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Linusorbs (LOs), significantly influence oil quality and sensory properties of flaxseed oil. Trp-containing LOs exhibit distinct oxidative behavior when γ-tocopherol (γ-T) is present. Polar fractions of crude flaxseed oil were stripped via silica absorption, and reintroduced (LO and γ-T) separately into the oil matrix to investigate their interaction during storage. Compared with crude oil, LOs account for 18.49% reduction of p-anisidine value, while LOs with γ-T contributed to most of the endogenous antioxidant effect in crude oil. γ-T was found to suppress oxidation of Trp-containing LO at early stage (Met form), while facilitate oxidation while at their mid-stage (MetO form, Methionine sulfoxide). In vitro oxidation shows that CLD more likely cleaved into peptide fragments, while few products retain intact ring structures. LC-MS/MS analysis and silicon simulation revealed proximity between MetO and Trp residues, facilitating inter- or intra-molecular reactions and ring structure rupture. Remarkably, the presence of γ-T facilitate these phenomena., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

As a surveillance mechanism, the activated spindle assembly checkpoint (SAC) potently inhibits the E3 ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) to ensure accurate chromosome segregation. Although the protein phosphatase 2A (PP2A) has been proposed to be both, directly and indirectly, involved in spindle assembly checkpoint inactivation in mammalian cells, whether it is similarly operating in the fission yeast Schizosaccharomycer pombe has never been demonstrated. Here, we investigated whether fission yeast PP2A is involved in SAC silencing by following the rate of cyclin B (Cdc13) destruction at SPBs during the recovery phase in nda3-KM311 cells released from the inhibition of APC/C by the activated spindle checkpoint. The timing of the SAC inactivation is only slightly delayed when two B56 regulatory subunits (Par1 and Par2) of fission yeast PP2A are absent. Overproduction of individual PP2A subunits either globally in the nda3-KM311 arrest-and-release system or locally in the synthetic spindle checkpoint activation system only slightly suppresses the SAC silencing defects in PP1 deletion (dis2Δ) cells. Our study thus demonstrates that the fission yeast PP2A is not a key regulator actively involved in SAC inactivation., (© 2022 Federation of American Societies for Experimental Biology.)

Rationale & Objective: Coronavirus disease 2019 (COVID-19) disproportionately affects people with chronic diseases such as chronic kidney disease (CKD). We assessed the incidence and outcomes of COVID-19 in people with CKD., Study Design: Systematic review and meta-analysis by searching MEDLINE, EMBASE, and PubMed through February 2021., Setting & Study Populations: People with CKD with or without COVID-19., Selection Criteria for Studies: Cohort and case-control studies., Data Extraction: Incidences of COVID-19, death, respiratory failure, dyspnea, recovery, intensive care admission, hospital admission, need for supplemental oxygen, hospital discharge, sepsis, short-term dialysis, acute kidney injury, and fatigue., Analytical Approach: Random-effects meta-analysis and evidence certainty adjudicated using an adapted version of GRADE (Grading of Recommendations Assessment, Development and Evaluation)., Results: 348 studies (382,407 participants with COVID-19 and CKD; 1,139,979 total participants with CKD) were included. Based on low-certainty evidence, the incidence of COVID-19 was higher in people with CKD treated with dialysis (105 per 10,000 person-weeks; 95% CI, 91-120; 95% prediction interval [PrI], 25-235; 59 studies; 468,233 participants) than in those with CKD not requiring kidney replacement therapy (16 per 10,000 person-weeks; 95% CI, 4-33; 95% PrI, 0-92; 5 studies; 70,683 participants) or in kidney or pancreas/kidney transplant recipients (23 per 10,000 person-weeks; 95% CI, 18-30; 95% PrI, 2-67; 29 studies; 120,281 participants). Based on low-certainty evidence, the incidence of death in people with CKD and COVID-19 was 32 per 1,000 person-weeks (95% CI, 30-35; 95% PrI, 4-81; 229 studies; 70,922 participants), which may be higher than in people with CKD without COVID-19 (incidence rate ratio, 10.26; 95% CI, 6.78-15.53; 95% PrI, 2.62-40.15; 4 studies; 18,347 participants)., Limitations: Analyses were generally based on low-certainty evidence. Few studies reported outcomes in people with CKD without COVID-19 to calculate the excess risk attributable to COVID-19, and potential confounders were not adjusted for in most studies., Conclusions: The incidence of COVID-19 may be higher in people receiving maintenance dialysis than in those with CKD not requiring kidney replacement therapy or those who are kidney or pancreas/kidney transplant recipients. People with CKD and COVID-19 may have a higher incidence of death than people with CKD without COVID-19., (Copyright © 2021 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Using genetic mutations to study protein functions in vivo is a central paradigm of modern biology. Single-domain camelid antibodies generated against GFP have been engineered as nanobodies or GFP-binding proteins (GBPs) that can bind GFP as well as some GFP variants with high affinity and selectivity. In this study, we have used GBP-mCherry fusion protein as a tool to perturb the natural functions of a few kinetochore proteins in the fission yeast Schizosaccharomyces pombe. We found that cells simultaneously expressing GBP-mCherry and the GFP-tagged inner kinetochore protein Cnp1 are sensitive to high temperature and microtubule drug thiabendazole (TBZ). In addition, kinetochore-targeted GBP-mCherry by a few major kinetochore proteins with GFP tags causes defects in faithful chromosome segregation. Thus, this setting compromises the functions of kinetochores and renders cells to behave like conditional mutants. Our study highlights the potential of using GBP as a general tool to perturb the function of some GFP-tagged proteins in vivo with the objective of understanding their functional relevance to certain physiological processes, not only in yeasts, but also potentially in other model systems., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Background: Acute myelogenous leukemia (AML) is a heterogeneous disease with recurrent gene mutations and variations in disease-associated gene expression, which may be useful for prognostic prediction. Methods: RNA matrix and clinical data of AML were downloaded from GEO, TCGA, and TARGET databases. Prognostic metabolic genes were identified by LASSO analysis to establish a metabolic model. Prognostic accuracy of the model was quantified by time-dependent receiver operating characteristic curves and the area under the curve (AUC). Survival analysis was performed by log-rank tests. Enriched pathways in different metabolic risk statuses were evaluated by gene set enrichment analyses (GSEA). Results: We identified nine genes to construct a prognostic model of shorter survival in the high-risk vs. low-risk group. The prognostic model showed good predictive efficacy, with AUCs for 5-year overall survival of 0.78 (0.73-0.83), 0.76 (0.62-0.89), and 0.66 (0.57-0.75) in the training, adult external, and pediatric external cohorts, respectively. Multivariable analysis demonstrated that the metabolic signature had independent prognostic value with hazard ratios of 2.75 (2.06-3.66), 1.89 (1.09-3.29), and 1.96 (1.00-3.84) in the training, adult external, and pediatric external cohorts, respectively. Combining metabolic signatures and classic prognostic factors improved 5-year overall survival prediction compared to the prediction by classic prognostic factors ( p < 0.05). GSEA revealed that most pathways were metabolism-related, indicating potential mechanisms. Conclusion: We identified dysregulated metabolic features in AML and constructed a prognostic model to predict the survival of patients with AML., (Copyright © 2020 Wang, Hu, Li, Nie, Chen, Cai, Shu, Deng, Xu and Liang.)

