Search

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: Studies examining the association between hyperoxia exposure after resuscitation from cardiac arrest and clinical outcomes have reported conflicting results. Our objective was to test the hypothesis that early postresuscitation hyperoxia is associated with poor neurological outcome. Methods: This was a multicenter prospective cohort study. We included adult patients with cardiac arrest who were mechanically ventilated and received targeted temperature management after return of spontaneous circulation. We excluded patients with cardiac arrest caused by trauma or sepsis. Per protocol, partial pressure of arterial oxygen (Pa o 2 ) was measured at 1 and 6 hours after return of spontaneous circulation. Hyperoxia was defined as a Pa o 2 >300 mm Hg during the initial 6 hours after return of spontaneous circulation. The primary outcome was poor neurological function at hospital discharge, defined as a modified Rankin Scale score >3. Multivariable generalized linear regression with a log link was used to test the association between Pa o 2 and poor neurological outcome. To assess whether there was an association between other supranormal Pa o 2 levels and poor neurological outcome, we used other Pa o 2 cut points to define hyperoxia (ie, 100, 150, 200, 250, 350, 400 mm Hg). Results: Of the 280 patients included, 105 (38%) had exposure to hyperoxia. Poor neurological function at hospital discharge occurred in 70% of patients in the entire cohort and in 77% versus 65% among patients with versus without exposure to hyperoxia respectively (absolute risk difference, 12%; 95% confidence interval, 1–23). Hyperoxia was independently associated with poor neurological function (relative risk, 1.23; 95% confidence interval, 1.11–1.35). On multivariable analysis, a 1-hour-longer duration of hyperoxia exposure was associated with a 3% increase in risk of poor neurological outcome (relative risk, 1.03; 95% confidence interval, 1.02–1.05). We found that the association with poor neurological outcome began at ≥300 mm Hg. Conclusions: Early hyperoxia exposure after resuscitation from cardiac arrest was independently associated with poor neurological function at hospital discharge.

The Precambrian geology of eastern Zambia and Malawi is highly complex due to multiple episodes of rifting and collision, particularly during the formation of Greater Gondwana as a product of the Neoproterozoic Pan-African Orogeny. The lithospheric structure and extent of known Precambrian tectonic entities of the region are poorly known as there have been to date few detailed geophysical studies to probe them. Herein, we present results from electromagnetic lithospheric imaging across Zambia into southern Malawi using the magnetotelluric method complemented by high-resolution aeromagnetic data of the upper crust in order to explore the extent and geometry of Precambrian structures in the region. We focus particularly on determining the extent of subcontinental lithospheric mantle (SCLM) beneath the Archean-Paleoproterozoic cratonic Bangweulu Block and the Mesoproterozoic-Neoproterozoic Irumide and Southern Irumide Orogenic Belts. We also focus on imaging the boundaries between these tectonic entities, particularly the boundary between the Irumide and Southern Irumide Belts. The thickest and most resistive lithosphere is found beneath the Bangweulu Block, as anticipated for stable cratonic lithosphere. Whereas the lithospheric thickness estimates beneath the Irumide Belt match those determined for other orogenic belts, the Southern Irumide Belt lithosphere is substantially thicker similar to that of the Bangweulu Block to the north. We interpret the thicker lithosphere beneath the Southern Irumide Belt as due to preservation of a cratonic nucleus (the pre-Mesoproterozoic Niassa Craton). A conductive mantle discontinuity is observed between the Irumide and Southern Irumide Belts directly beneath the Mwembeshi Shear Zone. We interpret this discontinuity as modified SCLM relating to a major suture zone. The lithospheric geometries determined from our study reveal tectonic features inferred from surficial studies and provide important details for the tectonothermal history of the region.

The scope of an unexpected Mitsunobu cyclisation to prepare N-arylated Fsp3-enriched azacycles was investigated. In the current study, we have identified whether a pKa-dependent Mitsunobu cyclodehydration or a pKa-independent Mitsunobu intramolecular reaction was in operation. A Mitsunobu reaction, creating a leaving group, followed by intramolecular nucleophilic displacement was determined to be the dominant pathway.

