Analysis of prion strains by PrPSc profiling in sporadic Creutzfeldt-Jakob disease.

BACKGROUND: Prion diseases are a group of invariably fatal neurodegenerative disorders affecting humans and a wide range of mammals. An essential part of the infectious agent, termed the prion, is composed of an abnormal isoform (PrPSc) of a host-encoded normal cellular protein (PrPC). The conversion of PrPC to PrPSc is thought to play a crucial role in the development of prion diseases and leads to PrPSc deposition, mainly in the central nervous system. Sporadic Creutzfeldt-Jakob disease (sCJD), the most common form of human prion disease, presents with a marked clinical heterogeneity. This diversity is accompanied by a molecular signature which can be defined by histological, biochemical, and genetic means. The molecular classification of sCJD is an important tool to aid in the understanding of underlying disease mechanisms and the development of therapy protocols. Comparability of classifications is hampered by disparity of applied methods and inter-observer variability. METHODS AND FINDINGS: To overcome these difficulties, we developed a new quantification protocol for PrPSc by using internal standards on each Western blot, which allows for generation and direct comparison of individual PrPSc profiles. By studying PrPSc profiles and PrPSc type expression within nine defined central nervous system areas of 50 patients with sCJD, we were able to show distinct PrPSc distribution patterns in diverse subtypes of sCJD. Furthermore, we were able to demonstrate the co-existence of more than one PrPSc type in individuals with sCJD in about 20% of all patients and in more than 50% of patients heterozygous for a polymorphism on codon 129 of the gene encoding the prion protein (PRNP). CONCLUSION: PrPSc profiling represents a valuable tool for the molecular classification of human prion diseases and has important implications for their diagnosis by brain biopsy. Our results show that the co-existence of more than one PrPSc type might be influenced by genetic and brain region-specific determinants. These findings provide valuable insights into the generation of distinct PrPSc types.

Mapping the molecular characteristics of Brazilian human T-cell lymphotropic virus type 1 Env (gp46) and Pol amino acid sequences for vaccine design

This study was carried out to evaluate the molecular pattern of all available Brazilian human T-cell lymphotropic virus type 1 Env (n = 15) and Pol (n = 43) nucleotide sequences via epitope prediction, physico-chemical analysis, and protein potential sites identification, giving support to the Brazilian AIDS vaccine program. In 12 previously described peptides of the Env sequences we found 12 epitopes, while in 4 peptides of the Pol sequences we found 4 epitopes. The total variation on the amino acid composition was 9 and 17% for human leukocyte antigen (HLA) class I and class II Env epitopes, respectively. After analyzing the Pol sequences, results revealed a total amino acid variation of 0.75% for HLA-I and HLA-II epitopes. In 5 of the 12 Env epitopes the physico-chemical analysis demonstrated that the mutations magnified the antigenicity profile. The potential protein domain analysis of Env sequences showed the loss of a CK-2 phosphorylation site caused by D197N mutation in one epitope, and a N-glycosylation site caused by S246Y and V247I mutations in another epitope. Besides, the analysis of selection pressure have found 8 positive selected sites (w = 9.59) using the codon-based substitution models and maximum-likelihood methods. These studies underscore the importance of this Env region for the virus fitness, for the host immune response and, therefore, for the development of vaccine candidates.

Access of extracellular cations to their binding sites in Na,K-ATPase: role of the second extracellular loop of the alpha subunit.

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na(+) and K(+) gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na(+) ions and releases them on the extracellular side of the membrane, and then binds two extracellular K(+) ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the alpha subunit of Na,K-ATPase are known to be involved in Na(+) and K(+) binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat alpha1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K(+), and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K(+) to its binding site.

Polymorphisms of toll-like receptor 2 and 4 genes in Chagas disease

The aim of this study was to test the possible implication of toll-like receptor 2 (TLR2) and TLR4 gene polymorphisms in determining the susceptibility to Chagas' disease. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism in 475 individuals from Colombia, 143 seropositive with chagasic cardiomyopathy, 132 seropositive asymptomatic and 200 seronegative. The TLR2 arginine to glutamine substitution at residue 753(Arg753Gln) polymorphism was absent in the groups analyzed. The TLR4 Asp299Gly and Thr399Ile polymorphisms are in linkage disequilibrium and we observed a very low frequency of these polymorphisms in our study population (2.6% and 1.8% respectively). The overall TLR2 and TLR4 alleles and genotype distribution in seronegative and seropositive were not significantly different. We compared the frequencies between asymptomatic patients and those with chagasic cardiomyopathy and we did not observe any significant differences in the distribution of alleles or genotypes. In summary, this study corroborates the low frequency of TLR2 and TLR4 polymorphisms observed in other populations and suggest that these do not play an important role in Chagas' disease. The validation of these findings in independent cohorts is needed to firmly establish a role for TLR2 and TLR4 variants in Chagas' disease.

