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Abstract Background “Herbarium X-ray Fluorescence (XRF) Ionomics” is a new quantitative approach for extracting the elemental concentrations from herbarium specimens using handheld XRF devices. These instruments are principally designed for dense sample material of infinite thickness (such as rock or soil powder), and their built-in algorithms and factory calibrations perform poorly on the thin dry plant leaves encountered in herbaria. While empirical calibrations have been used for ‘correcting’ measured XRF values post hoc, this approach has major shortcomings. As such, a universal independent data analysis pipeline permitting full control and transparency throughout the quantification process is highly desirable. Here we have developed such a pipeline based on Dynamic Analysis as implemented in the GeoPIXE package, employing a Fundamental Parameters approach requiring only a description of the measurement hardware and derivation of the sample areal density, based on a universal standard. Results The new pipeline was tested on potassium, calcium, manganese, iron, cobalt, nickel, and zinc concentrations in dry plant leaves. The Dynamic Analysis method can correct for complex X-ray interactions and performs better than both the built-in instrument algorithms and the empirical calibration approach. The new pipeline is also able to identify and quantify elements that are not detected and reported by the device built-in algorithms and provides good estimates of elemental concentrations where empirical calibrations are not straightforward. Conclusions The new pipeline for processing XRF data of herbarium specimens has a greater accuracy and is more robust than the device built-in algorithms and empirical calibrations. It also gives access to all elements detected in the XRF spectrum. The new analysis pipeline has made Herbarium XRF approach even more powerful to study the metallome of existing plant collections.

Abstract Background Hyperaccumulation of trace elements is a rare trait among plants which is being investigated to advance our understanding of the regulation of metal accumulation and applications in phytotechnologies. Noccaea caerulescens (Brassicaceae) is an intensively studied hyperaccumulator model plant capable of attaining extremely high tissue concentrations of zinc and nickel with substantial genetic variation at the population-level. Micro-X-ray Fluorescence spectroscopy (µXRF) mapping is a sensitive high-resolution technique to obtain information of the spatial distribution of the plant metallome in hydrated samples. We used laboratory-based µXRF to characterize a collection of 86 genetically diverse Noccaea caerulescens accessions from across Europe. We developed an image-processing method to segment different plant substructures in the µXRF images. We introduced the concentration quotient (CQ) to quantify spatial patterns of metal accumulation and linked that to genetic variation. Results Image processing resulted in automated segmentation of µXRF plant images into petiole, leaf margin, leaf interveinal and leaf vasculature substructures. The harmonic means of recall and precision (F1 score) were 0.79, 0.80, 0.67, and 0.68, respectively. Spatial metal accumulation as determined by CQ is highly heritable in Noccaea caerulescens for all substructures, with broad-sense heritability (H2) ranging from 76 to 92%, and correlates only weakly with other heritable traits. Insertion of noise into the image segmentation algorithm barely decreases heritability scores of CQ for the segmented substructures, illustrating the robustness of the trait and the quantification method. Very low heritability was found for CQ if randomly generated substructures were compared, validating the approach. Conclusions A strategy for segmenting µXRF images of Noccaea caerulescens is proposed and the concentration quotient is developed to provide a quantitative measure of metal accumulation pattern, which can be used to determine genetic variation for such pattern. The metric is robust to segmentation error and provides reliable H2 estimates. This strategy provides an avenue for quantifying XRF data for analysis of the genetics of metal distribution patterns in plants and the subsequent discovery of new genes that regulate metal homeostasis and sequestration in plants.