Heterotrimeric GTP-binding proteins (G proteins), consisting of Gα, Gβ and Gγ subunits, transduce signals from a diverse range of extracellular stimuli, resulting in the regulation of numerous cellular and physiological functions in Eukaryotes. According to the classic G protein paradigm established in animal models, the bound guanine nucleotide on a Gα subunit, either guanosine diphosphate (GDP) or guanosine triphosphate (GTP) determines the inactive or active mode, respectively. In plants, there are two types of Gα subunits: canonical Gα subunits structurally similar to their animal counterparts and unconventional extra-large Gα subunits (XLGs) containing a C-terminal domain homologous to the canonical Gα along with an extended N-terminal domain. Both Gα and XLG subunits interact with Gβγ dimers and regulator of G protein signalling (RGS) protein. Plant G proteins are implicated directly or indirectly in developmental processes, stress responses, and innate immunity. It is established that despite the substantial overall similarity between plant and animal Gα subunits, they convey signalling differently including the mechanism by which they are activated. This review emphasizes the unique characteristics of plant Gα subunits and speculates on their unique signalling mechanisms.

The dominant mechanism operating in a recently disclosed ring contraction is revealed for the first time. New insight and supporting evidence into a classic ring-contraction strategy within a rigidified larger ring system are provided.

Patients with severe sepsis who experience rapid, early deterioration and death are of particular concern. Our objective was to identify predictors of early death in Emergency Department (ED) patients with severe sepsis.Secondary analysis of two prospective studies of adult ED patients with severe sepsis. The primary outcome was early death, defined as death within 24h of triage.Out of 410 severe sepsis admissions, 20 patients experienced early death. These patients demonstrated significantly higher initial lactate (7.3 versus 3.3mmol/L, p0.001) and modified SOFA (mSOFA) scores (10 vs 6, p0.001), were less likely to normalize their lactate (p0.001), had lower initial pH (p0.001), and more frequently had early positive blood cultures (p=0.021). Multivariable logistic regression identified initial serum lactate level (OR 1.19, 95% CI 1.06-1.35) and mSOFA score (OR 1.17, 95% CI 1.00-1.36) as independent predictors of early death. A repeat lactate≥5mmol/L had a sensitivity of 55% and specificity of 89% for early death. There were no significant treatment differences between groups.Initial serum lactate and mSOFA score were independent predictors of mortality within 24h of ED admission in patients with severe sepsis.

The deep mantle African Superswell is considered to contribute to the topographic uplift of the Southern African Plateau, but dynamic support estimates vary wildly depending on the approach and data used. One reason for these large disparities is that the role of lithospheric structure, key in modulating deep dynamic contributions to elevation, is commonly ignored or oversimplified in convection studies. We use multiple high-quality geophysical data coupled with xenolith-based geochemical constraints to compute the isostatic lithospheric contribution to the elevation of the Plateau, facilitating isolation of the current dynamic component from the total observed elevation. We employ a multi-observable stochastic algorithm to invert geoid anomaly, surface-wave dispersion data, magnetotelluric data and surface heat flow to predict elevation in a fully thermodynamically and internally-consistent manner. We find that a compositionally-layered 230 ±7 km thick lithosphere is required to simultaneously fit all four data types, in agreement with abundant independent xenolith evidence. Our stochastic modelling indicates a lithospheric contribution to elevation of the order of 670 m, which implies dynamic support arising from the convecting sub-lithospheric mantle of ∼650 m. Our results have important implications for the understanding of lithospheric-deep mantle feedback mechanisms and for calibrating dynamic topography estimates from global convection studies.

Understanding the architecture of the West African craton at depth is essential to be able to reconstruct its evolution. Our study focuses on the crustal imaging of structures and geometries characterizing the crust of the Leo-Man shield with broadband and long period magnetotelluric data collected in southern Burkina Faso and covering a 220 km long profile. The resulting 3D resistivity crustal model highlights the distribution of the granite-greenstone assemblages with depth showing excellent correlation with mapped surficial lithologies. The whole crust of southern Burkina Faso is resistive, with lateral as opposed to vertical major resistivity contrasts, reflecting the location of major-scale shear zones characterizing this part of the Baoule-Mossi domain. Ground gravity data acquired along the same line as the MT data were also modeled and show relatively good correspondence with the resistivity model. The new resistivity and gravity models compared with results from joint inversion of gravity and aeromagnetic data highlight significant changes between the greenstone belts and granitoid domains along the profile. The comparison of the geophysics with the geology enables us to define new depth constraints of the main tectonic features in the area. The observed large scale dipping shear zones favour the model of crustal building through major parallel thrust faults.

