Expansion and evolutionary patterns of GDSL-type esterases/lipases in Rosaceae genomes.

GDSL-type esterase/lipase (GELP) is mainly characterized by a conserved GDSL domain at N terminus, and is widely found in all living species, both prokaryotes and eukaryotes. GELP gene family consists of a wide range of members playing important roles in plant physiological processes, such as development, stress responses, and functional divergences. In our study, 597 GELP genes were identified from six Rosaceae genomes (i.e., Fragaria vesca, Prunus persica, Prunus avium, Prunus mume, Pyrus bretschneideri, and Malus domestica) by a comprehensive analysis. All GELP genes were further divided into ten subfamilies based on phylogenetic tree analysis. Subfamily D and subfamily E are the two largest subfamilies. Microcollinearity analysis suggested that WGD/segmental events contribute to the expansion of the GELP gene family in M. domestica and P. bretschneideri compared to F. vesca, P. persica, P. avium, and P. mume. Some PbGELPs were expressed during the fruit development of P. bretschneideri and pollen tubes, indicating their activity in these tissues. The expression divergence of PbGELP duplication gene pairs suggests that many mutations were allowed during evolution, although the structure of GELP genes was highly conserved. The current study results provided the feasibility to understand the expansion and evolution patterns of GELP in Rosaceae genomes, and highlight the function during P. bretschneideri fruits and pollen tubes development.

DqsIR quorum sensing-mediated gene regulation of the extremophilic bacterium Deinococcus radiodurans in response to oxidative stress.

Here, we show that AHLs can be employed by Deinococcus radiodurans, which belongs to the unique phylum Deinococcus-Thermus and is known for its cellular resistance to environmental stresses. An AHL-mediated quorum-sensing system (DqsI/DqsR) was identified in D. radiodurans. We found that under non-stress conditions, the AHL level was "shielded" by quorum quenching enzymes, whereas AHLs accumulated when D. radiodurans was exposed to oxidative stress. Upon exposure to H2 O2 , AHL synthetic enzymes (DqsI) were immediately induced, while the expression of quorum-quenching enzymes began to increase approximately 30 min after exposure to H2 O2 , as shown by time-course analyses of gene expression. Both dqsI mutant (DMDqsI) and dqsR mutant (MDqsR) were more sensitive to oxidative stress compared with the wild-type strain. Exogenous AHLs (5 µM) could completely restore the survival fraction of DMDqsI under oxidative stress. RNA-seq analysis showed that a number of genes involved in stress-response, cellular cleansing, and DNA repair had altered transcriptional levels in MDqsR. The DqsR, acting as a regulator of quorum sensing, controls gene expression along with AHLs. Hence, the DqsIR-mediated quorum sensing that mediates gene regulation is an adaptive strategy for D. radiodurans in response to oxidative stresses and is conserved in the extremophilic Deinococcus bacteria.

Autoinducer-2 signaling is involved in regulation of stress-related genes of Deinococcus radiodurans.

Autoinducer-2 (AI-2) serves as a quorum-sensing signaling molecule that mediates both intraspecies and interspecies communication among bacteria, and plays critical roles in regulating various bacterial behaviors. In the present study, we investigated the functions of AI-2 signaling in the extremophilic bacterium Deinococcus radiodurans R1 by construction of the LuxS gene disruption mutant, survival phenotype assay and gene transcription assay. The gene mutant (DRΔLuxS), which was unable to produce AI-2, was significantly more sensitive to both gamma radiation and H2O2 compared with the wild-type strain. Addition of the wild-type-derived spent medium into the cell culture of DRΔLuxS fully restored the radioresistance of D. radiodurans. A higher level of reactive oxygen species accumulated in the mutant compared with the wild type under normal or oxidative stress. Quantitative real-time PCR assays showed that transcriptional levels of stress-related proteins, including catalase, extracellular nuclease, Dps-1 and ABC transporters, were decreased in DRΔLuxS, indicating that AI-2 is involved in regulation of stress-related genes of D. radiodurans. Hence, AI-2 signaling may contribute to the extreme resistance of D. radiodurans to radiation and oxidative stresses.

