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Despite the identification of clubroot resistance genes in various Brassica crops our understanding of the genetic basis of immunity toPlasmodiophora brassicaeinfection in the model plantArabidopsis thalianaremains limited. To address this issue we performed a screen of 142 natural accessions and identified 11 clubroot resistant Arabidopsis lines. Genome wide association analysis identified several genetic loci significantly linked with resistance. Three genes from two of these loci were targeted for deletion by CRISPR/Cas9 mutation in resistant accessions Est-1 and Uod-1. Deletion ofResistance to Plasmodiophora brassicae 1(RPB1) rendered both lines susceptible to theP. brassicaepathotype P1+. Further analysis ofrpb1knock-out Est-1 and Uod-1 lines showed that the RPB1 protein is required for activation of downstream defence responses, such as the expression of phytoalexin biosynthesis geneCYP71A13. RPB1 has no known functional domains or homology to previously characterised proteins. The clubroot susceptible Arabidopsis accession Col-0 lacks a functionalRPB1gene; when Col-0 is transformed withRPB1expression driven by its native promoter it is capable of activatingRPB1expression in response to infection but this is not sufficient to confer resistance. Constitutive over-expression ofRPB1in Col-0 leads to drastically reduced growth and activation of stress-responsive genes. Furthermore, we found that transient expression ofRPB1inNicotiana tabacuminduced programmed cell death in leaves. We conclude that RPB1 is a critical component of the defence response toP. brassicaeinfection in Arabidopsis, acting downstream of pathogen recognition but required for the elaboration of effective resistance.

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

BackgroundOscillatory rhythms during sleep such as slow oscillations (SO) and spindles, and most importantly their coupling, are thought to underlie processes of memory consolidation. External slow oscillatory transcranial direct current stimulation (so-tDCS) with a frequency of 0.75 Hz has been shown to improve this coupling and memory consolidation, however, effects varied quite markedly between individuals, studies, and species. Here, we aimed to determine how precisely the frequency of stimulation has to match the naturally occurring SO frequency in individuals to optimally improve SO-spindle coupling. Moreover, we systematically tested stimulation durations necessary to induce changes.MethodsWe addressed these questions by comparing so-tDCS with individualized frequency to standardized frequency of 0.75 Hz in a within-subject design with 28 older participants during napping while systematically varying stimulation train durations between 30 s, 2 min, and 5 min.ResultsStimulation trains as short as 30 s were sufficient to modulate the coupling between SOs and spindle activity. Contrary to our expectations, so-tDCS with standardized frequency indicated stronger aftereffects with regard to SO-spindle coupling compared to individualized frequency. Angle and variance of spindle maxima occurrence during the SO cycle were similarly modulated.ConclusionIn sum, short stimulation trains were sufficient to induce significant changes in sleep physiology allowing for more trains of stimulation, which provides methodological advantages and possibly even larger behavioral effects in future studies. With regard to individualized stimulation frequency, further options of optimization need to be investigated, such as closed-loop stimulation to calibrate stimulation frequency to the SO frequency at time of stimulation onset.Significance statementApplication of slow oscillatory transcranial direct current stimulation during sleep has been shown to enhance specific memory-relevant sleep parameters and memory performance after sleep, albeit with a high degree of variability. Here, we systematically explored two major stimulation parameters possibly accounting for this variability in humans: frequency and duration of stimulation. We found, contrary to our expectations, standardized frequency stimulation with 0.75 Hz being superior to individualized frequency stimulation in enhancing specific sleep parameters. Moreover short stimulation trains of 30 seconds were as effective as 5 min in modulating aftereffects. These are encouraging findings, implying methodological advantages as larger quantity of aftereffects data can be obtained within the same time window, which may also lead to enhanced behavioral stimulation effects.

Certain neurophysiological characteristics of sleep, in particular slow oscillations (SO), sleep spindles, and their temporal coupling, have been well characterized and associated with human memory formation. Delta waves, which are somewhat higher in frequency and lower in amplitude compared to SO, have only recently been found to play a critical role in memory processing of rodents, through a competitive interplay between SO-spindle and delta-spindle coupling. However, human studies that comprehensively address delta waves, their interactions with spindles and SOs as well as their functional role for memory are still lacking.Electroencephalographic data were acquired across three naps of 33 healthy older human participants (17 female) to investigate delta-spindle coupling and the interplay between delta and SO-related activity. Additionally, we determined intra-individual stability of coupling measures and their potential link to the ability to form novel memories.Our results revealed weaker delta-spindle compared to SO-spindle coupling. Contrary to our initial hypothesis, we found that increased delta activity was accompanied by stronger SO-spindle coupling. Moreover, we identified the ratio between SO- and delta-nested spindles as the sleep parameter that predicted ability to form novel memories best.Our study suggests that SOs, delta waves and sleep spindles should be jointly considered when aiming to link sleep physiology and memory formation in aging.Significance statementInterrelations between delta waves, slow oscillations and sleep spindles have recently been causally linked to the balance between consolidation and forgetting in rats using optogenetics. In humans, SO-spindle coupling has been intensively investigated, but delta waves and their interaction with spindles were only studied jointly as SWA. Here we delineate the coupling of delta waves to spindles, investigate interactions of delta- and SO-related activity and investigate their role for the ability to form novel memories in healthy older individuals. Our results show differences as well as dependencies between SO- and delta-related activities including spindle coupling. Further, our results indicate that the ratio of SO- to delta-nested spindles might be the most informative sleep parameter for memory formation of older adults.