Summary This article presents a review of medicolegal investigations of drug-related deaths among various Hollywood celebrities and popular music icons. The movie stars included: Marilyn Monroe, River Phoenix, John Belushi, Chris Penn, Heath Ledger and Philip Seymour Hoffman. The musicians are exemplified by Elvis Presley, Janis Joplin, Jimi Hendrix, Keith Moon, Sid Vicious, Kurt Cobain, Amy Winehouse, Michael Jackson, Whitney Houston and Prince. The tragic drug-related death of Anna Nicole Smith, a sex-symbol and Playboy model, is also included. The illicit drugs mainly responsible for the fatalities were heroin and/or cocaine or a mixture of the two narcotics. Some of the celebrity deaths were caused by inappropriate use of prescription medications, mostly combined influences of one or more benzodiazepine together with an opiate or opioid pain medication. Polypharmacy increases the risk of adverse drug events and this sometimes causes a sudden and unexpected death. As tolerance to the pharmacological effects of drugs develop, the amounts taken (the dose) are increased, which enhances the risk of a fatal drug-drug interaction. Ethanol was implicated in some of the celebrity deaths, which underscores the dangers of excessive drinking when taking centrally acting drugs. In the case of Amy Winehouse, a talented jazz singer, she died from acute alcohol poisoning, because ethanol was the only psychoactive drug identified in postmortem blood.

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.

OBJECTIVE Vital sign trends are used in clinical practice to assess treatment response and aid in disposition, yet quantitative data to support this practice are lacking. This study aimed to determine the prognostic value of vital sign normalization. METHODS Secondary analysis of a prospective cohort of adult emergency department (ED) patients admitted a single urban tertiary care hospital. A random sample of 182 days was chosen, and a manual review of all admissions was undertaken. Persistent tachycardia or tachypnea was defined as failure to decrease to a normal value in the ED. Elevated upon admission was defined as an abnormal value at the last set of vital signs documented. The primary outcome was in-hospital mortality. RESULTS 4,878 patients were enrolled and 4.5 (±3.8) sets of vital signs were checked per patient. 1,770 patients were tachycardic and 1,499 were tachypneic. Among tachycardic patients, 941 (53%) were persistently tachycardic and 1,074 (61%) were tachycardic upon admission. Among tachypneic patients 639 (42%) were persistently tachypneic and 768 (51%) were tachypneic upon admission. Mortality was higher in patients persistently tachycardic (5.7% vs. 3.1%, P=0.008) or tachycardic upon admission (5.5% vs. 3.0%, P=0.014). Similar results were found in tachypneic patients (8.3% vs. 4.5%, P=0.003; 7.8% vs. 4.4%, P=0.006). CONCLUSION Persistent tachycardia and tachypnea are associated with an increased risk of mortality in ED patients admitted to the hospital. Further study is necessary to determine if improved recognition or earlier interventions can affect outcomes.

Objective:To provide an update to “Surviving Sepsis Campaign Guidelines for Management of Sepsis and Septic Shock: 2012.”Design:A consensus committee of 55 international experts representing 25 international organizations was convened. Nominal groups were assembled at key international meetings (for

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.