Abstract The objective of this study was to evaluate the performance of two commercial‐grade electromagnetic sensors in measuring soil water content for the U.S. Climate Reference Network (USCRN). One sensor was a 50‐MHz coaxial impedance dielectric sensor (model HydraProbe, Stevens Water Monitoring Systems). The second sensor was a 1‐GHz time domain reflectometry (TDR) sensor (model TDR‐315L, Acclima). There was no substantial difference between both probes in daily mean volumetric soil water content and soil temperature measurements at a 0.1‐m depth in a customized uniform loamy soil testbed in Oak Ridge, TN, from 2016 to 2018, and at select USCRN stations with a variety of soils. However, the TDR‐315L provided more representative soil water content measurements for stations with high‐clay‐content soils than the HydraProbe. These results confirm the necessity of using site‐specific soil properties for soil sensors like the HydraProbe for estimating soil water content in different soil environments. They also suggest that TDR sensors may provide an opportunity to improve USCRN soil moisture measurements in higher‐clay‐content soils.

Injuries sustained while performing electrical work are a significant threat to the health and safety of workers and occur frequently. In some jurisdictions, non-fatal serious incidents have increased in recent years. Although significant work has been carried out on electrical safety from a human factor perspective, reviews of this literature are sparse. Thus, the purpose of this review is to collate and summarize human factors implicated in electrical safety events. Articles were collected from three databases (Scopus, Web of Science, and Google Scholar), using the search terms: safety, electri*, human factors, and arc flash. Titles and abstracts were screened, full-text reviews were conducted, and 18 articles were included in the final review. Quality checks were undertaken using the Mixed Methods Appraisal Tool and the Critical Appraisal Skills Program. Environmental, individual, team, organizational, and macro factors were identified in the literature as factors which shape frontline electrical worker behavior, highlighting the complexity of injury prevention. The key contributions of this paper include: (1) a holistic and integrated summary of human factors implicated in electrical safety events, (2) the application of an established theoretical model to explain dynamic forces implicated in electrical safety incidents, and (3) several practical implications and recommendations to improve electrical safety. It is recommended that this framework is used to develop and test future interventions at the individual, team, organizational, and regulator level to mitigate risk and create meaningful and sustainable change in the electrical safety space.

This article outlines and evaluates the US perspective on how treaty and customary international law protect the natural environment during international armed conflict. It surveys the relevant treaties to which the United States is a party and examines US views on their pertinent provisions. It then assesses claims that the environmental obligations residing in the 1977 Additional Protocol I to the 1949 Geneva Conventions have attained customary status, outlines the United States' rejection of those claims, and evaluates the reasonableness thereof. Finally, it highlights ambiguities in certain US environmental positions, the resolution of which would bring much-needed clarity to the law. [ABSTRACT FROM AUTHOR]

ABSTRACT Streptococcus pyogenes (group A Streptococcus [GAS]) causes ~700 million human infections/year, resulting in >500,000 deaths. There is no commercial GAS vaccine available. The GAS surface protein arginine deiminase (ADI) protects mice against a lethal challenge. ADI is an enzyme that converts arginine to citrulline and ammonia. Administration of a GAS vaccine preparation containing wild-type ADI, a protein with inherent enzymatic activity, may present a safety risk. In an approach intended to maximize the vaccine safety of GAS ADI, X-ray crystallography and structural immunogenic epitope mapping were used to inform vaccine design. This study aimed to knock out ADI enzyme activity without disrupting the three-dimensional structure or the recognition of immunogenic epitopes. We determined the crystal structure of ADI at 2.5 Å resolution and used it to select a number of amino acid residues for mutagenesis to alanine (D166, E220, H275, D277, and C401). Each mutant protein displayed abrogated activity, and three of the mutant proteins (those with the D166A, H275A, and D277A mutations) possessed a secondary structure and oligomerization state equivalent to those of the wild type, produced high-titer antisera, and avoided disruption of B-cell epitopes of ADI. In addition, antisera raised against the D166A and D277A mutant proteins bound to the GAS cell surface. The inactivated D166A and D277A mutant ADIs are ideal for inclusion in a GAS vaccine preparation. There is no human ortholog of ADI, and we confirm that despite limited structural similarity in the active-site region to human peptidyl ADI 4 (PAD4), ADI does not functionally mimic PAD4 and antiserum raised against GAS ADI does not recognize human PAD4. IMPORTANCE We present an example of structural biology informing human vaccine design. We previously showed that the administration of the enzyme arginine deiminase (ADI) to mice protected the mice against infection with multiple GAS serotypes. In this study, we determined the structure of GAS ADI and used this information to improve the vaccine safety of GAS ADI. Catalytically inactive mutant forms of ADI retained structure, recognition by antisera, and immunogenic epitopes, rendering them ideal for inclusion in GAS vaccine preparations. This example of structural biology informing vaccine design may underpin the formulation of a safe and efficacious GAS vaccine.