Gall formation on the belowground parts of plants infected with Plasmodiophora brassicae is the result of extensive host cellular reprogramming. The development of these structures is a consequence of increased cell proliferation followed by massive enlargement of cells colonized with the pathogen. Drastic changes in cellular growth patterns create local deformities in the roots and hypocotyl giving rise to mechanical tensions within the tissue of these organs. Host cell wall extensibility and recomposition accompany the growth of the gall and influence pathogen spread and also pathogen life cycle progression. Demethylation of pectin within the extracellular matrix may play an important role in P. brassicae-driven hypertrophy of host underground organs. Through proteomic analysis of the cell wall, we identified proteins accumulating in the galls developing on the underground parts of Arabidopsis thaliana plants infected with P. brassicae. One of the key proteins identified was the pectin methylesterase (PME18); we further characterized its expression and conducted functional and anatomic studies in the knockout mutant and used Raman spectroscopy to study the status of pectin in P. brassicae-infected galls. We found that late stages of gall formation are accompanied with increased levels of PME18. We have also shown that the massive enlargement of cells colonized with P. brassicae coincides with decreases in pectin methylation. In pme18-2 knockout mutants, P. brassicae could still induce demethylation; however, the galls in this line were smaller and cellular expansion was less pronounced. Alteration in pectin demethylation in the host resulted in changes in pathogen distribution and slowed down disease progression. To conclude, P. brassicae-driven host organ hypertrophy observed during clubroot disease is accompanied by pectin demethylation in the extracellular matrix. The pathogen hijacks endogenous host mechanisms involved in cell wall loosening to create an optimal cellular environment for completion of its life cycle and eventual release of resting spores facilitated by degradation of demethylated pectin polymers.

When plants are infected by Plasmodiophora brassicae, their developmental programs are subjected to extensive changes and the resultant clubroot disease is associated with formation of large galls on underground tissue. The pathogen’s need to build an efficient feeding site as the disease progresses drives these changes, ensuring successful production of resting spores. This developmental reprogramming is an outcome of interactions between the pathogen and the infected host. During disease progression, we can observe alteration of growth regulator dynamics, patterns of cell proliferation and differentiation, increased cell expansion, and eventual cell wall degradation as well as the redirection of nutrients toward the pathogen. Recently, detailed studies of anatomical changes occurring during infection and studies profiling transcriptional responses have come together to provide a clearer understanding of the sequence of events and processes underlying clubroot disease. Additionally, genome sequencing projects have revealed P. brassicae’s potential for the production of signaling molecules and effectors as well as its requirements and capacities with respect to taking up host nutrients. Integration of these new findings together with physiological studies can significantly advance our understanding of how P. brassicae brings about reprogramming of host development. This article summarizes the current state of knowledge on cellular changes induced by P. brassicae infection and aims to explain their impact and importance for both the host and the pathogen.

OBJECTIVE An intrinsic pain regulatory system is modulated by both cardiovascular dynamics that influence baroreflex sensitivity (BRS) and is diminished in fibromyalgia (FM). Baroreceptors relay cardiovascular output to the dorsal medial nucleus tractus solitarius reflex arcs that regulate pain, sleep, anxiety, and blood pressure. The aim of this study was to evaluate the effects of systolic extinction training (SET), which combines operant treatment (OT) with baroreflex training (BRT). BRT delivers peripheral electrical stimulation within a few milliseconds of the systolic or diastolic peak in the cardiac cycle. In addition, we compared SET to OT-transcutaneous electrical stimulation (TENS) independent of the cardiac cycle and aerobic exercise (AE)-BRT in FM patients with elevated blood pressure responses to stress. METHODS Sixty-two female patients with FM were randomized to receive either SET (n = 21), OT-TENS (n = 20), or AE-BRT (n = 21). Outcome assessments were performed before treatment (T1), after 5 weeks of treatment (T2), and after the 12-month follow-up (T3). RESULTS In contrast to patients receiving OT-TENS or AE-BRT, those receiving SET reported a significantly greater reduction in pain and pain interference (all P < 0.01) that was maintained at the 12-month follow-up. Clinically meaningful pain reduction at T3 was achieved in 82% of patients in the SET group, 39% of those in the OT-TENS group, and only 14% of those in the AE-BRT group. Patients in the SET group showed a significant increase (57%) in BRS following treatment, while neither the AE-BRT group or the OT-TENS group showed significant changes over time. CONCLUSION SET resulted in statistically significant, clinically meaningful, and long-lasting pain remission and interference compared to OT-TENS and AE-BRT. These results suggest that BRS modification is the primary mechanism of improvement. Replication of our results using larger samples and extension to other chronic pain conditions appear to be warranted.

