Somatostatin receptor 1, a novel EBV-associated CpG hypermethylated gene, contributes to the pathogenesis of EBV-associated gastric cancer.

BACKGROUND: Somatostatin receptor 1 (SSTR1) was preferentially methylated in Epstein-Barr virus (EBV)-positive gastric cancer using promoter methylation array. We aimed to analyse the epigenetic alteration and biological function of SSTR1 in EBV-associated gastric cancer (EBVaGC). METHODS: Promoter methylation was examined by combined bisulphite restriction analysis (COBRA) and pyrosequencing. The biological functions of SSTR1 were evaluated by loss- and gain-of-function assays. RESULTS: Promoter hypermethylation of SSTR1 was detected in EBV-positive gastric cancer cell lines (AGS-EBV) with SSTR1 transcriptional silence, but not in EBV-negative gastric cancer cell lines with SSTR1 expression. Expression level of SSTR1 was restored in AGS-EBV by exposure to demethylating agent. Moreover, methylation level of SSTR1 was significantly higher in EBV-positive primary gastric cancers compared with EBV-negative gastric cancers (P=0.004). Knock-down of SSTR1 in gastric cancer cell lines (AGS and BGC823) increased cell proliferation and colony formation ability, and promoted G1 to S-phase transition, enhanced cell migration and invasive ability. In contrast, ectopic expression of SSTR1 in gastric cancer cell lines (MKN28 and MGC803) significantly suppressed cell growth in culture conditions and reduced tumour size in nude mice. The tumour suppressive effect of SSTR1 was associated with upregulation of cyclin-dependent kinase inhibitors (p16, p15, p27 and p21); downregulation of oncogenes (MYC and MDM2), key cell proliferation and pro-survival regulators (PI3KR1, AKT, BCL-XL and MET); and inhibition of the migration/invasion-related genes (integrins, MMP1 (matrix metallopeptidase 1), PLAUR (plasminogen activator urokinase receptor) and IL8 (interleukin 8)). CONCLUSION: Somatostatin receptor 1 is a novel methylated gene driven by EBV infection in gastric cancer cells and acts as a potential tumour suppressor.

Epigenetic inactivation of paired box gene 5, a novel tumor suppressor gene, through direct upregulation of p53 is associated with prognosis in gastric cancer patients.

Using genome-wide methylation screening, we identified that paired box gene 5 (PAX5) is involved in human cancer development. However, the function of PAX5 in gastric cancer (GC) development is largely unclear. We analyzed its epigenetic inactivation, biological functions and clinical application in GC. PAX5 was silenced in seven out of eight GC cell lines. A significant downregulation was also detected in paired gastric tumors compared with adjacent non-cancerous tissues. The downregulation of PAX5 was closely linked to the promoter hypermethylation status and could be restored with demethylation treatment. Ectopic expression of PAX5 in silenced GC cell lines (AGS and BGC823) inhibited colony formation and cell viability, arrested cell cycle, induced apoptosis, suppressed cell migration and invasion and repressed tumorigenicity in nude mice. Consistent with the induction of apoptosis by PAX5 in vitro, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining showed significantly enhanced apoptotic cells in PAX5-expressed tumors compared with the vector control tumors. On the other hand, knockdown of PAX5 by PAX5-short hairpin RNA increased the cell viability and proliferation. The anti-tumorigenic function of PAX5 was revealed to be mediated by upregulating downstream targets of tumor protein 53 (p53), p21, BCL2-associated X protein, metastasis suppressor 1 and tissue inhibitors of metalloproteinase 1, and downregulating BCL2, cyclin D1, mesenchymal-epithelial transition factor (MET) and matrix metalloproteinase 1. Immunoprecipitation assay demonstrated that PAX5 directly bound to the promoters of p53 and MET. Moreover, PAX5 hypermethylation was detected in 77% (144 of 187) of primary GCs compared with 10.5% (2/19) of normal gastric tissues (P<0.0001). GC patients with PAX5 methylation had a significant poor survival compared with the unmethylated cases as demonstrated by Cox regression model and log-rank test. In conclusion, PAX5 is a novel functional tumor suppressor in gastric carcinogenesis. Detection of methylated PAX5 can be utilized as an independent prognostic factor in GC.

PMN-PT single crystal focusing transducer fabricated using a mechanical dimpling technique.

