**2.6. RT‐PCR**

istics of the patients are shown in **Table 1**, including mean age, TNM phase, histopathology, lymph node status, metastasis and so on. All these patients were incipient and were treated by systemic therapy, including 12 patients cured by cytokine‐induced killer cells therapy (CIK).

Carcinoma cell line SKBR‐3 (breast) was used to estimate the sensitivity and specificity of the

Antibodies, which were used for multiparameter flow cytometry, were as follows: anti‐CD45‐ PerCP, anti‐CD44‐APC (allophycocyanin, clone G44‐26, catalog number 559942) and anti‐ CD24‐FITC (fluorescein isothiocyanate, clone ML5, catalog number 555427) were from Becton Dickison Crop., USA. Ep‐CAM (epithelial‐cell adhesion molecule) and CK‐8, CK‐18, CK‐19

Every patient with breast cancer drew 20 ml blood for CTCs detection and three healthy volunteers. The blood of healthy volunteers was treated as negative control. About 5 ml blood was discarded to avoid contamination with skin cells as previously described [26]. We separated mononucleocytes from 15 ml blood by Ficoll‐Paque (Haoyang Biological Production Limited Company, Tianjin, China) for 20 min with 1800 × *g* at 25°C. One half of the mononu‐ clear cells was resuspended in phosphate‐buffered saline (PBS) for multiparameter flow cytometry on the account of at least 2–3 × 106 cells for each sample and the other half was kept

Mononucleocytes were enriched and washed twice with PBS and then labelled antibodies that

SKBR‐3 breast cancer cells were used to evaluate the sensitivity of the flow cytometry (tumor cells recovery). Tumor cells were resuspended and counted. 1, 10, 50, 500 cells were spiked, respectively, into 7.5 ml of blood from healthy person. And then, it was processed as described as earlier and control the preparation for the same volume of the cell suspension and cells were counted to accurately estimate the number of cells spiked into the blood. The average number of Ep‐CAM and CK‐8, CK‐18, CK‐19 positive cells on FACS Caliber™ was used to calculate

), kept in dark at 4°C for 30 min. We added 20 μl monoclonal antibodies for each sample. Cell pellets were resuspended in 250 μl PBS and enumerated by FACS Caliber™ (Becton

, CK‐8, CK‐18, CK‐

flow cytometry, which was maintained in RPMI 1640 plus 10% fetal calf serum.

(CK‐8, CK‐18, CK‐19 phycoerythrin staining) were from Abcam, USA.

in Trizol reagent (Invitrogen, UK) at -70°C until RNA extraction for RT‐PCR.

target white cell antigens and epithelial cell antigens (CD45‐, Ep‐CAM+

And all the patients were drawn blood for the detection of CTCs.

**2.2. Cell line**

198 Tumor Metastasis

**2.3. Antibodies**

**2.4. Preparation of samples**

**2.5. Flow cytometry**

the cells recovery.

Dickison Crop., USA) at last.

19+

We extract RNA with 1 ml trizol from the mononucleocytes, which were separated from 7.5 ml blood and then kept at -70°C. Add 0.2 ml chloroform and then centrifuge the sample at 12,000 × *g* for 15 min at 4°C. The intact RNA, which was contained in the supernatants, was removed into a new tube. RNA was dissolved in 10 μl RNase‐free water after precipitating RNA with isopropyl alcohol and washing with 75% ethanol. Then, RNA was transcribed to cDNA by a reverse transcriptase in a total 10 μl RT reaction solution, which was contained of 2 μl 5x Reverse Transcriptase Buffer, 1 μl dNTP (10 mM each), 0.25 μl RNase inhibitor (10 U), 1 μl oligo (dT)15 primer (25 pmol), 5.25 μl RNase‐free water containing RNA (>0.5 μg), 0.5 μl avian myeloblastosis virus (AMV) reverse transcriptase (5U) (TaKaRa, China). The resulting cDNA was subjected to PCR amplification. PCR was composed of 2 μl cDNA, 10 μl Mix, 2 μl primer of EpCAM, 6 μl H2O in a total volume of 20 μl. The primer of EpCAM was as follows: 5'‐GGACCTGACAGTAAATGGGGAAC‐3'; 5'‐CTCTTCTTTCTGGAAATAACCAGCAC‐3' [18]. GAPDH mRNA primer detail was as follows: 5'‐TGCACCACCAACTGCTTAGC‐3'; 5'‐ GGAGGCAGGGATGATGTTCT‐3' which was designed by Primer 5.0. The reaction condition was 95°C for 2 min to activate Taq DNA polymerase and it finally elongated 72°C for 7 min with 35 cycles. We detected the PCR products by ethidium bromide staining on a 1% agarose gel.