Septic shock is a critical clinical condition with a high mortality rate. Previous observations have suggested that circulating complement C3 fragments, released during septic shock, might contribute to the development of complications such as profound hypotension and disseminated intravascular coagulation, resulting in a more severe disease course and a poorer outcome. However, there are conflicting reports regarding the role of C3 in the course of septic shock. Because circulating C3 exists in different sizes through enzymatic processing or spontaneous hydrolysis, these previous studies, which used ELISA-based methods, would not have distinguished among different C3 species. We have used Western blotting, with its associated protein size differentiation feature, to investigate the forms of circulating C3 in two cohorts of septic shock patients: a discovery cohort of 24 patents and a validation cohort of 181 patients. In both cohorts there were significantly lower levels of the C3-alpha chain in non-survivors than in survivors. In addition, the levels of the C3-alpha chain significantly correlated with days of survival. ROC and AUC analyses showed an AUC=0.65 (95% confidence interval [CI]: 0.56–0.75). At a best cutoff value (Youden) of 970.6 mg/ml, the test demonstrated a sensitivity of 68.5% and specificity of 61.5%. At this cutoff point, Kaplan-Meier survival analysis showed that patients with lower levels of C3-alpha chain had significantly lower survival than those with higher levels (p < 0.001). Thus, circulating C3-alpha chain levels in septic shock patients may predict survival rates.

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.

Sulfation of the amino acid residues of proteins is a significant post-translational modification, the functions of which are yet to be fully understood. Current sulfation methods are limited mainly to O-tyrosine (sY), which requires negatively charged species around the desired amino acid residue and a specific sulfotransferase enzyme. Alternatively, for solid-phase peptide synthesis, a de novo protected sY is required. Therefore, synthetic routes that go beyond O-sulfation are required. We have developed a novel route to N-sulfamation and can dial-in/out O-sulfation (without S-sulfurothiolation), mimicking the initiation step of the ping-pong sulfation mechanism identified in structural biology. This rapid, low-temperature and non-racemising method is applicable to a range of amines, amides, amino acids, and peptide sequences.

This article commemorates the life and contributions of one of the most influential sports surgeons in the history of orthopedic surgery, Dr. Robert Jackson.

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.

As molecular on-off switches, heterotrimeric G protein complexes, comprised of a Gα subunit and an obligate Gβγ dimer, transmit extracellular signals received by G protein– coupled receptors (GPCRs) to cytoplasmic targets that respond to biotic and abiotic stimuli. Signal transduction is modulated by phosphorylation of GPCRs and G protein complexes. In Arabidopsis thaliana, the Gα subunit AtGPA1 is phosphorylated by the receptor-like kinase (RLK) BRI1-ASSOCIATED Kinase 1 (BAK1), but the extent that other RLKs phosphorylates AtGPA1 is unknown. We mapped 22 trans-phosphorylation sites on AtGPA1 by 12 RLKs hypothesized to act in the Arabidopsis G protein signaling pathway. Cis-phosphorylation sites on these RLKs were also identified. BRI1, BAK1, and SERK1 have been reported as Ser/Thr and Tyr dual specificity kinases. We identified 4 more dual specificity kinases: IOS1, PSY1R, PEPR1, and AT2G37050. Multiple sites are present in the core AtGPA1 functional units, including pSer52 and pThr53 of the conserved P-loop that directly binds nucleotide/phosphate, pThr164 and pSer175 from αE helix in the intramolecular domain interface for nucleotide exchange and GTP hydrolysis, and pThr193 or pThr194 in Switch I (SwI) that coordinates nucleotide exchange and protein partner binding. Several AtGPA1 S/T phosphorylation sites are nucleotide-dependent phosphorylation patterns, such as S52/T53 in the P-loop and T193 and/or T194 in SwI.

As molecular on-off switches, heterotrimeric G protein complexes, comprised of a Gα subunit and an obligate Gβγ dimer, transmit extracellular signals received by G protein-coupled receptors (GPCRs) to cytoplasmic targets that respond to biotic and abiotic stimuli. Signal transduction is modulated by phosphorylation of GPCRs and G protein complexes. In Arabidopsis thaliana, the Gα subunit AtGPA1 is phosphorylated by the receptor-like kinase (RLK) BRI1-associated Kinase 1 (BAK1), but the extent that other RLKs phosphorylates AtGPA1 is unknown. Twenty-two trans-phosphorylation sites on AtGPA1 are mapped by 12 RLKs hypothesized to act in the Arabidopsis G protein signaling pathway. Cis-phosphorylation sites are also identified on these RLKs, some newly shown to be dual specific kinases. Multiple sites are present in the core AtGPA1 functional units, including pSer52 and/or pThr53 of the conserved P-loop that directly binds nucleotide/phosphate, pThr164, and pSer175 from αE helix in the intramolecular domain interface for nucleotide exchange and GTP hydrolysis, and pThr193 and/or pThr194 in Switch I (SwI) that coordinates nucleotide exchange and protein partner binding. Several AtGPA1 S/T phosphorylation sites are potentially nucleotide-dependent phosphorylation patterns, such as Ser52/Thr53 in the P-loop and Thr193 and/or Thr194 in SwI.

