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Background : Hereditary transthyretin-related amyloidosis is a clinically heterogeneous autosomal dominant disease caused by pathogenic variants in the TTR gene (hATTR amyloidosis). Objective: The current study describes the demographic, clinical, and genetic characteristics of patients with suspected hATTR amyloidosis. Methods : This study is part of the "Hereditary transthyretin-related amyloidosis and longitudinal monitoring of TTR -positive patients" (TRAMmoni TTR ) study. This study included 3167 participants, along with their clinical details. Principal component (PC) analysis was used to analyze their clinical symptomatology. Next-generation sequencing of the TTR gene was performed and genotype-phenotype relationships were investigated. We compared the demographic and clinical characteristics using the principal components (PCs) and also compared participants with and without the TTR pathogenic variants. Results: We identified five main clinical phenotypes out of 22 single symptoms that explained 49% of the variation. The first two PCs referred to polyneuropathy and cardiomyopathy. We found significant differences between gender and PC-polyneuropathy and PC-cardiomyopathy, with male over-representation in the higher quantiles of PC-polyneuropathy and male under-representation in the lowest quantiles of PC-cardiomyopathy. We identified 92 participants with hATTR (3%), exhibiting 17 unique heterozygous TTR variants. The p.Val50Met variant was the most frequent. Furthermore, 503 participants (20%) were identified with ATTR and no relevant TTR variants (ATTRwt). We detected significant differences between the ATTRwt and hATTR groups, with male gender predominance in only the ATTRwt group and a positive family history of polyneuropathy and/or cardiomyopathy among the hATTR participants. Conclusions : The current clinical and genetic characterization of this cohort serves as a foundation for further longitudinal monitoring and assessment.

Gaucher disease (GD) is a rare autosomal recessive disorder arising from bi-allelic variants in the GBA1 gene, encoding glucocerebrosidase. Deficiency of this enzyme leads to progressive accumulation of the sphingolipid glucosylsphingosine (lyso-Gb1). The international, multicenter, observational "Lyso-Gb1 as a Long-term Prognostic Biomarker in Gaucher Disease"-LYSO-PROOF study succeeded in enrolling a cohort of 160 treatment-naïve GD patients from diverse geographic regions and evaluated the potential of lyso-Gb1 as a specific biomarker for GD. Using genotypes based on established classifications for clinical presentation, patients were stratified into type 1 GD ( n = 114) and further subdivided into mild ( n = 66) and severe type 1 GD ( n = 48). Due to having previously unreported genotypes, 46 patients could not be classified. Though lyso-Gb1 values at enrollment were widely distributed, they displayed a moderate and statistically highly significant correlation with disease severity measured by the GD-DS3 scoring system in all GD patients (r = 0.602, p < 0.0001). These findings support the utility of lyso-Gb1 as a sensitive biomarker for GD and indicate that it could help to predict the clinical course of patients with undescribed genotypes to improve personalized care in the future.

Increasing evidence suggests that induction of lethal macroautophagy/autophagy carries potential significance for the treatment of glioblastoma (GBM). In continuation of previous work, we demonstrate that pimozide and loperamide trigger an ATG5- and ATG7 (autophagy related 5 and 7)-dependent type of cell death that is significantly reduced with cathepsin inhibitors and the lipid reactive oxygen species (ROS) scavenger α-tocopherol in MZ-54 GBM cells. Global proteomic analysis after treatment with both drugs also revealed an increase of proteins related to lipid and cholesterol metabolic processes. These changes were accompanied by a massive accumulation of cholesterol and other lipids in the lysosomal compartment, indicative of impaired lipid transport/degradation. In line with these observations, pimozide and loperamide treatment were associated with a pronounced increase of bioactive sphingolipids including ceramides, glucosylceramides and sphingoid bases measured by targeted lipidomic analysis. Furthermore, pimozide and loperamide inhibited the activity of SMPD1/ASM (sphingomyelin phosphodiesterase 1) and promoted induction of lysosomal membrane permeabilization (LMP), as well as release of CTSB (cathepsin B) into the cytosol in MZ-54 wild-type (WT) cells. Whereas LMP and cell death were significantly attenuated in ATG5 and ATG7 knockout (KO) cells, both events were enhanced by depletion of the lysophagy receptor VCP (valosin containing protein), supporting a pro-survival function of lysophagy under these conditions. Collectively, our data suggest that pimozide and loperamide-driven autophagy and lipotoxicity synergize to induce LMP and cell death. The results also support the notion that simultaneous overactivation of autophagy and induction of LMP represents a promising approach for the treatment of GBM. Abbreviations : ACD: autophagic cell death; AKT1: AKT serine/threonine kinase 1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG14: autophagy related 14; CERS1: ceramide synthase 1; CTSB: cathepsin B; CYBB/NOX2: cytochrome b-245 beta chain; ER: endoplasmatic reticulum; FBS: fetal bovine serum; GBM: glioblastoma; GO: gene ontology; HTR7/5-HT7: 5-hydroxytryptamine receptor 7; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAP: LC3-associated phagocytosis; LMP: lysosomal membrane permeabilization; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; RB1CC1: RB1 inducible coiled-coil 1; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SMPD1/ASM: sphingomyelin phosphodiesterase 1; VCP/p97: valosin containing protein; WT: wild-type.

