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Introduction
Current institution
Additional affiliations
October 2016 - present
Position
- PhD Student
Description
- Surface analysis of cholesterol metabolic flux in intact tissue
January 2013 - December 2016
Education
September 2008 - August 2011
Publications
Publications (65)
Disability worsening in multiple sclerosis (MS) is linked to neurodegeneration. Cholesterol homeostasis is essential for normal brain function. CYP46A1, crucial for brain cholesterol turnover and reduced in some neurodegenerative diseases, is a potential neuroprotective target. We hypothesized that CYP46A1 is downregulated in MS brains and linked t...
Mass spectrometry imaging (MSI) is a new technique in the toolbox of the analytical biochemist. It allows the generation of a compound-specific image from a tissue slice where a measure of compound abundance is given pixel by pixel, usually displayed on a color scale. As mass spectra are recorded at each pixel, the data can be interrogated to gener...
Dysregulated cholesterol metabolism has been linked to neurodegeneration. We previously found that free, non-esterified, 7α,(25R)26-dihydroxycholesterol (7α,26-diHC), was significantly elevated in the cerebrospinal fluid of patients with Parkinson’s disease (PD). In this study we investigated the role of 7α,26-diHC in midbrain dopamine (mDA) neuron...
Most biologically active oxysterols have a 3β-hydroxy-5-ene function in the ring system with an additional site of oxidation at C-7 or on the side-chain. In blood plasma oxysterols with a 7α-hydroxy group are also observed with the alternative 3-oxo-4-ene function in the ring system formed by ubiquitously expressed 3β-hydroxy-Δ5-C27-steroid oxidore...
Sterol lipids were one of the first categories of biomolecules to be investigated using mass spectrometry. They represent a broad range of molecules starting with C 30 lanosterol, or cycloartenol in organisms of photosynthetic linage, proceeding through cholesterol in animals, ergosterol in yeast, and sitosterol, campesterol, and brassicasterol in...
The aim of this study was to identify oxysterols and any down-stream metabolites in placenta, umbilical cord blood plasma, maternal plasma and amniotic fluid to enhance our knowledge of the involvement of these molecules in pregnancy. We confirm the identification of 20S-hydroxycholesterol in human placenta, previously reported in a single publicat...
Alterations in brain cholesterol homeostasis have been broadly implicated in neurological disorders. Notwithstanding the complexity by which cholesterol biology is governed in the mammalian brain, excess neuronal cholesterol is primarily eliminated by metabolic clearance via cytochrome P450 46A1 (CYP46A1). No methods are currently available for vis...
Most biologically active oxysterols have a 3β-hydroxy-5-ene function in the ring system with an additional site of oxidation at C-7 or on the side-chain. In blood plasma oxysterols with a 7α-hydroxy group are also observed with the alternative 3-oxo-4-ene function in the ring system formed by ubiquitously expressed 3β-hydroxy-Δ5-C27-steroid oxidore...
The aim of this study was to identify sterols, oxysterols and any down-stream metabolites in placenta, umbilical cord plasma, maternal plasma and amniotic fluid to enhance our knowledge of the involvement of these molecules in pregnancy. We confirm the identification of 20S-hydroxycholesterol in human placenta, previously reported in a single publi...
Disordered cholesterol metabolism is linked to neurodegeneration. In this study we investigated the profile of cholesterol metabolites found in the cerebrospinal fluid (CSF) of Parkinson’s disease (PD) patients. When adjustments were made for confounding variables of age and sex, 7α,(25R)26-dihydroxycholesterol and a second oxysterol 7α,x,y-trihydr...
Cerebrotendinous xanthomatosis (CTX) is caused by autosomal recessive loss-of-function mutations in CYP27A1, a gene encoding cytochrome p450 oxidase essential for bile acid synthesis, resulting in altered bile acid and lipid metabolism. Here, we aimed to identify metabolic aberrations that drive ongoing neurodegeneration in some patients with CTX d...
