Targeting succinate: ubiquinone reductase potentiates the efficacy of anticancer therapy

Mitochondria play a pivotal role in apoptosis: permeabilization of the outer mitochondrial membrane and the release of pro-apoptotic proteins from the intermembrane space of mitochondria are regarded as the key event in apoptosis induction. Here we demonstrate how non-toxic doses of the mitochondrial Complex II inhibitor thenoyltrifluoroacetone (TTFA), which specifically inhibits the ubiquinone-binding site of succinate dehydrogenase (SDH), synergistically stimulated cell death, induced by harmless doses of cisplatin in a panel of chemoresistant neuroblastoma cell lines. Apoptotic cell death was confirmed by cytochrome c release from the mitochondria, cleavage of poly ADP-ribose polymerase, processing of caspase-3, which is an important executive enzyme in apoptosis, and caspase-3-like activity. Methyl malonate, an inhibitor of the SDHA subunit partially reversed apoptosis stimulated by TTFA in SK-N-BE(2) neuroblastoma cells (NB), indicating that sensitization requires oxidation of succinate. In contrast, in IMR-32 NB cells, the same concentrations of TTFA markedly suppressed cisplatin-induced apoptosis. Comparison of oxygen consumption in cisplatin-resistant SK-N-BE(2) and cisplatin-sensitive IMR-32 cells clearly demonstrated impaired Complex II activity in IMR-32 cells. We also found that in SK-N-BE(2) cells co-treatment with cisplatin and TTFA markedly stimulated formation of reactive oxygen species (ROS), whereas in IMR cells, cisplatin-mediated ROS production was attenuated by TTFA, which explains apoptosis suppression in these cells. Thus, functionally active SDH is a prerequisite for the ROS-mediated sensitization to treatment by TTFA

Video observation of encounters between the automated shuttles and other traffic participants along an approach to right-hand priority T-intersection

Introduction: Increasing numbers of deployment projects of automated shuttles have been taking place worldwide. Safety is one of the main concerns for their successful implementation. Therefore, it is vital to gain the knowledge about interactions between these shuttles and other traffic participants. Method: Given the lack of behavioural observational studies under regular traffic conditions, the presented study applies external video recordings to explore encounters between the shuttles approaching a T-intersection and other traffic participants. The encounters of interest included a vulnerable road user in the bicycle lane, a pedestrian on the zebra crossing and a road user overtaking the shuttle. The shuttles were identified from the video by RUBA software. We analysed the encounters using T-Analyst software together with the manual observation of traffic participants' behaviour. Results: From 220 h of video, 318 unique manoeuvres of the shuttle were observed and 83 encounters with other traffic participants were identified and explored. Several types of risks and behavioural patterns were identified, such as road users misusing the defensive style of the shuttles or cyclists in the bicycle lane not being sure about the shuttle’s intention. Frequent hard stops of the shuttles might be dangerous for the passengers inside and can increase the risk of rear end accidents. Conclusions: The findings provide a valuable insight into the interactions between automated shuttles and other traffic participants under regular traffic conditions on one location in Oslo, Norway. The study showed that introducing automated shuttles into regular traffic can lead to the emergence of new types of interactions between the shuttles and other traffic participants.Transport and Plannin

Genetic structure and diversity of European chestnut (Castanea sativa Mill.) populations in western Balkans: On a crossroad between east and west

European chestnut (Castanea sativa Mill.) is highly valued in the western Balkans as a source of timber and fruit, but also as an important source of nectar and pollen for the production of honey. In this study, four chestnut populations, covering a major portion of the western Balkans, and a reference population from the northern Italy were examined using 21 microsatellite markers. The highest genetic diversity was detected within the populations geographically closest to the Italian Peninsula, which also displayed the highest level of admixture with the samples from Italy. The strongest genetic differentiation was noted among the southern and eastern chestnut populations from Bosnia and Herzegovina (B&H) (Gst=12.05%). This pronounced differentiation is probably caused by the genetic adaptations to notably different climatic conditions present in the south (Mediterranean climate) and east (Continental climate) of B&H. The clear genetic differentiation of the southern and eastern B&H chestnut populations from the Italian population, determined by pairwise Gst, FCA and Bayesian Structure analyses, indicates that these populations most likely originated from independent shelter zones (refugia), after the last glaciation period. Based on these results we propose a presence of an introgression zone in the northwestern Balkans, established through gene flow from the Italian and the Balkan Peninsula. The obtained insights into the structure of all analyzed populations will significantly contribute towards establishing a regional conservation and utilization strategy for European chestnut in western Balkans

