Accuracy of Emergency Department Clinical Findings for Diagnosis of Coronavirus Disease 2019.

STUDY OBJECTIVE: We seek to describe the medical history and clinical findings of patients attending the emergency department (ED) with suspected coronavirus disease 2019 (COVID-19) and estimate the diagnostic accuracy of patients' characteristics for predicting COVID-19. METHODS: We prospectively enrolled all patients tested for severe acute respiratory syndrome coronavirus 2 by reverse-transcriptase polymerase chain reaction in our ED from March 9, 2020, to April 4, 2020. We abstracted medical history, physical examination findings, and the clinical probability of COVID-19 (low, moderate, and high) rated by emergency physicians, depending on their clinical judgment. We assessed diagnostic accuracy of these characteristics for COVID-19 by calculating positive and negative likelihood ratios. RESULTS: We included 391 patients, of whom 225 had positive test results for severe acute respiratory syndrome coronavirus 2. Reverse-transcriptase polymerase chain reaction result was more likely to be negative when the emergency physician thought that clinical probability was low, and more likely to be positive when he or she thought that it was high. Patient-reported anosmia and the presence of bilateral B lines on lung ultrasonography had the highest positive likelihood ratio for the diagnosis of COVID-19 (7.58, 95% confidence interval [CI] 2.36 to 24.36; and 7.09, 95% CI 2.77 to 18.12, respectively). The absence of a high clinical probability determined by the emergency physician and the absence of bilateral B lines on lung ultrasonography had the lowest negative likelihood ratio for the diagnosis of COVID-19 (0.33, 95% CI 0.25 to 0.43; and 0.26, 95% CI 0.15 to 0.45, respectively). CONCLUSION: Anosmia, emergency physician estimate of high clinical probability, and bilateral B lines on lung ultrasonography increased the likelihood of identifying COVID-19 in patients presenting to the ED.

Nuclear magnetic resonance metabolic fingerprint of bevacizumab in mutant IDH1 glioma cells.

Background Malignant gliomas are rapidly growing tumours that extensively invade the brain and have bad prognosis. Our study was performed to assess the metabolic effects of bevacizumab on the glioma cells carrying the IDH1 mutation, a mutation, associated with better prognosis and treatment outcome. Bevacizumab is known to inhibit tumour growth by neutralizing the biological activity of vascular endothelial growth factor (VEGF). However, the direct effects of bevacizumab on tumour cells metabolism remain poorly known. Materials and methods The immunoassay and MTT assay were used to assess the concentration of secreted VEGF and cell viability after bevacizumab exposure. Metabolomic studies on cells were performed using high resolution magic angle spinning spectroscopy (HRMAS). Results mIDH1-U87 cells secreted VEGF (13 ng/mL). Regardless, bevacizumab had no cytotoxic effect, even after a 72h exposure and with doses as high as 1 mg/mL. Yet, HRMAS analysis showed a significant effect of bevacizumab (0.1 mg/mL) on the metabolic phenotype of mIDH1-U87 cells with elevation of 2-hydroxyglutarate and changes in glutamine group metabolites (alanine, glutamate, glycine) and lipids (polyunsaturated fatty acids [PUFA], glycerophosphocholine, and phosphocholine). Conclusions In mIDH1-U87 cells, changes in glutamine group metabolites and lipids were identified as metabolic markers of bevacizumab treatment. These data support the possibility of a functional tricarboxylic acid cycle that runs in reductive manner, as a probable mechanism of action of bevacizumab in IDH1 mutated gliomas and propose a new target pathway for effective treatment of malignant gliomas.

Caveolin-1 and doxorubicin-induced P-glycoprotein modulate plasma cholesterol membrane accessibility in erythrolymphoblastic cell line.