Background: Hereditary Angioedema (HAE) is a genetic disorder that leads to frequent angioedema attacks in various parts of the body. In most cases it is caused by pathogenic variants in the SERPING1 gene, coding for C1-Inhibitor (C1-INH). The pathogenic variants in the gene result in reduced C1-INH levels and/or activity, which causes aberrant bradykinin production and enhanced vascular permeability. The standard-of-care diagnostic test is performed biochemically via measuring C1-INH level and activity as well as the C4 level. This, however, does not allow for the diagnosis of HAE types with normal C1-INH. There is an urgent need to identify and characterize HAE biomarkers for facilitating diagnostics and personalizing the treatment. The Hereditary Angioedema Kininogen Assay (HAEKA) study aims to measure the dynamics of cleaved High Molecular Weight Kininogen (HKa) and other metabolite levels during the angioedema and non-angioedema state of the disease. The metabolites will be analyzed and verified by liquid chromatography ion mobility high resolution mass spectrometry (LC/IM-QToF MS) of dried blood spot (DBS) cards upon the study completion. The study design is truly innovative: 100 enrolled participants provide blood samples via DBS: (1) every 3 months within 2 years during regular study site visits and (2) by at-home self-sampling during HAE attacks via finger pricking. We are presenting a project design that permits clinical study activities during pandemic contact restrictions and opens the door for other clinical studies during COVID-19., Results: As of October 2020, there are 41 patients from 5 sites in Germany enrolled. 90 blood samples were collected during the regular visits, and 19 of the participants also performed self-sampling during the HAE attacks from which a total of 286 attack blood samples were collected. Participating patients rate the study procedures as easy to implement in their daily lives. The concept of home self-sampling is effective, reproducible, and convenient especially in times of contact restrictions due to the COVID-19 pandemic., Conclusions: It is the hope that the HAEKA study will complete in 2023, reveal biomarker(s) for monitoring HAE disease activity, and may help to avoid HAE attacks via applying medication prior to the symptom onset., (© 2021. The Author(s).)

Selective degradation of the endoplasmic reticulum (ER; reticulophagy) is a type of autophagy involved in the removal of ER fragments. So far, amino acid starvation as well as ER stress have been described as inducers of reticulophagy, which in turn restores cellular energy levels and ER homeostasis. Here, we explored the autophagy-inducing mechanisms that underlie the autophagic cell death (ACD)-triggering compound loperamide (LOP) in glioblastoma cells. Interestingly, LOP triggers upregulation of the transcription factor ATF4, which is accompanied by the induction of additional ER stress markers. Notably, knockout of ATF4 significantly attenuated LOP-induced autophagy and ACD. Functionally, LOP also specifically induces the engulfment of large ER fragments within autophagosomes and lysosomes as determined by electron and fluorescence microscopy. LOP-induced reticulophagy and cell death are predominantly mediated through the reticulophagy receptor RETREG1/FAM134B and, to a lesser extent, TEX264, confirming that reticulophagy receptors can promote ACD. Strikingly, apart from triggering LOP-induced autophagy and ACD, ATF4 is also required for LOP-induced reticulophagy. These observations highlight a key role for ATF4, RETREG1 and TEX264 in response to LOP-induced ER stress, reticulophagy and ACD, and establish a novel mechanistic link between ER stress and reticulophagy, with possible implications for additional models of drug-induced ER stress. Abbreviations : ACD: autophagic cell death; ATF6: activating transcription factor 6; ATL3: atlastin 3; BafA 1 : bafilomycin A 1 ; CCPG1: cell cycle progression gene 1; co-IP: co-immunoprecipitation; DDIT3/CHOP: DNA damage inducible transcript 3; ER: endoplasmic reticulum; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; GABARAP: GABA type A receptor-associated protein; GBM: glioblastoma multiforme; HSPA5/BiP: heat shock protein family (Hsp70) member 5; LOP: loperamide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; RETREG1/FAM134B: reticulophagy regulator 1; RTN3L: reticulon 3 long; SEC62: SEC62 homolog, protein translocation factor; TEX264: testis-expressed 264, reticulophagy receptor; UPR: unfolded protein response.