Genetic variability in the 5' UTR and NS5A regions of hepatitis C virus RNA isolated from non-responding and responding patients with chronic HCV genotype 1 infection

Sequence variation among different hepatitis C virus (HCV) isolates has adaptive significance and reflects the modes and intensities of selection mechanisms operating on the virus. In this work, we sought to investigate using classical population genetics parameters, the genetic variability of HCV genotype 1 using the 5' UTR and NS5A regions from treatment non-responding and responding groups of patients. Both regions showed low genetic varia-bility and the 5' UTR showed neutral deviation. No differences were observed in the nonsynonymous/synonymous nucleotide substitution ratio among groups for NS5A. The analysis of molecular variance test of the 5' UTR region showed an 11.94% variation among groups. Phylogenetic analysis showed no correlation between sequence variations and therapeutic responses.

Frequency of Fabry disease in male and female haemodialysis patients in Spain

BACKGROUND: Fabry disease (FD), an X-linked lysosomal storage disorder, is caused by a reduced activity of the lysosomal enzyme alpha-galactosidase A. The disorder ultimately leads to organ damage (including renal failure) in males and females. However, heterozygous females usually present a milder phenotype with a later onset and a slower progression. METHODS: A combined enzymatic and genetic strategy was used, measuring the activity of alpha-galactosidase A and genotyping the alpha-galactosidase A gene (GLA) in dried blood samples (DBS) of 911 patients undergoing haemodialysis in centers across Spain. RESULTS: GLA alterations were found in seven unrelated patients (4 males and 3 females). Two novel mutations (p.Gly346AlafsX347 and p.Val199GlyfsX203) were identified as well as a previously described mutation, R118C. The R118C mutation was present in 60% of unrelated patients with GLA causal mutations. The D313Y alteration, considered by some authors as a pseudo-deficiency allele, was also found in two out of seven patients. CONCLUSIONS: Excluding the controversial D313Y alteration, FD presents a frequency of one in 182 individuals (0.55%) within this population of males and females undergoing haemodialysis. Moreover, our findings suggest that a number of patients with unexplained and atypical symptoms of renal disease may have FD. Screening programmes for FD in populations of individuals presenting severe kidney dysfunction, cardiac alterations or cerebrovascular disease may lead to the diagnosis of FD in those patients, the study of their families and eventually the implementation of a specific therapy.

Difficulties associated with out-patient management of drug abusers by general practitioners. A cross sectional survey of general practitioners with and without methadone patients in Switzerland

Résumé de l'article Contexte : En Suisse, les médecins de premier recours traitent la plupart des patients dépendants aux opiacés méthadone en tant que traitement de substitution. Méthode : Nous avons étudié les difficultés rencontrées dans la prise en charge des patients toxicodépendants en envoyant par poste un questionnaire d'enquête. Nous avons envoyé ce questionnaire à tous les médecins de premier recours de Suisse Romande prescrivant de la méthadone (556 médecins). Nous avons envoyé un autre questionnaire, plus court, à des médecins de premier recours du Canton de Vaud qui ne prescrivent pas de méthadone. Résultats : le taux de réponse global est de 63,3 %. La plus haute dose de méthadone donnée par les médecins de premier recours est de 120,4 mg/j (moyenne). Questionnés au sujet de l'aide qu'ils désireraient recevoir face à ces patients, les médecins de premier recours avec patients substitués par méthadone ont mentionné premièrement l'importance d'un meilleur remboursement des services prodigués. Les autres éléments demandés ont été une meilleure formation, de meilleures connaissances des pathologies psychiatriques et des groupes de discussion de cas cliniques. Les médecins sans patients sous méthadone refusent de traiter ces patients surtout pour des raisons émotionnelles et relationnelles. En conclusion : les médecins acceptant des patients sous méthadone rencontrent des difficultés relationnelles et émotionnelles. Ils désirent un meilleur remboursement pour les services prodigués.

