GC/MSn analysis of the crude reaction mixtures from Friedel–Crafts acylation: Unambiguous identification and differentiation of 3-aroylbenzofurans from their 4- and 6-regioisomers

Rationale: 3-Aroylbenzofurans and their 2-nitrophenyl derivatives constitute fundamental intermediates for the synthesis of target compounds with pharmaceutical properties. However, their preparation via the Friedel–Crafts acylation of 2-phenylbenzofurans, using Lewis acid as catalyst, often leads to mixtures of regioisomeric aroylbenzofurans that can be challenging to distinguish, thus preventing the reaction characterization. Method: We report a method for the unambiguous identification and differentiation of the desired 3-benzoyl isomers from their 4- and 6-regioisomers in a crude reaction mixture using gas chromatography coupled to multiple-stage mass spectrometric (GC/MSn) analysis performed in collision-induced dissociation (CID) mode. Results: Upon electron ionization, each set of isomers displayed nearly identical mass spectra. MSn revealed fragmentation patterns that varied in the location of the benzoyl group on the benzofuran scaffold: CID experiments performed on the molecular ion allowed the distinction of the 3-acyl isomers from the 4- and 6-regioisomers; CID experiments on the [M − Ar]+ ion allowed the distinction of the 4-benzoyl from the 6-benzoyl regioisomer, when the nitro group is located on the 2-phenyl ring. Moreover, the unusual loss of OH• radical allowed ascertaining the position of the nitro group in 3-acyl regioisomers bearing the NO2 group. The origin of the diagnostic OH• loss was investigated through MSn experiments using 18O-labelled 3-benzoyl derivatives. Conclusions: The method allows the rapid characterization of crude reaction mixtures of benzoylbenzofurans using solely GC/MSn analysis, simplifying the workflow of extensive isolation and purification for structure elucidationThe present work was partially supported by FIR (Fondo Integrativo per la Ricerca – annualità 2018 and 2019), University of CagliariS

A novel algorithm for dynamic student profile adaptation based on learning styles

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.E-learning recommendation systems are used to enhance student performance and knowledge by providing tailor- made services based on the students’ preferences and learning styles, which are typically stored in student profiles. For such systems to remain effective, the profiles need to be able to adapt and reflect the students’ changing behaviour. In this paper, we introduce new algorithms that are designed to track student learning behaviour patterns, capture their learning styles, and maintain dynamic student profiles within a recommendation system (RS). This paper also proposes a new method to extract features that characterise student behaviour to identify students’ learning styles with respect to the Felder-Silverman learning style model (FSLSM). In order to test the efficiency of the proposed algorithm, we present a series of experiments that use a dataset of real students to demonstrate how our proposed algorithm can effectively model a dynamic student profile and adapt to different student learning behaviour. The results revealed that the students could effectively increase their learning efficiency and quality for the courses when the learning styles are identified, and proper recommendations are made by using our method

Functional characterization and analgesic effects of mixed cannabinoid receptor/T-type channel ligands

<p>Abstract</p> <p>Background</p> <p>Both T-type calcium channels and cannabinoid receptors modulate signalling in the primary afferent pain pathway. Here, we investigate the analgesics activities of a series of novel cannabinoid receptor ligands with T-type calcium channel blocking activity.</p> <p>Results</p> <p>Novel compounds were characterized in radioligand binding assays and <it>in vitro </it>functional assays at human and rat CB1 and CB2 receptors. The inhibitory effects of these compounds on transient expressed human T-type calcium channels were examined in tsA-201 cells using standard whole-cell voltage clamp techniques, and their analgesic effects in response to various administration routes (intrathecally, intraplantarly, intraperitoneally) assessed in the formalin model. A series of compounds were synthesized and evaluated for channel and receptor activity. Compound NMP-7 acted as non-selective CB1/CB2 agonist while NMP4 was found to be a CB1 partial agonist and CB2 inverse agonist. Furthermore, NMP-144 behaved as a selective CB2 inverse agonist. All of these three compounds completely inhibited peak Cav3.2 currents with IC₅₀values in the low micromolar range. All compounds mediated analgesic effects in the formalin model, but depending on the route of administration, could differentially affect phase 1 and phase 2 of the formalin response.</p> <p>Conclusions</p> <p>Our results reveal that a set of novel cannabinioid receptor ligands potently inhibit T-type calcium channels and show analgesic effects <it>in vivo</it>. Our findings suggest possible novel means of mediating pain relief through mixed T-type/cannabinoid receptor ligands.</p

Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells

Cannabinoid 1 receptors (CB1Rs) are expressed in peripheral tissues, including islets of Langerhans, where their function(s) is under scrutiny. Using mouse beta-cell lines, human islets and CB1R-null (CB1R(-/-)) mice, we have now investigated the role of CB1Rs in modulating beta-cell function and glucose responsiveness. Synthetic CB1R agonists diminished GLP-1-mediated cAMP accumulation and insulin secretion as well as glucose-stimulated insulin secretion in mouse beta-cell lines and human islets. In addition, silencing CB1R in mouse cells resulted in an increased expression of pro-insulin, glucokinase (GCK) and glucose transporter 2 (GLUT2), but this increase was lost in cells lacking insulin receptor. Furthermore, CB1R(-/-) mice had increased pro-insulin, GCK and GLUT2 expression in cells. Our results suggest that CB1R signalling in pancreatic islets may be harnessed to improve beta-cell glucose responsiveness and preserve their function. Thus, our findings further support that blocking peripheral CB1Rs would be beneficial to beta-cell function in type 2 diabetes

M1 muscarinic allosteric modulators slow prion neurodegeneration and restore memory loss

