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G-proteins are molecular on–off switches that are involved in transmitting a variety of extracellular signals to their intracellular targets. In animal and yeast systems, the switch property is encoded through nucleotides: a GDP-bound state is the “off-state” and the GTP-bound state is the “on-state”. The G-protein cycle consists of the switch turning on through nucleotide exchange facilitated by a G-protein coupled receptor and the switch turning off through hydrolysis of GTP back to GDP, facilitated by a protein designated REGULATOR OF G SIGNALING 1 (RGS). In plants, G-protein signaling dramatically differs from that in animals and yeast. Despite stringent conservation of the nucleotide binding and catalytic structures over the 1.6 billion years that separate the evolution of plants and animals, genetic and biochemical data indicate that nucleotide exchange is less critical for this switch to operate in plants. Also, the loss of the single RGS protein in Arabidopsis (Arabidopsis thaliana) confers unexpectedly weaker phenotypes consistent with a diminished role for the G cycle, at least under static conditions. However, under dynamic conditions, genetic ablation of RGS in Arabidopsis results in a strong phenotype. We explore explanations to this conundrum by formulating a mathematical model that takes into account the accruing evidence for the indispensable role of phosphorylation in G-protein signaling in plants and that the G-protein cycle is needed to process dynamic signal inputs. We speculate that the plant G-protein cycle and its attendant components evolved to process dynamic signals through signaling modulation rather than through on–off, switch-like regulation of signaling. This so-called change detection may impart greater fitness for plants due to their sessility in a dynamic light, temperature, and pest environment.

Aims The aim of this study was to determine the differences and potential mechanistic rationale for observed adverse drug reactions (ADRs) between four approved PARP inhibitors (PARPi). Methods The Medicines and Healthcare products Regulatory Authority (MHRA) Yellow Card drug analysis profiles and NHS secondary care medicines database enabled the identification of suspected ADRs associated with the PARPi in the UK from launch to 2020. The polypharmacology of the PARPi were data-mined from several public data sources. Results The overall ADRs per 100 000 Rx identified across the four PARPi are statistically significant (χ2 test, P rucaparib > olaparib tracked with the Vd trend. Hypertension is only associated with niraparib and could be explained by the therapeutically achievable inhibition of DYRK1A and/or transporters. Arrhythmia cases are potentially linked to the structural features of hERG ion-channel inhibition found in rucaparib and niraparib. Enhanced psychiatric/nervous disorders associated with niraparib can be interpreted from the diverse neurotransporter off-targets reported. Conclusions Despite their similar mode of action, the differential polypharmacology of PARP inhibitors influences their ADR profile.

The extra-large guanosine-5′-triphosphate (GTP)-binding protein 2, XLG2, is an unconventional Gα subunit of the Arabidopsis (Arabidopsis thaliana) heterotrimeric GTP-binding protein complex with a major role in plant defense. In vitro biochemical analyses and molecular dynamic simulations show that affinity of XLG2 for GTP is two orders of magnitude lower than that of the conventional Gα, AtGPA1. Here we tested the physiological relevance of GTP binding by XLG2. We generated an XLG2(T476N) variant with abolished GTP binding, as confirmed by in vitro GTPγS binding assay. Yeast three-hybrid, bimolecular fluorescence complementation, and split firefly-luciferase complementation assays revealed that the nucleotide-depleted XLG2(T476N) retained wild-type XLG2-like interactions with the Gβγ dimer and defense-related receptor-like kinases. Both wild-type and nucleotide-depleted XLG2(T476N) restored the defense responses against Fusarium oxysporum and Pseudomonas syringae compromised in the xlg2 xlg3 double mutant. Additionally, XLG2(T476N) was fully functional restoring stomatal density, root growth, and sensitivity to NaCl, but failed to complement impaired germination and vernalization-induced flowering. We conclude that XLG2 is able to function in a GTP-independent manner and discuss its possible mechanisms of action.

Study objective Enterobacteriaceae resistant to ceftriaxone, mediated through extended-spectrum β-lactamases (ESBLs), commonly cause urinary tract infections worldwide, but have been less prevalent in North America. Current US rates are unknown. We determine Enterobacteriaceae antimicrobial resistance rates among US emergency department (ED) patients hospitalized for urinary tract infection. Methods We prospectively enrolled adults hospitalized for urinary tract infection from 11 geographically diverse university-affiliated hospital EDs during 2018 to 2019. Among participants with culture-confirmed infection, we evaluated prevalence of antimicrobial resistance, including that caused by ESBL-producing Enterobacteriaceae, resistance risk factors, and time to in vitro–active antibiotics. Results Of 527 total participants, 444 (84%) had cultures that grew Enterobacteriaceae; 89 of 435 participants (20.5%; 95% confidence interval 16.9% to 24.5%; 4.6% to 45.4% by site) whose isolates had confirmatory testing had bacteria that were ESBL producing. The overall prevalence of ESBL-producing Enterobacteriaceae infection among all participants with urinary tract infection was 17.2% (95% confidence interval 14.0% to 20.7%). ESBL-producing Enterobacteriaceae infection risk factors were hospital, long-term care, antibiotic exposure within 90 days, and a fluoroquinolone- or ceftriaxone-resistant isolate within 1 year. Enterobacteriaceae resistance rates for other antimicrobials were fluoroquinolone 32.3%, gentamicin 13.7%, amikacin 1.3%, and meropenem 0.3%. Ceftriaxone was the most common empirical antibiotic. In vitro–active antibiotics were not administered within 12 hours of presentation to 48 participants (53.9%) with ESBL-producing Enterobacteriaceae infection, including 17 (58.6%) with sepsis. Compared with other Enterobacteriaceae infections, ESBL infections were associated with longer time to in vitro–active treatment (17.3 versus 3.5 hours). Conclusion Among adults hospitalized for urinary tract infection in many US locations, ESBL-producing Enterobacteriaceae have emerged as a common cause of infection that is often not initially treated with an in vitro–active antibiotic.

In animals, several long-chain N-acylethanolamines (NAEs) have been identified as endocannabinoids and are autocrine signals that operate through cell surface G-protein-coupled cannabinoid receptors. Despite the occurrence of NAEs in land plants, including nonvascular plants, their precise signaling properties and molecular targets are not well defined. Here we show that the activity of N-linolenoylethanolamine (NAE 18:3) requires an intact G-protein complex. Specifically, genetic ablation of the Gβγ dimer or loss of the full set of atypical Gα subunits strongly attenuates an NAE-18:3-induced degreening of cotyledons in Arabidopsis (Arabidopsis thaliana) seedlings. This effect involves, at least in part, transcriptional regulation of chlorophyll biosynthesis and catabolism genes. In addition, there is feedforward transcriptional control of G-protein signaling components and G-protein interactors. These results are consistent with NAE 18:3 being a lipid signaling molecule in plants with a requirement for G-proteins to mediate signal transduction, a situation similar, but not identical, to the action of NAE endocannabinoids in animal systems.

Procyon lotor (Raccoon) are a dominant predator on eggs of Malaclemys terrapin (Diamondback Terrapin), an estuarine specialist turtle endemic to saltmarshes of the eastern and gulf coasts of the United States. The purpose of this study was to determine if broadcast predator vocalizations could potentially deter Raccoons from foraging on Diamondback Terrapin eggs. We used artificial nests arrays to test 2 hypotheses: (1) predator vocalization playbacks (barking Canis lupus familiaris [Domestic Dog]) reduce number of artificial nests depredated by Raccoons compared to non-predator vocalization playbacks (Urocyon cinereoargenteus [Gray Fox]) or control treatments (no playbacks), and (2) predator vocalization playbacks reduce frequency of visits by Raccoons and overall foraging time compared to non-predator playbacks and control treatments. We randomly assigned sound treatments to each night of a 3-day trial, with 3 trials conducted at 4 experimental sites. We counted the number of depredated artificial nests and used trail cameras to document number of Raccoon visits and Raccoon foraging time following each sound treatment. On average, 51% of artificial nests were depredated in the predator vocalization treatments, 66% in the non-predator–vocalization treatments, and 81% of nests in the control treatments. Average number of Raccoon visits was about 3 times higher in the control treatment compared to the predator treatment but auditory treatment did not affect time spent foraging. Response to predator vocalization was attenuated by repeated exposure to recorded predator playbacks. Variation in type of predator vocalization, length, and frequency of playbacks may increase effectiveness of auditory deterrents on predators of Diamondback Terrapin nests.