Oxidative stress and hypoxia are two opposite microenvironments involved in HCC metastasis. Thioredoxin (TXN) and hypoxia-inducible factor 2α (HIF-2α) are typical proteins involved in these two different microenvironments, respectively. How these two factors interact to influence the fate on tumor cells remains unknown. Hypoxia facilitated HCC cells withstood oxidative stress and eventually promoted HCC cells metastasis, in which TXN and HIF-2α were mostly involved. Upregulation of TXN/HIF-2α correlated with poor HCC prognosis and promoted HCC metastasis both in vitro and in vivo. Epithelial-mesenchymal transition (EMT) process was involved in TXN/HIF-2α-enhanced invasiveness of HCC cells. Additionally, the stability and activity of HIF-2α were precisely regulated by TXN via SUMOylation and acetylation, which contributed to HCC metastasis. Our data revealed that the redox protein TXN and HIF-2α are both associated with HCC metastasis, and the fine regulation of TXN on HIF-2α contributes essentially during the process of metastasis. Our study provides new insight into the interaction mechanism between hypoxia and oxidative stress and implies potential therapeutic benefits by targeting both TXN and HIF-2α in the treatment of HCC metastasis., (© 2020 Federation of American Societies for Experimental Biology.)

Background: The expression of the membrane receptor protein GFRA1 is frequently upregulated in many cancers, which can promote cancer development by activating the classic RET-RAS-ERK and RET-RAS-PI3K-AKT pathways. Several therapeutic anti-GFRA1 antibody-drug conjugates are under development. Demethylation (or hypomethylation) of GFRA1 CpG islands (dmGFRA1) is associated with increased gene expression and metastasis risk of gastric cancer. However, it is unknown whether dmGFRA1 affects the metastasis of other cancers, including colon cancer (CC)., Aim: To study whether dmGFRA1 is a driver for CC metastasis and GFRA1 is a potential therapeutic target., Methods: CC and paired surgical margin tissue samples from 144 inpatients and normal colon mucosal biopsies from 21 noncancer patients were included in this study. The methylation status of GFRA1 islands was determined by MethyLight and denaturing high-performance liquid chromatography and bisulfite-sequencing. Kaplan-Meier analysis was used to explore the effect of dmGFRA1 on the survival of CC patients. Impacts of GFRA1 on CC cell proliferation and migration were evaluated by a battery of biological assays in vitro and in vivo . The phosphorylation of AKT and ERK proteins was examined by Western blot analysis., Results: The proportion of dmGFRA1 in CC, surgical margin, and normal colon tissues by MethyLight was 68.4%, 73.4%, and 35.9% (median; nonparametric test, P = 0.001 and < 0.001), respectively. Using the median value of dmGFRA1 peak area proportion as the cutoff, the proportion of dmGFRA1-high samples was much higher in poorly differentiated CC samples than in moderately or well-differentiated samples (92.3%% vs 55.8%, Chi-square test, P = 0.002) and significantly higher in CC samples with distant metastasis than in samples without (77.8% vs 46.0%, P = 0.021). The overall survival of patients with dmGFRA1-low CC was significantly longer than that of patients with dmGFRA1-high CC (adjusted hazard ratio = 0.49, 95% confidence interval: 0.24-0.98), especially for 89 CC patients with metastatic CC (adjusted hazard ratio = 0.41, 95% confidence interval: 0.18-0.91). These data were confirmed by the mining results from TCGA datasets. Furthermore, GFRA1 overexpression significantly promoted the proliferation/invasion of RKO and HCT116 cells and the growth of RKO cells in nude mice but did not affect their migration. GFRA1 overexpression markedly increased the phosphorylation levels of AKT and ERK proteins, two key molecules in two classic GFRA1 downstream pathways., Conclusion: GFRA1 expression is frequently reactivated by DNA demethylation in CC tissues and is significantly associated with a poor prognosis in patients with CC, especially those with metastatic CC. GFRA1 can promote the proliferation/growth of CC cells, probably by the activation of AKT and ERK pathways. GFRA1 might be a therapeutic target for CC patients, especially those with metastatic potential., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

Background: Telomeres have long been found to be involved in cancer development, while little was known about the dynamic changes of telomere length in carcinogenesis process. Methods: The present study longitudinally investigated telomere alterations of cell-free DNA (cfDNA) in 86 gastric cancer (GC) subjects recruited through a 16-year prospective cohort with 2-4 serums collected before each GC-diagnosis from baseline and three follow-up time-points (a total of 276 samples). As the control, 86 individual-matched cancer-free subjects were enrolled with 276 serums from the matched calendar year. Results: In the 73 pairs of baseline serums from GC and control subjects, shortened telomeres showed increased subsequent GC risk [odds ratio (OR) = 9.17, 95% CI: 2.72-31.25 for 1 unit shortening]. In each baseline gastric lesion category, higher risks of GC progression were also found with shortened cfDNA telomeres; ORs per 1 unit shortening were 6.99 (95% CI: 1.63-30.30) for mild gastric lesions, 6.06 (95% CI: 1.89-19.61) for intestinal metaplasia and 15.63 (95% CI: 1.91-125.00) for dysplasia. With all measurements from baseline and follow-up time-points, shortened telomeres also showed significant association with GC risk (OR = 7.37, 95% CI: 2.06-26.32 for 1 unit shortening). In temporal trend analysis, shortened telomeres were found in GC subjects compared to corresponding controls more than 3 years ahead of GC-diagnosis (most P < 0.05), while no significant difference was found between two groups within 3 years approaching to GC-diagnosis. Conclusion: Our findings suggest that telomere shortening may be associated with gastric carcinogenesis, which supports further etiological study and potential biomarker for risk stratification., (Copyright © 2019 Shi, Zhang, Zhang, Ma, Zhou, Li, Liu, Li, Deng, You and Pan.)

Objective: To analyze the clinical effect of spinal cord decompression and lavage therapy on chronic cervical spinal cord injury and explore the possible mechanism., Methods: Fifty-seven patients with chronic cervical spinal cord injury treated in our hospital from January, 2008 to January, 2015 were enrolled, including 17 with multilevel cervical disc herniation, 25 with long segmental ossification of the posterior longitudinal ligament, 13 with hypertrophy or calcification of neck ligamentum flavum, and 2 with old cervical fractures. Open-door spinal canal laminoplasty via a posterior approach and decompression in simple extramedullary decompression was performed in 31 cases (group A), and open-door spinal cord incision decompression via a posterior approach, saline irrigation, and spinal canal laminoplasty in intramedullary decompression was performed in 26 cases (group B). The pre-operative cerebrospinal fluid in group B patients was collected to examine the inflammatory factors. All the patients were followed up and evaluated for pre- and postoperative JOA scores to calculate the improvement rate with regular examinations by X-ray, CT or MRI., Results: Imaging examinations 2 weeks after the operation showed obvious relief of the primary lesion in both groups, and the improvement of high signals was better in group B than in group A. The mean improvement rate at 12 months after the operation was 52.33% in group A and 61.52% in group B (P<0.05), and the mean JOA score was significantly higher in group B than in group A (14.80∓1.51 vs 13.58∓0.56; P<0.05). Cerebrospinal fluid leakage occurred in 3 cases, epidural hematoma in 2 cases, internal fixation loosening in 1 case in group A; portal shaft fracture and internal fixation loosening occurred in 1 case in group B. Postoperative recovery time was shorter in group B and entered the platform phase in 3 months. The inflammatory factors IFN-γ, IL-17F, IL-6 and sCD40L were all significantly higher than the normal levels after spinal cord injury, and the increment of IL-6 was the most conspicuous (P<0.05)., Conclusion: Intramedullary and extramedullary decompression can achieve better outcomes than extramedullary decompression in patients with chronic cervical cord injury. This may be related not only to relieving adhesions and secondary compression by cutting the dura under the microscope, but also to removal of local inflammatory factors.