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus found in fish from oceans of the northern hemisphere and freshwaters of Europe. It has caused extensive losses of cultured and wild fish and has become established in the North American Great Lakes. Large die-offs of wild fish in the Great Lakes due to VHSV have alarmed the public and provoked government attention on the introduction and spread of aquatic animal pathogens in freshwaters. We investigated the relations between VHSV dispersion and shipping and boating activity in the Great Lakes by sampling fish and water at sites that were commercial shipping harbors, recreational boating centers, and open shorelines. Fish and water samples were individually analyzed for VHSV using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and cell culture assays. Of 1,221 fish of 17 species, 55 were VHSV positive with highly varied qRT-PCR titers (1 to 5,950,000 N gene copies). The detections of VHSV in fish and water samples were closely associated and the virus was detected in 21 of 30 sites sampled. The occurrence of VHSV was not related to type of site or shipping related invasion hotspots. Our results indicate that VHSV is widely dispersed in the Great Lakes and is both an enzootic and epizootic pathogen. We demonstrate that pathogen distribution information could be developed quickly and is clearly needed for aquatic ecosystem conservation, management of affected populations, and informed regulation of the worldwide trade of aquatic organisms.

Fluidization experiments were conducted on a small scale and with a rapid response (short duration) to enable corresponding simulations at low‐computational cost. Rise times are reported for four or fewer polyethylene particles (intruders) in an air‐fluidized bed of ~5000 group D glass beads. Experimental inputs were completely characterized—particle properties, system dimensions and operating conditions—which is necessary for validating computational fluid mechanics (CFD)‐discrete element method (DEM) including a comprehensive uncertainty quantification (UQ) analysis. Input uncertainties are reported as bounds or cumulative distribution functions of measured values. The staggering number of simulations required to complete a UQ analysis (~O[104] simulations corresponding to ~5 uncertain inputs) motivates this study. These segregating‐bed experiments are designed to permit analogous CFD‐DEM simulations to complete in less than a day on a single (~2.5 GHz) computational processor unit (CPU). Segregation times are reported for several operating conditions, intruder sizes, and initial configurations, providing a rich dataset for numerical model testing, validation and UQ. [ABSTRACT FROM AUTHOR]

St. Augustinegrass [Stenotaphrum secundatum (Walter) Kuntze] is a popular warm‐season turfgrass in the southern United States. Gray leaf spot (GLS), caused by the fungal pathogen Pyricularia oryzae Cavara, is one of the major diseases in St. Augustinegrass. Although previous studies have reported polyploid lines with resistance to GLS, no comprehensive evaluations of sources of resistance have been performed in the genus. Such evaluations will enable breeders to identify resistant parents for cultivar development. In this study, 58 genotypes of St. Augustinegrass and two genotypes of pembagrass [Stenotaphrum dimidiatum (L.) Brongn.] were screened for resistance to three different P. oryzae sources of inoculum under controlled environmental conditions. The parameters evaluated were incubation period, number of leaves with lesions, mean lesion length, area under the disease progress curve (AUDPC), and area under the lesion expansion curve (AULEC). Significant differences among genotypes were identified. Polyploid genotypes PI 365031, PI 290888, PI 300129, PI 300130, and cultivar 'FX‐10' and diploid genotype PI 410353 consistently showed high levels of resistance across trials, inoculum sources, and parameters. The identification of resistance genes in diploid genotypes is of great importance for future St. Augustinegrass breeding efforts, as this germplasm pool can be more readily exploited because of the lack of reproductive barriers with most commercial cultivars and plant introductions. [ABSTRACT FROM AUTHOR]