#### **2.7. Statistical analysis**

Correlation, regression analysis and a Mann‐Whitney rank sum test were performed on titration experiments. Chi‐square test was used to compare across CTC groups. Overall survival was performed to describe the condition of CTCs and prognosis and was estimated by the Kaplan‐Meier. Comparison of groups used the log‐rank test. All statistical tests were two‐sided, and *P* values < 0.05 were considered statistically significant. Analyses above were performed by using the SPSS 13.0.


**1. Introduction**

Although an atherogenic lipoprotein phenotype was reported as a predictor of cardiovascular/ cerebrovascular disease, numerous recent studies have recognized additional lipid-related markers as emerging biomarkers to identify patients with cardiovascular/cerebrovascular disease risk. Among them the most promising biomarkers for cardiovascular/cerebrovascular risk assessment is lipoprotein-associated phospholipase A2 (Lp-PLA2).

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**Abstract**

In this chapter, with renewal argument, we derive higher simple moments of the Discounted Compound Delay Renewal Risk Process (DCDRRP) when introducing dependence between the inter-occurrence time and the subsequent claim size. To illustrate our results, we assume that the inter-occurrence time is following a delay-Poisson process and the claim amounts is following a mixture of Exponential distribution, we then provide numerical results for the first two moments. The dependence structure between the inter-occurrence time and the subsequent claim size is defined by a Farlie-Gumbel-Morgenstern copula. Assuming that the claim distribution has finite moments, we obtain a general formula for all the moments of the DCDRRP process.

**Keywords:** compound delay-Poisson process, discounted aggregate claims, moments, FGM copula, constant interest rate


*2.1. Proliferation Inhibition and Apoptosis Induction of Tumoral B-Cells*

Anti-cancer properties of fucoidan (mainly with extracted fucoidan of high molecular weight) were almost exclusively studied on solid tumors and emphasized anti-proliferative and pro-apoptotic events [32]. In order to confirm this role on lymphoma B-cells, we performed cell cycle analysis by flow cytometry on EBV latency III B-cells (three LCLs) and DLBCL cells (two ABCs: U2932 and OCILy10 and two GCBs: SUDHL4 and SUDHL6 cell lines) treated with native or vLMW-F F1 and F2. Estimation of cell population percentage in the different phases of the cell cycle highlighted significant decrease in S phase after treatment with vLMW-F in contrast to the native form (Figure 1A, B). This was consistent with inhibition of cell proliferation. A significant increase in subG1 peak on DNA content histograms (subsequent to fragmentation of nuclear DNA in late apoptosis) was also

sometimes observed, depending on cell types, for treatment by vLMW-F, especially F2 (Figure 1C).

**Figure 1.** vLMW-F decrease the percentage of LCLs and DLBCLs cells in S-phase. LCLs (J1209, C0401, C1504) and DLBCLs (U2932, OCILy10, SUDHL4, SUDHL6) cells were treated or not (control) with 100μg/mL of native fucoidan or vLMW-F (F1 and F2) for 48 h. Flow cytometry analysis was performed to determine cell-cycle distribution in all tested conditions. Results were obtained from three independent experiments. (**A**) Examples of cell cycle profiles for C0401 (LCL) and SUDHL4 (DLBCL). (**B**) Percentage of LCLs or DLBCLs cells in S-phase: fewer cells are in S-phase after 48 h treatment of 100 μg/mL vLMW-F in contrast with the native form and compared to the control. (**C**) Percentage of LCLs or DLBCLs cells in SubG1 phase: the increase in cell percentage in SubG1 phase occasionally observed suggests apoptosis induction. NS: not significant; \* *p*< 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001.