Summary Plasmodiophora brassicae is a soil‐borne biotroph whose life cycle involves reprogramming host developmental processes leading to the formation of galls on its underground parts. Formation of such structures involves modification of the host cell cycle leading initially to hyperplasia, increasing the number of cells to be invaded, followed by overgrowth of cells colonised by the pathogen. Here we show that P. brassicae infection stimulates formation of the E2Fa/RBR1 complex and upregulation of MYB3R1,MYB3R4 and A‐ and B‐type cyclin expression. These factors were previously described as important regulators of the G2−M cell cycle checkpoint. As a consequence of this manipulation, a large population of host hypocotyl cells are delayed in cell cycle exit and maintained in the proliferative state. We also report that, during further maturation of galls, enlargement of host cells invaded by the pathogen involves endoreduplication leading to increased ploidy levels. This study characterises two aspects of the cell cycle reprogramming efforts of P. brassicae: systemic, related to the disturbance of host hypocotyl developmental programs by preventing cell cycle exit; and local, related to the stimulation of cell enlargement via increased endocycle activity., Significance Statement We describe the changes in host cell cycle gene expression in response to P. brassicae infection, and characterise the impact of key cell cycle regulators (MYB3R4, E2Fa, CCS52A1), establishing that cell proliferation during gall development stems from repression of cell cycle exit, whereas hypertrophy in later disease stages depends on stimulation of endoreduplication. This provides a molecular framework for understanding the progress of clubroot disease and gall formation.

IntroductionWith the worldwide increase of life expectancy leading to a higher proportion of older adults experiencing age-associated deterioration of cognitive abilities, the development of effective and widely accessible prevention and therapeutic measures has become a priority and challenge for modern medicine. Combined interventions of cognitive training and transcranial direct current stimulation (tDCS) have shown promising results for counteracting age-associated cognitive decline. However, access to clinical centres for repeated sessions is challenging, particularly in rural areas and for older adults with reduced mobility, and lack of clinical personnel and hospital space prevents extended interventions in larger cohorts. A home-based and remotely supervised application of tDCS would make the treatment more accessible for participants and relieve clinical resources. So far, studies assessing feasibility of combined interventions with a focus on cognition in a home-based setting are rare. With this study, we aim to provide evidence for the feasibility and the effects of a multisession home-based cognitive training in combination with tDCS on cognitive functions of healthy older adults.Methods and analysisThe TrainStim-Home trial is a monocentric, randomised, double-blind, placebo-controlled study. Thirty healthy participants, aged 60–80 years, will receive 2 weeks of combined cognitive training and anodal tDCS over left dorsolateral prefrontal cortex (target intervention), compared with cognitive training plus sham stimulation. The cognitive training will comprise a letter updating task, and the participants will be stimulated for 20 min with 1.5 mA. The intervention sessions will take place at the participants’ home, and primary outcome will be the feasibility, operationalised by two-thirds successfully completed sessions per participant. Additionally, performance in the training task and an untrained task will be analysed.Ethics and disseminationEthical approval was granted by the ethics committee of the University Medicine Greifswald. Results will be available through publications in peer-reviewed journals and presentations at national and international conferences.Trial registration numberNCT04817124.

Summary Drought stress impacts the quality and yield of Pisum sativum. Here, we show how short periods of limited water availability during the vegetative stage of pea alters phloem sap content and how these changes are connected to strategies used by plants to cope with water deficit. We have investigated the metabolic content of phloem sap exudates and explored how this reflects P. sativum physiological and developmental responses to drought. Our data show that drought is accompanied by phloem‐mediated redirection of the components that are necessary for cellular respiration and the proper maintenance of carbon/nitrogen balance during stress. The metabolic content of phloem sap reveals a shift from anabolic to catabolic processes as well as the developmental plasticity of P. sativum plants subjected to drought. Our study underlines the importance of phloem‐mediated transport for plant adaptation to unfavourable environmental conditions. We also show that phloem exudate analysis can be used as a useful proxy to study stress responses in plants. We propose that the decrease in oleic acid content within phloem sap could be considered as a potential marker of early signalling events mediating drought response., Significance Statement We describe the changes in phloem metabolic content of pea (Pisum sativum) plants subjected to drought and show how the observed patterns reflect physiological and developmental responses. Phloem exudate analysis itself gives us a clear and informative picture of the stress response strategy centred on above‐ground growth reduction and the reuse and redistribution of carbon and nitrogen. This provides evidence for the importance of phloem‐mediated long‐distance coordination in plant responses to drought. Fluctuations of particular metabolites in vascular sap can be used to monitor drought response in pea plants.

Remote localized vapor sources manipulate binary droplets in a nonmonotonic way on pristine surfaces for various applications., Droplet motion on surfaces influences phenomena as diverse as microfluidic liquid handling, printing technology, and energy harvesting. Typically, droplets are set in motion by inducing energy gradients on a substrate or flow on their free surface. Current configurations for controllable droplet manipulation have limited applicability as they rely on carefully tailored wettability gradients and/or bespoke substrates. Here, we demonstrate the nonmonotonic contactless long-range manipulation of binary droplets on pristine substrates due to the sensing of localized water vapor sources. The droplet-source system presents an unexpected off-centered equilibrium position. We capture the underlying mechanism behind this symmetry breaking with a simplified model based on the full two-dimensional functional form of the surface tension gradient induced by the source on the droplet’s free surface. This insight on the transport mechanism enables us to demonstrate its versatility for applications by printing, aligning, and reacting materials controllably in space and time on pristine substrates.

