Overexpression of WRAP53 is associated with development and progression of esophageal squamous cell carcinoma.

BACKGROUND:Esophageal squamous cell carcinoma (ESCC) is a highly aggressive cancer whose underlying molecular mechanisms are poorly understood. The natural antisense transcript (NAT) WRAP53 regulates p53 expression and WRAP53 protein is a component of telomerase. NATs play key roles in carcinogenesis, and although WRAP53 is known to increase cancer cell survival, its role in ESCC clinicopathology is unknown. The aim of this study was to investigate WRAP53 expression in ESCC and to correlate it with clinicopathological characteristics. METHODS:WRAP53 mRNA and protein expression was measured by quantitative PCR (qRT-PCR) and western blotting, respectively, in 4 ESSC cells lines and in 45 paired ESCC and non-neoplastic esophageal mucosa tissues. To correlate WRAP53 protein expression with clinicopathological characteristics, immunohistochemistry (IHC) was performed on 134 ESCC and 85 non-neoplastic esophageal mucosa tissues. RESULTS:Expression of WRAP53 was detected in all ESCC cell lines and was upregulated in the ESCC tissues compared with the corresponding non-neoplastic tissues (P<0.01). More cells expressed WRAP53 protein in the ESCC tissues than in the non-neoplastic tissues (P<0.01). Overexpression of WRAP53 was significantly correlated with tumor infiltration depth (P = 0.000), clinical stage (P = 0.001), and lymph node metastasis (P = 0.025). Wrap53 expression was not correlated with age, gender, or tumor differentiation. CONCLUSION:This report indicates increased expression of WRAP53 in ESCC and that WRAP53 overexpression is correlated with tumor progression. WRAP53 may play a significant role in ESCC; accordingly, WRAP53 could be a useful biomarker for ESCC

Effect of Structural Design on the Pore Structure, Water Resistance, and Mechanical Properties of Engineered Wood/Bamboo Laminated Composites

An important principle in rational manufacturing design is matching the properties of composites to their intended uses. Herein, six laminated composites (LCs) were manufactured using fibrous moso bamboo and poplar veneer units, and their pore structure, water resistance, and mechanical properties were evaluated. The LC density (640&ndash;1290 kg/m3) increased significantly with increasing bamboo veneer unit content. The LC surface texture and roughness depended on the density and type of surface layer. With increasing LC density, the water absorption rate (WAR), width swelling rate (WSR), and thickness swelling rate (TSR) decreased exponentially and the mechanical properties increased linearly. This behavior was closely related to the changes in pore structure caused by density. Notably, the water resistance and mechanical properties of the LCs with densities higher than 910 kg/m3 were superior to the highest levels specified in GB/T 20241&ndash;2006 for &lsquo;&lsquo;laminated veneer lumber&rsquo;&rsquo; and GB/T 30364&ndash;2013 for &ldquo;bamboo scrimber flooring&rdquo;. Thus, these engineered materials are promising for outdoor structures and flooring

A Novel Bioassay for the Activity Determination of Therapeutic Human Brain Natriuretic Peptide (BNP)

<div><h3>Background</h3><p>Recombinant human brain natriuretic peptide (rhBNP) is an important peptide-based therapeutic drug indicated for the treatment of acute heart failure. Accurate determination of the potency of therapeutic rhBNP is crucial for the safety and efficacy of the drug. The current bioassay involves use of rabbit aortic strips, with experiments being complicated and time-consuming and markedly variable in results. Animal-less methods with better precision and accuracy should be explored. We have therefore developed an alternative cell-based assay, which relies on the ability of BNP to induce cGMP production in HEK293 cells expressing BNP receptor guanylyl cyclase-A.</p> <h3>Methodology/Principal Findings</h3><p>An alternative assay based on the measurement of BNP-induced cGMP production was developed. Specifically, the bioassay employs cells engineered to express BNP receptor guanylyl cyclase-A (GCA). Upon rhBNP stimulation, the levels of the second messager cGMP in these cells drastically increased and subsequently secreted into culture supernatants. The quantity of cGMP, which corresponds to the rhBNP activity, was determined using a competitive ELISA developed by us. Compared with the traditional assay, the novel cell-based assay demonstrated better reproducibility and precision.</p> <h3>Conclusion/Significance</h3><p>The optimized cell-based assay is much simpler, more rapid and precise compared with the traditional assay using animal tissues. To our knowledge, this is the first report on a novel and viable alternative assay for rhBNP potency analysis.</p> </div

Stable transfection and colony selection.