To refine the apoptotic response, we evaluated percentage of apoptotic cells, from early to late stages, for the same cell lines and in the same treatment conditions. Annexin-V binding to externalized phosphatidylserine revealed apoptosis induction only with the two vLMW-F (Figure 2). The importance of the process depended on the cell lines (up to 50% for J1209 and SUDHL6) and was similar for the two fractions. Our results emphasized that, unlike the native form, vLMW-F possess anti-proliferative and pro-apoptotic properties for EBV latency III and DLBCL tumoral cells at the tested dose. This suggests a better

cytotoxic effect for vLMW-F. We verified that toxicity (viability and apoptosis) was due to the formulas and not L-Fucose since it is the smallest subunit of fucoidan skeleton (Figure S2).

**Figure 2.** vLMW-F induce apoptosis in LCLs and DLBCLs. LCLs (J1209, C0401, C1504) and DLBCLs (U2932, OCILy10, SUDHL4, SUDHL6) cells were treated or not (control) with 100μg/mL of native fucoidan or vLMW-F (F1 and F2) for 48 h, followed by apoptosis analysis (Annexin V/PI staining) by flow cytometry. Results were obtained from three independent experiments. (**A**) Examples of cell apoptosis for J1209 (LCL) and SUDHL6 (DLBCL) are shown (intact cells: green events–early apoptotic cells: blue events–late apoptotic cells: purple events). (**B**) Percentage of LCLs or DLBCLs total Annexin V+ cells. vLMW-F fractions induce similar apoptosis. NS: not significant; \* *p*< 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001.


**5. Conclusions**

Our data revealed that a novel SNP (C to T) in the coding region (exon 2) of the gene encoding cytosolic PPDK (cy*OsPPDK*) was responsible for the floury endosperm characteristics of low grain hardness, low starch damage, and fine particle size, and this mutation may be valuable in rice breeding programs. Co-segregation analysis with the developed CAPS marker (flo4-5\_F/flo4-5\_R) revealed co-segregation between the floury phenotype and the *flo4-5* using the segregation population and 44 japonica varieties. This CAPS marker could be applied directly to MAS. Real-time RT-PCR experiments revealed that PPDK was expressed at considerably higher levels in the *flo4-5* mutant than in the wild type during the grain filling stage. Plastids *AGPS2a*, *AGPS2b*, *SSIIb*, and *SSIIc* also exhibited enhanced expression in the *flo4-5* mutant. Although more studies are needed to fully understand the functions of PPDK, our data indicate that PPDK is involved in the endosperm development function through directly or indirectly regulating *AGPS2a*, *AGPS2b*, *SSIIb*, and *SSIIc*.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4425/11/4/465/s1, Table S1: Primers used for sequencing the whole genome sequence of *flo4-5* and qRT-PCR in this study, Table S2: Primer sequences of CAPS markers that developed for fine mapping, Table S3: Compare the whole genome sequences of *cyOsPPDK* gene among Nipponbare, *flo4-5* and its wild type Namil. InDel (Insertion or Deletion).

**Author Contributions:** H.W., T.-H.H. and S.-W.K.; developed ideas, designed and performed all experiments, and wrote the manuscript. Y.M. and J.-U.J.; developed the mutant lines and mapping population. D.-E.I. and J.L.; analyzed the whole genome resequencing dates. S.M.L. and S.-G.J.; produced rice plants for experiments. S.T.K.; support expression analysis. All authors read and approved the final manuscript.

**Funding:** This work was supported by the grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology and the research grant from Rural Development Administration, Republic of Korea.

**Acknowledgments:** This work was supported by the grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2018R1A4A1025158) and the research grant from the Rural Development Administration, Republic of Korea (PJ012890032020).

**Conflicts of Interest:** The authors declare no conflict of interest.


**9. Acknowledgements**

We would like to express our appreciation to the reviewers of this paper for their valuable suggestions. This work is supported by the Malaysian Ministry of Science, Technology and Innovation (MOSTI) under Grant No. 02-01-06-SF0230.

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