Copy number gains and increased expression levels of cellular Inhibitor of Apoptosis protein (cIAP)1 and cIAP2 have been identified in primary diffuse large B-cell lymphoma (DLBCL) tissues. Second mitochondria-derived activator of caspases (Smac) mimetics were designed to antagonize IAP proteins. However, since their effect as single agents is limited, combination treatment represents a strategy for their clinical development. Therefore, we investigated the Smac mimetic BV6 in combination with proteasome inhibitors and analyzed the molecular mechanisms of action. We discovered that BV6 treatment sensitizes DLBCL cells to proteasome inhibition. We show a synergistic decrease in cell viability and induction of apoptosis by BV6/Carfilzomib (CFZ) treatment, which was confirmed by calculation of combination index (CI) and Bliss score. BV6 and CFZ acted together to trigger activation of BAX and BAK, which facilitated cell death, as knockdown of BAX and BAK significantly reduced BV6/CFZ-mediated cell death. Activation of BAX and BAK was accompanied by loss of mitochondrial membrane potential (MMP) and activation of caspases. Pretreatment with the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) rescued BV6/CFZ-induced cell death, confirming caspase dependency. Treatment with CFZ alone or in combination with BV6 caused accumulation of NOXA, which was required for cell death, as gene silencing by siRNA or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated NOXA inactivation inhibited BV6/CFZ-induced cell death. Together, these experiments indicate that BV6 and CFZ cooperatively induce apoptotic cell death via the mitochondrial pathway. These findings emphasize the role of Smac mimetics for sensitizing DLBCL cells to proteasome inhibition with important implications for further (pre)clinical studies., (© 2020 UICC.)

Background: The prognostic implications of non-sustained ventricular tachycardia (NSVT) and their significance as therapeutic targets in patients without prior sustained ventricular arrhythmias remain undetermined. The aim of this study was to investigate the prognostic significance of asymptomatic NSVT in patients who had primary prevention implantable cardioverter-defibrillator (ICD) implantation due to ischemic or non-ischemic cardiomyopathy (ICM, NICM)., Methods: We enrolled 157 consecutive primary prevention ICD patients without previous appropriate ICD therapy (AIT). Patients were allocated to two groups depending on the presence or absence of NSVT in a 6-month period prior to enrollment. The incidence of AIT and unplanned hospitalization due to decompensated heart failure (HF) were assessed during follow-up., Results: In 51 patients (32%), precedent NSVT was documented. During a median follow-up of 1011 days, AIT occurred in 36 patients (23%) and unplanned HF hospitalization was observed in 32 patients (20%). In precedent NSVT patients, the incidence of AIT and unplanned HF hospitalization was significantly higher as compared to patients without precedent NSVT (AIT: 29/51 [57%] vs. 7/106 [7%], P < 0.001, log-rank; HF hospitalization: 16/51 [31%] vs. 16/106 [15%], P = 0.043, log-rank). Cox-regression demonstrated that precedent NSVT independently predicted AIT (P < 0.0001). In subgroup analyses, precedent NSVT predicted AIT in both ICM and NICM (P < 0.0001, P = 0.020), but predicted HF hospitalization only in patients with ICM (P = 0.0030)., Conclusions: Precedent non-sustained VT in patients with primary prevention ICDs is associated with subsequent appropriate ICD therapies, and is an independent predictor of unplanned heart failure hospitalizations in patients with ischemic cardiomyopathy.