Circulating methylated MINT2 promoter DNA is a potential poor prognostic factor in gastric cancer.

BACKGROUND AND AIM: Aberrant DNA methylation has been shown to be associated with the growth, development, metastasis, and prognosis of tumors. Methylated DNAs may be suitable biomarkers for cancer patients. Here, we investigated whether circulating methylated MINT2 DNAs represent a potential poor prognostic factor in gastric cancer (GC). METHODS: MINT2 methylation was detected by real-time methylation-specific PCR in tumor tissues, pairing preoperative peritoneal lavage fluid (PPLF) and blood from 92 GC patients. The theory meaning and clinical practicality value of MINT2 methylation in different specimens were analyzed. RESULTS: The methylation status of the MINT2 gene was found to be significantly higher in tumor tissues (44.6%, 41/92) than in adjacent normal tissues (3.3%, 3/92). No MINT2 methylation was found in healthy controls, and partial MINT2 methylation was observed in three (6.25%, 3/48) patients with chronic atrophic gastritis. The frequency of MINT2 methylation in pairing PPLF and blood samples from 92 GC patients was 40.2% (37/92) and 39.1% (36/92), respectively. Methylated MINT2 in tumor tissues, pairing PPLF, and blood samples were very approximate. Aberrant MINT2 methylation in tumor tissues and pairing PPLF or blood samples were closely related to peritoneal dissemination, tumor progression, and poor prognosis (all P < 0.0001). CONCLUSIONS: Aberrant MINT2 methylation in PPLF/blood may predict peritoneal micrometastasis for GC patients, which is a potential poor prognostic factor in GC.

Enhanced RegIV expression predicts the intrinsic 5-fluorouracil (5-FU) resistance in advanced gastric cancer.

AIM: RegIV, a member of the Regenerating (REG) gene family, may be a marker for the prediction of resistance to 5-fluorouracil (5-FU)-based chemotherapy. However, the relationship between the intrinsic drug resistance of gastric cancer (GC) cells to 5-FU used alone (single FU) or in multidrug therapeutic regimens (5-FU combinations) and RegIV expression has not been investigated. METHODS: The patient cohort comprised 45 patients with primary GC. The chemoresistance of GC cells to therapeutic regimens consisting of single 5-FU or FU combinations was investigated using the ATP-tumor chemosensitivity assay. The level of RegIV mRNA transcripts was determined by real-time reverse transcriptase-PCR. RegIV expression was evaluated as a novel predictive biomarker for the intrinsic drug resistance of primary GC cells to single 5-FU or 5-FU combinations. RESULTS: Upregulation of RegIV mRNA transcripts was observed in 36 of the 45 tumor specimens and was positively correlated with the invasive depth of the tumor cells (p = 0.000), the clinical stages (p = 0.000) and the in vitro intrinsic drug resistance of primary GC cells to 5-FU (p = 0.000) or 5-FU combinations. CONCLUSION: RegIV mRNA transcript level was strongly associated with the intrinsic resistance of GC cells to single 5-FU or 5-FU combinations, suggesting that RegIV may play an important role in the intrinsic resistance of GC cells to 5-FU and that targeted therapy against the RegIV gene could be applied to overcome 5-FU resistance in the treatment of GC.

Stepwise cumulation of RUNX3 methylation mediated by Helicobacter pylori infection contributes to gastric carcinoma progression.

