Initial chest radiograph scores inform COVID-19 status, intensive care unit admission and need for mechanical ventilation.

AIM: To evaluate whether portable chest radiography (CXR) scores are associated with coronavirus disease 2019 (COVID-19) status and various clinical outcomes. MATERIALS AND METHODS: This retrospective study included 500 initial CXR from COVID-19-suspected patients. Each CXR was scored based on geographic extent and degree of opacity as indicators of disease severity. COVID-19 status and clinical outcomes including intensive care unit (ICU) admission, mechanical ventilation, mortality, length of hospitalisation, and duration on ventilator were collected. Multivariable logistic regression analysis was performed to evaluate the relationship between CXR scores and COVID-19 status, CXR scores and clinical outcomes, adjusted for code status, age, gender and co-morbidities. RESULTS: The interclass correlation coefficients amongst raters were 0.94 and 0.90 for the extent score and opacity score, respectively. CXR scores were significantly (p < 0.01) associated with COVID-19 positivity (odd ratio [OR] = 1.49; 95% confidence interval [CI]: 1.27 - 1.75 for extent score and OR = 1.75; 95% CI: 1.42 - 2.15 for opacity score), ICU admission (OR = 1.19; 95% CI: 1.09 - 1.31 for extent score and OR = 1.26; 95% CI: 1.10 - 1.44 for opacity score), and invasive mechanical ventilation (OR = 1.22; 95% CI: 1.11 - 1.35 for geographic score and OR = 1.21; 95% CI: 1.05 - 1.38 for opacity score). CXR scores were not significantly different between survivors and non-survivors after adjusting for code status (p>0.05). CXR scores were not associated with length of hospitalisation or duration on ventilation (p>0.05). CONCLUSIONS: Initial CXR scores have prognostic value and are associated with COVID-19 positivity, ICU admission, and mechanical ventilation.

Implementation of influenza point-of-care testing and patient cohorting during a high-incidence season: a retrospective analysis of impact on infection prevention and control and clinical outcomes.

BACKGROUND: During high-incidence influenza seasons, a robust infection prevention and control policy is imperative to reduce nosocomial transmission of influenza. AIM: To assess the impact of influenza point-of-care testing (POCT) in an emergency department (ED) and patient cohorting on an influenza ward on infection prevention and control and clinical outcomes. METHODS: Influenza POCT was operational in the study ED from 21st January 2018 and patient cohorting was operational on an influenza ward from 25th January 2018. A retrospective 'before-after' analysis was performed with pre-intervention defined as 1st November 2017 to 20th January 2018 and post-intervention defined as 21st January 2018 to 30th April 2018. The primary outcome was the rate of hospital-acquired influenza. Secondary outcomes included antiviral prescription and length of stay. The length of time that inpatients remained influenza-positive was estimated by polymerase chain reaction (PCR). FINDINGS: There were 654 inpatients with confirmed influenza during the 2017/18 influenza season: 223 pre- and 431 post-intervention. Post-intervention, there were fewer cases of hospital-acquired influenza per day (0.66 vs 0.95, P < 0.0001), median length of stay was shorter (5.5 vs 7.5 days, P = 0.005) and antiviral prescription was more frequent (80% vs 64.1%, P < 0.0001). Cohorting released 779 single rooms for use elsewhere in the trust. The fixed probability of being PCR-negative by the next day (P) was 0.14 [95% confidence interval (CI) 0.12-0.16] for immunocompetent patients. This implies that half of immunocompetent patients are PCR-negative by five days post-diagnosis (95% CI 5-6). CONCLUSION: Influenza POCT in an ED and patient cohorting on an influenza ward were associated with reduced nosocomial transmission of influenza and improved patient flow. A policy of retesting immunocompetent patients five days post-diagnosis could allow half of these patients to come out of respiratory isolation earlier.

Implementation of the cobas Liat influenza point-of-care test into an emergency department during a high-incidence season: a retrospective evaluation following real-world implementation.

The cobas Liat influenza A/B and respiratory syncytial virus (RSV) assay (Liat) was used in the adult emergency department of a large London hospital from 21st January 2018 to 14th April 2018. Influenza was detected in 308 of 1027 (30%) samples tested; influenza A in 157 (15.3%), influenza B in 149 (14.5%) and RSV in 28 (2.7%). When compared against Fast Track Diagnostics Respiratory Pathogens 21 multiplex polymerase chain reaction and Cepheid Xpert Xpress Flu/RSV assay, Liat performance for the detection of influenza A or B was: sensitivity 85% [95% confidence interval (CI) 76-92)], specificity 98% (95% CI 97-99), negative predictive value 94% (95% CI 92-96) and positive predictive value 95% (95% CI 91-97).

