The emerging role of the a4(+)a4(-) interface as a determinant of functional signatures of a4ß2 nicotinic acetylcholine receptors

The a4~2 nicotinic acetylcholine receptor (nAChR) assembles in two alternate forms, (a4p2ha4 and (a4~2hP2, which display stoichiometry-specific agonist sensitivity. Being heteromeric pentameric ligand-gated ion channels (pLGIC), a4p2 receptors are activated by binding of agonist to sites located at the a4( + )/P2( -) interfaces. These interfaces are present in both stoichiometries they are unlikely to contain structural differences conferring specific properties to (a4~2)2a4 and (a4p2)2p2 receptors. In contrast, the auxiliary subunit can be either a4 or P2, leading to stoichiometry-specific ~2( + )/P2( -) and a4( + )/a4( -) interfaces. Using fully concatenated (a4p2ha4 nAChRs in conjunction with structural modelling, chimeric receptors and functional mutagenesis, this study identified an additional agonist site at the a4( + )/a4( -) interface that accounts for the stoichiometry-specific agonist sensitivity of the (a4p2ha4 receptor. The additional agonist site occupies a region that also contains a potentiating 2n2+ binding site. However, unlike 2n2+, the agonist binding influences agonist responses by directly contributing to channel gating. By engineering a receptor with a C226S mutation to provide a free cysteine in loop C in the + side of the a4(+)/a4(-) interface, this study found that the acetylcholine (ACh) responses of the additional agonist site are modified following modification of the substituted cysteine with sulfhydryl reagents. These findings suggested that agonist occupation of the site at the a4(+)/a4(-) interface leads to channel gating through a coupling mechanism involving a conformational switch in loop C. The sulfhydryl reagents had similar effects on substituted cysteines in the a4( + )/P2( -) interfaces. Further studies showed that the additional agonist site is less sensitive to desensitisation than the sites at the a4( + )/~2( -) interfaces, suggesting that the agonist sites are functionally non-equivalent. Non-functional equivalency was also indicated by the finding that the agonist selectivity of the site at the a4(+)/a4(-) differs from that of the sites at the a4(+)/p2(-) interface. The findings may have important consequences for drug discovery programs and the manner by which a4p2 receptor signalling in the brain can be modified in brain pathologies. XI

Modelling Private Wealth Accumulation and Spend-down in the Italian Microsimulation Model CAPP_DYN: A Life-Cycle Approach

In microsimulation literature a limited number of models include a module aimed at analyzing and projecting the evolution of privat e wealth over time. However, this issue appears crucial in order to comprehensively evaluate the li kely distributional effects of institutional reforms adopted to cope with population ageing. In this work we describe the implementation in the Italian dynamic micro simulation model CAPP_DYN of a new module in which households\u2019 savings and asset allocation are modelled. In parti cular, we aim to account for possible behavioural responses to pension reforms in househo ld savings. To this end, we rely on an approximate life cycle structural framework for est imating saving behaviour, while adopting a traditional stochastic micro simulation approach fo r asset allocation. In line with Ando and Nicoletti Altimari (2004), we emphasize the role of lifetime economic resources in households\u2019 consumption decisions, yet we further account for i nternal habit formation and subjective expectations on pension outcomes in the econometric stage. In addition, we model intergenerational transfers of private wealth in a probabilistic fashio

