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

Characterization of triptolide-induced hepatotoxicity by imaging and transcriptomics in a novel zebrafish model

Triptolide is a vine extract used in traditional Chinese medicines and associated with hepatotoxicity. In vitro data suggest that inhibition of RNA synthesis may be the mechanism of toxicity. For studying drug-induced liver injury the zebrafish has experimental, practical and financial advantages compared with rodents. The aim of this study was to explore the mechanism of triptolide toxicity using zebrafish as the model system. The effect of triptolide exposure on zebrafish larvae was determined with regard to mortality, histology, expression of liver specific microRNA-122 and liver volume. Fluorescent microscopy was used to track toxicity in the Tg(-2.8lfabp:GFP)as3 zebrafish line. Informed by microscopy, RNA-sequencing was used to explore the mechanism of toxicity. Triptolide exposure resulted in dose-dependent mortality, a reduction in the number of copies of microRNA-122 per larva, hepatocyte vacuolation, disarray and oncotic necrosis, and a reduction in liver volume. These findings were consistent across replicate experiments. Time-lapse imaging indicated the onset of injury was 6 h after the start of exposure, at which point, RNA-sequencing revealed that 88% of genes were down-regulated. Immune response associated genes were up-regulated in the triptolide-treated larvae including nitric oxide synthase. Inhibition of nitric oxide synthase increased mortality. Triptolide induces hepatotoxicity in zebrafish larvae. This represents a new model of drug-induced liver injury that complements rodents. RNA sequencing, guided by time-lapse microscopy, revealed early down-regulation of genes consistent with previous invitro studies, and facilitated the discovery of mechanistic inflammatory pathways

Validation of separate multi-atlases for auto segmentation of cardiac substructures in CT-scans acquired in deep inspiration breath hold and free breathing

Background and purpose: Developing NTCP-models for cardiac complications after breast cancer (BC) radiotherapy requires cardiac dose-volume parameters for many patients. These can be obtained by using multi-atlas based automatic segmentation (MABAS) of cardiac structures in planning CT scans. We investigated the relevance of separate multi-atlases for deep inspiration breath hold (DIBH) and free breathing (FB) CT scans. Materials and methods: BC patients scanned in DIBH (n = 10) and in FB (n = 20) were selected to create separate multi-atlases consisting of expert panel delineations of the whole heart, atria and ventricles. The accuracy of atlas-generated contours was validated with expert delineations in independent datasets (n = 10 for DIBH and FB) and reported as Dice coefficients, contour distances and dose-volume differences in relation to interobserver variability of manual contours. Dependency of MABAS contouring accuracy on breathing technique was assessed by validation of a FB atlas in DIBH patients and vice versa (cross validation). Results: For all structures the FB and DIBH atlases resulted in Dice coefficients with their respective reference contours > 0.8 and average contour distances < 2 mm smaller than slice thickness of (CTs). No significant differences were found for dose-volume parameters in volumes receiving relevant dose levels (WH, LV and RV). Accuracy of the DIBH atlas was at least similar to, and for the ventricles better than, the interobserver variation in manual delineation. Cross-validation between breathing techniques showed a reduced MABAS performance. Conclusion: Multi-atlas accuracy was at least similar to interobserver delineation variation. Separate atlases for scans made in DIBH and FB could benefit atlas performance because accuracy depends on breathing technique

Reduction of adverse effects from intravenous acetylcysteine treatment for paracetamol poisoning: a randomised controlled trial:a randomised controlled trial

Copyright © 2014 Bateman et al. Open Access article distributed under the terms of CC BY. Published by Elsevier Ltd. All rights reserved. The trial was funded by Chief Scientist Office, Scotland (award CZB/4/722).Peer reviewedPublisher PD