Induction of autophagy can have beneficial effects in several human diseases, e.g. cancer and neurodegenerative diseases (ND). Here, we therefore evaluated the potential of two novel autophagy-inducing compounds, i.e. STF-62247 and pimozide, to stimulate autophagy as well as autophagic cell death (ACD) using mouse embryonic fibroblasts (MEFs) as a cellular model. Importantly, both STF-62247 and pimozide triggered several hallmarks of autophagy in MEFs, i.e. enhanced levels of LC3B-II protein, its accumulation at distinct cytosolic sites and increase of the autophagic flux. Intriguingly, autophagy induction by STF-62247 and pimozide resulted in cell death that was significantly reduced in ATG5- or ATG7-deficient MEFs. Consistent with ACD induction, pharmacological inhibitors of apoptosis, necroptosis or ferroptosis failed to protect MEFs from STF-62247- or pimozide-triggered cell death. Interestingly, at subtoxic concentrations, pimozide stimulated fragmentation of the mitochondrial network, degradation of mitochondrial proteins (i.e. mitofusin-2 and cytochrome c oxidase IV (COXIV)) as well as a decrease of the mitochondrial mass, indicative of autophagic degradation of mitochondria by pimozide. In conclusion, this study provides novel insights into the induction of selective autophagy as well as ACD by STF-62247 and pimozide in MEFs.

In the absence of ligands, the nuclear receptor PPARβ/δ recruits the NCOR and SMRT corepressors, which form complexes with HDAC3, to canonical target genes. Agonistic ligands cause dissociation of corepressors and enable enhanced transcription. Vice versa, synthetic inverse agonists augment corepressor recruitment and repression. Both basal repression of the target gene ANGPTL4 and reinforced repression elicited by inverse agonists are partially insensitive to HDAC inhibition. This raises the question how PPARβ/δ represses transcription mechanistically. We show that the PPARβ/δ inverse agonist PT-S264 impairs transcription initiation by decreasing recruitment of activating Mediator subunits, RNA polymerase II, and TFIIB, but not of TFIIA, to the ANGPTL4 promoter. Mass spectrometry identifies NCOR as the main PT-S264-dependent interactor of PPARβ/δ. Reconstitution of knockout cells with PPARβ/δ mutants deficient in basal repression results in diminished recruitment of NCOR, SMRT, and HDAC3 to PPAR target genes, while occupancy by RNA polymerase II is increased. PT-S264 restores binding of NCOR, SMRT, and HDAC3 to the mutants, resulting in reduced polymerase II occupancy. Our findings corroborate deacetylase-dependent and -independent repressive functions of HDAC3-containing complexes, which act in parallel to downregulate transcription., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Autophagy is a well-described degradation mechanism that promotes cell survival upon nutrient starvation and other forms of cellular stresses. In addition, there is growing evidence showing that autophagy can exert a lethal function via autophagic cell death (ACD). As ACD has been implicated in apoptosis-resistant glioblastoma (GBM), there is a high medical need for identifying novel ACD-inducing drugs. Therefore, we screened a library containing 70 autophagy-inducing compounds to induce ATG5-dependent cell death in human MZ-54 GBM cells. Here, we identified three compounds, i.e. loperamide, pimozide, and STF-62247 that significantly induce cell death in several GBM cell lines compared to CRISPR/Cas9-generated ATG5- or ATG7-deficient cells, pointing to a death-promoting role of autophagy. Further cell death analyses conducted using pharmacological inhibitors revealed that apoptosis, ferroptosis, and necroptosis only play minor roles in loperamide-, pimozide- or STF-62247-induced cell death. Intriguingly, these three compounds induce massive lipidation of the autophagy marker protein LC3B as well as the formation of LC3B puncta, which are characteristic of autophagy. Furthermore, loperamide, pimozide, and STF-62247 enhance the autophagic flux in parental MZ-54 cells, but not in ATG5 or ATG7 knockout (KO) MZ-54 cells. In addition, loperamide- and pimozide-treated cells display a massive formation of autophagosomes and autolysosomes at the ultrastructural level. Finally, stimulation of autophagy by all three compounds is accompanied by dephosphorylation of mammalian target of rapamycin complex 1 (mTORC1), a well-known negative regulator of autophagy. In summary, our results indicate that loperamide, pimozide, and STF-62247 induce ATG5- and ATG7-dependent cell death in GBM cells, which is preceded by a massive induction of autophagy. These findings emphasize the lethal function and potential clinical relevance of hyperactivated autophagy in GBM.

