Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples

Background: Leishmania infantum infections in dogs play a crucial role in the transmission of pathogens causing visceral leishmaniasis to humans in the Gansu province, northwest China. To be able to control zoonotic transmission of the parasite to humans, a non-invasive loop-mediated isothermal amplification (LAMP) assay to specifically detect L. infantum infections in dogs was developed. Methods: The primers used in the LAMP assay were designed to target kinetoplast DNA minicircle sequences of the L. infantum isolate MCAN/CN/90/SC and tested using DNA isolated from promastigotes of different Leishmania species. The LAMP assay was evaluated with conjunctional swab samples obtained from 111 and 33 dogs living in an endemic and a non-endemic region of zoonotic visceral leishmaniasis in the Gansu province, respectively. The LAMP assay was also compared with conventional PCR, ELISA and microscopy using conjunctional swab, serum and bone marrow samples from the dogs, respectively. Results: The LAMP assay detected 1 fg of L. infantum DNA purified from cultured promastigotes which was 10-fold more sensitive than a conventional PCR test using Leishmania genus-specific primers. No cross reaction was observed with DNA isolated from promastigotes of L. donovani, L. major, L. tropica, and L. braziliensis, and the L. infantum reference strain MHOM/TN/80/IPT1. The L. infantum-positive rates obtained for field-collected samples were 61.3%, 58.6%, 40.5% and 10.8% by LAMP, PCR, ELISA and microscopy, respectively. As only one out of the 33 samples from control dogs from the non-endemic region of zoonotic visceral leishmaniasis was positive by the LAMP assay and the PCR test, the observed true negative rate (specificity) was 97% for both methods. Conclusion: This study has shown that the non-invasive, conjunctional swab-based LAMP assay developed was more sensitive in the detection of leishmaniasis in dogs than PCR, ELISA and microscopy. The findings indicate that the LAMP assay is a sensitive and specific method for the field surveillance of domestic dogs, particularly of asymptomatic canines, in ZVL-endemic areas in western China

Changes in energy expenditure associated with ingestion of high protein, high fat versus high protein, low fat meals among underweight, normal weight, and overweight females

Background: Metabolic rate is known to rise above basal levels after eating, especially following protein consumption. Yet, this postprandial rise in metabolism appears to vary among individuals. This study examined changes in energy expenditure in response to ingestion of a high protein, high fat (HPHF) meal versus an isocaloric high protein, low fat (HPLF) meal in underweight, normal weight, or overweight females (n = 21) aged 19–28 years. Methods: Energy expenditure, measured using indirect calorimetry, was assessed before and every 30 minutes for 3.5 hours following consumption of the meals on two separate occasions. Height and weight were measured using standard techniques. Body composition was measured using bioelectrical impedance analysis. Results: Significant positive correlations were found between body mass index (BMI) and baseline metabolic rate (MR) (r = 0.539; p = 0.017), between body weight and baseline MR (r = 0.567; p = 0.011), between BMI and average total change in MR (r = 0.591; p = 0.008), and between body weight and average total change in MR (r = 0.464; p = 0.045). Metabolic rate (kcal/min) was significantly higher in the overweight group than the normal weight group, which was significantly higher than the underweight group across all times and treatments. However, when metabolic rate was expressed per kg fat free mass (ffm), no significant difference was found in postprandial energy expenditure between the overweight and normal groups. Changes in MR (kcal/min and kcal/min/kg ffm) from the baseline rate did not significantly differ in the underweight (n = 3) or in the overweight subjects (n = 5) following consumption of either meal at any time. Changes in MR (kcal/min and kcal/min/kg ffm) from baseline were significantly higher in normal weight subjects (n = 11) across all times following consumption of the HPHF meal versus the HPLF meal. Conclusion: There is no diet-induced thermogenic advantage between the HPHF and HPLF meals in overweight and underweight subjects. In contrast, in normal weight subjects, ingestion of a HPHF meal significantly increases MR (69.3 kcal/3.5 hr) versus consumption of a HPLF meal and provides a short-term metabolic advantage

