The enantiomers of epiboxidine and of two related analogs: synthesis and estimation of their binding affinity at α4β2 and α7 neuronal nicotinic acetylcholine receptors

Epiboxidine hydrochlorides (+)-2 and (-)-2, which are the structural analogs of the antipodes of epibatidine (±)-1, as well as the enantiomeric pairs (+)-3/(-)-3 and (+)-4/(-)-4 were synthesized and tested for binding affinity at α4β2 and α7 nicotinic acetylcholine receptor (nAChR) subtypes. Final derivatives were prepared through the condensation of racemic N-Boc-7-azabicyclo[2.2.1]heptane-2-one (±)-5 with the resolving agent (R)-(+)-2-methyl-2-propanesulfinamide. The pharmacological analysis carried out on the three new enantiomeric pairs evidenced an overall negligible degree of enantioselectivity at both nAChRs subtypes, a result similar to that reported for both natural and unnatural epibatidine enantiomers at the same investigated receptor subtypes.     

Estimation of reference intervals of five endocannabinoids and endocannabinoid related compounds in human plasma by two dimensional-LC/MS/MS

The elucidation of the role of endocannabinoids in physiological and pathological conditions and the transferability of the importance of these mediators from basic evidence into clinical practice is still hampered by the indefiniteness of their circulating reference intervals. In this work, we developed and validated a two-dimensional LC/MS/MS method for the simultaneous measurement of plasma endocannabinoids and related compounds such as arachidonoyl-ethanolamide, palmitoyl-ethanolamide, and oleoyl-ethanolamide, belonging to the N-acyl-ethanolamide (NAE) family, and 2-arachidonoyl-glycerol and its inactive isomer 1-arachidonoyl-glycerol from the monoacyl-glycerol (MAG) family. We found that several pitfalls in the endocannabinoid measurement may occur, from blood withdrawal to plasma processing. Plasma extraction with toluene followed by on-line purification was chosen, allowing high-throughput and reliability. We estimated gender-specific reference intervals on 121 healthy normal weight subjects fulfilling rigorous anthropometric and hematic criteria. We observed no gender differences for NAEs, whereas significantly higher MAG levels were found in males compared with females. MAGs also significantly correlated with triglycerides. NAEs increased with age in females, and arachidonoyl-ethanolamide correlated with adiposity and metabolic parameters in females. This work paves the way to the establishment of definitive reference intervals for circulating endocannabinoids to help physicians move from the speculative research field into the clinical field.     

Contribution of SNP arrays in diagnosis of deletion 2p11.2-p12

Deletions of the short arm of chromosome 2 are exceedingly rare, having been reported in few patients. Furthermore most cases with deletion in 2p11.2-p12 have been studied using standard karyotype and so it is not possible to delineate the precise size of deletions.Here, we describe a 9-year-old girl with a 9.4 Mb de novo interstitial deletion of region 2p11.2-p12 identified by SNP array analysis.The deleted region encompasses over 40 known genes, including LRRTM1, CTNNA2 and REEP1, haploinsufficiency of which could explain some clinical features of this patient such as mental retardation, speech delay and gait abnormalities.A comparison of our case with previously reported patients who present deletions in 2p11.2-p12 was carried out.Our case adds new information to the deletion of 2p11.2-p12, improving the knowledge on this rearrangement.     

Synthesis and binding affinity at α4β2 and α7 nicotinic acetylcholine receptors of new analogs of epibatidine and epiboxidine containing the 7-azabicyclo[2.2.1]hept-2-ene ring system

A group of novel racemic nicotinic ligands structurally related to epibatidine or epiboxidine [(±)-10-(±)-17] was synthesized through a palladium-catalyzed cross-coupling between the appropriate vinyl triflate and a range of organometallic heterocycles. The target compounds were evaluated for binding affinity at the α4β2 and α7 neuronal nicotinic receptors (nAChRs). The set of 3-pyridinyl derivatives (±)-10, (±)-11 and (±)-12 exhibited an affinity for the α4β2 nAChR subtype in the subnanomolar range (K(i) values of 0.20, 0.40 and 0.50nM, respectively) and behaved as α4β2 versus α7 subtype selective ligands. Interestingly, the epiboxidine-related dimethylammonium iodide (±)-17, which retained a good affinity for the α4β2 nAChR (K(i)=13.30nM), tightly bound also to the α7 subtype (K(i)=1.60nM), thus displaying a reversal of the affinity trend among the reference and new nicotinic ligands under investigation.     