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.

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.

© Copyright by the Author(s). Published by Cell Physiol Biochem Press. BACKGROUND/AIMS: Vascular calcification represents a huge clinical problem contributing to adverse cardiovascular events, with no effective treatment currently available. Upregulation of hepatocyte growth factor has been linked with vascular calcification, and thus, represent a potential target in the development of a novel therapeutic strategy. Glycomimetics have been shown to interrupt HGF-receptor signalling, therefore this study investigated the effect of novel glycomimetics on osteogenic signalling and vascular calcification in vitro. METHODS: Primary human vascular smooth muscle cells (HVSMCs) were induced by β-glycerophosphate (β-GP) and treated with 4 glycomimetic compounds (C1-C4). The effect of β-GP and C1-C4 on alkaline phosphatase (ALP), osteogenic markers and c-Met/Notch3/HES1 signalling was determined using colorimetric assays, qRT-PCR and western blotting respectively. RESULTS: C1-C4 significantly attenuated β-GP-induced calcification, as shown by Alizarin Red S staining and calcium content by day 14. In addition, C1-C4 reduced ALP activity and prevented upregulation of the osteogenic markers, BMP-2, Runx2, Msx2 and OPN. Furthermore, β-GP increased c-Met phosphorylation at day 21, an effect ameliorated by C2 and C4 and the c-Met inhibitor, crizotinib. We next interrogated the effects of the Notch inhibitor DAPT and confirmed an inhibition of β-GP up-regulated Notch3 protein by C2, DAPT and crizotinib compared to controls. Hes-1 protein upregulation by β-GP, was also significantly downregulated by C2 and DAPT. GOLD docking analysis identified a potential binding interaction of C1-C4 to HGF which will be investigated further. CONCLUSION: These findings demonstrate that glycomimetics have potent anti-calcification properties acting via HGF/c-Met and Notch signalling.

L-carnitine is a candidate therapeutic for the treatment of septic shock, a condition that carries a ≥40% mortality. Responsiveness to L-carnitine may hinge on unique metabolic profiles that are not evident from the clinical phenotype. To define these profiles, we performed an untargeted metabolomic analysis of serum from 21 male sepsis patients enrolled in a placebo-controlled L-carnitine clinical trial. Although treatment with L-carnitine is known to induce changes in the sepsis metabolome, we found a distinct set of metabolites that differentiated 1-year survivors from non-survivors. Following feature alignment, we employed a new and innovative data reduction strategy followed by false discovery correction, and identified 63 metabolites that differentiated carnitine-treated 1-year survivors versus non-survivors. Following identification by MS/MS and database search, several metabolite markers of vascular inflammation were determined to be prominently elevated in the carnitine-treated non-survivor cohort, including fibrinopeptide A, allysine, and histamine. While preliminary, these results corroborate that metabolic profiles may be useful to differentiate L-carnitine treatment responsiveness. Furthermore, these data show that the metabolic signature of L-carnitine-treated non-survivors is associated with a severity of illness (e.g., vascular inflammation) that is not routinely clinically detected.

This article commemorates Dr. Adrian Ede Flatt (Figure 1) for his contributions to the world of hand surgery, helping to form it into the field it is today. He died on October 14, 2017, at the age ...

The study of organosulfates is a burgeoning area in biology, yet there are significant challenges with their synthesis. We report the development of a tributylsulfoammonium betaine as a high yielding route to organosulfates. The optimised reaction conditions were interrogated with a diverse range of alcohols, including natural products and amino acids.

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.

