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

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

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

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

Schematic of the working principle of SPSM color imaging.

<p>(a) While the target objects rest on the surface of the image sensor, we sequentially turn on each LED in the RGB LED array illumination above and take sequences of low-resolution images. (b) Each low-resolution sequence is reconstructed into three monochromatic high-resolution images using the pixel super-resolution algorithm. The red, green, and blue channels are then combined into a single color image.</p

Results of cell-type identification tests with conventional and SPSM images.

<p>Results of cell-type identification tests with conventional and SPSM images.</p

(a) Lens-less SPSM imaging system prototype.

<p>(b) A CMOS image sensor with a microfluidic chamber mounted on the sensor surface for sample loading. (c) A sample slide can be made directly on the image sensor.</p

SPSM and 20× objective microscope images of Giardia, Cryptosporidium, and Entamoeba cysts.

<p>We imaged unstained cysts, iodine-stained, and methylene blue-stained cysts.</p