In most cases, macroautophagy/autophagy serves to alleviate cellular stress and acts in a pro-survival manner. However, the effects of autophagy are highly contextual, and autophagic cell death (ACD) is emerging as an alternative paradigm of (stress- and drug-induced) cell demise. AT 101 ([-]-gossypol), a natural compound from cotton seeds, induces ACD in glioma cells as confirmed here by CRISPR/Cas9 knockout of ATG5 that partially, but significantly rescued cell survival following AT 101 treatment. Global proteomic analysis of AT 101-treated U87MG and U343 glioma cells revealed a robust decrease in mitochondrial protein clusters, whereas HMOX1 (heme oxygenase 1) was strongly upregulated. AT 101 rapidly triggered mitochondrial membrane depolarization, engulfment of mitochondria within autophagosomes and a significant reduction of mitochondrial mass and proteins that did not depend on the presence of BAX and BAK1. Conversely, AT 101-induced reduction of mitochondrial mass could be reversed by inhibiting autophagy with wortmannin, bafilomycin A 1 and chloroquine. Silencing of HMOX1 and the mitophagy receptors BNIP3 (BCL2 interacting protein 3) and BNIP3L (BCL2 interacting protein 3 like) significantly attenuated AT 101-dependent mitophagy and cell death. Collectively, these data suggest that early mitochondrial dysfunction and HMOX1 overactivation synergize to trigger lethal mitophagy, which contributes to the cell killing effects of AT 101 in glioma cells., Abbreviations: ACD, autophagic cell death; ACN, acetonitrile; AT 101, (-)-gossypol; BAF, bafilomycin A 1 ; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BH3, BCL2 homology region 3; BNIP3, BCL2 interacting protein 3; BNIP3L, BCL2 interacting protein 3 like; BP, Biological Process; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CC, Cellular Component; Con, control; CQ, chloroquine; CRISPR, clustered regularly interspaced short palindromic repeats; DMEM, Dulbecco's Modified Eagle Medium; DTT, 1,4-dithiothreitol; EM, electron microscopy; ER, endoplasmatic reticulum; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GO, Gene Ontology; HAcO, acetic acid; HMOX1, heme oxygenase 1; DKO, double knockout; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LPL, lipoprotein lipase, MEFs, mouse embryonic fibroblasts; mPTP, mitochondrial permeability transition pore; MTG, MitoTracker Green FM; mt-mKeima, mito-mKeima; MT-ND1, mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1; PBS, phosphate-buffered saline; PE, phosphatidylethanolamine; PI, propidium iodide; PRKN, parkin RBR E3 ubiquitin protein ligase; SDS, sodium dodecyl sulfate; SQSTM1/p62, sequestome 1; STS, staurosporine; sgRNA, single guide RNA; SILAC, stable isotope labeling with amino acids in cell culture; TFA, trifluoroacetic acid, TMRM, tetramethylrhodamine methyl ester perchlorate; WM, wortmannin; WT, wild-type.

Farber disease (FD) is a rare autosomal recessive disease caused by mutations in the acid ceramidase gene (ASAH1). Low ceramidase activity results in the accumulation of fatty substances, mainly ceramides. Hallmark symptoms at clinical level are periarticular nodules, lipogranulomas, swollen and painful joints and a hoarse voice. FD phenotypes are heterogeneous varying from mild to very severe cases, with the patients not surviving past their first year of life. The diagnostic aspects of FD are poorly developed due to the rarity of the disease. In the present study, the screening for ceramides and related molecules was performed in Farber affected patients (n = 10), carriers (n = 11) and control individuals (n = 192). This study has the highest number of enrolled Farber patients and carriers reported to present. Liquid chromatography multiple reaction mass spectrometry (LC/MRM-MS) studies revealed that the ceramide C26:0 and especially its isoform 1 is a highly sensitive and specific biomarker for FD (p < 0.0001). The new biomarker can be determined directly in the dried blood spot extracts with low sample consumption. This allows for easy sample preparation, high reproducibility and use in high throughput screenings.

