Evaluation of the performance of selected in-house and commercially available PCR and real-time PCR assays for the detection of Leishmania DNA in canine clinical samples

Protozoa of the genus Leishmania are the causative agents of leishmaniosis. Although the polymerase chain reaction (PCR) has proved very effective in the detection of Leishmania DNA, a standardized method does not exist. In this study we attempt a comparative evaluation between one real time PCR (Method D), two in-house (Methods A and C), and a commercially available PCR assay (Method B) for the detection of Leishmania DNA, in order to support reliable diagnostic investigation of leishmaniosis. This evaluation was performed in regard to relative specificity and sensitivity, minimum detection limit (MDL), repeatability and reproducibility using cultured isolates and clinical samples. All the methods under study produced the expected result with the positive and negative controls. However with regard to clinical samples, Method C showed a statistically significant higher level of positivity. Relative sensitivity and specificity of Methods A, B and D in comparison to C was calculated respectively at 50.7%, 43%, 40%, and 90.8%, 93.4% and 89.5%. The MDL for Methods A-D was defined respectively at 30.7, 5, 3.7, and 5 promastigotes/ml. Repeatability and reproducibility were excellent in all cases with only the exception of Method A regarding reproducibility with a different brand of PCR reagents. The results that were recorded indicate that evaluation of PCR assays before their application for research and clinical diagnosis can provide useful evidence for their reliable application. Within this context the use of internal amplification controls and the confirmation of the specificity of the amplification product is recommended.     

Impact of waist circumference on cardiac phenotype in hypertensives according to gender

Our aim was to assess the differential effect of waist circumference on left-ventricular (LV) structural and functional alterations, in hypertensive males and females. One thousand seven hundred and eighty nine consecutive, nondiabetic, essential hypertensives (aged 55.8 +/- 13.5 years, 966 females), included in the 3H Study, an ongoing registry of hypertension-related-target-organ damage, were classified to obese and nonobese groups according to Adult Treatment Panel III criteria. All participants underwent complete echocardiographic study including LV diastolic function evaluation by means of conventional and tissue Doppler imaging (TDI) methods, averaging early and late diastolic mitral annular peak velocities (Em, Am, Em/Am) from four separate sites of measurement. Hypertensive obese women compared with nonobese exhibited significantly greater LV mass index and prevalence of LV hypertrophy (by 5.5 g/m(2), P = 0.003, and 8.8%, P = 0.005, respectively), while such differences were not present among men. Obese women compared to nonobese ones were accompanied by lower transmitral E/A (by 0.08, P < 0.001), TDI-derived Em/Am (by 0.12, P < 0.001), and higher E/Em ratio (by 0.8, P = 0.016). In contrast, hypertensive obese men compared to nonobese ones exhibited lower E and Em (by 0.04 m/s and 0.6 cm/s, both P < 0.05). A significant interaction between sex and abdominal obesity was observed only regarding TDI-derived Am and Em/Am. Furthermore, waist circumference was a predictor of E/A (beta = -0.097, P = 0.002) and Em/Am (beta = -0.116, P = 0.001), independently of body size, in females but not in males. The adverse effect of abdominal obesity on LV alterations is more pronounced among female hypertensives, suggesting that routine measurement of waist circumference provides additional information on cardiac phenotype especially in women.     

Novel ENU-Induced Point Mutation in Scavenger Receptor Class B,Member 1, Results in Liver Specific Loss of SCARB1 Protein