In animals, endocytosis of a seven-transmembrane GPCR is mediated by arrestins to propagate or arrest cytoplasmic G protein-mediated signaling, depending on the bias of the receptor or ligand, which determines how much one transduction pathway is used compared to another. In Arabidopsis thaliana, GPCRs are not required for G protein-coupled signaling because the heterotrimeric G protein complex spontaneously exchanges nucleotide. Instead, the seven-transmembrane protein AtRGS1 modulates G protein signaling through ligand-dependent endocytosis, which initiates derepression of signaling without the involvement of canonical arrestins. Here, we found that endocytosis of AtRGS1 initiated from two separate pools of plasma membrane: sterol-dependent domains and a clathrin-accessible neighborhood, each with a select set of discriminators, activators, and candidate arrestin-like adaptors. Ligand identity (either the pathogen-associated molecular pattern flg22 or the sugar glucose) determined the origin of AtRGS1 endocytosis. Different trafficking origins and trajectories led to different cellular outcomes. Thus, in this system, compartmentation with its associated signalosome architecture drives biased signaling.

A role for the heterotrimeric G protein complex in the induction of a transient burst of reactive oxygen species (ROS) by the Microbial-Associated Molecular Pattern, flg22, a 22-amino acid peptide derived from bacterial flagella, is well established. However, the evidence for a negative or positive role for one component of the Arabidopsis G protein complex, namely, Regulator of G Signaling 1 (AtRGS1) leads to opposing conclusions. We show that the reason for this difference is due to the isolate of Col-0 ecotype used as the wildtype control in flg22-induced ROS and our data further support the idea that AtRGS1 is a negative regulator of the flg22-induced ROS response. Whole-genome genotyping led to the identification and validation of polymorphism in five genes between two Col-0 isolates that are candidates for the different ROS response relative to the rgs1 null mutant.

Summary T-cell acute lymphoblastic leukaemia (T-ALL) is a cancer of the immune system. Approximately 20% of paediatric and 50% of adult T-ALL patients relapse and die from the disease. To improve patient outcome new drugs are needed. With the aim to identify new therapeutic targets, we integrated transcriptomics and metabolomics data, including live-cell NMR-spectroscopy, of cell lines and patient samples. We found that T-ALL cells have limited energy availability, resulting in down-regulated mTOR-signalling and reduced autophagy. Despite this, mTOR kinase remains active and essential for the glutamine-uptake and rapid proliferation, as glutamine supplies three nitrogen atoms in purines and all atoms but one carbon in pyrimidines. We show that EAAT1, a glutamate-aspartate transporter normally only expressed in the CNS, is crucial for glutamine conversion to nucleotides and that T-ALL cell proliferation depends on EAAT1 function, identifying it as a target for T-ALL treatment. Finally, we performed an in silico screen and identified a novel EAAT1-specific allosteric inhibitor.

To evaluate the outcomes of proximal femoral derotation osteotomy (PFDO) on the hip and spine function of patients with abnormal femoral torsion.This retrospective study included patients who underwent PFDO to treat increased or decreased femoral torsion between July 2014 and February 2019. The exclusion criteria were: previous fracture, fixation of slipped capital femoral epiphysis or osteotomy in the ipsilateral femur; PFDO associated to varus or valgus osteotomy; Tönnis grade 2 or 3 osteoarthritis; and PFDO performed to treat knee abnormalities. Hip function was assessed through the modified Harris Hip Score (mHHS). A subgroup of consecutive patients with low back pain before the PFDO and operated after 2017 had the spine function assessed through the Oswestry disability index (ODI).A total of 37 hips (34 patients) were studied: 15 hips with increased femoral torsion and 22 with decreased femoral torsion. Eight patients were male and 26 were female. The average age at PFDO was 33 years (range, 15-54 years). At a mean follow-up of 24 months (range, 12-65 months), the mean mHHS improved from 58.1 ± 14.3 before PFDO to 82 ± 15.6 at the most recent follow-up (P.001). Improvement in the mHHS above the minimum clinically important difference (MCID) was observed in 33 hips (89%). In the subgroup of 14 consecutive patients with ODI available, the ODI improved from a mean of 45% ± 16% before the PFDO to 22% ± 17% at the most recent follow-up (P = .001). Nine (64.3%) of the 14 patients presented improvement in the ODI above the MCID. Revision procedure with a larger intramedullary nail was necessary in 2 hips to treat nonunion.Proximal femoral derotation osteotomy improves the hip and spine function in patients with increased or decreased femoral torsion and nonarthritic hips.Level IV, therapeutic case series.

Depending on the environment, sunlight can positively or negatively affect litter decomposition, through the ensemble of direct and indirect processes constituting photodegradation. Which of these processes predominate depends on the ecosystem studied and on the spectral composition of sunlight received. To examine the relevance of photodegradation for litter decomposition in forest understoreys, we filtered ultraviolet radiation (UV) and blue light from leaves of Fagus sylvatica and Bettda pendula at two different stages of senescence in both a controlled-environment experiment and outdoors in four different forest stands (Picea abies, Pagus sylvatica, Acer platanoides, Betula pendula). Controlling for leaf orientation and initial differences in leaf chlorophyll and flavonol concentrations; we measured mass loss at the end of each experiment and characterised the phenolic profile of the leaf litter following photodegradation. In most forest stands, less mass was lost from decomposing leaves that received solar UV radiation compared with those under UV-attenuating filters, while in the controlled environment UV-A radiation either slightly accelerated or had no significant effect on photodegradation, according to species identity. Only a few individual phenolic compounds were affected by our different filter treatments, but photodegradation did affect the phenolic profile. We can conclude that photodegradation has a small stand- and species-specific effect on the decomposition of surface leaf litter in forest understoreys during the winter following leaf fall in southern Finland. Photodegradation was wavelength-dependent and modulated by the canopy species filtering sunlight and likely creating different combinations of spectral composition, moisture, temperature and snowpack characteristics.

Nitrogen is a key nutrient for plant growth and development. Plants regulate nitrogen availability and uptake efficiency through controlling root architecture. While the heterotrimeric G protein complex is an important element to regulate root morphology, it remains unknown whether the G protein regulates the root architecture in response to nitrogen supply. We used rice and Arabidopsis G protein mutants to study the root architecture in response to different nitrogen concentrations. We found that nitrogen inhibits root horizontal projection area (network area), root perimeter, total length, but not root diameter (average root width). Nitrogen influenced bushiness and root spatial distribution by inhibiting horizontal growth and promoting vertical expansion. The dynamic changes of the rice G protein mutant DK22 at different concentrations of nitrogen from day 7 to day 9 were different from the wild type with regard to bushiness and spatial distribution. The agb1-2 mutant in Arabidopsis lacked the inhibitory effect of nitrate on root growth. The heterotrimeric G protein complex regulates the inhibitory effect on root growth caused by high nitrogen supply and root spatial distribution in response to different nitrogen concentrations.