Cyberpunk 2077 is a highly anticipated massive open-world video game, with a complex, branching narrative. This talk details new research and innovative workflow contributions, developed by jali, toward the generation of an unprecedented number of hours of realistic, expressive speech animation in ten languages, often with multiple languages interleaved within individual sentences. The speech animation workflow is largely automatic but remains under animator control, using a combination of audio and tagged text transcripts. We use insights from anatomy, perception, and the psycho-linguistic literature to develop independent and combined language models that drive procedural animation of the mouth and paralingual (speech supportive non-verbal expression) motion of the neck, brows and eyes. Directorial tags in the speech transcript further enable the integration of performance capture driven facial emotion. The entire workflow is animator-centric, allowing efficient key-frame customization and editing of the resulting facial animation on any typical facs-like face rig. The talk will focus equally on technical contributions and its integration and creative use within the animation pipeline of the highly anticipated aaa game title: Cyberpunk 2077.

Lolium multiflorum/Festuca arundinacea introgression forms have been proved several times to be good models to identify key components of grass metabolism involved in the mechanisms of tolerance to water deficit. Here, for the first time, a relationship between photosynthetic and antioxidant capacities with respect to drought tolerance of these forms was analyzed in detail. Two closely related L. multiflorum/F. arundinacea introgression forms distinct in their ability to re-grow after cessation of prolonged water deficit in the field were selected and subjected to short-term drought in pots to dissect precisely mechanisms of drought tolerance in this group of plants. The studies revealed that the form with higher drought tolerance was characterized by earlier and higher accumulation of abscisic acid, more stable cellular membranes, and more balanced reactive oxygen species metabolism associated with a higher capacity of the antioxidant system under drought conditions. On the other hand, both introgression forms revealed the same levels of stomatal conductance, CO2 assimilation, and consequently, intrinsic water use efficiency under drought and recovery conditions. However, simultaneous higher adjustment of the Calvin cycle to water deficit and reduced CO2 availability, with respect to the accumulation and activity of plastid fructose-1,6-bisphosphate aldolase, were clearly visible in the form with higher drought tolerance.

Droplets moving on solid surfaces are at the heart of many phenomena of fundamental and applied interest in chemistry, physics and materials science. On the fundamental side, as they are often subject to evaporation, these droplets are a beautiful and complex example of non-equilibrium physical chemistry, whose explanation and understanding still capture the imagination of multiple researchers around the world. In technology, droplets on solid surfaces are of widespread use for handling small amounts of matter, for harvesting energy, for manufacturing materials and for sensing chemical and biological analytes. A key underlying factor of their widespread applicability is the degree of control that can be achieved over their transport on surfaces. This tutorial review provides an overview of recent progress towards the programmable transport of droplets on solid surfaces. We will first present the physical principles behind the main experimental strategies for droplet transport. We will then review the most inspiring applications where these strategies have been employed in chemistry, materials science and engineering. Finally, we will outline possible future research directions for the programmable transport of droplets. Beyond projecting the reader at the forefront of this exciting field of physical chemistry, we believe that this tutorial review will inspire diverse, multidisciplinary scientific communities to devise novel ways of manipulating the flow of matter, energy and information on solid surfaces using programmable droplets as vessels.

The problem of road safety is an important problem that accompanies mankind from the very beginning of the development of communication. Based on the statistics, the article presents the analysis of road traffic safety in our country over the past few years and shows how it shapes compared to other European Union countries. The main reasons, which lie behind the situation, are also identified as well as the ways and directions of actions that are taken to minimize the effects of road accidents. The pace of reduction of the number of fatalities and injured on Polish roads is still unsatisfactory and therefore far-reaching measures to counteract such a situation have to be taken. These steps are based on normative provisions and long-term actions supported by the media and various types of advertising campaigns. The article also presents the issues related to the road transport of dangerous goods, which, due to the specific nature of the cargo carried, is subjected to special treatment and governed by international regulations and arrangements under the ADR Agreement.

Background Urinary disorders in cats often require subjective caregiver quantification of clinical signs to establish a diagnosis and monitor therapeutic outcomes. Objective To investigate use of a video recording system (VRS) to better assess and quantify urination behaviors in cats. Animals Eleven healthy cats and 8 cats with disorders potentially associated with abnormal urination patterns. Methods Prospective study design. Litter box urination behaviors were quantified with a VRS for 14 days and compared to daily caregiver observations. Video recordings were analyzed by a behavior analysis software program. Results The mean number of urinations per day detected by VRS (2.5 ± 0.7) was significantly higher compared with caregiver observations (0.6 ± 0.6; P < .0001). Five cats were never observed in the litter box by their caregivers. The mean number of urinations per day detected by VRS was significantly higher for abnormal cats (2.9 ± 0.7) compared with healthy cats (2.1 ± 0.7; P = .02); there were no apparent differences in frequency between these groups reported by caregivers (0.7 ± 1.0 and 0.5 ± 1.0, respectively). There were no differences in mean urination time between healthy and abnormal cats as determined by VRS or caregivers. Mean cover-up time determined by VRS was significantly longer in healthy cats (22.7 ± 12.9 seconds/urination) compared with abnormal cats (8.7 ± 12.9 seconds/urination; P = .03); differences in cover-up time were not detected by caregivers. Conclusions and Clinical Importance Caregivers commonly underestimate urination frequency in cats when compared to video-based observations. Video recording appears to facilitate objective assessment of urination behaviors and could be of value in future clinical studies of urinary disorders in cats.