A ∼5MHz focusing PMN-PT single crystal ultrasound transducer has been fabricated utilizing a mechanical dimpling technique, where the dimpled crystal wafer was used as an active element of the focusing transducer. For the dimpled focusing transducer, the effective electromechanical coupling coefficient was enhanced significantly from 0.42 to 0.56. The dimpled transducer also yields a -6dB bandwidth of 63.5% which is almost double the bandwidth of the plane transducer. An insertion loss of the dimpled transducer (-18.1dB) is much lower than that of the plane transducer. Finite element simulation also reveals specific focused beam from concave crystal surface. These promising results show that the dimpling technique can be used to develop high-resolution focusing single crystal transducers.

Epigenetic inactivation of T-box transcription factor 5, a novel tumor suppressor gene, is associated with colon cancer.

T-box transcription factor 5 (TBX5) is a member of a phylogenetically conserved family of genes involved in the regulation of developmental processes. The function of TBX5 in cancer development is largely unclear. We identified that TBX5 was preferentially methylated in cancer using methylation-sensitive arbitrarily primed PCR. We aim to clarify the epigenetic inactivation, biological function and clinical significance of TBX5 in colon cancer. Promoter methylation was evaluated by combined bisulfite restriction analysis and bisulfite genomic sequencing. Cell proliferation was examined by cell viability assay and colony formation assay, apoptosis by flow cytometry and cell migration by wound-healing assay. TBX5 target genes were identified by cDNA microarray analysis. Cox regression model and log-rank test were used to identify independent predictors of prognosis. TBX5 was silenced or downregulated in 88% (7/8) colon cancer cell lines, but was expressed in normal colon tissues. Loss of gene expression was associated with promoter methylation. The biological function of TBX5 in human colon cancer cells was examined. Re-expression of TBX5 in silenced colon cancer cell lines suppressed colony formation (P<0.001), proliferation (P<0.001), migration and induced apoptosis (P<0.01). Induction of apoptosis was mediated through cross-talk of extrinsic apoptosis pathway, apoptotic BCL2-associated X protein and Granzyme A signaling cascades. TBX5 suppressed tumor cell proliferation and metastasis through the upregulation of cyclin-dependent kinase inhibitor 2A, metastasis suppressor 1 and downregulation of synuclein gamma and metastasis-associated protein 1 family member 2. TBX5 methylation was detected in 68% (71/105) of primary colon tumors. Multivariate analysis showed that patients with TBX5 methylation had a significantly poor overall survival (P=0.0007). In conclusion, we identified a novel functional tumor suppressor gene TBX5 inactivated by promoter methylation in colon cancer. Detection of methylated TBX5 may serve as a potential biomarker for the prognosis of this malignancy.

Therapeutic efficacy of Traditional Chinese Medicine 319 recipe on hepatic fibrosis induced by carbon tetrachloride in rats.

BACKGROUND/AIMS: Hepatic fibrosis is a consequence of severe liver damage that occurs in many patients with chronic liver diseases. TCM 319 recipe is a Chinese Medicine formula which consists of six Chinese herbs. In this study, we investigated the anti-fibrotic efficacy and mechanisms of TCM 319 recipe. METHODS: Hepatic fibrosis in rats was induced by carbon tetrachloride (CCl4). 34 male adult SD rats were allocated into five groups (group 1-concomitant CCl4 and TCM 319 recipe for 8 weeks; group 2-CCl4 for 4 weeks and then CCl4 and TCM 319 recipe for 4 weeks; group 3-CCl4 alone for 8 weeks; group 4-TCM 319 recipe only for 8 weeks; group 5-untreated controls). After 8 weeks of treatment, serum ALT assay, liver tissue histological examination and immunostaining were carried out to examine the liver function and fibrosis degree. The expression levels of platelet derived growth factor (PDGF-B), PDGF-Rbeta, and transforming growth factor-beta 1 (TGF-beta1) were measured by quantitative RT-PCR and western blot. RESULTS: TCM 319 recipe reduced liver injury and attenuated hepatic fibrosis in group 1 compared with that in group 3. TCM 319 recipe suppressed the mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1). In addition, treatment with TCM 319 recipe significantly down-regulated mRNA expression of PDGF-B and PDGF-Rbeta, and it also suppressed protein expression of PDGF-Rbeta and TGF-beta1. CONCLUSIONS: TCM 319 recipe extracts could attenuate hepatic fibrosis induced by CCl4 in rats. The anti-fibrotic effect of TCM 319 recipe is associated with the down-regulation of mRNA expression of TIMP-1, PDGF-B and PDGF-Rbeta, and with the suppression of protein expression of PDGF-Rbeta and TGF-beta1.