Contents Summary 35 I Overview of animal and plant WNK kinases 35 II. Structure: domains and topology 36 III. Phylogeny-evolutionary relationships 41 IV. Plant WNK kinase distribution and regulation of WNK expression and activity 41 V. Functions of WNK family members in physiology and development 41 VI. Say no more? Still many questions to be answered 45 Acknowledgements 46 References 46 SUMMARY: WITH NO LYSINE (WNK) kinases are serine/threonine kinases uniquely characterized by an anomalous placement of a catalytic lysine, hence their moniker. In animals, WNK protein kinases play critical roles in protein trafficking of components that mediate renal ion transport processes and regulate osmoregulation of cell volume. In plants, the WNK kinase gene family is larger and more diverse. Recent studies revealed WNK kinase roles in orchestrating the trafficking of an ion channel, a lipid kinase complex in animals, and a heterotrimeric G protein signaling component in plants that is necessary for signal transduction. For this reason, new attention is geared toward investigating the mechanisms adopted by WNK kinases to nudge intracellular proteins to their destinations. In this review, the functions of WNK kinases in protein trafficking are derived from what we have learned from the model organism Arabidopsis thaliana. To place this new idea in context, we provide the predicted WNK kinase structures, their predicted expression patterns, a speculated evolutionary pathway, and the regulatory roles of plant WNKs in transport processes and other physiologies. We brazenly predict that the WNK kinases in both plants and animals will soon be recognized as a nexus for trafficking-based signal transduction.

Background Chlorophyll content decreases in plants under stress conditions, therefore it is used commonly as an indicator of plant health. Arabidopsis thaliana offers a convenient and fast way to test physiological phenotypes of mutations and treatments. However, chlorophyll measurements with conventional solvent extraction are not applicable to Arabidopsis leaves due to their small size, especially when grown on culture dishes. Results We provide a nondestructive method for chlorophyll measurement whereby the red, green and blue (RGB) values of a color leaf image is used to estimate the chlorophyll content from Arabidopsis leaves. The method accommodates different profiles of digital cameras by incorporating the ColorChecker chart to make the digital negative profiles, to adjust the white balance, and to calibrate the exposure rate differences caused by the environment so that this method is applicable in any environment. We chose an exponential function model to estimate chlorophyll content from the RGB values, and fitted the model parameters with physical measurements of chlorophyll contents. As proof of utility, this method was used to estimate chlorophyll content of G protein mutants grown on different sugar to nitrogen ratios. Conclusion This method is a simple, fast, inexpensive, and nondestructive estimation of chlorophyll content of Arabidopsis seedlings. This method lead to the discovery that G proteins are important in sensing the C/N balance to control chlorophyll content in Arabidopsis. Electronic supplementary material The online version of this article (doi:10.1186/s13007-017-0174-6) contains supplementary material, which is available to authorized users.

The study hypothesis is that administration of inhaled nitric oxide (NO) plus oxygen to subjects with submassive pulmonary embolism (PE) will improve right ventricular (RV) systolic function and reduce RV strain and necrosis, while improving patient dyspnea, more than treatment with oxygen alone.This article describes the rationale and protocol for a registered (NCT01939301), nearly completed phase II, 3-center, randomized, double-blind, controlled trial. Eligible patients have pulmonary imaging-proven acute PE. Subjects must be normotensive, and have RV dysfunction on echocardiography or elevated troponin or brain natriuretic peptide and no fibrinolytics. Subjects receive NO plus oxygen or placebo for 24 hours (±3 hours) with blood sampling before and after treatment, and mandatory echocardiography and high-sensitivity troponin posttreatment to assess the composite primary end point. The sample size of N=78 was predicated on 30% more NO-treated patients having a normal high-sensitivity troponin (14 pg/mL) and a normal RV on echocardiography at 24 hours with α=.05 and β=.20. Safety was ensured by continuous spectrophotometric monitoring of percentage of methemoglobinemia and a predefined protocol to respond to emergent changes in condition. Blinding was ensured by identical tanks, software, and physical shielding of the device display and query of the clinical care team to assess blinding efficacy.We have enrolled 78 patients over a 31-month period. No patient has been withdrawn as a result of a safety concern, and no patient has had a serious adverse event related to NO.We present methods and a protocol for the first double-blinded, randomized trial of inhaled NO to treat PE.

Plants tolerate large variations in the intensity of the light environment by controlling the efficiency of solar to chemical energy conversion. To do this, plants have a mechanism to detect the intensity, duration, and change in light as they experience moving shadows, flickering light, and cloud cover. Sugars are the primary products of CO2 fixation, a metabolic pathway that is rate limited by this solar energy conversion. We propose that sugar is a signal encoding information about the intensity, duration and change in the light environment. We previously showed that the Arabidopsis heterotrimeric G protein complex including its receptor-like Regulator of G signaling protein, AtRGS1, detects both the concentration and the exposure time of sugars (Fu et al., 2014. Cell 156: 1084-1095). This unique property, designated dose-duration reciprocity, is a behavior that emerges from the system architecture / system motif. Here, we show that another property of the signaling system is to detect large changes in light while at the same time, filtering types of fluctuation in light that do not affect photosynthesis efficiency. When AtRGS1 is genetically ablated, photosynthesis efficiency is reduced in a changing- but not a constant-light environment. Mathematical modeling revealed that information about changes in the light environment is encoded in the amount of free AtRGS1 that becomes compartmentalized following stimulation. We propose that this property determines when to adjust photosynthetic efficiency in an environment where light intensity changes abruptly caused by moving shadows on top of a background of light changing gradually from sun rise to sun set and fluctuating light such as that caused by fluttering leaves.

Plants uniquely have a family of proteins called extra-large G proteins (XLG) that share homology in their C-terminal half with the canonical Gα subunits; we carefully detail here that Arabidopsis XLG2 lacks critical residues requisite for nucleotide binding and hydrolysis which is consistent with our quantitative analyses. Based on microscale thermophoresis, Arabidopsis XLG2 binds GTPγS with an affinity 100 times lower than that to canonical Gα subunits. This means that given the concentration range of guanine nucleotide in plant cells, XLG2 is not likely bound by GTP in vivo. Homology modeling and molecular dynamics simulations provide a plausible mechanism for the poor nucleotide binding affinity of XLG2. Simulations indicate substantially stronger salt bridge networks formed by several key amino-acid residues of AtGPA1 which are either misplaced or missing in XLG2. These residues in AtGPA1 not only maintain the overall shape and integrity of the apoprotein cavity but also increase the frequency of favorable nucleotide-protein interactions in the nucleotide-bound state. Despite this loss of nucleotide dependency, XLG2 binds the RGS domain of AtRGS1 with an affinity similar to the Arabidopsis AtGPA1 in its apo-state and about 2 times lower than AtGPA1 in its transition state. In addition, XLG2 binds the Gβγ dimer with an affinity similar to that of AtGPA1 XLG2 likely acts as a dominant negative Gα protein to block G protein signaling. We propose that XLG2, independent of guanine nucleotide binding, regulates the active state of the canonical G protein pathway directly by sequestering Gβγ and indirectly by promoting heterodimer formation.

Sunlight can accelerate the decomposition process through an ensemble of direct and indirect processes known as photodegradation. Although photodegradation is widely studied in arid environments, there have been few studies in temperate regions. This experiment investigated how exposure to solar radiation, and specifically UV-B, UV-A, and blue light, affects leaf litter decomposition under a temperate forest canopy in France. For this purpose, we employed custom-made litterbags built using filters that attenuated different regions of the solar spectrum. Litter mass loss and carbon to nitrogen (C:N) ratio of three species: European ash (Fraxinus excelsior), European beech (Fagus sylvatica) and pedunculate oak (Quercus robur), differing in their leaf traits and decomposition rate, were analysed over a period of 7–10 months. Over the entire period, the effect of treatments attenuating blue light and solar UV radiation on leaf litter decomposition was similar to that of our dark treatment, where litter lost 20–30% less mass and had a lower C:N ratio than under the full-spectrum treatment. Moreover, decomposition was affected more by the filter treatment than mesh size, which controlled access by mesofauna. The effect of filter treatment differed among the three species and appeared to depend on litter quality (and especially C:N), producing the greatest effect in recalcitrant litter (F. sylvatica). Even under the reduced irradiance found in the understorey of a temperate forest, UV radiation and blue light remain important in accelerating surface litter decomposition. Electronic supplementary material The online version of this article (10.1007/s00442-019-04478-x) contains supplementary material, which is available to authorized users.