Phenotypic and molecular characterization of Bruck syndrome (Osteogenesis imperfecta with contractures of the large joints) caused by a recessive mutation in PLOD2

Le Syndrome de Bruck (Bruck Syndrome; BS) est une maladie autosomique récessive assemblant la combinaison inhabituelle de fragilité osseuse semblable à celle de l'Ostéogenèse Imparfaite (0I) avec des contractures congénitales tendineuses et cutanées des grandes articulations («ptérygia»). Les cas décrits jusqu'à ce jour mettent en évidence une grande hétérogénéité du tableau clinique, liée en partie au manque d'un diagnostic biochimique ou moléculaire. Nous savons que dans le BS les gènes codant pour le collagène 1 ne sont pas mutés, mais savons néanmoins, grâce à l'étude du collagène extrait de biopsies osseuses, qu'il y a un déficit d'hydroxylation des résidus de lysine dans les télopeptides du collagène 1 qui servent à la formation des liens intermoléculaires (crosslinks) et donc à la stabilisation des fibres de collagène. Un locus génétique du BS à été mappé sur 17q12, mais le gène responsable sur ce locus reste inconnu; plus récemment, deux mutations dans le gène de la lysyl hydroxylase 2 (PLOD2, position chromosomique 3q23-q24) ont été identifiées, démontrant l'hétérogénéité génétique du ES. La proportion de ES liée à 17p22 (BS type 1) et celle liée à une mutation dans PLOD2 (BS type 2) est encore incertaine et nous manquons de données sur la corrélation phenotype-génotype. Nous avons étudié le cas d'un garçon avec des contractures et des ptérygia dès la naissance, combinées à une ostéopénie sévère de type OI menant à des fractures multiples. Ses urines contenaient une quantité élevée d'hydroxyproline, indiquant un remaniement important du tissu osseux, mais peu de produits de dégradation des crosslinks du collagène, indiquant donc une réduction de la proportion de crosslinks dans le collagène in vivo. Nous avons pu démontrer chez lui la présence d'une nouvelle mutation homozygote dans le gène PLOD2 menant à une substitution Arg598His; les deux parents du sujet étaient hétérozygotes pour la mutation et celle-ci était absente dans notre population témoin. La mutation est adjacente aux deux mutations rapportées précédemment (Gly601Val et Thr608Ile), ce qui suggère la présence d'un ''hotspot'' mutationnel mais aussi d'une région de grande importance fonctionnelle sur PLOD2 : cette observation est importante pour la création d'inhibiteurs de PLOD2, recherchés en ce moment pour le traitement de la fibrose. La combinaison de ptérygia et de fragilité osseuse, comme illustrée par notre patient est apparemment contradictoire et donc difficilement explicable mais indique que l'hydroxylation des résidus lysyl des télopeptides est importante non seulement pour la stabilité osseuse mais aussi dans la morphogénèse et la formation des articulations dans la période prénatale. Finalement, la mesure des produits de dégradation du collagène dans l'urine et l'analyse de mutation de PLOD2 permet le diagnostic du syndrome de Bruck et permet de le différencier de l'Osteogénèse Imparfaite. -- Bruck syndrome (BS) is a recessively-inherited phenotypic disorder featuring the unusual combination of skeletal changes resembling osteogenesis imperfecta (0I) with congenital contractures of the large joints. Clinical heterogeneity is apparent in cases reported thus far. While the genes coding for collagen 1 chains are unaffected in BS, there is biochemical evidence for a defect in the hydroxylation of lysine residues in collagen 1 telopeptides. One BS locus has been mapped at 17p12, but more recently, two mutations in the lysyl hydroxylase 2 gene (PLOD2, 3q23-q24) have been identified in BS, showing genetic heterogeneity. The proportion of BS cases linked to 17p22 (BS type 1) or caused by mutations in PLOD2 (BS type 2) is still uncertain, and phenotypic correlations are lacking. We report on a boy who had congenital contractures with pterygia at birth and severe 0I-like osteopenia and multiple frac-tures. His urine contained high amounts of hydroxyproline but low amounts of collagen crosslinks degradation products; and he was shown to be homozygous for a novel mutation leading to an Arg598His substitution in PLOD2. The mutation is adjacent to the two mutations previously reported (Gly601Val and Thr608Ile), suggesting a functionally important hotspot in PLOD2. The combination of pterygia with bone fragility, as illustrated by this case, is difficult to explain; it suggests that telopeptide lysyl hydroxylation must be involved in prenatal joint formation and morphogenesis. Collagen degradation products in urine and mutation analysis ofPLOD2 maybe used to diagnose BS and differentiate it from M.