The catalytic oligomerization of ethylene with chromium-based complexes containing bis(benzimidazolemethyl)amine (BIMA) ligands results in alternating distributions of linear α-olefins (LAOs). Extremely high activities are obtained (>100 000 g mmol–1 h–1 bar–1) with N-alkyl-substituted BIMA ligands, whereas bulky groups on the central nitrogen or alternative central donors result in much lower activities. Variations in the ligand backbone, as well as methylation of the benzimidazole units, lead to reduction in activity. The alternating LAO distributions have been mathematically analyzed using second-order recurrence relations. The shape of the distributions is affected by ethylene pressure (1–4 bar) and by the cocatalyst to some degree. On the basis of the results and analysis presented herein, we propose that the alternating behavior originates from the ability of these chromium BIMA catalysts to undergo single as well as double ethylene insertion reactions. A minor second distribution (

Background This study evaluates the generalizability of an eight-station progress clinical skills examination and assesses the growth in performance for six clinical skills domains among first- and second-year medial students over four time points during the academic year. Methods We conducted a generalizability study for longitudinal and cross-sectional comparisons and assessed growth in six clinical skill domains via repeated measures ANOVA over the first and second year of medical school. Results The generalizability of the examination domain scores was low but consistent with previous studies of data gathering and communication skills. Variations in case difficulty across administrations of the examination made it difficult to assess longitudinal growth. It was possible to compare students at different training levels and the interaction of level of training and growth. Second-year students outperformed first-year students, but first-year students’ clinical skills performance grew faster than second-year students narrowing the gap in clinical skills over the students’ first year of medical school. Conclusions Case specificity limits the ability to assess longitudinal growth in clinical skills through progress testing. Providing students with early clinical skills training and authentic clinical experiences appears to result in the rapid growth of clinical skills during the first year of medical school.

Contains fulltext : 215333.pdf (Publisher’s version ) (Closed access) BACKGROUND: Patients with stable coronary artery disease and diabetes with previous percutaneous coronary intervention (PCI), particularly those with previous stenting, are at high risk of ischaemic events. These patients are generally treated with aspirin. In this trial, we aimed to investigate if these patients would benefit from treatment with aspirin plus ticagrelor. METHODS: The Effect of Ticagrelor on Health Outcomes in diabEtes Mellitus patients Intervention Study (THEMIS) was a phase 3 randomised, double-blinded, placebo-controlled trial, done in 1315 sites in 42 countries. Patients were eligible if 50 years or older, with type 2 diabetes, receiving anti-hyperglycaemic drugs for at least 6 months, with stable coronary artery disease, and one of three other mutually non-exclusive criteria: a history of previous PCI or of coronary artery bypass grafting, or documentation of angiographic stenosis of 50% or more in at least one coronary artery. Eligible patients were randomly assigned (1:1) to either ticagrelor or placebo, by use of an interactive voice-response or web-response system. The THEMIS-PCI trial comprised a prespecified subgroup of patients with previous PCI. The primary efficacy outcome was a composite of cardiovascular death, myocardial infarction, or stroke (measured in the intention-to-treat population). FINDINGS: Between Feb 17, 2014, and May 24, 2016, 11 154 patients (58% of the overall THEMIS trial) with a history of previous PCI were enrolled in the THEMIS-PCI trial. Median follow-up was 3.3 years (IQR 2.8-3.8). In the previous PCI group, fewer patients receiving ticagrelor had a primary efficacy outcome event than in the placebo group (404 [7.3%] of 5558 vs 480 [8.6%] of 5596; HR 0.85 [95% CI 0.74-0.97], p=0.013). The same effect was not observed in patients without PCI (p=0.76, pinteraction=0.16). The proportion of patients with cardiovascular death was similar in both treatment groups (174 [3.1%] with ticagrelor vs 183 (3.3%) with placebo; HR 0.96 [95% CI 0.78-1.18], p=0.68), as well as all-cause death (282 [5.1%] vs 323 [5.8%]; 0.88 [0.75-1.03], p=0.11). TIMI major bleeding occurred in 111 (2.0%) of 5536 patients receiving ticagrelor and 62 (1.1%) of 5564 patients receiving placebo (HR 2.03 [95% CI 1.48-2.76], p

Background This study evaluates the generalizability of an eight-station progress clinical skills examination and assesses the growth in performance for six clinical skills domains among first- and second-year medical students over four time points during the academic year. Methods We conducted a generalizability study for longitudinal and cross-sectional comparisons and assessed growth in six clinical skill domains via repeated measures ANOVA over the first and second year of medical school. Results The generalizability of the examination domain scores was low but consistent with previous studies of data gathering and communication skills. Variations in case difficulty across administrations of the examination made it difficult to assess longitudinal growth. It was possible to compare students at different training levels and the interaction of training level and growth. Second-year students outperformed first-year students, but first-year students’ clinical skills performance grew faster than second-year students narrowing the gap in clinical skills over the students’ first year of medical school. Conclusions Case specificity limits the ability to assess longitudinal growth in clinical skills through progress testing. Providing students with early clinical skills training and authentic clinical experiences appears to result in the rapid growth of clinical skills during the first year of medical school.

