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Nuffield Department of Women's & Reproductive Health sits within the Medical Sciences Division of the University of Oxford. The department encompasses multi-disciplinary research across four overarching themes; Cancer, Global Health, Maternal & Fetal Health and Reproductive Medicine & Genetics
Differential regulation of HIF‐mediated pathways increases mitochondrial metabolism and ATP production in hypoxic osteoclasts
AbstractInappropriate osteoclast activity instigates pathological bone loss in rheumatoid arthritis. We have investigated how osteoclasts generate sufficient ATP for the energy‐intensive process of bone resorption in the hypoxic microenvironment associated with this rheumatic condition. We show that in human osteoclasts differentiated from CD14+ monocytes, hypoxia (24 h, 2% O2): (a) increases ATP production and mitochondrial electron transport chain activity (Alamar blue, O2 consumption); (b) increases glycolytic flux (glucose consumption, lactate production); and (c) increases glutamine consumption. We demonstrate that glucose, rather than glutamine, is necessary for the hypoxic increase in ATP production and also for cell survival in hypoxia. Using siRNA targeting specific isoforms of the hypoxia‐inducible transcription factor HIF (HIF‐1α, HIF‐2α), we show that employment of selected components of the HIF‐1α‐mediated metabolic switch to anaerobic respiration enables osteoclasts to rapidly increase ATP production in hypoxia, while at the same time compromising long‐term survival. We propose this atypical HIF‐driven metabolic pathway to be an adaptive mechanism to permit rapid bone resorption in the short term while ensuring curtailment of the process in the absence of re‐oxygenation. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Separation of Different Sized Nanoparticles with Time Using a Rotational Flow
In this paper, we describe the development of a microfluidic centrifuge with two inlets and two outlets potentially capable of rapidly separating nanoparticles and nanovesicles. Compared with the microfluidic centrifuge with a single inlet and outlet, the 2 ×2 microfluidic centrifuge gives improved centrifugation performance by increasing momentum flux transfer, angular velocity, and centrifugal acceleration. The center of flow rotation and the symmetry of the horizontal velocity in the microchamber were examined numerically. On the basis of the determined maximum velocity, the angular velocity and centrifugal acceleration were also evaluated. The centrifugation time of three different nanoparticles was examined by calculating the time when the nanoparticles left the microchamber for the first time. For visual observation and quantitative measurement of nanoparticle centrifugation, a 2 ×2 microfluidic centrifuge was fabricated and the experimental results demonstrate similar physical behavior to those of a mechanical centrifuge. On the basis of a comparison of the centrifugation time of two different nanoparticle populations of 300 and 700 nm in diameter, we propose that nanoparticles of different sizes can be physically separated by time under a range of inlet volume flow rates.
From whole body to cellular models of hepatic triglyceride metabolism: man has got to know his limitations
The liver is a main metabolic organ in the human body and carries out a vital role in lipid metabolism. Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, encompassing a spectrum of conditions from simple fatty liver (hepatic steatosis) through to cirrhosis. Although obesity is a known risk factor for hepatic steatosis, it remains unclear what factor(s) is/are responsible for the primary event leading to retention of intrahepatocellular fat. Studying hepatic processes and the etiology and progression of disease in vivo in humans is challenging, not least as NAFLD may take years to develop. We present here a review of experimental models and approaches that have been used to assess liver triglyceride metabolism and discuss their usefulness in helping to understand the aetiology and development of NAFLD.
Oxygen-sensing scaffolds for 3-dimensional cell and tissue culture
Porous membrane scaffolds are widely used materials for three-dimensional cell cultures and tissue models. Additional functional modification of such scaffolds can significantly extend their use and operational performance. Here we describe hybrid microporous polystyrene-based scaffolds impregnated with a phosphorescent O2-sensitive dye PtTFPP, optimized for live cell fluorescence microscopy and imaging of O2 distribution in cultured cells. Modified scaffolds possess high brightness, convenient spectral characteristics (534 nm excitation, 650 nm emission), stable and robust response to pO2 in phosphorescence intensity and lifetime imaging modes (twofold response over 21/0% O2), such as confocal PLIM. They are suitable for prolonged use under standard culturing conditions without affecting cell viability, and for multi-parametric imaging analysis of cultured cells and tissue samples. We tested the O2 scaffolds with cultured cancer cells (HCT116), multicellular aggregates (PC12) and rat brain slices and showed that they can inform on tissue oxygenation at different depths and cell densities, changes in respiration activity, viability and responses to drug treatment. Using this method multiplexed with staining of dead cells (CellTox Green) and active mitochondria (TMRM), we demonstrated that decreased O2 (20 24 lM) in scaffold corresponds to highest expression of tyrosine hydroxylase in PC12 cells. Such hypoxia is also beneficial for action of hypoxia-specific anti-cancer drug tirapazamine (TPZ). Thus, O2 scaffolds allow for better control of conditions in 3D tissue cultures, and are useful for a broad range of biomaterials and physiological studies.