Objective: To study the association between the AKAP12 promoter methylation and recurrence of hepatocellular carcinoma. Methods: A total of 142 primary liver cancer patients underwent surgery in department of Hepatobiliary surgery in Peking University Cancer Hospital from 2003 to 2009 were selected as subjects in the survey; with the inclusion criteria as hepatocellular carcinoma, no cancer cells were observed in the surgical margin(SM) samples. All patients had neither lymph nor distant metastasis at the time of surgery, and receiving complete follow-up data for at least 3 years. By the end of May 2014, a total of 75 patients had relapsed of whom 71 died and there were no lost. All samples were acquired from the frozen surgical tissues. Genomic DNA was extracted using phenol/chloroform method and performed bisulfite modification following with polymerase chain reaction (PCR). AKAP12 methylation in hepatoma and the corresponding SM samples from 142 patients was determined by denature high-performance liquid chromatography (DHPLC) and bisulfite clone sequencing. Kaplan-Meier and Cox proportion hazard regression model were used to identify the factors related to the survival time. Results: In 142 cases, 125 patients (88.0%) were male and 17 (12.0%) cases were female. The median age was 52.5 years, ranging from 34 years to 76 years. AKAP12 methylation-positive rate was significantly higher in hepatomas than SMs (54.9% vs. 10.2%, P< 0.001). Patients with AKAP12 methylation-positive had less risk of the recurrence ( HR= 0.62, 95% CI : 0.39-0.99); with tumor diameter more than 5 cm ( HR= 1.53, 95 %CI: 1.00-2.50),portal vein invasion( HR= 4.53, 95% CI: 2.69-7.64) increased the recurrence risk. Moreover, portal vein invasion had a higher risk of death ( HR= 2.98, 95% CI: 1.73-4.98). Conclusion: There was significant association between AKAP12 DNA methylation and low risk of recurrence and long progression-free survival of hepatocellular carcinoma patients.

Objective: To investigate the effect of Shangbai ointment in relieving pain in patients with acute ankle joint lateral collateral ligament injury., Methods: Fifty patients with unilateral ankle injury were randomized into Shangbai ointment treatment group and control group (n=25). The Visual Analog Scale (VAS) pain scores, ankle joint circumference, Kofoed score and self-rated lower limb function were recorded before and at 3, 7, and 14 days after the treatment., Results: The baseline data were comparable between the two groups. The VAS pain score and swelling elimination in the treatment group were better than those in the control group at 3, 7, and 14 days after treatment; the Kofoed ankle score was higher in the treatment group than in the control group at 7 and 14 days after treatment. The patients in the treatment also reported better self-rated lower limb function than those in the control group at 7 and 14 days after the treatment., Conclusion: Shangbai ointment treatment can rapidly relieve pain in patients with acute ankle joint lateral collateral ligament injury and improve the functional scores of the joint.

Purpose: To investigate Helicobacter pylori (H.pylori) associated genome-wide aberrant methylation patterns in gastric mucosa and blood leukocyte DNA, a population-based study was conducted in Linqu County., Results: A total of 3000 and 386 CpGs were differentially methylated after successful H.pylori eradication in gastric mucosa and blood leukocyte DNA respectively, and 17 were the same alteration trend in the both tissues. The differentially methylated CpGs were located more frequently in promoters or CpG islands for gastric mucosa and gene body or open sea for blood leukocyte DNA. In eradicated gastric mucosa, the hypermethylated CpGs were enriched across inflammatory pathways, while the hypomethylated CpGs in tube morphogenesis, development and so on. The final validation found lower SPI1, PRIC285 and S1PR4 methylation levels in H.pylori positive subjects by case-control comparison, and increased methylation levels in H.pylori eradicated gastric mucosa by self-comparison. The Cancer Genome Atlas (TCGA) database analysis suggested that the up-regulation of the three genes by hypomethylation might be associated with gastric carcinogenesis., Experimental Design: Infinium HumanMethylation 450K BeadChip was used to compare methylation profiles prior to and after eradication treatment. The methylation levels of identified candidate differentially methylated genes before and after H.pylori eradication were further validated by two stages (Stage I: self-comparison of 16 subjects before and after anti-H.pylori treatment; Stage II: case-control comparison of 25 H.pylori positive and 25 negative subjects and self-comparison of 50 anti-H.pylori treated subjects)., Conclusions: Novel H.pylori associated aberrant methylated genes were identified across the whole genome both in gastric mucosa and blood leukocyte DNA., Competing Interests: All authors disclosed no potential conflicts.

Aim: To investigate the effect and mechanism of stimulation of the hypothalamic paraventricular nucleus with glutamate acid in rats with ulcerative colitis (UC)., Methods: The rats were anesthetized with 10% chloral hydrate via abdominal injection and treated with an equal volume of TNBS + 50% ethanol enema, injected into the upper section of the anus with the tail facing up. Colonic damage scores were calculated after injecting a certain dose of glutamic acid into the paraventricular nucleus (PVN), and the effect of the nucleus tractus solitarius (NTS) and vagus nerve in alleviating UC injury through chemical stimulation of the PVN was observed in rats. Expression changes of C-myc, Apaf-1, caspase-3, interleukin (IL)-6, and IL-17 during the protection against UC injury through chemical stimulation of the PVN in rats were detected by Western blot. Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in colon tissues of rats were measured by colorimetric methods., Results: Chemical stimulation of the PVN significantly reduced UC in rats in a dose-dependent manner. The protective effects of the chemical stimulation of the PVN on rats with UC were eliminated after chemical damage to the PVN. After glutamate receptor antagonist kynurenic acid was injected into the PVN, the protective effects of the chemical stimulation of the PVN were eliminated in rats with UC. After AVP-Vl receptor antagonist ([Deamino-penl, val4, D-Arg8]-vasopressin) was injected into NTS or bilateral chemical damage to NTS, the protective effect of the chemical stimulation of PVN on UC was also eliminated. After chemical stimulation of the PVN, SOD activity increased, MDA content decreased, C-myc protein expression significantly increased, caspase-3 and Apaf-1 protein expression significantly decreased, and IL-6 and IL-17 expression decreased in colon tissues in rats with UC., Conclusion: Chemical stimulation of the hypothalamic PVN provides a protective effect against UC injury in rats. Hypothalamic PVN, NTS and vagus nerve play key roles in this process.