Endothelial colony forming progenitor cell (ECFC) function is compromised in diabetes, leading to poor vascular endothelial repair, which contributes to impaired diabetic foot ulcer healing. We have generated novel glycomimetic drugs with protective effects against endothelial dysfunction. We investigated the effect of glycomimetic C3 on the functional capacity of diabetic ECFCs. ECFCs were isolated from healthy controls and patients with diabetes with neuroischaemic (NI) or neuropathic (NP) foot ulcers. Functionally, diabetic ECFCs demonstrated delayed colony formation (p in vitro in patient ECFCs (p

Key Points Question Do 1 or more doses of levocarnitine reduce organ failure in septic shock at 48 hours, and, if so, what is the likelihood of success in a phase 3 trial? Findings In an adaptive randomized, blinded clinical trial of 250 adults, the most efficacious dose of levocarnitine (18 g) demonstrated a posterior probability of efficacy of 0.78, which did not reach the a priori threshold of 0.90. Meaning Levocarnitine did not meaningfully reduce organ failure at 48 hours in patients with septic shock., Importance Sepsis induces profound metabolic derangements, while exogenous levocarnitine mitigates metabolic dysfunction by enhancing glucose and lactate oxidation and increasing fatty acid shuttling. Previous trials in sepsis suggest beneficial effects of levocarnitine on patient-centered outcomes. Objectives To test the hypothesis that levocarnitine reduces cumulative organ failure in patients with septic shock at 48 hours and, if present, to estimate the probability that the most efficacious dose will decrease 28-day mortality in a pivotal phase 3 clinical trial. Design, Setting, and Participants Multicenter adaptive, randomized, blinded, dose-finding, phase 2 clinical trial (Rapid Administration of Carnitine in Sepsis [RACE]). The setting was 16 urban US medical centers. Participants were patients aged 18 years or older admitted from March 5, 2013, to February 5, 2018, with septic shock and moderate organ dysfunction. Interventions Within 24 hours of identification, patients were assigned to 1 of the following 4 treatments: low (6 g), medium (12 g), or high (18 g) doses of levocarnitine or an equivalent volume of saline placebo administered as a 12-hour infusion. Main Outcomes and Measures The primary outcome required, first, a greater than 90% posterior probability that the most promising levocarnitine dose decreases the Sequential Organ Failure Assessment (SOFA) score at 48 hours and, second (given having met the first condition), at least a 30% predictive probability of success in reducing 28-day mortality in a subsequent traditional superiority trial to test efficacy. Results Of the 250 enrolled participants (mean [SD] age, 61.7 [14.8] years; 56.8% male), 35, 34, and 106 patients were adaptively randomized to the low, medium, and high levocarnitine doses, respectively, while 75 patients were randomized to placebo. In the intent-to-treat analysis, the fitted mean (SD) changes in the SOFA score for the low, medium, and high levocarnitine groups were −1.27 (0.49), −1.66 (0.38), and −1.97 (0.32), respectively, vs −1.63 (0.35) in the placebo group. The posterior probability that the 18-g dose is superior to placebo was 0.78, which did not meet the a priori threshold of 0.90. Mortality at 28 days was 45.9% (34 of 74) in the placebo group compared with 43.3% (45 of 104) for the most promising levocarnitine dose (18 g). Similar findings were noted in the per-protocol analysis. Conclusions and Relevance In this dose-finding, phase 2 adaptive randomized trial, the most efficacious dose of levocarnitine (18 g) did not meaningfully reduce cumulative organ failure at 48 hours. Trial Registration ClinicalTrials.gov Identifier: NCT01665092, This randomized clinical trial tests the hypothesis that levocarnitine reduces cumulative organ failure in patients with septic shock at 48 hours and estimates the probability that the most efficacious dose will decrease 28-day mortality.