BACKGROUND: Helicobacter pylori has been recognized as a definite carcinogen for gastric cancer (GC); however, the pathogenesis of H. pylori infection remains unclear. Runt-related transcription factor 3 (RUNX3) is a candidate tumor suppressor gene whose deficiency is causally related to GC. However, in H. pylori infection-associated GC, the role of RUNX3 has not been studied. METHODS: The authors used real-time methylation-specific polymerase chain reaction analysis to determine methylation status of the RUNX3 promoter in a spectrum of gastric lesions, including 220 samples of chronic atrophic gastritis, 196 samples of intestinal metaplasia, 134 samples of gastric adenoma, 102 samples of dysplasia, and 202 samples of GC with paired noncancerous mucosa tissues and corresponding blood specimens. The association of abnormal methylation with precancerous gastric lesions was evaluated along with the association between RUNX3 methylation and H. pylori infection, and the concordance of methylation levels was investigated between serum and tissues. RESULTS: The results indicated that increasing RUNX3 promoter methylation was correlated with distinct stages of GC progression. GC tissues had the highest methylation proportion (75.2%) compared with precancerous gastric lesions, including chronic atrophic gastritis (15.9%), intestinal metaplasia (36.7%), gastric adenoma (41.8%), and dysplasia (54.9%). H. pylori infection, a major risk factor for GC, contributed to the inactivation of RUNX3 in gastric epithelial cells through promoter hypermethylation. The levels of RUNX3 methylation in serum were in significant concordance with the methylation levels observed in GC tissues (P = .887). CONCLUSIONS: The current findings supported RUNX3 methylation as a risk factors for the carcinogenesis of chronic atrophic gastritis with H. pylori infection and indicated that circulating RUNX3 methylation is a valuable biomarker for the detection of early GC.

CDH1 methylation in preoperative peritoneal washes is an independent prognostic factor for gastric cancer.

BACKGROUND AND OBJECTIVES: To investigate the clinical value of CDH1 methylation in preoperative peritoneal washes (PPW) from gastric cancer patients. METHODS: CDH1 methylation was detected by real-time methylation specific-PCR in tumor tissues and corresponding PPW from 92 gastric cancer patients, gastric mucosa from 40 chronic gastritis patients and 48 normal persons. RESULTS: CDH1 methylation was found in 75 of 92 (81.5%) gastric cancer tissues, which significantly correlated with size, growth pattern, differentiation, lymphatic invasion, venous invasion, invasion depth, lymph node metastasis, distant metastasis, and TNM stage of tumor (all P < 0.05), but its relationship to age, gender, tumor site, and H. pylori infection was not found (all P > 0.05). The percentage of CDH1 methylation in PPW was 48.9%, of which the Aζ value of ROC curve was 0.8 compared to that in gastric cancer tissues. Kaplan-Meier analysis showed that there was a significant difference in disease-free survival (DFS) between the patients with or without methylated CDH1 in their PPW (χ(2) = 109.64, P < 0.000). Cox regression analysis revealed CDH1 methylation in PPW was an independent risk factor for gastric cancer patients, with a remarkable decrease in DFS after postoperative 30 months. CONCLUSIONS: Methylated CDH1 in PPW predicts poor prognosis for gastric cancer patients.

[Expression of epidermal growth factor receptor in salivary adenoid cystic carcinoma and its role in cancer invasion].

OBJECTIVE: To assess the epidermal growth factor receptor (EGFR) status in salivary adenoid cystic carcinoma and explore its role in cancer invasion. METHODS: Fifty-four patients with pathologically confirmed salivary adenoid cystic carcinoma (SACC) were divided into invasion group and non-invasion group. The EGFR expression was determined by immunohistochemstry (SP staining). The relations between the EGFR expression and the SACC clinical pathological characteristics were analyzed. RESULTS: EGFR were mainly expressed in the cell membrane and cytoplasm in the tissue of SACC. The positive rate of EGFR expression in the tumor tissue was 75.9% (41/54), and EGFR was over-expressed in the cytoplasm. The positive rate of EGFR expression in invasion group was higher than that in the non-invasion group (10.0%, P < 0.05). EGFR expression were related with the SACC T stages, histological types, distant metastasis, lymph node metastasis, and nerve invasion (P < 0.05). CONCLUSIONS: A higher expression of EGFR gene in the cytoplasm may have important effect on the progression of invasive carcinoma. Further investigations are required to develop new strategy in the treatment of salivary adenoid cystic carcinoma.

