Absolute measurement of the tissue origins of cell-free DNA in the healthy state and following paracetamol overdose.

BACKGROUND: Despite the emergence of cell-free DNA (cfDNA) as a clinical biomarker in cancer, the tissue origins of cfDNA in healthy individuals have to date been inferred only by indirect and relative measurement methods, such as tissue-specific methylation and nucleosomal profiling. METHODS: We performed the first direct, absolute measurement of the tissue origins of cfDNA, using tissue-specific knockout mouse strains, in both healthy mice and following paracetamol (APAP) overdose. We then investigated the utility of total cfDNA and the percentage of liver-specific cfDNA as clinical biomarkers in patients presenting with APAP overdose. RESULTS: Analysis of cfDNA from healthy tissue-specific knockout mice showed that cfDNA originates predominantly from white and red blood cell lineages, with minor contribution from hepatocytes, and no detectable contribution from skeletal and cardiac muscle. Following APAP overdose in mice, total plasma cfDNA and the percentage fraction originating from hepatocytes increased by ~ 100 and ~ 19-fold respectively. Total cfDNA increased by an average of more than 236-fold in clinical samples from APAP overdose patients with biochemical evidence of liver injury, and 18-fold in patients without biochemically apparent liver injury. Measurement of liver-specific cfDNA, using droplet digital PCR and methylation analysis, revealed that the contribution of liver to cfDNA was increased by an average of 175-fold in APAP overdose patients with biochemically apparent liver injury compared to healthy subjects, but was not increased in overdose patients with normal liver function tests. CONCLUSIONS: We present a novel method for measurement of the tissue origins of cfDNA in healthy and disease states and demonstrate the potential of cfDNA as a clinical biomarker in APAP overdose.

Transcriptomics Analysis of Porcine Caudal Dorsal Root Ganglia in Tail Amputated Pigs Shows Long-Term Effects on Many Pain-Associated Genes.

Tail amputation by tail docking or as an extreme consequence of tail biting in commercial pig production potentially has serious implications for animal welfare. Tail amputation causes peripheral nerve injury that might be associated with lasting chronic pain. The aim of this study was to investigate the short- and long-term effects of tail amputation in pigs on caudal DRG gene expression at different stages of development, particularly in relation to genes associated with nociception and pain. Microarrays were used to analyse whole DRG transcriptomes from tail amputated and sham-treated pigs 1, 8, and 16 weeks following tail treatment at either 3 or 63 days of age (8 pigs/treatment/age/time after treatment; n = 96). Tail amputation induced marked changes in gene expression (up and down) compared to sham-treated intact controls for all treatment ages and time points after tail treatment. Sustained changes in gene expression in tail amputated pigs were still evident 4 months after tail injury. Gene correlation network analysis revealed two co-expression clusters associated with amputation: Cluster A (759 down-regulated) and Cluster B (273 up-regulated) genes. Gene ontology (GO) enrichment analysis identified 124 genes in Cluster A and 61 genes in Cluster B associated with both "inflammatory pain" and "neuropathic pain." In Cluster A, gene family members of ion channels e.g., voltage-gated potassium channels (VGPC) and receptors e.g., GABA receptors, were significantly down-regulated compared to shams, both of which are linked to increased peripheral nerve excitability after axotomy. Up-regulated gene families in Cluster B were linked to transcriptional regulation, inflammation, tissue remodeling, and regulatory neuropeptide activity. These findings, demonstrate that tail amputation causes sustained transcriptomic expression changes in caudal DRG cells involved in inflammatory and neuropathic pain pathways.

Barriers to healthcare for female patients in Papua New Guinea.

A 25-year-old woman presented to hospital in the remote highlands of Papua New Guinea (PNG) with a 3-year history of increasing abdominal distension, amenorrhoea and syncope. Ultrasound showed a large unilocular ovarian cyst. During her work-up, she was found to be HIV positive. She was treated with antiretroviral therapy, and once her CD4 count improved, she underwent a laparotomy and removal of the ovarian cyst with immediate improvement in symptoms. PNG has high levels of HIV particularly in young women and children.1 This is partly due to a lack of screening and treatment facilities and partly due to significant gender discrimination. PNG is considered one of the most dangerous places in the world for females; women are treated as second-class citizens with few human rights or access to services such as healthcare.2 Rape, sexual assault and domestic violence are common, and their lives are dictated to them by their husbands or male relatives.2 3 The lack of healthcare resources and significant levels of gender discrimination meant that this patient had a delayed presentation resulting in potentially grave complications.

Determination of stable reference genes for RT-qPCR expression data in mechanistic pain studies on pig dorsal root ganglia and spinal cord.

RNA expression levels for genes of interest must be normalised with appropriate reference or "housekeeping" genes that are stably expressed across samples and treatments. This study determined the most stable reference genes from a panel of 6 porcine candidate genes: beta actin (ACTB), beta-2-microglobulin (B2M), eukaryotic elongation factor 1 gamma-like protein (eEF-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex subunit A (SDHA), Ubiquitin C (UBC) in sacral dorsal root ganglia and spinal cord samples collected from 16 tail docked pigs (2/3rds of tail amputated) 1, 4, 8 and 16weeks after tail injury (4 pigs/time point). Total RNA from pooled samples was measured by SYBRgreen real-time quantitative PCR. Cycle threshold values were analysed using geNorm, BestKeeper and NormFinder PCR analysis software. Average expression stability and pairwise variation values were calculated for each candidate reference gene. GeNorm analysis identified the most stable genes for normalisation of gene expression data to be GAPDH>eEF-1>UBC>B2M>ACTB>SDHA for dorsal root ganglia and ACTB>SDHA>UBC>B2M>GAPDH>eEF-1 for spinal cord samples. Expression stability estimates were verified by BestKeeper and NormFinder analysis. Expression stability varied between genes within and between tissues. Validation of most stably expressed reference genes was performed by normalisation of calcitonin gene related polypeptide beta (CALCB). The results show similar patterns of CALCB expression when the best reference genes selected by all three programs were used. GAPDH, eEF-1 and UBC are suitable reference genes for porcine dorsal root ganglia samples, whereas ACTB, SDHA and UBC are more appropriate for spinal cord samples.