SUMMARY Biased signaling occurs when different ligands that are directed at the same receptor launch different cellular outcomes. Because of their pharmacological importance, we know the most about biased ligands and little is known about other mechanisms to achieve signaling bias. In the canonical animal G protein system, endocytosis of a 7-transmembrane GPCR desensitizes a cell from its extracellular signal. β-arrestins facilitate GPCR endocytosis but also propagate cytoplasmic signaling depending on the bias. In Arabidopsis, GPCRs are not required for G protein coupled signaling because the heterotrimeric G protein complex spontaneously exchanges nucleotide. Instead, the prototype 7-transmembrane Regulator of G Signaling 1 protein AtRGS1 modulates G signaling and through ligand-dependent endocytosis, de-repression of signaling is initiated but canonical arrestins are not involved. Endocytosis initiates from two separate pools of plasma membrane: microdomains and a clathrin-accessible neighborhood, each with a select set of discriminators, activators, and newly-discovered arrestin-like adaptors. Different trafficking origins and trajectories lead to different cellular outcomes. Thus, compartmentation with its attendant signalosome architecture is a previously unknown mechanism to drive biased signaling.

We examined the use of previously observed restriction fragment length polymorphisms (RFLPs) of a polymerase chain reaction (PCR)-amplified fragment of plasmid pEA29 for differentiating strains of Erwinia amylovora. The PCR fragment from E. amylovora strain CA11 contains a region of 8-bp tandem repeats which is predicted to cause the RFLPs. Examination of a collection of 93 strains revealed the repeat sequence GATTACA(GAATTACA)nGAATTACA in pEA29 with n ranging from 3 to 14. Selected strains were examined after growth in liquid culture to establish the stability of this character. Four strains originally with n = 14, 13, 7, and 3 repeats were grown overnight in liquid culture and streaked onto agar plates to produce individual colonies. Respectively, 4, 10, 1, and 0 out of 17 colonies per strain had an altered copy number when retested. Considering the instability in the number of repeats, it is concluded that the polymorphism in this region of pEA29 is not useful as a marker for following the migration of E. amylovora.

Isolates of Venturia inaequalis were sampled from apple orchards during 1990 to 1996 and isolate sensitivities to dodine were determined by testing the relative growth (RG) of mycelial colonies at a discriminatory dose of 0.2 μg ml-1. Sensitivities were not significantly different for a wild-type population and several populations sampled from orchards never or rarely treated with dodine, and respective data were combined to provide a reference for baseline populations. The baseline sensitivity was compared with sensitivities determined for four orchards with evidence for practical dodine resistance. At these sites, increases of phenotype frequencies were most pronounced for isolates with RG values >90; they had increased from a baseline level of 0.9 to >30% and, therefore, were rated dodine-resistant. For two orchards with confirmed cases of previous dodine resistance, frequencies of resistant isolates had declined to 11 and 14% after dodine use was discontinued for 13 and 4 years, respectively. Sensitivities had not returned to baseline levels, and indirect evidence suggested that practical dodine resistance could recur rapidly in response to resumed dodine usage. Monitoring of commercial orchards in New York and Michigan revealed that dodine sensitivities were not uniform throughout regions where dodine resistance was widespread in the late 1970s. Sensitivities ranged from baseline to dodine-resistant and appeared to reflect the dodine use history at particular sites. Because the history of dodine use is not always known to growers, applications of dodine remain risky when accurate historical records are not available.

A detached leaf disk assay was developed for screening sour cherry (Prunus cerasus) cultivars for resistance to cherry leaf spot (Blumeriella jaapii). This assay was used to characterize the events occurring in cv. Montmorency (susceptible) and cv. GiSelA 6 [GI 148-1] (resistant) host-pathogen interactions, and to develop a disease scoring scheme to categorize disease severity in sour cherry trees. Forty-three seedlings grown from seeds collected in Russia were screened for resistance using the scoring scheme. Cross infection studies were also carried out with leaf spot isolates from four other Prunus species. These studies were made possible by the development of a new method of culturing B. jaapii isolates, using cherry fruit agar. This method enabled large amounts of inoculum to be produced within 2 weeks rather than a couple of months as required previously. In ‘Montmorency’, disease symptoms were first observed 4 days postinoculation (dpi), with the appearance of small white spots on the undersides of inoculated leaves. These spots gradually grew, producing erumpent spore masses in acervuli approximately 7 dpi. In ‘GI 148-1’, disease symptoms were also first observed 4 dpi, with the appearance of small transparent lesions. In the majority of cases, these lesions did not increase in size; however, acervuli occasionally were formed approximately 8 dpi. These contained small cirrhi and were often surrounded by an abscission zone. Of the sour cherry seedlings tested, only ‘Almaz’ open pollinated (o.p.) R1(1) was rated as resistant. ‘Almaz’ o.p. R1(1) and ‘GI 148-1’ were also rated as resistant to leaf spot isolates from other Prunus species. These results and the implications for breeding resistant sour cherry cultivars are discussed.

Plants and some protists have heterotrimeric G protein complexes that activate spontaneously without canonical G protein-coupled receptors (GPCRs). In Arabidopsis, the sole 7-transmembrane regulator of G protein signaling 1 (AtRGS1) modulates the G protein complex by keeping it in the resting state (GDP-bound). However, it remains unknown how a myriad of biological responses is achieved with a single G protein modulator. We propose that in complete contrast to G protein activation in animals, plant leucine-rich repeat receptor-like kinases (LRR RLKs), not GPCRs, provide this discrimination through phosphorylation of AtRGS1 in a ligand-dependent manner. G protein signaling is directly activated by the pathogen-associated molecular pattern flagellin peptide 22 through its LRR RLK, FLS2, and co-receptor BAK1.

Plants lack 7-transmembrane, G-protein coupled receptors (GPCRs) because the G alpha subunit of the heterotrimeric G protein complex is “self-activating”—meaning that it spontaneously exchanges bound GDP for GTP without the need of a GPCR. In lieu of GPCRs, most plants have a seven transmembrane receptor-like regulator of G-protein signaling (RGS) protein, a component of the complex that keeps G-protein signaling in its non-activated state. The addition of glucose physically uncouples AtRGS1 from the complex through specific endocytosis leaving the activated G protein at the plasma membrane. The complement of proteins in the AtRGS1/G-protein complex over time from glucose-induced endocytosis was profiled by immunoprecipitation coupled to mass spectrometry (IP-MS). A total of 119 proteins in the AtRGS1 complex were identified. Several known interactors of the complex were identified, thus validating the approach, but the vast majority (93/119) were not known previously. AtRGS1 protein interactions were dynamically modulated by d -glucose. At low glucose levels, the AtRGS1 complex is comprised of proteins involved in transport, stress and metabolism. After glucose application, the AtRGS1 complex rapidly sheds many of these proteins and recruits other proteins involved in vesicular trafficking and signal transduction. The profile of the AtRGS1 components answers several questions about the type of coat protein and vesicular trafficking GTPases used in AtRGS1 endocytosis and the function of endocytic AtRGS1.

