Biochemistry PhD Seminar | Eiren Sweetman
Comprehensive molecular analysis of different classes of molecules in an ME/CFS pilot study group, and investigation of RNA-activated Protein Kinase R (PKR) as a diagnostic biomarker
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a poorly understood illness characterised by severe debilitating fatigue, affecting approximately 1% of the global population and disproportionately affecting females. There are no agreed diagnostic markers or definitive clinical tests, and the causative agent and disease pathophysiology are ill-defined. However, immune dysfunction, chronic viral infection and, recently, metabolic and mitochondrial dysregulation are proposed causes of ME/CFS. Further, abnormal upregulation and activation of a protein in the innate antiviral immune response, Protein Kinase R (PKR), has been found in ME/CFS patients.
This study aimed to identify biological pathways affected by the disease by comprehensive analysis of different classes of molecules, including micro-RNA, expressed mRNA, and cellular proteomes in a group of ten ME/CFS patients and ten age-gender matched controls. For a potential ELISA based diagnostic test, antibodies against (i) a nonphosphorylated PKR fragment and (ii) a phosphorylated PKR peptide were raised and purified by antigen affinity chromatography to investigate the ratio of active:inactive PKR in ME/CFS patients. Identification of cellular RNA and miRNA regulators of PKR was studied using a known RNA regulator of PKR as a control.
Principal Component Analysis and t-tests were used to identify significant differences in micro-RNA, gene transcript and protein expression between the two study groups. Regulators of metabolic, immune and oxidative pathways were significantly increased and there was dysregulation in immune, inflammation, apoptosis, oxidative stress, metabolic and mitochondrial pathways in the ME/CFS group. Western immunoblots of lymphocyte and neutrophil protein extracts detected an increased ratio of active:inactive PKR in ME/CFS patients. Finally, MiSeq analysis found 14 miRNAs that bound to PKR. The small RNAs Y5 RNA and 5S ribosomal RNA, and the mRNA of PKR itself were also identified as potential PKR regulators. This study of a range of biologically important molecules within a small, well-characterised patient group has given significant insight into the disease ME/CFS.
|Date||Tuesday, 17 October 2017|
|Time||12:00pm - 1:00am|
|Event Category||Health Sciences|
|Location||Biochemistry seminar rm 231|