torsdag den 18. oktober 2018

H2AFY and ME

H2AFY (also known as macroH2A1) is a histone H2A variant. It replaces conventional H2A histones in a subset of nucleosomes.

This gene encodes two splice isoforms, H2AFY1.1 and H2AFY1.2 (also known as mH2A1.1 and mH2A1.2). mH2A1.1 and mH2A1.2 are produced by mutually exclusive use of exons 6b and 6a.  The isoforms have distinct regulatory properties.

H2AFY, ZFR and interferon signaling

ZFR (zinc finger RNA binding protein)  coordinates crosstalk between RNA decay and transcription in innate immunity.

ZFR protein is required for H2AFY expression. ZFR regulates alternative splicing and decay of H2AFY mRNA.

ZFR controls interferon signaling by preventing aberrant splicing and nonsense-mediated decay of histone variant macroH2A1/H2AFY mRNAs (1).



Figure from reference 1: Model for ZFR-mediated regulation of IFNβ. ZFR is required for productive splicing of mH2A1. In the absence of ZFR, mH2A1 pre-mRNA is mis-spliced and resulting transcript is degraded by NMD. In the presence of ZFR, mH2A1 protein is expressed and directly represses the IFNB1 promoter (1).

MacroH2A1.1 regulates mitochondrial respiration

MacroH2A1.1 contains a macrodomain capable of binding NAD+-derived metabolites.

MacroH2A1.1 is rapidly induced during myogenic differentiation through a switch in alternative splicing, and  myotubes that lack macroH2A1.1 have a defect in mitochondrial respiratory capacity (2).


H2AFY and B-cells

The H2AFY1.1 isoform is important for B cell development. Reduction in this isoform may contribute to abnormal hematopoiesis (3).

H2AFY in ME

DNA methylation pattern in the gene H2AFY in peripheral blood mononuclear cells (PBMC) from ME patients:

de Vega 2017(table S7 in ref 4): TSS200, TSS1500 and body are hypermethylated

de Vega 2018 (table S3 in ref 5): Five different probes (rank 96, 229, 277, 711 and 965) show the gene is differentially methylated in ME patients subtypes

Trivedi 2018 (table S6 in ref 6): The gene promoter is hypomethylated

Trivedi 2018 (table S4 in ref 6): Thirteen different probes show that TSS1500 and 3'UTR are hypomethylated

Why is H2AFY hypermethylated in de Vega's study and hypomethylated in Trivedi's study?

H2AFY isoform 2 content in cerebrospinal fluid (7):
1) Controls: no value
2) ME patients: 3
3) Post treatment Lyme patients: 5

What does H2AFY do in ME?

References: 

1) Hauque et al. ZFR coordinates crosstalk between RNA decay and transcription in innate immunity. Nature Communicationsvolume 9, Article number: 1145 (2018)

2)  Marjanovic et al. MacroH2A1.1 regulates mitochondrial respiration by limiting nuclear NAD+ consumption. Nature Structural & Molecular Biology,  volume 24, pages902–910 (2017)
https://www.nature.com/articles/nsmb.3481

3) Sanghyun Kim, Monique Chavez, Cara Shirai and Matt Walter. Abstract 5108: The role of H2afy in normal and malignant hematopoiesis.  DOI: 10.1158/1538-7445.AM2018-5108 Published July 2018 http://cancerres.aacrjournals.org/content/78/13_Supplement/5108.short

4) de Vega et al: Epigenetic modifications and glucocorticoid sensitivity in ME/CFS. BMC Medical Genomics, 2017, 10, 11 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5324230/

5) de Vega et al: Integration of DNA methylation & health scores identifies subtypes in ME/CFS. Epigenomics 2018, 10, 5 https://www.futuremedicine.com/doi/full/10.2217/epi-2017-015

6) Trivedi et al: Identification of ME/CFS - associated DNA methylation patterns.

7) Schutzer et al: Distinct Cerebrospinal Fluid Proteomes Differentiate Post- Treatment Lyme Disease from Chronic Fatigue Syndrome. PLOS One February 2011, volume 6, Issue  https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017287

fredag den 12. oktober 2018

CLYBL, itaconate and B12 - involved in ME?