A Worldwide Phylogeography for the Human X Chromosome

BACKGROUND: We reasoned that by identifying genetic markers on human X chromosome regions where recombination is rare or absent, we should be able to construct X chromosome genealogies analogous to those based on Y chromosome and mitochondrial DNA polymorphisms, with the advantage of providing information about both male and female components of the population. METHODOLOGY/PRINCIPAL FINDINGS: We identified a 47 Kb interval containing an Alu insertion polymorphism (DXS225) and four microsatellites in complete linkage disequilibrium in a low recombination rate region of the long arm of the human X chromosome. This haplotype block was studied in 667 males from the HGDP-CEPH Human Genome Diversity Panel. The haplotypic diversity was highest in Africa (0.992+/-0.0025) and lowest in the Americas (0.839+/-0.0378), where no insertion alleles of DXS225 were observed. Africa shared few haplotypes with other geographical areas, while those exhibited significant sharing among themselves. Median joining networks revealed that the African haplotypes were numerous, occupied the periphery of the graph and had low frequency, whereas those from the other continents were few, central and had high frequency. Altogether, our data support a single origin of modern man in Africa and migration to occupy the other continents by serial founder effects. Coalescent analysis permitted estimation of the time of the most recent common ancestor as 182,000 years (56,700-479,000) and the estimated time of the DXS225 Alu insertion of 94,400 years (24,300-310,000). These dates are fully compatible with the current widely accepted scenario of the origin of modern mankind in Africa within the last 195,000 years and migration out-of-Africa circa 55,000-65,000 years ago. CONCLUSIONS/SIGNIFICANCE: A haplotypic block combining an Alu insertion polymorphism and four microsatellite markers on the human X chromosome is a useful marker to evaluate genetic diversity of human populations and provides a highly informative tool for evolutionary studies

Genetic variants associated with fasting blood lipids in the U.S. population: Third National Health and Nutrition Examination Survey

<p>Abstract</p> <p>Background</p> <p>The identification of genetic variants related to blood lipid levels within a large, population-based and nationally representative study might lead to a better understanding of the genetic contribution to serum lipid levels in the major race/ethnic groups in the U.S. population.</p> <p>Methods</p> <p>Using data from the second phase (1991-1994) of the Third National Health and Nutrition Examination Survey (NHANES III), we examined associations between 22 polymorphisms in 13 candidate genes and four serum lipids: high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triglycerides (TG). Univariate and multivariable linear regression and within-gene haplotype trend regression were used to test for genetic associations assuming an additive mode of inheritance for each of the three major race/ethnic groups in the United States (non-Hispanic white, non-Hispanic black, and Mexican American).</p> <p>Results</p> <p>Variants within <it>APOE </it>(rs7412, rs429358), <it>PON1 </it>(rs854560), <it>ITGB3 </it>(rs5918), and <it>NOS3 </it>(rs2070744) were found to be associated with one or more blood lipids in at least one race/ethnic group in crude and adjusted analyses. In non-Hispanic whites, no individual polymorphisms were associated with any lipid trait. However, the <it>PON1 </it>A-G haplotype was significantly associated with LDL-C and TC. In non-Hispanic blacks, <it>APOE </it>variant rs7412 and haplotype T-T were strongly associated with LDL-C and TC; whereas, rs5918 of <it>ITGB3 </it>was significantly associated with TG. Several variants and haplotypes of three genes were significantly related to lipids in Mexican Americans: <it>PON1 </it>in relation to HDL-C; <it>APOE </it>and <it>NOS3 </it>in relation to LDL-C; and <it>APOE </it>in relation to TC.</p> <p>Conclusions</p> <p>We report the significant associations of blood lipids with variants and haplotypes in <it>APOE</it>, <it>ITGB3, NOS3</it>, and <it>PON1 </it>in the three main race/ethnic groups in the U.S. population using a large, nationally representative and population-based sample survey. Results from our study contribute to a growing body of literature identifying key determinants of plasma lipoprotein concentrations and could provide insight into the biological mechanisms underlying serum lipid and cholesterol concentrations.</p

Missed, not missing: Phylogenomic evidence for the existence of Avian FoxP3

The Forkhead box transcription factor FoxP3 is pivotal to the development and function of regulatory T cells (Tregs), which make a major contribution to peripheral tolerance. FoxP3 is believed to perform a regulatory role in all the vertebrate species in which it has been detected. The prevailing view is that FoxP3 is absent in birds and that avian Tregs rely on alternative developmental and suppressive pathways. Prompted by the automated annotation of foxp3 in the ground tit (Parus humilis) genome, we have questioned this assumption. Our analysis of all available avian genomes has revealed that the foxp3 locus is missing, incomplete or of poor quality in the relevant genomic assemblies for nearly all avian species. Nevertheless, in two species, the peregrine falcon (Falco peregrinus) and the saker falcon (F. cherrug), there is compelling evidence for the existence of exons showing synteny with foxp3 in the ground tit. A broader phylogenomic analysis has shown that FoxP3 sequences from these three species are similar to crocodilian sequences, the closest living relatives of birds. In both birds and crocodilians, we have also identified a highly proline-enriched region at the N terminus of FoxP3, a region previously identified only in mammals