Bismuthene on a SiC substrate: A candidate for a high-temperature quantum spin Hall material.

Quantum spin Hall materials hold the promise of revolutionary devices with dissipationless spin currents but have required cryogenic temperatures owing to small energy gaps. Here we show theoretically that a room-temperature regime with a large energy gap may be achievable within a paradigm that exploits the atomic spin-orbit coupling. The concept is based on a substrate-supported monolayer of a high-atomic number element and is experimentally realized as a bismuth honeycomb lattice on top of the insulating silicon carbide substrate SiC(0001). Using scanning tunneling spectroscopy, we detect a gap of ~0.8 electron volt and conductive edge states consistent with theory. Our combined theoretical and experimental results demonstrate a concept for a quantum spin Hall wide-gap scenario, where the chemical potential resides in the global system gap, ensuring robust edge conductance.

Correlation of Bandgap Reduction with Inversion Response in (Si)GeSn/High-k/Metal Stacks.

The bandgap tunability of (Si)GeSn group IV semiconductors opens a new era in Si-technology. Depending on the Si/Sn contents, direct and indirect bandgaps in the range of 0.4-0.8 eV can be obtained, offering a broad spectrum of both photonic and low power electronic applications. In this work, we systematically studied capacitance-voltage characteristics of high-k/metal gate stacks formed on GeSn and SiGeSn alloys with Sn-contents ranging from 0 to 14 at. % and Si-contents from 0 to 10 at. % particularly focusing on the minority carrier inversion response. A clear correlation between the Sn-induced shrinkage of the bandgap energy and enhanced minority carrier response was confirmed using temperature and frequency dependent capacitance voltage-measurements, in good agreement with k.p theory predictions and photoluminescence measurements of the analyzed epilayers as reported earlier. The enhanced minority generation rate for higher Sn-contents can be firmly linked to the bandgap reduction in the GeSn epilayer without significant influence of substrate/interface effects. It thus offers a unique possibility to analyze intrinsic defects in (Si)GeSn epilayers. The extracted dominant defect level for minority carrier inversion lies approximately 0.4 eV above the valence band edge in the studied Sn-content range (0-12.5 at. %). This finding is of critical importance since it shows that the presence of Sn by itself does not impair the minority carrier lifetime. Therefore, the continuous improvement of (Si)GeSn material quality should yield longer nonradiative recombination times which are required for the fabrication of efficient light detectors and to obtain room temperature lasing action.

Low Temperature Deposition of High-k/Metal Gate Stacks on High-Sn Content (Si)GeSn-Alloys.

(Si)GeSn is an emerging group IV alloy system offering new exciting properties, with great potential for low power electronics due to the fundamental direct band gap and prospects as high mobility material. In this Article, we present a systematic study of HfO2/TaN high-k/metal gate stacks on (Si)GeSn ternary alloys and low temperature processes for large scale integration of Sn based alloys. Our investigations indicate that SiGeSn ternaries show enhanced thermal stability compared to GeSn binaries, allowing the use of the existing Si technology. Despite the multielemental interface and large Sn content of up to 14 atom %, the HfO2/(Si)GeSn capacitors show small frequency dispersion and stretch-out. The formed TaN/HfO2/(Si)GeSn capacitors present a low leakage current of 2 × 10(-8) A/cm(2) at -1 V and a high breakdown field of ∼8 MV/cm. For large Sn content SiGeSn/GeSn direct band gap heterostructures, process temperatures below 350 °C are required for integration. We developed an atomic vapor deposition process for TaN metal gate on HfO2 high-k dielectric and validated it by resistivity as well as temperature and frequency dependent capacitance-voltage measurements of capacitors on SiGeSn and GeSn. The densities of interface traps are deduced to be in the low 10(12) cm(-2) eV(-1) range and do not depend on the Sn-concentration. The new processes developed here are compatible with (Si)GeSn integration in large scale applications.

GS-9857 in patients with chronic hepatitis C virus genotype 1-4 infection: a randomized, double-blind, dose-ranging phase 1 study.