Objective: To investigate the possibility for human papillomavirus (HPV) infection to be a predictable signal for the carcinogenesis of oral mucosa by comparing the prevalences of HPV in each stage of oral mucosal carcinogenesis and to compare the sensitivity differences of the two methods in detecting HPV infection in oral cavity., Methods: The hybrid capture (HC-II) was used to detect infection of HPV in 255 samples taken from 12 cases of healthy oral mucosa, 211 cases of patients with pathological diagnosis and 32 cases of patients with clinical diagnosis. The diagnosed cases included 8 cases of benign lesions of the oral mucosa, precancerous lesions [74 cases of oral leukoplakia (OLK) with hyperplasia and 42 cases of OLK with oral epithelial dysplasia (OED)], 91 cases of precancerous condition [oral lichen planus (OLP)] and 28 cases of oral squamous cell carcinoma (OSCC). And in situ hybridization (ISH) was used to detect infection of HPV in 33 cases of OSCC and 76 cases of OLK, including 30 cases of hyperplasia, 15 cases of mild OED, 15 cases of moderate OED and 16 cases of severe OED., Results: The prevalence of HPV in OLP samples was higher (12.12%, 8/66) than that of OLK (2.59%, 3/116) (χ(2)=4.666, P=0.031) and OSCC(7.14%, 2/28, χ(2)=0.513, P=0.474). The prevalence of HPV in OSCC (7.14%, 2/28) was higher than that of OLK (2.59%, 3/116), and no significant difference was found. There was only one case of smoke spot and statistical analysis was not carried out. ISH was used to detect type 16/18 and type 31/33 HPV DNA in 109 cases of oral mucosal lesions in paraffin sections and only one case of OSCC was HPV positive. Thirty-seven cases were detected by HC-II and ISH methods at the same time. The same negative results by the two methods were found in 94.6% samples (35/37). In the other two samples, one was OSCC with early infiltration and the other was OLK with hyperplasia, The HC-II results were positive while the ISH results were negative. The patients with OLP and HPV testing results were followed up and the average follow-up period was (36.2 ± 10.5) months. It was found that three of them had a malignant transformation, and the malignant transformation rate of HPV positive patients was 12.50% (1/8), which was higher than that of HPV negative patients (3.45%, 2/58), and the difference was not statistically significant, P=0.249., Conclusion: HC-II assay was more sensitive in detecting HPV infection of oral mucosal lesions than ISH. The results of this study showed that there was insufficient evidence for taking HPV infection as a predictor of OLK carcinogenesis. Patients suffering from OLP were in a precancerous condition. The prevalence of HPV in OLP patients of this study was higher than that in OLK and OSCC patients, suggesting that for some reason, OLP patients were susceptible to HPV. HPV testing can be considered as routine in patients with OLP, and HC-II assay was recommended. And patients with OLP and HPV positive should be followed up regularly.

Common mycorrhizal network (CMN) allows nutrients and signals to pass between two or more plants. In this study, trifoliate orange (Poncirus trifoliata) and white clover (Trifolium repens) were planted in a two-compartmented rootbox, separated by a 37-μm nylon mesh and then inoculated with an arbuscular mycorrhizal fungus (AMF), Diversispora spurca. Inoculation with D. spurca resulted in formation of a CMN between trifoliate orange and white clover, whilst the best AM colonization occurred in the donor trifoliate orange-receptor white clover association. In the trifoliate orange-white clover association, the mycorrhizal colonization of receptor plant by extraradical hyphae originated from the donor plant significantly increased shoot and root fresh weight and chlorophyll concentration of the receptor plant. Enzymatic activity of soil β-glucoside hydrolase, protease, acid and neutral phosphatase, water-stable aggregate percentage at 2-4 and 0.5-1 mm size, and mean weight diameter in the rhizosphere of the receptor plant also increased. The hyphae of CMN released more easily-extractable glomalin-related soil protein and total glomalin-related soil protein into the receptor rhizosphere, which represented a significantly positive correlation with aggregate stability. AMF inoculation exhibited diverse changes in leaf and root sucrose concentration in the donor plant, and AM colonization by CMN conferred a significant increase of root glucose in the receptor plant. These results suggested that CMN formed in the trifoliate orange-white clover association, and root AM colonization by CMN promoted plant growth, root glucose accumulation, and rhizospheric soil properties in the receptor plant.

This paper investigates a novel joint problem of routing, scheduling, and channel allocation for single-radio multichannel wireless mesh networks in which multiple channel widths can be adjusted dynamically through a new software technology so that more concurrent transmissions and suppressed overlapping channel interference can be achieved. Although the previous works have studied this joint problem, their linear programming models for the problem were not incorporated with some delicate constraints. As a result, this paper first constructs a linear programming model with more practical concerns and then proposes a simulated annealing approach with a novel encoding mechanism, in which the configurations of multiple time slots are devised to characterize the dynamic transmission process. Experimental results show that our approach can find the same or similar solutions as the optimal solutions for smaller-scale problems and can efficiently find good-quality solutions for a variety of larger-scale problems.

Objective: To investigate the relationship between chromobox protein homolog 7 (cbx7) expression and the occurrence and development of colorectal carcinoma (CRC), gastric carcinoma (GC) and hepatocarcinoma (HCC) tissues., Methods: The samples of neoplastic tissues and the corresponding cutting-edge normal tissues from 22 cases of CRC, 20 cases of GC, 30 cases of HCC were surgically collected. Level of cbx7 mRNA was detected with a fluorescent quantitative RT-PCR assay, and the correlationship among expression of cbx7 mRNA, the patients' clinicopathologic features and the surviving time after surgery was analyzed., Results: The relative copy number of cbx7 mRNA in carcinomas and the normal tissues was 0.010 ± 0.015 vs 0.053 ± 0.042 for CRCs, 0.197 ± 0.195 vs 1.891 ± 1.254 for GCs, and 0.008 ± 0.008 vs 0.030 ± 0.021 for HCCs, respectively. Compared with the corresponding normal tissues, cbx7 expression was significantly downregulated in CRCs, GCs, and HCCs (t = -7.351, -5.417 and -6.680, respectively, P < 0.01). The expression of cbx7 mRNA in CRCs had significant differences not only between two age groups (the relative copy number of cbx7 mRNA in age > 55 group was 0.007 ± 0.015, but 0.017 ± 0.012 in age ≤ 55 group, t = -2.586, P = 0.022); but also between vascular embolus-positive and negative groups (the level of cbx7 mRNA in positive and negative group was 0.022 ± 0.021 vs 0.006 ± 0.011, t = -3.175, P = 0.010). The area under the receiver operating characteristics (ROC) curve is 0.769 (P = 0.033). when the Cut-off value of the relative copy number of cbx7 mRNA was 0.002 in CRCs. The values less-than 0.002 were defined as low expression. The CRC patients with low expression of cbx7 had a shorter overall survival time; whose 5 years survival rate was only 30.8% (4/13); while the rate was 77.8% (7/9) in high expression of cbx7 group. The difference had statistical significance (χ(2) = 4.329, P = 0.037). The similar differences could not be found among GC and HCC patients., Conclusion: Downregulation of cbx7 expression was very common among multiple carcinomas cases, and the downregulation influenced the prognosis of CRC patients.

