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Aims: The aim of this study was to explore the suspected adverse drug reaction (ADR) data of five licensed statins in the UK: atorvastatin, fluvastatin, pravastatin, rosuvastatin, and simvastatin. A secondary aim was to determine if there are any associations between the polypharmacological properties of the statins and their associated muscle-related side effects. Methods: The chemical database of bioactive molecules with drug-like properties, European Molecular Biology Laboratory (ChEMBL), was used to obtain data on the pharmacological interactions of statins with human proteins. The Medicines and Healthcare Products Regulatory Agency’s (MHRA) Yellow Card scheme was used to obtain reports of suspected ADRs from 2018 to 2022. The OpenPrescribing database was used to obtain the prescribing rates for statistical interpretation. Results: The study found no significant difference between the statins association with suspected ADRs across all organ classes (X2, p > 0.05). Fluvastatin was found to have a higher incidence of ADRs/100,000 Rx across multiple system organ classes. Conclusions: No significant difference was found between the suspected ADR incidence of the statins across all system organ classes.

Abstract The development of electrochemical approaches to the valorization of abundant natural products into high value medications and metabolites is of pharmaceutical interest. In this study, we explored the electrosynthetic behavior of the abundant legal psychoactive, caffeine, a representative member of the purine alkaloid class. Initial screening of the cyclic voltammetric behavior of eleven exemplar purine alkaloids revealed a structure electroactivity relationship (SeAR) for determining the initial oxidation site of caffeine. Optimization of the current controlled electrochemical (CCE) reaction enabled the dialing‐in/out of differential oxidative metabolic products using both undivided and divided cells. Sequential desmethylation around the purine ring was observed both by isolation and comparison to authentic metabolite reference standards via HPLC measurements. Amide, imide, and a novel N‐methyl heteroaryl oxidation mechanism were observed. Tractable quantities of the high‐value medication, theophylline, and the dietary supplement, paraxanthine, were isolated in 17 % and 8 % b.r.s.m. This approach offers a marked improvement compared to the best‐in‐class techniques (chemical 0.8 % and enzymatic 0.97 % yields) and may have potential in other natural product and drug discovery settings to prepare valuable metabolites.

Abstract Probing the surface of proteins to predict the binding site and binding affinity for a given small molecule is a critical but challenging task in drug discovery. Blind docking addresses this issue by performing docking on binding regions randomly sampled from the entire protein surface. However, compared with local docking, blind docking is less accurate and reliable because the docking space is too largetly sampled. Cavity detection-guided blind docking methods improved the accuracy by using cavity detection (also known as binding site detection) tools to guide the docking procedure. However, it is worth noting that the performance of these methods heavily relies on the quality of the cavity detection tool. This constraint, namely the dependence on a single cavity detection tool, significantly impacts the overall performance of cavity detection-guided methods. To overcome this limitation, we proposed Consensus Blind Dock (CoBDock), a novel blind, parallel docking method that uses machine learning algorithms to integrate docking and cavity detection results to improve not only binding site identification but also pose prediction accuracy. Our experiments on several datasets, including PDBBind 2020, ADS, MTi, DUD-E, and CASF-2016, showed that CoBDock has better binding site and binding mode performance than other state-of-the-art cavity detector tools and blind docking methods.

The tractable preparation of Phase I drug metabolites is a critical step to understand the first-pass behaviour of novel chemical entities (NCEs) in drug discovery. In this study, we have developed a structure–electroactivity relationship (SeAR)-informed electrochemical reaction of the parent 2-chlorophenothiazine and the antipsychotic medication, chlorpromazine. With the ability to dial-in under current controlled conditions, the formation of S-oxide and novel S,S-dioxide metabolites has been achieved for the first time on a multi-milligram scale using a direct batch electrode platform. A potential rationale for the electrochemical formation of these metabolites in situ is proposed using molecular docking to a cytochrome P450 enzyme.

In this article, we introduce a proof-of-concept strategy, Computational Predictive and Electrochemical Detection of Metabolites (CP-EDM), to expedite the discovery of drug metabolites. The use of a bioactive natural product, piperine, that has a well-curated metabolite profile but an unpredictable computational metabolism (Biotransformer v3.0) was selected. We developed an electrochemical reaction to oxidize piperine into a range of metabolites, which were detected by LC-MS. A series of chemically plausible metabolites were predicted based on ion fragmentation patterns. These metabolites were docked into the active site of CYP3A4 using Autodock4.2. From the clustered low-energy profile of piperine in the active site, it can be inferred that the most likely metabolic position of piperine (based on intermolecular distances to the Fe-oxo active site) is the benzo[d][1,3]dioxole motif. The metabolic profile was confirmed by comparison with the literature, and the electrochemical reaction delivered plausible metabolites, vide infra, thus, demonstrating the power of the hyphenated technique of tandem electrochemical detection and computational evaluation of binding poses. Taken together, we outline a novel approach where diverse data sources are combined to predict and confirm a metabolic outcome for a bioactive structure.

The Beckmann rearrangement of ketoximes to their corresponding amides, using a Brønsted acid-mediated fragmentation and migration sequence, has found wide-spread industrial application. We postulated that the development of a methodology to access ylideneamino sulfates using tributylsulfoammonium betaine (TBSAB) would afford isolable Beckmann-type intermediates and competent partners for subsequent rearrangement cascades. The ylideneamino sulfates generated, isolated as their tributylammonium salts, are sufficiently activated to undergo Beckmann rearrangement without additional reagent activation. The generation of sulfuric acid in situ from the ylideneamino sulfate giving rise to a routine Beckmann rearrangement and additional amide bond cleavage to the corresponding aniline was detrimental to reaction success. The screening of bases revealed inexpensive sodium bicarbonate to be an effective additive to prevent classic Brønsted acid-mediated fragmentation and achieve optimal conversions of up to 99%.

Objectives: Effective and widely available therapies are still needed for outpatients with COVID-19. We aimed to evaluate the efficacy and safety of lopinavir/ritonavir (LPV/r) for early treatment of non-hospitalized individuals diagnosed with COVID-19. Methods: This randomized, placebo (Plb)-controlled, double-blind, multi-site decentralized clinical trial enrolled non-hospitalized adults with confirmed SARS-CoV-2 infection and six or fewer days of acute respiratory infection symptoms who were randomized to either twice-daily oral LPV/r (400 mg/100 mg) or Plb for 14 days. Daily surveys on study days 1 through 16 and again on study day 28 evaluated symptoms, daily activities, and hospitalization status. The primary outcome was longitudinal change in an ordinal scale based on a combination of symptoms, activity, and hospitalization status through day 15 and was analyzed by use of a Bayesian longitudinal proportional odds logistic regression model for estimating the probability of a superior recovery for LPV/r over Plb (odds ratio >1). Results: Between June 2020 and December 2021, 448 participants were randomized to receive either LPV/r (n = 216) or Plb (n = 221). The mean symptom duration before randomization was 4.3 days (SD 1.3). There were no differences between treatment groups through the first 15 days for the ordinal primary outcome (odds ratio 0.96; 95% credible interval: 0.66 to 1.41). There were 3.2% (n = 7) of LPV/r and 2.7% (n = 6) of Plb participants hospitalized by day 28. Serious adverse events did not differ between groups. Conclusion: LPV/r did not significantly improve symptom resolution or reduce hospitalization in non-hospitalized participants with COVID-19. Trial registration: ClinicalTrials.gov Identifier: NCT04372628

