Antibacterial Effects of a Cell-Penetrating Peptide Isolated from Kefir

Kefir is a traditional fermented milk beverage used throughout the world for centuries. A cell-penetrating peptide, F3, was isolated from kefir by Sephadex G-50 gel filtration, DEAE-52 ion exchange, and reverse-phase high-performance liquid chromatography. F3 was determined to be a low molecular weight peptide containing one leucine and one tyrosine with two phosphate radicals. This peptide displayed antimicrobial activity across a broad spectrum of organisms including several Gram-positive and Gram-negative bacteria as well as fungi, with minimal inhibitory concentration (MIC) values ranging from 125 to 500 μg/mL. Cellular penetration and accumulation of F3 were determined by confocal laser scanning microscopy. The peptide was able to penetrate the cellular membrane of Escherichia coli and Staphylococcus aureus. Changes in cell morphology were observed by scanning electron microscopy (SEM). The results indicate that peptide F3 may be a good candidate for use as an effective biological preservative in agriculture and the food industry

DCLK1 protein levels are elevated in the serum of stage I and II PDAC patients.

<p>A: DCLK1 protein levels in the serum samples were determined by ELISA. DCLK1 levels were grouped based on the cancer stage and compared to normal group. **p<0.05. n = 12, 12, 19, 16 and 15 for normal, stage I, II, III, and IV respectively. B: Receiver operating characteristic (ROC) curve analysis for the use of DCLK1 as serum marker for stage I and II PDAC (AUC = 0.74, and p = 0.029). C: DCLK1 protein levels in the serum samples were detected by western blotting. D: Percentage of DCLK1 levels in each stage relative to control based on western blotting analysis. **p<0.002.</p

General information of human subjects used in this study.

<p>General information of human subjects used in this study.</p

CA 19–9 levels are increased in the serum of stage II, III, and IV PDAC patients.

<p>CA 19–9 in the same serum samples was detected by ELISA. CA 19–9 levels were grouped based on the cancer stage and compared to normal group. **p<0.05. n = 12, 12, 19, 16, and 15 for normal, stage I, II, III, and IV respectively.</p

DCLK1 staining scores in epithelial and stromal cells of PDAC patients.

<p>DCLK1 staining scores in epithelial and stromal cells of PDAC patients.</p

Dclk1 expression levels were evaluated in mouse models.

<p>A: Distinct expression of Dclk1 is associated with ADM. Dclk1 immunostaining in pancreas of C57Bl/6 mice treated with caerulein on day 0 (control), 1, 3, and 5 post injection. B: Dclk1 protein levels are upregulated in the serum of pre-cancerous and cancerous KPC mice. The serum of 5-, 15- (pre-cancerous) and 25- (cancerous) week old control (con) and KPC mice (n = 5 for each group) was purified and subjected to SDS-PAGE and immuno-blotted with anti-Dclk1 antibody. Dclk1 levels are upregulated in serum of pre-cancerous and cancerous KPC mice relative to controls (<i>p</i><0.01). C: Dclk1 is expressed on the surface of circulating tumor cells in KPCY mice. Whole blood was collected from 4-month old KPCY mice (n = 3), incubated with anti-Dclk1 antibody conjugated with AlexaFluor-546, and subjected to flow cytometry. Among the YFP+ circulating cells, 52% were positive for Dclk1. D: Dclk1 is co-localized with YFP in both primary and metastatic tumors. Immuno-staining demonstrating both primary PDAC tissue and metastatic PDAC tissue (colon) of KPCY mice express Dclk1 (red) co-localized with YFP+ tumor cells (green). Dclk1/YFP double positive cells appear yellow-orange.</p

DCLK1 expression level is higher in stromal relative to epithelial cells in human PDAC.

<p>A: Paraffin-embedded PDAC tumor sections were immuno-stained with anti-cytokeratin antibody (yellow brown) and anti-DCLK1 antibody (purple) to detect tumor epithelial cells and DCLK1 expression. B: The intensity of DCLK1 expression is stronger in stromal relative to epithelial cells in human PDAC (p = 0.009). The intensity of DCLK1 expression in epithelial cells and stromal cells was scored separately for each specimen and grouped based on cancer stage (n = 10, 12, 12, and 10 for stage I, II, III, and IV respectively). C: DCLK1 mRNA levels are elevated in human pancreatic tumor tissue relative to normal pancreas tissue. DCLK1 mRNA expression levels were summarized and grouped using a published DNA microarray data [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118933#pone.0118933.ref027" target="_blank">27</a>]. p<0.05.</p