Objective: To understand the changing trends of non-communicable diseases (NCDs) in Xinjiang Production and Construction Corps from 1998 to 2008., Methods: A stratified-cluster random sampling based cross-sectional NCDs survey was carried out in 2008, and using the data of NCDs from the health service surveys in 1998 and 2004, in Xinjiang Production and Construction Corps. The prevalence rate of NCDs was standardization according to age proportion of the population being surveyed in 1998., Results: In 1998, 2004 and 2008, the prevalence rates of NCDs in Xinjiang Production and Construction Corps were 17.26%, 25.61%, 24.85% while the Standardized rates of NCDs were 17.26%, 23.54% and 20.49% respectively. The prevalence rates of NCDs were statistically significant different in 35-, 45-, 55- and over 65 age groups in 1998, 2004 and 2008 which showed an consecutive upward trend. The prevalence rates of hypertension, diabetes, cerebrovascular disease, coronary heart disease and chronic obstructive pulmonary disease increased significantly from 1998 to 2008. The prevalence rate of hypertensive disease among 25- age group, diabetes among 35- age group, cerebrovascular disease and coronary heart disease among 45- age groups showed an increasing trend., Conclusion: Cardiovascular and cerebrovascular diseases, together with diabetes were the fastest increasing ones over the past 10 years and becoming the major diseases, making the Xinjiang Production and Construction Corps an aging population. NCDs should be prioritized in the health development plan. Targeted health education should be carried out in the whole population, together with other interventions as well as management programs on chronic diseases to reduce the prevalence of NCDs.

Aim: To understand the implication of GATA-4 and GATA-5 methylation in gastric carcinogenesis., Methods: Methylation status of GATA-4 and GATA-5 CpG islands in human gastric mucosa samples, including normal gastric biopsies from 45 outpatients, gastric dysplasia [low-grade gastric intraepithelial neoplasia (GIN), n = 30; indefinite, n = 77], and 80 paired sporadic gastric carcinomas (SGC) as well as the adjacent non-neoplastic gastric tissues was analyzed by methylation specific polymerase chain reaction (MSP) and confirmed by denatured high performance liquid chromatography (DHPLC). Immunohistochemical staining was used to detect protein expression. The correlation between GATA-4 and GATA-5 methylation and clinicopathological characteristics of patients including Helicobacter pylori (H. pylori) infection was analyzed., Results: GATA-4 and GATA-5 methylation was frequently observed in SGCs (53.8% and 61.3%, respectively) and their corresponding normal tissues (41.3% and 46.3%) by MSP. The result of MSP was consistent with that of DHPLC. Loss of both GATA-4 and GATA-5 proteins was associated with their methylation in SGCs (P = 0.01). Moreover, a high frequency of GATA-4 and GATA-5 methylation was found in both gastric low-grade GIN (57.1% and 69.0%) and indefinite for dysplasia (42.9% and 46.7%), respectively. However, GATA-4 and GATA-5 methylation was detected only in 4/32 (12.5%) and 3/39 (7.7%) of normal gastric biopsies. GATA-4 methylation in both normal gastric mucosa and low-grade GIN was also significantly associated with H. pylori infection (P = 0.023 and 0.027, two-sides)., Conclusion: Epigenetic inactivation of GATA-4 (and GATA-5) by methylation of CpG islands is an early frequent event during gastric carcinogenesis and is significantly correlated with H. pylori infection.

Objective: To investigate the effects of methylation status of CpG islands of endogenous E-cadherin (CDH1) gene on the promoter activity of corresponding genes in reporter assays., Methods: The methylation statuses of CpG island of CDH1 in 8 different cell lines were detected by methylation-specific PCR. CDH1 protein was analyzed by Western blotting. Two sets of pGL3 reporter vectors with different genotypes/haplotypes of the CDH1 promoter were constructed [ pGL3-A(-73)/-C(-73) pGL3-H1/-H4] and used to transfect these cell lines. The differences between these promoter reporter vectors were analyzed by t-test., Results: (1) CDH1 CpG island was unmethylated in AGS, MCF7, MKN74, and PC-3 cell lines, expressed in MCF7, MKN74, and PC-3, but not in AGS. Expression of CDH1 was silenced by methylation in HeLa, BGC823, A549, and RKO cell lines. (2) In the four CDH1-unmethylated MCF7, MKN74, PC-3, and AGS cell lines, the promoter activities of pGL3-C(-73), (as 0.78 +/- 0.10, 0.17 +/- 0.01, 0.11 +/- 0.01, 1.19 +/- 0.18) were significantly higher than those of pGL3-A(-73) (as 0.30 +/- 0.08, 0.07 +/- 0.01, 0.07 +/- 0.01, 0.39 +/- 0.04) (t values are -6.298, - 12.349, -8.128, -7.388, and P <. 0.1). However, in the four CDH1-methylated HeLa, BGC823, A549, and RKO cell lines, the promoter activity of pGL3-C(-73) (as 0.09 +/- 0.02, 0.13 +/- 0.02, 0.05 +/- 0.01, 0.01 +/- 0.00) was significantly lower than that of pGL3-A(-73) (as 0.16 +/- 0.01, 0.25 +/- 0.01, 0.11 +/- 0.03, 0.03 +/- 0.00) (t valued at 5.958, 11.189, 3.661, 13.866, and P<0.05). (3) In the unmethylated MKN74 and methylated RKO cell lines, the promoter activities of pGL3-H1/-H4 were obviously and contrarily different (as 1.57 +/- 0.23/0.94 +/- 0.06 and 0.38 +/- 0.02/0.50 +/- 0.04, t values were 4.577 and -4.915, P values were 0.010 and 0.003)., Conclusion: The methylation status of CpG island of the target gene in the tested cell lines affects the promoter activity in Reporter Assay significantly. The most active one may be the most suppressive one.

Objective: To observe the alterations of saliva nitrate and nitrite level in patients with oral candidiasis., Methods: Parotid saliva and whole saliva were collected from 33 patients and 34 healthy volunteers. Concentrations of nitrate and nitrite in saliva were determined by high-performance liquid chromatography. Follow-up observation was performed on 10 patients after treatment. The data were statistically analyzed with independent-samples t test or paired-samples t test at alpha = 0.05., Results: There was significant increase of the concentrations and secretion rate of parotid saliva nitrate in patient group as compared with controls: (49.70 +/- 0.50) vs (21.51 +/- 0.60) mg/L (t = 2.692, P = 0.009) and (27.71 +/- 0.50) vs (12.55 +/- 0.60) microg/min (t = 2.554, P = 0.013), respectively. Significantly increased concentrations and secretion rate of nitrate and nitrite [nitrate: (6.46 +/- 0.94) vs (1.11 +/- 0.70) mg/L (t = 3.792, P = 0.000); nitrite: (8.48 +/- 0.58) vs (3.39 +/- 0.53) mg/L (t = 2.888, P = 0.005); nitrate secretion rate: (10.57 +/- 0.91) vs (2.10 +/- 0.74) microg/min (t = 3.464, P= 0.001); nitrite secretion rate: (13.91 +/- 0.55) vs (6.42 +/- 0.58) microg/min (t = 2.397, P = 0.020)] were revealed in whole saliva of patients group. Significantly decreased nitrate and nitrite levels were also observed in patients after treatment, especially the changes of parotid saliva nitrate secretion rate [(37.50 +/- 0.50) vs (14.34 +/- 0.64) microg/min (t = 3.142, P = 0.012)], whole saliva nitrate [(14.29 +/- 1.01) vs (2.59 +/- 1.03) mg/L (t = 3.475, P = 0.007)] and whole saliva nitrate secretion rate [(25.97 +/- 0.93) vs (4.12 +/- 1.00) microg/min (t = 3.922, P = 0.003)]., Conclusion: The present study revealed the significant increase of salivary nitrate and nitrite level in patients with oral candidiasis is considered to be associated with the host defense reaction.