Despite being a critical molecule in the brain, mass spectrometry imaging (MSI) of cholesterol has been under-reported compared to other lipids due to the difficulty in ionizing the sterol molecule. In the present work, we have employed an on-tissue enzyme-assisted derivatization strategy to improve detection of cholesterol in brain tissue sections...
Every cell in vertebrates possesses the machinery to synthesise cholesterol and to metabolise it. The major route of cholesterol metabolism is conversion to bile acids. Bile acids themselves are interesting molecules being ligands to nuclear and G protein‐coupled receptors, but perhaps the intermediates in the bile acid biosynthesis pathways are ev...
Bile acids are the end products of cholesterol metabolism secreted into bile. They are essential for the absorption of lipids and lipid soluble compounds from the intestine. Here we have identified a series of unusual Δ⁵-unsaturated bile acids in plasma and urine of patients with Smith-Lemli-Opitz syndrome (SLOS), a defect in cholesterol biosynthes...
Both plasma and cerebrospinal fluid (CSF) are rich in cholesterol and its metabolites. Here we describe in detail a methodology for the identification and quantification of multiple sterols including oxysterols and sterol-acids found in these fluids. The method is translatable to any laboratory with access to liquid chromatography – tandem mass spe...
Introduction: We present our views on the current application of mass spectrometry (MS) based lipidomics and how lipidomics can develop in the next decade to be most practical use to society. That is not to say that lipidomics has not already been of value. In-fact, in its earlier guise as metabolite profiling most of the pathways of steroid biosyn...
Significance
The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiological agent of coronavirus disease-2019 (COVID-19), has swept the world in unprecedented speed. In a few months, SARS-CoV-2 has infected millions of people and caused tens of thousands of deaths. There are no Food and Drug Administration-approved antivira...
Despite being a critical molecule for neurobiology and brain health, mass spectrometry imaging (MSI) of cholesterol has been under reported compared to other lipids, due to the difficulty in ionising the sterol molecule. In the present work we have employed an on-tissue enzyme-assisted derivatisation strategy to improve detection of cholesterol in...
The brain is the most cholesterol rich organ in the body containing about 25% of the body's free cholesterol. Cholesterol cannot pass the blood–brain barrier and be imported or exported; instead, it is synthesised in situ and metabolised to oxysterols, oxidised forms of cholesterol, which can pass the blood–brain barrier. 24S‐Hydroxycholesterol is...
Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better unders...
Background:
This study used a genome-wide screen of gene expression to better understand the metabolic and functional differences between commercially valuable intramuscular fat (IMF) and commercially wasteful subcutaneous (SC) fat depots in Bos taurus beef cattle.
Results:
We confirmed many findings previously made at the biochemical level and...
Lipidomics can be regarded as the quantitative profiling of the entire lipid composition of a defined system, be that a cell, tissue, biofluid or intact organism. Lipidomics is a descendent of what was previously known as “metabolite profiling” and is a relative of the related “omic” disciplines of genomics, transcriptomics and proteomics. Lipidomi...
Background
This study used a genome-wide screen of gene expression to better understand the metabolic and functional differences between commercially valuable intramuscular fat (IMF) and commercially wasteful subcutaneous (SC) fat depots in Bos taurus beef cattle.
Results
We confirmed many findings previously made at the biochemical level and made...
Background This study used a genome-wide screen of gene expression to better understand the metabolic and functional differences between commercially valuable intramuscular fat (IMF) and commercially wasteful subcutaneous (SC) fat depots in Bos taurus beef cattle.Results We confirmed many findings previously made at the biochemical level and made n...
While the presence and abundance of the major oxysterols and cholestenoic acids in the circulation is well established, minor cholesterol metabolites may also have biological importance and be of value to investigate. In this study by observing the metabolism of deuterium-labelled cholesterol in the pdgfbret/ret mouse, a mouse model with increased...
Deficiency in cytochrome P450 (CYP) 7B1, also known as oxysterol 7α-hydroxylase, in humans leads to hereditary spastic paraplegia type 5 (SPG5) and in some cases in infants to liver disease. SPG5 is medically characterized by loss of motor neurons in the corticospinal tract. In an effort to gain a better understanding of the fundamental biochemistr...
