, a comprehensive understanding of whisker growth remains elusive. This report describes recent research on characterization of Sn whiskers with the aim of understanding the underlying whisker growth mechanism(s). The report is divided into four sections and an Appendix. In Section 1, the Sn plating process is summarized. Specifically, the Sn plating parameters that were successful in producing samples with whiskers will be reviewed. In Section 2, the scanning electron microscopy (SEM) of Sn whiskers and time-lapse SEM studies of whisker growth will be discussed. This discussion includes the characterization of straight as well as kinked whiskers. In Section 3, a detailed discussion is given of SEM/EBSD (electron backscatter diffraction) techniques developed to determine the crystallography of Sn whiskers. In Section 4, these SEM/EBSD methods are employed to determine the crystallography of Sn whiskers, with a statistically significant number of whiskers analyzed. This is the largest study of Sn whisker crystallography ever reported. This section includes a review of previous literature on Sn whisker crystallography. The overall texture of the Sn films was also analyzed by EBSD. Finally, a short Appendix is included at the end of this report, in which the X-Ray diffraction (XRD) results are discussed and compared to the EBSD analyses of the overall textures of the Sn films. Sections 2, 3, and 4 have been or will be submitted as stand-alone papers in peer-reviewed technical journals. A bibliography of recent Sandia Sn whisker publications and presentations is included at the end of the report.

The Proton-21 Laboratory in the Ukraine has been publishing results on shock-induced transmutation of several elements, including Cobalt 60 into non-radioactive elements. This report documents exploratory characterization of a shock-compressed Aluminum-6061 sample, which is the only available surrogate for the high-purity copper samples in the Proton-21 experiments. The goal was to determine Sandia's ability to detect possible shock-wave-induced transmutation products and to unambiguously validate or invalidate the claims in collaboration with the Proton-21 Laboratory. We have developed a suitable characterization process and tested it on the surrogate sample. Using trace elemental analysis capabilities, we found elevated and localized concentrations of impurity elements like the Ukrainians report. All our results, however, are consistent with the ejection of impurities that were not in solution in our alloy or were deposited from the cathode during irradiation or possibly storage. Based on the detection capabilities demonstrated and additional techniques available, we are positioned to test samples from Proton-21 if funded to do so.

Although several models have been proposed to account for how cytoskeleton polymerization drives protrusion in cell motility, the precise mechanism remains controversial. Here, we show that, in addition to force exerted directly against the membrane by growing filaments, the way elongating filaments pack also contributes to protrusion by generating an expansion of the cytoskeleton gel. Tomography shows that filament packing in the major sperm protein (MSP) -based nematode sperm-motility machinery resembles that observed with rigid rods. Maximum rod-packing density decreases dramatically as the rods lengthen. Therefore, as filaments elongate, the cytoskeleton gel expands to accommodate their packing less densely. This volume expansion combines with polymerization to drive protrusion. Consistent with this hypothesis, an engineered MSP mutant that generates shorter filaments shows higher filament-packing density and slower movement.

Nuclear abundant poly(A) RNA-binding protein 2 (Nab2) is an essential yeast heterogeneous nuclear ribonucleoprotein that modulates both mRNA nuclear export and poly(A) tail length. The N-terminal domain of Nab2 (residues 1–97) mediates interactions with both the C-terminal globular domain of the nuclear pore-associated protein, myosin-like protein 1 (Mlp1), and the mRNA export factor, Gfd1. The solution and crystal structures of the Nab2 N-terminal domain show a primarily helical fold that is analogous to the PWI fold found in several other RNA-binding proteins. In contrast to other PWI-containing proteins, we find no evidence that the Nab2 N-terminal domain binds to nucleic acids. Instead, this domain appears to mediate protein:protein interactions that facilitate the nuclear export of mRNA. The Nab2 N-terminal domain has a distinctive hydrophobic patch centered on Phe73, consistent with this region of the surface being a protein:protein interaction site. Engineered mutations within this hydrophobic patch attenuate the interaction with the Mlp1 C-terminal domain but do not alter the interaction with Gfd1, indicating that this patch forms a crucial component of the interface between Nab2 and Mlp1.