Much confusion exists on whether force‐ or energy‐based descriptions of cohesive‐particle interactions are more appropriate. We hypothesize a force‐based description is appropriate when enduring‐contacts dominate and an energy‐based description when contacts are brief in nature. Specifically, momentum is transferred through force‐chains when enduring‐contacts dominate and particles need to overcome a cohesive force to induce relative motion, whereas particles experiencing brief contacts transfer momentum through collisions and must overcome cohesion‐enhanced energy losses to avoid agglomeration. This hypothesis is tested via an attempt to collapse the dimensionless, dependent variable characterizing a given system against two dimensionless numbers: A generalized bond number, BoG–ratio of maximum cohesive force to the force driving flow, and a new Agglomerate number, Ag–ratio of critical cohesive energy to the granular energy. A gamut of experimental and simulation systems (fluidized bed, hopper, etc.), and cohesion sources (van der Waals, humidity, etc.), are considered. For enduring‐contact systems, collapse occurs with BoG but not Ag, and vice versa for brief‐contact systems, thereby providing support for the hypothesis. An apparent discrepancy with past work is resolved, and new insight into Geldart's classification is gleaned. [ABSTRACT FROM AUTHOR]

This work investigates how one can postprocess a series of flash-generated X-ray radiographs with known point spread functions and collection geometries to produce radiographs with enhanced image quality and/or to approximate a multiframe X-ray movie by spatially correlating temporally sequenced images. To produce images with enhanced quality, one collects multiple individual projections, simultaneously, and then uses the known point spread function and acquisition geometry to correlate and integrate the numerous projections as if they were acquired from approximately the same perspective. If using sources of similar spectral characteristics, the outcome correlates with that which would be produced from a multiflash integration, where one benefits from an increase in flux of X-ray photons. If using sources of different spectral characteristics, a composite radiograph can be produced where the unique source spectrum can be matched to known material absorption cross-sections to accentuate radiographic features within the target material. To produce multiframe movies, similar mathematics are applied to images collected throughout a timespan over which the object changes. The aforementioned processes' mathematics and examples are demonstrated for the case in which the individual projections are completely separable on the detector with no overlap. Finally, a potential method is discussed for separation of images in the case where multiple radiograph projections are overlapped and entangled on the detector. [ABSTRACT FROM AUTHOR]

The magnetization reversal of patterned NiFe nanolines, which have a universal dependence of a switching field, is examined. The coercivity of the narrowest lines increases rapidly and the inverse-width trend is recovered when the temperature declines.

Bermudagrass [Cynodon spp. (L.) Rich.] is a high-quality, durable turfgrass with excellent heat and drought tolerance. However, its lack of freezing tolerance limits its use in the transition zone. The development of cultivars with enhanced freezing tolerance would constitute a significant improvement in the management of bermudagrass in this region and could extend its area of adaptation further north. There has been substantial work on screening of common-type bermudagrass [Cynodon dactylon (L.) Pers.] germplasm for freezing tolerance, but not for the African (Cynodon transvaalensis Burtt-Davy) germplasm. The purpose of this research was to conduct multiyear field testing and laboratory-based freezing test evaluations of winter hardiness and freezing tolerance, respectively, of an African and common bermudagrass germplasm collection. A high level of cold hardiness was observed among the germplasm in this study. In field evaluations, plant introductions (PIs) PI 290905, PI 647879, PI 255447, PI 289923, and PI 615161 were the top performers, having consistently greater spring green-up and reduced winterkill compared with 'Patriot', 'Tifsport', 'Quickstand', and 'Tifway', though not always significantly. A comparison between field-based ratings and calculated lethal temperatures for 50% death (LT50) from laboratory-based freezing tests showed significant correlations of -0.26 and -0.24 for spring green-up and winterkill, respectively, suggesting that these controlled freeze experiments could be used to prescreen materials prior to field testing. Overall, results indicate that some of the PIs evaluated in this study can be used as additional sources of cold hardiness in bermudagrass breeding. [ABSTRACT FROM AUTHOR]