Objectives: We validate a novel CT coronary angiography (CCTA) coronary calcium scoring system., Methods: Calcium was quantified on CCTA images using a new patient-specific attenuation threshold: mean + 2SD of intra-coronary contrast density (HU). Using 335 patient data sets a conversion factor (CF) for predicting CACS from CCTA scores (CCTAS) was derived and validated in a separate cohort (n = 168). Bland-Altman analysis and weighted kappa for MESA centiles and Agatston risk groupings were calculated., Results: Multivariable linear regression yielded a CF: CACS = (1.185 × CCTAS) + (0.002 × CCTAS × attenuation threshold). When applied to CCTA data sets there was excellent correlation (r = 0.95; p < 0.0001) and agreement (mean difference -10.4 [95% limits of agreement -258.9 to 238.1]) with traditional calcium scores. Agreement was better for calcium scores below 500; however, MESA percentile agreement was better for high risk patients. Risk stratification was excellent (Agatston groups k = 0.88 and MESA centiles k = 0.91). Eliminating the dedicated CACS scan decreased patient radiation exposure by approximately one-third., Conclusion: CCTA calcium scores can accurately predict CACS using a simple, individualized, semiautomated approach reducing acquisition time and radiation exposure when evaluating patients for CAD. This method is not affected by the ROI location, imaging protocol, or tube voltage strengthening its clinical applicability., Key Points: • Coronary calcium scores can be reliably determined on contrast-enhanced cardiac CT • This score can accurately risk stratify patients • Elimination of a dedicated calcium scan reduces patient radiation by a third.

We validate a method of calcium scoring on CT coronary angiography (CTCA) and propose an algorithm for the assessment of patients with stable chest pain. 503 consecutive patients undergoing coronary artery calcium score (CACS) and CTCA were included. A 0.1 cm2 region of interest was used to determine the mean contrast density on CTCA images either in the left main stem (LM) or right coronary artery. Axial 3 mm CTCA images were scored for calcium using conventional software with a modified threshold: mean LM contrast density (HU) + 2SD. A conversion factor (CF) for predicting CACS from raw CTCA scores (rCTCAS) was determined using a multivariable regression model adjusted for model over-optimism (1,000 bootstrap samples). Accuracy of this method was determined using weighted kappa for NICE recommended CACS groupings (0, 1-400, >400) and Bland-Altman analysis for absolute score. With the CF applied: CACS = (1.183 × rCTCAS) + (0.002 × rCTCAS × threshold), there was excellent agreement between methods for absolute score (mean difference 5.44 [95% limits of agreement -207.0 to 217.8]). The method discriminated between high (>400) and low risk (<400) calcium scores with a sensitivity and specificity of 85 and 99%, and a PPV and NPV of 92 and 98%, respectively, and led to a significant reduction in radiation exposure (6.9 [5.1-10.2] vs. 5.2 [6.3-8.7] mSv; p < 0.0001). Our proposed method allows a comprehensive assessment of coronary artery pathology through the use of an individualised, semi-automated approach. If incorporated into stable chest pain guidelines the need for further functional testing or invasive angiography could be determined from CTCA alone, supporting a change to the current guidelines.

Background: Variability has been described between different echo machines and different modalities when measuring tissue velocities. We assessed the consistency of tissue velocity measurements across different modalities and different manufacturers in an in vitro model and in patients. Furthermore, we present freely available software tools to repeat these evaluations., Methods and Results: We constructed a simple setup to generate reproducible motion and used it to compare velocities measured using three echocardiographic modalities: M-mode, speckle tracking, and tissue Doppler, with a straightforward, non-ultrasound, optical gold standard. In the clinical phase, 25 patients underwent M-mode, speckle tracking, and tissue Doppler measurements of s', e', and a' velocities. In vitro, the M-mode and speckle tracking velocities agreed with optical assessment. Of the three possible tissue Doppler measurement conventions (outer, middle, and inner edge) only the middle agreed with optical assessment (discrepancy -0.20 (95% CI -0.44 to 0.03) cm/s, P = 0.11, outer +5.19 (4.65 to 5.73) cm/s, P < 0.0001, inner -6.26 (-6.87 to -5.65) cm/s, P < 0.0001). A similar pattern occurred across all four studied manufacturers. M-mode was therefore chosen as the in vivo gold standard. Clinical measurements of s' velocities by speckle tracking and the middle line of the tissue Doppler showed concordance with M-mode, while the outer line overestimated significantly (+1.27(0.96 to 1.59) cm/s, P < 0.0001) and the inner line underestimated (-1.82 (-2.11 to -1.52) cm/s, P < 0.0001)., Conclusions: Echocardiographic velocity measurements can be more consistent than previously suspected. The statistically modal velocity, found at the centre of the spectral pulsed wave tissue Doppler envelope, most closely represents true tissue velocity. This article includes downloadable, vendor-independent software enabling calibration of echocardiographic machines using a simple, inexpensive in vitro setup., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

