Targeting allosteric sites of the M
muscarinic acetylcholine receptor (mAChR) is an enticing approach to overcome the lack of receptor subtype selectivity observed with orthosteric ligands. This is a ...promising strategy for obtaining novel therapeutics to treat cognitive deficits observed in Alzheimer's disease and schizophrenia, while reducing the peripheral side effects such as seen in the current treatment regimes, which are non-subtype selective. We previously described compound 2, the first positive allosteric modulator (PAM) of the M
mAChR based on a 6-phenylpyrimidin-4-one scaffold, which has been further developed in this study. Herein, we present the synthesis, characterization, and pharmacological evaluation of a series of 6-phenylpyrimidin-4-ones with modifications to the 4-(1-methylpyrazol-4-yl)benzyl pendant. Selected compounds, BQCA, 1, 2, 9i, 13, 14b, 15c, and 15d, were further profiled in terms of their allosteric affinity, cooperativity with acetylcholine (ACh), and intrinsic efficacy. Additionally, 2 and 9i were tested in mouse primary cortical neurons, displaying various degrees of intrinsic agonism and potentiation of the acetylcholine response. Overall, the results suggest that the pendant moiety is important for allosteric binding affinity and the direct agonistic efficacy of the 6-phenylpyrimidin-4-one based M
mAChR PAMs.
Cure of hypertension after adrenalectomy for primary aldosteronism is no certainty and therefore preoperative patient counseling is essential. The Primary Aldosteronism Surgical Outcome (PASO) Score ...is a useful prediction model with an area under the curve (AUC) of 0.839. The PASO Score includes ‘Target Organ Damage’ (TOD) (i.e., left ventricular hypertrophy and/or microalbuminuria), which is often unavailable during preoperative counseling and might therefore limit its use in clinical practice. We hypothesized that the PASO score would still be useful if TOD is unknown at time of counseling. Therefore, we aimed to examine the predictive performance of the simplified PASO Score, without taking TOD into account.
In this retrospective cohort study, patients who underwent unilateral adrenalectomy between 2010 and 2016 in 16 medical centers from North America, Europe and Australia were included. TOD was unknown in our database and therefore assigned as absent. Patients were classified as complete, partial or absent clinical success using the PASO consensus criteria.
A total of 380 (73.9%) patients were eligible for analysis. Complete, partial and absent clinical success were observed in 29.5%, 55.8% and 14.7% of patients, respectively. The simplified PASO Score had an AUC of 0.730 (95% confidence interval 0.674–0.785) in our total cohort.
Without taking TOD into account, the simplified PASO Score had a lower predictive value as compared to the original derivation cohort. Ideally, the complete PASO Score should be used, but when data on TOD are not readily available, the simplified PASO Score is a useful and reasonable alternative.
•We aimed to examine the predictive performance of the PASO Score, without taking ‘target organ damage’ (TOD) into account.•This simplified PASO Score had a lower predictive value as compared to the PASO Score in the original derivation cohort.•The simplified PASO Score increases the applicability of the model and is reasonable for clinicians to use in daily practice.•Ideally, the complete PASO Score should be used, but the simplified PASO Score is a useful and reasonable alternative.
Tyrosine hydroxylase (TH) catalyzes the conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine, the first and rate-limiting step in catecholamine biosynthesis. The cAMP-dependent protein kinase ...(PKA) phosphorylates and activates the TH enzyme and is thought to mediate transcriptional induction of the TH gene. To better understand the functional role of PKA in TH gene regulation, we studied TH gene expression at the transcriptional, translational, and post-translational levels in several PKA-deficient cell lines derived from rat PC12 pheochromocytoma cells. Strikingly, all PKA-deficient cell lines analyzed in this study showed substantial deficits in basal TH expression as measured by TH enzymatic activity, level of TH immunoreactivity, TH protein level, and steady-state mRNA level. Interestingly, the steady-state level of mRNA correlated well with levels of TH activity, immunoreactivity, and protein. In addition, PKA-deficient cell lines lacked transcriptional induction of the TH gene following treatment with dibutyryl cAMP. Cotransfection of PKA-deficient cells with an expression plasmid for the catalytic subunit of PKA fully reversed transcriptional defect, as indicated by robust transcriptional induction of a reporter construct containing 2400 bp of TH upstream sequence in all PC12 cells tested. These data indicate that the PKA system regulates both the basal and the cAMP-inducible expression of the TH gene primarily at the transcriptional level in PC12 cells.
