發作性血色素尿症ニ就テ

The α4β2 nicotinic acetylcholine receptor (nAChR) is the most abundant subtype in the brain and exists in two functional stoichiometries: (α4)3(β2)2 and (α4)2(β2)3. A distinct feature of the (α4)3(β2)2 receptor is the biphasic activation response to the endogenous agonist acetylcholine, where it is activated with high potency and low efficacy when two α4-β2 binding sites are occupied and with low potency/high efficacy when a third α4-α4 binding site is occupied. Further, exogenous ligands can bind to the third α4-α4 binding site and potentiate the activation of the receptor by ACh that is bound at the two α4-β2 sites. We propose that perturbations of the recently described pre-activation step when a third binding site is occupied are a key driver of these distinct activation properties. To investigate this, we used a combination of simple linear kinetic models and voltage clamp electrophysiology to determine whether transitions into the pre-activated state were increased when three binding sites were occupied. We separated the binding at the two different sites with ligands selective for the α4-β2 site (Sazetidine-A and TC-2559) and the α4-α4 site (NS9283) and identified that when a third binding site was occupied, changes in the concentration-response curves were best explained by an increase in transitions into a pre-activated state. We propose that perturbations of transitions into a pre-activated state are essential to explain the activation properties of the (α4)3(β2)2 receptor by acetylcholine and other ligands. Considering the widespread clinical use of benzodiazepines, this discovery of a conserved mechanism that benzodiazepines and ACh potentiate receptor activation via a third binding site can be exploited to develop therapeutics with similar properties at other cys-loop receptors

Prognostic Factors in Dedifferentiated Chondrosarcoma: A Retrospective Analysis of a Large Series Treated at a Single Institution.

Background:Dedifferentiated chondrosarcomas (DDCSs) are highly malignant tumors with a dismal prognosis and present a significant challenge in clinical management. Methods:In an IRB approved retrospective protocol, we identified 72 patients with DDCS treated at our institution between 1993 and 2017 and reviewed clinicopathological characteristics, treatment modalities, and outcomes to analyze prognostic factors. Results:Femur (44.4%), pelvis (22.2%), and humerus (12.5%) were most commonly involved sites. Twenty-three patients (31.9%) presented with distant metastasis, and 3 (4.2%) of them also had regional lymph node involvement. The median overall survival (OS) was 13.9 months. On multivariate analysis, pathological fracture, larger tumor size, lymph node involvement, metastasis at diagnosis, extraosseous extension, and undifferentiated pleomorphic sarcoma component correlated with worse OS, whereas surgical resection and chemotherapy were associated with improved OS. For progression-free survival (PFS), pathological fracture and metastasis at diagnosis showed increased risk, while chemotherapy was associated with decreased risk. Among patients who received chemotherapy, doxorubicin and cisplatin were significantly associated with improved PFS but not OS. Among patients without metastasis at diagnosis, 17 (34.7%) developed local recurrence. Thirty-one (63.3%) developed distant metastases at a median interval of 18.1 months. On multivariate analysis, R1/R2 resection was related with local recurrence, while macroscopic dedifferentiated component was associated with distant metastasis. Conclusions:The prognosis of DDCS is poor. Complete resection remains a significant prognostic factor for local control. Chemotherapy with doxorubicin and cisplatin seems to have better PFS. More prognostic, multicenter trials are warranted to further explore the effectiveness of chemotherapy in selected DDCS patients

Investigating the role of loop c hydrophilic residue 'T244' in the binding site of ρ1 GABAC receptors via site mutation and partial agonism

