Table_2_Comparative Proteomic and Morphological Change Analyses of Staphylococcus aureus During Resuscitation From Prolonged Freezing.XLSX

<p>When frozen, Staphylococcus aureus survives in a sublethally injured state. However, S. aureus can recover at a suitable temperature, which poses a threat to food safety. To elucidate the resuscitation mechanism of freezing survived S. aureus, we used cells stored at -18°C for 90 days as controls. After resuscitating the survived cells at 37°C, the viable cell numbers were determined on tryptic soy agar with 0.6% yeast extract (TSAYE), and the non-injured-cell numbers were determined on TSAYE supplemented with 10% NaCl. The results showed that the total viable cell number did not increase within the first 3 h of resuscitation, but the osmotic regulation ability of freezing survived cells gradually recovered to the level of healthy cells, which was evidenced by the lack of difference between the two samples seen by differential cell enumeration. Scanning electron microscopy (SEM) showed that, compared to late exponential stage cells, some frozen survived cells underwent splitting and cell lysis due to deep distortion and membrane rupture. Transmission electron microscopy (TEM) showed that, in most of the frozen survived cells, the nucleoids (low electronic density area) were loose, and the cytoplasmic matrices (high electronic density area) were sparse. Additionally, a gap was seen to form between the cytoplasmic membranes and the cell walls in the frozen survived cells. The morphological changes were restored when the survived cells were resuscitated at 37°C. We also analyzed the differential proteome after resuscitation using non-labeled high-performance liquid chromatography–mass spectrometry (HPLC-MS). The results showed that, compared with freezing survived S. aureus cells, the cells resuscitated for 1 h had 45 upregulated and 73 downregulated proteins. The differentially expressed proteins were functionally categorized by gene ontology enrichment, KEGG pathway, and STRING analyses. Cell membrane synthesis-related proteins, oxidative stress resistance-related proteins, metabolism-related proteins, and virulence factors exhibited distinct expression patterns during resuscitation. These findings have implications in the understanding of the resuscitation mechanism of freezing survived S. aureus, which may facilitate the development of novel technologies for improved detection and control of foodborne pathogens in frozen food.</p

Table_1_Comparative Proteomic and Morphological Change Analyses of Staphylococcus aureus During Resuscitation From Prolonged Freezing.XLSX

<p>When frozen, Staphylococcus aureus survives in a sublethally injured state. However, S. aureus can recover at a suitable temperature, which poses a threat to food safety. To elucidate the resuscitation mechanism of freezing survived S. aureus, we used cells stored at -18°C for 90 days as controls. After resuscitating the survived cells at 37°C, the viable cell numbers were determined on tryptic soy agar with 0.6% yeast extract (TSAYE), and the non-injured-cell numbers were determined on TSAYE supplemented with 10% NaCl. The results showed that the total viable cell number did not increase within the first 3 h of resuscitation, but the osmotic regulation ability of freezing survived cells gradually recovered to the level of healthy cells, which was evidenced by the lack of difference between the two samples seen by differential cell enumeration. Scanning electron microscopy (SEM) showed that, compared to late exponential stage cells, some frozen survived cells underwent splitting and cell lysis due to deep distortion and membrane rupture. Transmission electron microscopy (TEM) showed that, in most of the frozen survived cells, the nucleoids (low electronic density area) were loose, and the cytoplasmic matrices (high electronic density area) were sparse. Additionally, a gap was seen to form between the cytoplasmic membranes and the cell walls in the frozen survived cells. The morphological changes were restored when the survived cells were resuscitated at 37°C. We also analyzed the differential proteome after resuscitation using non-labeled high-performance liquid chromatography–mass spectrometry (HPLC-MS). The results showed that, compared with freezing survived S. aureus cells, the cells resuscitated for 1 h had 45 upregulated and 73 downregulated proteins. The differentially expressed proteins were functionally categorized by gene ontology enrichment, KEGG pathway, and STRING analyses. Cell membrane synthesis-related proteins, oxidative stress resistance-related proteins, metabolism-related proteins, and virulence factors exhibited distinct expression patterns during resuscitation. These findings have implications in the understanding of the resuscitation mechanism of freezing survived S. aureus, which may facilitate the development of novel technologies for improved detection and control of foodborne pathogens in frozen food.</p

