The M4 Helix Is Involved in α7 nACh Receptor Function.

Nicotinic acetylcholine receptors (nAChR) are the archetypal members of the pentameric ligand-gated ion channel (pLGIC) family, an important class of cell signaling proteins. In all members of this family, each of the five subunits has four transmembrane α-helices (M1-M4) with M2 lining the pore and then M1 and M3, with M4 outermost and adjacent to the membrane lipids. M4 has a variety of roles: its interaction with neighboring M1 and M3 helices is important for receptor assembly, it can a transmit information on the lipid content of the membrane to the gating mechanism, and it may form a vital link to the extracellular domain via the Cys-loop. This study examines the role of M4 receptor residues in the α7 nAChR using site-directed mutagenesis and subsequent expression in Xenopus oocytes. The data indicate that many of the residues in M4 play a role in receptor function, as substitution with Ala can modify functional parameters; 11 of 24 mutants showed a small gain of function (<10-fold decrease in EC50), and 1 (D446A) did not respond to the agonist; it was also not expressed at the cell surface. Removal or addition of aromatic residues had small or no effects. These results demonstrate the α7 nAChR M4 has a role in receptor function, and a structural model suggests possible interactions of some of these residues with their neighbors.

Evaluation of two methods for computational HLA haplotypes inference using a real dataset.

BACKGROUND: HLA haplotype analysis has been used in population genetics and in the investigation of disease-susceptibility locus, due to its high polymorphism. Several methods for inferring haplotype genotypic data have been proposed, but it is unclear how accurate each of the methods is or which method is superior. The accuracy of two of the leading methods of computational haplotype inference--Expectation-Maximization algorithm based (implemented in Arlequin V3.0) and Bayesian algorithm based (implemented in PHASE V2.1.1)--was compared using a set of 122 HLA haplotypes (A-B-Cw-DQB1-DRB1) determined through direct counting. The accuracy was measured with the Mean Squared Error (MSE), Similarity Index (IF) and Haplotype Identification Index (IH). RESULTS: None of the methods inferred all of the known haplotypes and some differences were observed in the accuracy of the two methods in terms of both haplotype determination and haplotype frequencies estimation. Working with haplotypes composed by low polymorphic sites, present in more than one individual, increased the confidence in the assignment of haplotypes and in the estimation of the haplotype frequencies generated by both programs. CONCLUSION: The PHASE v2.1.1 implemented method had the best overall performance both in haplotype construction and frequency calculation, although the differences between the two methods were insubstantial. To our knowledge this was the first work aiming to test statistical methods using real haplotypic data from the HLA region.

Effectiveness and safety of infliximab in two cases of severe chondrocalcinosis: nine years of follow-up.

Objectives. To investigate the efficacy of infliximab in the treatment of severe calcium pyrophosphate deposition diseases (CPPD). Methods. Two patients with severe CPPD and diffuse idiopathic skeletal hyperostosis- (DISH-) like phenotype are described. Both patients were resistant to therapy with nonsteroidal anti-inflammatory drugs (NSAIDs). Both patients were treated with infliximab, a TNF-α receptor antagonist, for nine years. Results. Treatment with infliximab resulted in major clinical and laboratory improvements without relevant side effects. Conclusions. These results suggest that infliximab may be an effective treatment of severe CPDD.

Characterisation of human papillomavirus (HPV) genotypes in the Azorean population, Terceira island.

BACKGROUND: Human papillomavirus detection is very important for the evaluation of prevention strategies in cervical cancer. In the Azorean population, the virus prevalence has never been studied, and there is no data available to preview a successful outcome with HPV vaccination. In this article, our objective is to characterise the HPV genotypes in Terceira Island, contributing for the epidemiological knowledge on the virus infection. RESULTS: Cervical samples were collected from 289 women aged 16-81 in the Gynaecological Outpatient Clinic of the Hospital de Santo Espírito de Angra do Heroísmo (HSEAH). HPV DNA was amplified by Polymerase Chain Reaction using the general consensus primers PGMYO9/PGMY11. Commercially available Papillomavirus Clinical Arrays kits (Genomica) were used to perform HPV genotyping. 30 women were HPV positive, with a median age of 41 years old. Our results show that the overall HPV prevalence was 10.49%. Seventeen genotypes were identified, including 58.82% high risk, 17.65% low risk and 23.53% undetermined risk. CONCLUSION: Unlike other epidemiological studies, HPV31 was the most frequent type (26.67%) in Terceira Island, followed by HPV16 (10.00%), HPV51, HPV53, HPV70 and HPV82 (6.67%). Further studies are needed to investigate if the HPV types found in our population are associated with the risk of progression to high-grade squamous intraepithelial lesions or cervical cancer.

A novel LEMD3 mutation common to patients with osteopoikilosis with and without melorheostosis.

Recent studies have reported loss of function mutations in the LEMD3 gene, encoding an inner nuclear membrane protein that influences Smad signaling, as a cause of osteopoikilosis, Buschke-Ollendorff syndrome, and melorheostosis. We investigated LEMD3 in a three-generation family with osteopoikilosis from the Azores, an affected father and daughter from Ireland with osteopoikilosis (the daughter also had melorheostosis), and two other individuals from the UK with isolated melorheostosis. We found a novel C to T substitution at position 2032 bp (cDNA) in exon 8 of LEMD3, resulting in a premature stop codon at amino acid position 678. This mutation co-segregates with the osteopoikilosis phenotype in both the Azorean family and the Irish family. It was not detected in any of the six unaffected family members or in 342 healthy Caucasian individuals. No LEMD3 mutations were detected in the two patients with sporadic melorheostosis. The LEMD3 mutation reported was clearly the cause of osteopoikilosis in the two families but its relationship to melorheostosis in one of the family members is still unclear. Perhaps unsurprisingly in what is a segmental disease, we did not find LEMD3 mutations in peripheral-blood-derived DNA from the two other individuals with sporadic melorheostosis. The nature of the additional genetic and/or environmental influences required for the development of melorheostosis in those with osteopoikilosis requires further investigation.