The Introduction of a Private Wealth Module in CAPP_DYN: an Overview

Household saving rate in Italy declined over the last two decades.This trend still persists despite three pension reforms have been enacted since the beginning of the nineties. In this paper we search further evidence of general macroeconomic effects through the analysis of households behaviour. In the first part of the paper we use data from five surveys of the Bank of Italy Surveys of Household Income and Wealth (SHIW) to estimate the lifetime profiles of saving and wealth accumulation. Estimates show that the age profile of the propensity to save has been influenced more by cohort effects than by general trend effects; whereas the age profile of the ratios of financial assets to disposable income has been subject to relevant trend effects. In the second part of the paper we analyse the effects of pension reforms on saving behaviour of Italian Households. Firstly we use a difference-in-difference estimator in order to test whether the groups more severely hit by the reforms actually increased their saving rate relative to the other groups. Then we estimate the Social Security Net Wealth (SSWN) for each individual in the SHIW in the analysed period (1989-2000). Finally we estimate the substitution coefficient between SSWN and private wealth taking into account that the reaction of saving to a change in SSWN depends also on age of the individual. Our results show that the reduction of SSWN is unequally distributed across individuals. The cut is stronger for self employed, young workers and women. Most of the groups more severely hit by the reforms did not increase their saving rate relative to the control group: younger households, in particular, did not increase the saving rate. On the whole a reduction of one Euro in SSWN seems to induce, on the average, a compensating increase in private wealth by about fifty cents. The substitution coefficient between private and social security wealth is higher for the richest and oldest part of the sample. Finally when we split the sample observations by year we find that the more dramatised is the impact of the reform, the higher is the substitution coefficient.Pension reform; household saving; social security wealth; difference-in-difference

Modelling Private Wealth Accumulation and Spend-down in the Italian Microsimulation Model CAPP_DYN: A Life-Cycle Approach

In microsimulation literature a limited number of models are provided with a module aimed at analyzing and projecting the evolution of private wealth over time. However, this issue appears crucial in order to get a comprehensive evaluation of the likely distributional effects of institutional reforms adopted to cope with population ageing. In this work we describe the implementation in the Italian dynamic micro simulation model CAPP_DYN of a new module in which household’s savings and asset allocation are modelled. In particular, our efforts are addressed at accounting for some possible behavioural responses to pension reforms in household savings. To this end, we rely on an approximate life cycle structural framework for estimating saving behaviour, while adopting the traditional stochastic micro simulation approach for assets allocation. In line with Ando and Nicoletti Altimari (2004), we emphasize the role of lifetime economic resources in households’ consumption decisions, yet we further account for internal habit formation and subjective expectations on pension outcomes in the econometric stage. In addition, we model intergenerational transfers of private wealth in a probabilistic fashion. Despite possible saving responses to pension reforms, simulated results for the period 2008-2050 suggest a rising dispersion in saving propensity and intergenerational transfers received are largely responsible for the predicted increase in disposable income inequality in the next decades which, differently from the recent past, will also affect the group of elderly.household consumption, habit formation, pension expectations, social security, intergenerational transfers, income and wealth distribution, microsimulation

alpha 4 beta 2 Nicotinic Acetylcholine Receptors: Relationships Between Subunit Stoichiometry and Function at the Single Channel Level

Acetylcholine receptors comprising alpha 4 and beta 2 subunits are the most abundant class of nicotinic acetylcholine receptor in the brain. They contribute to cognition, reward, mood, and nociception and are implicated in a range of neurological disorders. Previous measurements of whole-cell macroscopic currents showed that alpha 4 and beta 2 subunits assemble in two predominant pentameric stoichiometries, which differ in their sensitivity to agonists, antagonists, and allosteric modulators. Here we compare agonist-elicited single channel currents from receptors assembled with an excess of either the alpha 4 or beta 2 subunit, forming receptor populations biased toward one or the other stoichiometry, with currents from receptors composed of five concatemeric subunits in which the subunit stoichiometry is predetermined. Our results associate each subunit stoichiometry with a unique single channel conductance, mean open channel lifetime, and sensitivity to the allosteric potentiator 3-[3-(3-pyridinyl)-1,2,4-oxadiazol-5-yl] benzonitrile (NS-9283). Receptors with the composition (alpha 4 beta 2)(2)alpha 4 exhibit high single channel conductance, brief mean open lifetime, and strong potentiation by NS-9283, whereas receptors with the composition (alpha 4 beta 2)(2)beta 2 exhibit low single channel conductance and long mean open lifetime and are not potentiated by NS-9283. Thus single channel current measurements reveal bases for the distinct functional and pharmacological properties endowed by different stoichiometries of alpha 4 and beta 2 subunits and establish pentameric concatemers as a means to delineate interactions between subunits that confer these properties