[Expression and significance of E-cadherin in adenoid cystic carcinoma of salivary glands].

OBJECTIVE: To examine the expression of E-cadherin and the methylation status of CDH1 and explore their clinical significance in salivary adenoid cystic carcinoma (SACC). METHODS: The expression of E-cadherin was detected by the immunohistochemical method. And the methylation of CDH1 gene promoter 5'-CpG island was analyzed by real-time methylation-specific polymerase chain reaction (real-time MSP) in salivary adenoid cystic carcinoma and normal salivary gland tissue respectively. RESULTS: The expression rate of E-cadherin was lower in SACC than that in normal salivary gland tissue (100.0% vs 55.6%, P < 0.05). And the expression of E-cadherin was associated with different histopathological types, T-stage, nerve invasion, lymphatic and distant metastasis (P < 0.05). However, there was no correlation between the expression of E-cadherin and gender, age and tumor location. Partial methylation of CDH1 was detected in 3 of 30 cases with a positive expression of E-cadherin and full methylation of CDH1 in 23 of 24 cases with a negative expression of E-cadherin. There was a negative correlation between the expression of E-cadherin and the methylation of CDH1 in salivary adenoid cystic carcinoma (r = -0.483, P < 0.001). CONCLUSION: The down-regulation of E-cadherin, as modulated by the methylation of CDH1, may contribute to nerve invasion, lymphatic and distant metastasis in SACC. Thus it may be used as a biological indicator of malignancy and prognosis.

Construction of an AI-2 quorum sensing induced heterologous protein expression system in Escherichia coli.

BACKGROUND: The pET expression system based on T7 promoter which is induced by isopropyl-ß-D-1-thiogalactopyranoside (IPTG) is by far the most commonly used system for production of heterogeneous proteins in Escherichia coli. However, this system was limited by obvious drawbacks including the host toxicity and metabolic burden imposed by the presence of IPTG. METHODS: In this study, we incorporated the autoinducer-2 (AI-2) quorum sensing system to realize autoinduction of the pET expression system. The autoinduction expression vector pXWZ1 was constructed by inserting the lsr promoter regions into the pET28a(+) vector. The expression efficiency of the reporter genes gfpuv and lacZ by the pXWZ1 and pET28a(+) vectors were compared. RESULTS: The results showed that the expression levels of the both report genes in the cells transformed with pXWZ1 without any addition of exogenous inducer were higher than that transformed with pET28a(+) vectors by the induction of IPTG. CONCLUSION: This new auto-induction system will exclude the limitations of the IPTG induction including toxic to host and increasing formation of inclusion body and will become a more economical and convenient tool for recombinant protein expression.

Aspirin Positively Contributes to Drosophila Intestinal Homeostasis and Delays Aging through Targeting Imd.

The intestine, a high-turnover tissue, plays a critical role in regulating aging and health in both vertebrates and invertebrates. Maintaining the epithelial barrier function of the intestine by preserving innate immune homeostasis significantly delays aging and prevents mortality. In an effort to explore effective chemicals and materials that can improve intestinal integrity, we performed a nonbiased screen utilizing Drosophila as an animal model. We showed that long-term uptake of aspirin markedly prevented age-onset gut leakage, the over-proliferation of intestinal stem cells, and the dysbiosis of commensal microbiota in fruit flies. Mechanistically, aspirin efficiently downregulated chronic activation of intestinal immune deficiency signaling during aging. Furthermore, our in vivo and in vitro biochemical analyses indicated that aspirin is a negative modulator in control of the K63-linked ubiquitination of Imd. Our findings uncover a novel regulatory mechanism by which aspirin positively modulates intestinal homeostasis, thus delaying aging, in Drosophila.