A method to measure interfacial mechanical properties at high temperatures and in a controlled atmosphere has been developed to study anodized aluminum surface coatings at temperatures where the interior aluminum alloy is molten. This is the first time that the coating strength has been studied under these conditions. We have investigated the effects of ambient atmosphere, temperature, and surface finish on coating strength for samples of aluminum alloy 7075. Surprisingly, the effective Young's modulus or strength of the coating when tested in air was twice as high as when samples were tested in an inert nitrogen or argon atmosphere. Additionally, the effective Young's modulus of the anodized coating increased with temperature in an air atmosphere but was independent of temperature in an inert atmosphere. The effect of surface finish was also examined. Sandblasting the surface prior to anodization was found to increase the strength of the anodized coating with the greatest enhancement noted for a nitrogen atmosphere. Machining marks were not found to significantly affect the strength.

Thermally-activated (or molten salt) batteries utilize high melting temperature (> 300 ºC) salts as the electrolyte. Since the electrolyte is solid at room temperature, an advantage of these types of batteries is that there is no self-discharge prior to activation, which significantly increases the shelf life compared to conventional electrochemical batteries. To activate the battery, a heat source (usually an internal pyrotechnic) is required to melt the electrolyte and enable current flow. During the melting/activation process, the separator in each cell thins until the oxide binder supports the cell compaction force. An open question is what becomes of the melted electrolyte within the separator and electrodes during activation. The transport and infiltration of the electrolyte into the electrodes can reduce the internal impedance of the cell; however, excess electrolyte could cause shortage and failure of the battery. Therefore, an improved fundamental understanding of the governing transport properties of the electrolyte is needed to better predict the performance of thermal batteries during activation. In this work, we performed a series of fundamental studies to probe the ionic mobility and conductivity of the electrolyte. First, by utilizing a series of single cells frozen at various activated times, we performed electron probe microanalysis (EPMA) on the cell cross-sections to quantify the transport of bromine tracers. Through the use of image analysis techniques, we mapped out of the concentration of bromine throughout each cell and elucidated the complex process of electrolyte diffusion and flow. Next, using a custom-built pressure vessel cell, we quantified the permeability of the electrolyte through the oxide binder during and after activation. Furthermore, we investigated the effects of compaction pressure on both the permeability and internal impedance of the separator. Collectively, these experimental studies provide quantitative transport information about the electrolyte, which can be used to inform predictive models for battery activation and improve the understanding of the impact of separator compaction on electrolyte transport properties. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.

The stainless steel alloy 17-4PH contains a martensitic microstructure and second phase delta ({delta}) ferrite. Strengthening of 17-4PH is attributed to Cu-rich precipitates produced during age hardening treatments at 900-1150 F (H900-H1150). For wrought 17-4PH, the effects of heat treatment and microstructure on mechanical properties are well-documented [for example, Ref. 1]. Fewer studies are available on cast 17-4PH, although it has been a popular casting alloy for high strength applications where moderate corrosion resistance is needed. Microstructural features and defects particular to castings may have adverse effects on properties, especially when the alloy is heat treated to high strength. The objective of this work was to outline the effects of microstructural features specific to castings, such as shrinkage/solidification porosity, on the mechanical behavior of investment cast 17-4PH. Besides heat treatment effects, the results of metallography and SEM studies showed that the largest effect on mechanical properties is from shrinkage/solidification porosity. Figure 1a shows stress-strain curves obtained from samples machined from castings in the H925 condition. The strength levels were fairly similar but the ductility varied significantly. Figure 1b shows an example of porosity on a fracture surface from a room-temperature, quasi-static tensile test. The rounded features represent the surfaces ofmore » dendrites which did not fuse or only partially fused together during solidification. Some evidence of local areas of fracture is found on some dendrite surfaces. The shrinkage pores are due to inadequate backfilling of liquid metal and simultaneous solidification shrinkage during casting. A summary of percent elongation results is displayed in Figure 2a. It was found that higher amounts of porosity generally result in lower ductility. Note that the porosity content was measured on the fracture surfaces. The results are qualitatively similar to those found by Gokhale et al. and Surappa et al. in cast A356 Al and by Gokhale et al. for a cast Mg alloys. The quantitative fractography and metallography work by Gokhale et al. illustrated the strong preference for fracture in regions of porosity in cast material. That is, the fracture process is not correlated to the average microstructure in the material but is related to the extremes in microstructure (local regions of high void content). In the present study, image analysis on random cross-sections of several heats indicated an overall porosity content of 0.03%. In contrast, the area % porosity was as high as 16% when measured on fracture surfaces of tensile specimens using stereology techniques. The results confirm that the fracture properties of cast 17-4PH cannot be predicted based on the overall 'average' porosity content in the castings.« less

