Insights into N-calls of mitochondrial DNA sequencing using MitoChip v2.0

Developments in DNA resequencing microarrays include mitochondrial DNA (mtDNA) sequencing and mutation detection. Failure by the microarray to identify a base, compared to the reference sequence, is designated an 'N-call.' This study re-examined the N-call distribution of mtDNA samples sequenced by the Affymetrix MitoChip v.2.0, based on the hypothesis that N-calls may represent insertions or deletions (indels) in mtDNA.We analysed 16 patient mtDNA samples using MitoChip. N-calls by the proprietary GSEQ software were significantly reduced when either of the freeware on-line algorithms ResqMi or sPROFILER was utilized. With sPROFILER, this decrease in N-calls had no effect on the homoplasmic or heteroplasmic mutation levels compared to GSEQ software, but ResqMi produced a significant change in mutation load, as well as producing longer N-cell stretches. For these reasons, further analysis using ResqMi was not attempted. Conventional DNA sequencing of the longer N-calls stretches from sPROFILER revealed 7 insertions and 12 point mutations. Moreover, analysis of single-base N-calls of one mtDNA sample found 3 other point mutations.Our study is the first to analyse N-calls produced from GSEQ software for the MitoChipv2.0. By narrowing the focus to longer stretches of N-calls revealed by sPROFILER, conventional sequencing was able to identify unique insertions and point mutations. Shorter N-calls also harboured point mutations, but the absence of deletions among N-calls suggests that probe confirmation affects binding and thus N-calling. This study supports the contention that the GSEQ is more capable of assigning bases when used in conjunction with sPROFILER

Glucocorticoids alleviate intestinal ER stress by enhancing protein folding and degradation of misfolded proteins

Endoplasmic reticulum (ER) stress in intestinal secretory cells has been linked with colitis in mice and inflammatory bowel disease (IBD). Endogenous intestinal glucocorticoids are important for homeostasis and glucocorticoid drugs are efficacious in IBD. In Winnie mice with intestinal ER stress caused by misfolding of the Muc2 mucin, the glucocorticoid dexamethasone (DEX) suppressed ER stress and activation of the unfolded protein response (UPR), substantially restoring goblet cell Muc2 production. In mice lacking inflammation, a glucocorticoid receptor antagonist increased ER stress, and DEX suppressed ER stress induced by the N-glycosylation inhibitor, tunicamycin (Tm). In cultured human intestinal secretory cells, in a glucocorticoid receptor-dependent manner, DEX suppressed ER stress and UPR activation induced by blocking N-glycosylation, reducing ER Ca2+ or depleting glucose. DEX up-regulated genes encoding chaperones and elements of ER-associated degradation (ERAD), including EDEM1. Silencing EDEM1 partially inhibited DEX's suppression of misfolding-induced ER stress, showing that DEX enhances ERAD. DEX inhibited Tm-induced MUC2 precursor accumulation, promoted production of mature mucin, and restored ER exit and secretion of Winnie mutant recombinant Muc2 domains, consistent with enhanced protein folding. In IBD, glucocorticoids are likely to ameliorate ER stress by promoting correct folding of secreted proteins and enhancing removal of misfolded proteins from the ER

Improving Patient Outcomes Through Better Understanding of Thiopurine Metabolism

The thiopurine pro-drugs, azathioprine (AZA) and 6-mercaptopurine (6MP), are employed to maintain remission in inflammatory bowel diseases (IBD) because they are effective and economical treatments in 60% of patients. However, they are frequently associated with treatment-limiting adverse drug reactions (ADR), and therefore 6-thioguanine (6TG) has been proposed as an alternative thiopurine therapy in the treatment of IBD. 6TG has a faster onset of action, less dose-independent ADR and it exerts a good clinical response, but it has been frequently implicated with the serious ADR of hepatic nodular regenerative hyperplasia and/or veno-occlusive disease. The term, sinusoidal obstructive syndrome (SOS), was recently coined to describe the underlying vascular pathology. SOS is usually clinically silent until development of portal hypertension. Animal models with SOS have been previously described, in rats after monocrotaline (MC) administration and in dogs after treatment with a generation II proton pump inhibitor, but no animal model of SOS after thiopurine treatment is known. SOS has been described with AZA/6MP, and some clinicians have suggested that it is a thiopurine class effect, but mainly was associated with 6TG. There is controversy about whether thiopurine associated SOS is: i) 6TG specific or a thiopurine class effect; ii) idiosyncratic or 6TG dose-related. We report on a novel model of SOS due to 6TG. We show that the SOS pathology is dose-related, is not a thiopurine class effect, is mediated principally by thioguanosine nucleotides, and is amplified by inflammatory leukocytes entering the liver. A novel scoring system defining SOS was created based on pathology of the central vein, sinusoids and perivenular parenchyma. Wildtype, hypoxanthine-guanine phosphoribosyltransferase (Hprt) -/-, PE-selectin -/-, Winnie (Muc2 single nucleotide mutation) or RaW (cross Winnie /Rag1 -/-) male or female C57Bl/6 mice, were gavaged daily with 100µL of tap water (vehicle control) or drugs in the same volume of water for periods up to 28 days. The administered drugs were 6TG (0-5 mg/kg/d) or 6MP, methyl-6MP or methyl-6TG (≤ 5 mg/kg/d). Furthermore, 6TG effects were also characterised in vivo in different haematopoietic organs and in vitro on hepatic and endothelial cell lines. The safe administration of 6TG in IBD patients would translate into a huge therapeutic value, providing both a faster onset of action and an efficacious treatment

Microbiome shifts with 6-Thioguanine provide insights about IBD and its treatment

The microbiome is a major determinant of colonic health. Shifts in the microbiome community (dysbiosis) have been reported in IBD but interpretation of dysbiosis is fraught by confounding factors that include a large interindividual variation due to many factors including the genetics of the host glycobiome and mucosal immune system, stochastic microbiota founder effects and exogenous substrate.SCIRO Preventative Health National Research Flagship and Division of Livestock Industries; The University of Queensland; Mater Medical Research InstituteN

The role of IL-22 in the resolution of sterile and nonsterile inflammation

In a broad sense, inflammation can be conveniently characterised by two phases: the first phase, which is a pro-inflammatory, has evolved to clear infection and/or injured tissue; and the second phase concerns regeneration of normal tissue and restitution of normal physiology. Innate immune cell-derived pro-inflammatory cytokines and chemokines activate and recruit nonresident immune cells to the site of infection, thereby amplifying the inflammatory responses to clear infection or injury. This phase is followed by a cytokine milieu that promotes tissue regeneration. There is no absolute temporal distinction between these two phases, and cytokines may have dual pleiotropic effects depending on the timing of release, inflammatory microenvironment or concentrations. IL-22 is a cytokine with reported pro- and anti-inflammatory roles; in this review, we contend that this protein has primarily a function in restitution of normal tissue and physiology