In order to quantitatively investigate the interplay between shape anisotropy (magnetostatics) and exchange, patterned zigzag structures were prepared with sizes varying over two orders of magnitude. The magnetic state is a balance between shape anisotropy, which causes the magnetization to follow the serrated edge of the structure, and exchange, which prefers uniform magnetization. In intermediate-sized structures, we find that the magnetization along the center of the zigzag oscillates between approximately ±32° and is relatively independent of the device size. As the dimensions are reduced to less than a critical length λcr, the oscillation magnitude drops rapidly. On the other hand, the largest structures develop extended film properties such as multiple vortices and ripple. [ABSTRACT FROM AUTHOR]

We have successfully demonstrated the construction of patterned magnetic nanostructures down to 50 nm dimensions using electron-beam lithography. Two types of patterned structures have been fabricated for this work: nanostructured arrays consisting of isolated identical elements and nanostructured chains of alternating elements with different widths. Small patterned arrays (∼200 &micron;m X 200 &micron;m) have been characterized using a variable temperature (5 to 325 K) focused magneto-optic Kerr effect (MOKE) measurement system. The switching field of the isolated element arrays is found to be inversely proportional to the element width. However, as the elements of two different widths are connected to form a chain, magnetization reversal is essentially controlled by the wider constituent, indicating that the switching of chains starts in the wider elements. Domain walls in these wide elements then propagate through the narrower components, requiring a lower field than domain nucleation in the narrow elements. Magnetic force microscopy (MFM) images show that in the virgin state the narrower elements are domain wall free but complex domain patterns exist in the wider elements. Both field-dependent MFM imaging and micromagnetic simulations agree very well with the MOKE measurements. [ABSTRACT FROM AUTHOR]

Hypervelocity impacts of copper rods into liquid filled channels circumferentially confined by steel cylinders were investigated using 2D axisymmetric hydrocode simulations. The confined liquid was utilized as a means to investigate the interplay of eroded target material and the remaining projectile in order to elucidate relevant penetration behaviors in solid materials. This led to a better understanding of two significant contributing parameters in the flow characteristics: the density (how difficult the material is to accelerate) and the channel diameter (how far the material has to move). While the liquid eliminates direct strength effects on penetration of the rods, varying the channel width allows a survey of parameter space that is typically a function of both material strength and density. Three successive copper rods with varying velocities and delays were used to model a penetrator representative of select sections of a shaped charge jet. For each target material, the residual penetration in solid rolled homogeneous armor (RHA) was then measured as a function of the liquid channel width. A strong dependence on both the material density and channel width was observed in regards to the efficiency of the material against penetration. [ABSTRACT FROM AUTHOR]

Bermudagrass, Cynodon spp. is one of the most commonly grown turfgrass genera in the southern United States having excellent drought tolerance, but poor tolerance to shade. Developing cultivars tolerant to shade would allow bermudagrass to become more prevalent in home lawns or other recreational areas in the southeast, where trees dominate the landscape. In this field study, nine accessions collected from Pretoria, South Africa were evaluated for their ability to grow under shade with varying fertility treatments. These accessions and cultivars 'Celebration', 'TifGrand', and 'Tifway' were evaluated under 0%, 63%, and 80% continuous shade during 2011-12. For both years, significant differences among shade levels, genotypes, and the interaction of the two were observed. As expected, the progression from 0% to 63% to 80% shade reduced normalized difference vegetation index (NDVI), percent turfgrass cover (TC), and turf quality (TQ) readings for all accessions. Some genotypes, however, were able to maintain adequate quality and aggressiveness under 63% shade. 'Celebration', WIN10F, and STIL03 performed better than 'Tifway' (P ≤ 0.05), the susceptible control. Overall, our results indicate that there are promising genotypes among the bermudagrass materials collected from South Africa. These accessions represent additional sources of shade hardiness to be used in bermudagrass breeding. Furthermore, higher nitrogen fertility provided increased NDVI and TQ in some instances suggesting an added benefit of fertility under low-light conditions. However, the increased economic value attributed to the added inputs associated with these increases is outweighed by the low impacts offered. [ABSTRACT FROM AUTHOR]