The gene citrate lyase subunit beta-like (CLYBL) is a mitochondrial enzyme (1).




Figure 1. A Novel Pathway Linked to Vitamin B12 Metabolism. 

The metabolic role of CLYBL is linked to B12 metabolism and the immunomodulatory metabolite, itaconate (1).

CLYBL in ME

The gene CLYBL (body) is hypermethylated in peripheral blood mononuclear cells (PBMC) from ME patients (table S1 in ref 2).

Some ME patients have single nucleotide polymorphism  (SNP) in the gene CLYBL (3). 


CLYBL, itaconate and B12

CLYBL participates in a relatively unexplored human C5 metabolic pathway.

CLYBL is required for maintaining mitochondrial B12 function.

Immunoresponsive gene 1 (IRG1) is expressed exclusively in activated macrophages. IRG1 produce itaconate through decarboxylation of cis-aconitate, a TCA-cycle intermediate.

In vitro test showed that itaconate added to human B-lymphocytes were converted to itaconyl-CoA inside the cells and dramatically lowered B12.

High concentrations of macrophage-derived itaconate might have autocrine and paracrine effects and poison B12 in nearby tissues, raising the possibility for a localized vitamin deficiency in the setting of inflammation (4). 

The B12 regulation may have important implications for other metabolic pathways and pathophysiological contexts. Serine, glycine and one-carbon (SGOC) metabolism, which requires B12 to couple the methionine and folate cycles, is important for nucleotide biosynthesis, redox homeostasis and methylation reactions (1, 4).

SGOC metabolism in ME

Naviaux et al have described  dysregulated SGOC metabolism in ME patients (ref 5 - do take a look at figure S6 in the article supplementary).

B12 in ME

Some ME patients respond to B12/folic acid support (6).

Further reading about STING and itaconate: 

Is STING involved in ME? http://followmeindenmark.blogspot.com/2018/10/is-sting-involved-in-me.html



References: 

1) A Missing Link to Vitamin B12 Metabolism.
https://www.semanticscholar.org/paper/A-Missing-Link-to-Vitamin-B12-Metabolism.-Reid-Paik/aec86264a4fb0a28865ef026ace7d772b25f1114/figure/0

2) de Vega et al: Epigenetic modifications and glucocorticoid sensitivity in ME/CFS. BMC Medical Genomics, 2017, 10, 11 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5324230/

3) Smith et al: Convergent genomic studies identify association of GRIK2 and NPAS2 with chronic fatigue syndrome.  2011;64(4):183-94. doi: 10.1159/000326692. Epub 2011 Sep 9.
 https://www.ncbi.nlm.nih.gov/pubmed/21912186 

4) Shen et al: The Human Knockout Gene CLYBL Connects Itaconate to Vitamin B12. Cell, 171, 771-782, 2017
https://www.sciencedirect.com/science/article/pii/S0092867417311820

5) Naviaux RK, Naviaux JC, Li K, Bright AT, Alaynick WA, Wang L, Baxter A, Nathan N et al (2016) Metabolic features of chronic fatigue syndrome. Proc Natl Acad Sci U S A 113:E5472–E5480.   https://doi.org/10.1073/pnas.1607571113 

6) Response to Vitamin B12 and Folic Acid in Myalgic Encephalomyelitis and Fibromyalgia
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0124648



onsdag den 3. oktober 2018

Is STING involved in ME?

NEW RESEARCH
Nitro-fatty acids are formed in response to virus infection and are potent inhibitors of STING palmitoylation and signaling
Abstract:The adaptor molecule stimulator of IFN genes (STING) is central to production of type I IFNs in response to infection with DNA viruses and to presence of host DNA in the cytosol. Excessive release of type I IFNs through STING-dependent mechanisms has emerged as a central driver of several interferonopathies, including systemic lupus erythematosus (SLE), Aicardi–Goutières syndrome (AGS), and stimulator of IFN genes-associated vasculopathy with onset in infancy (SAVI). The involvement of STING in these diseases points to an unmet need for the development of agents that inhibit STING signaling. Here, we report that endogenously formed nitro-fatty acids can covalently modify STING by nitro-alkylation. These nitro-alkylations inhibit STING palmitoylation, STING signaling, and subsequently, the release of type I IFN in both human and murine cells. Furthermore, treatment with nitro-fatty acids was sufficient to inhibit production of type I IFN in fibroblasts derived from SAVI patients with a gain-of-function mutation in STING. In conclusion, we have identified nitro-fatty acids as endogenously formed inhibitors of STING signaling and propose for these lipids to be considered in the treatment of STING-dependent inflammatory disease (1).
The gene TMEM173 encodes STING.
TMEM173 is hypermethylated (genic location: body, p-value = 4,53E-05, FDR =  0,000816) in peripheral blood mononuclear cells (PBMC) from ME patients compared to controls (2).