To investigate the relationship between p16 methylation and Helicobacter pylori infection in precancerous gastric lesions, a population-based study was conducted in Linqu County, a high-risk area of gastric cancer in China. Methylation status of p16 was evaluated by methylation-specific polymerase chain reaction in 920 subjects with precancerous gastric lesions. H. pylori status was determined by 13C-urea breath test and the density of H. pylori in biopsy specimens used for detecting methylation status was assessed by the modified Giemsa stain. The frequency of p16 methylation was significantly higher in subjects with H. pylori positive than those with H. pylori negative in each category of gastric lesion (p<0.001, respectively). Compared with H. pylori negative, the odds ratios (ORs) of p16 methylation were markedly elevated in subjects with H. pylori positive for superficial gastritis (OR, 9.45; 95% confidence interval [CI]: 2.94-30.41), chronic atrophic gastritis (OR, 15.92; 95%CI: 7.60-33.36), intestinal metaplasia (OR, 4.46; 95%CI: 2.44-8.13), indefinite dysplasia (OR, 3.67; 95%CI: 1.90-7.10), and dysplasia (OR, 2.48; 95%CI: 1.02-5.99). Moreover, the frequencies of p16 methylation increased steadily with the severity of H. pylori density in gastric mucosa. Compared with H. pylori negative, the OR of p16 methylation was 1.02-16.13 times higher in subjects with mild H. pylori infection, and 2.69-38.73 times higher in those with moderate/severe infection, respectively. Our findings indicate that p16 methylation was significantly associated with H. pylori infection in precancerous gastric lesions, suggesting that H. pylori infection could potently induce methylation of p16 CpG island., (Copyright (c) 2008 Wiley-Liss, Inc.)

The paper considers the connected target coverage (CTC) problem in wireless heterogeneous sensor networks (WHSNs) with multiple sensing units, termed MU-CTC problem. MU-CTC problem can be reduced to a connected set cover problem and further formulated as an integer linear programming (ILP) problem. However, the ILP problem is an NP-complete problem. Therefore, two distributed heuristic schemes, REFS (remaining energy first scheme) and EEFS (energy efficiency first scheme), are proposed. In REFS, each sensor considers its remaining energy and its neighbors' decisions to enable its sensing units and communication unit such that all targets can be covered for the required attributes and the sensed data can be delivered to the sink. The advantages of REFS are its simplicity and reduced communication overhead. However, to utilize sensors' energy efficiently, EEFS is proposed. A sensor in EEFS considers its contribution to the coverage and the connectivity to make a better decision. To our best knowledge, this paper is the first to consider target coverage and connectivity jointly for WHSNs with multiple sensing units. Simulation results show that REFS and EEFS can both prolong the network lifetime effectively. EEFS outperforms REFS in network lifetime, but REFS is simpler.

Objective: To investigate the relationship between point mutation of penicillin-binding protein gene (pbp) and amoxicillin resistance (AMOgamma) of Helicobacter pylori (H. pylori) as well as to compare the protein profiles under proteomic technology to get the candidate resistance-related proteins., Methods: (1) AMOgamma strains were selected from the sensitive H. pylori strain 26695 by serial passage technique in vitro. (2) Point mutations of five putative resistance genes (HP0597, HP1565, HP1542, HP1556, and HP0160) were analyzed by denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing. (3) Proteins samples were separated by two-dimensional electrophoresis (2-DE). Protein profiles were compared between the AMOgamma strain obtained in vitro and its sensitive parent strain 26695. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was performed to identify the proteins of interest. The proteins were searched by software MASCOT and identified by peptide fingerprint map using the program MS-FIT of Protein Prospect., Results: (1) An AMOgamma strain (MIC 8 microg/ml) was obtained. Complete loss of the resistant phenotype was observed after cultivation in the absence of AMO or storage at - 80 degrees C. (2) DHPLC and Sequencing result showed no point mutations in five pbp genes in the AMOgamma strain when compared with the corresponding PCR products from its parent strain 26695. (3) Protein profiling showed that eleven protein spots were differently expressed between 26695 and the AMOgamma strain. Of these protein spots in the AMOgamma strain, two new spots (Spot 1 and Spot 2) were observed with one (Spot 3) was up-regulated three-fold and the remained ones (Spot 4-11) were down-regulated., Conclusion: AMO resistance of H. pylori might be resulted from, unstable phenotype change rather than point mutations of pbp genes. These differentially regulated proteins in AMOgamma strain might play a role in development of resistance to AMO in H. pylori.

Objective: To detect the therapeutic effect of selenium-enriched garlic (SeG) on chronic gastritis., Methods: Chronic gastritis was induced of the glandular stomach of male Mongolian Gerbils via gastric instillation of H. pylori TN2 strain once every 4 days for 5 consecutive times followed by random classification into six groups. Fresh SeG suspension was administrated daily at dosages of 4.70, 1.5, 0.47, 0.15 g.kg(-1).d(-1) for four weeks. The gerbils in the positive control group were treated with omeprazole, clarithromycin, and amoxicillin for one week. The gerbils were killed for pathological examination four weeks after SeG-treatment., Results: Chronic gastritis (CAG), low-grade dysplasia or gastric intraepithelial neoplasia (DYS/GIN) were observed among 77% and 38.5% of the 13 H. pylori-treated animals in the negative control group, respectively; whereas 40% and 26.7% in the positive control group (n = 15), respectively. The incidences of CAG and DYS/GIN in the SeG groups (n = 21 - 27) were reduced dose-dependently, 16.7% - 38.7% and 11.1% - 14.3% for CAG and DYS/GIN, respectively., Conclusion: SeG administration inhibits the development and progression of CAG induced by H. pylori remarkably.

Objective: To establish a sensitive assay to detect methylation status of the critical 7862nt CpG site related to transcription of HPV16 E6 and E7 genes., Methods: Genomic DNA of two HPV16-infected cell line SiHa and CaSki was extracted and modified by sodium bisulfite to convert the unmethylated Cs to Us (Ts in PCR products). The target sequence of HPV16 including the 7862nt CpG site was pre-amplified by PCR. Then, the methylation status of the 7862nt site was differentiated in a primer extension reaction with an HPV16-specific primer, and separated by DHPLC at 80 degrees C., Results: The primers without extension and with extension, whether matched to CpG or TpG, could be separated by DHPLC completely. The peak for ddTTP-extension products corresponding to the demethylated CpG site was observed at retention time 6.7 min in both cell lines. However, the peak for ddCTP-extension products representing the methylated CpG site could be detected at retention time 6.3 min in CaSki cell line only, which integrated with 499 methylated and one demethylated HPV16 copies., Conclusion: The established DHPLC-primer extension assay can be used to detect methylated and demethylated HPV16 copies simultaneously with a sensitivity up to 1/500 (0.2%).