Candidate gene study of TRAIL and TRAIL receptors: association with response to interferon beta therapy in multiple sclerosis patients.

TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO) and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10(-4), pc = 0.048, OR = 0.30). This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A), a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells) were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS.

Postischemic recovery of heart metabolism and function: role of mitochondrial fatty acid transfer.

Postischemic recovery of contractile function is better in hearts from fasted rats than in hearts from fed rats. In this study, we examined whether feeding-induced inhibition of palmitate oxidation at the level of carnitine palmitoyl transferase I is involved in the mechanism underlying impaired recovery of contractile function. Hearts isolated from fasted or fed rats were submitted to no-flow ischemia followed by reperfusion with buffer containing 8 mM glucose and either 0.4 mM palmitate or 0.8 mM octanoate. During reperfusion, oxidation of palmitate was higher after fasting than after feeding, whereas oxidation of octanoate was not influenced by the nutritional state. In the presence of palmitate, recovery of left ventricular developed pressure was better in hearts from fasted rats. Substitution of octanoate for palmitate during reperfusion enhanced recovery of left ventricular developed pressure in hearts from fed rats. However, the chain length of the fatty acid did not influence diastolic contracture. The results suggest that nutritional variation of mitochondrial fatty acid transfer may influence postischemic recovery of contractile function.

Lack of association between the protein tyrosine phosphatase non-receptor type 22 R263Q and R620W functional genetic variants and endogenous non-anterior uveitis.

OBJECTIVE Endogenous uveitis is a major cause of visual loss mediated by the immune system. The protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene encodes a lymphoid-specific phosphatase that plays a key role in T-cell receptor (TCR) signaling. Two independent functional missense single nucleotide polymorphisms (SNPs) located within the PTPN22 gene (R263Q and R620W) have been associated with different autoimmune disorders. We aimed to analyze for the first time the influence of these PTPN22 genetic variants on endogenous non-anterior uveitis susceptibility. METHODS We performed a case-control study of 217 patients with endogenous non-anterior uveitis and 718 healthy controls from a Spanish population. The PTPN22 polymorphisms (rs33996649 and rs2476601) were genotyped using TaqMan allelic discrimination assays. The allele, genotype, carriers, and allelic combination frequencies were compared between cases and controls with χ(2) analysis or Fisher's exact test. RESULTS Our results showed no influence of the studied SNPs in the global susceptibility analysis (rs33996649: allelic P- value=0.92, odds ratio=0.97, 95% confidence interval=0.54-1.75; rs2476601: allelic P- value=0.86, odds ratio=1.04, 95% confidence interval=0.68-1.59). Similarly, the allelic combination analysis did not provide additional information. CONCLUSIONS Our results suggest that the studied polymorphisms of the PTPN22 gene do not play an important role in the pathophysiology of endogenous non-anterior uveitis.

Combined simulation and mutagenesis analyses reveal the involvement of key residues for peroxisome proliferator-activated receptor alpha helix 12 dynamic behavior.

The dynamic properties of helix 12 in the ligand binding domain of nuclear receptors are a major determinant of AF-2 domain activity. We investigated the molecular and structural basis of helix 12 mobility, as well as the involvement of individual residues with regard to peroxisome proliferator-activated receptor alpha (PPARalpha) constitutive and ligand-dependent transcriptional activity. Functional assays of the activity of PPARalpha helix 12 mutants were combined with free energy molecular dynamics simulations. The agreement between the results from these approaches allows us to make robust claims concerning the mechanisms that govern helix 12 functions. Our data support a model in which PPARalpha helix 12 transiently adopts a relatively stable active conformation even in the absence of a ligand. This conformation provides the interface for the recruitment of a coactivator and results in constitutive activity. The receptor agonists stabilize this conformation and increase PPARalpha transcription activation potential. Finally, we disclose important functions of residues in PPARalpha AF-2, which determine the positioning of helix 12 in the active conformation in the absence of a ligand. Substitution of these residues suppresses PPARalpha constitutive activity, without changing PPARalpha ligand-dependent activation potential.

