Development of circulating microRNA in drug-induced liver injury: studies in humans and zebrafish

The aim of these studies was to identify circulating miRNAs that can be used as biomarkers in patients with paracetamol-induced liver injury. Whether the miRNAs discovered in humans could be back-translated to zebrafish with the aim of developing a liver toxicity model to replace rodent use was also investigated. First, the miRNA signature of DILI induced by paracetamol was defined. Plasma miRNAs were quantified in paracetamol overdose patients. A signature of 16 miRNAs was discovered that best separated patients with liver injury from those without liver injury. This signature was tested in a second cohort and resulted in the detection of paracetamol-induced liver injury with high specificity and sensitivity. At first presentation to hospital miR-122-5p was the most sensitive single miRNA and superior to ALT activity in predicting liver injury. In order to further qualify miR-122-5p, three detailed studies relevant to possible clinical scenarios were performed. The effect of acute alcohol ingestion (commonly co-ingested with paracetamol overdose) on circulating concentrations of miR-122-5p in healthy volunteers was investigated. Alcohol ingestion induced a small, non-clinically relevant, increase in miR-122-5p. The effect of chronic kidney disease (CKD) and haemodialysis (HD) on circulating miR-122-5p concentrations was explored because kidney dysfunction has been associated with a reduction in the concentration of circulating miRNAs. HD patients had lower concentrations of miR- 122-5p compared to healthy volunteers and CKD patients. To facilitate miRNA measurement outwith hospitals, miR-122-5p was measured in a blood drop from a finger prick. miR-122-5p was readily measurable in finger prick samples and concentrations were significantly higher in the blood drop from DILI patients compared with healthy volunteers. To complement miR-122-5p as a marker of toxicity, circulating paracetamol metabolites were measured in plasma samples from paracetamol overdose patients. A higher percentage of circulating metabolites formed by cytochrome P450 enzymes were present in patients with liver injury and these metabolites were superior to both ALT and paracetamol concentration with regard to early patient stratification. To reduce need for rodent studies, miRNAs were back-translated into zebrafish. In order to study circulating miR-122-5p in adult zebrafish, a bloodletting method by collecting blood retro-orbitally was developed. After studying different dosing regimens of paracetamol in adult and larvae zebrafish the model was determined to be too variable with regard to liver injury. A new drug, triptolide, originating from traditional Chinese medicine and responsible for DILI in China, was tested as an alternative model for drug-induced liver injury in zebrafish larvae. miRNA-122-5p decreased in zebrafish larvae after triptolide treatment and triptolide-induced liver injury could be tracked by fluorescent microscopy. Selective plane illumination microscopy was able to track the decrease in liver volume during triptolide exposure. In order to identify the toxic pathways involved in triptolide-induced liver injury, RNA-sequencing was performed. This identified KEGG pathways including ribosome, spliceosome and notch signalling as pathways affected by triptolide. In summary, miRNAs can be used as highly sensitive biomarkers to detect acute liver injury in patients and zebrafish. Zebrafish may represent an alternative model species to study DILI, further work is needed

Dynamic Myocardial Perfusion CT for the Detection of Hemodynamically Significant Coronary Artery Disease

OBJECTIVES In this international, multicenter study, using third-generation dual-source computed tomography (CT), we investigated the diagnostic performance of dynamic stress CT myocardial perfusion imaging (CT-MPI) in addition to coronary CT angiography (CTA) compared to invasive coronary angiography (ICA) and invasive fractional flow reserve (FFR). BACKGROUND CT-MPI combined with coronary CTA integrates coronary artery anatomy with inducible myocardial ischemia, showing promising results for the diagnosis of hemodynamically significant coronary artery disease in single-center studies. METHODS At 9 centers in Europe, Japan, and the United States, 132 patients scheduled for ICA were enrolled; 114 patients successfully completed coronary CTA, adenosine-stress dynamic CT-MPI, and ICA. Invasive FFR was performed in vessels with 25% to 90% stenosis. Data were analyzed by independent core laboratories. For the primary analysis, for each coronary artery the presence of hemodynamically significant obstruction was interpreted by coronary CTA with CT-MPI compared to coronary CTA alone, using an FFR of ≤0.80 and angiographic severity as reference. Territorial absolute myocardial blood flow (MBF) and relative MBF were compared using C-statistics. RESULTS ICA and FFR identified hemodynamically significant stenoses in 74 of 289 coronary vessels (26%). Coronary CTA with ≥50% stenosis demonstrated a per-vessel sensitivity, specificity, and accuracy for the detection of hemodynamically significant stenosis of 96% (95% CI: 91-100), 72% (95% CI: 66-78), and 78% (95% CI: 73-83), respectively. Coronary CTA with CT-MPI showed a lower sensitivity (84%; 95% CI: 75-92) but higher specificity (89%; 95% CI: 85-93) and accuracy (88%; 95% CI: 84-92). The areas under the receiver-operating characteristic curve of absolute MBF and relative MBF were 0.79 (95% CI: 0.71-0.86) and 0.82 (95% CI: 0.74-0.88), respectively. The median dose-length product of CT-MPI and coronary CTA were 313 mGy·cm and 138 mGy·cm, respectively. CONCLUSIONS Dynamic CT-MPI offers incremental diagnostic value over coronary CTA alone for the identification of hemodynamically significant coronary artery disease. Generalized results from this multicenter study encourage broader consideration of dynamic CT-MPI in clinical practice. (Dynamic Stress Perfusion CT for Detection of Inducible Myocardial Ischemia [SPECIFIC]; NCT02810795)

Mechanical Deformation of Spherical Viruses with Icosahedral Symmetry

Virus capsids and crystalline surfactant vesicles are two examples of self-assembled shells in the nano- to micrometer size range. Virus capsids are particularly interesting since they have to sustain large internal pressures while encapsulating and protecting the viral DNA. We therefore study the mechanical properties of crystalline shells of icosahedral symmetry on a substrate under a uniaxial applied force by computer simulations. We predict the elastic response for small deformations, and the buckling transitions at large deformations. Both are found to depend strongly on the number of elementary building blocks N (the capsomers in the case of viral shells), the Föppl-von Kármán number γ (which characterizes the relative importance of shear and bending elasticity), and the confining geometry. In particular, we show that whereas large shells are well described by continuum elasticity-theory, small shells of the size of typical viral capsids behave differently already for small deformations. Our results are essential to extract quantitative information about the elastic properties of viruses and vesicles from deformation experiments