Objective: To investigate relationship between methylation status of the APC and Bikunin CpG islands and clinicopathological characteristics of breast carcinomas., Methods: The methylation status of APC and Bikunin CpG islands in 152 sporadic breast carcinoma samples were analyzed by methylation specific PCR., Results: 40.8% of breast carcinomas examined showed methylated signals for the APC. The methylation frequency of APC was significantly correlated to the tumor size (chi(2) = 4.041; P = 0.044), but not to patients' age, pathologic type of tumor, clinical stage, histological grade, lymph node metastasis and the status of estrogen or progestogen receptor. In addition, 24.6% of carcinoma samples examined revealed strong methylated signals for Bikunin. No significant correlation was found between the aberrant methylation of Bikunin and the clinicopathological characteristics of sporadic breast carcinomas., Conclusion: The aberrant methylations of APC and Bikunin are frequent events during breast carcinogenesis. APC methylation might play a role in the progression of breast cancer.

Objective: To compare binding activity of different zinc finger domain of human Kaiso with methylated CpG., Methods: pGEX constructs with different human Kaiso domain were generated and then corresponding fusion proteins were induced and purified. Electrophoretic mobility shift assays were applied to evaluate the binding activity of fusion proteins with methylated CpG., Results: The purified GST-KaisoZF fusion protein (without the POZ protein binding domain) could bind with methylated CpG probe specifically, but not for three or two zinc fingers without flanking domains., Conclusion: Human zinc finger protein Kaiso could bind with methylated CpG specifically, only in the assistance of the neighboring flank sequence of the zinc finger domain.

Objective: To setup a quantitative assay for detection of methylation of SNCG CpG island in human tissue samples., Methods: Methylation status of the 16 tested CpG sites within the CpG island was analyzed by bisulfite-clone-sequencing for 2 gastric carcinoma cell lines, 2 normal gastric mucosa samples, and 2 pairs of primary gastric carcinomas and their corresponding non-neoplastic tissues, respectively., Results: The methylation of -88 and other four CpG sites was well correlated with the methylation of the overall CpG island. Thus, a combined bisulfite-restriction assay (COBRA) was developed based on the enzyme AciI, which digested the only one GCGG sequence in the PCR products of the methylated CpG island, but not the GTGG in the demethylated one. The digested fragments (144 bp and 85 bp) and undigested fragment (229 bp) could be completely separated by denaturing high performance liquid chromatography (DHPLC). According to the peak areas of these fragments, the proportion of the methylated copies of the SNCG CpG island was calculated easily. The result of the COBRA-DHPLC assay was reproducible and consistent with that of clone-sequencing., Conclusion: A COBRA-DHPLC assay is setup successfully for quantification of methylation of the SNCG CpG island.

Objective: To confirm whether there is myocytes proliferation in the adult rat with heart failure or not, and to investigate the relationship between myocyte proliferation and heart function., Methods: Descending anterior branch of left coronary artery was ligated in 20 adult male SD rats so as to establish an heart failure models. Eight rats were used as controls. Hemodynamic parameters, blood pressure (BP), left ventricle end systolic pressure (LVESP), left ventricle end diastolic pressure (LVEDP), +LVdp/dt(max), and -LVdp/dt(max), were measured 30 days after the coronary occlusion. Based on the results of heart function examination, the heart infarct rats were divided into 2 subgroups: cardiac functional compensation subgroup (8 rats), and cardiac functional decompensation subgroup (6 rats). Then the rats were killed and their hearts were taken out and stained with propidium iodide (PI) and antibody to alpha-sarcomeric actin. Immunohistochemistry was used to detect the proliferation cell nuclear antigen (PCNA). Confocal microscopy was used to observe the mitotic image. Light microscopy was used to observe the PCNA positive rate in the myocardium., Results: (1) Mitotic images of myocytes could be identified by confocal microscopy in the left ventricle of all rats. (2) PCNA expression was detected in the nuclei of both infarct and normal hearts. The PCNA positive rate of the cardiac functional compensation subgroup was 7.2% +/- 1.4%, significantly higher than that of the control group (2.2% +/- 0.8%, P = 0.648). However, the PCNA positive rate of the cardiac functional decompensation subgroup was 3.0% +/- 1.3%, not significantly different from that of the control group (P = 0.648). (3) The correlation coefficient between PCNA-positivity of cardiomyocytes and +LVdp/dt(max) in the infarct rats were 0.80 (P < 0.01) and the correlation coefficient between PCNA-positivity of cardiomyocytes and -LVdp/dt(max) was -0.76 (P = 0.01)., Conclusion: (1) There is myocyte proliferation in the adult rat heart. (2) Myocyte proliferation is positively correlated with heart systolic function, and negatively correlated with heart diastolic function in chronic heart failure.

We have previously cloned and identified a novel esophageal cancer related gene 2 (ECRG2; GenBank Accession Number AF268198), which is down-regulated in esophageal squamous cell carcinoma (ESCC) and involved in the induction of the apoptosis in esophageal cancer cell lines. In the present study, we have found a short tandem repeat (STR) polymorphism in the noncoding region of the exon 4 of the ECRG2 gene by using PCR-denaturing high-performance liquid chromatography (DHPLC). Three STR genotypes, TCA3/TCA3, TCA3/TCA4 and TCA4/TCA4 were revealed and confirmed by DNA sequencing analysis. A total of 661 objects including 228 patients with ESCC and 373 normal controls were analyzed to investigate the impact of this ECRG2 STR polymorphism on risk of ESCC in case-control studies. Genotypes were determined in 231 controls and 162 cases from Beijing, which is a low risk area of ESCC, and in 142 controls and 126 cases from Linxian, a well-known high-risk area of ESCC. In both of the Beijing and Linxian population, subjects who carried the TCA3/TCA3 genotype were at an increased risk of ESCC compared to those carrying the TCA4/TCA4 genotype, with the adjusted odds ratios (ORs) being 2.05 [95% confidence interval (CI), 1.02-4.06] for the subjects from Beijing and 4.40 (95% CI, 1.93-10.01) for the subjects from Linxian. Furthermore, comparison of the genotype distributions among other cancer sites might suggest that risk of the ECRG2 STR polymorphism might be specific to the esophagus. These findings indicate for the first time that the ECRG2 STR is a genetic susceptibility factor for ESCC and the TCA3/TCA3 allele might play a role in the development of this cancer., (Copyright 2003 Wiley-Liss, Inc.)