The loop C hydrophilic residue, threonine 244 lines the orthosteric binding site of ρ1 GABAC receptors was studied by point mutation into serine, alanine and cysteine, and tested with GABA, some representative partial agonists and antagonists. Thr244 has a hydroxyl group essential for GABA activity that is constrained by the threonine methyl group, orienting it toward the binding site. Significant decreases in activation effects of the studied ligands at ρ1 T244S mutant receptors, suggests a critical role for this residue. Results of aliphatic and heteroaromatic partial agonists demonstrate different pharmacological effects at ρ1 T244S mutant receptors when co-applied with GABA EC50 responses. ρ1 T244A and ρ1 T244C mutant receptors have minimal sensitivity to GABA at high mM concentrations, whereas, the ρ1 WT partial agonists, β-alanine and MTSEA demonstrate more efficacy and potency, respectively, than GABA at these mutant receptors. This study explores the role of Thr244 in the binding of agonists as an initial step during channel gating by moving loop C towards the ligand

Characterization of different DLC and DLN electrodes for biosensor design

International audienceDiamond-Like Carbon and Carbon-Like Nanocomposite electrodes, novel materials in the field of biosensors, made with different ratio of sp3/sp2 carbon hybridization or doped with elements such as Ni, Si and W, were characterized electrochemically by cyclic voltammetry and by amperometric measurements towards hydrogen peroxide. SiCAr1 and SiCNi5% were chosen as sensitive transducers for elaboration of amperometric glucose biosensors. Immobilization of glucose oxidase was carried out by cross-linking with glutareldehyde. Measurements were made at a fixed potential + 1.0 V in 40 mM phosphate buffer pH 7.4. SiCAr1 seems to be more sensitive for glucose (0.6875 µA/mM) then SiCNi5% (0.3654 µA/mM). Detections limits were respectively 20 µM and 30 µM. Michaelis-Menten constants for the two electrodes were found around 3 mM. 48% and 79% of the original response for 0.5 mM glucose remained respectively for both electrodes after 10 days

Highly connected 3D chromatin networks established by an oncogenic fusion protein shape tumor cell identity.

Cell fate transitions observed in embryonic development involve changes in three-dimensional genomic organization that provide proper lineage specification. Whether similar events occur within tumor cells and contribute to cancer evolution remains largely unexplored. We modeled this process in the pediatric cancer Ewing sarcoma and investigated high-resolution looping and large-scale nuclear conformation changes associated with the oncogenic fusion protein EWS-FLI1. We show that chromatin interactions in tumor cells are dominated by highly connected looping hubs centered on EWS-FLI1 binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Conversely, EWS-FLI1 depletion led to the disassembly of these looping networks and a widespread nuclear reorganization through the establishment of new looping patterns and large-scale compartment configuration matching those observed in mesenchymal stem cells, a candidate Ewing sarcoma progenitor. Our data demonstrate that major architectural features of nuclear organization in cancer cells can depend on single oncogenes and are readily reversed to reestablish latent differentiation programs

LIN28B Underlies the Pathogenesis of a Subclass of Ewing Sarcoma

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GABAA Receptors Containing ρ1 Subunits Contribute to In Vivo Effects of Ethanol in Mice

Yuri A. Blednov, Jillian M. Benavidez, Mendy Black, Courtney R. Leiter, Elizabeth Osterndorff-Kahanek, David Johnson, Cecilia M. Borghese, R. Adron Harris, Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, Texas, United States of AmericaJane R. Hanrahan, Mary Chebib, Faculty of Pharmacy, The University of Sydney, Sydney NSW, AustraliaGraham A. R. Johnston, Department of Pharmacology, The University of Sydney, Sydney NSW, AustraliaGABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ1” antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ2” antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo.This work was supported by NIH grants AA013520 and AA06399. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Waggoner Center for Alcohol and Addiction ResearchEmail: [email protected]

γ-Aminobutyric acid (GABA) signalling in human pancreatic islets is altered in type 2 diabetes

