Mapping the expression of an ANK3 isoform associated with bipolar disorder in the human brain

The gene ankyrin-3 (ANK3) has been consistently associated with bipolar disorder (BD) in several genome-wide association studies (GWAS). The exact molecular mechanisms underlying this genetic association remain unknown. The discovery of a loss-of-function variant (rs41283526*G) in an alternatively spliced exon (ENSE00001786716) with a protective effect, suggested that elevated expression of this particular isoform could be a risk factor for developing the disorder. We developed a novel approach for measuring the expression level of all splice forms at a challenging genetic locus using a combination of droplet digital PCR and high-throughput sequencing of indexed PCR amplicons. The combined method was performed on a large collection of 568 postmortem brain samples of BD and SCZ cases and controls. We also studied the expression of the splice forms in a child-development cohort of 41 healthy males. We found that our approach can quantify the splice forms in brain samples, although with less precision than ddPCR. We detected highly significant differences in expression of splice forms and transcription start sites between brain regions, notably with higher expression of the BD-associated isoform in the corpus callosum compared to frontal tissue (mean fold change = 1.80, p < 1e-4). Although the patients in our sample expressed the BD-associated splice form at a similar level to controls, adolescents in our child-development cohort had a clearly higher expression level than younger children (mean fold change = 1.97, p = 5e-3). These results suggest that this ANK3 splice form may play a role in the myelin maturation of the human brain.publishedVersio

Identification of essential residues for binding and activation in the human 5-HT7(a) serotonin receptor by molecular modeling and site-directed mutagenesis

The human 5-HT7 receptor is expressed in both the central nervous system and peripheral tissues and is a potential drug target in behavioral and psychiatric disorders. We examined molecular determinants of ligand binding and G protein activation by the human 5-HT7(a) receptor. The role of several key residues in the 7th transmembrane domain (TMD) and helix 8 were elucidated combining in silico and experimental mutagenesis. Several single and two double point mutations of the 5-HT7(a) wild type receptor were made (W7.33V, E7.35T, E7.35R, E7.35D, E7.35A, R7.36V, Y7.43A, Y7.43F, Y7.43T, R8.52D, D8.53K; E7.35T-R7.36V, R8.52D-D8.53K), and their effects upon ligand binding were assessed by radioligand binding using a potent agonist (5-CT) and a potent antagonist (SB269970). In addition, the ability of the mutated 5-HT7(a) receptors to activate G protein after 5-HT-stimulation was determined through activation of adenylyl cyclase. In silico investigation on mutated receptors substantiated the predicted importance of TM7 and showed critical roles of residues E7.35, W7.33, R7.36 and Y7.43 in agonist and antagonist binding and conformational changes of receptor structure affecting adenylyl cyclase activation. Experimental data showed that mutants E7.35T and E7.35R were incapable of ligand binding and adenylyl cyclase activation, consistent with a requirement for a negatively charged residue at this position. The mutant R8.52D was unable to activate adenylyl cyclase, despite unaffected ligand binding, consistent with the R8.52 residue playing an important role in the receptor-G protein interface. The mutants Y7.43A and Y7.43T displayed reduced agonist binding and AC agonist potency, not seen in Y7.43F, consistent with a requirement for an aromatic residue at this position. Knowledge of the molecular interactions important in h5-HT7 receptor ligand binding and G protein activation will aid the design of selective h5-HT7 receptor ligands for potential pharmacological use

Mapping the expression of an ANK3 isoform associated with bipolar disorder in the human brain