TMEM173 is differentially methylated (genic location: 3'UTR, p-value = 7,25E-05, FDR = 0,000184) in PBMC from ME patient subtypes (3).

This DNA methylation pattern is related to degree of disease and quality of life in the ME patients (2, 3).

Is STING (TMEM173) involved in the pathomechanism in ME patient subtypes?

ME patents are metabolic reprogrammed (4, 5, 6, 7). Is a downregulated Nrf2 involved in a STING-pathomechanism in ME? Read about Nrf2 and STING in:


Nrf2 negatively regulates STING indicating a link between antiviral sensing and metabolic reprogramming
Abstract:

The transcription factor Nrf2 is a critical regulator of inflammatory responses. If and how Nrf2 also affects cytosolic nucleic acid sensing is currently unknown. Here we identify Nrf2 as an important negative regulator of STING and suggest a link between metabolic reprogramming and antiviral cytosolic DNA sensing in human cells. Here, Nrf2 activation decreases STING expression and responsiveness to STING agonists while increasing susceptibility to infection with DNA viruses. Mechanistically, Nrf2 regulates STING expression by decreasing STING mRNA stability. Repression of STING by Nrf2 occurs in metabolically reprogrammed cells following TLR4/7 engagement, and is inducible by a cell-permeable derivative of the TCA-cycle-derived metabolite itaconate (4-octyl-itaconate, 4-OI). Additionally, engagement of this pathway by 4-OI or the Nrf2 inducer sulforaphane is sufficient to repress STING expression and type I IFN production in cells from patients with STING-dependent interferonopathies. We propose Nrf2 inducers as a future treatment option in STING-dependent inflammatory diseases (8).

Om STING: 
Fedtstof kan stoppe immunforsvar, der løber løbsk
https://ing.dk/artikel/fedtstof-kan-stoppe-immunforsvar-loeber-loebsk-214342


References:

1) Anne Louise Hansen et al: Nitro-fatty acids are formed in response to virus infection and are potent inhibitors of STING palmitoylation and signaling
PNAS published ahead of print July 30, 2018 https://doi.org/10.1073/pnas.1806239115

2) de Vega et al: Epigenetic modifications and glucocorticoid sensitivity in ME/CFS. BMC Medical Genomics, 2017, 10, 11 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5324230/

3) de Vega et al: Integration of DNA methylation & health scores identifies subtypes in ME/CFS. Epigenomics 2018, 10, 5 https://www.futuremedicine.com/doi/full/10.2217/epi-2017-015
    4)  Naviaux et al: Metabolic features of CFS. www.pnas.org/cgi/doi/10.1073/pnas.1607571113

    5) Germain et al: Metabolic profiling of a ME/CFS discovery cohort reveals disturbances in fatty acid and lipid metabolism. Mol. BioSyst. 2017, 13, 371.
      6) Nagy-Szakal et al: Insights into ME/CFS phenotypes through comprehensive metabolomics. Nat Sci Rep 2018, 8.
        7) Reuter and Evans: Long-chain acylcarnitine deficiency in patients with CFS. Potential involvement of altered carnitine palmitoyltransferase-I-activity. J. Int. Med. 2011, 270.

        8)  David Olangnier et al: Nrf2 negatively regulates STING indicating a link between antiviral sensing and metabolic reprogramming
        Nature Communicationsvolume 9, Article number: 3506 (2018)
        https://www.nature.com/articles/s41467-018-05861-7

        søndag den 30. september 2018

        RPS6KB1, EIF4G1 and CD79A in ME/CFS

        RPS6KB1 (5'UTR) is the most hypermethylated gene in peripheral blood mononuclear cells (PBMC) from ME patients with a mean beta-difference: 0,353 compared to controls (table S4 in ref 1).