The heterotrimeric G protein complex, comprising Gα, Gγ and Gγ subunits, is an evolutionarily conserved signaling molecular machine that transmits signals from transmembrane receptors to downstream target proteins. Plants conserved the core G protein elements, while developing their own regulatory systems differently from animals. Genetic evidence supports the conclusion that the heterotrimeric G proteins regulate shoot, root and epidermis development, as well as sugar sensing, hormone responsiveness and abiotic and biotic stress tolerance. This review is a compendium of the known morphological changes conferred by loss- and gain-of-function mutations of the G protein subunit genes across three higher land plant models, namely Arabidopsis, rice and maize.

Plant exposure to enhanced UV-B radiation typically induces changes in leaf secondary metabolite profiles which will be inherited in litter, affecting litter breakdown and the carbon (C) dynamics of sensitive plant communities. A key enzyme in the decomposition process is phenol oxidase which is influenced by litter quality and, hence, a decomposition bioindicator. Here we investigated dwarf shrub litter decomposition following experimental community exposure to enhanced UV-B over two decades in the Swedish sub-Arctic. We examined the hypothesis that foliar UV-B exposure would alter litter quality to elevate phenol oxidase activity. This was tested in the field by measuring phenol oxidase activity in freshly collected mixed-community litter from under our experimental vegetation. A laboratory mesocosm was next used in a decomposition assay to investigate individual species responses over eight weeks, with an emphasis on the quality of leachate outputs from decomposing litter (from Empetrum hermaphroditum, Vaccinium vitis-idaea, Vaccinium uliginosum). In the assay bi-weekly collections of leachate were analysed for phenol oxidase activity, together with total phenolics and dissolved organic C (DOC). At the end of the assay litter mass loss and respired C were also determined. The initial assessment on field mixed-community litter found no enhanced UV-B treatment (henceforth: ‘UV-B treatment’) effect on phenol oxidase activity. However, in the controlled laboratory mesocosm assay, significant species-specific effects of the UV-B treatment were evident, with increased phenol oxidase activity in V. vitis-idaea leachate (P

The G-protein complex is a cytoplasmic on-off molecular switch that is set by plasma membrane receptors that activate upon binding of its cognate extracellular agonist. In animals, the default setting is the "off" resting state, while in plants, the default state is constitutively "on" but repressed by a plasma membrane receptor-like protein. De-repression appears to involve specific phosphorylation of key elements of the G-protein complex and possibly target proteins that are positioned downstream of this complex. To address this possibility, protein abundance and phosphorylation state are quantified in wild type and G-protein deficient Arabidopsis roots in the unstimulated resting state. A total of 3246 phosphorylated and 8141 non-modified protein groups are identified. It has been found that 428 phosphorylation sites decrease and 509 sites increase in abundance in the G-protein quadrupole mutant lacking an operable G-protein-complex. Kinases with known roles in G-protein signaling including MAP KINASE 6 and FERONIA are differentially phosphorylated along with many other proteins now implicated in the control of G-protein signaling. Taken together, these datasets will enable the discovery of novel proteins and biological processes dependent on G-protein signaling.

Genetic ablation of the β subunit of the heterotrimeric G protein complex in agb1-2 confers defective activation of microbe-associated molecular pattern (MAMP)-triggered immunity, resulting in agb1-2 enhanced susceptibility to pathogens like the fungus Plectosphaerella cucumerina BMM. A mutant screen for suppressors of agb1-2 susceptibility (sgb) to P. cucumerina BMM identified sgb10, a new null allele (mkp1-2) of the mitogen-activated protein kinase phosphatase 1 (MKP1). The enhanced susceptibility of agb1-2 to the bacterium Pseudomonas syringae pv. tomato DC3000 and the oomycete Hyaloperonospora arabidopsidis is also abrogated by mkp1-2. MKP1 negatively balances production of reactive oxygen species (ROS) triggered by MAMPs, since ROS levels are enhanced in mkp1. The expression of RBOHD, encoding a NADPH oxidase–producing ROS, is upregulated in mkp1 upon MAMP treatment or pathogen infection. Moreover, MKP1 negatively regulates RBOHD activity, because ROS levels upon MAMP treatment are increased in mkp1 plants constitutively overexpressing RBOHD (35S::RBOHD mkp1). A significant reprograming of mkp1 metabolic profile occurs with more than 170 metabolites, including antimicrobial compounds, showing differential accumulation in comparison with wild-type plants. These results suggest that MKP1 functions downstream of the heterotrimeric G protein during MAMP-triggered immunity, directly regulating the activity of RBOHD and ROS production as well as other immune responses.

The heterotrimeric G protein complex, composed of Gα, Gβ, and Gγ subunits, plays some role in structural development in plants but this role could be indirect because loss-of-function mutations do not alter the body plan and post-embryonic organs differ only morphologically and not in their identity. This uncertainty has been compounded by the fact that loss of the Gβ subunit in cereals, but not Arabidopsis, is seedling lethal and that loss of maize Gα subunit confers prolificacy of a reproductive organ. In this study, we comprehensively profiled the root and shoot structural traits of rice Gα-null and viable Gβ-RNAi "knockdown" mutants, and found anomalous morphologies caused by Gβ-RNAi that are distinct from the Arabidopsis orthologue. The rice Gβ-RNAi mutant exhibited reduced radial growth of aerial parts as well as a more compact root architecture, among which smaller root mass seems mainly due to increased necrosis when grown on soil. In addition, three dimensional analyses of rice root system architecture revealed that the smaller root architecture of Gβ-RNAi plant is also due to both reduced root elongation and adventitious root formation. This contrasts to the Arabidopsis Gβ-null mutation that promotes cell proliferation. There is elevated cell senescence activity both visualized by Evans Blue staining and inferred from an expression analysis of cell-death marker genes. We propose that the morphological phenotypes of rice Gβ-RNAi plants are predominantly associated with the mediation of various stresses and cell senescence, consistent with an indirect role for Arabidopsis Gβ in development where the orthologous gene ablation mainly confers altered cell proliferation. We also elaborate our speculative working hypothesis that cell division is a type of stress and as such due to impairment in responding to stress in the G protein mutants, manifests as altered morphology and architecture but not an altered body plan or organ identities.

In plant cells, heterotrimeric G protein signaling mediates development, biotic/abiotic stress responsiveness, hormone signaling, and extracellular sugar sensing. The amount of sugar in plant cells fluctuates from nanomolar to high millimolar concentrations over time depending on changes in the light environment. Arabidopsis thaliana Regulator of G Signaling protein 1 (AtRGS1) modulates G protein activation and detects the concentration and the exposure time of sugars. This is called dose–duration reciprocity in sugar sensing and occurs through AtRGS1 internalization which is directly proportional to G protein activation. One source of sugars is from CO 2 fixation by photosynthesis. Through a simple set of experiments, we show that sugars made in cotyledons that are undergoing photomorphogenesis activate G signaling in cells distal to the nascent photosynthesis center. This occurs with sufficient speed to enable distal cells to monitor changes in photosynthetic activity in the leaves.

