Diversity of nicotinic acetylcholine receptor positive allosteric modulators revealed by mutagenesis and a revised structural model

By combining electrophysiological and computational approaches we have examined a series of positive allosteric modulators (PAMs) acting on the human α7 nicotinic acetylcholine receptor (nAChR). Electrophysiological studies have focussed on three α7-selective PAMs (A-867744, TBS-516 and TQS) that display similar effects on wild-type α7 nAChRs. In addition to potentiating agonist-evoked responses, all three compounds reduce receptor desensitisation and, consequently, are classed as type II PAMs. Despite having similar effects on wild-type receptors, A-867744 was found to have profoundly differing effects to TBS-516 and TQS on mutated receptors, a finding that is consistent with previous studies indicating that A-867744 may have a different mechanism of action to other α7-selective type II PAMs. Due to evidence that these PAMs bind within the α7 nAChR transmembrane region, we generated and validated new structural models of α7. Importantly, we have corrected a previously identified error in the transmembrane region of the original cryo-EM Torpedo model; the only pentameric ligand-gated ion channel imaged in a native lipid membrane. Real-space refinement was used to generate closed and open conformations on which the α7 models were based. Consensus docking with an extended series of PAMs with chemical similarity to A-867744, TBS-516 and TQS suggests that all bind to a broadly similar inter-subunit transmembrane site. However, differences in the predicted binding of A-867744, compared with TBS-516 and TQS, may help to explain the distinct functional effects of A-867744. Thus, our revised structural models may provide a useful tool for interpreting functional effects of PAMs

Cabbage and fermented vegetables : From death rate heterogeneity in countries to candidates for mitigation strategies of severe COVID-19

Large differences in COVID-19 death rates exist between countries and between regions of the same country. Some very low death rate countries such as Eastern Asia, Central Europe, or the Balkans have a common feature of eating large quantities of fermented foods. Although biases exist when examining ecological studies, fermented vegetables or cabbage have been associated with low death rates in European countries. SARS-CoV-2 binds to its receptor, the angiotensin-converting enzyme 2 (ACE2). As a result of SARS-CoV-2 binding, ACE2 downregulation enhances the angiotensin II receptor type 1 (AT(1)R) axis associated with oxidative stress. This leads to insulin resistance as well as lung and endothelial damage, two severe outcomes of COVID-19. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is the most potent antioxidant in humans and can block in particular the AT(1)R axis. Cabbage contains precursors of sulforaphane, the most active natural activator of Nrf2. Fermented vegetables contain many lactobacilli, which are also potent Nrf2 activators. Three examples are: kimchi in Korea, westernized foods, and the slum paradox. It is proposed that fermented cabbage is a proof-of-concept of dietary manipulations that may enhance Nrf2-associated antioxidant effects, helpful in mitigating COVID-19 severity.Peer reviewe

Nrf2-interacting nutrients and COVID-19 : time for research to develop adaptation strategies

There are large between- and within-country variations in COVID-19 death rates. Some very low death rate settings such as Eastern Asia, Central Europe, the Balkans and Africa have a common feature of eating large quantities of fermented foods whose intake is associated with the activation of the Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) anti-oxidant transcription factor. There are many Nrf2-interacting nutrients (berberine, curcumin, epigallocatechin gallate, genistein, quercetin, resveratrol, sulforaphane) that all act similarly to reduce insulin resistance, endothelial damage, lung injury and cytokine storm. They also act on the same mechanisms (mTOR: Mammalian target of rapamycin, PPAR gamma:Peroxisome proliferator-activated receptor, NF kappa B: Nuclear factor kappa B, ERK: Extracellular signal-regulated kinases and eIF2 alpha:Elongation initiation factor 2 alpha). They may as a result be important in mitigating the severity of COVID-19, acting through the endoplasmic reticulum stress or ACE-Angiotensin-II-AT(1)R axis (AT(1)R) pathway. Many Nrf2-interacting nutrients are also interacting with TRPA1 and/or TRPV1. Interestingly, geographical areas with very low COVID-19 mortality are those with the lowest prevalence of obesity (Sub-Saharan Africa and Asia). It is tempting to propose that Nrf2-interacting foods and nutrients can re-balance insulin resistance and have a significant effect on COVID-19 severity. It is therefore possible that the intake of these foods may restore an optimal natural balance for the Nrf2 pathway and may be of interest in the mitigation of COVID-19 severity

The Introduction of Open and Free Academic Courses in Conservation of Cultural Heritage in Greece

The 4-year Bachelor Degree in Conservation-Restoration at the Technological Educational Institute (TEI) of Athens has the difficult task of preparing its students to become professional Conservator-Restorers with knowledge and skills in six specializations of either archaeological (objects) conservation or in conservation works of art. Thus, the academic staff of the Department has the challenge to prepare its students to be qualified in practice and knowledge of many specializations of conservation of Cultural Heritage (CH) within its 4 year program. To meet this demand, many members of the academic staff are participating in a project co-financed by Greece and the European Union (European Social Fund) called ‘Open Academic Courses’ (2013-2015). The goal is to prepare their course material in Greek as freely accessible digital educational content in the form of thirteen PowerPoint lectures with or without video lectures per course offered via the open source eLearning platform ‘Open eClass’ hosted by the TEI of Athens. The paper describes how the courses were prepared, the challenges involved in its preparation, as well as the benefits to the students

Pharmacological Characterisation of Nicotinic Acetylcholine Receptors Expressed in Human iPSC-Derived Neurons.