The simplicity and specialization of the cell motility machinery of Ascaris sperm provides a powerful system in which to probe the basic molecular mechanism of amoeboid cell motility. Although Ascaris sperm locomotion closely resembles that seen in many other types of crawling cell, movement is generated by modulation of a cytoskeleton based on the major sperm protein (MSP) rather than the actin present in other cell types. The Ascaris motility machinery can be studied conveniently in a cell-free in vitro system based on the movement of plasma membrane vesicles by fibres constructed from bundles of MSP filaments. In addition to ATP, MSP and a plasma membrane protein, reconstitution of MSP motility in this cell-free extract requires cytosolic proteins to orchestrate the site-specific assembly and bundling of MSP filaments that generates locomotion. One of these proteins, MFP2, accelerates the rate of movement in this assay. Here, we describe crystal structures of two isoforms of MFP2 and show that both are constructed from two domains that have the same fold based on a novel, compact beta sheet arrangement. Patterns of conservation observed in a structure-based analysis of MFP2 sequences from different nematode species identified regions that may be putative functional interfaces involved both in interactions between MFP2 domains and also with other components of the sperm motility machinery. Analysis of the growth of fibres in vitro in the presence of added MFP2 indicated that MFP2 increases the rate of locomotion by enhancing the effective rate of MSP filament polymerization. This observation, together with the structural data, suggests that MFP2 may function in a manner analogous to formins in actin-based motility.

The mRNA nuclear export function of Tap/NXF1 requires interactions with nuclear pore proteins (nucleoporins) that contain characteristic Phe-Gly repeats based on FG, GLFG or FxFG cores separated by hydrophilic linkers. FG-nucleoporins bind the two most C-terminal domains of Tap, which have NTF2 and UBA folds, respectively. We used a combination of NMR and X-ray crystallography to define the interaction interface between Tap UBA and FxFG nucleoporins and show that it involves primarily the two aromatic rings of the FxFG core that bind in a hydrophobic surface depression centred on Tap Cys588. NMR evidence indicates that the same depression mediates the binding of GLFG nucleoporins, which we confirmed by demonstrating competition between the two classes of repeat for binding to Tap UBA. Moreover, modification of Cys588 reduced the binding of Tap UBA to both GLFG and FxFG nucleoporins as well as to nuclear envelopes. These data underscore the central role of the conserved FG-nucleoporin repeat cores in binding to Tap UBA and indicate that functional differences between different classes of nucleoporins depend more on their spatial distribution in nuclear pores than on their binding to different sites on Tap UBA.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

The trafficking of macromolecules between cytoplasm and nucleus through nuclear pore complexes is mediated by specific carrier molecules such as members of the importin-β family. Nuclear pore proteins (nucleoporins) frequently contain sequence repeats based on FG cores and carriers appear to move their cargo through the pores by hopping between successive FG cores. A major question is why some macromolecules are transported while others are not. This selectivity may be generated by the ability to bind FG repeats, a local concentration of carrier–cargo complexes near the entrance to the pore channel, and steric hindrance produced by high concentrations of nucleoporins in the channel.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

The beta subunit cytoplasmic domains of integrin adhesion receptors are necessary for the connection of these receptors to the actin cytoskeleton. The cytoplasmic protein, talin, binds to beta integrin cytoplasmic tails and actin filaments, hence forming an integrin-cytoskeletal linkage. We used recombinant structural mimics of beta(1)A, beta(1)D and beta(3) integrin cytoplasmic tails to characterize integrin-binding sites within talin. Here we report that an integrin-binding site is localized within the N-terminal talin head domain. The binding of the talin head domain to integrin beta tails is specific in that it is abrogated by a single point mutation that disrupts integrin localization to talin-rich focal adhesions. Integrin-cytoskeletal interactions regulate integrin affinity for ligands (activation). Overexpression of a fragment of talin containing the head domain led to activation of integrin alpha(IIb)beta(3); activation was dependent on the presence of both the talin head domain and the integrin beta(3) cytoplasmic tail. The head domain of talin thus binds to integrins to form a link to the actin cytoskeleton and can thus regulate integrin function.