Abstract Background Maize lethal necrosis (MLN) disease is a significant constraint for maize producers in sub-Saharan Africa (SSA). The disease decimates the maize crop, in some cases, causing total crop failure with far-reaching impacts on regional food security. Results In this review, we analyze the impacts of MLN in Africa, finding that resource-poor farmers and consumers are the most vulnerable populations. We examine the molecular mechanism of MLN virus transmission, role of vectors and host plant resistance identifying a range of potential opportunities for genetic and phytosanitary interventions to control MLN. We discuss the likely exacerbating effects of climate change on the MLN menace and describe a sobering example of negative genetic association between tolerance to heat/drought and susceptibility to viral infection. We also review role of microRNAs in host plant response to MLN causing viruses as well as heat/drought stress that can be carefully engineered to develop resistant varieties using novel molecular techniques. Conclusions With the dual drivers of increased crop loss due to MLN and increased demand of maize for food, the development and deployment of simple and safe technologies, like resistant cultivars developed through accelerated breeding or emerging gene editing technologies, will have substantial positive impact on livelihoods in the region. We have summarized the available genetic resources and identified a few large-effect QTLs that can be further exploited to accelerate conversion of existing farmer-preferred varieties into resistant cultivars.

The C(sp2)-aryl sulfonate functional group is found in bioactive molecules, but their synthesis can involve extreme temperatures (>190 °C or flash vacuum pyrolysis) and strongly acidic reaction conditions. Inspired by the 1917 Tyrer industrial process for a sulfa dye that involved an aniline N(sp2)-SO3 intermediate en route to a C(sp2)-SO3 rearranged product, we investigated tributylsulfoammonium betaine (TBSAB) as a milder N-sulfamation to C-sulfonate relay reagent. Initial investigations of a stepwise route involving TBSAB on selected anilines at room temperature enabled the isolation of N(sp2)-sulfamate. Subsequent thermal rearrangement demonstrated the intermediary of a sulfamate en route to the sulfonate; however, it was low-yielding. Investigation of the N-sulfamate to C--sulfonate mechanism through control experiments with variation at the heteroatom positions and kinetic isotope experiments (KIEH/D) confirmed the formation of a key N(sp2)-SO3 intermediate and further confirmed an intermolecular mechanism. Furthermore, compounds without an accessible nitrogen (or oxygen) lone pair did not undergo sulfamation- (or sulfation) -to-sulfonation under these conditions. A one-pot sulfamation and thermal sulfonation reaction was ultimately developed and explored on a range of aniline and heterocyclic scaffolds with high conversions, including N(sp2)-sulfamates (O(sp2)-sulfates) and C(sp2)-sulfonates, in up to 99 and 80% (and 88% for a phenolic example) isolated yield, respectively. Encouragingly, the ability to modulate the ortho-para selectivity of the products obtained was observed under thermal control. A sulfonated analog of the intravenous anesthetic propofol was isolated (88% yield), demonstrating a proof-of-concept modification of a licensed drug alongside a range of nitrogen- and sulfur-containing heterocyclic fragments used in drug discovery.

Heparan sulphate (HS) can act as a co-receptor on the cell surface and alterations in this process underpin many pathological conditions. We have previously described the usefulness of mimics of HS (glycomimetics) in protection against β-glycerophosphate-induced vascular calcification and in the restoration of the functional capacity of diabetic endothelial colony-forming cells in vitro. This study aims to investigate whether our novel glycomimetic compounds can attenuate glycated low-density lipoprotein (g-LDL)-induced calcification by inhibiting RAGE signalling within the context of critical limb ischemia (CLI). We used an established osteogenic in vitro vascular smooth muscle cell (VSMC) model. Osteoprotegerin (OPG), sclerostin and glycation levels were all significantly increased in CLI serum compared to healthy controls, while the vascular calcification marker osteocalcin (OCN) was down-regulated in CLI patients vs. controls. Incubation with both CLI serum and g-LDL (10 µg/mL) significantly increased VSMC calcification vs. controls after 21 days, with CLI serum-induced calcification apparent after only 10 days. Glycomimetics (C2 and C3) significantly inhibited g-LDL and CLI serum-induced mineralisation, as shown by a reduction in alizarin red (AR) staining and alkaline phosphatase (ALP) activity. Furthermore, secretion of the osteogenic marker OCN was significantly reduced in VSMCs incubated with CLI serum in the presence of glycomimetics. Phosphorylation of cyclic AMP response element-binding protein (CREB) was significantly increased in g-LDL-treated cells vs. untreated controls, which was attenuated with glycomimetics. Blocking CREB activation with a pharmacological inhibitor 666-15 replicated the protective effects of glycomimetics, evidenced by elevated AR staining. In silico molecular docking simulations revealed the binding affinity of the glycomimetics C2 and C3 with the V domain of RAGE. In conclusion, these findings demonstrate that novel glycomimetics, C2 and C3 have potent anti-calcification properties in vitro, inhibiting both g-LDL and CLI serum-induced VSMC mineralisation via the inhibition of LDLR, RAGE, CREB and subsequent expression of the downstream osteogenic markers, ALP and OCN.

We present a model for the effects of ligands on information transmission in G-Protein Coupled Receptor (GPCR) complexes. The model is built ab initio entirely on principles of statistical mechanics and tenets of information transmission theory and was validated in part using agonist-induced effector activity and signaling bias for the angiotensin- and adrenergic-mediated signaling pathways, with in vitro observations of phosphorylation sites on the C tail of the GPCR complex, and single-cell information-transmission experiments. The model extends traditional kinetic models that form the basis for many existing models of GPCR signaling. It is based on maximizing the rates of entropy production and information transmission through the GPCR complex. The model predicts that (1) phosphatase-catalyzed reactions, as opposed to kinase-catalyzed reactions, on the C-tail and internal loops of the GPCR are responsible for controlling the signaling activity, (2) signaling favors the statistical balance of the number of switches in the ON state and the number in the OFF state, and (3) biased-signaling response depends discontinuously on ligand concentration.