Role of McbR in the regulation of antibiotic susceptibility in avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes a variety of bacterial infectious diseases known as avian colibacillosis leading to significant economic losses in the poultry industry worldwide and restricting the development of the poultry industry. The development of efflux pumps is one important bacterial antibiotic resistance mechanism. Efflux pumps are capable of extruding a wide range of antibiotics out of the cytoplasm of some bacterial species, including ß-lactams, polymyxins, tetracyclines, fluoroquinolones, aminoglycosides, novobiocin, nalidixic acid, and fosfomycin. In the present study, we constructed the mcbR mutant and the mcbR-overexpressing strain of E. coli strain APECX40 and performed antimicrobial susceptibility testing, antibacterial activity assays, real-time reverse transcription PCR, and electrophoretic mobility shift assays (EMSA) to investigate the molecular regulatory mechanism of McbR on the genes encoding efflux pumps. Our results showed that McbR positively regulates cell susceptibility to 12 antibiotics, including clindamycin, lincomycin, cefotaxime, cefalexin, doxycycline, tetracycline, gentamicin, kanamycin, norfloxacin, ofloxacin, erythromycin, and rifampicin by activating the transcription of acrAB, acrD, emrD, and mdtD (P < 0.01). Additionally, EMSA indicated that McbR specifically binds to the promoter regions of acrAB, acrD, acrR, emrD, and mdtD. This study suggests that, in APECX40, McbR plays an important role in the regulation of bacterial susceptibility by directly activating the transcription of efflux pumps genes.

BamA is a pivotal protein in cell envelope synthesis and cell division in Deinococcus radiodurans.

The beta-barrel assembly machinery (BAM) is an indispensable complex for protein transportation located at the outer membrane of bacteria. BAM is composed of five subunits (BamA-E) in the model bacterium Escherichia coli. DR_0379 is a BamA homolog in Deinococcus radiodurans, but the other subunits have not been detected in this species. In the present study, deletion of bamA resulted in decreased growth rate and altered morphology of D. radiodurans. ΔbamA cells underwent abnormal cell division, leading to aggregated bacteria of diverse size and shape, and the cell envelope was detached from the cell surface, resulting in reduced resistance to high ionic strength. Oxidative stress resistance was significantly enhanced in the mutant, which may be attributed to increased manganese ion concentration and Mn/Fe ratio. Numerous proteins were released into the medium from ΔbamA cells, including surface layer (S-layer) proteins and various transporters located in the periplasm and outer membrane. These results indicate that BamA affects the synthesis and assembly of the outer membrane and S-layer, and thereby influences material transport and cell division. The findings highlight the special functions of BamA in D. radiodurans, and promote our understanding of the multi-layer structure of the D. radiodurans cell envelope.

Optimization of quercetin extraction method in Dendrobium officinale by response surface methodology.

Ultrasonic-assisted extraction of quercetin from Dendrobium officinale was optimized by response surface methodology (RSM) using high-performance liquid chromatography as a separative method. Based on single-factor experiments and two-level factorial analysis, the ethanol concentration, solid-to-liquid ratio and ultrasonic power were selected as significant response factors. The amount of quercetin that we extracted from Dendrobium officinale was 2.506-2.594 µg/g under the extraction conditions, which showed that optimization could improve the extration rate of quercetin from Dendrobium officinale. Quercetin was extracted and detected within 12 consecutive months after the germination of Dendrobium officinale by optimizing the extraction process to analyze the accumulation of quercetin. The UV-B exposure experiments showed that the Dendrobium officinale leaves have different responses to low- and high-dose UV light. The results showed that the quercetin content in Dendrobium officinale could be changed by UV-B radiation, and the response of distinct tissue parts to varying intensities of UV-B radiation was different.

Rapid, quantitative and ultra-sensitive detection of cancer biomarker by a SERRS-based lateral flow immunoassay using bovine serum albumin coated Au nanorods.