The expression of two genes encoding xyloglucan endotransglucosylase/hydrolases (XTHs), Cs-XTH1 and Cs-XTH3, was upregulated during the onset of cucumber somatic embryogenesis. As a means of characterising the developmental regulation of these genes, the activity of the respective upstream regulatory regions was investigated in seedlings and somatic embryos of Arabidopsis thaliana and Cucumis sativus. GUS assays revealed that both genes are under developmental control. In addition, elevated promoter activity was found in the tension-bearing regions of the plant and in response to touch and wounding, which is consistent with the existence of numerous stress-related cis elements in the 5′-regulatory regions. In vivo xyloglucan endotransglucosylase (XET) action assays were performed to gain an overview on the role of XTHs during somatic embryogenesis. The highest XET action was observed in the external cell layers of somatic embryos in the cotyledonary region and in the presumptive region of peg formation. Based on the results, we propose a dual mechanism (one developmental and the second adaptive) for the regulation of Cs-XTH1 and Cs-XTH3 activity wherein the developmental pattern can be modified by mechanical stimuli.

Successful biotrophic plant pathogens can divert host nutrition toward infection sites. Here we describe how the protist Plasmodiophora brassicae establishes a long-term feeding relationship with its host by stimulating phloem differentiation and phloem-specific expression of sugar transporters within developing galls. Development of galls in infected Arabidopsis (Arabidopsis thaliana) plants is accompanied by stimulation of host BREVIS RADIX, COTYLEDON VASCULAR PATTERN, and OCTOPUS gene expression leading to an increase in phloem complexity. We characterized how the arrest of this developmental reprogramming influences both the host and the invading pathogen. Furthermore, we found that infection leads to phloem-specific accumulation of SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS11 and 12 facilitating local distribution of sugars toward the pathogen. Utilizing Fourier-transform infrared microspectroscopy to monitor spatial distribution of carbohydrates, we found that infection leads to the formation of a strong physiological sink at the site of infection. High resolution metabolic and structural imaging of sucrose distributions revealed that sweet11 sweet12 double mutants are impaired in sugar transport toward the pathogen, delaying disease progression. This work highlights the importance of precise regulation of sugar partitioning for plant–pathogen interactions and the dependence of P. brassicae’s performance on its capacity to induce a phloem sink at the feeding site.

Background The protist Plasmodiophora brassicae is a soil-borne pathogen of cruciferous species and the causal agent of clubroot disease of Brassicas including agriculturally important crops such as canola/rapeseed (Brassica napus). P. brassicae has remained an enigmatic plant pathogen and is a rare example of an obligate biotroph that resides entirely inside the host plant cell. The pathogen is the cause of severe yield losses and can render infested fields unsuitable for Brassica crop growth due to the persistence of resting spores in the soil for up to 20 years. Results To provide insight into the biology of the pathogen and its interaction with its primary host B. napus, we produced a draft genome of P. brassicae pathotypes 3 and 6 (Pb3 and Pb6) that differ in their host range. Pb3 is highly virulent on B. napus (but also infects other Brassica species) while Pb6 infects only vegetable Brassica crops. Both the Pb3 and Pb6 genomes are highly compact, each with a total size of 24.2 Mb, and contain less than 2 % repetitive DNA. Clustering of genome-wide single nucleotide polymorphisms (SNP) of Pb3, Pb6 and three additional re-sequenced pathotypes (Pb2, Pb5 and Pb8) shows a high degree of correlation of cluster grouping with host range. The Pb3 genome features significant reduction of intergenic space with multiple examples of overlapping untranslated regions (UTRs). Dependency on the host for essential nutrients is evident from the loss of genes for the biosynthesis of thiamine and some amino acids and the presence of a wide range of transport proteins, including some unique to P. brassicae. The annotated genes of Pb3 include those with a potential role in the regulation of the plant growth hormones cytokinin and auxin. The expression profile of Pb3 genes, including putative effectors, during infection and their potential role in manipulation of host defence is discussed. Conclusion The P. brassicae genome sequence reveals a compact genome, a dependency of the pathogen on its host for some essential nutrients and a potential role in the regulation of host plant cytokinin and auxin. Genome annotation supported by RNA sequencing reveals significant reduction in intergenic space which, in addition to low repeat content, has likely contributed to the P. brassicae compact genome. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2597-2) contains supplementary material, which is available to authorized users.