OBJECTIVES:. Perturbed host metabolism is increasingly recognized as a pillar of sepsis pathogenesis, yet the dynamic alterations in metabolism and its relationship to other components of the host response remain incompletely understood. We sought to identify the early host-metabolic response in patients with septic shock and to explore biophysiological phenotyping and differences in clinical outcomes among metabolic subgroups. DESIGN:. We measured serum metabolites and proteins reflective of the host-immune and endothelial response in patients with septic shock. SETTING:. We considered patients from the placebo arm of a completed phase II, randomized controlled trial conducted at 16 U.S. medical centers. Serum was collected at baseline (within 24 hr of the identification of septic shock), 24-hour, and 48-hour postenrollment. Linear mixed models were built to assess the early trajectory of protein analytes and metabolites stratified by 28-day mortality status. Unsupervised clustering of baseline metabolomics data was conducted to identify subgroups of patients. PATIENTS:. Patients with vasopressor-dependent septic shock and moderate organ dysfunction that were enrolled in the placebo arm of a clinical trial. INTERVENTIONS:. None. MEASUREMENTS AND MAIN RESULTS:. Fifty-one metabolites and 10 protein analytes were measured longitudinally in 72 patients with septic shock. In the 30 patients (41.7%) who died prior to 28 days, systemic concentrations of acylcarnitines and interleukin (IL)-8 were elevated at baseline and persisted at T24 and T48 throughout early resuscitation. Concentrations of pyruvate, IL-6, tumor necrosis factor-α, and angiopoietin-2 decreased at a slower rate in patients who died. Two groups emerged from clustering of baseline metabolites. Group 1 was characterized by higher levels of acylcarnitines, greater organ dysfunction at baseline and postresuscitation (p < 0.05), and greater mortality over 1 year (p < 0.001). CONCLUSIONS:. Among patients with septic shock, nonsurvivors exhibited a more profound and persistent dysregulation in protein analytes attributable to neutrophil activation and disruption of mitochondrial-related metabolism than survivors.

This article presents a bibliometric evaluation of Forensic Science International (FSI) as a scholarly journal within the “legal medicine” subject category. Citation data were retrieved from Science Citation Index (SCI) and Journal Citation Reports (JCR), both of which are part of the Web-of-Science (WOS) database. The most cited articles in FSI were identified along with the most prolific authors. The current journal impact factor (JIF) of FSI is 2.2, which was in good agreement with the 5-year JIF of 2.3. FSI was ranked fourth among 17 journals within the legal medicine subject category. Since 1979, a total of 209 FSI articles were cited over 100 times and the H-index for times cited was 125. Although widely used in academia, bibliometric methods might also prove useful in jurisprudence, such as when evaluating the research and publications of people proposed as expert witnesses.

Abstract Sepsis‐induced metabolic dysfunction contributes to organ failure and death. L‐carnitine has shown promise for septic shock, but a recent phase II study of patients with vasopressor‐dependent septic shock demonstrated a non‐significant reduction in mortality. We undertook a pharmacometabolomics study of these patients (n = 250) to identify metabolic profiles predictive of a 90‐day mortality benefit from L‐carnitine. The independent predictive value of each pretreatment metabolite concentration, adjusted for L‐carnitine dose, on 90‐day mortality was determined by logistic regression. A grid‐search analysis maximizing the Z‐statistic from a binomial proportion test identified specific metabolite threshold levels that discriminated L‐carnitine responsive patients. Threshold concentrations were further assessed by hazard ratio and Kaplan‐Meier estimate. Accounting for L‐carnitine treatment and dose, 11 1H‐NMR metabolites and 12 acylcarnitines were independent predictors of 90‐day mortality. Based on the grid‐search analysis numerous acylcarnitines and valine were identified as candidate metabolites of drug response. Acetylcarnitine emerged as highly viable for the prediction of an L‐carnitine mortality benefit due to its abundance and biological relevance. Using its most statistically significant threshold concentration, patients with pretreatment acetylcarnitine greater than or equal to 35 µM were less likely to die at 90 days if treated with L‐carnitine (18 g) versus placebo (p = 0.01 by log rank test). Metabolomics also identified independent predictors of 90‐day sepsis mortality. Our proof‐of‐concept approach shows how pharmacometabolomics could be useful for tackling the heterogeneity of sepsis and informing clinical trial design. In addition, metabolomics can help understand mechanisms of sepsis heterogeneity and variable drug response, because sepsis induces alterations in numerous metabolite concentrations.

Abstract To interpret the relationship between the polypharmacology of dipeptidyl‐peptidase IV inhibitors (DPP4i) and their suspected adverse drug reaction (ADR) profiles using a national registry. A retrospective investigation into the suspected ADR profile of four licensed DPP4i in the United Kingdom using the National MHRA Yellow Card Scheme and OpenPrescribing databases. Experimental data from the ChEMBL database alongside physiochemical (PC) and pharmacokinetic (PK) profiles were extracted and interpreted. DPP4i show limited polypharmacology alongside low suspected ADR rates. We found a minimal statistical difference between the unique ADR profiles ascribed to the DPP4i except for total ADRs (χ2; p .05). We confirmed the Phase III clinical trial data that showed gastrointestinal and skin reactions are the most reported ADRs across the DPP4i class and postulated underlying mechanisms for this based on possible drug interactions. The main pharmacological mechanism behind the ADRs is attributed to interactions with DPP4 activity and/or structure homolog (DASH) proteins which augment the immune‐inflammatory modulation of DPP4.

Amyloid aggregates arise from either the partial or complete loss of the native protein structure or the inability of proteins to attain their native conformation. These aggregates have been linked to several diseases, including Alzheimer’s, Parkinson’s, and lysozyme amyloidosis. A comprehensive dataset was recently reported, demonstrating the critical role of the protein’s surrounding environment in amyloid formation. In this study, we investigated the formation of lysozyme amyloid fibrils induced by sodium dodecyl sulfate (SDS) and the effect of solvents in the medium. Experimental data obtained through fluorescence spectroscopy revealed a notable lag phase in amyloid formation when acetone solution was present. This finding suggested that the presence of acetone in the reaction medium created an unfavorable microenvironment for amyloid fibril formation and impeded the organization of the denatured protein into the fibril form. The in silico data provided insights into the molecular mechanism of the interaction between acetone molecules and the lysozyme protofibril, once acetone presented the best experimental results. It was observed that the lysozyme protofibril became highly unstable in the presence of acetone, leading to the complete loss of its β-sheet conformation and resulting in an open structure. Furthermore, the solvation layer of the protofibril in acetone solution was significantly reduced compared to that in other solvents, resulting in fewer hydrogen bonds. Consequently, the presence of acetone facilitated the exposure of the hydrophobic portion of the protofibril, precluding the amyloid fibril formation. In summary, our study underscores the pivotal role the surrounding environment plays in influencing amyloid formation.

Abstract Background Convalescent plasma is being used widely as a treatment for coronavirus disease 2019 (COVID-19). However, the clinical efficacy of COVID-19 convalescent plasma is unclear. Methods The Passive Immunity Trial for Our Nation (PassITON) is a multicenter, placebo-controlled, blinded, randomized clinical trial being conducted in the USA to provide high-quality evidence on the efficacy of COVID-19 convalescent plasma as a treatment for adults hospitalized with symptomatic disease. Adults hospitalized with COVID-19 with respiratory symptoms for less than 14 days are eligible. Enrolled patients are randomized in a 1:1 ratio to 1 unit (200–399 mL) of COVID-19 convalescent plasma that has demonstrated neutralizing function using a SARS-CoV-2 chimeric virus neutralization assay. Study treatments are administered in a blinded fashion and patients are followed for 28 days. The primary outcome is clinical status 14 days after study treatment as measured on a 7-category ordinal scale assessing mortality, respiratory support, and return to normal activities of daily living. Key secondary outcomes include mortality and oxygen-free days. The trial is projected to enroll 1000 patients and is designed to detect an odds ratio ≤ 0.73 for the primary outcome. Discussion This trial will provide the most robust data available to date on the efficacy of COVID-19 convalescent plasma for the treatment of adults hospitalized with acute moderate to severe COVID-19. These data will be useful to guide the treatment of COVID-19 patients in the current pandemic and for informing decisions about whether developing a standardized infrastructure for collecting and disseminating convalescent plasma to prepare for future viral pandemics is indicated. Trial registration ClinicalTrials.gov NCT04362176 . Registered on 24 April 2020.