        EIF4G1 (body) is the second most hypermethylated gene in PBMC from ME patients (table 2, page 5 in ref 2). EIF4G1 mRNA expression is upregulated in PBMC from ME patients (figure 1 in ref 3).

        CD79A is the gene with lowest expression in whole blood from adolescent CFS patients (table S3 in ref 4).

        These three gene products interact (5):

        Gene Cards STRING Interaction network RPS6KB1 (5)

        RPS6KB1: Ribosomal protein S6 kinase, 70kDa, polypeptide 1; Serine/threonine-protein kinase that acts downstream of mTOR signaling in response to growth factors and nutrients to promote cell proliferation, cell growth and cell cycle progression. Regulates protein synthesis through phosphorylation of EIF4B, RPS6 and EEF2K, and contributes to cell survival by repressing the pro-apoptotic function of BAD. Under conditions of nutrient depletion, the inactive form associates with the EIF3 translation initiation complex.

        EIF4G1: Eukaryotic translation initiation factor 4 gamma, 1; Component of the protein complex eIF4F, which is involved in the recognition of the mRNA cap, ATP-dependent unwinding of 5’-terminal secondary structure and recruitment of mRNA to the ribosome.

        CD79A: CD79a molecule, immunoglobulin-associated alpha; Required in cooperation with CD79B for initiation of the signal transduction cascade activated by binding of antigen to the B-cell antigen receptor complex (BCR) which leads to internalization of the complex, trafficking to late endosomes and antigen presentation. Also required for BCR surface expression and for efficient differentiation of pro- and pre-B-cells. Stimulates SYK autophosphorylation and activation. Binds to BLNK, bringing BLNK into proximity with SYK and allowing SYK to phosphorylate BLNK.


        SYK and BLNK are also in the RPS6KB1 network (5):



        Gene Cards STRING Interaction network CMTM8 (5)


        Use the link below to see how the B cell Receptor (BCR) functions with CD79A, SYK and BLNK(6):
        https://media.springernature.com/m685/nature-assets/nrd/journal/v12/n3/images/nrd3937-f1.jpg


        References:

        1) Trivedi et al: Identification of ME/CFS - associated DNA methylation patterns.
         Plos One 2018, 13, 7   https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201066

        2) de Vega et al: Epigenetic modifications and glucocorticoid sensitivity in ME/CFS. BMC Medical Genomics, 2017, 10, 11 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5324230/

        3) Kerr et al:  Assessment of a 44 Gene Classifier for the Evaluation of Chronic Fatigue Syndrome from Peripheral Blood Mononuclear Cell Gene Expression . Plos One 2011 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016872

        4)  Nguyen et al: Whole blood gene expression in adolescent CFS: an exploratory crosssectional study suggesting altered B cell differentiation and survival. J Transl Med. 2017,15,102.  https://www.ncbi.nlm.nih.gov/pubmed/28494812

        5) Gene Cards    https://www.genecards.org/

        Gene Cards RPS6KB1

        Gene Cards CMTM8

        6 )Young and Staudt: Targeting pathological B cell receptor signalling in lymphoid malignancies. Nature Reviews Drug Discovery volume12, pages229–243 (2013)
        https://www.nature.com/articles/nrd3937

        lørdag den 1. september 2018

        HOX-genes and MIR10A in ME

        The genes HOXB3, HOXB4, HOXB5, HOXB-AS3 (LOC404266) and MIR10A are located together on chromosome 17.

        MIR10A is hypermethylated, HOXB5 and LOC404266 are hypomethylated in CD4+ T-cells from ME patients (1).

        The HOXB4 gene promoter is hypomethylated in peripheral blood mononuclear cells (PBMC) from ME patients (table S7 in ref 2).

        HOXB3 (5'UTR), HOXB4 (1stExon, 5'UTR), LOC404266, HOXB5 (5'UTR), LOC404266 (TSS200, TSS1500) and MIR10A (1stExon, TSS1500) are hypomethylated in PBMC from ME patients (table S4 in ref 2).