N-Acetylcysteine (H2NAC) is a potent antioxidant, a precursor for cysteine and glutathione, and a potential antidote against certain metal ions such as cadmium and mercury. Little is known about the structural aspects of complexes formed between Hg(II) and N-acetylcysteine, despite many biological tests on its ability to bind to organic and inorganic mercury, and a few reports on formation constants for Hg(NAC)n (n = 1-3) complexes. We have combined several techniques, including Hg L3-edge EXAFS (extended X-ray absorption fine structure), (199)Hg NMR and Raman spectroscopy, to investigate the nature and structure of Hg(II) N-acetylcysteine complexes formed in aqueous solution at pH 7.5 and 10.5. To allow measurements on the same samples, rather concentrated solutions containing CHg(II) = 0.1 M and variable H2NAC/Hg(II) mole ratios = 2.0-10.0 were used. At physiological pH, Hg(NAC)2(2-) and Hg(NAC)3(4-) complexes form, while in ligand excess and at alkaline pH (H2NAC/Hg(II) > 4), a novel tetra-thiolate species Hg(NAC)4(6-) dominates. Comparison between the Hg(II) complex formation with cysteine, penicillamine and N-acetylcysteine in alkaline aqueous solution has been made to elucidate the influence of the blocked amino group of N-acetylcysteine.

The transient receptor potential (TRP) family of ion channels comprises receptors that are activated by a vast variety of physical as well as chemical stimuli. TRP channels interact in a complex manner with several intracellular signaling cascades, both up- and downstream of receptor activation. Investigating cascades stimulated downstream of the cold and menthol receptor TRPM8, we found evidence for both, functional and structural interaction of TRPM8 with Gαq. We demonstrated menthol-evoked increase in intracellular Ca(2+) under extracellular Ca(2+)-free conditions, which was blocked by the PLC inhibitors U73122 or edelfosine. This metabotropic Ca(2+) signal could be observed also in cells expressing a channel-dead (i.e. non-conducting) or a chloride-conducting TRPM8 pore mutant. However, this intracellular metabotropic Ca(2+) signal could not be detected in Gαq deficient cells or in the presence of dominant-negative GαqX. Evidence for a close spatial proximity necessary for physical interaction of TRPM8 and Gαq was provided by acceptor bleaching experiments demonstrating FRET between TRPM8-CFP and Gαq-YFP. A Gαq-YFP mobility assay (FRAP) revealed a restricted diffusion of Gαq-YFP under conditions when TRPM8 is immobilized in the plasma membrane. Moreover, a menthol-induced and TRPM8-mediated G protein activation could be demonstrated by FRET experiments monitoring the dissociation of Gαq-YFP from a Gβ/Gγ-CFP complex, and by the exchange of radioactive [(35)S]GTPγS for GDP. Our observations lead to a view that extends the operational range of the TRPM8 receptor from its function as a pure ion channel to a molecular switch with additional metabotropic capacity.

Telomeres are the repetitive sequences of DNA and associated proteins that cap the ends of eukaryotic chromosomes and play an essential role in maintaining chromosome stability. Compromised telomeres can lead to cell cycle arrest, senescence, apoptosis, or genetic instability, whereas maintenance of telomeres can endow cells with the capacity for indefinite self-renewal. Telomere integrity is maintained in most cells by the activity of telomerase, a ribonucleoprotein that can catalyze the addition of repeat sequences onto chromosome ends. Using the telomeric repeat amplification protocol (TRAP) assay, we detected telomerase activity in host nuclear extracts prepared from two scleractinian corals, Madracis auretenra and Madracis decactis, and also in cultured Symbiodinium, the symbiotic algae that live within corals. Sequencing the TRAP reaction products indicated that the telomeric DNA repeat sequence was TTAGGG for coral and TTTAGGG for Symbiodinium. Using this sequence information, we estimated telomere lengths by terminal restriction fragment (TRF) analysis to be greater than 19 kb for several species of coral and their associated Symbiodinium. Maintenance of coral telomeres by telomerase activity may be a mechanism that confers continuous growth and reproductive plasticity to these long-lived organisms.