Aim: To study the mechanisms responsible for inactivation of a novel esophageal cancer related gene 4 (ECRG4) in esophageal squamous cell carcinoma (ESCC)., Methods: A pair of primers was designed to amplify a 220 bp fragment, which contains 16 CpG sites in the core promoter region of the ECRG 4 gene. PCR products of bisulfite-modified CpG islands were analyzed by denaturing high-performance liquid chromatography (DHPLC), which were confirmed by DNA sequencing. The methylation status of ECRG 4 promoter in 20 cases of esophageal cancer and the adjacent normal tissues, 5 human tumor cell lines (esophageal cancer cell line-NEC, EC109, EC9706; gastric cancer cell line- GLC; human embryo kidney cell line-Hek293) and 2 normal esophagus tissues were detected. The expression level of the ECRG 4 gene in these samples was examined by RT-PCR., Results: The expression level of ECRG 4 gene was varied. Of 20 esophageal cancer tissues, nine were unexpressed, six were lowly expressed and five were highly expressed compared with the adjacent tissues and the 2 normal esophageal epithelia. In addition, 4 out of the 5 human cell lines were also unexpressed. A high frequency of methylation was revealed in 12 (8 unexpressed and 4 lowly expressed) of the 15 (80 %) downregulated cancer tissues and 3 of the 4 unexpressed cell lines. No methylation peak was observed in the two highly expressed normal esophageal epithelia and the methylation frequency was low (3/20) among the 20 cases in the highly expressed adjacent tissues. The methylation status of the samples was consistent with the result of DNA sequencing., Conclusion: These results indicate that the inactivation of ECRG 4 gene by hypermethylation is a frequent molecular event in ESCC and may be involved in the carcinogenesis of this cancer.

The role of salivary glands in nitrate and nitrite metabolism is poorly understood. The aim of the present study was to investigate the effect of parotid gland ablation on dynamic metabolism of nitrate and nitrite in miniature pigs. The parotid glands of 5 healthy miniature pigs were bilaterally ablated by methyl violet. Concentrations of nitrate and nitrite of whole saliva, serum, and urine samples were analyzed by high-performance liquid chromatography. Results showed that bilateral ablation of the parotid glands led to a significant decrease of nitrate secretion from blood to saliva (P < 0.05) and thus low nitrite levels. Dysfunction of the parotid glands temporarily increased the systemic level of nitrate in miniature pigs after nitrate loading. This study suggests that the parotid glands play an important role in the balance of nitrate and nitrite levels in both whole saliva and the body.

Aim: To investigate CpG methylation and single nucleotide polymorphism (SNP) of a specific promoter region of hMLH1 in primary gastric carcinoma., Methods: Primary gastric carcinomas (n=80), their corresponding normal mucosal samples, and gastric mucosal biopsies from normal/gastritis control patients (n=54) were used. Hypermethylation at -253 nt and -251 nt in relation with the translational start site and SNP of a silencing specific region (-339 nt-46 nt) in the hMLH1 promoter were analyzed by Bst UI-combined bisulfite assay (COBRA), denaturing high performance liquid chromatogram (DHPLC), and sequencing., Results: (A) The specific methylation at -253 nt and -251 nt was observed in 2 of 60 primary gastric carcinomas, but neither in all of the corresponding mucosa nor in normal/gastritis samples, by Bst UI-COBRA and DHPLC. (B) The hMLH1 promoter was methylated homogeneously in the xenograft of the primary gastric carcinoma with the methylated and unmethylated hMLH1. (C) The pattern of SNP at -93 nt of the hMLH1 promoter in 54 Chinese patients with gastric carcinoma was the same as that in the control patients: 51 % was A/G heteroalleles, 34 % and 15 % were A/A and G/G homoalleles, respectively., Conclusion: Biallelic inactivation of hMLH1 by epigenetic silencing existed in human primary gastric carcinoma homogeneously. Hypermethylation of hMLH1 may play a role in the early stage of development of a few gastric carcinomas. The SNP at -93 nt is not related to the susceptibility of gastric carcinomas.

Aim: To optimize conditions of DHPLC and analyze the effectiveness of various DNA polymerases on DHPLC resolution, and evaluate the sensitivity of DHPLC in the mutation screening of mitochondrial DNA (mtDNA)., Methods: Two fragments of 16s gene of mitochondrial DNA (one of them F2 is a mutant fragment) and an A3243G mutated fragment were used to analyze the UV detection limit and determine the minimum percentage of mutant PCR products for DHPLC and evaluate effects of DNA polymerases on resolution of DHPLC. Under the optimal conditions, we analyzed the mtDNA mutations from muscle tissues of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and screened blindly for variances in D-loop region of mtDNA from human gastric tumor specimen., Results: Ten A3243G variants were detected in 12 cases of MELAS, no alterations were detected in controls and these results were consistent with the results obtained by analysis of RFLP with ApaI. We also identified 26 D-loop variances in 46 cases of human gastric cancer tissues and 38 alterations in 13 gastric cancer cell lines. The mutation of mtDNA at 80 ng PCR products containing a minimum of 5% mutant sequences could be detected by using DHPLC with UV detector. Moreover, Ampli-Taq Gold polymerase was equally as good as the proofreading DNA polymerase (e.g., Pfu) in eliminating the false positive produced by Taq DNA polymerases., Conclusion: DHPLC is a powerful, rapid and sensitive mutation screening method for mtDNA. Proofreading DNA polymerase is more suitable for DHPLC analysis than Taq polymerase.

N-(nitrosomethyl) urea (NMU) was characterized in the carcinogenic nitrosated fish sauce (NFS) with high performance liquid chromatography (HPLC)-photohydrolysis-pyrolysis-thermal energy analysis recently. We used HPLC-electronic spray ionization-mass spectrometry and HPLC-diode array detection to confirm NMU in NFS further. It was observed that the corresponding chromatographic fraction of NMU of NFS showed the same mass spectrum (m/z 64, 102, and 145) and ultraviolet-absorbance (lambda max = 230 nm) as those of authentic NMU. These results confirmed that the component of NFS was NMU.

Adenocarcinoma, adenoma and dysplasia of the stomach of adult Wistar rats were induced following administration of MNNG for 10 days by gavage when they were new-born. The incidence of the three types of pathological lesions at the dose of 0.4 mg MNNG per rat being 39%, 50% and 100% respectively. Induction of gastric adenocarcinoma is dose-dependent. The incidence of adenocarcinoma in male rats being significantly higher then that of female rats (P < 0.02). 23 of 33 (70%) induced cancers were found in the gastric antrum. By the use of DNA cotransfection technique and the assay of carcinogenicity in the Balb/c nude mice, it was also found that the DNA of 4 of the 6 induced gastric carcinomas could transform NIH/3T3 cells into malignant cells, an indication that the induced cancer DNA contains transforming gene.

Most of anticancer drugs are mutagenic. A possible exception is diallyl trisulfide (DAT), a component of garlic. It is an antimutagenic anticancer chemical although it is mainly used as antibiotic. Its modifying effect on induction of UDS by mutagenic mitomycin C (MMC), cyclophosphamide (CP) and cis-diamine dichloroplatin (DDP) was investigated with the UDS assay in the primary cultures of Wistar rat hepatocytes (hpc) using the autoradiographic technique. Results showed that 1.0-4.0 nmol/ml of DAT did not induce UDS and that MMC, CP and DDP resulted in a significant induction of dose-dependent UDS. DAT enhanced induction of UDS by these drugs. A dose-effect relationship was observed between dose of DAT and enhancement of induction of UDS. However, the mechanism of the enhancement is not clear.