        HOXB3 (5'UTR), HOXB4 (body), HOXB5 (3'UTR), LOC404266 (TSS200, TSS1500, body) and MIR10A (TSS1500) are hypermethylated in PBMC from ME patients (table S7 in ref 3).

        HOXB3, HOXB4, LOC404266 and MIR10A are differentially methylated in ME subtypes (table S3 in ref 4).

        Why are HOX-genes hypomethylated in Trivedi's study and hypermethylated in de Vega's study?

        DNA methyltransferase DNMT3A and DNMT3B are responsible for de novo methylation, while DNMT1 is responsible for maintaining methylation signatures.

        DNMT3A (TSS1500) is hypomethylated in the Trivedi study (table S4 in ref 2).

        DNMT3A (body) and DNMT3B (5'UTR) are hypermethylated in the de Vega (2017) study (table S7 in ref 3).

        DNMT3A (body) and DNMT3B (5'UTR) are differentially methylated in ME subtypes (table S3 in ref 4).

        References:
        1. Brenu et al: Methylation profile of CD4+ T cells in CFS/ME. J. Clin Cell Immunol 5, 228  
        2. Trivedi et al: Identification of ME/CFS - associated DNA methylation patterns. Plos One 2018, 13, 7 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201066
        3. de Vega et al: Epigenetic modifications and glucocorticoid sensitivity in ME/CFS. BMC Medical Genomics, 2017, 10, 11 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5324230/
        4. de Vega et al: Integration of DNA methylation & health scores identifies subtypes in ME/CFS. Epigenomics 2018, 10, 5 https://www.futuremedicine.com/doi/full/10.2217/epi-2017-0150

        lørdag den 25. august 2018

        FAM13A, fatty acid oxidation and ME

        Lipid metabolism is dysregulated in ME patients (1, 2, 3, 4).

        Family with sequence similarity 13 number A (FAM13A) regulates fatty acid oxidation (FAO) (5).

        The gene FAM13A is hypomethylated (TSS200 and TSS1500) in peripheral blood mononuclear cells (PBMC) from ME patients (table S4 in ref 6).

        The gene FAM13A is hypermethylated (1stExon/5'UTR), CPT1A is hypermethylated (5'UTR) and CPT1B is hypermethylated (TSS200/5'UTR) in PBMC from ME patients. This DNA methylation pattern is related to quality of life in the ME patients (table S7 in ref 7).

        In whole blood from adolescent CFS patients FAM13A gene expression is elevated (table S3 in ref 8).

        Use the link to see the figure:
        Figure. FAM13A regulates the CPT1A–FAO pathway in chronic obstructive pulmonary disease (COPD).  (Ref. 5)

        FAM13A regulates the CPT1A–FAO pathway in chronic obstructive pulmonary disease (COPD). FAM13 locus has been shown to be associated with increased expression of FAM13A and higher risk for COPD. FAM13 in association with SIRT1 induces the expression of CPT1A, a key enzyme that regulates fatty acid oxidation (FAO) in the mitochondria. Up-regulation of CPT1A promotes FAO and high production of ROS, leading to lung epithelial cell death; this is an important pathogenic mechanism that might lead to COPD. CAT, carnitine translocase; CoA, coenzyme A; CPT1A, carnitine palmitoyltransferase 1; CPT2, carnitine palmitoyltransferase 2; FACS, fatty acyl-CoA synthase; FADH2, flavin adenin dinucleotide; FAM13A, family with sequence similarity 13 member A; NADH, nicotinamide adenine dinucleotide reduced; ROS, reactive oxygen species; SIRT1, sirtuin 1; TCA, tricarboxylic acid cycle.  (Ref. 5)


        Is FAM13A and CPT1A/CPT1B involved in the ME/CFS pathomechanism?