UNLABELLED: AIM/ BACKGROUND: Various interactions between Caveolae membrane domains, multidrug resistance transporter P-glycoprotein (P-gp) and cholesterol have been suggested. We tested the assumption that anthracycline-induced P-gp and Caveolin-1 have correlated effects on cholesterol distribution in plasma membrane. MATERIALS AND METHODS: The present study was performed in four lymphoblastic K562 cell lines expressing none (KS), one (Cav and KR cells) or both P-gp and caveolin-1 proteins (CavKR cells). RESULTS: The CavKR cell line exhibits a significantly higher free cholesterol content than the other cell lines. Cholesterol distribution at the outer leaflet was distinct from the total cellular cholesterol by its accessibility to cholesterol oxidase (COase). When cells were ATP-deprived, cholesterol accessibility to oxidation was significantly delayed in CavKR cells. Caveolin-1 or P-gp expression did not induce detectable changes in membrane cholesterol accessibility to COase. CONCLUSION: Combination of functional P-gp, caveolae presence and lasting effect of anthracycline treatment appear determinant in free membrane cholesterol homeostasis and likely modulate cholesterol membrane order.

Norepinephrine weaning in septic shock patients by closed loop control based on fuzzy logic.

INTRODUCTION: The rate of weaning of vasopressors drugs is usually an empirical choice made by the treating in critically ill patients. We applied fuzzy logic principles to modify intravenous norepinephrine (noradrenaline) infusion rates during norepinephrine infusion in septic patients in order to reduce the duration of shock. METHODS: Septic patients were randomly assigned to norepinephrine infused either at the clinician's discretion (control group) or under closed-loop control based on fuzzy logic (fuzzy group). The infusion rate changed automatically after analysis of mean arterial pressure in the fuzzy group. The primary end-point was time to cessation of norepinephrine. The secondary end-points were 28-day survival, total amount of norepinephine infused and duration of mechanical ventilation. RESULTS: Nineteen patients were randomly assigned to fuzzy group and 20 to control group. Weaning of norepinephrine was achieved in 18 of the 20 control patients and in all 19 fuzzy group patients. Median (interquartile range) duration of shock was significantly shorter in the fuzzy group than in the control group (28.5 [20.5 to 42] hours versus 57.5 [43.7 to 117.5] hours; P < 0.0001). There was no significant difference in duration of mechanical ventilation or survival at 28 days between the two groups. The median (interquartile range) total amount of norepinephrine infused during shock was significantly lower in the fuzzy group than in the control group (0.6 [0.2 to 1.0] microg/kg versus 1.4 [0.6 to 2.7] microg/kg; P < 0.01). CONCLUSIONS: Our study has shown a reduction in norepinephrine weaning duration in septic patients enrolled in the fuzzy group. We attribute this reduction to fuzzy control of norepinephrine infusion. TRIAL REGISTRATION: Trial registration: Clinicaltrials.gov NCT00763906.

Perturbation of membrane microdomains in GLC4 multidrug-resistant lung cancer cells--modification of ABCC1 (MRP1) localization and functionality.

The multidrug resistance-associated protein transporter ABCC1 (MRP1) is an integral plasma membrane protein involved in the multidrug resistance phenotype. It actively expels a number of cytotoxic molecules from cells. To gain insight into the modulation of the functional properties of this integral membrane protein by cholesterol, a main component of the lipid bilayer, we used multidrug-resistant GLC4/ADR cells, which overexpress MRP1. Upon altering the plasma membrane cholesterol content of these cells, membrane localization and the activity of MRP1 were analyzed. A detergent-free methodology was used to separate "light" and "heavy" plasma membrane fractions. Our data show that MRP1 was exclusively found in "light" fractions known as L0 phase membrane microdomains, together with 23% of gangliosides GM1 and 40% of caveolin-1. Depletion of the membrane cholesterol level to 40% by treatment with the cholesterol-chelating agent methyl-beta-cyclodextrin did not modify MRP1 activity, as evidenced either by the rate of efflux of pirarubicin or that of glutathione. Further cholesterol depletion below 40% yielded both a partial shift of MRP1 to the high-density fraction and a decrease of its functionality. Taken together, these data suggest that MRP1 functionality depends on its localization in cholesterol-rich membrane microdomains.

