Unique Identifcation of research resources in studies in Reproducibility Project: Cancer Biology

<p>Reproducibility Project: Cancer Biology (https://osf.io/e81xl/wiki/home/) aims to reproduce the key experiments from 50 landmark papers in cancer research. As a follow up to the previously published study, which showed a lack of indentifiability of research resources in the published biomedical literature (Vasilevsky, et al. 2014, PeerJ 1:e148), we analyzed 6 resource types reported in these papers to determine the identifiability of these resources. The resource types included antibodies, cell lines, constructs, knockdown reagents, model organisms and software. The results showed an average 85% of the resources were identifiable, and the ability to identify the resources varied amongst the resource types.</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

Unique Identification of research resources in studies in Reproducibility Project: Cancer Biology

<p>Reproducibility Project: Cancer Biology (https://osf.io/e81xl/wiki/home/) aims to reproduce the key experiments from 50 landmark papers in cancer research. As a follow up to the previously published study, which showed a lack of indentifiability of research resources in the published biomedical literature (Vasilevsky, et al. 2014, PeerJ 1:e148), we analyzed 6 resource types reported in these papers to determine the identifiability of these resources. The resource types included antibodies, cell lines, constructs, knockdown reagents, model organisms and software. The results showed an average 85% of the resources were identifiable, and the ability to identify the resources varied amongst the resource types.</p> <p> </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 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

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

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

A New Mouse Model for the Study of Human Breast Cancer Metastasis

<div><p>Breast cancer is the most common cancer in women, and this prevalence has a major impact on health worldwide. Localized breast cancer has an excellent prognosis, with a 5-year relative survival rate of 85%. However, the survival rate drops to only 23% for women with distant metastases. To date, the study of breast cancer metastasis has been hampered by a lack of reliable metastatic models. Here we describe a novel in vivo model using human breast cancer xenografts in NOD <em>scid</em> gamma (NSG) mice; in this model human breast cancer cells reliably metastasize to distant organs from primary tumors grown within the mammary fat pad. This model enables the study of the entire metastatic process from the proper anatomical site, providing an important new approach to examine the mechanisms underlying breast cancer metastasis. We used this model to identify gene expression changes that occur at metastatic sites relative to the primary mammary fat pad tumor. By comparing multiple metastatic sites and independent cell lines, we have identified several gene expression changes that may be important for tumor growth at distant sites.</p> </div

NSG mice consistently develop macro-metastases when MDA-MB-436 cells are injected orthotopically into the mammary fat pad.

<p><b>A.</b> Volumes of tumors in mammary fat pads of NSG mice injected with MDA-MB-436 cells. Each data point is the mean value (+/− s.e.m) of 12 primary tumors. <b>B.</b> Micrographs of haematoxylin and eosin (H&E), CK18, EGFR and Her2 IHC staining of harvested MDA-MB-436 primary tumor tissue. MDA-MB-436 primary tumors are CK18 positive, EGFR positive, Her2 negative. <b>C.</b> Quantification of the percentage of mice bearing macro-metastasis in each organ observed at the time of necropsy (43 days post injection). Macro-metastases were frequently observed in the lung (92% of mice) and left axillary lymph node (75% of mice), as well as sporadically in other organs. LN = lymph node. <b>D.</b> Photographs of representative common MDA-MB-436 metastases are shown, along with micrographs of H&E and CK18 IHC staining of harvested tissue.</p