Exploring the impact of varying definitions of exacerbations of chronic obstructive pulmonary disease in routinely collected electronic medical records

Background: Validity of exposure and outcome measures in electronic medical records is vital to ensure robust, comparable study findings however, despite validation studies, definitions of variables used often differ. Using exacerbations of chronic obstructive pulmonary disease (COPD) as an example, we investigated the impact of potential misclassification of different definitions commonly used in publications on study findings. Methods: A retrospective cohort study was performed. English primary care data from the Clinical Practice Research Datalink Aurum database with linked secondary care data were used to define a population of COPD patients ≥40 years old registered at a general practice. Index date was the date eligibility criteria were met and end of follow-up was 30/12/19, death or end of data collection. Exacerbations were defined using 6 algorithms based on definitions commonly used in the literature, including one validated definition. For each algorithm, the proportion of frequent exacerbators (≥2 exacerbations/year) and exacerbation rates were described. Cox proportional hazard regression was used to investigate each algorithm on the association between heart failure and risk of COPD exacerbation. Findings: A total of 315,184 patients were included. Baseline proportion of frequent exacerbators varied from 2.7% to 15.3% depending on the algorithm. Rates of exacerbations over follow-up varied from 19.3 to 66.6 events/100 person-years. The adjusted hazard ratio for the association between heart failure and exacerbation varied from 1.45, 95% confidence intervals 1.42–1.49, to 1.01, 0.98–1.04. Interpretation: The use of high validity definitions and standardisation of definitions in electronic medical records is crucial to generating high quality, robust evidence

The expression of P-glycoprotein does influence the distribution of novel fluorescent compounds in solid tumour models

Solid tumours display a complex drug resistance phenotype that involves inherent and acquired mechanisms. Multicellular resistance is an inherent feature of solid tumours and is known to present significant barriers to drug permeation in tumours. Given this barrier, do acquired resistance mechanisms such as P-glycoprotein (P-gp) contribute significantly to resistance? To address this question, the multicellular tumour spheroid (MCTS) model was used to examine the influence of P-gp on drug distribution in solid tissue. Tumour spheroids (TS) were generated from either drug-sensitive MCF7WT cells or a drug-resistant, P-gp-expressing derivative MCF7Adr. Confocal microscopy was used to measure time courses and distribution patterns of three fluorescent compounds; calcein-AM, rhodamine123 and BODIPY-taxol. These compounds were chosen because they are all substrates for P-gp-mediated transport, exhibit high fluorescence and are chemically dissimilar. For example, BODIPY-taxol and rhodamine 123 showed high accumulation and distributed extensively throughout the TSWT, whereas calcein-AM accumulation was restricted to the outermost layers. The presence of P-gp in TSAdr resulted in negligible accumulation, regardless of the compound. Moreover, the inhibition of P-gp by nicardipine restored intracellular accumulation and distribution patterns to levels observed in TSWT. The results demonstrate the effectiveness of P-gp in modulating drug distribution in solid tumour models. However, the penetration of agents throughout the tissue is strongly determined by the physico-chemical properties of the individual compounds

The seventies in Australia: the first decade of the 21 st century

While usually referred to as the ‘anything goes’ decade, the importance of the seventies in Australian art has been overlooked. From the perspective of the 21st century, the seventies appear to provide the link needed to connect the modern and the contemporary eras. This essay examines a few of the ideas and theories that made the seventies a presage to the art of the future

Relationship between COPD exacerbations and cardiovascular risk

Chronic obstructive pulmonary disease (COPD), is associated with an increased risk of myocardial infarction (MI), and additionally, cardiovascular disease is responsible for up to 1/3 of deaths in people with COPD. This may be attributable to the fact people with COPD are managed differently and have higher mortality after MI compared to people without COPD. One reason for the differences in management may be that prognostic risk scores after MI do not perform well in those with COPD. Another reason may be that acute exacerbations of COPD (AECOPD) are thought to be associated with a transiently increased risk of MI. The aims of this thesis are to: 1)systematically review the evidence for an increased risk of MI associated with COPD and AECOPD, and increased risk of death following MI for those with COPD; 2) investigate the potential contribution of differences in management after MI on differences in mortality; 3) investigate the performance of prognostic risk scores after MI for those with COPD; 4) validate the recording of AECOPD in UK electronic healthcare records (EHR); 5) investigate the recording of hospitalisations for AECOPD in UK primary and secondary care EHR; and 6) to conduct a self-controlled case series to investigate the risk of MI associated with AECOPD. This work showed an increased risk of MI associated with COPD independent of smoking, and evidence for an increased risk of death following hospital discharge for people with compared to those without COPD. This work demonstrated that differences in recognition and management of MI for those with COPD may explain some of the higher risk of death for COPD patients following MI. Additionally, the GRACE score (commonly used for risk stratification following MI) does not perform as well for COPD patients and may explain some of the differences in management. A validated algorithm was developed for identifying AECOPD both in primary care and resulting in hospital admission in electronic health records. Finally, using a self-controlled case series analysis, data showed that AECOPD is associated with increased risk of MI for approximately four weeks following AECOPD onset, and that the risk is modified by important patient characteristics