Impact of exogenous lysolipids on sensitive and multidrug resistant K562 cells: 1H NMR studies.

The ability of lysolipids to enter into a membrane bi-layer and disturb the membrane structure was used to study the behavior of K562 erythroleukemic cells, K562 wild type (K562wt) as well as the multidrug resistant cells K562adr. Both types of cells, when analyzed by proton NMR spectroscopy exhibit the high resolution signals assigned to so-called "mobile lipid" signals, which, in most cases, are located outside the lipid bi-layer as lipid droplets. In order to perform these studies, the K562wt and K562adr cells were treated for 48h with lysophosphatidylcholine oleoyl (LPC18), lysophosphatidylcholine palmitoyl (LPC16) and L-alpha-lysophosphatidyslerine (LPS). After evaluating toxicity of lysolipids, proton NMR of whole treated cells was used to analyze the mobile lipid content. Nile red staining and fluorescence microscopy were used to detect the presence of intracellular lipid droplets. Membrane lipid asymmetry perturbation was estimated by annexin V staining with use of flow cytometry. Using fluorescence spectroscopy the functioning of P-glycoprotein (P-gp) responsible for multidrug resistance was also evaluated after the treatment with lysolipids. Lysolipids were found to be more toxic for K562wt than for K562adr cells. LPS and LPC16 produced an increased of a mobile lipid NMR signal and amount of lipid droplets in K562wt cells only. LPC18, with the lowest toxicity, has shown more intense effects on NMR spectra with a large increase of lipid NMR signal without changes in lipid droplet staining. The functioning of the P-gp pump and membrane asymmetry were not modified by any of the lysolipids used.

Decrease of P-glycoprotein activity in K562/ADR cells by MbetaCD and filipin and lack of effect induced by cholesterol oxidase indicate that this transporter is not located in rafts.

The effect of low-density membrane domains on function of the plasma membrane transporter P-glycoprotéine (P-gp), involved in multidrug resistance (MDR) phenotype, has been investigated in K562/ADR cells. To this end we reversibly altered the cholesterol content of K562/ADR cells by using methyl-beta-cyclodextrin as a cholesterol chelator and conversely we repleted them through incubation with cholesterol in culture medium. We also used the cholesterol-binding fluorochrome filipin and cholesterol oxidase. Our data show that either cholesterol depletion or complex formation with filipin resulted in a strong decrease of P-gp activity. However, when cells were incubated with cholesterol oxidase that are known to disrupt rafts, no modification of the P-gp activity was observed. In addition, using a free-detergent methodology to separate by ultracentrifugation, "light," "heavy," and "extra heavy" fractions we show that no P-gp is found in the "light" fraction where rafts are usually detected. Altogether, our data strongly suggest that, in this cell line, P-gp is not localized in rafts.

New insights into the P-glycoprotein-mediated effluxes of rhodamines.

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma drug transporter P-glycoprotein (P-gp). This protein is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Despite the advances of recent years, we still have an unclear view of the molecular mechanism by which P-gp transports such a wide diversity of compounds across the membrane. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. The aim of the present study was to further characterize the transport of several rhodamine analogues, either positively charged or zwitterionic. We took advantage of the intrinsic fluorescence of rhodamines and performed a flow-cytometric analysis of dye accumulation in the wild-type drug sensitive K562 that do not express P-gp and its MDR subline that display high levels of MDR. The measurements were made in real time using intact cells. The kinetic parameter, ka = VM/km, which is a measure of the efficiency of the P-gp-mediated efflux of a substrate was similar for almost all the rhodamine analogues tested. In addition these values were compared with those determined previously for the P-gp-mediated efflux of anthracycline. Our conclusion is that the compounds of these two classes of molecules, anthracyclines and rhodamines, are substrates of P-gp and that their pumping rates at limiting low substrate concentration are similar. The findings presented here are the first to show quantitative information about the kinetic parameters for P-gp-mediated efflux of rhodamine analogues in intact cells.