![Fig. 5. LC-MS RICs or MRM chromatograms of [ 2 H 0 ]GP derivatised...](publication/332535208/figure/fig4/AS:865448857980938@1583350495440/LC-MS-RICs-or-MRM-chromatograms-of-2-H-0-GP-derivatised-oxysterols-and-bile-acids-from_Q320.jpg)
Cholestane-3β,5α,6β-triol (3β,5α,6β-triol) is formed from cholestan-5,6-epoxide (5,6-EC) in a reaction catalysed by cholesterol epoxide hydrolase, following formation of 5,6-EC through free radical oxidation of cholesterol. 7-Oxocholesterol (7-OC) and 7β-hydroxycholesterol (7β-HC) can also be formed by free radical oxidation of cholesterol. Here we...
![Figure 2. (a) RIC and (b) MS 2 ([M] + ), (c) MS 3 ([M] + [M-Py] + ),...](publication/330948869/figure/fig1/AS:723863570759680@1549593932823/a-RIC-and-b-MS-2-M-c-MS-3-M-M-Py-d-M-M-Py-18_Q320.jpg)
![Figure 3. RICs and MS 3 ([M] + [M-Py] + ) spectra of cholesterol...](publication/330948869/figure/fig2/AS:723863570771969@1549593932904/RICs-and-MS-3-M-M-Py-spectra-of-cholesterol-oxidase-oxidized-GP-derivatized_Q320.jpg)
Enzyme-assisted derivatization for sterol analysis (EADSA) is a technology designed to enhance sensitivity and specificity for sterol analysis using electrospray ionization–mass spectrometry. To date it has only been exploited on sterols with a 3β-hydroxy-5-ene or 3β-hydroxy-5α-hydrogen structure, using bacterial cholesterol oxidase enzyme to conve...
The liver X receptors Lxrα/NR1H3 and Lxrβ/NR1H2 are ligand-dependent nuclear receptors critical for midbrain dopaminergic (mDA) neuron development. We previously found that 24(S),25-epoxycholesterol (24,25-EC), the most potent and abundant Lxr ligand in the developing mouse midbrain, promotes mDA neurogenesis in vitro. In this study, we demonstrate...
Using liquid chromatography – mass spectrometry in combination with derivatisation chemistry we profiled the oxysterol and cholestenoic acid content of cerebrospinal fluid from patients with Alzheimer's disease (n = 21), vascular dementia (n = 11), other neurodegenerative diseases (n = 15, Lewy bodies dementia, n = 3, Frontotemporal dementia, n = 1...
Cytochrome P450 (CYP) 27A1 is a key enzyme in both the acidic and neutral pathways of bile acid biosynthesis accepting cholesterol and ring-hydroxylated sterols as substrates introducing a (25R)26-hydroxy and ultimately a (25R)26-acid group to the sterol side-chain. In human, mutations in the CYP27A1 gene are the cause of the autosomal recessive di...
Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better unders...
Bile acids are the end products of cholesterol metabolism secreted into bile. They are essential for the absorption of lipids and lipid soluble compounds from the intestine. Here we have investigated the bile acid content of plasma and urine from patients with a defect in cholesterol biosynthesis, i.e. Smith-Lemli-Opitz syndrome (SLOS), resulting i...
![Fig. 3. LC-HRMS RICs (585.4059 ± 10 ppm) corresponding to [M] þ ions of...](publication/326037919/figure/fig3/AS:864440295632897@1583110035644/LC-HRMS-RICs-5854059-10-ppm-corresponding-to-M-th-ions-of-2-H-5-GP-derivatised_Q320.jpg)
![Fig. 4. MS 3 ([M] þ /[M-Py] þ /, i.e. 585.4/501.3/) spectra acquired in...](publication/326037919/figure/fig4/AS:864440295620610@1583110035976/MS-3-M-th-M-Py-th-ie-5854-5013-spectra-acquired-in-the-analyses-of-control_Q320.jpg)
Dihydroxyoxocholestenoic acids are intermediates in bile acid biosynthesis. Here, using liquid chromatography - mass spectrometry, we confirm the identification of 7α,24-dihydroxy-3-oxocholest-4-en-26-oic and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic acids in cerebrospinal fluid (CSF) based on comparisons to authentic standards and of 7α,12α-dihydro...
