516 research outputs found
NMR Spectroscopy of Cell Culture, Tissues, and Other Biofluids
NMR spectroscopy can provide a wealth of information on cellular metabolism and is frequently used in metabolomics application that use cultured cells, tissues, and whole organisms. Central to these analyses are the protocols for sample harvest, which incorporate procedures for quenching metabolic processes to preserve samples in a state that is representative of their source. In this chapter, the main considerations are discussed with reference to literature exemplars. In the latter half of the chapter, less commonly studied biofluids that also have specific sample preparation requirements are discussed, with a focus on cerebrospinal fluid, faeces, bile, seminal fluid, and milk.</jats:p
Opportunities at the interface of network science and metabolic modeling
Metabolism plays a central role in cell physiology because it provides the molecular machinery for growth. At the genome-scale, metabolism is made up of thousands of reactions interacting with one another. Untangling this complexity is key to understand how cells respond to genetic, environmental, or therapeutic perturbations. Here we discuss the roles of two complementary strategies for the analysis of genome-scale metabolic models: Flux Balance Analysis (FBA) and network science. While FBA estimates metabolic flux on the basis of an optimization principle, network approaches reveal emergent properties of the global metabolic connectivity. We highlight how the integration of both approaches promises to deliver insights on the structure and function of metabolic systems with wide-ranging implications in discovery science, precision medicine and industrial biotechnology
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Metabonomics study of the effects of single copy mutant KRAS in the presence or absence of WT allele using human HCT116 isogenic cell lines.
INTRODUCTION: KRAS was one of the earliest human oncogenes to be described and is one of the most commonly mutated genes in different human cancers, including colorectal cancer. Despite KRAS mutants being known driver mutations, KRAS has proved difficult to target therapeutically, necessitating a comprehensive understanding of the molecular mechanisms underlying KRAS-driven cellular transformation. OBJECTIVES: To investigate the metabolic signatures associated with single copy mutant KRAS in isogenic human colorectal cancer cells and to determine what metabolic pathways are affected. METHODS: Using NMR-based metabonomics, we compared wildtype (WT)-KRAS and mutant KRAS effects on cancer cell metabolism using metabolic profiling of the parental KRAS G13D/+ HCT116 cell line and its isogenic, derivative cell lines KRAS +/- and KRAS G13D/-. RESULTS: Mutation in the KRAS oncogene leads to a general metabolic remodelling to sustain growth and counter stress, including alterations in the metabolism of amino acids and enhanced glutathione biosynthesis. Additionally, we show that KRASG13D/+ and KRASG13D/- cells have a distinct metabolic profile characterized by dysregulation of TCA cycle, up-regulation of glycolysis and glutathione metabolism pathway as well as increased glutamine uptake and acetate utilization. CONCLUSIONS: Our study showed the effect of a single point mutation in one KRAS allele and KRAS allele loss in an isogenic genetic background, hence avoiding confounding genetic factors. Metabolic differences among different KRAS mutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenic KRAS
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Metabolic characterization of colorectal cancer cells harbouring different KRAS mutations in codon 12, 13, 61 and 146 using human SW48 isogenic cell lines
Introduction:
KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) mutations occur in approximately one-third of colorectal (CRC) tumours and have been associated with poor prognosis and resistance to some therapeutics. In addition to the well-documented pro-tumorigenic role of mutant Ras alleles, there is some evidence suggesting that not all KRAS mutations are equal and the position and type of amino acid substitutions regulate biochemical activity and transforming capacity of KRAS mutations.
Objectives:
To investigate the metabolic signatures associated with different KRAS mutations in codons 12, 13, 61 and 146 and to determine what metabolic pathways are affected by different KRAS mutations.
Methods:
We applied an NMR-based metabonomics approach to compare the metabolic profiles of the intracellular extracts and the extracellular media from isogenic human SW48 CRC cell lines with different KRAS mutations in codons 12 (G12D, G12A, G12C, G12S, G12R, G12V), 13 (G13D), 61 (Q61H) and 146 (A146T) with their wild-type counterpart. We used false discovery rate (FDR)-corrected analysis of variance (ANOVA) to determine metabolites that were statistically significantly different in concentration between the different mutants.
Results:
CRC cells carrying distinct KRAS mutations exhibited differential metabolic remodelling, including differences in glycolysis, glutamine utilization and in amino acid, nucleotide and hexosamine metabolism.