Potentiation of a neuronal nicotinic receptor via pseudo-agonist site

Neuronal nicotinic receptors containing α4 and β2 subunits assemble in two pentameric stoichiometries, (α4)3(β2)2 and (α4)2(β2)3, each with distinct pharmacological signatures; (α4)3(β2)2 receptors are strongly potentiated by the drug NS9283, whereas (α4)2(β2)3 receptors are unaffected. Despite this stoichiometry-selective pharmacology, the molecular identity of the target for NS9283 remains elusive. Here, studying (α4)3(β2)2 receptors, we show that mutations at either the principal face of the β2 subunit or the complementary face of the α4 subunit prevent NS9283 potentiation of ACh-elicited single-channel currents, suggesting the drug targets the β2–α4 pseudo-agonist sites, the α4–α4 agonist site, or both sites. To distinguish among these possibilities, we generated concatemeric receptors with mutations at specified subunit interfaces, and monitored the ability of NS9283 to potentiate ACh-elicited single-channel currents. We find that a mutation at the principal face of the β2 subunit at either β2–α4 pseudo-agonist site suppresses potentiation, whereas mutation at the complementary face of the α4 subunit at the α4–α4 agonist site allows a significant potentiation. Thus, monitoring potentiation of single concatemeric receptor channels reveals that the β2–α4 pseudo-agonist sites are required for stoichiometry-selective drug action. Together with the recently determined structure of the (α4)3(β2)2 receptor, the findings have implications for structure-guided drug design

An analogue of the Prolactin Releasing Peptide reduces obesity and promotes adult neurogenesis

Hypothalamic Adult Neurogenesis (hAN) has been implicated in regulating energy homeostasis. Adult-generated neurons and adult Neural Stem Cells (aNSCs) in the hypothalamus control food intake and body weight. Conversely, diet-induced obesity (DIO) by high fat diets (HFD) exerts adverse influence on hAN. However, the effects of anti-obesity compounds on hAN are not known. To address this, we administered a lipidized analogue of an anti-obesity neuropeptide, Prolactin Releasing Peptide (PrRP), so-called LiPR, to mice. In the HFD context, LiPR rescued the survival of adult-born hypothalamic neurons and increased the number of aNSCs by reducing their activation. LiPR also rescued the reduction of immature hippocampal neurons and modulated calcium dynamics in iPSC-derived human neurons. In addition, some of these neurogenic effects were exerted by another anti-obesity compound, Liraglutide. These results show for the first time that anti-obesity neuropeptides influence adult neurogenesis and suggest that the neurogenic process can serve as a target of anti-obesity pharmacotherapy

Mortality after Transplantation for Hepatocellular Carcinoma: A Study from the European Liver Transplant Registry

Background and Aims: Prognosis after liver transplantation differs between hepatocellular carcinoma (HCC) arising in cirrhotic and non-cirrhotic livers and aetiology is poorly understood. The aim was to investigate differences in mortality after liver transplantation between these patients. Methods: We included patients from the European Liver Transplant Registry transplanted due to HCC from 1990 to November 2016 and compared cirrhotic and non-cirrhotic patients using propensity score (PS) calibration of Cox regression estimates to adjust for unmeasured confounding. Results: We included 22,787 patients, of whom 96.5% had cirrhosis. In the unadjusted analysis, non-cirrhotic patients had an increased risk of overall mortality with a hazard ratio (HR) of 1.37 (95% confidence interval [CI] 1.23-1.52). However, the HR approached unity with increasing adjustment and was 1.11 (95% CI 0.99-1.25) when adjusted for unmeasured confounding. Unadjusted, non-cirrhotic patients had an increased risk of HCC-specific mortality (HR 2.62, 95% CI 2.21-3.12). After adjustment for unmeasured confounding, the risk remained significantly increased (HR 1.62, 95% CI 1.31-2.00). Conclusions: Using PS calibration, we showed that HCC in non-cirrhotic liver has similar overall mortality, but higher HCC-specific mortality. This may be a result of a more aggressive cancer form in the non-cirrhotic liver as higher mortality could not be explained by tumour characteristics or other prognostic variables