The antibiotic resistant threat is continuing to grow, due in part to the overuse of antibiotics in livestock feed. Many nations in Europe have banned the use of antibiotics in feed, leading to higher rates of infection in livestock animals and reduced productivity for the food market. Increasingly, researchers are looking into the efficacy of phytopreparations to replace antibiotics in feed, allowing for increased animal health without the development of resistance. Macleaya cordata, or Chinese plume poppy, shows promise as a food additive. To evaluate the antimicrobial efficacy of this plant, we tested in vitro activity of M. cordata extract, as well as pure compounds sanguinarine and chelerythrine against wild-type, methicillin-resistant, and multiply-resistant strains of Staphylococcus aureus (SA1199, AH1263, and IA116, respectively). Combination tests to evaluate synergy, additivity, and antagonism within the extract were also completed for the first time. Sanguinarine and chelerythrine showed complete growth inhibition of all strains of S. aureus at concentrations ranging from 3–10 μg/mL, and were equal in activity or were more potent than the reference compound chloramphenicol. Combination studies of pure sanguinarine and chelerythrine with M. cordata extract revealed additivity or indifference of mixture components with these compounds. Because sanguinarine and chelerythrine represent the major active constituents of M. cordata, the pooled amounts of these two compounds may be useful for establishing potency for quality control purposes. This is the first report of activity of chelerythrine and sanguinarine against methicillin-resistant S. aureus AH1263 and multiply-resistant S. aureus IA116, and illustrates the promise of M. cordata extract as an alternative to antibiotics in feed additives.

Serum is a common sample of convenience for metabolomics studies. Its processing time can be lengthy and may result in the loss of metabolites including those of red blood cells (RBCs). Unlike serum, whole blood (WB) is quickly processed, minimizing the influence of variable hemolysis while including RBC metabolites. To determine differences between serum and WB metabolomes, both sample types, collected from healthy volunteers, were assayed by H-NMR (proton nuclear magnetic resonance) spectroscopy. A total of 34 and 50 aqueous metabolites were quantified from serum and WB, respectively. Free hemoglobin (Hgb) levels in serum were measured, and the correlation between Hgb and metabolite concentrations was determined. Most metabolites detected in serum were at higher concentrations in WB with the exception of acetoacetate and propylene glycol. The 18 unique metabolites of WB included adenosine, AMP, ADP, and ATP, which are associated with RBC metabolism. The use of serum results in the underrepresentation of a number of metabolic pathways including branched-chain amino acid degradation and glycolysis and gluconeogenesis. The range of free Hgb in serum was 0.03 to 0.01 g/dL, and eight metabolites were associated (P ≤ 0.05) with free Hgb. The range of free Hgb in serum samples from 18 sepsis patients was 0.02 to 0.46 g/dL. Whole blood and serum have unique aqueous metabolite profiles, but the use of serum may introduce potential pathway bias. Use of WB for metabolomics may be particularly important for studies in diseases such as sepsis in which RBC metabolism is altered, and mechanical and sepsis-induced hemolysis contributes to variance in the metabolome.

Highlight Maize COMPACT PLANT 2, the alpha subunit of the heterotrimeric G protein complex, is important for some plastic developmental traits: shoot and root system architectures, and ear production., Plasticity in plant development is controlled by environmental signals through largely unknown signalling networks. Signalling coupled by the heterotrimeric G protein complex underlies various developmental pathways in plants. The morphology of two plastic developmental pathways, root system architecture and female inflorescence formation, was quantitatively assessed in a mutant compact plant 2 (ct2) lacking the alpha subunit of the heterotrimeric G protein complex in maize. The ct2 mutant partially compensated for a reduced shoot height by increased total leaf number, and had far more ears, even in the presence of pollination signals. The maize heterotrimeric G protein complex is important in some plastic developmental traits in maize. In particular, the maize Gα subunit is required to dampen the overproduction of female inflorescences.

Rates of decomposition in Arctic soils are regulated by temperature and moisture, but substrate availability is dictated by vegetation inputs, which are also subject to biotic influences. Here, we examine how leaf and litter inputs from individual dwarf shrub species influence soil enzyme activity in a sub-Arctic heath community in Abisko, Sweden. We further consider how foliar damage via insect herbivory (and outbreak) affects the soil community and decomposition. During the peak growing season (July 2011), we assessed how shrub community composition (Empetrum hermaphroditum, Vaccinium myrtillus, V. uliginosum and V. vitis-idaea) determined litter and soil phenol oxidase activity. A periodic severe outbreak of autumn moth larvae (Epirrita autumnata) affected this community in the following year (July 2012), and we used this to investigate its impact on relationships with phenol oxidase activity, soil respiration, soluble NH4 + and soluble phenolics; the soluble factors being directly associated with inputs from insect larval waste (frass). Pre-outbreak (2011), the strongest relationship observed was higher phenol oxidase activity with E. hermaphroditum cover. In the outbreak year (2012), phenol oxidase activity had the strongest relationship with damage to the deciduous species V. myrtillus, with greater herbivory lowering activity. For the other deciduous species, V. uliginosum, soil NH4 + and phenolics were negatively correlated with foliar larval damage. Phenol oxidase activity was not affected by herbivory of the evergreen species, but there was a strong positive relationship observed between E. hermaphroditum community abundance and soil respiration. We highlight the dominant role of E. hermaphroditum in such sub-Arctic shrub communities and show that even during insect outbreaks, it can dictate soil processes.

The three-member family of Arabidopsis extra-large G proteins (XLG1-3) defines the prototype of an atypical Gα subunit in the heterotrimeric G protein complex. Recent evidence indicate that XLG subunits operate along with its Gβγ dimer in root morphology, stress responsiveness and cytokinin induced development, however downstream targets of activated XLG proteins in the stress pathways are rarely known. To assemble a set of candidate XLG-targeted proteins, a yeast two-hybrid complementation-based screen was performed using XLG protein baits to query interactions between XLG and partner protein found in glucose-treated seedlings, roots, and Arabidopsis cells in culture. Seventy two interactors were identified and greater than 60% of a test set displayed in vivo interaction with XLG proteins. Gene co-expression analysis shows that greater than 70 % of the interactors are positively correlated with the corresponding XLG partners. Gene Ontology enrichment for all the candidates indicates stress responses and posits a molecular mechanism involving a specific set of transcription factor partners to XLG. Genes encoding two of these transcription factors, SZF1 and 2, require XLG proteins for full NaCl-induced expression. The subcellular localization of the XLG proteins in the nucleus, endosome, and plasma membrane is dependent on the specific interacting partner.

Arabidopsis heterotrimeric G-protein complex modulates pathogen-associated molecular pattern-triggered immunity (PTI) and disease resistance responses to different types of pathogens. It also plays a role in plant cell wall integrity as mutants impaired in the Gβ- (agb1-2) or Gγ-subunits have an altered wall composition compared with wild-type plants. Here we performed a mutant screen to identify suppressors of agb1-2 (sgb) that restore susceptibility to pathogens to wild-type levels. Out of the four sgb mutants (sgb10–sgb13) identified, sgb11 is a new mutant allele of ESKIMO1 (ESK1), which encodes a plant-specific polysaccharide O-acetyltransferase involved in xylan acetylation. Null alleles (sgb11/esk1-7) of ESK1 restore to wild-type levels the enhanced susceptibility of agb1-2 to the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM), but not to the bacterium Pseudomonas syringae pv. tomato DC3000 or to the oomycete Hyaloperonospora arabidopsidis. The enhanced resistance to PcBMM of the agb1-2 esk1-7 double mutant was not the result of the re-activation of deficient PTI responses in agb1-2. Alteration of cell wall xylan acetylation caused by ESK1 impairment was accompanied by an enhanced accumulation of abscisic acid, the constitutive expression of genes encoding antibiotic peptides and enzymes involved in the biosynthesis of tryptophan-derived metabolites, and the accumulation of disease resistance-related secondary metabolites and different osmolites. These esk1-mediated responses counterbalance the defective PTI and PcBMM susceptibility of agb1-2 plants, and explain the enhanced drought resistance of esk1 plants. These results suggest that a deficient PTI-mediated resistance is partially compensated by the activation of specific cell-wall-triggered immune responses., The Plant Journal, 92 (3), ISSN:0960-7412, ISSN:1365-313X

In animal cells, activation of heterotrimeric G protein signaling generally occurs when the system’s cognate signal exceeds a threshold, whereas in plant cells, both the amount and the exposure time of at least one signal, D-glucose, are used toward activation. This unusual signaling property called Dose-Duration Reciprocity, first elucidated in the genetic model Arabidopsis thaliana, is achieved by a complex that is comprised of a 7-transmembrane REGULATOR OF G SIGNALING (RGS) protein (AtRGS1), a Gα subunit that binds and hydrolyzes nucleotide, a Gβγ dimer, and three WITH NO LYSINE (WNK) kinases. D-glucose is one of several signals such as salt and pathogen-derived molecular patterns that operates through this protein complex to activate G protein signaling by WNK kinase transphosphorylation of AtRGS1. Because WNK kinases compete for the same substrate, AtRGS1, we hypothesize that activation is sensitive to the AtRGS1 amount and that modulation of the AtRGS1 pool affects the response to the stimulant. Mathematical simulation revealed that the ratio of AtRGS1 to the kinase affects system sensitivity to D-glucose, and therefore illustrates how modulation of the cellular AtRGS1 level is a means to change signal-induced activation. AtRGS1 levels change under tested conditions that mimic physiological conditions therefore, we propose a previously-unknown mechanism by which plants react to changes in their environment.