        References
        1. Naviaux et al: Metabolic features of CFS. www.pnas.org/cgi/doi/10.1073/pnas.1607571113
        2. Germain et al: Metabolic profiling of a ME/CFS discovery cohort reveals disturbances in fatty acid and lipid metabolism. Mol. BioSyst. 2017, 13, 371.
        3. Nagy-Szakal et al: Insights into ME/CFS phenotypes through comprehensive metabolomics. Nat Sci Rep 2018, 8.
        4. Reuter and Evans: Long-chain acylcarnitine deficiency in patients with CFS. Potential involvement of altered carnitine palmitoyltransferase-I-activity. J. Int. Med. 2011, 270.
        5. Hawkins et al. FAM13A, A Fatty Acid Oxidation Switch in Mitochondria. Friend or Foe in Chronic Obstructive Pulmonary Disease Pathogenesis?  Am J Respir Cell Mol Biol. 2017 Jun; 56(6): 689–691.doi: 10.1165/rcmb.2017-0080ED  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5516298/
        6. Trivedi et al: Identification of ME/CFS - associated DNA methylation patterns. Plos One 2018, 13, 7.
        7. de Vega et al: Epigenetic modifications and glucocorticoid sensitivity in ME/CFS. BMC Medical Genomics, 2017, 10, 11.
        8. Nguyen et al: Whole blood gene expression in adolescent CFS: an exploratory crosssectional study suggesting altered B cell differentiation and survival. J Transl Med. 2017,15,102

        søndag den 19. august 2018

        ACSL3, GPD2 og PDHX i ME

        Forskere har anvendt en ny forbedret teknologi til at undersøge epigenetiske ændringer i blodceller fra ME patienter. Resultatet blev valideret på ME patienter fra både USA og Spanien (1).

        De supplerende tabeller i artiklen rummer værdifulde informationer. Jeg vil nævne tre eksempler (ACSL3, GPD2 og PDHX) fra Tabel 7.

        Tabel S7 er en liste over 144 hypomethylerede gen promotorer. Promotoren er det område på DNA'et, hvorfra transkriptionen af et gen starter. Ændret DNA methylering af promotoren har stor betydning for genekspressionen. Man kan formode, at de 144 hypomethylerede gener er "på overarbejde", og at dette er relateret til ME sygdomsmekanismen. Vi ved ikke hvordan. Måske er de på overarbejde for at kompensere for noget, der er gået galt i cellens metabolisme?

        ACSL3
        Acyl-CoA synthetase long-chain family member 3 (ACSL3) aktiverer langkædede fedtsyrer til både syntese af cellulære lipider og til nedbrydning via beta-oxidation. ACSL3 er nødvendig for at bygge fedtsyrer ind i phosphatidylcholine.

        ACSL3 interagerer med proteiner kodet af generne: CPT1A, CPT1B, PNPLA2 og MGLL. Disse proteiner har en central rolle i lipidmetabolismen. Et tidligere epigenetisk studie har vist, at disse gener er påvirkede hos ME patienter (2).

        GPD2
        Glycerol-3-phosphate dehydrogenase 2 (GPD2) er et mitokondrie enzym, som er komponent i den respiratoriske kæde og i glycerophosphate-shuttlen. Det er et nøgleenzym i skæringspunktet mellem glykolyse, oxidativ fosforylering og fedtsyrer metabolismen (3).

        PDHX
        Pyruvate dehydrogenase complex component X (PDHX) er en delkomponent i PDH-komplekset. Hvis der opstår fejl i dette gen, eller PDHX-proteinet udsættes for et autoimmunt angreb bliver man alvorlig syg. Læs om genet: https://www.ncbi.nlm.nih.gov/gene/8050

        Spørgsmålet er hvorfor er dette gen epigenetisk ændret hos ME patienter? Et nedreguleret PDH-kompleks er allerede sat i centrum af ME sygdoms-mekanismen (4),

        Referencer:
        1. Trivedi et al: Identification of ME/CFS - associated DNA methylation patterns. Plos One 2018, 13, 7 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201066
        2. de Vega et al: Epigenetic modifications and glucocorticoid sensitivity in ME/CFS. BMC Medical Genomics, 2017, 10, 11
        3. Mracek et al: The function and the role of the mitochondrial glycerol-3-phosphate dehydrogenase in mammalian tissue. Biochimica et Biophysica Acta 1827, 2013, 401-410.
        4. Fluge et al: Metabolic profiling indicates impaired pyruvate dehydrogenase function in myalgic encephalopathy / chronic fatigue syndrome. JCI Insight. 2016; 1(21):e89376. Doi 10.1172/jci.insight.89276