Neurons derived from human induced pluripotent stem cells (iPSCs) represent a potentially valuable tool for the characterisation of neuronal receptors and ion channels. Previous studies on iPSC-derived neuronal cells have reported the functional characterisation of a variety of receptors and ion channels, including glutamate receptors, γ-aminobutyric acid (GABA) receptors and several voltage-gated ion channels. In the present study we have examined the expression and functional properties of nicotinic acetylcholine receptors (nAChRs) in human iPSC-derived neurons. Gene expression analysis indicated the presence of transcripts encoding several nAChR subunits, with highest levels detected for α3-α7, β1, β2 and β4 subunits (encoded by CHRNA3-CHRNA7, CHRNB1, CHRNB2 and CHRNB4 genes). In addition, similarly high transcript levels were detected for the truncated dupα7 subunit transcript, encoded by the partially duplicated gene CHRFAM7A, which has been associated with psychiatric disorders such as schizophrenia. The functional properties of these nAChRs have been examined by calcium fluorescence and by patch-clamp recordings. The data obtained suggest that the majority of functional nAChRs expressed in these cells have pharmacological properties typical of α7 receptors. Large responses were induced by a selective α7 agonist (compound B), in the presence of the α7-selective positive allosteric modulator (PAM) PNU-120596, which were blocked by the α7-selective antagonist methyllycaconitine (MLA). In addition, a small proportion of the neurons express nAChRs with properties typical of heteromeric (non-α7 containing) nAChR subtypes. These cells therefore represent a great tool to advance our understanding of the properties of native human nAChRs, α7 in particular

Characterisation of native nAChRs in rat primary hippocampal cells and human induced pluripotent stem cell-derived neurons, examined by fluorescence-based intracellular calcium imaging.

<p>A) Pseudocolour images of rat hippocampal neurons corresponding to low initial resting calcium levels (Left) and higher calcium levels, after application of 30 µM 4BP-TQS (Right). B) Single cell traces (cyan) for all neurons present in the optical field. The average response is shown in red (n = 89 cells). C) Histogram illustrating the percentage of cells that responded to compound B (1 µM), compound B co-applied with TQS (1 µM and 10 µM, respectively) and 4BP-TQS (30 µM) in rat primary hippocampal cells (blue) and in iCell neurons (red). Data were normalised to the total number of cells that responded to KCl (50 mM) (n = 3–31).</p

Pharmacological properties of TQS and 4BP-TQS on nAChR subtypes.

*<p>Fold potentiation of response to ACh (100 µM) by TQS (100 µM).</p>**<p>Percentage inhibition of response to ACh (100 µM) by TQS (100 µM).</p>†<p>Agonist response of 4BP-TQS (100 µM) expressed as a fold response normalised to maximal concentration of ACh (3 mM).</p>††<p>Percentage inhibition of response to ACh (100 µM) by 4BP-TQS (100 µM). Data are means ± SEM.</p

Characterisation of 4BP-TQS on α7 nAChRs examined by patch-clamp recording of rat primary hippocampal cells.

<p>A) Representative recordings showing responses to the application of compound B (30 µM; Left; black bar) and of 4BP-TQS (30 µM; Right; grey bar). B) Prolonged exposure of α7 nAChRs to compound B (1 µM; black bar) results in receptor activation, followed by rapid desensitisation. In continued presence of compound B (1 µM; black bar), co-application of either TQS (1 µM; Left; grey bar) or 4BP-TQS (30 µM; Right; black bar) resulted in reactivation of desensitised receptors. C) Responses to 4BP-TQS (30 µM; Left; black bar) are blocked by MLA (10 nM; grey bar) when MLA was pre-applied for 15 s and then co-applied with 4BP-TQS (30 µM; black bar).</p

Block of 4BP-TQS responses by TQS in rat primary hippocampal cells examined by fluorescence-based intracellular calcium imaging.

<p>Dose response data are presented for a range of concentrations of TQS (0.3 to 30 µM) in the presence of 4BP-TQS (10 µM). In all cases TQS was pre-applied for 30 s and then co-applied with 4BP-TQS. Responses were normalised to 4BP-TQS (10 µM). Data are means ± SEM of 3 independent experiments.</p

Chemical structure of α7 nAChR orthosteric and allosteric ligands examined in the present study.

<p>Chemical structure of α7 nAChR orthosteric and allosteric ligands examined in the present study.</p