A case of acute intoxication due to combined use of fentanyl and 3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide (U-47700)

&lt;p&gt;A 30-year old man was found dead in his home after inhaling fumes of a powder burned on aluminum foil. Blood and urine were taken by the medical examiner during the external body examination and submitted to the laboratory for a comprehensive systematic toxicological analysis. A toxic fentanyl level of 10.9μg/L was measured in the subclavian blood. Police investigation revealed that the man searched the internet for information on new psychotropic substances, among others including U-47700. A powder found in the victims&#039; home was transferred to the laboratory for analysis, in which trace amounts of fentanyl (0.0035%, m/m) and U-47700 (0.0012%, m/m) were identified by gas chromatography mass spectrometry. U-47700 is an opioid analgesic drug, considered to have a potency of approximately 7.5 times that of morphine. A target analysis on U-47700 was performed using liquid-liquid extraction and ultra performance liquid chromatography tandem mass spectrometry operating in multiple reaction monitoring mode. The method validation was based on the Scientific Working Group of Forensic Toxicology document &#039;Standard Practices of Method Validation in Forensic Toxicology&#039;. In blood and urine the U-47700 concentration was 13.8 and 71.0μg/L, respectively. To the author&#039;s knowledge, this is the first case report of a fatal intoxication involving U-47700 abused as a new psychotropic substance.&lt;/p&gt;</p

Towards understanding the child’s experience in the process of parentification: young adults’ reflections on growing up with a depressed parent

This article reports on a qualitative study with 21 young adults who grew up with a depressed parent. We examined how young adults make sense of their childhood experiences of parental depression and how their retrospective reflections help us to understand the experiences of children and the processes of parentification. Participants recounted that their childhood consisted mainly of actions in the service of family well-being. At that time, they reflected on their own experiences only rarely. In adolescence, there was an evolution toward a greater consideration for oneself and a repositioning within the family. In the discussion, we explore the therapeutic implications of this studyand in particularthe meaningfulness of silence in the family process of parentification

RhoA GTPase switch controls Cx43-hemichannel activity through the contractile system

ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine. In this study we investigated the involvement of RhoA activation in the inhibition of hemichannel-mediated ATP release in BCEC. We found that RhoA activation occurs rapidly and transiently upon thrombin treatment of BCEC. The RhoA activity correlated with the onset of actomyosin contractility that is involved in the inhibition of Cx43 hemichannels. RhoA activation and inhibition of Cx43-hemichannel activity were both prevented by pre-treatment of the cells with C3-toxin as well as knock down of RhoA by siRNA. These findings provide evidence that RhoA activation is a key player in thrombin-induced inhibition of Cx43-hemichannel activity. This study demonstrates that RhoA GTPase activity is involved in the acute inhibition of ATP-dependent paracrine signaling, mediated by Cx43 hemichannels, in response to the inflammatory mediator thrombin. Therefore, RhoA appears to be an important molecular switch that controls Cx43 hemichannel openings and hemichannel-mediated ATP-dependent paracrine intercellular communication under (patho) physiological conditions of stress

The type 2 inositol 1,4,5-trisphosphate receptor, emerging functions for an intriguing Ca2+-release channel

AbstractThe inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) type 2 (IP3R2) is an intracellular Ca2+-release channel located on the endoplasmic reticulum (ER). IP3R2 is characterized by a high sensitivity to both IP3 and ATP and is biphasically regulated by Ca2+. Furthermore, IP3R2 is modulated by various protein kinases. In addition to its regulation by protein kinase A, IP3R2 forms a complex with adenylate cyclase 6 and is directly regulated by cAMP. Finally, in the ER, IP3R2 is less mobile than the other IP3R isoforms, while its functional properties appear dominant in heterotetramers. These properties make the IP3R2 a Ca2+ channel with exquisite properties for setting up intracellular Ca2+ signals with unique characteristics. IP3R2 plays a crucial role in the function of secretory cell types (e.g. pancreatic acinar cells, hepatocytes, salivary gland, eccrine sweat gland). In cardiac myocytes, the role of IP3R2 appears more complex, because, together with IP3R1, it is needed for normal cardiogenesis, while its aberrant activity is implicated in cardiac hypertrophy and arrhythmias. Most importantly, its high sensitivity to IP3 makes IP3R2 a target for anti-apoptotic proteins (e.g. Bcl-2) in B-cell cancers. Disrupting IP3R/Bcl-2 interaction therefore leads in those cells to increased Ca2+ release and apoptosis. Intriguingly, IP3R2 is not only implicated in apoptosis but also in the induction of senescence, another tumour-suppressive mechanism. These results were the first to unravel the physiological and pathophysiological role of IP3R2 and we anticipate that further progress will soon be made in understanding the function of IP3R2 in various tissues and organs

Highlights Analysis System (HAnS) for low dynamic range to high dynamic range conversion of cinematic low dynamic range content

We propose a novel and efficient algorithm for detection of specular reflections and light sources (highlights) in cinematic content. The detection of highlights is important for reconstructing them properly in the conversion of the low dynamic range (LDR) to high dynamic range (HDR) content. Highlights are often difficult to be distinguished from bright diffuse surfaces, due to their brightness being reduced in the conventional LDR content production. Moreover, the cinematic LDR content is subject to the artistic use of effects that change the apparent brightness of certain image regions (e.g. limiting depth of field, grading, complex multi-lighting setup, etc.). To ensure the robustness of highlights detection to these effects, the proposed algorithm goes beyond considering only absolute brightness and considers five different features. These features are: the size of the highlight relative to the size of the surrounding image structures, the relative contrast in the surrounding of the highlight, its absolute brightness expressed through the luminance (luma feature), through the saturation in the color space (maxRGB feature) and through the saturation in white (minRGB feature). We evaluate the algorithm on two different image data-sets. The first one is a publicly available LDR image data-set without cinematic content, which allows comparison to the broader State of the art. Additionally, for the evaluation on cinematic content, we create an image data-set consisted of manually annotated cinematic frames and real-world images. For the purpose of demonstrating the proposed highlights detection algorithm in a complete LDR-to-HDR conversion pipeline, we additionally propose a simple inverse-tone-mapping algorithm. The experimental analysis shows that the proposed approach outperforms conventional highlights detection algorithms on both image data-sets, achieves high quality reconstruction of the HDR content and is suited for use in LDR-to-HDR conversion