![Figure 3. Patterns of MS 3 ([M] + [M-Py] + ) neutral-losses which...](profile/Simon-Jones-13/publication/324271047/figure/fig1/AS:631598538051596@1527596234966/Patterns-of-MS-3-M-M-Py-neutral-losses-which-distinguish-between-or-are_Q320.jpg)
![Figure 4. MRM chromatograms [M] + [M-Py] + [M-Py-59] + reveal...](profile/Simon-Jones-13/publication/324271047/figure/fig2/AS:631598538031185@1527596234992/MRM-chromatograms-M-M-Py-M-Py-59-reveal-3bH-7O-CA-and-3bH-24-diH-7O-CA-in_Q320.jpg)
7-Oxocholesterol (7-OC), 5,6-epoxycholesterol (5,6-EC) and its hydrolysis product cholestane-3β,5α,6β-triol (3β,5α,6β-triol) are normally minor oxysterols in human samples, however, in disease their levels may be greatly elevated. This is the case in plasma from patients suffering from some lysosomal storage disorders e.g. Niemann Pick disease type...
![Fig. 3. MS 3 ([M + ! [M-Py] + ! ) spectra of (A) 7b-HC, (B) 7a-HC and...](publication/316040196/figure/fig1/AS:862592402743298@1582669463651/MS-3-M-M-Py-spectra-of-A-7b-HC-B-7a-HC-and-C-7-OC-Aliquots-of_Q320.jpg)
The introduction of a hydroxy group to the cholesterol skeleton introduces not only the possibility for positional isomers but also diastereoisomers, where two or more isomers have different configurations at one or more of the stereocentres but are not mirror images. The differentiation of diastereoisomers is important as differing isomers can hav...
Cholesterolomics can be regarded as the identification and quantification of cholesterol, its precursors post squalene, and metabolites of cholesterol and of its precursors, in a biological sample. These molecules include 1,25-dihydroxyvitamin D3, steroid hormones and bile acids and intermediates in their respective biosynthetic pathways. In this s...
As neurons die cholesterol is released in the central nervous system (CNS), hence this sterol and its metabolites may represent a biomarker of neurodegeneration, including in amyotrophic lateral sclerosis (ALS) in which altered cholesterol levels have been linked to prognosis. More than 40 different sterols were quantified in serum and cerebrospina...
In this short review we provide a synopsis of recent developments in oxysterol research highlighting topics of current interest to the community. These include the involvement of oxysterols in neuronal development and survival, their participation in the immune system, particularly with respect to bacterial and viral infection and to Th17-cell deve...
In invertebrates, small interfering RNAs are at the vanguard of cell-autonomous antiviral immunity. In contrast, antiviral mechanisms initiated by interferon (IFN) signaling predominate in mammals. Whilst mammalian IFN-induced miRNA are known to inhibit specific viruses, it is not known whether host-directed microRNAs, downstream of IFN-signaling,...
MiR-342-5p regulates intracellular cholesterol by multiple mechanisms.
(A) Quantitation of miR-342-5p effects on total intracellular cholesterol in NIH/3T3 fibroblasts. Data are mean +/− SEM (n = 3) * p ≤ 0.05. (B) Quantitation of miR-342-5p inhibitor effects on total intracellular cholesterol in NIH/3T3 fibroblasts. Data are mean +/− SEM (n = 2)....
MiR-342-5p regulation of MCMV in vitro and in vivo.