The Belle II experiment is evaluating an upgrade program aiming at the time frame 2026–2027 for the installation of the upgraded detectors, to improve the detector performance and robustness against ...beam-induced backgrounds. The VTX concept, a fully-pixelated system based on depleted monolithic active pixel sensors, has been developed as a replacement of the current pixel-and-strip vertex detector. A simulation framework for the VTX has been developed and integrated with the standard Belle II simulation, facilitating the comparison among different layouts. Simulation results (such as tracking efficiency and vertex resolution) on benchmark physics channels are presented, showing a significant improvement with respect to the current detector.
Abstract
Background
Impairment of myocardial mitochondrial function is regarded as an established pathomechanism in heart failure. Enhanced oxidation of ketone bodies may potentially exert protective ...effects on myocardial function. High-resolution respirometry (HRR) resembles a gold-standard methodology to determine myocardial mitochondrial metabolism and oxidative function but has not been validated for ketone substrates yet.
Purpose
We hypothesized that (1) quantification of ketone body oxidative capacity (OC) in myocardium utilizing ex-vivo HRR is feasible and that (2) ketone-associated OC is elevated after fasting and under conditions of chronic mechanical ventricular unloading.
Methods
We established new HRR (Oxygraph-2k) protocols, measuring oxygen flux generated by oxidation of the ketone substrates beta-hydroxybutyrate (HBA) and acetoacetate (ACA). Ketone protocols were then applied to twelve C57BL/6 mice' (of which six were fasted for 16h) left ventricular and right liver lobe tissue, as well as to eleven terminal heart failure patients' left ventricular tissue, harvested at heart transplantation. Heart transplant recipients were subdivided into patients with left ventricular assist device prior to transplantation (LVAD group, n=6) or no unloading prior to transplantation (HTX group, n=5).
Results
In non-fasted rodent hearts, HBA yielded an OC of 25±4 pmol/(s*mg tissue) above basal respiration, when applied as sole substrate (21±11 pmol/(s*mg) in liver). ACA alone did not induce oxygen flux, but ACA+succinate yielded 229% higher oxygen flux than succinate alone in state III (146±32 vs 44±12 pmol/(s*mg); p=0.0003). When titrated after succinate, ACA increased OC by 93±25 pmol/(s*mg) (p=0.0003). In 16h-fasted rodent hearts, HBA-supported OC was 27% higher (41±3 vs 52±9 pmol/(s*mg); p=0.04), while OC with ACA+succinate was unchanged (p=0.60). In rodent liver, no oxygen flux was induced by ACA, reflecting absence of 3-oxoacid CoA-transferase. However, HBA-supported OC was 118% higher in fasted liver (37±13 vs 57±13 pmol/(s*mg); p=0.03). In humans, left ventricular unloading was not associated with altered myocardial OC for fatty acids and glycolytic substrates (standard protocol, p=0.13), but HBA-supported OC was 39% higher in the LVAD group compared to the HTX group (54±12 vs 39±9 pmol/(s*mg), p=0.04).
Conclusion
Quantification of ketone body OC with HRR is feasible in permeabilized myocardial fibers. Applying this novel method revealed increased HBA-supported myocardial mitochondrial respiration after fasting and chronic left ventricular unloading. These data support a concept of enhanced ketone oxidation following ventricular unloading in myocardial mitochondria. Our findings facilitate new studies on myocardial ketone turnover and the interaction of mitochondrial ketone metabolism with cardiac performance.