Receptor of activated C kinase1 (RACK1) is a versatile scaffold protein that binds to numerous proteins to regulate diverse cellular pathways in mammals. In Arabidopsis (Arabidopsis thaliana), RACK1 has been shown to regulate plant hormone signaling, stress responses, and multiple processes of growth and development. However, little is known about the molecular mechanism underlying these regulations. Here, we show that an atypical serine (Ser)/threonine (Thr) protein kinase, WITH NO LYSINE8 (WNK8), phosphorylates RACK1. WNK8 physically interacted with and phosphorylated RACK1 proteins at two residues: Ser-122 and Thr-162. Genetic epistasis analysis of rack1 wnk8 double mutants indicated that RACK1 acts downstream of WNK8 in the glucose responsiveness and flowering pathways. The phosphorylation-dead form, RACK1A(S122A/T162A), but not the phosphomimetic form, RACK1A(S122D/T162E), rescued the rack1a null mutant, implying that phosphorylation at Ser-122 and Thr-162 negatively regulates RACK1A function. The transcript of RACK1A(S122D/T162E) accumulated at similar levels as those of RACK1(S122A/T162A). However, although the steady-state level of the RACK1A(S122A/T162A) protein was similar to wild-type RACK1A protein, the RACK1A(S122D/T162E) protein was nearly undetectable, suggesting that phosphorylation affects the stability of RACK1A proteins. Taken together, these results suggest that RACK1 is phosphorylated by WNK8 and that phosphorylation negatively regulates RACK1 function by influencing its protein stability.

Highlight text This work reveals two distinct functions of Gα in NaCl stress in rice and maize: attenuation of leaf senescence caused by sodium toxicity in leaves, and cell cycle regulation by osmotic/ionic stress., The plant G-protein network, comprising Gα, Gβ, and Gγ core subunits, regulates development, senses sugar, and mediates biotic and abiotic stress responses. Here, we report G-protein signalling in the salt stress response using two crop models, rice and maize. Loss-of-function mutations in the corresponding genes encoding the Gα subunit attenuate growth inhibition and cellular senescence caused by sodium chloride (NaCl). Gα null mutations conferred reduced leaf senescence, chlorophyll degradation, and cytoplasm electrolyte leakage under NaCl stress. Sodium accumulated in both wild-type and Gα-mutant shoots to the same levels, suggesting that Gα signalling controls cell death in leaves rather than sodium exclusion in roots. Growth inhibition is probably initiated by osmotic change around root cells, because KCl and MgSO4 also suppressed seedling growth equally as well as NaCl. NaCl lowered rates of cell division and elongation in the wild-type leaf sheath to the level of the Gα-null mutants; however there was no NaCl-induced decrease in cell division in the Gα mutant, implying that the osmotic phase of salt stress suppresses cell proliferation through the inhibition of Gα-coupled signalling. These results reveal two distinct functions of Gα in NaCl stress in these grasses: attenuation of leaf senescence caused by sodium toxicity in leaves, and cell cycle regulation by osmotic/ionic stress.

Objectives This study sought to determine the clinical and biological significance of elevated cardiac troponin T (cTnT) in patients with neuromuscular diseases. Background Practice guidelines regard cTnT and cardiac troponin I (cTnI) as equally sensitive and specific for the diagnosis of myocardial injury. Although cTnI is unique to myocardium, cTnT can be re-expressed in skeletal muscle in response to injury. The commercial cTnT assay is claimed to be cardiac specific. Methods Fifty-two patients with 20 different types of acquired and inherited neuromuscular diseases underwent full clinical assessment, cardiac investigations, and measurements of serum cTnT, cTnI, creatine kinase (CK), creatine kinase myocardial band (CK-MB), and N-terminal pro-B–type natriuretic peptide (NT-proBNP). Results Serial measurements (265 samples) in 25 initially hospitalized patients taken during a mean of 2.4 years showed persistent elevation of cTnT (median: 0.08 μg/l; interquartile range: 0.06 to 0.14 μg/l), CK (582 U/l; 303 to 3,662 U/l), and CK-MB (24 μg/l; 8 to 34 μg/l). In contrast, cTnI, measured using 2 sensitive assays, was persistently normal throughout the study in 22 patients. Electrocardiograms (ECGs) and echocardiograms were normal in 16 and 17 patients, respectively, and no serial changes were observed. Therapeutic interventions in patients with reversible myopathies normalized cTnT, CK, and CK-MB in unison. Single measurements in 27 ambulatory patients showed elevated CK (953 U/l; 562 to 1,320 U/l), CK-MB (18 μg/l; 11 to 28 μg/l), and cTnT (0.03 μg/l; 0.02 to 0.05 μg/l) in 21, 22, and 18 patients respectively. cTnI was abnormal in only 1 patient. NT-proBNP (41 pg/ml; 35 to 97 pg/ml) was normal in all but 2 patients. ECGs were normal in 15 patients. No patients with elevated cTnT, but with normal cTnI, had any cardiovascular events in either group during follow-up. Conclusions Patients with a wide spectrum of neuromuscular diseases commonly have persistent elevation of cTnT and CK-MB in the absence of clinical and cTnI evidence of myocardial injury. Re-expressed cTnT in diseased skeletal muscle appears to be the source of the elevated cTnT detected in the circulation of these patients.

Plant and soil bio(chemical) indicators are increasingly used to provide information on N deposition inputs and effects in a wide range of ecosystem types. However, many factors, including climate and site management history, have the potential to influence bioindicator relationships with N due to nutrient export and changing vegetation nutrient demands. We surveyed 33 heathlands in England, along a gradient of background N deposition (7.2-24.5 kg ha(-1) year(-1)), using Calluna vulgaris growth phase as a proxy for time since last management. Our survey confirmed soil nutrient accumulation with increasing time since management. Foliar N and phosphorus (P) concentrations in pioneer- and mature-phase vegetation significantly increased with N deposition. Significant interactions between climate and N deposition were also evident with, for example, higher foliar P concentrations in pioneer-phase vegetation at sites with higher temperatures and N deposition rates. Although oxidized N appeared more significant than reduced N, overall there were more, stronger relationships with total N deposition; suggesting efforts to control all emissions of N (i.e., both oxidized and reduced forms) will have ecological benefits.

A Different Route The plant hormone auxin regulates a variety of developmental processes and responses to environmental inputs, often via changes in gene transcription. Xu et al. (p. 1025 ) analyzed a signaling pathway involving ABP1 (auxin-binding protein 1) that affects the cytoskeleton and endocytosis in Arabidopsis without changing gene transcription. Instead, ABP1 functions at the cell surface to bind auxin and a family of membrane kinases, thereby activating intracellular guanosine triphosphatases to initiate important developmental changes in cell shape.

