Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines

This protocol describes a highly reproducible antibody-based method that provides protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV-activated linking chemistry to detect changes in phosphorylation status. As an example application, we describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adaptor protein ​CrkL, a major substrate of the oncogenic tyrosine kinase ​BCR-​ABL in chronic myeloid leukemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5 pg/nl limit of protein detection (<0.2% of a stem cell sample containing <104 cells). Additional assays are described for phosphorylated tyrosine 207 (pTyr207)-​CrkL and the protein tyrosine phosphatase ​PTPRC/​CD45; these assays were developed using this protocol and applied to CML patient samples. This method is of high throughput, and it can act as a screen for in vitro cancer stem cell response to drugs and novel agents

Variance reduction in randomised trials by inverse probability weighting using the propensity score.

In individually randomised controlled trials, adjustment for baseline characteristics is often undertaken to increase precision of the treatment effect estimate. This is usually performed using covariate adjustment in outcome regression models. An alternative method of adjustment is to use inverse probability-of-treatment weighting (IPTW), on the basis of estimated propensity scores. We calculate the large-sample marginal variance of IPTW estimators of the mean difference for continuous outcomes, and risk difference, risk ratio or odds ratio for binary outcomes. We show that IPTW adjustment always increases the precision of the treatment effect estimate. For continuous outcomes, we demonstrate that the IPTW estimator has the same large-sample marginal variance as the standard analysis of covariance estimator. However, ignoring the estimation of the propensity score in the calculation of the variance leads to the erroneous conclusion that the IPTW treatment effect estimator has the same variance as an unadjusted estimator; thus, it is important to use a variance estimator that correctly takes into account the estimation of the propensity score. The IPTW approach has particular advantages when estimating risk differences or risk ratios. In this case, non-convergence of covariate-adjusted outcome regression models frequently occurs. Such problems can be circumvented by using the IPTW adjustment approach

Simulation of combustion noise of premixed flames in OpenFOAM

Advancements in combustion which have increased efficiency and reduced greenhouse gas emissions, such as premixed combustion and lean injection, have led to increased noise emission due to higher turbulence and mixing fluctuations. This study explored the viability of a hybrid CFD and acoustic analogy method for simulating noise in residential furnaces. This was done by exploring the validity of various chemical mechanisms to reduce computational time requirements, validating the acoustic analogy in enclosed spaces with an expansion chamber silencer, and finally testing the method on a laboratory scale furnace geometry. The WD2, SG35, Z42, DRM22 and GRI-3.0 combustion mechanisms were compared against the experimental data of the Sandia D Flame. The computational cost of these mechanisms had a power relation to the number reaction as predicted by theory, except for the global mechanism which performed worse than both the skeletal mechanisms. The libAcoustics library and Ffwocs-Williams Hawkings (FWH) acoustic analogy was validated for confined flow using the expansion chamber silencer and plane wave-theory. The calculated transmission loss compared well with the plane-wave theory and the sine-sweep method was determined to be an effective method for simulating the acoustic source. Larger time steps smoothed the sound pressure level (SPL) frequency spectrum, but this did not result in a significantly depressed the overall sound pressure level (OASPL). A laboratory scale furnace was built, and the SPL frequency spectrum was measured with SPL meter and microphones. The equivalence ratio and ramping the flow rate did not significantly alter the SPL spectrum or OASPL. However, the flow rate had a linear relationship with the SPL amplitude and peak frequencies. A simulation of the laboratory scale furnace was performed using both LES and URANS turbulence modeling. The LES-FWH simulation of the furnace predicted the combustion peak frequency and amplitude, and the OASPL. The URANS simulation underpredicted the amplitude of the SPL spectrum

The Clinical Frailty Scale can be used retrospectively to assess the frailty of patients with hip fracture:a validation study

PURPOSE: Frailty is a common clinical syndrome affecting hip fracture patients. Recognising and accurately assessing frailty status is important in clinical and research settings. The Rockwood Clinical Frailty Scale (CFS) is a commonly used instrument and demonstrates a strong correlation with mortality and length of hospital admission following hip fracture. What is not understood, however, is the validity of retrospectively assigned CFS scores in hip fracture patients. The aim of this study was to assess the validity of retrospective non-orthogeriatrician assigned CFS scores in hip fracture patients. METHODS: Hip fracture patients from a single major trauma centre were assessed and CFS scores were assigned prospectively by non-orthogeriatric clinicians (n = 57). A subset of these patients were also assigned a prospective CFS score by a specialist orthogeriatrician (n = 27). Two separate blinded observers (non-orthogeriatric clinicians) assigned CFS scores retrospectively using electronic patient records alone. Agreement and precision was examined using the Bland–Altman plot, accuracy was assessed using R(2) statistic and inter-rater reliability was assessed using quadratic weighted Cohen’s kappa. RESULTS: Seventy percent of the cohort were female with an average age of 83. Agreement was high between prospective non-orthogeriatrician assigned CFS scores and retrospective non-orthogeriatrician assigned CFS scores, with a low bias (0.046) and good accuracy (R(2) = 73%). Good agreement was also seen in comparisons between prospective orthogeriatrician assigned CFS scores versus retrospective non-orthogeriatrician assigned scores, with a low bias (0.23) and good accuracy (R(2) = 78%). Good inter-rater reliability was seen between blinded observers with a quadratic weighted Cohen’s kappa of 0.76. CONCLUSIONS: Retrospective CFS scores assigned by non-orthogeriatricians are a valid means of assessing frailty status in hip fracture patients. However, our results suggest a tendency for non-orthogeriatricians to marginally overestimate frailty status when assigning CFS scores retrospectively. LEVEL OF EVIDENCE: 3

Berezinskii-Kosterlitz-Thouless transitions in an easy-plane ferromagnetic superfluid

A two-dimensional (2D) spin-1 Bose gas exhibits two Berezenskii-Kosterlitz-Thouless (BKT) transitions in the easy-plane ferromagnetic phase. The higher temperature transition is associated with superfluidity of the mass current determined predominantly by a single spin component. The lower temperature transition is associated with superfluidity of the axial spin current, quasi-long range order of the transverse spin density and binding of polar-core spin vortices (PCVs). Above the spin BKT temperature, the component circulations that make up each PCV spatially separate, suggesting possible deconfinement analogous to quark deconfinement in high energy physics. Intercomponent interactions give rise to superfluid drag between the spin components, which we calculate analytically at zero temperature. We present the mass/spin superfluid phase diagram as a function of quadratic Zeeman energy $q$. At $q=0$ the system is in an isotropic spin phase with $\mathrm{SO}(3)$ symmetry. Here the fluid response exhibits a system size dependence, suggesting the absence of a BKT transition. Despite this, for finite systems the decay of spin correlations changes from exponential to algebraic as the temperature is decreased.Comment: 4 pages + refs, 3 figures. Interpretation of Fig. 3 results has changed since v