Terrestrial locomotion can impose substantial loads on vertebrate limbs. Previous studies have shown that limb bones from cursorial species of eutherian mammals experience high bending loads with minimal torsion, whereas the limb bones of non- avian reptiles (and amphibians) exhibit considerable torsion in addition to bending. It has been hypothesized that these differences in loading regime are related to the difference in limb posture between upright mammals and sprawling reptiles, and that the loading patterns observed in non-avian reptiles may be ancestral for tetrapod vertebrates. To evaluate whether non- cursorial mammals show loading patterns more similar to those of sprawling lineages, we measured in vivo strains in the femur during terrestrial locomotion of the Virginia opossum (Dideiphis virginiana), a marsupial that uses more crouched limb posture than most mammals from which bone strains have been recorded, and which belongs to a dade phylogenetically between reptiles and the eutherian mammals studied previously. The presence of substantial torsion in the femur of opossums, similar to non- avian reptiles, would suggest that this loading regime likely reflects an ancestral condition for tetrapod limb bone design. Strain recordings indicate the presence of both bending and appreciable torsion (shear strain: 419.1±212.8μϵ) in the opossum femur, with planar strain analyses showing neutral axis orientations that placed the lateral aspect of the femur in tension at the time of peak strains. Such mediolateral bending was unexpected for a mammal running with near-parasagittal limb kinematics. Shear strains were similar in magnitude to peak compressive axial strains, with opossum femora experiencing similar bending loads but higher levels of torsion compared with most previously studied mammals. Analyses of peak femoral strains led to estimated safety factor ranges of 5.1-7.2 in bending and 5.5-7.3 in torsion, somewhat higher than typical mammalian values for bending, but approaching typical reptilian values for shear. Loading patterns of opossum limb bones therefore appear intermediate in some respects between those of eutherian mammals and non-avian reptiles, providing further support for hypotheses that high torsion and elevated limb bone safety factors may represent persistent ancestral conditions in the evolution of tetrapod limb bone loading and design. [ABSTRACT FROM AUTHOR]

• An electrothermal research gun capable of launching small projectiles (<1.0 g) at hypervelocity (2 – 4 km/s) was designed. • Limiting chamber expansion was key to improving performance. • Performance has some dependence on anode point shape, however if sufficient copper vapor is produced, a plateau in velocity is achieved. • ALEGRA proved to be an effective tool to predict and diagnose efficiency mechanisms including comparisons to PDV, chamber expansion, and soft captured projectiles. An electrothermal research gun capable of firing small cylindrical projectiles up to 4 km/s has been developed. While electromagnetic forces certainly exist in the system, it is not an electromagnetic launch system, but rather utilizes an electrical arc to produce a rapidly expanding gas that is used as the working fluid. A pointed copper anode is insulated such that the arc initiates only at the very tip, while the electrical circuit is completed through both the projectile and the barrel. Experiments show that copper vapor from the eroding copper electrode is the most likely working fluid of the gun, however, a portion of the aluminum projectile is also eroded. The design was directly aided by simulations performed using the multiphysics hydrocode ALEGRA developed by Sandia National Laboratories. Simulations included investigating the anode tip shape as well as limiting the expansion of the chamber due to the rapid expansion of gasses caused by the arc. Comparisons of projectile velocity profiles from Photon Doppler Velocimetry, barrel expansion, and pellet erosion with the ALEGRA results were utilized as benchmarks, leading to significant velocity gains for given energy inputs. [ABSTRACT FROM AUTHOR]