Summary Cells continuously adjust their behavior in response to changing environmental conditions. Both intensity and duration of external signals are critical factors in determining what response is initiated. To understand how intracellular signaling networks process such multidimensional information, we studied the AtRGS1-mediated glucose response system of Arabidopsis . By combining experiments with mathematical modeling, we discovered a reciprocal dose and duration response relying on the orchestrated action of three kinases (AtWNK1, AtWNK8, and AtWNK10) acting on distinct timescales and activation thresholds. Specifically, we find that high concentrations of D-glucose rapidly signal through AtWNK8 and AtWNK10, whereas low, sustained sugar concentration slowly activate the pathway through AtWNK1, allowing the cells to respond similarly to transient, high-intensity signals and sustained, low-intensity signals. This "dose-duration reciprocity" allows encoding of both the intensity and persistence of glucose as an important energy resource and signaling molecule.

Heterotrimeric G-proteins, composed of Gα, Gβ, and Gγ subunits, regulate many fundamental processes in plants. In animals, ligand binding to seven transmembrane (7TM) cell surface receptors designated G-protein coupled receptors (GPCR) leads to heterotrimeric G-protein activation. Because the plant G-protein complex is constitutively active, the exact role of plant 7TM proteins in this process is unclear. Members of the mildew resistance locus O (MLO) family represent the best-characterized 7TM plant proteins. Although genetic ablation of either MLO2 or G-proteins alters susceptibility to pathogens in Arabidopsis thaliana, it is unknown whether G-proteins directly couple signaling through MLO2. Here, we exploited two well-documented phenotypes of mlo2 mutants, broad-spectrum powdery mildew resistance and spontaneous callose deposition in leaf mesophyll cells, to assess the relationship of MLO2 proteins to the G-protein complex. Although our data reveal modulation of antifungal defense responses by Gβ and Gγ subunits and a role for the Gγ1 subunit in mlo2-conditioned callose deposition, our findings overall are inconsistent with a role of MLO2 as a canonical GPCR. We discovered that mutants lacking the Gβ subunit show delayed accumulation of a subset of defense-associated genes following exposure to the microbe-associated molecular pattern flg22. Moreover, Gβ mutants were found to be hypersusceptible to spray inoculation with the bacterial pathogen Pseudomonas syringae.

PURPOSE:The objective of this study was to investigate the association of endothelial-related markers with organ dysfunction and in-hospital mortality to validate our earlier findings in a multicenter study. We hypothesize that (i) endothelial biomarkers will be associated with organ dysfunction and mortality in sepsis and that (ii) soluble fms-like tyrosine kinase 1 (sFlt-1) holds promise as a novel prognostic marker in sepsis.METHODS:This was a prospective, multicenter, observational study of a convenience sample of emergency department (ED) patients with a suspected infection presenting to one of four urban, academic medical center EDs between January 2009 and January 2010. We collected plasma while the patients were in the ED and subsequently assayed endothelial-related biomarkers, namely, sFlt-1, soluble E-selectin, soluble intercellular adhesion molecule 1, soluble vascular cell adhesion molecule 1, and plasminogen activator inhibitor 1 (PAI-1). Outcomes were organ dysfunction and in-hospital mortality.RESULTS:We enrolled a total of 166 patients: 63 with sepsis (38%), 61 with severe sepsis (37%), and 42 with septic shock (25%). All endothelial biomarkers were significantly associated with sepsis severity, P < 0.002. We found a significant intercorrelation between all biomarkers, strongest between sFlt-1 and PAI-1 (r = 0.61, P < 0.001) and PAI-1 and soluble E-selectin and soluble intercellular adhesion molecule 1 (r = 0.49, P < 0.001). Among the endothelial biomarkers, sFlt-1 had the strongest association with Sequential Organ Failure Assessment score (r = 0.58, P < 0.001). Soluble fms-like tyrosine kinase 1 and PAI-1 had the highest area under the operating receiver characteristic curve for mortality of 0.87.CONCLUSIONS:This multicenter validation study confirms that markers of endothelial activation are associated with sepsis severity, organ dysfunction, and mortality in sepsis. This supports the hypothesis that the endothelium plays a central role in the pathophysiology of sepsis and may serve as a more accurate prediction tool and a target for therapies aimed at ameliorating endothelial cell dysfunction. In addition, sFLT-1 holds promise as a novel sepsis severity biomarker. PURPOSE: The objective of this study was to investigate the association of endothelial-related markers with organ dysfunction and in-hospital mortality to validate our earlier findings in a multicenter study. We hypothesize that (i) endothelial biomarkers will be associated with organ dysfunction and mortality in sepsis and that (ii) soluble fms-like tyrosine kinase 1 (sFlt-1) holds promise as a novel prognostic marker in sepsis. METHODS: This was a prospective, multicenter, observational study of a convenience sample of emergency department (ED) patients with a suspected infection presenting to one of four urban, academic medical center EDs between January 2009 and January 2010. We collected plasma while the patients were in the ED and subsequently assayed endothelial-related biomarkers, namely, sFlt-1, soluble E-selectin, soluble intercellular adhesion molecule 1, soluble vascular cell adhesion molecule 1, and plasminogen activator inhibitor 1 (PAI-1). Outcomes were organ dysfunction and in-hospital mortality. RESULTS: We enrolled a total of 166 patients: 63 with sepsis (38%), 61 with severe sepsis (37%), and 42 with septic shock (25%). All endothelial biomarkers were significantly associated with sepsis severity, P

Checkpoint kinase (CHK) inhibitors offer the promise of enhancing the effectiveness of widely prescribed cancer chemotherapies and radiotherapy by inhibiting the DNA damage response, as well as the potential for single agent efficacy.This article surveys structural insights into the checkpoint kinases CHK1 and CHK2 that have been exploited to enhance the selectivity and potency of small molecule inhibitors. Furthermore, the authors review the use of mechanistic cellular assays to guide the optimisation of inhibitors. Finally, the authors discuss the status of the current clinical candidates and emerging new clinical contexts for CHK1 and CHK2 inhibitors, including the prospects for single agent efficacy.Protein-bound water molecules play key roles in structural features that can be targeted to gain high selectivity for either enzyme. The results of early phase clinical trials of checkpoint inhibitors have been mixed, but significant progress has been made in testing the combination of CHK1 inhibitors with genotoxic chemotherapy. Second-generation CHK1 inhibitors are likely to benefit from increased selectivity and oral bioavailability. While the optimum therapeutic context for CHK2 inhibition remains unclear, the emergence of single agent preclinical efficacy for CHK1 inhibitors in specific tumour types exhibiting constitutive replication stress represents exciting progress in exploring the therapeutic potential of these agents.

In the classic 1942 movie Casablanca, Vichy Police Captain Louis Renault obfuscated the truth by commanding his lieutenants to “round up the usual suspects” knowing well that the culprit with the gun stood in plain view [1]. Something similar has happened in the plant G protein field. This Scientific Correspondence was written to shed light on the source of misunderstanding and to pre-empt further confusion. Plant heterotrimeric G proteins are self-activating and therefore do not need and do not utilize G protein-coupled receptors (GPCR). This conclusion was reached previously from biochemical analyses of plant G proteins [2,3]; here, we buttress this point of view using an evolutionary argument. Proteins suspected as plant GPCRs were “rounded up” because they have predicted topology of animal GPCRs and/or have been mis-annotated as such, however these proteins are highly conserved in organisms that lack heterotrimeric G proteins. Therefore they have functions unrelated to G-coupled signaling. Instead, the culprit protein standing in plain view is a receptor GTPase-accelerating protein (GAP), a receptor-GAP called AtRGS1.