(A) Effects of IFN-regulated miRNA on MCMV-GFP replication. Data are normalised to values obtained with non-targeting siRNA and are mean +/− SEM (n = 2). (B) Effects of IFN-γ treatment (100 pg/ml for 6 h) on MCMV-GFP replication in negative control transfected NIH/3T3 cells. N = 3, *p ≤ 0.05. (C a...
Schematic showing IFN-activated 25-HC and miR-342-5p pathways regulating sterol metabolism.
(EPS)
MiR-342- 5p is predicted to target Herpesvirus but not Influenza virus transcripts.
MiRanda version 3.0 was used to scan viral coding sequences for predicted miRNA binding sites. Results were extracted as key-value pairs and sorted according to total score and free energy.
(DOCX)
IFN up-regulates the expression of EVL/ miR-342.
(A) IFN-γ or -β effects on mature miR-342-5p abundance in BMDM 7 h or 24 h after treatment. Values are normalised to mock treated cells. Data are mean +/− SEM (n = 3), * p ≤ 0.05. (B) IFN-γ or -β effects on mature miR-155 abundance in BMDM 7 h or 24 h after treatment. Values are normalised to mock tr...
MiR-342-5p targets Srebf2 and miR-33 and regulates intracellular cholesterol by multiple mechanisms.
(A and B) Effects of miR-342-5p on relative SREBF1 or SREBF2 RNA abundance in mouse and human cells. * p ≤0.05, ** p ≤0.01 (n > 2). (C) IFN-γ or -β effects on mature miR-33 abundance in BMDM 7 h or 24 h after treatment. Values are normalised to mock...
MiR-342-5p does not affect cell viability.
(A and B) Analysis of miR-342-5p effects on cell viability. Primary MEFs (A), 3T3, MRC-5 and HeLa (B) cells were transfected with the indicated doses of control siRNA/ miRNA or miR-342-5p mimics 48 h before analysis using the CellTiter-Blue viability assay. (C) Schematic showing sterol biosynthesis pathway...
MiR-342-5p is predicted to target the 3’UTR of SREBF2 and multiple cholesterol pathway transcripts.
The database TargetScan was queried using mouse Entrez gene IDs for cholesterol biosynthesis pathway members. Target predictions for mouse miRNAs were then tabulated.
(DOCX)
Labelling and isolation of newly transcribed RNA.
(EPS)
Ifng down-regulates synthesis of sterol pathway transcripts.
Upper Graphs: Sequential analysis of sterol pathway-related transcript synthesis (every 30 min) in IFN-γ-treated BMDM (relative to mock). Log2 fold change values were calculated by subtracting the Mock from the IFN-γ-treated Log2 scale signal value. Lower graphs: Sequential analysis of SR...
Ifng down-regulates abundance of sterol pathway transcripts.
Upper graphs: Abundance of sterol biosynthesis pathway-related transcripts in IFN-γ-treated BMDM (relative to mock). Log2 fold change values were calculated by subtracting the Mock from the IFN-γ treated Log2 scale signal value. Lower graphs: Abundance of SREBF1, SREBF2, INSIG1, INSIG2, a...
Extended descriptions of materials and methods.
(DOCX)
Numerical values underlying summary data displayed in the figures.
(XLSX)
IFN directly regulates EVL/ miR-342 transcription.
(A) Predicted ISRE and IRF7 binding sites in EVL promoter derived from the software Toucan. (B) Chromatin immunoprecipitation analysis of STAT1 and IRF1 transcription factor binding to ACTB (negative control), CXCL10 (positive control) or the EVL promoter in BMDM cultured with IFN-γ (10 U/ml) for 2...
SREBF2 siRNA and miR-342-5p effects on cholesterol pathway transcript abundance.
SREBF2 siRNA (upper panel) and miR-342-5p (lower panel) effects on cholesterol pathway transcript abundance in pMEF. Data are mean +/− SEM (n = 6), ** p ≤ 0.01.
(EPS)
Oxysterols are oxygenated forms of cholesterol or its precursors. They are formed enzymatically and via reactive oxygen species. Oxysterols are intermediates in bile acid and steroid hormone biosynthetic pathways and are also bioactive molecules in their own right, being ligands to nuclear receptors and also regulators of the processing of steroid...