Interaction of Dietary Fatty Acids with Tumour Necrosis Factor Family Cytokines during Colon Inflammation and Cancer

Intestinal homeostasis is precisely regulated by a number of endogenous regulatory molecules but significantly influenced by dietary compounds. Malfunction of this system may result in chronic inflammation and cancer. Dietary essential n-3 polyunsaturated fatty acids (PUFAs) and short-chain fatty acid butyrate produced from fibre display anti-inflammatory and anticancer activities. Both compounds were shown to modulate the production and activities of TNF family cytokines. Cytokines from the TNF family (TNF-α, TRAIL, and FasL) have potent inflammatory activities and can also regulate apoptosis, which plays an important role in cancer development. The results of our own research showed enhancement of apoptosis in colon cancer cells by a combination of either docosahexaenoic acid (DHA) or butyrate with TNF family cytokines, especially by promotion of the mitochondrial apoptotic pathway and modulation of NFκB activity. This review is focused mainly on the interaction of dietary PUFAs and butyrate with these cytokines during colon inflammation and cancer development. We summarised recent knowledge about the cellular and molecular mechanisms involved in such effects and outcomes for intestinal cell behaviour and pathologies. Finally, the possible application for the prevention and therapy of colon inflammation and cancer is also outlined

Lowering levels of reelin in entorhinal cortex layer II-neurons results in lowered levels of intracellular amyloid-β

Projection neurons in the anteriolateral part of entorhinal cortex layer II are the predominant cortical site for hyper-phosphorylation of tau and formation of neurofibrillary tangles in prodromal Alzheimer's disease. A majority of layer II projection neurons in anteriolateral entorhinal cortex are unique among cortical excitatory neurons by expressing the protein reelin. In prodromal Alzheimer's disease, these reelin-expressing neurons are prone to accumulate intracellular amyloid-β, which is mimicked in a rat model that replicates the spatio-temporal cascade of the disease. Two important findings in relation to this are that reelin-signalling downregulates tau phosphorylation, and that oligomeric amyloid-β interferes with reelin-signalling. Taking advantage of this rat model, we used proximity ligation assay to assess whether reelin and intracellular amyloid-β directly interact during early, pre-plaque stages in anteriolateral entorhinal cortex layer II reelin-expressing neurons. We next made a viral vector delivering micro-RNA against reelin, along with a control vector, and infected reelin-expressing anteriolateral entorhinal cortex layer II-neurons to test whether reelin levels affect levels of intracellular amyloid-β and/or amyloid precursor protein. We analysed 25.548 neurons from 24 animals, which results in three important findings. First, in reelin-expressing anteriolateral entorhinal cortex layer II-neurons, reelin and intracellular amyloid-β engage in a direct protein-protein interaction. Second, injecting micro-RNA against reelin lowers reelin levels in these neurons, amounting to an effect size of 1.3-4.5 (Bayesian estimation of Cohen's d effect size, 95% credible interval). This causes a concomitant reduction of intracellular amyloid-β ranging across three levels of aggregation, including a reduction of Aβ42 monomers/dimers amounting to an effect size of 0.5-3.1, a reduction of Aβ prefibrils amounting to an effect size of 1.1-3.5 and a reduction of protofibrils amounting to an effect size of 0.05-2.1. Analysing these data using Bayesian estimation of mutual information furthermore reveals that levels of amyloid-β are dependent on levels of reelin. Third, the reduction of intracellular amyloid-β occurs without any substantial associated changes in levels of amyloid precursor protein. We conclude that reelin and amyloid-β directly interact at the intracellular level in the uniquely reelin-expressing projection neurons in anteriolateral entorhinal cortex layer II, where levels of amyloid-β are dependent on levels of reelin. Since amyloid-β is known to impair reelin-signalling causing upregulated phosphorylation of tau, our findings are likely relevant to the vulnerability for neurofibrillary tangle-formation of this entorhinal neuronal population