Plasmodiophora brassicae (clubroot) infection leads to reprogramming of host development resulting in the formation of characteristic galls. In this work we explored the cellular events that underly gall formation in Arabidopsis thaliana with the help of molecular markers of cell division (CYCB1:GUS) and meristematic activity (ANT:GUS). Our results show that gall development involved the amplification of existing meristematic activities within the vascular cambium (VC) and phloem parenchyma (PP) cells in the region of the hypocotyl. Additionally we found that the increase in VC activity and prolonged maintenance of cambial-derived cells in a meristematic state was crucial for gall formation; disruption of the VC activity significantly decreased the gall size. Gall formation also perturbed vascular development with a significant reduction in xylem and increase in PP in infected plants. This situation was reflected in a decrease in transcripts of key factors promoting xylogenesis (VND6, VND7 and MYB46) and an increase in those promoting phloem formation and function (APL, SUC2). Finally we show, using the cell cycle inhibitor ICK1/KRP1 and a cle41 mutant with altered regulation of cambial stem cell maintenance and differentiation, that a decrease in gall formation did not prevent pathogen development. This finding demonstrates that although gall formation is a typical symptom of the disease and influences numbers of spores produced, it is not required for completion of the pathogen life cycle. Together, these results provide an insight into the relationship of the cellular events that accompany Plasmodiophora infection and their role in disease progression.

A classical view is that leaf shape is the result of local promotion of growth linked to cell proliferation. However, an alternative hypothesis is that leaf form is the result of local repression of growth in an otherwise growing system. Here we show that leaf form can indeed be manipulated in a directed fashion by local repression of growth. We show that targeting expression of an inhibitor of a cyclin-dependent kinase (KRP1) to the sinus area of developing leaves of Arabidopsis leads to local growth repression and the formation of organs with extreme lobing, including generation of leaflet-like organs. Directing KRP1 expression to other regions of the leaf using an miRNA target sequence tagging approach also leads to predictable novel leaf forms, and repression of growth in the leaf margin blocks the outgrowth of lobes, leading to a smoother perimeter. In addition, we show that decreased growth around the perimeter and across the leaf abaxial surface leads to a change in 3D form, as predicted by mechanical models of leaf growth. Our analysis provides experimental evidence that local repression of growth influences leaf shape, suggesting that it could be part of the mechanism of morphogenesis in plants in the context of an otherwise growing system.

Trifolium alexandrinum L. was transformed with the Arabidopsis HARDY gene that belongs to the stress-related AP2/ERF (APETALA2/ethylene responsive element binding factors) superfamily of transcription factors. The fresh weights of the transgenic lines L2 and L3 were improved by 42 and 55% under drought stress and by 38 and 95% under salt stress compared to the wild type, respectively. The dry weights were similarly improved. Overexpression of HARDY improved the instantaneous water use efficiency (WUE) under drought stress by reducing transpiration (E) and under salt stress by improving photosynthesis (A), through reducing Na+ accumulation in leaves, and reducing E. However, HARDY improved the growth of drought-stressed transgenic plants as compared to the wild type by delaying water depletion from soil and preventing rapid decline in A. L2 and L3 had thicker stems and in case of L3, more xylem rows per vascular bundle, which may have made L3 more resistant to lodging in the field. Field performance of L2 and L3 under combined drought and salt stress was significantly better than that of the wild type in terms of fresh and dry weights (40%, 46% and 31%, 40%, respectively). The results provide further evidence for the efficiency of overexpression of a single gene in improving tolerance to abiotic stress under field conditions.

Expansins are cell wall proteins associated with the process of plant growth. However, investigations in which expansin gene expression has been manipulated throughout the plant have often led to inconclusive results. In this article, we report on a series of experiments in which overexpression of expansin was targeted to specific phases of leaf growth using an inducible promoter system. The data indicate that there is a restricted window of sensitivity when increased expansin gene expression leads to increased endogenous expansin activity and an increase in leaf growth. This phase of maximum expansin efficacy corresponds to the mid phase of leaf growth. We propose that the effectiveness of expansin action depends on the presence of other modulating factors in the leaf and we suggest that it is the control of expression of these factors (in conjunction with expansin gene expression) that defines the extent of leaf growth. These data help to explain some of the previously observed variation in growth response following manipulation of expansin gene expression and highlight a potential linkage of the expression of modifiers of expansin activity with the process of exit from cell division.

The tomato wound signal systemin is perceived by a specific high-affinity, saturable, and reversible cell surface receptor. This receptor was identified as the receptor-like kinase SR160, which turned out to be identical to the brassinosteroid receptor BRI1. Recently, it has been shown that the tomato bri1 null mutant cu3 is as sensitive to systemin as wild type plants. Here we explored these contradictory findings by studying the responses of tobacco plants (Nicotiana tabacum) to systemin. A fluorescently-labeled systemin analog bound specifically to plasma membranes of tobacco suspension-cultured cells that expressed the tomato BRI1-FLAG transgene, but not to wild type tobacco cells. On the other hand, signaling responses to systemin, such as activation of mitogen-activated protein kinases and medium alkalinization, were neither increased in BRI1-FLAG-overexpressing tobacco cells nor decreased in BRI1-silenced cells as compared to levels in untransformed control cells. Furthermore, in transgenic tobacco plants BRI1-FLAG became phosphorylated on threonine residues in response to brassinolide application, but not in response to systemin. When BRI1 transcript levels were reduced by virus-induced gene silencing in tomato plants, the silenced plants displayed a phenotype characteristic of bri1 mutants. However, their response to overexpression of the Prosystemin transgene was the same as in control plants. Taken together, our data suggest that BRI1 can function as a systemin binding protein, but that binding of the ligand does not transduce the signal into the cell. This unusual behavior and the nature of the elusive systemin receptor will be discussed.