Improving public engagement in ecological research improves the visibility of science and educates a wider audience about the value of ecology and its study. To this end, we assess the success of two simple activities, designed to track forest cover and understorey conditions, implemented at Lammi Biological Station Science Trail, Finland, in terms of effective public participation and useability of the data generated. We consider how best to engage participants in the activities, and we validate the data obtained by comparison of its reliability and useability against standard ecological approaches. It is also increasingly timely for researchers to utilise the large datasets that can be generated through effective public engagement. If experiments are effectively designed, these data can provide information at a larger scale than is attainable with the resources typically available to individual research projects. Consequently, given high enough uptake, such activities hold the potential for upscaling or generalisation from their findings. Both activities proved useful to collect more intensive data than would otherwise have been feasible. The quadrat vegetation survey (Activity 1) provided useable data to determine species phenology but not species composition. The canopy disk observations (Activity 2) reliably tracked seasonal changes in canopy cover when calibrated against baseline data. Training in these activities fostered engagement in how climate change affects forest ecology, improving the quality of data collected, and engaging participants eager to learn about and contribute to research into these processes.

Variability in the blood–breath ratio (BBR) of alcohol is important, because it relates a measurement of the blood-alcohol concentration (BAC) with the co-existing breath-alcohol concentration (BrAC). The BBR is also used to establish the statutory BrAC limit for driving from the existing statutory BAC limits in different countries. The in-vivo BBR depends on a host of analytical, sampling and physiological factors, including subject demographics, time after end of drinking (rising or falling BAC), the nature of the blood draw (whether venous or arterial) and the subject’s breathing pattern prior to exhalation into the breath analyzer. The results from a controlled drinking study involving healthy volunteers (85 men and 15 women) from three ethnic groups (Caucasians, Hispanics and African Americans) were used to evaluate various factors influencing the BBR. Ethanol in breath was determined with a quantitative infrared analyzer (Intoxilyzer 8000) and BAC was determined by headspace gas chromatography (HS-GC). The BAC and BrAC were highly correlated (r = 0.948) and the BBR in the post-absorptive state was 2 382 ± 119 (mean ± SD). The BBR did not depend on gender (female: 2 396 ± 101 and male: 2 380 ± 123, P > 0.05) nor on racial group (Caucasians 2 398 ± 124, African Americans 2 344 ± 119 and Hispanics 2 364 ± 104, P > 0.05). The BBR was lower in subjects with higher breath- and body-temperatures (P

Abstract Background The BinaxNOW coronavirus disease 2019 (COVID‐19) Ag Card test (Abbott Diagnostics Scarborough, Inc.) is a lateral flow immunochromatographic point‐of‐care test for the qualitative detection of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) nucleocapsid protein antigen. It provides results from nasal swabs in 15 minutes. Our purpose was to determine its sensitivity and specificity for a COVID‐19 diagnosis. Methods Eligible patients had symptoms of COVID‐19 or suspected exposure. After consent, 2 nasal swabs were collected; 1 was tested using the Abbott RealTime SARS‐CoV‐2 (ie, the gold standard polymerase chain reaction test) and the second run on the BinaxNOW point of care platform by emergency department staff. Results From July 20 to October 28, 2020, 767 patients were enrolled, of which 735 had evaluable samples. Their mean (SD) age was 46.8 (16.6) years, and 422 (57.4%) were women. A total of 623 (84.8%) patients had COVID‐19 symptoms, most commonly shortness of breath (n = 404; 55.0%), cough (n = 314; 42.7%), and fever (n = 253; 34.4%). Although 460 (62.6%) had symptoms ≤7 days, the mean (SD) time since symptom onset was 8.1 (14.0) days. Positive tests occurred in 173 (23.5%) and 141 (19.2%) with the gold standard versus BinaxNOW test, respectively. Those with symptoms >2 weeks had a positive test rate roughly half of those with earlier presentations. In patients with symptoms ≤7 days, the sensitivity, specificity, and negative and positive predictive values for the BinaxNOW test were 84.6%, 98.5%, 94.9%, and 95.2%, respectively. Conclusions The BinaxNOW point‐of‐care test has good sensitivity and excellent specificity for the detection of COVID‐19. We recommend using the BinasNOW for patients with symptoms up to 2 weeks.

Plant growth and grain filling are the key agronomical traits for grain weight and yield of rice. The continuous improvement in rice yield is required for a future sustainable global economy and food security. The heterotrimeric G protein complex containing a canonical α subunit (RGA1) couples extracellular signals perceived by receptors to modulate cell function including plant development and grain weight. We hypothesized that, besides RGA1, three atypical, extra-large GTP-binding protein (XLG) subunits also regulate panicle architecture, plant growth, development, grain weight, and disease resistance. Here, we identified a role of XLGs in agronomic traits and stress tolerance by genetically ablating all three rice XLGs individually and in combination using the CRISPR/Cas9 genome editing in rice. For this study, eight (three single, two double, and three triple) null mutants were selected. Three XLG proteins combinatorically regulate seed filling, because loss confers a decrease in grain weight from 14% with loss of one XLG and loss of three to 32% decrease in grain weight. Null mutations in XLG2 and XLG4 increase grain size. The mutants showed significantly reduced panicle length and number per plant including lesser number of grains per panicle compared to the controls. Loss-of-function of all individual XLGs contributed to 9% more aerial biomass compared to wild type (WT). The double mutant showed improved salinity tolerance. Moreover, loss of the XLG gene family confers hypersensitivity to pathogens. Our findings suggest that the non-canonical XLGs play important roles in regulating rice plant growth, grain filling, panicle phenotype, stress tolerance, and disease resistance. Genetic manipulation of XLGs has the potential to improve agronomic properties in rice.

The treatment of common steroids: estrone, estradiol, cortisol, and pregnenolone with tributylsulfoammonium betaine (TBSAB) provides a convenient chemoselective conversion of the steroids alcohol/phenol moiety to the corresponding steroidal organosulfate. An important feature of the disclosed methodology is the millimolar scale of the reaction, and the isolation of the corresponding steroid sulfates as their biologically relevant sodium salts without the need for ion-exchange chromatography. The scope of the method was further explored in the estradiol and pregnanediol steroid systems with the bis-sulfated derivatives. Ultimately, a method to install an isotopic label, deuterium (2H) combined with estrone sulfation is a valuable tool for its mass-spectrometric quantification in biological studies.