Cardiovascular disease (CVD) is the largest cause of premature death in human populations throughout the world. Circulating plasma lipid levels, specifically high levels of LDL or low levels of HDL, are predictive of susceptibility to CVD. The scavenger receptor class B member 1 (SCARB1) is the primary receptor for the selective uptake of HDL cholesterol by liver and steroidogenic tissues. Hepatic SCARB1 influences plasma HDL-cholesterol levels and is vital for reverse cholesterol transport. Here we describe the mapping of a novel N-ethyl-N-nitrosourea (ENU) induced point mutation in the Scarb1 gene identified in a C57BL/6J background. The mutation is located in a highly conserved amino acid in the extracellular loop and leads to the conversion of an isoleucine to an asparagine (I179N). Homozygous mutant mice express normal Scarb1 mRNA levels and are fertile. SCARB1 protein levels are markedly reduced in liver (∼90%), but not in steroidogenic tissues. This leads to ∼70% increased plasma HDL levels due to reduced HDL cholesteryl ester selective uptake. Pdzk1 knockout mice have liver-specific reduction of SCARB1 protein as does this mutant; however, in vitro analysis of the mutation indicates that the regulation of SCARB1 protein in this mutant is independent of PDZK1. This new Scarb1 model may help further our understanding of post-translational and tissue-specific regulation of SCARB1 that may aid the important clinical goal of raising functional HDL.     

Unprecedented long-term genetic monomorphism in an endangered relict butterfly species

Multi-locus monomorphism in microsatellites is practically non-existent, with one notable exception, the island fox (Urocyon littoralis dickeyi) population on San Nicolas island off the coast of southern California, having been called “the most monomorphic sexually reproducing animal population yet reported”. Here, we present the unprecedented long-term monomorphism in relict populations of the highly endangered Parnassius apollo butterfly, which is protected by CITES and classified as “threatened” by the IUCN. The species is disjunctly distributed throughout the western Palaearctic and has occurred in several small remnant populations outside its main distribution area. We screened 78 individuals from 1 such relict area (Mosel valley, Germany) at 16 allozyme and 6 microsatellite loci with the latter known to be polymorphic in this species elsewhere. From the same area, we also genotyped 55 museum specimens sampled from 1895 to 1989 to compare historical and present levels of genetic diversity. However, none of all these temporal populations yielded any polymorphism. Thus, present and historical butterflies were completely monomorphic for the same fixed allele. This is the second study to report multi-locus monomorphism for microsatellites in an animal population and the first one to prove this monomorphism not to be the consequence of recent factors. Possible explanations for our results are a very low long-term effective population size and/or a strong historic bottleneck or founder event. Since the studied population has just recovered from a recent population breakdown (second half of twentieth century) and no signs of inbreeding depression have been detected, natural selection might have purged the population of weakly deleterious alleles, thus rendering it less susceptible to the usual negative corollaries of high levels of homozygosity and low effective population size.     

Calprotectin: A protein related to cardiovascular risk in adult patients with obstructive sleep apnea

IntroductionIncreased levels of inflammatory mediators, such as hs-CRP, have been detected in patients with obstructive sleep apnea (OSA) and used as cardiovascular risk and disease outcome predictors. Calprotectin is an inflammatory marker regulating atherogenic processes not investigated in adult OSA patients. The aim of the present study as primary objective was to examine the role of calprotectin as an inflammatory molecule, acting through a distinct pathway to the atherogenic process in adult OSA patients and its associations with hs-CRP and the lipidemic profile of the patients. As a secondary objective was the evaluation of the atherogenic markers post-CPAP treatment.Materials and methodsSeventy-four participants underwent full overnight polysomnography. Blood samples were collected for calprotectin, hs-CRP, total cholesterol, triglycerides, LDL, HDL and glucose levels. Thirty-two OSA patients were reexamined 6 months post-CPAP treatment.ResultsOut of 74 participants included in the study, 33 had moderate OSA, 27 had severe OSA and 14 were controls. Calprotectin and hs-CRP were significantly increased in patients with moderate and severe OSA compared to controls (p < 0.0001). Calprotectin and hs-CRP levels were positively correlated with apnea-hypopnea index, BMI and total time of sleep with SaO₂ <90% and inversely correlated with SaO₂ minimum and mean values. Calprotectin and hs-CRP levels were significantly improved post-CPAP treatment (p < 0.0001).DiscussionCalprotectin may serve as a novel and reliable, biomarker of cardiovascular risk severity in OSA patients. The decrease of calprotectin levels post-CPAP treatment combined with hs-CRP amelioration could provide evidence for reduction of cardiovascular risk post CPAP treatment.     

