Genetics of obesity

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Genetics of obesity

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Replication Attempt: “Effect of BMAP-28 Antimicrobial Peptides on Leishmania Major Promastigote and Amastigote Growth: Role of Leishmanolysin in Parasite Survival”

<div><p>This study describes an attempt to replicate experiments from the paper “Effect of BMAP-28 Antimicrobial Peptides on <i>Leishmania major</i> Promastigote and Amastigote Growth: Role of Leishmanolysin in Parasite Survival,” which was submitted to the Reproducibility Initiative for independent validation. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) and its isomers were previously shown to have potent antiparasitic activity against <i>Leishmania major.</i> We tested the effectiveness of L-BMAP-28 and two of its isomers, the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), in both unamidated and amidated forms, as anti-leishmanial agents against <i>Leishmania major</i> promastigotes <i>in vitro</i>. We observed that L-BMAP-28, as well as its D and RI isomers, demonstrate anti-leishmanial activity against <i>L. major</i> promastigotes <i>in vitro</i>. The inhibitory effect was lower than what was seen in the original study. At 2 µM of amidated peptides, the viability was 94%, 36%, and 66% with L-, D- and RI-peptides, versus 57%, 6%, and 18% in the original study.</p></div

Endocannabinoid receptor 1 gene variations increase risk for obesity and modulate body mass index in European populations.

The therapeutic effects of cannabinoid receptor blockade on obesity-associated phenotypes underline the importance of the endocannabinoid pathway on the energy balance. Using a staged-approach, we examined the contribution of the endocannabinoid receptor 1 gene (CNR1) on obesity and body mass index (BMI) in the European population. With the input of CNR1 exons and 3' and 5' regions sequencing and HapMap database, we selected and genotyped 26 tagging single-nucleotide polymorphisms (SNPs) in 1932 obese cases and 1173 non-obese controls of French European origin. Variants that showed significant associations (P 0.5) with these two SNPs in the initial case-control study, identified two better associated SNPs (rs6454674 and rs10485170). Our study of 5750 subjects shows that CNR1 variations increase the risk for obesity and modulate BMI in our European population. As CB1 is a drug target for obesity, a pharmacogenetic analysis of the endocannabinoid blockade obesity treatment may be of interest to identify best responders

Promastigote viability assay of <i>Leishmania major</i> when treated with amidated BMAP-28 variants.

<p><i>L. major</i> MHOM/SN/74/SD strain was treated with L-, RI- and D-BMAP-28 peptides for 4 hours. Viability was expressed as a percentage of untreated control cells. Three complete biological replicates were performed and the standard errors are shown.</p

Cohen's d, 95% confidence intervals of the original and replication studies and their combination.

<p>Unpaired t-tests untreated vs treated indicated significance, where *p<0.05, **p<0.005, ***p<0.0005. The combined study p-values were generated using Fisher's combined probability test. The Forest plot was generated using GraphPad Prism version 6.</p

Promastigote viability assay of <i>Leishmania major</i> when treated with 0.5 µM or 2 µM BMAP-28 variants.

<p><i>L. major</i> MHOM/SN/74/SD strain was treated with L-, RI- and D-BMAP-28 peptides for 4 hours. Viability was expressed as a percentage of untreated control cells. Three complete biological replicates were performed and the mean and standard deviation are shown. The percentage viability at 0.5 µM or 2 µM of each BMAP-28 peptide was calculated from the dose response curve performed (replication). The percentage viability at 0.5 µM or 2 µM of each BMAP-28 peptide was determined from the bar graph reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114614#pone-0114614-g001" target="_blank">Fig. 1A</a> of Lynn <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114614#pone.0114614-Lynn1" target="_blank">[5]</a> (original). Unpaired t-tests untreated vs treated indicated significance.</p><p>Promastigote viability assay of <i>Leishmania major</i> when treated with 0.5 µM or 2 µM BMAP-28 variants.</p

HPLC profile and mass spectrum of synthesized amidated BMAP-28 peptides.

<p>The peptides (Sequence: GGLRSLGRKILRAWKKYGPIIVPIIRI-NH₂; M.W: 3131.92; Formula: C147H252N44O31) were isolated and purified by high-performance liquid chromatography (HPLC) to greater than 95% purity. The purity and molecular weight of the respective peptides were confirmed by matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry. Left panels: HPLC profile, right panels: mass spectrum. A) L-BMAP-28-NH₂, purity 95.64%; B) D-BMAP-28-NH₂, purity 95.35%; C) RI-BMAP-28-NH₂, purity 95.85%; D) Scrambled-BMAP-28-NH₂, purity 95.19%.</p

HPLC profile and mass spectrum of synthesized unmodified BMAP-28 peptides.

<p>The peptides (Sequence: GGLRSLGRKILRAWKKYGPIIVPIIRIG; M.W: 3131.92; Formula: C147H252N44O31) were isolated and purified by high-performance liquid chromatography (HPLC) to greater than 95% purity. The purity and molecular weight of the respective peptides were confirmed by matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry. Left panels: HPLC profile, right panels: mass spectrum. A) L-BMAP-28, purity 95.61%; B) D-BMAP-28, purity 96.67%; C) RI-BMAP-28, purity 95.62%; D) Scrambled-BMAP-28, purity 95.34%.</p

Genome-wide linkage analysis for severe obesity in french caucasians finds significant susceptibility locus on chromosome 19q.

To ascertain whether distinct chromosomal loci existed that were linked to severe obesity, as well as to utilize the increased heritability of this excessive phenotype, we performed a genome-wide scan in severely obese French Caucasians. The 109 selected pedigrees, totaling 447 individuals, required both the proband and a sibling to be severely obese (BMI >or=35 kg/m(2)), and 84.8% of the nuclear families possessed >or=1 morbidly obese sibling (BMI >or=40). Severe and morbid obesity are still relatively rare in France, with rates of 2.5 and 0.6%, respectively. The initial genome scan consisted of 395 evenly spaced microsatellite markers. Six regions were found to have suggestive linkage on 4q, 6cen-q, 17q, and 19q for a BMI >or=35 phenotypic subset, and 5q and 10q for an inclusive BMI >or=27 group. The highest peak on chromosome 19q (logarithm of odds [LOD] = 3.59) was significant by genome scan simulation testing (P = 0.042). These regions then underwent second-stage mapping with an additional set of 42 markers. BMI >or=35 analysis defined regions on 17q23.3-25.1 and 19q13.33-13.43 with an maximum likelihood score LOD of 3.16 and 3.21, respectively. Subsequent pooled data analysis with an additional previous population of 66 BMI >or=35 sib-pairs led to a significant LOD score of 3.8 at the 19q locus (empirical P = 0.023). For more moderate obesity and overweight susceptibility loci, BMI >or=27 analysis confirmed suggestive linkage to chromosome regions 5q14.3-q21.3 (LOD = 2.68) and 10q24.32-26.2 (LOD = 2.47). Plausible positional candidate genes include NR1H2 and TULP2