• Stochastic variations were included in material parameters of a shaped charge jet. • Eulerian simulations impart modest lateral drift without material variations. • Density variations in shaped charge jet liners material significantly affect drift. • Asymmetries in a simulated shaped charge jet are visually apparent. This report investigates the ability of the shock multiphysics hydrocode, ALEGRA, to simulate the lateral drift of a shaped charge jet through stochastic variations of simulation parameters. Stochastic variations in the explosive density and the liner initial yield strength, fracture pressure, and density were introduced, and a method was developed to compare the magnitude of lateral drift between the simulations. The extent to which each of the material parameter variations affected lateral drift is presented and compared to a baseline simulation. Variations in liner yield strength, liner fracture pressure, and explosive density were found to impart a modest amount of lateral drift, while variations in the liner density imparted a significant amount of lateral drift. [ABSTRACT FROM AUTHOR]

We report a universal dependence of switching field of patterned magnetic nanolines as a function of the linewidth for Co and NiFe films of various thicknesses. This dependence is shown to be consistent with a nucleation picture in which the magnetization reversal is controlled only by a small nucleus equivalent to a particle with an aspect ratio of 1.25, which spreads across the width of the nanoline. Micromagnetic simulation, taking into account of the edge roughness, agrees well with the observed results. © 2004 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Abstract Objective Using a physician‐directed, patient “opt‐out” approach to prescriptive smoking cessation in the emergency department (ED) setting, we set out to describe patient actions as they related to smoking cessation behaviors. Methods A convenience sample of smokers at 2 Pennsylvania hospital EDs who met inclusion/exclusion criteria were approached to participate in a brief intervention known as screening, treatment initiation, and referral (STIR) counseling that included phone follow‐up. Demographic information, current smoking status, and specific physician prescription and follow‐up recommendations were collected. Approximately 3 months later, patients were contacted to determine current smoking status and actions taken since their ED visit. Results One hundred six patients were approached and 7 (6.6%) opted out of the intervention. Patients who did not opt out were evaluated for appropriate use of smoking cessation‐related medications; 35 (35.4%) opted out of the prescription(s) and 6 (6.1%) were not indicated. Twenty‐one (21.2%) patients opted out of ambulatory referral follow‐ups with primary care and/or tobacco treatment program; one (1.0%) was not indicated for referral. Nineteen (32.8%) patients who received prescription(s) for smoking cessation‐related medications initially also followed the prescription(s). Seventeen (22.1%) patients participated in referral follow‐up. Conclusion In this small ED pilot, using the STIR concepts in an opt‐out method, few smokers opted out of the smoking cessation intervention. About one‐third of the patients declined prescriptions for smoking cessation‐related medications and less than one‐quarter declined ambulatory referrals for follow‐up. These findings support a willingness of patients to participate in STIR and the benefits of intervention in this setting.

The N-terminal Toll/interleukin-1 receptor/resistance protein (TIR) domain has been shown to be both necessary and sufficient for defence signalling in the model plants flax and Arabidopsis. In examples from these organisms, TIR domain self-association is required for signalling function, albeit through distinct interfaces. Here, we investigate these properties in the TIR domain containing resistance protein RPV1 from the wild grapevine Muscadinia rotundifolia. The RPV1 TIR domain, without additional flanking sequence present, is autoactive when transiently expressed in tobacco, demonstrating that the TIR domain alone is capable of cell-death signalling. We determined the crystal structure of the RPV1 TIR domain at 2.3 Å resolution. In the crystals, the RPV1 TIR domain forms a dimer, mediated predominantly through residues in the αA and αE helices (AE interface). This interface is shared with the interface discovered in the dimeric complex of the TIR domains from the Arabidopsis RPS4/RRS1 resistance protein pair. We show that surface-exposed residues in the AE interface that mediate the dimer interaction in the crystals are highly conserved among plant TIR domain-containing proteins. While we were unable to demonstrate self-association of the RPV1 TIR domain in solution or using yeast 2-hybrid, mutations of surface-exposed residues in the AE interface prevent the cell-death autoactive phenotype. In addition, mutation of residues known to be important in the cell-death signalling function of the flax L6 TIR domain were also shown to be required for RPV1 TIR domain mediated cell-death. Our data demonstrate that multiple TIR domain surfaces control the cell-death function of the RPV1 TIR domain and we suggest that the conserved AE interface may have a general function in TIR-NLR signalling.