Basal ryanodine receptor activity suppresses autophagic flux

The inositol 1,4,5-trisphosphate receptors (IP3Rs) and intracellular Ca2+ signaling are critically involved in regulating different steps of autophagy, a lysosomal degradation pathway. The ryanodine receptors (RyR), intracellular Ca2+-release channels mainly expressed in excitable cell types including muscle and neurons, have however not yet been extensively studied in relation to autophagy. Yet, aberrant expression and excessive activity of RyRs in these tissues has been implicated in the onset of several diseases including Alzheimer’s disease, where impaired autophagy regulation contributes to the pathology. In this study, we determined whether pharmacological RyR inhibition could modulate autophagic flux in ectopic RyR-expressing models, like HEK293 cells and in cell types that endogenously express RyRs, like C2C12 myoblasts and primary hippocampal neurons. Importantly, RyR3 overexpression in HEK293 cells impaired the autophagic flux. Conversely, in all cell models tested, pharmacological inhibition of endogenous or ectopically expressed RyRs, using dantrolene or ryanodine, augmented autophagic flux by increasing lysosomal turn-over (number of autophagosomes and autolysosomes measured as mCherry-LC3 punctae/cell increased from 70.37 ± 7.81 in control HEK RyR3 cells to 111.18 ± 7.72 and 98.14 ± 7.31 after dantrolene and ryanodine treatments, respectively). Moreover, in differentiated C2C12 cells, transmission electron microscopy demonstrated that dantrolene treatment decreased the number of early autophagic vacuoles from 5.9 ± 2.97 to 1.8 ± 1.03 per cellular cross section. The modulation of the autophagic flux could be linked to the functional inhibition of RyR channels as both RyR inhibitors efficiently diminished the number of cells showing spontaneous RyR3 activity in the HEK293 cell model (from 41.14% ± 2.12 in control cells to 18.70% ± 2.25 and 9.74% ± 2.67 after dantrolene and ryanodine treatments, respectively). In conclusion, basal RyR-mediated Ca2+-release events suppress autophagic flux at the level of the lysosomes

Calcium puffs are generic InsP₃-activated elementary calcium signals and are downregulated by prolonged hormonal stimulation to inhibit cellular calcium responses

Elementary Ca2+ signals, such as "Ca2+ puffs", which arise from the activation of inositol 1,4,5-trisphosphate receptors, are building blocks for local and global Ca2+ signalling. We characterized Ca2+ puffs in six cell types that expressed differing ratios of the three inositol 1,4,5-trisphosphate receptor isoforms. The amplitudes, spatial spreads and kinetics of the events were similar in each of the cell types. The resemblance of Ca2+ puffs in these cell types suggests that they are a generic elementary Ca2+ signal and, furthermore, that the different inositol 1,4,5-trisphosphate isoforms are functionally redundant at the level of subcellular Ca2+ signalling. Hormonal stimulation of SH-SY5Y neuroblastoma cells and HeLa cells for several hours downregulated inositol 1,4,5-trisphosphate expression and concomitantly altered the properties of the Ca2+ puffs. The amplitude and duration of Ca2+ puffs were substantially reduced. In addition, the number of Ca2+ puff sites active during the onset of a Ca2+ wave declined. The consequence of the changes in Ca2+ puff properties was that cells displayed a lower propensity to trigger regenerative Ca2+ waves. Therefore, Ca2+ puffs underlie inositol 1,4,5-trisphosphate signalling in diverse cell types and are focal points for regulation of cellular responses

Ryanodine receptors are targeted by anti-apoptotic Bcl-X-L involving its BH4 domain and Lys87 from its BH3 domain

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) family members target several intracellular Ca2+-transport systems. Bcl-2, via its N-terminal Bcl-2 homology (BH) 4 domain, inhibits both inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs), while Bcl-X-L, likely independently of its BH4 domain, sensitizes IP3Rs. It remains elusive whether Bcl-XL can also target and modulate RyRs. Here, Bcl-X-L co-immunoprecipitated with RyR3 expressed in HEK293 cells. Mammalian protein-protein interaction trap (MAPPIT) and surface plasmon resonance (SPR) showed that Bcl-XL bound to the central domain of RyR3 via its BH4 domain, although to a lesser extent compared to the BH4 domain of Bcl-2. Consistent with the ability of the BH4 domain of Bcl-X-L to bind to RyRs, loading the BH4-Bcl-X-L peptide into RyR3-overexpressing HEK293 cells or in rat hippocampal neurons suppressed RyR-mediated Ca2+ release. In silico superposition of the 3D-structures of Bcl-2 and Bcl-XL indicated that Lys87 of the BH3 domain of Bcl-XL could be important for interacting with RyRs. In contrast to Bcl-X-L, the Bcl-X-L(K87D) mutant displayed lower binding affinity for RyR3 and a reduced inhibition of RyR-mediated Ca2+ release. These data suggest that Bcl-X-L binds to RyR channels via its BH4 domain, but also its BH3 domain, more specific Lys87, contributes to the interaction