Early diagnosis of cancer biomarkers is the key to guiding treatments and improving the survival rate of patients. Herein, we report a novel surface-enhanced resonance Raman scattering (SERRS)-based lateral flow immunoassay (LFIA) for quantitative and ultra-sensitive analysis of alpha-fetoprotein (AFP). Gold nanorods (AuNRs) were fabricated to be in resonance with 785 nm laser excitation, that is, the excitation level that can maximize SERRS activity. The AuNRs were modified with 5,5'-dithiobis(2-nitrobenzoic acid), bovine serum albumin (BSA), and AFP detection antibody successively as the SERRS nanotags for the LFIA system. Modification of the BSA layer guaranteed good stability and biocompatibility of the SERRS nanotags in complex samples. The SERRS-LFIA strip for AFP detection showed a low detection limit of 9.2 pg mL-1 and a broad detection range from 10 pg mL-1 to 500 ng mL-1. By comparison, the detection limit of the proposed assay is about 100 and 10 times lower than those of the Au nanoparticle-based SERS-strip and conventional enzyme-linked immunosorbent assay, respectively. Moreover, the potential clinical applications of the assay were evaluated by detecting 10 actual serum samples. Results showed 100% accuracy based on the clinical tests.

A tamB homolog is involved in maintenance of cell envelope integrity and stress resistance of Deinococcus radiodurans.

The translocation and assembly module (TAM) in bacteria consists of TamA and TamB that form a complex to control the transport and secretion of outer membrane proteins. Herein, we demonstrated that the DR_1462-DR_1461-DR_1460 gene loci on chromosome 1 of Deinococcus radiodurans, which lacks tamA homologs, is a tamB homolog (DR_146T) with two tamB motifs and a DUF490 motif. Mutation of DR_146T resulted in cell envelope peeling and a decrease in resistance to shear stress and osmotic pressure, as well as an increase in oxidative stress resistance, consistent with the phenotype of a surface layer (S-layer) protein SlpA (DR_2577) mutant, demonstrating the involvement of DR_146T in maintenance of cell envelope integrity. The 123 kDa SlpA was absent and only its fragments were present in the cell envelope of DR_146T mutant, suggesting that DR_146T might be involved in maintenance of the S-layer. A mutant lacking the DUF490 motif displayed only a slight alteration in phenotype compared with the wild type, suggesting DUF490 is less important than tamB motif for the function of DR_146T. These findings enhance our understanding of the properties of the multilayered envelope in extremophilic D. radiodurans, as well as the diversity and functions of TAMs in bacteria.

Deinococcus radiodurans Toxin-Antitoxin MazEF-dr Mediates Cell Death in Response to DNA Damage Stress.

Here we identified a functional MazEF-dr system in the exceptionally stress-resistant bacterium D. radiodurans. We showed that overexpression of the toxin MazF-dr inhibited the growth of Escherichia coli. The toxic effect of MazF-dr was due to its sequence-specific endoribonuclease activity on RNAs containing a consensus 5'ACA3', and it could be neutralized by MazE-dr. The MazF-dr showed a special cleavage preference for the nucleotide present before the ACA sequence with the order by U>A>G>C. MazEF-dr mediated the death of D. radiodurans cells under sub-lethal dose of stresses. The characteristics of programmed cell death (PCD) including membrane blebbing, loss of membrane integrity and cytoplasm condensation occurred in a fraction of the wild-type population at sub-lethal concentration of the DNA damaging agent mitomycin C (MMC); however, a MazEF-dr mutation relieved the cell death, suggesting that MazEF-dr mediated cell death through its endoribonuclease activity in response to DNA damage stress. The MazEF-dr-mediated cell death of a fraction of the population might serve as a survival strategy for the remaining population of D. radiodurans under DNA damage stress.