Abstract Sodium glucose co‐transporter 2 inhibitors (SGLT2i) are a promising second‐line treatment strategy for type 2 diabetes mellitus (T2DM) with a developing landscape of both beneficial cardio‐ and nephroprotective properties and emerging adverse drug reactions (ADRs) including diabetic ketoacidosis (DKA), genetic mycotic infections, and amputations among others. A national register study (MHRA Yellow Card, UK) was used to quantify the SGLT2i's suspected ADRs relative to their Rx rate (OpenPrescribing, UK). The polypharmacology profiles of SGLT2i were data‐mined (ChEMBL) for the first time. The ADR reports (n = 3629) and prescribing numbers (Rx n = 5,813,325) for each SGLT2i in the United Kingdom (from launch date to the beginning December 2019) were determined. Empagliflozin possesses the most selective SGLT2/SGLT1 inhibition profile at ~2500‐fold, ~10‐fold more selective than cangliflozin (~260‐fold). Canagliflozin was found to also inhibit CYP at clinically achievable concentrations. We find that for overall ADR rates, empagliflozin versus dapagliflozin and empagliflozin versus canagliflozin are statistically significant (χ2, p dapagliflozin > empagliflozin. For fatalities, there is a greater relative rate for dapagliflozin > canagliflozin > empagliflozin. An organ classification that resulted in a statistically significant difference between SGLT2i was suspected infection/infestation ADRs between empagliflozin and dapagliflozin. Our findings at this stage of SGLT2i usage in the United Kingdom suggest that empagliflozin, the most selective SGLT2i, had the lowest suspected ADR incident rate (relative to prescribing) and in all reported classes of ADRs identified including infections, amputations, and DKA.

Abstract Objective The IIA isoform of phospholipase A2 is an acute phase reactant that increases in sepsis, although data regarding its prognostic value are limited. We hypothesized that group IIA secretory phospholipase A2 (sPLA2‐IIA) predicts sepsis mortality and positive cultures and sought to compare its predictive characteristics to lactate and procalcitonin. Methods sPLA2‐IIA and procalcitonin levels were measured at enrollment in emergency department patients with early severe sepsis and compared with lactate levels. The primary outcome was in‐hospital mortality. The secondary outcome was any positive culture with a sub‐group analysis of only blood‐culture positive patients. Optimum cut‐point was determined using receiver operating characteristics curves. A multivariable model was developed to test the independent prognostic value of elevated sPLA2‐IIA to predict mortality. Results Of the 192 patients in the cohort, 160, 153, and 158 had samples available for analysis of sPLA2‐IIA, procalcitonin, and lactate, respectively. A total of 21% of patients met the primary outcome of in‐hospital mortality. At a 100 ng/mL threshold for sPLA2‐IIA, adjusted odds to predict mortality were 3.78 (95% confidence interval = 1.14–12.56, P = 0.03). sPLA2‐IIA and procalcitonin were both elevated in culture‐positive patients; however, the difference was not statistically significant. sPLA2‐IIA was significantly higher in blood culture‐positive patients. Conclusion An elevated level of sPLA2‐IIA was associated with increased mortality in sepsis patients. sPLA2‐IIA levels, unlike procalcitonin, also were significantly higher in blood culture‐positive patients.

Introduction In the USA, many emergency departments (EDs) have established protocols to treat patients with newly diagnosed deep vein thrombosis (DVT) as outpatients. Similar treatment of patients with pulmonary embolism (PE) has been proposed, but no large-scale study has been published to evaluate a comprehensive, integrated protocol that employs monotherapy anticoagulation to treat patients diagnosed with DVT and PE in the ED.Methods and analysis This protocol describes the implementation of the Monotherapy Anticoagulation To expedite Home treatment of Venous ThromboEmbolism (MATH-VTE) study at 33 hospitals in the USA. The study was designed and executed to meet the requirements for the Standards for Reporting Implementation Studies guideline. The study was funded by investigator-initiated awards from industry, with Indiana University as the sponsor. The study principal investigator and study associates travelled to each site to provide on-site training. The protocol identically screens patients with both DVT or PE to determine low risk of death using either the modified Hestia criteria or physician judgement plus a negative result from the simplified PE severity index. Patients must be discharged from the ED within 24 hours of triage and treated with either apixaban or rivaroxaban. Overall effectiveness is based upon the primary efficacy and safety outcomes of recurrent VTE and bleeding requiring hospitalisation respectively. Target enrolment of 1300 patients was estimated with efficacy success defined as the upper limit of the 95% CI for the 30-day frequency of VTE recurrence below 2.0%. Thirty-three hospitals in 17 states were initiated in 2016–2017.Ethics and dissemination All sites had Institutional Review Board approval. We anticipate completion of enrolment in June 2020; study data will be available after peer-reviewed publication. MATH-VTE will provide information from a large multicentre sample of US patients about the efficacy and safety of home treatment of VTE with monotherapy anticoagulation.

Abstract Direct oral anticoagulants (DOACs) have rapidly become the drug class of choice for anticoagulation therapy in secondary care. It is known that gastrointestinal hemorrhage are potential side effects of the DOAC drug class. In this study we have investigated the relevance of molecular structure and on/off‐target pharmacology as a predictor of adverse drug reactions (ADRs) for the DOAC drug class. Use of the Reaxys MedChem module allowed for data mining of all possible reported off‐target effects of the DOAC class members. For the first time, the MHRA Yellow card database in combination with prescribing rates in the United Kingdom (data for n = 30 566 936 DOAC Rx (up to 2017) and ADR data n = 22 275 (up to 2018)) were used for our data comparison of DOACs. From the underlying reported data, we were able to rank the DOACs in terms of the likely adverse events we would expect to observe. We identified potential risks of ADRs based on the DOACs pharmacology including the expected GI hemorrhage, but also the unexpected risk of stroke, pulmonary embolism and kidney injury. Statistically significant (P

Abstract Chronic kidney disease is highly prevalent, affecting 10% to 15% of the adult population worldwide and is associated with increased cardiovascular morbidity and mortality. As chronic kidney disease worsens, a unique cardiovascular phenotype develops characterized by heart muscle disease, increased arterial stiffness, atherosclerosis, and hypertension. Cardiovascular risk is multifaceted, but most cardiovascular deaths in patients with advanced chronic kidney disease are caused by heart failure and sudden cardiac death. While the exact drivers of these deaths are unknown, they are believed to be caused by uremic cardiomyopathy: a specific pattern of myocardial hypertrophy, fibrosis, with both diastolic and systolic dysfunction. Although the pathogenesis of uremic cardiomyopathy is likely to be multifactorial, accumulating evidence suggests increased production of fibroblast growth factor‐23 and αKlotho deficiency as potential major drivers of cardiac remodeling in patients with uremic cardiomyopathy. In this article we review the increasing understanding of the physiology and clinical aspects of uremic cardiomyopathy and the rapidly increasing knowledge of the biology of both fibroblast growth factor‐23 and αKlotho. Finally, we discuss how dissection of these pathological processes is aiding the development of therapeutic options, including small molecules and antibodies, directly aimed at improving the cardiovascular outcomes of patients with chronic kidney disease and end‐stage renal disease.