Forensics investigations of bio-crime or bio-terrorism incidents require careful analysis of collected evidentiary material. Although the biological markers in the evidentiary material are important (e.g. genomic signatures, protein markers), the elemental make-up of the organisms themselves and the surrounding non-biological material is extremely useful for attributing a specific process and, perhaps, specific persons to the production of the biological agent. This talk will describe the coordinated use of microanalytical techniques such as SEM-EDX, STEM-EDX, and NanoSIMS for generating compositional signatures for bio-forensics investigations. These analytical techniques span length scales from the 50 {micro}m range to the 5nm range. The range of analytical sensitivities spans from {approx}.5wt% for EDX down to parts per billion for SIMS techniques. In addition, we will discuss the use of spectrum imaging techniques for rapidly extracting the key elemental signatures from large scale data sets. Spectrum imaging techniques combined with multivariate statistical analysis allow for the collection and interrogation or enormous quantities of data without pre-biasing the answer.[1] Spectrum imaging has been used successfully in EDX microanalysis[1] (both in the SEM and TEM) and TOF-SIMS[2]. In this study, a set of test biological agents, ?-irradiated Bacillus thuringiensis (Bt), were examined using the aforementioned microanalytical techniques. The sample set included a number of processing conditions to gauge the ability of these techniques to identify the production methods of these simulated agents. Complementary but distinct forensic signatures were obtained by all three analytical techniques. Figure 1 shows two types of silicate particles observed among the spore material itself. At this length scale, the spores themselves cannot be resolved, but the presence of these silicates is key marker for distinguishing this production route. A STEM-EDX spectrum image from the same material does not show these large silicates but instead shows the segregation of elements such as sulfur and silicon to the extra-cellular material between spores, again a result of the specific process used to produce this simulated agent (Figure 2). NanoSIMS data from the same material also shows the segregation of Si in this preparation. The NanoSIMS data also displays and quantifies the distribution of elements such as fluorine at levels which were not detectable in the STEM-EDX measurements (Figure 3).

Recent reports have described transformation- and tumour-specific expression of fibronectin isoforms generated by alternative splicing of the fibronectin pre-mRNA. We have investigated the expression and distribution of EDIIIA+ and EDIIIB+ fibronectin splice variants in tumorigenic and non-tumorigenic somatic cell hybrids made by fusing fibrosarcoma-derived cells (HT1080) and normal fibroblasts (GM00097). Alternative splicing of EDIIIA and EDIIIB was assessed quantitatively by S1 nuclease analyses. The levels of EDIIIA+ and EDIIIB+ fibronectin mRNAs were similar in the parental and hybrid cells. Domain-specific monoclonal antibodies were used in immunohistochemical studies to identify EDIIIA+ and EDIIIB+ fibronectins in fixed cells. GM00097 and the non-tumorigenic hybrid (clone G3) showed high levels of both EDIIIA+ and EDIIIB+ fibronectin staining. The tumorigenic hybrid (clone C1) showed reduced amounts of EDIIIA+ fibronectin, but no detectable EDIIIB+ fibronectin. No fibronectin was detected on the surface of HT1080 cells. Western blots of protein extracted from culture supernatants and extracellular matrices revealed that GM00097 and G3 cells incorporated most of the EDIIIA+ and EDIIIB+ fibronectin into the extracellular matrix whereas C1 cells released a large proportion of the EDIIIA+ fibronectin, and almost all of the EDIIIB+ fibronectin, into the supernatant. We conclude that there are differences in the presence of EDIIIA+ and EDIIIB+ FNs on the surface of tumorigenic and non-tumorigenic cells and that these differences are due to differential incorporation of FN variants into the ECM.