DiSSCo Prepare WP7 –D7.3 Assessment tools and direction map to the implementation of common DiSSCo policies

The Distributed System for Scientific Collections (DiSSCo) Research Infrastructure will operate a number of e-services, all of which will have policy requirements for participating institutions. These policies include those related to digital and physical access to specimens, digital image and specimen metadata, and FAIR / Open Data. Previous projects have shown that the policy landscape is complex, and Task 7.3 has developed a policy self-assessment tool that will allow DiSSCo to assess policy alignment across the consortium. This deliverable describes the development of the policy self-assessment tool and provides a walkthrough of the key features. The same technical framework was used to create a digital maturity tool, which was initially proposed by Task 3.1, and this is also described within this document. A set of recommendations are included that outline the future direction for the development of the policy tool.The Distributed System for Scientific Collections (DiSSCo) Research Infrastructure will operate a number´of e-services, all of which will have policy requirements for participating institutions. These policies include those related to digital and physical access to specimens, digital image and specimen metadata, and FAIR / Open Data. Previous projects have shown that the policy landscape is complex, and Task 7.3 has developed a policy self-assessment tool that will allow DiSSCo to assess policy alignment across the consortium. This deliverable describes the development of the policy self-assessment tool and provides a walkthrough of the key features. The same technical framework was used to create a digital maturity tool, which was initially proposed by Task 3.1, and this is also described within this document. A set of recommendations are included that outline the future direction for the development of the policy tool

Chapitre 8. La presse périodique

L’évolution de la grande presse commerciale mary vipond Pour la majeure partie du XXe siècle, l’univers de la presse périodique au Canada est dominé par des questions de propriété et de concurrence. En ce qui concerne les journaux, l’essor des chaînes et des conglomérats médiatiques qui conduira les villes à n’être desservies que par un seul journal soulève l’inquiétude et déclenche la tenue d’enquêtes publiques. Pour ce qui est des périodiques, qui se heurtent à la concurrence serrée des mag..

Voltage-Clamp Fluorometry in the Local Environment of the C255–C511 Disulfide Bridge of the Na(+)/Glucose Cotransporter

We recently identified a functionally important disulfide bridge between C255 and C511 of the human Na(+)/glucose cotransporter SGLT1. In this study, voltage-clamp fluorometry was used to characterize the fluorescence of four different dyes attached to C255 and C511 under various ionic and substrate/inhibitor conditions. State-dependent fluorescence changes (ΔF) were observed when TMR5M or TMR6M dyes were attached to C255 and C511 or when Alexa488 was bound to C511. TMR5M-C511 was extremely sensitive to membrane potential (V(m)) and to external Na(+) and αMG (a nonmetabolizable glucose analog) concentrations. A progressive increase in αMG concentration drastically changed the maximal voltage-dependent ΔF and produced a positive shift in the midpoint of the ΔF-V(m) curve. By determining specific fluorescence intensity for each state of the cotransporter, our steady-state fluorescence data could be reproduced using the rate constants previously proposed for a five-state kinetic model exclusively derived from electrophysiological measurements. Our results bring an independent support to the proposed kinetic model and show that the binding of αMG substrate significantly modifies the environment of C255 and C511

Effect of Substrate on the Pre-Steady-State Kinetics of the Na(+)/Glucose Cotransporter

When measuring Na(+)/glucose cotransporter (SGLT1) activity in Xenopus oocytes with the two-electrode voltage-clamp technique, pre-steady-state currents dissipate completely in the presence of saturating α-methyl-glucose (αMG, a nonhydrolyzable glucose analog) concentrations. In sharp contrast, two SGLT1 mutants (C255A and C511A) that lack a recently identified disulfide bridge express the pre-steady-state currents in the presence of αMG. The dose-dependent effects of αMG on pre-steady-state currents were studied for wild-type (wt) SGLT1 and for the two mutants. Increases in αMG concentration reduced the total transferred charge (partially for the mutants, totally for wt SGLT1), shifted the transferred charge versus membrane potential (Q-V) curve toward positive potentials, and significantly modified the time constants of the pre-steady-state currents. A five-state kinetic model is proposed to quantitatively explain the effect of αMG on pre-steady-state currents. This analysis reveals that the reorientation of free transporter is the slowest step for wt SGLT1 either in the presence or in the absence of αMG. In contrast, the conformational change of the fully loaded mutant transporters constitutes their rate-limiting step in the presence of substrate and explains the persistence of pre-steady-state currents in this situation

Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies*

Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to ∼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications

Copy number variations alter methylation and parallel IGF2 overexpression in adrenal tumors

Overexpression of insulin growth factor 2 (IGF2) is a hallmark of adrenocortical carcinomas and pheochromocytomas. Previous studies investigating the IGF2/H19 locus have mainly focused on a single molecular level such as genomic alterations or altered DNA methylation levels and the causal changes underlying IGF2 overexpression are still not fully established. In the current study, we analyzed 62 tumors of the adrenal gland from patients with Conn's adenoma (CA, n=12), pheochromocytomas (PCC, n=10), adrenocortical benign tumors (ACBT, n=20), and adrenocortical carcinomas (ACC, n=20). Gene expression, somatic copy number variation of chr11p15.5, and DNA methylation status of three differential methylated regions of the IGF2/H19 locus including the H19 imprinting control region were integratively analyzed. IGF2 overexpression was found in 85% of the ACCs and 100% of the PCCs compared to 23% observed in CAs and ACBTs. Copy number aberrations of chr11p15.5 were abundant in both PCCs and ACCs but while PCCs retained a diploid state, ACCs were frequently tetraploid (7/19). Loss of either a single allele or loss of two alleles of the same parental origin in tetraploid samples resulted in a uniparental disomy-like genotype. These copy number changes correlated with hypermethylation of the H19 ICR suggesting that the lost alleles were the unmethylated maternal alleles. Our data provide conclusive evidence that loss of the maternal allele correlates with IGF2 overexpression in adrenal tumors and that hypermethylation of the H19 ICR is a consequence thereof

Effect of Attachment Site on Stability of Cleavable Antibody Drug Conjugates

The systemic stability of the antibody–drug linker is crucial for delivery of an intact antibody–drug conjugate (ADC) to target-expressing tumors. Linkers stable in circulation but readily processed in the target cell are necessary for both safety and potency of the delivered conjugate. Here, we report a range of stabilities for an auristatin-based payload site-specifically attached through a cleavable valine-citrulline-<i>p</i>-aminobenzylcarbamate (VC-PABC) linker across various sites on an antibody. We demonstrate that the conjugation site plays an important role in determining VC-PABC linker stability in mouse plasma, and that the stability of the linker positively correlates with ADC cytotoxic potency both in vitro and in vivo. Furthermore, we show that the VC-PABC cleavage in mouse plasma is not mediated by Cathepsin B, the protease thought to be primarily responsible for linker processing in the lysosomal degradation pathway. Although the VC-PABC cleavage is not detected in primate plasma in vitro, linker stabilization in the mouse is an essential prerequisite for designing successful efficacy and safety studies in rodents during preclinical stages of ADC programs. The divergence of linker metabolism in mouse plasma and its intracellular cleavage offers an opportunity for linker optimization in the circulation without compromising its efficient payload release in the target cell