Characterization of lipid metabolism in a novel immortalized human hepatocyte cell line
The development of hepatocyte cell models that represent fatty acid partitioning within the human liver would be beneficial for the study of the development and progression of nonalcoholic fatty liver disease (NAFLD). We sought to develop and characterize a novel human liver cell line (LIV0APOLY) to establish a model of lipid accumulation using a physiological mixture of fatty acids under low- and high-glucose conditions. LIV0APOLY cells were compared with a well-established cell line (HepG2) and, where possible, primary human hepatocytes. LIV0APOLY cells were found to proliferate and express some mature liver markers and were wild type for the PNPLA3 (rs738409) gene, whereas HepG2 cells carried the Ile148Met variant that is positively associated with liver fat content. Intracellular triglyceride content was higher in HepG2 than in LIV0APOLY cells; exposure to high glucose and/or exogenous fatty acids increased intracellular triglyceride in both cell lines. Triglyceride concentrations in media were higher from LIV0APOLY compared with HepG2 cells. Culturing LIV0APOLY cells in high glucose increased a marker of endoplasmic reticulum stress and attenuated insulin-stimulated Akt phosphorylation whereas low glucose and exogenous fatty acids increased AMPK phosphorylation. Although LIV0APOLY cells and primary hepatocytes stored similar amounts of exogenous fatty acids as triglyceride, more exogenous fatty acids were partitioned toward oxidation in the LIV0APOLY cells than in primary hepatocytes. LIV0APOLY cells offer the potential to be a renewable cellular model for studying the effects of exogenous metabolic substrates on fatty acid partitioning; however, their usefulness as a model of lipoprotein metabolism needs to be further explored.
Magnetophoretic velocities of superparamagnetic particles, agglomerates and complexes
A study into the magnetically induced mobility of four types of superparamagnetic particles (SMPs) was conducted using a video camera, an inverted light microscope and ImageJ tracking software. The objective is to improve the understanding of how SMP-capture assays perform by measuring mobilities of SMPs, when aggregated together or attached to non-magnetic beads (NMB). The magnetically induced velocities of self-assembled SMP chains were measured and found to meet the proposed models. A study into the zeta potential of the SMPs was completed to determine a scenario for maximal electrostatic interactions and efficient capture of the SMPs to a target. SMPs were bound to biotinylated NMBs, representing attachment to a disease biomarker. The drift velocity of SMP chains and SMP-NMB complexes in a gradient magnetic field was compared. It is expected that the observable changes to the magnetophoretic mobility of SMPs attached to a disease biomarker will lead to new biosensor technology.
The over-expression of cell migratory genes in alveolar rhabdomyosarcoma could contribute to metastatic spread
Alveolar (ARMS) and Embryonal (ERMS) rhabdomyosarcoma differ in their response to current treatments. The ARMS subtype has a less favourable prognosis and often presents with widespread metastases, while the less metastatic ERMS has a 5 year survival rate of more than 80 %. In this study we investigate gene expression differences that could contribute to the high frequency of metastasis in ARMS. Microarray analysis identified significant differences in DNA repair, cell cycle and cell migration between the two RMS subtypes. Two genes up regulated in ARMS and involved in cell migration; the engulfment and cell motility gene 1 (ELMO1) and NEL-like 1 gene (NELL1) were selected for further nvestigation. Over-expression of ELMO1 significantly increased cell invasion from 24.70 ± 7 % to 93 ± 5.4 % in primary myoblasts and from 29.43 ± 2.1 % to 87.33 ± 4.1 % in the ERMS cell line RD. siRNA knockout of ELMO1 in the ARMS cell line RH30 significantly reduced cell invasion from 88.2 ± 3.8 % to 35.2 ± 2.5 %. Over-expression of NELL1 significantly increased myoblast invasion from 23.6 ± 6.9 % to 100 ± 0.1 %, but had no effect on invasion of the ERMS cell line RD. These findings suggest that ELMO1 may play a key role in ARMS metastasis. NELL1 increased invasion in primary myoblasts, but other factors required for it to enhance motility were not present in the RD ERMS cell line. Impairing ELMO1 function by pharmacological or siRNA knockdown could be a highly effective approach to reduce the metastatic spread of RMS. © Springer Science+Business Media B.V. 2012.