Public information affects breeding dispersal in a colonial bird: kittiwakes cue on neighbours

Habitat selection and dispersal behaviour are key processes in evolutionary ecology. Recent studies have suggested that individuals may use the reproductive performance of conspecifics as a source of public information on breeding patch quality for dispersal decisions, but experimental evidence is still limited for species breeding in aggregates, i.e. colonial species. We addressed this issue by manipulating the local breeding success of marked individuals and that of their neighbours on a series of breeding patches of a colonial seabird, the black-legged kittiwake (Rissa tridactyla). Based on previous observations in this species, we predicted that individuals that lost their eggs on successful patches would attend their nest and come back to it the year after at a higher rate than individuals that lost their eggs on patches where their neighbours were also in failure. As predicted, the attendance of breeders and prospectors was strongly affected by the local level of breeding success, resulting in differential site fidelity and recruitment. This suggests that individuals used information conveyed by conspecific breeding performance to make decisions relative to breeding site selection. This process can amplify the response of these populations to environmental change and may have contributed to the evolution of colonial breeding.     

Variation in the bitter-taste receptor gene TAS2R38, and adiposity in a genetically isolated population in Southern Italy

Objective: Variation in the bitter‐taste receptor gene, TAS2R38 confers the ability to taste 6‐n‐propylthiouracil (PROP). The objective of this study was to relate TAS2R38 haplotypes and PROP‐tasting phenotypes to adiposity in a genetically isolated population. We hypothesized that the nontaster phenotype would be associated with higher BMI and waist circumference (WC) in females, and that dietary restraint would mediate this relationship. Methods and Procedures: Participants were 540 healthy inhabitants of the genetically isolated village of Carlantino in southern Italy who were 15–89 years of age at the time of the study. Haplotype analyses were performed and PROP tasting was assessed using a filter paper method. Height, weight, and WC were measured and restrained eating was assessed using a brief questionnaire. Results: Nontaster females had higher BMI and WC than females who were phenotypic tasters, and this relationship was specific to females with low dietary restraint. Regression analysis showed that BMI declined by 1.7 units across taster groups in females when the model included the PROP by restraint interaction. PROP phenotype was not significantly associated with WC in the regression models. Polymorphisms in TAS2R38 were not associated with BMI or WC in females. Neither TAS2R38 haplotype nor PROP phenotype was strongly related to BMI or WC in males. Discussion: These data support previous findings of a relation between the nontaster phenotype and higher BMI in females that is modified by dietary restraint. Assessment of PROP phenotypes might provide unique information about adiposity that is not captured by haplotype analysis alone.     

Use of a real-time polymerase chain reaction thermocycler to study bacterial cell permeabilization by antimicrobial peptides

Antimicrobial peptides are good leads to develop new antibiotics, but knowledge of their mode of action is a prerequisite. Destruction of the microbial membranes through a detergent-like mechanism is one of these modes of action. This is usually studied by using a fluorescent nucleic acid stain such as SYTOX Green, which is impermeable to living cells. Using a simple protocol based on the use of a standard real-time thermocycler, we confirmed that the actions of the antimicrobial peptides LL-37 and magainin 2 on bacterial cells are different.     

Growth inhibition by glucose oxidase system of enterotoxic Escherichia coli and Salmonella derby: in vitro studies

The antibacterial effect of different glucose oxidase (GOX)/glucose combinations was studied on two food-poisoning organisms, enterotoxic Escherichia coli PM 015 and Salmonella derby BP 177. Growth of E. coli in nutrient broth (NB) was clearly inhibited by 4.0 mg/ml glucose after 24 h when combined with 2.0 U/ml GOX and after 48 h when combined with 0.5 or 1.0 U/ml GOX. Salmonella derby was more resistant than E. coli, but showed clear growth inhibition only after 48 h when inoculated in tubes where 2 mg glucose/ml and 2 U GOX/ml (or 4 mg glucose/ml and 1 U GOX/ml) were combined. In order to understand if the enzyme effect on microbial growth can be attributed to hydrogen peroxide or to pH decrease as a result of the production of gluconic acid, catalase (CAT) was added to GOX/glucose system. Since CAT decomposes H₂O₂ to H₂O and O₂, the antibacterial effect was ascribed to a pH decrease as a result of gluconic acid in the system.     

Asymptotic distributions of kappa statistics and their differences with many raters,many rating categories and two conditions

In clinical research and in more general classification problems, a frequent concern is the reliability of a rating system. In the absence of a gold standard, agreement may be considered as an indication of reliability. When dealing with categorical data, the well‐known kappa statistic is often used to measure agreement. The aim of this paper is to obtain a theoretical result about the asymptotic distribution of the kappa statistic with multiple items, multiple raters, multiple conditions, and multiple rating categories (more than two), based on recent work. The result settles a long lasting quest for the asymptotic variance of the kappa statistic in this situation and allows for the construction of asymptotic confidence intervals. A recent application to clinical endoscopy and to the diagnosis of inflammatory bowel diseases (IBDs) is shortly presented to complement the theoretical perspective.