Biosynthesis of gold nanoparticles by the extreme bacterium Deinococcus radiodurans and an evaluation of their antibacterial properties.

Deinococcus radiodurans is an extreme bacterium known for its high resistance to stresses including radiation and oxidants. The ability of D. radiodurans to reduce Au(III) and biosynthesize gold nanoparticles (AuNPs) was investigated in aqueous solution by ultraviolet and visible (UV/Vis) absorption spectroscopy, electron microscopy, X-ray diffraction (XRD), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). D. radiodurans efficiently synthesized AuNPs from 1 mM Au(III) solution in 8 h. The AuNPs were of spherical, triangular and irregular shapes with an average size of 43.75 nm and a polydispersity index of 0.23 as measured by DLS. AuNPs were distributed in the cell envelope, across the cytosol and in the extracellular space. XRD analysis confirmed the crystallite nature of the AuNPs from the cell supernatant. Data from the FTIR and XPS showed that upon binding to proteins or compounds through interactions with carboxyl, amine, phospho and hydroxyl groups, Au(III) may be reduced to Au(I), and further reduced to Au(0) with the capping groups to stabilize the AuNPs. Biosynthesis of AuNPs was optimized with respect to the initial concentration of gold salt, bacterial growth period, solution pH and temperature. The purified AuNPs exhibited significant antibacterial activity against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria by damaging their cytoplasmic membrane. Therefore, the extreme bacterium D. radiodurans can be used as a novel bacterial candidate for efficient biosynthesis of AuNPs, which exhibited potential in biomedical application as an antibacterial agent.

[Screening of specific miRNA in early gastric cancer].

OBJECTIVE: To examine the micro-RNA (mirna) expression profile in tissues of early gastric cancer and to screen the specific mirna associated with gastric cancer. METHODS: Gene chip technology was used to detect the expression of mirna in early gastric cancer tissues and adjacent normal tissues. RESULTS: Compared to adjacent normal tissues, a total of 36 mirnas were down-regulated, such as mir-9-1, mir-103 and mir-141, while 12 mirnas were up-regulated, such as mir-196a, mir-142-3p and mir-25, etc. CONCLUSION: Abnormal mirna expression level in early gastric cancer tissues may be associated to the development of gastric cancer.

Methylated TIMP-3 DNA in body fluids is an independent prognostic factor for gastric cancer.

CONTEXT: Fluid methylated DNA may be a suitable biomarker for cancer patients. OBJECTIVE: To investigate whether circulating methylated tissue inhibitor of metalloproteinase 3 (TIMP-3) DNA in body fluids is a useful prognostic biomarker in gastric cancer (GC). DESIGN: TIMP-3 methylation was detected by real-time methylation-specific polymerase chain reaction in tumor tissues, paired preoperative peritoneal washes (PPWs), and paired serum samples from 92 GC patients. RESULTS: The frequency of TIMP-3 methylation was significantly elevated in GC tissues (63.04%; 58 of 92) compared with that in paired adjacent normal tissue (4.3%; 4 of 92) (P < .001). TIMP-3 methylation correlated closely with peritoneal metastasis and TNM stage (all P < .001). The frequency of TIMP-3 methylation in preoperative peritoneal washes and serum samples was 53.3% (49 of 92) and 58.7% (54 of 92), respectively. The Aζ values of the receiver operator characteristic curve for methylated TIMP-3 were 0.966 and 0.922 for serum and preoperative peritoneal washes, respectively, compared with those in GC tissues. The patients with elevated methylated TIMP-3 levels in body fluids had poorer disease-free survival rates than those without (all P < .001). Cox regression analysis showed that detection of methylated TIMP-3 DNA in body fluids was an independent risk factor for GC patients, with a remarkable decrease in disease-free survival 30 months after surgical resection of the gastric tumor. CONCLUSION: Presence of methylated TIMP-3 DNA in body fluids is a useful biomarker for predicting the progression and prognosis of GC patients.