GS-9857, an inhibitor of the hepatitis C virus (HCV) nonstructural protein (NS) 3/4A, demonstrates potent activity against HCV genotypes 1-6 and improved coverage against commonly encountered NS3 resistance-associated variants (RAVs). In this study, the safety, tolerability, antiviral activity and pharmacokinetics (PK) of GS-9857 were evaluated in patients with chronic HCV genotype 1-4 infection. Patients with genotype 1-4 infection received placebo or once-daily GS-9857 at doses ranging from 50 to 300 mg for 3 days under fasting conditions. GS-9857 was well tolerated; all reported adverse events (AEs) were mild or moderate in severity. Diarrhoea and headache were the most commonly reported AEs. Grade 3 or 4 laboratory abnormalities were observed in 17% of patients receiving GS-9857; there were no Grade 3 or 4 abnormalities in alanine aminotransferase, aspartate aminotransferase or alkaline phosphatase levels. GS-9857 demonstrated potent antiviral activity in patients with chronic HCV infection, achieving mean and median maximum reductions in HCV RNA of ≥3 log10 IU/mL following administration of a 100-mg dose in patients with HCV genotype 1a, 1b, 2, 3 or 4 infection. The antiviral activity of GS-9857 was unaffected by the presence of pretreatment NS3 RAVs. In patients with genotype 1-4 infection, GS-9857 exhibited linear PK and was associated with a median half-life of 29-42 h, supporting once-daily dosing. Thus, the tolerability, efficacy and pharmacokinetic profile of GS-9857 support its further evaluation for treatment of patients with chronic HCV infection.

Carboplatin/taxane-induced gastrointestinal toxicity: a pharmacogenomics study on the SCOTROC1 trial.

Carboplatin/taxane combination is first-line therapy for ovarian cancer. However, patients can encounter treatment delays, impaired quality of life, even death because of chemotherapy-induced gastrointestinal (GI) toxicity. A candidate gene study was conducted to assess potential association of genetic variants with GI toxicity in 808 patients who received carboplatin/taxane in the Scottish Randomized Trial in Ovarian Cancer 1 (SCOTROC1). Patients were randomized into discovery and validation cohorts consisting of 404 patients each. Clinical covariates and genetic variants associated with grade III/IV GI toxicity in discovery cohort were evaluated in replication cohort. Chemotherapy-induced GI toxicity was significantly associated with seven single-nucleotide polymorphisms in the ATP7B, GSR, VEGFA and SCN10A genes. Patients with risk genotypes were at 1.53 to 18.01 higher odds to develop carboplatin/taxane-induced GI toxicity (P<0.01). Variants in the VEGF gene were marginally associated with survival time. Our data provide potential targets for modulation/inhibition of GI toxicity in ovarian cancer patients.

Triangular Spin-Orbit-Coupled Lattice with Strong Coulomb Correlations: Sn Atoms on a SiC(0001) Substrate.

Two-dimensional (2D) atom lattices provide model setups with Coulomb correlations that induce competing ground states. Here, SiC emerges as a wide-gap substrate with reduced screening. We report the first artificial high-Z atom lattice on SiC(0001) by Sn adatoms, based on experimental realization and theoretical modeling. Density-functional theory of our triangular structure model closely reproduces the scanning tunneling microscopy. Photoemission data show a deeply gapped state (∼2 eV gap), and, based on our calculations including dynamic mean-field theory, we argue that this reflects a pronounced Mott-insulating scenario. We also find indications that the system is susceptible to antiferromagnetic superstructures. Such artificial lattices on SiC(0001) thus offer a novel platform for coexisting Coulomb correlations and spin-orbit coupling, with bearing for unusual magnetic phases and proposed topological quantum states of matter.

EphrinB3 blocks EphB3 dependence receptor functions to prevent cell death following traumatic brain injury.

Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, have a variety of roles in the developing and adult central nervous system that require direct cell-cell interactions; including regulating axon path finding, cell proliferation, migration and synaptic plasticity. Recently, we identified a novel pro-survival role for ephrins in the adult subventricular zone, where ephrinB3 blocks Eph-mediated cell death during adult neurogenesis. Here, we examined whether EphB3 mediates cell death in the adult forebrain following traumatic brain injury and whether ephrinB3 infusion could limit this effect. We show that EphB3 co-labels with microtubule-associated protein 2-positive neurons in the adult cortex and is closely associated with ephrinB3 ligand, which is reduced following controlled cortical impact (CCI) injury. In the complete absence of EphB3 (EphB3(-/-)), we observed reduced terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL), and functional improvements in motor deficits after CCI injury as compared with wild-type and ephrinB3(-/-) mice. We also demonstrated that EphB3 exhibits dependence receptor characteristics as it is cleaved by caspases and induces cell death, which is not observed in the presence of ephrinB3. Following trauma, infusion of pre-clustered ephrinB3-Fc molecules (eB3-Fc) into the contralateral ventricle reduced cortical infarct volume and TUNEL staining in the cortex, dentate gyrus and CA3 hippocampus of wild-type and ephrinB3(-/-) mice, but not EphB3(-/-) mice. Similarly, application of eB3-Fc improved motor functions after CCI injury. We conclude that EphB3 mediates cell death in the adult cortex through a novel dependence receptor-mediated cell death mechanism in the injured adult cortex and is attenuated following ephrinB3 stimulation.

Effects of a learning trial on self-regulation of exercise.

Heart rate (HR) and ratings of perceived exertion (RPE) are common exercise intensity regulatory strategies, however, some individuals are unable to use these strategies effectively. Alternative or conjunctive strategies may aid in the transition to self-guided programs. The purpose of the present study was to examine the value of a brief, field-based exercise intensity learning trial on self-regulation of intensity during a weeklong exercise program. Forty-two males were randomly assigned to either a paced learning trial (P) or non-paced control (NP), and then one of three intensity feedback groups: HR, HR + RPE, or no feedback (CONT). The paced trial consisted of an 800-m trial at 75 % of maximal heart rate reserve (MHRR) on day one. Subjects then completed four 800-m trials each day for four days and received feedback on deviation from target HR (THR) after each 800-m trial. Four-way MANOVA (pacing x feedback x trials x days) was used to assess the influence of the learning trial on THR deviation scores across the week. The pacing x feedback x trials interaction was significant (Pillai's Trace = 0.36, approximately F (6,70) = 2.56, p = 0.03) and thus, the influence of the learning trial was assessed within feedback group. There were no significant differences in THR deviation scores for P vs. NP within the HR or HR + RPE feedback groups. However, P was significantly more accurate then NP (p < 0.05) within the CONT feedback groups during each trial averaged across the week (T (1) = - 2.6 vs. 5.3; T (2) = 2.6 vs. 14.2; T (3) = 4.6 vs. 16.2; T (4) = 5.3 vs. 20.5 beats . min (-1)). These results demonstrate that a brief intensity learning trial, in the absence of HR or HR+RPE feedback, provided for accurate self-regulation of vigorous exercise training. These results would support the efficacy of a brief intensity learning trial within the context of transitioning an individual to a self-guided exercise program.

The effects of intravenous tobramycin on renal tubular function in children with cystic fibrosis.

BACKGROUND: Tobramycin, used to treat respiratory exacerbations in cystic fibrosis (CF), is also a renal tubular toxin. Tubular dysfunction leads to increased urinary levels of the proximal tubular lysosomal enzyme, N-acetyl-beta-D-glucosaminidase (NAG) and the proximal tubular protein, retinol-binding protein (RBP). Hypermagnesuria and resulting hypomagnesaemia are indicative of more severe tubular damage, occasionally seen following repeated courses of intravenous tobramycin. Using these biochemical markers we studied the effect of a 2-week course of this agent on tubular function. METHODS: Twenty-two children (11 boys) with CF were studied. Median age = 10.9 years, range 3.1-16.4 years. All had a normal predicted glomerular filtration rate (pGFR). They received tobramycin 3 mg/kg/dose tds. Urinary NAG, RBP, creatinine and plasma magnesium and creatinine were assayed: a) immediately before commencing tobramycin, b) immediately following the course, c) 4 weeks after the end of the course. RESULTS: Mean log UrNAG and UrRBP rose significantly between time points a) and b) before falling to almost pre-treatment levels by time c). Using two way ANOVA analysis the results for UrNAG and UrRBP were both highly statistically significant (p<0.0001). Paired t-tests on the logged values revealed highly significant differences between all time points for UrNAG and in the case of UrRBP for all other than a) compared to c). In all patients plasma magnesium and pGFR remained within normal limits. CONCLUSIONS: Intravenous tobramycin produces acute tubular injury, which showed evidence of almost complete recovery after 4 weeks. The insult to the tubules was not sufficient to produce hypomagnesaemia in our study group. To assess cumulative tubular damage in more detail it would be necessary to repeat this study after further courses of tobramycin. We recommend monitoring plasma magnesium during courses of intravenous tobramycin.

Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors.

Specific binding of the anticoagulants heparin and antithrombin III to the blood clotting cascade factor human thrombin was recorded as a function of time with a Love-wave biosensor array consisting of five sensor elements. Two of the sensor elements were used as references. Three sensor elements were coated with RNA or DNA aptamers for specific binding of human thrombin. The affinity between the aptamers and thrombin, measured using the biosensor, was within the same range as the value of K(D) measured by filter binding experiments. Consecutive binding of the thrombin inhibitors heparin, antithrombin III or the heparin-antithrombin III complex to the immobilized thrombin molecules, and binding of a ternary complex of heparin, anithrombin III, and thrombin to aptamers was evaluated. The experiments showed attenuation of binding to thrombin due to heparin-antithrombin III complex formation. Binding of heparin activated the formation of the inhibitory complex of antithrombin III with thrombin about 2.7-fold. Binding of the DNA aptamer to exosite II appeared to inhibit heparin binding to exosite I.

Continuous monitoring of Mg oxidation by internal exoemission.

Thin-film Mg/Si(111) Schottky diodes are exposed to oxygen to detect chemicurrents in the devices. The detected charge is created by nonadiabatic energy dissipation and due to either internal exoemission currents or surface chemiluminescence induced photocurrents. Both contributions can be distinguished by changing the metal film thickness of the device. Auger electron spectroscopy to study the oxygen uptake demonstrates that the chemicurrent transients represent truly the time dependent reaction rate at the surface. Model calculations indicate that the current monitors Mg oxide island nucleation and growth.

Determination of olanzapine in human blood by liquid chromatography-tandem mass spectrometry.

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) assay was developed and validated to quantitatively determine olanzapine (OLZ) concentrations in human blood. Liquid-liquid extraction, using n-butanol:cyclohexane (3:47, v/v), was used to isolate OLZ and its internal standard, LY170158, from the biological matrix. Chromatographic resolution of OLZ from endogenous interferences and known metabolites was accomplished with a MetaChem Monochrom HPLC column (4.6 x 150 mm, d(p) 5 microm). Detection occurred using a Perkin-Elmer Sciex API III Plus triple quadrupole mass spectrometer using positive ion APCI and multiple reaction monitoring (MRM). The linear dynamic range was from 5 to 500 ng ml(-1) based on a 0.25-ml aliquot of human blood. The inter-day precision (%RSD) and accuracy (%RE) ranged from 3.65 to 10.64 and from -2.14 to 3.07, respectively. Modifications to an existing assay for the determination of OLZ in human plasma were necessary. A different structural analog was used as the internal standard due to instability observed for the original analog when using human blood as the matrix. A second modification was the addition of the anti-oxidant sodium ascorbate to inhibit degradation of OLZ in human blood, as has been noted by other investigators. Upon fortification of human blood with sodium ascorbate (final concentration, 0.33 mM), OLZ was found to be stable for at least 1 week at -70 degrees C as well as through two freeze-thaw cycles. This assay, which will be used to investigate the distribution of OLZ in human blood, grants insight into the proper sample handling conditions needed to perform valid determinations of OLZ in human blood.

Preferred exertion across three common modes of exercise training.