Acknowledgement/Funding
CRC 1116, Research commission of the University Hospital Düsseldorf
A numerical evolutionary procedure for the structural optimisation for stress reduction of two-dimensional structures is presented in this paper. The proposed procedure couples the biological growth ...method (BGM) with the boundary element method (BEM). The boundary-only intrinsic characteristic of BEM together with its accuracy in the boundary displacement and stress solutions make BEM especially attractive for solving shape-optimisation problems. Two formulations of BEM are used in this work: the standard for two-dimensional elastostatics for the stress analysis and the dual reciprocity method (DRM), which is used to model the swelling or shrinking of the material. Two examples are analysed to illustrate the proposed methodology and to demonstrate its versatility and robustness.
The pattern of neuronal activation in the rat nucleus of the solitary tract (NTS) in response to a standard gustatory stimulus was examined using c-Fos-like immunoreactivity (c-FLI) before and after ...conditioned taste aversion (CTA) formation. While unconditioned oral infusions of sucrose solution did not induce c-FLI in the NTS, after three pairings of sucrose with lithium chloride injections, sucrose induced c-FLI in the medial intermediate NTS 1 h after oral infusion. Extinction of the CTA by repeated infusions of sucrose alone reversed the induction of c-FLI.
Background and Purpose Affinity‐based, selective orthosteric ligands for the muscarinic acetylcholine receptors (mAChRs) are difficult to develop due to high sequence homology across the five ...subtypes. Selectivity can also be achieved via the selective activation of a particular subtype or signalling pathway. Promisingly, a prior study identified compounds 6A and 7A as functionally selective and G i biased compounds at the M 2 mAChR. Here, we have investigated the activation of individual G protein subfamilies and the downstream signalling profiles of 6A and 7A at the M 2 mAChR. Experimental Approach G protein activation was measured with the TRUPATH assay in M 2 mAChR FlpIn CHO cells. Activity in downstream signalling pathways was determined using the cAMP CAMYEL BRET sensor and assay of ERK 1/2 phosphorylation. Key Results M 2 mAChRs coupled to Gɑ i1 , Gɑ oA and Gɑ s , but not Gɑ q , in response to canonical orthosteric agonists. Compounds 6A and 7A did not elicit any G protein activation, cAMP inhibition or stimulation, or ERK 1/2 phosphorylation. Instead, a Schild analysis indicates a competitive, antagonistic interaction of compounds 6A and 7A with ACh in the Gɑ i1 activation assay. Overexpression of the M 2 mAChR may suggest an expression‐dependent activation profile of compounds 6A and 7A. Conclusions and Implications These data confirm that the M 2 mAChR preferentially couples to Gɑ i/o and to a lesser extent to Gɑ s in response to canonical orthosteric ligands. However, this study was not able to detect Gɑ i bias of compounds 6A and 7A, highlighting the importance of cellular background when classifying new ligands. LINKED ARTICLES This article is part of a themed issue Therapeutic Targeting of G Protein‐Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc
Due to the non-commercial, research-oriented context, software in medical informatics research projects is often developed by researchers as a proof-of-concept without applying structured software ...development process models. A guideline for software development can bring sufficient structure to the development process while avoiding the complexity of industry-standard methods.
We adapted the common evidence-based guideline development process from medicine to build a guideline for software development in our medical informatics teaching and research project.
Our guideline development used the six steps of problem identification, first proposal, review, revision, gaining consensus and periodic guideline review. Since the developers had taken part in guideline development, our guideline clearly states the consensus of the development team over critical topics. The guideline improved the quality of our source code in structure and understandability.
A software development guideline that is developed following a consensus panel approach is a good instrument for basic software quality assurance in domains where complex, industry-standard software development methods cannot be applied. This is especially the case in non-commercial, research-oriented medical informatics projects where mainly non-software engineers like students do the development work.
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