The gene ankyrin-3 (ANK3) has been consistently associated with bipolar disorder (BD) in several genome-wide association studies (GWAS). The exact molecular mechanisms underlying this genetic association remain unknown. The discovery of a loss-of-function variant (rs41283526*G) in an alternatively spliced exon (ENSE00001786716) with a protective effect, suggested that elevated expression of this particular isoform could be a risk factor for developing the disorder. We developed a novel approach for measuring the expression level of all splice forms at a challenging genetic locus using a combination of droplet digital PCR and high-throughput sequencing of indexed PCR amplicons. The combined method was performed on a large collection of 568 postmortem brain samples of BD and SCZ cases and controls. We also studied the expression of the splice forms in a child-development cohort of 41 healthy males. We found that our approach can quantify the splice forms in brain samples, although with less precision than ddPCR. We detected highly significant differences in expression of splice forms and transcription start sites between brain regions, notably with higher expression of the BD-associated isoform in the corpus callosum compared to frontal tissue (mean fold change = 1.80, p < 1e-4). Although the patients in our sample expressed the BD-associated splice form at a similar level to controls, adolescents in our child-development cohort had a clearly higher expression level than younger children (mean fold change = 1.97, p = 5e-3). These results suggest that this ANK3 splice form may play a role in the myelin maturation of the human brain

Deletion of beta-strands 9 and 10 converts VDAC1 voltage-dependence in an asymmetrical process

Voltage-dependent anion selective channel isoform1 maintains the permeability of the outer mitochondrial membrane. Its voltage-gating properties are relevant in bioenergetic metabolism and apoptosis. The N-terminal domain is suspected to be involved in voltage-gating, due to its peculiar localization. However this issue is still controversial. In this work we exchanged or deleted the beta-strands that take contact with the N-terminal domain. The exchange of the whole hVDAC1 beta-barrel with the homologous hVDAC3 beta-barrel produces a chimeric protein that, in reconstituted systems, loses completely voltage-dependence. hVDAC3 beta-barrel has most residues in common with hVDAC1, including V143 and L150 considered anchor points for the N-terminus. hVDAC1 mutants completely lacking either the beta-strand 9 or both beta-strands 9 and 10 were expressed, refolded and reconstituted in artificial bilayers. The mutants formed smaller pores. Molecular dynamics simulations of the mutant structure supported its ability to form smaller pores. The mutant lacking both beta-strands 9 and 10 showed a new voltage-dependence feature resulting in a fully asymmetric behavior. These data indicate that a network of beta-strands in the pore-walls, and not single residues, are required for voltage-gating in addition to the N-terminus. (C) 2013 Elsevier B.V. All rights reserved

The antineoplastic drug flavopiridol reverses memory impairment induced by Amyloid-ß1-42 oligomers in mice

The ectopic re-activation of cell cycle in neurons is an early event in the pathogenesis of Alzheimer's disease (AD), which could lead to synaptic failure and ensuing cognitive deficits before frank neuronal death. Cytostatic drugs that act as cyclin-dependent kinase (CDK) inhibitors have been poorly investigated in animal models of AD. In the present study, we examined the effects of flavopiridol, an inhibitor of CDKs currently used as antineoplastic drug, against cell cycle reactivation and memory loss induced by intracerebroventricular injection of A beta(1-42) oligomers in CD1 mice. Cycling neurons, scored as NeuN-positive cells expressing cyclin A, were found both in the frontal cortex and in the hippocampus of A beta-injected mice, paralleling memory deficits. Starting from three days after A beta injection, flavopiridol (0.5, 1 and 3 mg/kg) was intraperitoneally injected daily, for eleven days. Here we show that a treatment with flavopiridol (0.5 and 1 mg/kg) was able to rescue the loss of memory induced by A beta(1-42), and to prevent the occurrence of ectopic cell-cycle events in the mouse frontal cortex and hippocampus. This is the first evidence that a cytostatic drug can prevent cognitive deficits in a non-transgenic animal model of AD. (C) 2016 Elsevier Ltd. All rights reserved

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 licenseBackground: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licenseBackground: Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide. Methods: A multimethods analysis was performed as part of the GlobalSurg 3 study—a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital. Findings: Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3·85 [95% CI 2·58–5·75]; p<0·0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63·0% vs 82·7%; OR 0·35 [0·23–0·53]; p<0·0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer. Interpretation: Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised. Funding: National Institute for Health and Care Research