Background and aims: Pancreatic cyst fluid (PCF) CEA has been shown to be the most accurate preoperative test for detection of cystic mucinous neoplasms (CMNs). This study aimed to assess the added value of PCF KRAS mutational analysis to CEA for diagnosis of CMNs. Patients and methods: This is a retrospective study of prospectively collected endoscopic ultrasonography (EUS) fine-needle aspiration (FNA) data. KRAS mutation was determined by direct sequencing or equivalent methods. Cysts were classified histologically (surgical cohort) or by clinical (EUS or FNA) findings (clinical cohort). Performance characteristics of KRAS, CEA and their combination for detection of a cystic mucinous neoplasm (CMN) and malignancy were calculated. Results: The study cohort consisted of 943 patients: 147 in the surgical cohort and 796 in the clinical cohort. Overall, KRAS and CEA each had high specificity (100 % and 93.2 %), but low sensitivity (48.3 % and 56.3 %) for the diagnosis of a CMN. The positivity of KRAS or CEA increased the diagnostic accuracy (80.8 %) and AUC (0.84) significantly compared to KRAS (65.3 % and 0.74) or CEA (65.8 % and 0.74) alone, but only in the clinical cohort (P

Group A Streptococcus (GAS) is a human pathogen that has the potential to cause invasive disease by binding and activating human plasmin(ogen). Streptococcal surface enolase (SEN) is an octameric α-enolase that is localized at the GAS cell surface. In addition to its glycolytic role inside the cell, SEN functions as a receptor for plasmin(ogen) on the bacterial surface, but the understanding of the molecular basis of plasmin(ogen) binding is limited. In this study, we determined the crystal and solution structures of GAS SEN and characterized the increased plasminogen binding by two SEN mutants. The plasminogen binding ability of SENK312A and SENK362A is ~2- and ~3.4-fold greater than for the wild-type protein. A combination of thermal stability assays, native mass spectrometry and X-ray crystallography approaches shows that increased plasminogen binding ability correlates with decreased stability of the octamer. We propose that decreased stability of the octameric structure facilitates the access of plasmin(ogen) to its binding sites, leading to more efficient plasmin(ogen) binding and activation.

Lipopolysaccharide (LPS), a surface polymer of Gram-negative bacteria, helps bacteria survive in different environments and acts as a virulence determinant of host infection. The O-antigen (Oag) component of LPS exhibits a modal chain-length distribution that is controlled by polysaccharide co-polymerases (PCPs). The molecular basis of the regulation of Oag chain-lengths remains unclear, despite extensive mutagenesis and structural studies of PCPs from Escherichia coli and Shigella. Here, we identified a single mutation (A107P) of the Shigella flexneri WzzBSF, by a random mutagenesis approach, that causes a shortened Oag chain-length distribution in bacteria. We determined the crystal structures of the periplasmic domains of wild-type WzzBSF and the A107P mutant. Both structures form a highly similar open trimeric assembly in the crystals, and show a similar tendency to self-associate in solution. Binding studies by bio-layer interferometry reveal cooperative binding of very short (VS)-core-plus-O-antigen polysaccharide (COPS) to the periplasmic domains of both proteins, but with decreased affinity for the A107P mutant. Our studies reveal that subtle and localized structural differences in PCPs can have dramatic effects on LPS chain-length distribution in bacteria, for example by altering the affinity for the substrate, which supports the role of the structure of the growing Oag polymer in this process.