The use of self-selected intensities of exercise may increase adherence to exercise programs by allowing the participant more freedom to choose activities that are enjoyable. However, self-selected intensities may vary across exercise modes, and participants may not choose an intensity that is adequate to produce health benefits. The purpose of this study was to determine influence of exercise mode on self-selected exercise intensities. Eighteen subjects (12 men and 6 women) between the ages of 18 and 25 participated in this study. Preferred intensity tests were performed for 3 modes of exercise (treadmill, cycle ergometer, and stairstepper). .V(O)(2) values were obtained continuously and 1-minute averages were recorded at minutes 5, 10, 15, and 20 for each submaximal test. Comparisons were made using a repeated-measures analysis of variance (mode, time, mode x time). Scheffe's F test was used to test simple effects. There was a significant increase in the relative .V(O)(2) for all 3 modes of exercise across the 20-minute trials (p = 0.0001). Relative .V(O)(2) (%.V(O)(2)peak) increased from 52.20 to 64.71% for cycle exercise, 43.27 to 63.25% for the treadmill, and 47.16 to 61.17% for stairstepping. The average relative .V(O)(2) for the cycle ergometer (60.40 +/- 15.55%) was significantly higher (p = 0.02) than both the treadmill (53.65 +/- 18.36%) and stairstepper (54.66 +/- 11.98%). Relative heart rate (HR) (%HRR) for the stairstepper (80.21 +/- 9.67%) and the cycle ergometer (80.03 +/- 10.59%) were significantly higher than the treadmill (74.77 +/- 13.13%) (p = 0.0003). There were no significant differences in rating of perceived exertion (RPE) among the 3 modes of exercise. Similar RPE values were reported for the stairstepper (12.79 +/- 2.97), cycle ergometer (12.57 +/- 2.90), and the treadmill (12.50 +/- 2.87). The results indicate that subjects allowed to choose exercise intensity by self-selection chose work rates that were within the moderate range of American College of Sports Medicine guidelines of 50-85% .V(O)(2)max for treadmill, cycle ergometer, and stairstepping exercise.

Characterization of partially sintered ceramic powder compacts using fluorinated gas NMR imaging.

We use nuclear magnetic resonance (NMR) imaging of C2F6 gas to characterize porosity, mean pore size, and permeability of partially sintered ceramic (Y-TZP Yttria-stabilized tetragonal-zirconia polycrystal) samples. Conventional measurements of these parameters gave porosity values from 0.18 to 0.4, mean pore sizes from 10 nm to 40 nm, and permeability from 4 nm(2) to 25 nm(2). The NMR methods are based on relaxation time measurements (T(1)) and the time dependent diffusion coefficient D(Delta). The relaxation time of C2F6 gas is longer in pores than in bulk gas and it increases as the pore sizes decrease. NMR yielded accurate porosity values after correcting for surface adsorption effects. A model for T(1) dependence on pore size that accounts for collisions between gas molecules and walls as well as surface adsorption effects is proposed. The model fits the experimental data well. Finally, the long time limit of D(Delta)/D(o), where D(o) is the bulk gas diffusion coefficient is useful for measuring tortuosity, while the short time limit was not achieved experimentally and could not be used for calculating surface-area to volume (S/V) ratios.

Development of a ubiquitin transfer assay for high throughput screening by fluorescence resonance energy transfer.

An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu(3+), and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc). When Rsc is ubiquitinated, Eu(3+) and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wavelength of APC, almost entirely free of nonspecific fluorescence from Eu(3+) and APC. The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 microg/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening.

SCF(beta-TRCP) and phosphorylation dependent ubiquitinationof I kappa B alpha catalyzed by Ubc3 and Ubc4.

NF kappa B is an important transcriptional regulator of multiple pro-inflammatory genes. In non-stimulated cells NF kappa B is anchored in the cytoplasm via the inhibitory protein I kappa B alpha. Following exposure to diverse pro-inflammatory signals (e.g. TNF alpha, IL1, LPS) various signal transduction cascades are initiated converging on the I kappa B kinase (IKK). IKK phosphorylates I kappa B alpha on serines 32 and 36 signaling the inhibitory protein for ubiquitin-mediated degradation. The SCF beta-TRCP complex is the ubiquitin ligase responsible for mediating phosphorylation dependent ubiquitination of I kappa B alpha. Here we reconstitute phosphorylation dependent ubiquitination of I kappa B alpha using recombinant components. Our results suggest that the cullin specificity of the SCF complex may reflect its ability to associate with Rbx1. We demonstrate specific ubiquitination of I kappa B alpha by Ubc3 and Ubc4 in a phosphorylation and SCF beta-TRCP dependent manner and that both are capable of associating with the SCF beta-TRCP complex isolated from human cells. Finally, we show that Ubc4 is in excess to Ubc3 in THP.1 cells and 19 times more efficient in catalyzing the reaction, suggesting that Ubc4 is the preferentially used Ubc in this reaction in vivo. Our results also suggest that ubiquitin is transferred directly from the Ubc to phospho-I kappa B alpha in a SCF beta-TRCP dependent reaction. Oncogene (2000) 19, 3529 - 3536