Table_3_Comparative Proteomic and Morphological Change Analyses of Staphylococcus aureus During Resuscitation From Prolonged Freezing.DOCX

<p>When frozen, Staphylococcus aureus survives in a sublethally injured state. However, S. aureus can recover at a suitable temperature, which poses a threat to food safety. To elucidate the resuscitation mechanism of freezing survived S. aureus, we used cells stored at -18°C for 90 days as controls. After resuscitating the survived cells at 37°C, the viable cell numbers were determined on tryptic soy agar with 0.6% yeast extract (TSAYE), and the non-injured-cell numbers were determined on TSAYE supplemented with 10% NaCl. The results showed that the total viable cell number did not increase within the first 3 h of resuscitation, but the osmotic regulation ability of freezing survived cells gradually recovered to the level of healthy cells, which was evidenced by the lack of difference between the two samples seen by differential cell enumeration. Scanning electron microscopy (SEM) showed that, compared to late exponential stage cells, some frozen survived cells underwent splitting and cell lysis due to deep distortion and membrane rupture. Transmission electron microscopy (TEM) showed that, in most of the frozen survived cells, the nucleoids (low electronic density area) were loose, and the cytoplasmic matrices (high electronic density area) were sparse. Additionally, a gap was seen to form between the cytoplasmic membranes and the cell walls in the frozen survived cells. The morphological changes were restored when the survived cells were resuscitated at 37°C. We also analyzed the differential proteome after resuscitation using non-labeled high-performance liquid chromatography–mass spectrometry (HPLC-MS). The results showed that, compared with freezing survived S. aureus cells, the cells resuscitated for 1 h had 45 upregulated and 73 downregulated proteins. The differentially expressed proteins were functionally categorized by gene ontology enrichment, KEGG pathway, and STRING analyses. Cell membrane synthesis-related proteins, oxidative stress resistance-related proteins, metabolism-related proteins, and virulence factors exhibited distinct expression patterns during resuscitation. These findings have implications in the understanding of the resuscitation mechanism of freezing survived S. aureus, which may facilitate the development of novel technologies for improved detection and control of foodborne pathogens in frozen food.</p

Protein-Bound Polysaccharide from <i>Corbicula fluminea</i> Inhibits Cell Growth in MCF-7 and MDA-MB-231 Human Breast Cancer Cells

<div><p>A novel protein-bound polysaccharide, CFPS-1, isolated from <i>Corbicula fluminea</i>, is composed predominantly of mannose (Man) and glucose (Glc) in a molar ratio of 3.1:12.7. The polysaccharide, with an average molecular weight of about 283 kDa, also contains 10.8% protein. Atomic force microscopy, high-performance liquid chromatography, Fourier transform infrared spectroscopy, gas chromatography/mass spectrometry, and nuclear magnetic resonance spectroscopy analyses revealed that CFPS-1 has a backbone of 1,6-linked and 1,4,6-linked-α-D-Glc, which is terminated with a 1-linked-α-D-Man residue at the O-4 position of 1,4,6-linked-α-D-Glc, in a molar ratio of 3:1:1. Preliminary <i>in vitro</i> bioactivity tests revealed that CFPS-1 effectively and dose-dependently inhibits human breast cancer MCF-7 and MDA-MB-231 cell growth, with an IC₅₀ of 243 ± 6.79 and 1142 ± 14.84 μg/mL, respectively. In MCF-7, CFPS-1 produced a significant up-regulation of p53, p21, Bax and cleaved caspase-7 and down-regulation of Cdk4, cyclin D1, Bcl-2 and caspase-7. These effects resulted in cell cycle blockade at the S-phase and apoptosis induction. In contrast, in MDA-MB-231, with limited degree of change in cell cycle distribution, CFPS-1 increases the proportion of cells in apoptotic sub-G1 phase executed by down-regulation of Bcl-2 and caspase-7 and up-regulation of Bax and cleaved caspase-7. This study extends our understanding of the anticancer mechanism of <i>C</i>. <i>fluminea</i> protein-bound polysaccharide.</p></div

Effect of the <i>C</i>. <i>fluminea</i> protein-bound polysaccharide CFPS-1 on MCF-7 and MDA-MB-231 cell cycle progression.

