Absinthin, an agonist of the bitter taste receptor hTAS2R46, uncovers an ER-to-mitochondria Ca2–shuttling event

Type 2 taste receptors (TAS2R) are G protein-coupled receptors first described in the gustatory system, but have also been shown to have extra-oral localizations, including airway smooth muscle (ASM) cells, in which TAS2R have been reported to induce relaxation. TAS2R46 is an unexplored subtype that responds to its highly specific agonist absinthin. Here, we first demonstrate that, unlike other bitter-taste receptor agonists, absinthin alone (1 μM) in ASM cells does not induce Ca2+ signals, but reduces histamine-induced cytosolic Ca2+ increases. To investigate this mechanism, we introduced into ASM cells aequorin-based Ca2+ probes targeted to the cytosol, sub-plasma membrane domain, or the mitochondrial matrix. We show that absinthin reduces cytosolic histamine-induced Ca2+-rises and simultaneously increases Ca2+-influx into mitochondria. We found that this effect is inhibited by the potent human TAS2R46 (hTAS2R46) antagonist 3β-hydroxydihydrocostunolide and is no longer evident in hTAS2R46-silenced ASM cells, indicating that it is hTAS2R46-dependent. Furthermore, these changes were sensitive to the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP); the mitochondrial calcium uniporter inhibitor KB-R7943 (carbamimidothioic-acid); the cytoskeletal disrupter latrunculin; and an inhibitor of the exchange protein directly activated by cAMP (EPAC), ESI-09. Similarly, the β2 agonist salbutamol also could induce Ca2+ shuttling from cytoplasm to mitochondria, suggesting that this new mechanism might be generalizable. Moreover, forskolin and an EPAC activator mimicked this effect in HeLa cells. Our findings support the hypothesis that plasma membrane receptors can positively regulate mitochondrial Ca2+ uptake, adding a further facet to the ability of cells to encode complex Ca2+ signals

PP272—Migraine and parthenolide inhibition of transient receptor potential ankyrin 1

2013 e103 emerged as a major complication of bortezomib therapy, which usually appears in the first courses of therapy with a number of sensory and painful symptoms, including reduced threshold to mechanical and cold stimuli. No satisfactory explanation or effective treatment exists for bortezomib-evoked CIPN. Patients (or Materials) and Methods: In this study, we evaluated whether TRPA1 acted as a critical mediator of CIPN by bortezomib or oxaliplatin in a mouse model system. Results: Our data demonstrated that CIPN hypersensitivity phenotype that was stably established by bortezomib could be transiently reverted by systemic or local treatment with the TRPA1 antagonist HC-030031. A similar effect was produced by the oxidative stress scavenger α -lipoic acid. Notably, the CIPN phenotype was abolished completely in mice that were genetically deficient in TRPA1, highlighting its essential role. Administration of bortezomib or oxaliplatin, which also elicits TRPA1-dependent hypersensitivity, produced a rapid, transient increase in plasma of carboxy-methyllysine, a byproduct of oxidative stress. Short-term systemic treatment with either HC-030031 or α -lipoic acid could completely prevent hypersensitivity if administered before the cytotoxic drug. Conclusion: Our findings highlight a key role for early activation/ sensitization of TRPA1 by oxidative stress by-products in producing CIPN. Furthermore, they suggest prevention strategies for CIPN in patients through the use of early, short-term treatments with TRPA1 antagonists. Disclosure of Interest: None declared

First Report of Coniella granati as a Causal Agent of Pomegranate Crown Rot in Southern Italy

Pomegranate (Punica granatum L.), grown in Italy since ancient times, recently became an important crop for the human health benefits associated with fruit consumption. In the summer 2014 and 2015, crown rot symptoms were observed on 20 to 30% of the pomegranate trees (cultivars Wonderful One, Ako, and Mollar de Elche) in 20 different orchards in Apulia (North-East of Bari and South Salento), Basilicata (Metapontum area), and Calabria (Catanzaro province) and, rarely, a fruit rot was also observed. Crown rot-affected plants showed reduced growth, small, yellowish leaves and decline and ultimately death. In seven orchards at least three 2- to 5-year-old plants were sampled, and in two orchards sampling was carried out twice in 2014 and 2015. A brown-black wood discoloration was extensive in longitudinal sections and affected up to 50% of the cross-sectional area. Samples of decayed wood were placed on Water Agar in petri dishes and cultures kept 7 days at 25 ± 1°C in the dark. After 10 days, the pure colonies obtained on potato dextrose agar (PDA) of 33 selected isolates, were 80 to 90 mm in diameter and the mycelium was white to light yellow. Pycnidia were solitary, globose, and brownish to black. Hyphae were septate and conidia were hyaline, one-celled, ellipsoid to fusiform (12.1 to 15.3 × 4.0 to 4.5 μm). All the isolates showed morphological features were consistent with Coniella granati (Sacc.) Petr. & Syd (syn. Pilidiella granati Sacc.) (Van Niekerk et al. 2004). ITS genomic regions were sequenced for molecular identification. DNA was extracted from 3-day-old mycelium of a representative isolate, amplified by PCR using the universal ITS5/ITS4 primers, and the amplicon was sequenced by external service (Macrogen, Seoul, South Korea). BLASTn analysis of the ITS sequence (562 bps) obtained showed 99 to 100% of identity (E value = 0.0, coverage = 86 to 99%) with Pilidiella (= Coniella) granati (i.e., GenBank Accession Nos. JN815312.1 and KT852449.1). Consequently, the pathogen was ascribed to C. granati and its ITS sequence was deposited in GenBank (KU147239). Pathogenicity tests were carried out on 10 replicated 5-month-old potted plants of each of the pomegranate cultivars Wonderful One and Mollar de Elche. Five-millimeter mycelial plugs from the edge of 15-day-old colonies on PDA were placed in artificial wounds (5 mm long and 3 mm in deep) under crown bark and then protected with a layer of parafilm. PDA medium plugs were used as the noninoculated control. Plants were maintained in the greenhouse (25 ± 2°C; 16-h daylight photoperiod). Six months after inoculation, a wood decay extending 3.0 to 3.5 cm from the inoculation site was observed only on inoculated plants. C. granati was always reisolated only from decayed bark of inoculated plants, hence satisfying the Koch’s postulates. The pathogen was recently reported on pomegranate associated with a fruit rot in China, Iran, and Spain, and with a crown rot in Greece (Thomidis and Exadaktylou 2011) and Turkey (Çeliker et al. 2012). As far as we are aware, this is the first report of C. granati in Italy associated with a severe crown rot decline. It can be expected that severe damage will be caused by the pathogen owing to its rapid spreading to new pomegranate orchards. References: Çeliker, N. M., et al. 2012. Australas. Plant Dis. Notes 7:161. 10.1007/s13314-012-0074-6 Thomidis, T., and Exadaktylou, E. 2011. Plant Dis. 95:79. 10.1094/PDIS-07-10-0514 Van Niekerk, J. M., et al. 2004. Mycol. Res. 108:283. 10.1017/S095375620400926