Questions
Questions (5)
Also, does this block all subsequent protein transports (to include not only cytokines but also surface proteins) at the point of treatment?
I would like to get your opinion on how to normalise measured metabolites from cell culture supernatants.
I often culture and activate T cells at different cell densities (eg. 1x10^6, 2x10^6 cells per mL, etc). then measure metabolites (in nanograms) produced both intracellularly and secreted in the media.
Logically, I have normalised my data using 'ng per million of cells' but I found that this may not be ideal with measurements made from culture media.
For example, 3x10^6 cells per 3mL were seeded and activated, upon harvest, cells were pelleted to separate media supernatants. Cells were counted and media volumes noted.
If am going to use 1 million cells for metabolite analysis and the other 2 million for other analysis, any metabolite I measured from the 1 million cells can just be normalised with 1 million cells. Easy enough.
In terms of media, 3mls will be obtained. I will use 1 mL for metabolite and 2ml for others.
Should I normalise the metabolites measured in 1mL media in 1million cells since this is the equivalent based on cell density?
To complicate things a bit, longer cultures will result to some cell losses due to cell death or gains due to cell proliferation and some evaporation of media.
Again, at harvest, 1mL was taken for analysis. Due to changes in cell number and media volume, I wont be logical to normalise to 1million cells anymore.
If there was proliferation and final total cell count was 5million in 2.5mL media and 1mL was for analysis, should I normalise to 2million cells?
If there were cell losses and final total cell count was 1million in 2.5mL media, and 1mL was for analysis, should I normalise to 0.4million cells?
Any help, comments, or suggestions would be welcome. Apologies for the long read.
I did four treatments and cultured T cells for three days then assessed cell death by 7AAD single staining. the cells were harvested, washed in PBS then stained straight without fixation. Why did the Treatment 4 Stained (boxed red) produce such two positive 7AAD peaks?
I have some assumptions but not sure of them. Could the higher intensity peak be dead cells that have passed S phase thus have double the amount of DNA to be stained?
Both treatment 3 and 4 had the same drug but at 0.1uM and 1uM respectively. I can see a slight shoulder in treatment 3, could my drug have induce fluorescence?
any other explanations would be a great help. many thanks.
I am new in cell line cultures but I recently tried to culture and check the proliferation of THP-1 cell line (passage 13) using a Cell Proliferation Dye (like CFSE) from eBioscience. I have previously used the cell proliferation dye in CD4 human primary cells and got very good parent/daughter peaks by flow cytometry.
THP-1 was first maintained in 10%HI-FBS in RPMI before it was handed to me, I cultured them at 0.2million cells/ml in serum-free X-Vivo-20 (Lonza) for three days after staining them intracellularly with the proliferation dye. At day three I analysed the cells but I only saw one peak. I have attached the overlay of day 0 and day 3 THP-1 cells and also day 0/3 of a previous primary CD4 t cell culture.
The results suggests that:
- the all the THP-1 cells have divided and none of the original seeded cells remained;
- or, none of the cells have divided and just lost the stain in culture.
Is number 1 normal for THP-1 cells? Is this as expected? or my conditions were not right for the cells to divide?
The activated T cell data shows a clear parent/daughter peaks up to 3 generations which I expected.
Many thanks for the help.
I will be getting some knockout mice in a few months and I have some WT mice spleen frozen down. I would like to do a dry run on how to get the most CD4 T cell from the mice spleens. I have very little experience in dissecting mice and collecting spleens so I would leave that to the lab I'm getting them from. Question is, what's the best way to transport them?
Once I receive the spleen, I then need to isolate CD4 T cells. I spoke to some scientific reps and they suggest using the rubber tip end of a syringe plunger and gently push the spleen into a 70um cell strainer, then rinse with buffer.
The instruction seems straightforward but I'm not too sure about the cells viability afterwards. Is there a better way to do this?