<p>Cells were cultured for 24 h with CFPS-1 (0, 50, 150 and 250 μg/mL, respectively). Untreated cells (0 μg/mL) were used as control. Cell cycle distribution (%) of MCF-7 (A) and MDA-MB-231 (C) cells were determined by flow cytometry after CFPS-1 treatment; Apoptotic rate (%) of MCF-7 (B) and MDA-MB-231 (D) cells treated with CFPS-1. Apoptotic cells were identified with a TUNEL technique and counted with a light microscope (magnification, ×40). All data were expressed as mean ± SD of three experiments and each experiment included triplicate repeats. Values marked with * are significantly different from the control (<i>p</i> < 0.05).</p

Molecular and morphological characterization of <i>C</i>. <i>fluminea</i> protein-bound polysaccharide CFPS-1.

<p>(A and B) The atomic force microscopy (AFM) images of CFPS-1. The CFPS-1 concentration was 1 μg/mL (A; B1), 10 μg/mL (B2) and 100 μg/mL (B3), respectively. Arrow: (a) single linear chain; (b) branched chain; (C) FTIR spectra of CFPS-1 (4000 cm⁻¹ to 400 cm⁻¹) were obtained from solid samples by KBr disc method using a Nicolet 5700 FT-IR spectrophotometer. FTIR spectra showed that CFPS-1 mainly contained three types of groups, namely hydroxyl group, amino group and sulfate group.</p

Effect of the <i>C</i>. <i>fluminea</i> protein-bound polysaccharide CFPS-1 on growth of human breast cancer MCF-7 and MDA-MB-231 cells.

<p>The inhibitory effect of CFPS-1 on the cell growth of MCF-7 (A) and MDA-MB-231(C) cells at 24, 48, 72 and 96 h. Proliferation and cytotoxicity against MCF-7 (B) and MDA-MB-231 (D) cells by the CFPS-1 at 72 h. CK, Control of proliferation or cytotoxicity. All data were expressed as mean ± SD of three experiments and each experiment included triplicate repeats. Values marked with * are significantly different from the control (<i>p</i> < 0.05).</p

¹H and ¹³C NMR chemical shifts for the protein-bound polysaccharide CFPS-1 isolated from <i>C</i>. <i>fluminea</i> in D₂O ^a.

<p>¹H and ¹³C NMR chemical shifts for the protein-bound polysaccharide CFPS-1 isolated from <i>C</i>. <i>fluminea</i> in D₂O <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167889#t004fn001" target="_blank">^a</a>.</p

Yield, chemical compositions and contents of carbohydrate, protein, <i>M</i>_W and sulfonic acid in the <i>C</i>. <i>fluminea</i> protein-bound polysaccharide CFPS-1.

<p>Yield, chemical compositions and contents of carbohydrate, protein, <i>M</i>_W and sulfonic acid in the <i>C</i>. <i>fluminea</i> protein-bound polysaccharide CFPS-1.</p

Structural elucidation of the <i>C</i>. <i>fluminea</i> protein-bound polysaccharide CFPS-1.

<p><b>(A)</b>^<b>1</b><b>H NMR and (B)</b>^<b>13</b><b>C NMR spectra of CFPS-1 in D</b>_<b>2</b><b>O.</b> Chemical shifts were reported relative to internal acetone at δH 2.23 ppm for ¹H spectrum and δC 32.45 ppm for ¹³C spectrum; (C) A diagram showing the partial structures of polysaccharide portions of the protein-bound polysaccharide CFPS-1 from C. fluminea. (a): 1,6-linked -α-D-Glc; (b): 1,4,6-linked-α-D-Glc; (c): 1-linked-α-D-Man.</p