Sublethal necroptosis signaling promotes inflammation and liver cancer

It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged "sublethal" state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1. This triggered hepatic cell proliferation as well as activation of procarcinogenic monocyte-derived macrophage cell clusters, contributing to hepatocarcinogenesis. In contrast, necrosome activation in hepatocytes with inactive NF-κB-signaling caused an accelerated execution of necroptosis, limiting alarmin release, and thereby preventing inflammation and hepatocarcinogenesis. Consistently, intratumoral NF-κB-necroptosis signatures were associated with poor prognosis in human hepatocarcinogenesis. Therefore, pharmacological reprogramming between these distinct forms of necroptosis may represent a promising strategy against hepatocellular carcinoma

Development and Validation of a Comprehensive Model to Estimate Early Allograft Failure among Patients Requiring Early Liver Retransplant

Importance: Expansion of donor acceptance criteria for liver transplant increased the risk for early allograft failure (EAF), and although EAF prediction is pivotal to optimize transplant outcomes, there is no consensus on specific EAF indicators or timing to evaluate EAF. Recently, the Liver Graft Assessment Following Transplantation (L-GrAFT) algorithm, based on aspartate transaminase, bilirubin, platelet, and international normalized ratio kinetics, was developed from a single-center database gathered from 2002 to 2015. Objective: To develop and validate a simplified comprehensive model estimating at day 10 after liver transplant the EAF risk at day 90 (the Early Allograft Failure Simplified Estimation [EASE] score) and, secondarily, to identify early those patients with unsustainable EAF risk who are suitable for retransplant. Design, Setting, and Participants: This multicenter cohort study was designed to develop a score capturing a continuum from normal graft function to nonfunction after transplant. Both parenchymal and vascular factors, which provide an indication to list for retransplant, were included among the EAF determinants. The L-GrAFT kinetic approach was adopted and modified with fewer data entries and novel variables. The population included 1609 patients in Italy for the derivation set and 538 patients in the UK for the validation set; all were patients who underwent transplant in 2016 and 2017. Main Outcomes and Measures: Early allograft failure was defined as graft failure (codified by retransplant or death) for any reason within 90 days after transplant. Results: At day 90 after transplant, the incidence of EAF was 110 of 1609 patients (6.8%) in the derivation set and 41 of 538 patients (7.6%) in the external validation set. Median (interquartile range) ages were 57 (51-62) years in the derivation data set and 56 (49-62) years in the validation data set. The EASE score was developed through 17 entries derived from 8 variables, including the Model for End-stage Liver Disease score, blood transfusion, early thrombosis of hepatic vessels, and kinetic parameters of transaminases, platelet count, and bilirubin. Donor parameters (age, donation after cardiac death, and machine perfusion) were not associated with EAF risk. Results were adjusted for transplant center volume. In receiver operating characteristic curve analyses, the EASE score outperformed L-GrAFT, Model for Early Allograft Function, Early Allograft Dysfunction, Eurotransplant Donor Risk Index, donor age × Model for End-stage Liver Disease, and Donor Risk Index scores, estimating day 90 EAF in 87% (95% CI, 83%-91%) of cases in both the derivation data set and the internal validation data set. Patients could be stratified in 5 classes, with those in the highest class exhibiting unsustainable EAF risk. Conclusions and Relevance: This study found that the developed EASE score reliably estimated EAF risk. Knowledge of contributing factors may help clinicians to mitigate risk factors and guide them through the challenging clinical decision to allocate patients to early liver retransplant. The EASE score may be used in translational research across transplant centers