Conclusions:
Metabolic differences among different KRAS mutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenic KRAS
The potential and challenges of targeting MTAP-negative cancers beyond synthetic lethality
Approximately 15% of cancers exhibit loss of the chromosomal locus 9p21.3 â the genomic location of the tumour suppressor gene CDKN2A and the methionine salvage gene methylthioadenosine phosphorylase (MTAP). A loss of MTAP increases the pool of its substrate methylthioadenosine (MTA), which binds to and inhibits activity of protein arginine methyltransferase 5 (PRMT5). PRMT5 utilises the universal methyl donor S-adenosylmethionine (SAM) to methylate arginine residues of protein substrates and regulate their activity, notably histones to regulate transcription. Recently, targeting PRMT5, or MAT2A that impacts PRMT5 activity by producing SAM, has shown promise as a therapeutic strategy in oncology, generating synthetic lethality in MTAP-negative cancers. However, clinical development of PRMT5 and MAT2A inhibitors has been challenging and highlights the need for further understanding of the downstream mediators of drug effects. Here, we discuss the rationale and methods for targeting the MAT2A/PRMT5 axis for cancer therapy. We evaluate the current limitations in our understanding of the mechanism of MAT2A/PRMT5 inhibitors and identify the challenges that must be addressed to maximise the potential of these drugs. In addition, we review the current literature defining downstream effectors of PRMT5 activity that could determine sensitivity to MAT2A/PRMT5 inhibition and therefore present a rationale for novel combination therapies that may not rely on synthetic lethality with MTAP loss
The 14q32 maternally imprinted locus is a major source of longitudinally stable circulating microRNAs as measured by small RNA sequencing.
Understanding the normal temporal variation of serum molecules is a critical factor for identifying useful candidate biomarkers for the diagnosis and prognosis of chronic disease. Using small RNA sequencing in a longitudinal study of 66 women with no history of cancer, we determined the distribution and dynamics (via intraclass correlation coefficients, ICCs) of the miRNA profile over 3 time points sampled across 2-5 years in the course of the screening trial, UKCTOCS. We were able to define a subset of longitudinally stable miRNAs (ICC >0.75) that were individually discriminating of women who had no cancer over the study period. These miRNAs were dominated by those originating from the C14MC cluster that is subject to maternal imprinting. This assessment was not significantly affected by common confounders such as age, BMI or time to centrifugation nor alternative methods to data normalisation. Our analysis provides important benchmark data supporting the development of miRNA biomarkers for the impact of life-course exposure as well as diagnosis and prognostication of chronic disease
Intracellular Staphylococcus aureus modulates host central carbon metabolism to activate autophagy
Staphylococcus aureus is a facultative intracellular pathogen that invades and replicates within many types of phagocytic and nonphagocytic cells. During intracellular infection, S. aureus is capable of subverting xenophagy and escaping to the cytosol of the host cell. Furthermore, drug-induced autophagy facilitates the intracellular replication of S. aureus, but the reasons behind this are unclear. Here, we have studied the host central carbon metabolism during S. aureus intracellular infection. We found extensive metabolic rerouting and detected several distinct metabolic changes that suggested starvation-induced autophagic flux in infected cells. These changes included increased uptake but lower intracellular levels of glucose and low abundance of several essential amino acids, as well as markedly upregulated glutaminolysis. Furthermore, we show that AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) phosphorylation levels are significantly increased in infected cells. Interestingly, while autophagy was activated in response to S. aureus invasion, most of the autophagosomes detected in infected cells did not contain bacteria, suggesting that S. aureus induces the autophagic flux during cell invasion for energy generation and nutrient scavenging. Accordingly, AMPK inhibition halted S. aureus intracellular proliferation
Assessment of metabolic phenotypic variability in children's urine using 1H NMR spectroscopy
The application of metabolic phenotyping in clinical and
epidemiological studies is limited by a poor understanding of
inter-individual, intra-individual and temporal variability in
metabolic phenotypes. Using 1H NMR spectroscopy we characterised
short-term variability in urinary metabolites measured from 20
children aged 8-9 years old. Daily spot morning, night-time and
pooled (50:50 morning and night-time) urine samples across six
days (18 samples per child) were analysed, and 44 metabolites
quantified. Intraclass correlation coefficients (ICC) and mixed
effect models were applied to assess the reproducibility and
biological variance of metabolic phenotypes. Excellent
analytical reproducibility and precision was demonstrated for
the 1H NMR spectroscopic platform (median CV 7.2%). Pooled
samples captured the best inter-individual variability with an
ICC of 0.40 (median). Trimethylamine, N-acetyl neuraminic acid,
3-hydroxyisobutyrate, 3-hydroxybutyrate/3-aminoisobutyrate,
tyrosine, valine and 3-hydroxyisovalerate exhibited the highest
stability with over 50% of variance specific to the child. The
pooled sample was shown to capture the most inter-individual
variance in the metabolic phenotype, which is of importance for
molecular epidemiology study design. A substantial proportion of
the variation in the urinary metabolome of children is specific
to the individual, underlining the potential of such data to
inform clinical and exposome studies conducted early in life
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