Modes of programmed cell death during Ceratitis capitata oogenesis

In the present study, we demonstrate the existence of two distinct apoptotic patterns in nurse cells during Ceratitis capitata oogenesis. One is developmentally regulated and normally occurs during stages 12 and 13, and the other is stage specific and is sporadically observed during stages 7 and 8. The pre-apoptotic manifestation of the first pattern begins at stage 11 and is characterized by the formation of actin bundles. Subsequently, at stages 12 and 13, the nurse cell nuclei exhibit condensed chromatin and contain fragmented DNA, as revealed by TUNEL assay. The apoptotic nurse cell remnants are phagocytosed by the neighboring follicle cells at the end of oogenesis during stages 13 and 14. In the second apoptotic pattern, which occurs sporadically during stages 7 and 8, the nurse cells degenerate and are phagocytosed by the follicular epithelium that contains apoptotic cell bodies. The data presented herein, compared to previous reported results in Drosophila melanogaster and Dacus oleae (Nezis et al., 2000, 2001), strongly suggest that nurse cell apoptosis is a developmentally regulated and phylogenetically conserved mechanism in higher Dipteran. They also suggest that, the sporadic apoptotic pattern consists of a possible protective mechanism throughout oogenesis when damaged or abnormal egg chambers, are eliminated before they reach maturity.     

Calculation of helix packing angles in protein structures

Software is presented for the calculation of packing angles and geometry of helical secondary structure elements in protein structures. AVAILABILITY: C language source code and documentation is available from http://www.bioinformatics.leeds.ac.uk.     

Isotropic-nematic phase equilibrium and phase separation of kappa-carrageenan in aqueous salt solution: experimental and theoretical approaches

The behavior of chiral-nematic and isotropic phases of helical kappa-carrageenan in aqueous solution of sodium iodide was compared with that of the anisotropic biphasic phase that contains both these phases. On the basis of birefringence, rheology, chemical analysis, average molecular weight, and polydispersity index measurements, we derived a number of characteristic differences as well as similarities between these phases, over a range of polysaccharide concentrations obtained by the dilution of each phase. For example, we assessed the critical concentration of an isotropic-anisotropic transition (C(i)), the temperature of the anisotropic-isotropic phase shift during thermal heating-cooling cycles, and the viscosity changes due to the phase shift and due to the diminishing of the helical conformation. We also demonstrated how the different phases and their dilutions behave under the effect of shear and frequency of oscillation and how the viscoelastic properties vary in each phase and discussed the isotropic and anisotropic liquid crystal controlling behavior mechanisms. From a theoretical point of view, we propose to combine the wormlike chain model for semiflexible polyelectrolytes interacting via both hard-core and electrostatic repulsion to assess the concentration of isotropic-nematic transition, to assess the coexistence concentration range, and to determine the effects of charge by applying the effective diameter and a twisting effect.     

Spiro[pyrrolidine-2,2'-adamantanes]: synthesis,anti-influenza virus activity and conformational properties

Synthetic spiro[pyrrolidine-2,2'-adamantanes] 2, 3, 11, 15, 12, 16, 18, 20 were evaluated in vitro and found to be active anti-influenza virus A compounds; the effect of the position of C-Me pyrrolidine ring substituent on antiviral activity was examined. Pyrrolidine 5-Me substitution appears to be optimal for H(2)N(2) strain activity. From the four different possible protonated conformers, experimental observation using NMR spectroscopy and molecular mechanics calculations demonstrated only a pair of conformers A(+)H (N-Me (ps-ax), C-Me (ps-eq)) and B(+)H ((N-Me ps-ax, C-Me ps-ax)) which can contribute to the biological activity of C-Me, N-Me protonated derivatives 15(+)H, 16(+)H and 20(+)H. The relative populations were calculated from NMR spectra. For compounds 15(+)H and 20(+)H conformer A(+)H (cis dimethyl orientation) is the major one whereas a similar population of conformers A(+)H and B(+)H (trans dimethyl orientation) was observed for compound 16(+)H. Since this new series is characterized by a lipophilic part, that is the pyrrolidine ring, in addition to adamantane, that can interact with influenza A M2 protein, an ultimate future goal would be the in vitro mapping of M2 lipophilic pocket.