Markov chain montecarlo method applied to rounding zeros of compositional data: first approach

As stated in Aitchison (1986), a proper study of relative variation in a compositional data set should be based on logratios, and dealing with logratios excludes dealing with zeros. Nevertheless, it is clear that zero observations might be present in real data sets, either because the corresponding part is completelyabsent –essential zeros– or because it is below detection limit –rounded zeros. Because the second kind of zeros is usually understood as “a trace too small to measure”, it seems reasonable to replace them by a suitable small value, and this has been the traditional approach. As stated, e.g. by Tauber (1999) and byMartín-Fernández, Barceló-Vidal, and Pawlowsky-Glahn (2000), the principal problem in compositional data analysis is related to rounded zeros. One should be careful to use a replacement strategy that does not seriously distort the general structure of the data. In particular, the covariance structure of the involvedparts –and thus the metric properties– should be preserved, as otherwise further analysis on subpopulations could be misleading. Following this point of view, a non-parametric imputation method isintroduced in Martín-Fernández, Barceló-Vidal, and Pawlowsky-Glahn (2000). This method is analyzed in depth by Martín-Fernández, Barceló-Vidal, and Pawlowsky-Glahn (2003) where it is shown that thetheoretical drawbacks of the additive zero replacement method proposed in Aitchison (1986) can be overcome using a new multiplicative approach on the non-zero parts of a composition. The new approachhas reasonable properties from a compositional point of view. In particular, it is “natural” in the sense thatit recovers the “true” composition if replacement values are identical to the missing values, and it is coherent with the basic operations on the simplex. This coherence implies that the covariance structure of subcompositions with no zeros is preserved. As a generalization of the multiplicative replacement, in thesame paper a substitution method for missing values on compositional data sets is introduced

Efficacy of prescribed injectable diacetylmorphine in the Andalusian trial: Bayesian analysis of responders and non-responders according to a multi domain outcome index.

BACKGROUND The objective of this research was to evaluate data from a randomized clinical trial that tested injectable diacetylmorphine (DAM) and oral methadone (MMT) for substitution treatment, using a multi-domain dichotomous index, with a Bayesian approach. METHODS Sixty two long-term, socially-excluded heroin injectors, not benefiting from available treatments were randomized to receive either DAM or MMT for 9 months in Granada, Spain. Completers were 44 and data at the end of the study period was obtained for 50. Participants were determined to be responders or non responders using a multi-domain outcome index accounting for their physical and mental health and psychosocial integration, used in a previous trial. Data was analyzed with Bayesian methods, using information from a similar study conducted in The Netherlands to select a priori distributions. On adding the data from the present study to update the a priori information, the distribution of the difference in response rates were obtained and used to build credibility intervals and relevant probability computations. RESULTS In the experimental group (n = 27), the rate of responders to treatment was 70.4% (95% CI 53.287.6), and in the control group (n = 23), it was 34.8% (95% CI 15.354.3). The probability of success in the experimental group using the a posteriori distributions was higher after a proper sensitivity analysis. Almost the whole distribution of the rates difference (the one for diacetylmorphine minus methadone) was located to the right of the zero, indicating the superiority of the experimental treatment. CONCLUSION The present analysis suggests a clinical superiority of injectable diacetylmorphine compared to oral methadone in the treatment of severely affected heroin injectors not benefiting sufficiently from the available treatments. TRIAL REGISTRATION Current Controlled Trials ISRCTN52023186.

Detection of possible variant forms of hemoglobin in HbA1c measurements by chromatography liquid ion exchange high resolution (HPLC)

Background: The glycosylated hemoglobin (HbA1c) is used to help monitor the degree of a diabetic’s hyperglycemia. Security and accuracy of the methods used in its detection are affected by variants forms of Hb or elevations in levels of Fetal Hb (HbF). These interference are the result of a change in the haemoglobin total net charge of the variant due of a substitution of one amino acid in the remaining amino terminal of the beta chain. International Standardization for HbA1c values (NGSP) not include interference assessment as part of the certification program. Therefore, the effect of each variant or the lifting of the HbF on HbA1c result should be examined in each sample depending on the detected variant and the method used for the detection of the same. The objectives were: to describe the possible variants of Hb and their interference in HbA1c measurement by our method, after the implementation of a computer program for their detection. To identify some variants detected by chromatography liquid ion exchange high resolution (HPLC) with DNA molecular sequencing.