The oligomerization of ethylene produces α-olefin distributions ranging from Schulz–Flory distributions to alternating and selective oligomer distributions that can be mathematically analyzed and characterized by recurrence relations.

Veterinarians in the United States and abroad are faced with growing public concern for the welfare of animals, particularly those in production. To prepare veterinarians to exert the leadership expected by the public and industry, steps should be taken to provide instruction in animal welfare at veterinary colleges. The ultimate goal is to offer courses in animal welfare in a consistent manner on a global scale, utilizing existing expertise in an efficient and cost-effective manner. Given the intense curricula of veterinary schools and the scarcity of instructors trained in animal welfare, a nontraditional approach is needed to educate veterinary students in the United States and abroad in animal welfare. Michigan State University (MSU) is developing a graduate-level, online interactive course in animal welfare assessment. The course will approach the topic of animal welfare education from a holistic, multidisciplinary standpoint (encompassing ethics, economics, and behavior) and address issues important to the general public and the international community. The MSU course will draw on renowned international animal welfare experts, allowing students to receive high-quality education that would be difficult in any other circumstance. The course will bridge an important gap in the veterinary curriculum and offer a complete and congruous education in animal welfare to veterinarians worldwide. The MSU course will also serve as a model for collaboration in content assembly and course delivery, by using technology to leverage global expertise in the interests of educational equity. In addition to innovative technology, such as the use of Web-collaboration software to create the course, a variety of media that enable students to interact with the material will also be incorporated throughout the course. Thus, the course will not only utilize the Internet to provide access to high-quality material, but will also require the active participation of the student, which is needed for effective learning.

Defined changes in the cell wall directed by many proteins accompany every morphogenetic process in plants. Xyloglucan endotransglucosylase/hydrolase proteins (XTH; EC 2.4.1.207) have the potential to modify the hemicellulose matrix within the cell wall. Cs-XTH1 and Cs-XTH3 genes, which encode XTH proteins, were found among numerous genes that are differentially expressed after the induction of cucumber somatic embryogenesis. The expression of these genes increased during somatic embryogenesis. The Cs-XTH1 gene was localized on the second chromosome near the centromere region, whereas Cs-XTH3 was found in the middle of the fifth chromosome's longer arm. Northern blot hybridization showed that both genes were preferentially expressed in roots. We also observed higher accumulation of both transcripts in somatic embryos than in the proembryogenic mass. The localization of mRNA by in situ hybridization revealed that the Cs-XTH1 transcripts were largely accumulated in the presumptive cotyledon primordia of somatic embryos. The XTH gene family consists of a number of genes with a high degree of structural similarity. Screening a cucumber genomic library has identified other members of this gene family. The intron/exon structure, sequence similarities and the close chromosomal distance between some members suggest their common evolutionary origin. The involvement of XTH-related genes in somatic embryo formation is discussed.

Plasmodiophora brassicae (clubroot) infection leads to reprogramming of host development resulting in the formation of characteristic galls. In this work we explored the cellular events that underly gall formation in Arabidopsis thaliana with the help of molecular markers of cell division (CYCB1:GUS) and meristematic activity (ANT:GUS). Our results show that gall development involved the amplification of existing meristematic activities within the vascular cambium (VC) and phloem parenchyma (PP) cells in the region of the hypocotyl. Additionally we found that the increase in VC activity and prolonged maintenance of cambial-derived cells in a meristematic state was crucial for gall formation; disruption of the VC activity significantly decreased the gall size. Gall formation also perturbed vascular development with a significant reduction in xylem and increase in PP in infected plants. This situation was reflected in a decrease in transcripts of key factors promoting xylogenesis (VND6, VND7 and MYB46) and an increase in those promoting phloem formation and function (APL, SUC2). Finally we show, using the cell cycle inhibitor ICK1/KRP1 and a cle41 mutant with altered regulation of cambial stem cell maintenance and differentiation, that a decrease in gall formation did not prevent pathogen development. This finding demonstrates that although gall formation is a typical symptom of the disease and influences numbers of spores produced, it is not required for completion of the pathogen life cycle. Together, these results provide an insight into the relationship of the cellular events that accompany Plasmodiophora infection and their role in disease progression.

Understanding the relationship of the size and shape of an organism to the size, shape, and number of its constituent cells is a basic problem in biology; however, numerous studies indicate that the relationship is complex and often nonintuitive. To investigate this problem, we used a system for the inducible expression of genes involved in the G1/S transition of the plant cell cycle and analyzed the outcome on leaf shape. By combining a careful developmental staging with a quantitative analysis of the temporal and spatial response of cell division pattern and leaf shape to these manipulations, we found that changes in cell division frequency occurred much later than the observed changes in leaf shape. These data indicate that altered cell division frequency cannot be causally involved in the observed change of shape. Rather, a shift to a smaller cell size as a result of the genetic manipulations performed correlated with the formation of a smoother leaf perimeter, i.e. appeared to be the primary cellular driver influencing form. These data are discussed in the context of the relationship of cell division, growth, and leaf size and shape.