OBJECTIVE: To test the hypothesis that adjunctive inhaled NO would improve RV function and viability in acute PE. METHODS: This was a randomized, placebo-controlled, double blind trial conducted at four academic hospitals. Eligible patients had acute PE without systemic arterial hypotension but had RV dysfunction and a treatment plan of standard anticoagulation. Subjects received either oxygen plus 50 parts per million nitrogen (placebo) or oxygen plus 50 ppm NO for 24 hours. The primary composite endpoint required a normal RV on echocardiography and a plasma troponin T concentration

Simple Summary Ovarian cancer is the fifth most common cancer in the developing world, with many front-line treatments combining paclitaxel with platinum-based anticancer agents cisplatin/carboplatin. However, increased incidence of platinum resistance demands the development of new chemotherapeutic agents. The aim of our study was to explore a new family of organoruthenium(II) pyrithione complexes for their efficacy towards platinum-resistant ovarian cancer cells. We confirmed that this new class of compounds remain highly potent towards platinum-insensitive cells and appear to work by a mechanism that is not common to platinum agents. Abstract Drug resistance to existing anticancer agents is a growing clinical concern, with many first line treatments showing poor efficacy in treatment plans of some cancers. Resistance to platinum agents, such as cisplatin, is particularly prevalent in the treatment of ovarian cancer, one of the most common cancers amongst women in the developing world. Therefore, there is an urgent need to develop next generation of anticancer agents which can overcome resistance to existing therapies. We report a new series of organoruthenium(II) complexes bearing structurally modified pyrithione ligands with extended aromatic scaffold, which overcome platinum and adriamycin resistance in human ovarian cancer cells. The mechanism of action of such complexes appears to be unique from that of cisplatin, involving G1 cell cycle arrest without generation of cellular ROS, as is typically associated with similar ruthenium complexes. The complexes inhibit the enzyme thioredoxin reductase (TrxR) in a model system and reduce cell motility towards wound healing. Importantly, this work highlights further development in our understanding of the multi-targeting mechanism of action exhibited by transition metal complexes.

Thermal groundwater is currently being exploited for district-scale heating in many locations world-wide. The chemical compositions of these thermal waters reflect the provenance and circulation patterns of the groundwater, which are controlled by recharge, rock type and geological structure. Exploring the provenance of these waters using multivariate statistical analysis (MSA) techniques increases our understanding of the hydrothermal circulation systems, and provides a reliable tool for assessing these resources.Hydrochemical data from thermal springs situated in the Carboniferous Dublin Basin in east-central Ireland were explored using MSA, including hierarchical cluster analysis (HCA) and principal component analysis (PCA), to investigate the source aquifers of the thermal groundwaters. To take into account the compositional nature of the hydrochemical data, compositional data analysis (CoDa) techniques were used to process the data prior to the MSA.The results of the MSA were examined alongside detailed time-lapse temperature measurements from several of the springs, and indicate the influence of three important hydrogeological processes on the hydrochemistry of the thermal waters: 1) salinity and increased water-rock interaction; 2) dissolution of carbonates; and 3) dissolution of sulfides, sulfates and oxides associated with mineral deposits. The use of MSA within the CoDa framework identified subtle temporal variations in the hydrochemistry of the thermal springs, which could not be identified with more traditional graphing methods, or with a standard statistical approach. The MSA was successful in distinguishing different geological settings and different annual behaviours within the group of springs. This study demonstrates the usefulness of the application of MSA within the CoDa framework in order to better understand the underlying controlling processes governing the hydrochemistry of a group of thermal springs in a low-enthalpy setting.

Despite extensive effort over many decades to understand the tectonic evolution of the Tibetan Plateau, the geodynamic processes creating the iconic south-eastward expansion of the plateau at the Eastern Himalayan Syntaxis (EHS) are still unclear and are hotly debated. Two popular (but not necessarily exclusive) geodynamic models, namely crustal flow at mid-to-lower crustal depths and coherent deformation between the crust and lithospheric mantle, are commonly invoked to explain the expansion mechanism. However, neither of these is able to reconcile all of the abundant geological and geophysical data. Here we present a three-dimensional (3D) geo-electrical model, derived from new SINOPROBE magnetotelluric (MT) array data, that reveals the geo-electrical, and by inference rheological, structure of southeast Tibet. Instead of NW–SE conductive channels proposed in prior two-dimensional (2D) MT studies, distinct NNE–SSW directed quasi-linear conductive anomalies are identified in the mid-to-lower crust, which are separated by a large-scale electrically resistive structure that extends from the crust to the upper mantle. This argues against the prior proposed model of south-eastward conductive anomalies, and hence against the southeast lower crust flow of material. To interpret our observations and resultant model, a new mechanism of “extensional extrusion” is proposed to address the lithospheric deformation of the south-eastward expansion of Tibetan Plateau.