YRITYKSEN KASVUSTRATEGIAN SUUNNITTELU

Työ on tehty ulkovalaistuksen asiantuntijayritykselle nimeltä FIXULUX OY, jonka toiminta on käynnistynyt muutamia vuosia sitten. Yritys on vakiinnuttanut asemansa toimialalla ja ajatuksena on kasvattaa ja kehittää liiketoimintaa. Yrityksen päätuotteita ovat valmistajista riippumattomat, puolueettomat valaistussuunnittelu- ja asiantuntijapalvelut kattaen koko ulkovalaistuksen. Työssä kerättiin teoriatietoa liiketoiminnan kehittämisestä ja kasvattamisesta strategisen johtamisen keinoin ja kerätyistä tiedoista koottiin teoriaosuuteen referaatti. Tutkimuksessa selvitettiin mihin asioihin kannattava kasvu mikroyrityksessä voi perustua ja mitkä ovat yrityksen keinot kannattavan kasvun mahdollistamiseksi. Tutkimuksen perusteella mikroyrityksessä kasvu kannattaa perustaa olemassa olevaan osaamiseen, innovaatioihin, asiakaslähtöiseen liiketoimintaan sekä toiminnan sijoittamiseen oikeaan ympäristöön. Yrityksen keinot kannattavan kasvun mahdollistamiseksi ovat tämän tutkimuksen mukaan seuraavat: asiakkuuksien johtaminen, toiminnan ja osaamisen jatkuva kehittäminen, kilpailukykyinen hinnoittelu, ydinliiketoiminnan lähialueelle siirtyminen sekä yritysostot. Teoriapohjaksi on valittu strateginen johtaminen, mutta myös operatiivinen johtaminen nousi tärkeään asemaan työn edetessä. Tässä työssä on hyödynnetty tutkimuksellisen kehitystyön menetelmiä ja lähestymistapana on konstruktiivinen tutkimus. Aineistona on käytetty strategiseen johtamiseen sekä yritystoiminnan kehittämiseen liittyvää kirjallisuutta sekä digitaalisia lähteitä. Konstruktiivisen tutkimuksen päätuloksena tuotettiin kolme kasvuvaihtoehtoa sisältävä palveluliiketoiminnan kasvusuunnitelma. Sisällön perusteella yrityksen johto voi muodostaa oman näkemyksensä, tehdä tarvittavia päätöksiä sekä lähteä kehittämään tarkempaa kasvusuunnitelmaa.This thesis was made as an assignment to an outdoor lighting company called FIXULUX OY which was established a few years ago. The company has established its position in the industry with the idea of expanding and growing its business. The main products of the company are manufacturerindependent and neutral lighting design solutions and expert services throughout all kinds of outdoor lighting. The theory part of this thesis was made by gathering and summarizing information about developing and growing businesses with the help of strategic leadership. Thesis gives answers to questions such as which aspects should be taken into consideration when trying to find profitable growth in a micro sized company and what the means to enable that growth are. Research showed that in a micro sized company growth should be based on existing know-how, innovation, clientbased business and by placing the operations into the right environment. The means to enable profitable growth according to this research are as follows: managing customerships, continuous improvement of operations and know-how, competitive pricing, moving the business close enough to core business areas and business buy-outs. Strategic leadership was selected as the basis of this thesis but approaches from the perspective of operative leadership became equally important once the research progressed. The thesis utilized the research method of development through research with the approach of constructive research. The material used includes literature on strategic leadership and business development as well as digital sources. The main outcome of this thesis is a growth plan for service business including three different approaches for growth. With the help of this content the company management can make up their own opinions, make necessary decisions and start to develop a more precise growth plan

Characterization of triptolide-induced hepatotoxicity by imaging and transcriptomics in a novel zebrafish model

Triptolide is a vine extract used in traditional Chinese medicines and associated with hepatotoxicity. In vitro data suggest that inhibition of RNA synthesis may be the mechanism of toxicity. For studying drug-induced liver injury the zebrafish has experimental, practical and financial advantages compared with rodents. The aim of this study was to explore the mechanism of triptolide toxicity using zebrafish as the model system. The effect of triptolide exposure on zebrafish larvae was determined with regard to mortality, histology, expression of liver specific microRNA-122 and liver volume. Fluorescent microscopy was used to track toxicity in the Tg(-2.8lfabp:GFP)as3 zebrafish line. Informed by microscopy, RNA-sequencing was used to explore the mechanism of toxicity. Triptolide exposure resulted in dose-dependent mortality, a reduction in the number of copies of microRNA-122 per larva, hepatocyte vacuolation, disarray and oncotic necrosis, and a reduction in liver volume. These findings were consistent across replicate experiments. Time-lapse imaging indicated the onset of injury was 6 h after the start of exposure, at which point, RNA-sequencing revealed that 88% of genes were down-regulated. Immune response associated genes were up-regulated in the triptolide-treated larvae including nitric oxide synthase. Inhibition of nitric oxide synthase increased mortality. Triptolide induces hepatotoxicity in zebrafish larvae. This represents a new model of drug-induced liver injury that complements rodents. RNA sequencing, guided by time-lapse microscopy, revealed early down-regulation of genes consistent with previous invitro studies, and facilitated the discovery of mechanistic inflammatory pathways

Sequences of capture probe (1) and miR-122 target (2).

<p>Sequences of capture probe (1) and miR-122 target (2).</p