This is the final version of the article. Available from American Society for Clinical Investigation via the DOI in this record.The current frontline symptomatic treatment for Alzheimer’s disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR–selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.ABT, AC, and PMS received funding from a Wellcome Trust Collaborative Award (201529/Z/16/Z). ABT, SJB, AJB, and TMH were funded through a Medical Research Council programme leader grant provided by the MRC Toxicology Unit. CCF, LMB, AJM, and HES were funded by the Eli Lilly Company. JMB received funding through a Lilly Research Award Program (LRAP) grant (Eli Lilly). RP received funding from the Marie Curie grant “Extrabrain” (European Commission). AC is a senior principal research fellow and PMS a principal research fellow of the National Health and Medical Research Council of Australia. Tissue samples were from Randy Woltjer at the Oregon Alzheimer’s Disease Center. The Oregon Alzheimer’s Disease Center is supported by NIH grant P30AG008017

The M3 muscarinic receptor Is required for optimal adaptive immunity to Helminth and bacterial infection

Innate immunity is regulated by cholinergic signalling through nicotinic acetylcholine receptors. We show here that signalling through the M3 muscarinic acetylcholine receptor (M3R) plays an important role in adaptive immunity to both Nippostrongylus brasiliensis and Salmonella enterica serovar Typhimurium, as M3R-/- mice were impaired in their ability to resolve infection with either pathogen. CD4 T cell activation and cytokine production were reduced in M3R-/- mice. Immunity to secondary infection with N. brasiliensis was severely impaired, with reduced cytokine responses in M3R-/- mice accompanied by lower numbers of mucus-producing goblet cells and alternatively activated macrophages in the lungs. Ex vivo lymphocyte stimulation of cells from intact BALB/c mice infected with N. brasiliensis and S. typhimurium with muscarinic agonists resulted in enhanced production of IL-13 and IFN-γ respectively, which was blocked by an M3R-selective antagonist. Our data therefore indicate that cholinergic signalling via the M3R is essential for optimal Th1 and Th2 adaptive immunity to infection

Cannabinoid Receptor 2 Signaling Does Not Modulate Atherogenesis in Mice

BACKGROUND:Strong evidence supports a protective role of the cannabinoid receptor 2 (CB(2)) in inflammation and atherosclerosis. However, direct proof of its involvement in lesion formation is lacking. Therefore, the present study aimed to characterize the role of the CB(2) receptor in Murine atherogenesis. METHODS AND FINDINGS:Low density lipoprotein receptor-deficient (LDLR(-/-)) mice subjected to intraperitoneal injections of the selective CB(2) receptor agonist JWH-133 or vehicle three times per week consumed high cholesterol diet (HCD) for 16 weeks. Surprisingly, intimal lesion size did not differ between both groups in sections of the aortic roots and arches, suggesting that CB(2) activation does not modulate atherogenesis in vivo. Plaque content of lipids, macrophages, smooth muscle cells, T cells, and collagen were also similar between both groups. Moreover, CB(2) (-/-)/LDLR(-/-) mice developed lesions of similar size containing more macrophages and lipids but similar amounts of smooth muscle cells and collagen fibers compared with CB(2) (+/+)/LDLR(-/-) controls. While JWH-133 treatment reduced intraperitoneal macrophage accumulation in thioglycollate-elicited peritonitis, neither genetic deficiency nor pharmacologic activation of the CB(2) receptor altered inflammatory cytokine expression in vivo or inflammatory cell adhesion in the flow chamber in vitro. CONCLUSION:Our study demonstrates that both activation and deletion of the CB(2) receptor do not relevantly modulate atherogenesis in mice. Our data do not challenge the multiple reports involving CB(2) in other inflammatory processes. However, in the context of atherosclerosis, CB(2) does not appear to be a suitable therapeutic target for reduction of the atherosclerotic plaque

Molecular Evolution of the Two-Component System BvgAS Involved in Virulence Regulation in Bordetella

The whooping cough agent Bordetella pertussis is closely related to Bordetella bronchiseptica, which is responsible for chronic respiratory infections in various mammals and is occasionally found in humans, and to Bordetella parapertussis, one lineage of which causes mild whooping cough in humans and the other ovine respiratory infections. All three species produce similar sets of virulence factors that are co-regulated by the two-component system BvgAS. We characterized the molecular diversity of BvgAS in Bordetella by sequencing the two genes from a large number of diverse isolates. The response regulator BvgA is virtually invariant, indicating strong functional constraints. In contrast, the multi-domain sensor kinase BvgS has evolved into two different types. The pertussis type is found in B. pertussis and in a lineage of essentially human-associated B. bronchiseptica, while the bronchiseptica type is associated with the majority of B. bronchiseptica and both ovine and human B. parapertussis. BvgS is monomorphic in B. pertussis, suggesting optimal adaptation or a recent population bottleneck. The degree of diversity of the bronchiseptica type BvgS is markedly different between domains, indicating distinct evolutionary pressures. Thus, absolute conservation of the putative solute-binding cavities of the two periplasmic Venus Fly Trap (VFT) domains suggests that common signals are perceived in all three species, while the external surfaces of these domains vary more extensively. Co-evolution of the surfaces of the two VFT domains in each type and domain swapping experiments indicate that signal transduction in the periplasmic region may be type-specific. The two distinct evolutionary solutions for BvgS confirm that B. pertussis has emerged from a specific B. bronchiseptica lineage. The invariant regions of BvgS point to essential parts for its molecular mechanism, while the variable regions may indicate adaptations to different lifestyles. The repertoire of BvgS sequences will pave the way for functional analyses of this prototypic system