In this work, we show that valve-based bioprinting induces no measurable detrimental effects on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The aim of the current study was three-fold: first, to assess the response of hiPSC-CMs to several hydrogel formulations by measuring electrophysiological function; second, to customise a new microvalve-based cell printing mechanism in order to deliver hiPSC-CMs suspensions, and third, to compare the traditional manual pipetting cell-culture method and cardiomyocytes dispensed with the bioprinter. To achieve the first and third objectives, iCell2 (Cellular Dynamics International) hiPSC-CMs were used. The effects of well-known drugs were tested on iCell2 cultured by manual pipetting and bioprinting. Despite the results showing that hydrogels and their cross-linkers significantly reduced the electrophysiological performance of the cells compared with those cultured on fibronectin, the bio-ink droplets containing a liquid suspension of live cardiomyocytes proved to be an alternative to standard manual handling and could reduce the number of cells required for drug testing, with no significant differences in drug-sensitivity between both approaches. These results provide a basis for the development of a novel bioprinter with nanolitre resolution to decrease the required number of cells and to automate the cell plating process.

Heat Shock Protein 70s (HSP70s) are key molecular chaperones that are overexpressed in many cancers and often associated with metastasis and poor prognosis. It has proven difficult to develop ATP-competitive, drug-like small molecule inhibitors of HSP70s due to the flexible and hydrophilic nature of the HSP70 ATP-binding site and its high affinity for endogenous nucleotides. The aim of this study was to explore the potential for the inhibition of HSP70 through alternative binding sites using fragment-based approaches. A surface plasmon resonance (SPR) fragment screen designed to detect secondary binding sites in HSP70 led to the identification by X-ray crystallography of a cryptic binding site in the nucleotide-binding domain (NBD) of HSP70 adjacent to the ATP-binding site. Fragment binding was confirmed and characterized as ATP-competitive using SPR and ligand-observed NMR methods. Molecular dynamics simulations were applied to understand the interactions with the protein upon ligand binding, and local secondary structure changes consistent with interconversion between the observed crystal structures with and without the cryptic pocket were detected. A virtual high-throughput screen (vHTS) against the cryptic pocket was conducted, and five compounds with diverse chemical scaffolds were confirmed to bind to HSP70 with micromolar affinity by SPR. These results identified and characterized a new targetable site on HSP70. While targeting HSP70 remains challenging, the new site may provide opportunities to develop allosteric ATP-competitive inhibitors with differentiated physicochemical properties from current series.

Piperlongumine (PPL) is an alkaloid extracted from several pepper species that exhibits anti-inflammatory and anti-carcinogenic properties. Nevertheless, the molecular mode of action of PPL that confers such powerful pharmacological properties remains unknown. From this perspective, spectroscopic methods aided by computational modeling were employed to characterize the interaction between PPL and nucleotide-binding domain of heat shock protein 70 (NBD/HSP70), which is involved in the pathogenesis of several diseases. Steady-state fluorescence spectroscopy along with time-resolved fluorescence revealed the complex formation based on a static quenching mechanism. Van’t Hoff analyses showed that the binding of PPL toward NBD is driven by equivalent contributions of entropic and enthalpic factors. Furthermore, IDF and Scatchard methods applied to fluorescence intensities determined two cooperative binding sites with Kb of (6.3 ± 0.2) × 104 M−1. Circular dichroism determined the thermal stability of the NBD domain and showed that PPL caused minor changes in the protein secondary structure. Computational simulations elucidated the microenvironment of these interactions, showing that the binding sites are composed mainly of polar amino acids and the predominant interaction of PPL with NBD is Van der Waals in nature.

Abstract 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.

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.

Medical errors are of economic importance and can contribute to serious adverse events for patients. Medical errors refer to preventable events resulting from healthcare interactions, whether these events harm the patient or not. In Kuwait, there is a paucity literature detailing the causes, forms, and risks of medical errors in their state-funded healthcare facilities. This study aimed to explore medical errors, their causes and preventive strategies in a Kuwait tertiary hospital based on the perceptions and experience of a cross-section of healthcare professionals using a questionnaire with 27 open (n = 10) and closed (n = 17) questions. The recruited healthcare professionals in this study included pharmacists, nurses, physicians, dentists, radiographers, hospital administrators, surgeons, nutritionists, and physiotherapists. The collected data were analysed quantitatively using descriptive statistics. A total of 203 participants filled and completed the survey questionnaire. The frequency of medical errors in Kuwait was found to be high at 60.3% ranging from incidences of prolonged hospital stays (32.9%), adverse events and life-threatening complications (32.3%), and fatalities (20.9%). The common medical errors result from incomplete instructions, incorrect dosage, and incorrect route of administration, diagnosis errors, and labelling errors. The perceived causes of these medical errors include high workload, lack of support systems, stress, medical negligence, inadequate training, miscommunication, poor collaboration, and non-adherence to safety guidelines among the healthcare professionals.

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 crystal structures of three cyclopenta[c]furans with various substituents at the 4-, 5- and 6-positions of the ring system are reported, namely, (±)-(3aR,4S,5S,6aS)-4-methyl-5-phenylhexahydro-1H-cyclopenta[c]furan-4,5-diol, C14H18O3, (I), (±)-(3aR,4S,5S,6aS)-4-benzyloxy-4-methyl-5-phenylhexahydro-1H-cyclopenta[c]furan-5-ol, C21H24O3, (II), and (±)-(1aR,1bS,4aR,5S,5aR)-5-benzyloxy-5-methyl-5a-phenylhexahydro-2H-oxireno[2′,3′:3,4]cyclopenta[1,2-c]furan, C21H22O3, (III). The dominant interaction in (I) and (II) is an O—H...O hydrogen bond across the bicyclic 5,5-ring system between the non-functionalized hydroxy group and the tetrahydrofuran O atom, which appears to influence the envelope conformations of the fused five-membered rings, whereas in (III), the rings have different conformations. A weak intramolecular C—H...O interaction appears to influence the degree of tilt of the phenyl ring attached to the 5-position and is different in (I) compared to (II) and (III).

Electron transfer plays a vital role in drug metabolism and underlying toxicity mechanisms. Currently, pharmaceutical research relies on pharmacokinetics (PK) and absorption, distribution, metabolism, elimination and toxicity (ADMET) measurements to understand and predict drug reactions in the body. Metabolic stability (and toxicity) prediction in the early phases of the drug discovery and development process is key in identifying a suitable lead compound for optimisation. Voltammetric methods have the potential to overcome the significant barrier of new drug failure rates, by giving insight into phase I metabolism events which can have a direct bearing on the stability and toxicity of the parent drug being dosed. Herein, we report for the first time a data-mining investigation into the voltammetric behaviour of reported drug molecules and their correlation with metabolic stability (indirectly measured via t½), as a potential predictor of drug stability/toxicity in vivo. We observed an inverse relationship between oxidation potential and drug stability. Furthermore, we selected and prepared short- (2 h) drug molecules to prospectively survey the relationship between oxidation potential and stability.

