Sequence determination of phosphorothioated oligonucleotides using MALDI-TOF mass spectrometry for controlling gene doping in equestrian sports.

In human and equestrian sporting events, one method of gene doping is the illegal use of therapeutic oligonucleotides to alter gene expression. In this study, we aimed to identify therapeutic oligonucleotides via sequencing using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). As a model of therapeutic oligonucleotides, 22 bp-long phosphorothioated oligonucleotides (PSOs) were used. By using a Clarity OTX kit for extracting short-length oligonucleotides, a spectrum of singly charged PSO with a mean intensity of 6.08 × 104 (standard deviation: 4.34 × 103 ) was detected from 500 pmol PSO in 1 ml horse plasma using the linear negative mode of MALDI-TOF MS. In addition, a 17 bp sequence was determined using in-source decay (ISD) mode, indicating that 500 pmol of a PSO in 1 ml plasma is the detection limit for sequencing. Using the determined sequences (17 bp), a targeted gene for PSO was singly identified on the horse reference genome, EquCab2.0, via a GGGenome search. These procedures can be potentially used to identify therapeutic oligonucleotides, whose nucleotides are unknown, for gene doping control.

Digital PCR detection of plasmid DNA administered to the skeletal muscle of a microminipig: a model case study for gene doping detection.

OBJECTIVE: Doping control is an important and indispensable aspect of fair horse racing; genetic doping has been recently included to this. In this study, we aimed to develop a detection method of gene doping. A plasmid cloned with human erythropoietin gene (p.hEPO, 250 µg/head) was intramuscularly injected into a microminipig. Subsequently, p.hEPO was extracted from 1 mL of plasma and detected by droplet digital polymerase chain reaction. RESULTS: The results confirmed that the maximum amount of plasmid was detected at 15 min after administration and the majority of the plasmid was degraded in the bloodstream within 1-2 days after administration. In contrast, low amounts of p.hEPO were detected at 2-3 weeks after administration. These results suggest that the proposed method to detect gene doping can help obtain information for experiments using horses.

Urinary neutrophil gelatinase-associated lipocalin: a useful biomarker for tacrolimus-induced acute kidney injury in liver transplant patients.

Tacrolimus is widely used as an immunosuppressant in liver transplantation, and tacrolimus-induced acute kidney injury (AKI) is a serious complication of liver transplantation. For early detection of AKI, various urinary biomarkers such as monocyte chemotactic protein-1, liver-type fatty acid-binding protein, interleukin-18, osteopontin, cystatin C, clusterin and neutrophil gelatinase-associated lipocalin (NGAL) have been identified. Here, we attempt to identify urinary biomarkers for the early detection of tacrolimus-induced AKI in liver transplant patients. Urine samples were collected from 31 patients after living-donor liver transplantation (LDLT). Twenty recipients developed tacrolimus-induced AKI. After the initiation of tacrolimus therapy, urine samples were collected on postoperative days 7, 14, and 21. In patients who experienced AKI during postoperative day 21, additional spot urine samples were collected on postoperative days 28, 35, 42, 49, and 58. The 8 healthy volunteers, whose renal and liver functions were normal, were asked to collect their blood and spot urine samples. The urinary levels of NGAL, monocyte chemotactic protein-1 and liver-type fatty acid-binding protein were significantly higher in patients with AKI than in those without, while those of interleukin-18, osteopontin, cystatin C and clusterin did not differ between the 2 groups. The area under the receiver operating characteristics curve of urinary NGAL was 0.876 (95% confidence interval, 0.800-0.951; P<0.0001), which was better than those of the other six urinary biomarkers. In addition, the urinary levels of NGAL at postoperative day 1 (p = 0.0446) and day 7 (p = 0.0006) can be a good predictive marker for tacrolimus-induced AKI within next 6 days, respectively. In conclusion, urinary NGAL is a sensitive biomarker for tacrolimus-induced AKI, and may help predict renal event caused by tacrolimus therapy in liver transplant patients.

Impact of cytochrome P450 3A5 polymorphism in graft livers on the frequency of acute cellular rejection in living-donor liver transplantation.

OBJECTIVE: We investigated whether the cytochrome P450 3A5*3 (CYP3A5*3) genotype affects tacrolimus pharmacokinetics and the risk of acute cellular rejection in living-donor liver transplant patients in Japan. MATERIALS AND METHODS: Between July 2004 and June 2011, we enrolled 410 living-donor liver transplant patients receiving tacrolimus. Biopsy specimens of intestinal mucosa and graft liver at surgery were obtained to examine the mRNA expression of CYP3A subfamilies as well as the genotyping of CYP3A5*3 polymorphism. RESULTS: The CYP3A5 genotype in the native intestine had no significant effect on the occurrence of acute cellular rejection between postoperative days 14 and 23 in cases with identical or compatible ABO blood types (11.5% for the CYP3A5*1 allele vs. 7.4% for CYP3A5*3/*3; P=0.2643), although the concentration/dose ratio of tacrolimus was significantly higher in patients with the intestinal CYP3A5*3/*3 genotype than in those with the CYP3A5*1 allele for 5 post-transplant weeks. However, patients who received a graft liver with the CYP3A5*1 allele showed a higher rate of acute cellular rejection than those who received a graft liver with the CYP3A5*3/*3 genotype (14.5 vs. 5.7%; P=0.0134). The relative risk for acute cellular rejection associated with the CYP3A5*1 liver allele was 2.629 (P=0.018, Cox regression model). Consequently, graft liver CYP3A5*1 genotype might increase the risk for acute cellular rejection after living-donor liver transplantation, possibly by associating with the local hepatic tacrolimus concentration. CONCLUSIONS: The target level of tacrolimus may be affected by the CYP3A5*3 genotype of the liver, rather than by that of the small intestine, after postoperative day 14.