AIMS/HYPOTHESIS: γ-Aminobutyric acid (GABA) is a signalling molecule in the interstitial space in pancreatic islets. We examined the expression and function of the GABA signalling system components in human pancreatic islets from normoglycaemic and type 2 diabetic individuals. METHODS: Expression of GABA signalling system components was studied by microarray, quantitative PCR analysis, immunohistochemistry and patch-clamp experiments on cells in intact islets. Hormone release was measured from intact islets. RESULTS: The GABA signalling system was compromised in islets from type 2 diabetic individuals, where the expression of the genes encoding the α1, α2, β2 and β3 GABA(A) channel subunits was downregulated. GABA originating within the islets evoked tonic currents in the cells. The currents were enhanced by pentobarbital and inhibited by the GABA(A) receptor antagonist, SR95531. The effects of SR95531 on hormone release revealed that activation of GABA(A) channels (GABA(A) receptors) decreased both insulin and glucagon secretion. The GABA(B) receptor antagonist, CPG55845, increased insulin release in islets (16.7 mmol/l glucose) from normoglycaemic and type 2 diabetic individuals. CONCLUSIONS/INTERPRETATION: Interstitial GABA activates GABA(A) channels and GABA(B) receptors and effectively modulates hormone release in islets from type 2 diabetic and normoglycaemic individuals

EWSR1-ATF1 dependent 3D connectivity regulates oncogenic and differentiation programs in Clear Cell Sarcoma.

Oncogenic fusion proteins generated by chromosomal translocations play major roles in cancer. Among them, fusions between EWSR1 and transcription factors generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs in permissive precursor cells. Here we define the epigenetic and 3D connectivity landscape of Clear Cell Sarcoma, an aggressive cancer driven by the EWSR1-ATF1 fusion gene. We find that EWSR1-ATF1 displays a distinct DNA binding pattern that requires the EWSR1 domain and promotes ATF1 retargeting to new distal sites, leading to chromatin activation and the establishment of a 3D network that controls oncogenic and differentiation signatures observed in primary CCS tumors. Conversely, EWSR1-ATF1 depletion results in a marked reconfiguration of 3D connectivity, including the emergence of regulatory circuits that promote neural crest-related developmental programs. Taken together, our study elucidates the epigenetic mechanisms utilized by EWSR1-ATF1 to establish regulatory networks in CCS, and points to precursor cells in the neural crest lineage as candidate cells of origin for these tumors

Clinical spectrum, prognosis and estimated prevalence of DNAJB11-kidney disease

Monoallelic mutations of DNAJB11 were recently described in seven pedigrees with atypical clinical presentations of autosomal dominant polycystic kidney disease. DNAJB11 encodes one of the main cofactors of the endoplasmic reticulum chaperon BiP, a heat-shock protein required for efficient protein folding and trafficking. Here we conducted an international collaborative study to better characterize the DNAJB11-associated phenotype. Thirteen different loss-of-function variants were identified in 20 new pedigrees (54 affected individuals) by targeted next-generation sequencing, whole-exome sequencing or whole-genome sequencing. Amongst the 77 patients (27 pedigrees) now in total reported, 32 reached end stage kidney disease (range, 55-89 years, median age 75); without a significant difference between males and females. While a majority of patients presented with non-enlarged polycystic kidneys, renal cysts were inconsistently identified in patients under age 45. Vascular phenotypes, including intracranial aneurysms, dilatation of the thoracic aorta and dissection of a carotid artery were present in four pedigrees. We accessed Genomics England 100,000 genomes project data, and identified pathogenic variants of DNAJB11 in nine of 3934 probands with various kidney and urinary tract disorders. The clinical diagnosis was cystic kidney disease for eight probands and nephrocalcinosis for one proband. No additional pathogenic variants likely explaining the kidney disease were identified. Using the publicly available GnomAD database, DNAJB11 genetic prevalence was calculated at 0.85/10.000 individuals. Thus, establishing a precise diagnosis in atypical cystic or interstitial kidney disease is crucial, with important implications in terms of follow-up, genetic counseling, prognostic evaluation, therapeutic management, and for selection of living kidney donors