To ensure scientific reproducibility of metabolomics data, alternative statistical methods are needed. A paradigm shift away from the p-value toward an embracement of uncertainty and interval estimation of a metabolite’s true effect size may lead to improved study design and greater reproducibility. Multilevel Bayesian models are one approach that offer the added opportunity of incorporating imputed value uncertainty when missing data are present. We designed simulations of metabolomics data to compare multilevel Bayesian models to standard logistic regression with corrections for multiple hypothesis testing. Our simulations altered the sample size and the fraction of significant metabolites truly different between two outcome groups. We then introduced missingness to further assess model performance. Across simulations, the multilevel Bayesian approach more accurately estimated the effect size of metabolites that were significantly different between groups. Bayesian models also had greater power and mitigated the false discovery rate. In the presence of increased missing data, Bayesian models were able to accurately impute the true concentration and incorporating the uncertainty of these estimates improved overall prediction. In summary, our simulations demonstrate that a multilevel Bayesian approach accurately quantifies the estimated effect size of metabolite predictors in regression modeling, particularly in the presence of missing data.

Inspired by the remarkable bioactivities exhibited by the natural products, piperine and piperlongumine, we synthesised eight natural product-inspired analogues to further investigate their structures. For the first time, we confirmed the structure of the key cyclised dihydropyrazolecarbothioamide piperine analogues including the use of two-dimensional (2D) 15N-based spectroscopy nuclear magnetic resonance (NMR) spectroscopy. Prior investigations demonstrated promising results from these scaffolds for the inhibition of inflammatory response via downregulation of the IL-1β and NF-κB pathway. However, the molecular interaction of these molecules with their protein targets remains unknown. Ab initio calculations revealed the electronic density function map of the molecules, showing the effects of structural modification in the electronic structure. Finally, molecular interactions between the synthesized molecules and the proteins IL-1β and NF-κB were achieved. Docking results showed that all the analogues interact in the DNA binding site of NF-κB with higher affinity compared to the natural products and, with the exception of 9a and 9b, have higher affinity than the natural products for the binding site of IL-1β. Specificity for the molecular recognition of 3a, 3c and 9b with IL-1β through cation–π interactions was determined. These results revealed 3a, 3c, 4a, 4c and 10 as the most promising molecules to be evaluated as IL-1β and NF-κB inhibitors.

Background: Sepsis-induced alterations in mitochondrial function contribute to organ dysfunction and mortality. Measuring mitochondrial function in vital organs is neither feasible nor practical, highlighting the need for non-invasive approaches. Mitochondrial function may be reflected in the concentrations of metabolites found in platelets and whole blood (WB) samples. We proposed to use these as alternates to indirectly estimate platelet mitochondrial oxygen consumption rate (mOCR) in sepsis patients. Methods: We determined the relationships between platelet mOCR and metabolites in both platelets and WB, as measured by quantitative 1H-NMR metabolomics. The associations were identified by building multiple linear regression models with stepwise forward-backward variable selection. We considered the models to be significant with an ANOVA test (p-value ≤ 0.05) and a positive predicted-R2. Results: The differences in adjusted-R2 and ANOVA p-values (platelet adj-R2: 0.836 (0.0003), 0.711 (0.0004) vs. WB adj-R2: 0.428 (0.0079)) from the significant models indicate the platelet models were more associated with platelet mOCR. Conclusions: Our data suggest there are groups of metabolites in WB (leucine, acetylcarnitine) and platelets (creatine, ADP, glucose, taurine) that are associated with platelet mOCR. Thus, WB and platelet metabolites could be used to estimate platelet mOCR.

OBJECTIVE:High density lipoprotein (HDL) is important for defense against sepsis but becomes dysfunctional (Dys-HDL) during inflammation. We hypothesize that Dys-HDL correlates with organ dysfunction (sequential organ failure assessment (SOFA) score) early sepsis. METHODS:A prospective cohort study of adult ED sepsis patients enrolled within 24 hours. RESULTS:Eighty eight patients were analyzed. Dys-HDL (expressed as HDL inflammatory index (HII)) correlated with SOFA at enrollment (r = 0.23, p = 0.024) and at 48 hours (r = 0.24, p = 0.026) but HII change over the first 48 hours did not correlate with change in SOFA (r = 0.06, p = 0.56). Enrollment HII was significantly different in patients with most severe organ failure (2.31, IQR 1.33-5.2) compared to less severe organ failure (1.81, IQR 1.23-2.64, p = 0.043). Change in HII over 48 hours was significantly different for in-hospital non-survivors (-0.45, IQR-2.6, -0.14 p = 0.015) and for 28-day non-survivors (-1.12, IQR -1.52, 0.12, p = 0.044). In a multivariable linear regression equation (R2 = 0.13), for each unit HII increase, 48-hour SOFA increased by 0.72 (p = 0.009). CONCLUSION:HII correlated with SOFA and predicted 48-hour SOFA score in early sepsis. Future studies are needed to delineate potential mechanisms. TRIAL REGISTRATION:NCT02370186. Registered February 24, 2015.

Objective To describe the effect of liver disease (LD) on lactate clearance during early sepsis resuscitation. Methods This is a multicenter randomized clinical trial. An initial lactate >2 mmol/L and subsequent serum lactate measurement within 6 hours were required for inclusion. LD was categorized by two methods: 1) past medical history (PMH) categorized as no LD, mild LD (no Child’s score criteria, but PMH of hepatitis B/C), cirrhosis; and 2) measurable liver dysfunction determined by the liver component of the sequential organ failure assessment (L-SOFA) score as no dysfunction (L-SOFA score 0), mild dysfunction (score 1), moderate-severe dysfunction (score 2 to 4). Primary outcome was the rate of lactate normalization. Results One hundred eighty-seven patients were included. When categorized by PMH, 169 patients had no LD, 6 mild LD, and 12 cirrhosis. 63/169 (37%) of patients with no LD achieved lactate normalization, compared to 4/6 (67%) with mild LD, and 1/12 (8%) with cirrhosis (P

Objective: To externally validate the diagnostic and prognostic value of three previously identified microRNAs in emergency department patients with sepsis. Methods: Patients meeting consensus criteria for sepsis and septic shock were compared to controls. Three microRNAs (miR-150, miR-146a, and miR-223) were measured using real-time quantitative PCR, and levels of miRNAs were compared among the three cohorts. The association between miRNAs and both inflammatory markers and Sequential Organ Failure Assessment (SOFA) score were compared. To assess the prognostic value of each miRNA, unadjusted and adjusted logistic regression models were constructed using in-hospital mortality as the dependent variable. Results: Ninety-three patients were enrolled; 24 controls, 29 with sepsis, and 40 with septic shock. We found no difference in serum plasma miR-146a or miR-223 between cohorts, and found no association among these microRNAs and either inflammatory markers or SOFA score. miR-150 demonstrated a significant correlation with SOFA score (P= 0.31, P=0.01) and IL-10 (P=0.37, P=0.001), but no IL-6 or TNF-α (P=0.046, P=0.59). Logistic regression demonstrated miR-150 to be independently associated with mortality, even after adjusting for SOFA score (P=0.003) or initial lactate (P=0.01). Conclusions: miR-146a and miR-223 demonstrated no significantly diagnostic or prognostic ability in this cohort. miR-150 was associated with inflammation, severity of illness, and mortality. Given the independent predictive value of miR-150, additional research regarding its role in sepsis is warranted.