<p>GCA expression in 293GCAC3. The expression of DDK-tagged GCA in 293GCAC3 and parental HEK293 were analyzed by indirect immunofluorescence staining and flow cytometry. The results indicated that almost all of the 293GCAC3 cells over-expressed DDK-tagged GCA.</p

Optimized parameters for the 293GCAC3-based bioassay.

<p>Various parameters of the assay were optimized, including cell number, cell culture time, cGMP-HRP dilution rate and so on as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049934#pone-0049934-t001" target="_blank">Table 1</a>.</p

Immunohistochemical detection of WRAP53 protein expression in esophageal carcinoma and in adjacent non-neoplastic esophageal mucosa.

<p>(a) WRAP53 protein is visualized by yellow or brownish yellow staining in ESCC tissues. (b) Nuclear WRAP53 expression in ESCC tissues and weak or absent WRAP53 expression in the adjacent muscularis. (c) Strong expression of WRAP53 protein in poorly differentiated ESCC tissue. (d) and (e) Positive expression of WRAP53 in the well-differentiated ESCC tissue. (f) Negative WRAP53 expression in ESCC tissue. (g) and (h) Expression of WRAP53 is weak in adjacent non-neoplastic esophageal mucosa and is limited in basal and/or suprabasal layer cells. Scale bar  = 25 micron in c and 100 micron in all other figures.</p

mRNA and protein expression of WRAP53 in ESCC and non-neoplastic tissues.

<p>(<b>a</b>) Relative expression of WRAP53 mRNA in ESCC tissues and non-neoplastic mucosa. <i>GADPH</i> was used as an internal control gene in the qRT-PCR. (<b>b</b>) Western blot analysis of WRAP53 protein expression in esophageal carcinoma tissues and non-neoplastic mucosa tissues. Representative blots are shown for the 75-kDa WRAP53 protein. The upper panel is representative of two paired ESCC tissues (marked “T”) and their corresponding non-neoplastic esophageal mucosa tissues (marked “N”); β-actin was used as a control. (<b>c</b>) Densitometric values were determined by normalization to β-actin protein levels. **p<0.01.</p

Comparison between 293GCAC3-based test and rabbit aortic strips test (RAST).

<p>RhBNP references were tested by both 293GCAC3-based assay and RAST. Various items including curve fitness, intra-assay RSD, inter-assay RSD, 95% CI of EC₅₀, and linear detection range were compared between the two assays. The 293GCAC3-assay is better than RAST assay in curve fitness, intra- and inter-assay variability.</p

Test of the stability of the cell lines.

<p>293GCAC3 cells at three different stages (passage # 5, 18, 65) were tested in optimized condition, which involved 3 replicates for each dose. EC₅₀, mean, SD (standard deviation) and RSD (relative standard deviation) values of them were listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049934#pone-0049934-t003" target="_blank">Table 3</a>. No significant difference existed among the EC₅₀ means of three stages (One-way analysis of variance, p = 0.3788).</p

Transfection and detection of the response to rhBNP.

<p><b>Panel A.</b> GCA over-expression in transiently transfected cells. Western blot was used to detect the expression of GCA. The primary antibodies are GCA antibody and DDK antibody; GAPDH serves as internal control. <b>Panel B.</b> HEK293 cells were transiently transfected with pCMV6-ENTRY-GCA and stimulated with different concentrations of rhBNP for 2 hours. Then cGMP was quantified by competitive ELISA. Each point represents the mean of 3 replicates.</p