Kilbrook spring is a thermal spring in east-central Ireland. The temperatures in the spring are the highest recorded for any thermal spring in Ireland (maximum of 25 °C). The temperature is elevated with respect to average Irish groundwater temperatures (9.5–10.5 °C), and represents a geothermal energy potential, which is currently under evaluation. A multi-disciplinary investigation based upon an audio-magnetotelluric (AMT) survey, and hydrochemical analysis including time-lapse temperature and chemistry measurements, has been undertaken with the aims of investigating the provenance of the thermal groundwater and characterising the geological structures facilitating groundwater circulation in the bedrock. The three-dimensional (3-D) electrical resistivity model of the subsurface at Kilbrook spring was obtained by the inversion of AMT impedances and vertical magnetic transfer functions. The model is interpreted alongside high resolution temperature and electrical conductivity measurements, and a previous hydrochemical analysis. The hydrochemical analysis and time-lapse measurements suggest that the thermal waters have a relatively stable temperature and major ion hydrochemistry, and flow within the limestones of the Carboniferous Dublin Basin at all times. The 3-D resistivity model of the subsurface reveals a prominent NNW aligned structure within a highly resistive limestone lithology that is interpreted as a dissolutionally enhanced strike-slip fault, of Cenozoic age. The karstification of this structure, which extends to depths of at least 500 m directly beneath the spring, has provided conduits that facilitate the operation of a relatively deep hydrothermal circulation pattern (likely estimated depths between 560 and 1000 m) within the limestone succession of the Dublin Basin. The results of this study support the hypothesis that the winter thermal maximum and simultaneous increased discharge at Kilbrook spring is the result of rapid infiltration, heating and re-circulation of meteoric waters within this structurally controlled hydrothermal circulation system. This paper illustrates how AMT may be useful in a multi-disciplinary investigation of an intermediate-depth (100–1000 m), low-enthalpy, geothermal target, and shows how the different strands of inquiry from a multi-disciplinary investigation may be woven together to gain a deeper understanding of a complex hydrothermal system.

Ultrafine particles (diameter

The study purposes were to prospectively evaluate occurrence of posttraumatic stress (PTS) symptoms at hospital admission and 6 months later in patients with orthopaedic injury; to explore differences in PTS symptoms in those with and without orthopaedic injury; and to determine whether PTS symptoms are influenced by orthopaedic injury type.Prospective, longitudinal observational study.Level 1 Trauma Center.Two hundred fifty-nine participants admitted for at least 24 hours.The Primary Care Posttraumatic Stress Disorder (PTSD) Screen (PC-PTSD) measured PTSD symptoms during hospitalization. The PTSD Checklist-Civilian Version (PCL-C) measured PTS symptoms at 6 months.In orthopaedic patients, 28% had PTS at 6 months, compared with 34% of nonorthopaedic patients. Odds ratios (ORs) were calculated to determine the influence of pain, physical and mental function, depression, and work status. At 6 months, if the pain score was 5 or higher, the odds of PTS symptoms increased to 8.38 (3.55, 19.8) (P0.0001). Those scoring below average in physical function were significantly more likely to have PTS symptoms [OR = 7.60 (2.99, 19.32), P0.0001]. The same held true for mental functioning and PTS [OR = 11.4 (4.16, 30.9), P0.0001]. Participants who screened positive for depression had a 38.9 (14.5, 104) greater odds (P0.0001). Participants who did not return to work after injury at 6 months were significantly more likely to have PTS [OR = 16.5 (1.87, 146), P = 0.012].PTSD is common in patients after injury, including those with orthopaedic trauma. At 6 months, pain of 5 or greater, poor physical and mental function, depression, and/or not returning to work seem to be predictive of PTSD. Orthopaedic surgeons should identify and refer for PTSD treatment given the high incidence postinjury.Prognostic Level I. See Instructions for Authors for a complete description of levels of evidence.

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.