A cyclam (1,4,8,11-tetraazacyclotetradecane)-based macrocycle bearing two benzyl and two 2-hydroxy-3,5-di-tert-butylbenzyl pendent arms was synthesized and characterized using spectroscopic techniques and single crystal X-ray diffraction. The macrocycle crystallizes in the triclinic space group P-1, with the asymmetric unit containing one-half of the molecule. The structure is stabilized by hydrogen-bonding which exists between the phenolic protons and the nitrogen atoms of the macrocyclic ring. The presence of this hydrogen bonding is observed in the 1H-NMR due to the deshielded nature of the phenolic OH peak (δ 9.99). Cyclic voltammetry of the ligand revealed a single quasi-reversible peak at −0.58 V (Epc = −0.48 V and Epa = −0.68 V), which is due to the electrochemical oxidation of the phenol to the phenoxyl radical.

Heterotrimeric G proteins function in development, biotic, and abiotic stress responses, hormone signaling as well as sugar sensing. We previously proposed that discrimination of these various external signals in the G protein pathway is accomplished in plants by membrane-localized receptor-like kinases (RLKs) rather than G-protein-coupled receptors. Arabidopsis thaliana Regulator of G Signaling protein 1 (AtRGS1) modulates G protein activation and is phosphorylated by several RLKs and by WITH-NO-LYSINE kinases (WNKs). Here, a combination of in vitro kinase assays, mass spectrometry, and computational bioinformatics identified and functionally prioritized phosphorylation sites in AtRGS1. Phosphosites for two more RLKs (BRL3 and PEPR1) were identified and added to the AtRGS1 phosphorylation profile. Bioinformatics analyses revealed that RLKs and WNK kinases phosphorylate plant RGS proteins within regions that are conserved across eukaryotes and at a high frequency. Four phospho-sites among 14 identified are proximal to equivalent mammalian phosphosites that impact RGS function, including: pS437 and pT267 in GmRGS2, and pS339 and pS436 in AtRGS1. Based on these analyses, we propose that pS437 and pS436 regulate GmRGS2 and AtRGS1 protein interactions and/or localization, whereas pT267 is important for modulation of GmRGS2 GAP activity and localization. Moreover, pS339 most likely affects AtRGS1 activation.

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 >60% of a test set displayed in vivo interaction with XLG proteins. Gene co-expression analysis shows that >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.

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.

BACKGROUND:Arabidopsis, 7-transmembrane Regulator of G signaling protein 1 (AtRGS1) modulates canonical G protein signaling by promoting the inactive state of heterotrimeric G protein complex on the plasma membrane. It is known that plant leucine-rich repeat receptor-like kinases (LRR RLKs) phosphorylate AtRGS1 in vitro but little is known about the in vivo interaction, molecular dynamics, or the cellular consequences of this interaction. METHODS:Therefore, a subset of the known RLKs that phosphorylate AtRGS1 were selected for elucidation, namely, BAK1, BIR1, FLS2. Several microscopies for both static and dynamic protein-protein interactions were used to follow in vivo interactions between the RLKs and AtRGS1 after the presentation of the Pathogen-associated Molecular Pattern, Flagellin 22 (Flg22). These microscopies included Förster Resonance Energy Transfer, Bimolecular Fluoresence Complementation, and Cross Number and Brightness Fluorescence Correlation Spectroscopy. In addition, reactive oxygen species and calcium changes in living cells were quantitated using luminometry and R-GECO1 microscopy. RESULTS:The LRR RLKs BAK1 and BIR1, interact with AtRGS1 at the plasma membrane. The RLK ligand flg22 sets BAK1 in motion toward AtRGS1 and BIR1 away, both returning to the baseline orientations by 10 minutes. The C-terminal tail of AtRGS1 is important for the interaction with BAK1 and for the tempo of the AtRGS1/BIR1 dynamics. This window of time corresponds to the flg22-induced transient production of reactive oxygen species and calcium release which are both attenuated in the rgs1 and the bak1 null mutants. CONCLUSIONS:A temporal model of these interactions is proposed. flg22 binding induces nearly instantaneous dimerization between FLS2 and BAK1. Phosphorylated BAK1 interacts with and enables AtRGS1 to move away from BIR1 and AtRGS1 becomes phosphorylated leading to its endocytosis thus leading to de-repression by permitting AtGPA1 to exchange GDP for GTP. Finally, the G protein complex becomes dissociated thus AGB1 interacts with its effector proteins leading to changes in reactive oxygen species and calcium.

Plasma membrane-localized leucine-rich repeat receptor-like kinases directly activates G protein complex via interaction with seven transmembrane domain Regulator of G-protein Signaling 1 (AtRGS1) protein. Brassinosteroid insensitive 1 (BRI1) LIKE3 (BRL3) phosphorylates AtRGS1 in vitro. FRET analysis showed that BRL3 and AtRGS1 interaction dynamics change in response to glucose and flg22. Both BRL3 and AtRGS1 function in glucose sensing and brl3 and rgs1-2 single mutants are hyposensitive to high glucose as well as the brl3/rgs1 double mutant. BRL3 and AtRGS1 function in the same pathway linked to high glucose sensing. Hypocotyl elongation, another sugar-mediated pathway, is also implicated to be partially mediated by BRL3 and AtRGS1 because rgs1-2, brl3-2 and brl3-2/ rgs1-2 mutants share the long hypocotyl phenotype. BRL3 and AtRGS1 modulate the flg22-induced ROS burst and block one or more components positively regulating ROS production because the brl3/rgs1 double mutant has ~60% more ROS production than wild type while rgs1-2 has a partial ROS burst impairment and brl3 has slightly more ROS production. Here, we proposed a simple model where both BRL3 and AtRGS1 are part of a fine-tuning mechanism sensing glucose and flg22 to prevent excess ROS burst and control growth inhibition.

N-acyliminium ions are useful reactive synthetic intermediates in a variety of important carbon–carbon bond forming and cyclisation strategies in organic chemistry. The advent of an electrochemical anodic oxidation of unfunctionalised amides, more commonly known as the Shono oxidation, has provided a complementary route to the C–H activation of low reactivity intermediates. In this article, containing over 100 references, we highlight the development of the Shono-type oxidations from the original direct electrolysis methods, to the use of electroauxiliaries before arriving at indirect electrolysis methodologies. We also highlight new technologies and techniques applied to this area of electrosynthesis. We conclude with the use of this electrosynthetic approach to challenging syntheses of natural products and other complex structures for biological evaluation discussing recent technological developments in electroorganic techniques and future directions.