Functional expression of Multidrug Resistance Protein 4 MRP4/ABCC4

To study the function and structure of membrane proteins, high quantities of pure and stable protein are needed. One of the first hurdles in accomplishing this is expression of the membrane protein at high levels and in a functional state. Membrane proteins are naturally expressed at low levels, so finding a suitable host for overexpression is imperative. Multidrug resistance protein 4 (MRP4) or ATP-binding cassette subfamily C member 4 (ABCC4) is a multi-transmembrane protein that is able to transport a range of organic anionic compounds (both endogenous and xenobiotic) out of the cell. This versatile transporter has been linked with extracellular signaling pathways and cellular protection, along with conferring drug resistance in cancers. Here we report the use of MRP4 as a case study to be expressed in three different expression systems: mammalian, insect, and yeast cells, to gain the highest yield possible. Interestingly, using the baculovirus expression system with Sf9 insect cells produced the highest protein yields. Vesicular transport assays were used to confirm that MRP4 expressed in Sf9 was functional using a fluorescent cAMP analogue (fluo-cAMP) instead of the traditional radiolabeled substrates. MRP4 transported fluo-cAMP in an ATP-dependent manner. The specificity of functional expression of MRP4 was validated by the use of nonhydrolyzable ATP analogues and MRP4 inhibitor MK571. Functionally expressed MRP4 in Sf9 cells can now be used in downstream processes such as solubilization and purification in order to better understand its function and structure

Stabilization of human Multidrug Resistance Protein 4 (MRP4/ABCC4) using novel solubilization agents.

Membrane proteins (MPs) are important drug discovery targets for a wide range of diseases. However, elucidating the structure and function of native MP is notoriously challenging as their original structure has to be maintained once removed from the lipid bilayer. Conventionally, detergents have been used to solubilize MP with varying degrees of success concerning MP stability. To try to address this, new, more stabilizing agents have been developed, such as calixarene-based detergents and styrene–maleic acid (SMA) copolymer. Calixarene-based detergents exhibit enhanced solubilizing and stabilizing properties compared with conventional detergents, whereas SMA is able to extract MPs with their surrounding lipids, forming a nanodisc structure. Here we report a comparative study using classical detergents, calixarene-based detergents, and SMA to assess the solubilization and stabilization of the human ABC transporter MRP4 (multidrug resistance protein 4/ABCC4). We show that both SMA and calixarene-based detergents have a higher solubility efficiency (at least 80%) than conventional detergents, and show striking overstabilization features of MRP4 (up to 70 °C) with at least 30 °C stability improvement in comparison with the best conventional detergents. These solubilizing agents were successfully used to purify aggregate-free, homogenous and stable MRP4, with sevenfold higher yield for C4C7 calixarene detergent in comparison with SMA. This work paves the way to MRP4 structural and functional investigations and illustrates once more the high value of using calixarene-based detergent or SMA as versatile and efficient tools to study MP, and eventually enable drug discovery of challenging and highly druggable targets

Hsc70-induced changes in clathrin-auxilin cage structure suggest a role for clathrin light chains in cage disassembly

The molecular chaperone, Hsc70, together with its co-factor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin401-910 and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map, we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly

Roles of ABCC1 and ABCC4 in Proliferation and Migration of Breast Cancer Cell Lines

ABCC1 and ABCC4 utilize energy from ATP hydrolysis to transport many different molecules, including drugs, out of the cell and, as such, have been implicated in causing drug resistance. However recently, because of their ability to transport signaling molecules and inflammatory mediators, it has been proposed that ABCC1 and ABCC4 may play a role in the hallmarks of cancer development and progression, independent of their drug efflux capabilities. Breast cancer is the most common cancer affecting women. In this study, the aim was to investigate whether ABCC1 or ABCC4 play a role in the proliferation or migration of breast cancer cell lines MCF-7 (luminal-type, receptor-positive) and MDA-MB-231 (basal-type, triple-negative). The effects of small molecule inhibitors or siRNA-mediated knockdown of ABCC1 or ABCCC4 were measured. Colony formation assays were used to assess the clonogenic capacity, MTT assays to measure the proliferation, and scratch assays and Transwell assays to monitor the cellular migration. The results showed a role for ABCC1 in cellular proliferation, whilst ABCC4 appeared to be more important for cellular migration. ELISA studies implicated cAMP and/or sphingosine-1-phosphate efflux in the mechanism by which these transporters mediate their effects. However, this needs to be investigated further, as it is key to understand the mechanisms before they can be considered as targets for treatment