Intracellular levels of hepatitis B virus DNA and pregenomic RNA in peripheral blood mononuclear cells of chronically infected patients.

It remains uncertain whether hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and pregenomic RNA (pgRNA) can be detected in the serum or peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis B (CHB) infection. We examined HBV cccDNA and pgRNA in the serum and PBMC, and investigated the effect of lamivudine therapy on the viral loads in the PBMC of CHB patients. Paired serum and PBMC samples from 50 treatment-naïve CHB patients [25 hepatitis B e antigen (HBeAg) positive and 25 HBeAg negative] were quantified for total HBV DNA, cccDNA and pgRNA by real time polymerase chain reaction. HBV cccDNA and pgRNA were below the lower detection limit in all serum samples, and in 84% of PBMC. HBV DNA (r = 0.889, P < 0.001) and pgRNA (r = 0.696, P < 0.001) in PBMC correlated with the HBV DNA in serum. In the longitudinal study, 30 patients treated with lamivudine therapy for a median duration of 34 weeks (range 12-48 weeks) were examined. The median HBV DNA reduction in PBMC before and after treatment was 1.318 (range -0.471 to 3.846) log units, which was significantly lower than serum HBV DNA reduction [3.371 (range -0.883 to 9.454) log units, P < 0.05]. HBV cccDNA and pgRNA were undetectable in the serum of CHB patients. HBV viral loads in PBMC correlated with serum HBV DNA. Lamivudine therapy had less effect on the HBV viral loads in PBMC compared with the serum viral loads.

Cell cycle-dependent expression of potassium channels and cell proliferation in rat mesenchymal stem cells from bone marrow.

OBJECTIVE: Recently, our team has demonstrated that voltage-gated delayed rectifier K(+) current (IK(DR)) and Ca(2+)-activated K(+) current (I(KCa)) are present in rat bone marrow-derived mesenchymal stem cells; however, little is known of their physiological roles. The present study was designed to investigate whether functional expression of IK(DR) and I(KCa) would change with cell cycle progression, and whether they could regulate proliferation in undifferentiated rat mesenchymal stem cells (MSCs). MATERIALS AND METHODS: Membrane potentials and ionic currents were recorded using whole-cell patch clamp technique, cell cycling was analysed by flow cytometry, cell proliferation was assayed with DNA incorporation method and the related genes were down-regulated by RNA interference (RNAi) and examined using RT-PCR. RESULTS: It was found that membrane potential hyperpolarized, and cell size increased during the cell cycle. In addition, IK(DR) decreased, while I(KCa) increased during progress from G(1) to S phase. RT-PCR revealed that the mRNA levels of Kv1.2 and Kv2.1 (likely responsible for IK(DR)) reduced, whereas the mRNA level of KCa3.1 (responsible for intermediate-conductance I(KCa)) increased with the cell cycle progression. Down-regulation of Kv1.2, Kv2.1 or KCa3.1 with the specific RNAi, targeted to corresponding gene inhibited proliferation of rat MSCs. CONCLUSION: These results demonstrate that membrane potential, IK(DR) and I(KCa) channels change with cell cycle progression and corresponding alteration of gene expression. IK(DR) and intermediate-conductance I(KCa) play an important role in maintaining membrane potential and they participate in modulation of proliferation in rat MSCs.

Incorporating production rules with spatial information onto a neocognitron neural network.

Rule-embedded neocognitron (REN) is proposed where the knowledge base of a neocognitron is constructed through incorporating production rules into its interlayer connections. Prototype patterns training is not required. The semantic of interlayer connections is established. The resulting network can now be analyzed according to the rule structure and problematic portions can be corrected. We demonstrate the ease with which performance can be improved by applying REN on handwritten numeral recognition. The same set of handwritten numerals initiated by Fukushima is used to test this methodology. It is found that the performance is comparable with that of Fukushima's neocognitron with supervised training.

Competency-based evaluation of case-management skills in child sexual abuse intervention.

A model training protocol for case management of child sexual abuse cases, with a concomitant competency-based evaluation of these skills, is presented. Findings suggest that relative to skills in problem identification, child protective service workers need training in formulating goals and objectives and negotiating contracts with sexually abusive families.

Optical-processor architectures for alternating-projection neural networks.

Optical-processor architectures for various forms of the alternating-projection neural network are considered. Required iteration is performed by passive optical feedback. No electronics or slow optics (e.g., phase conjugators) are used in the feedback path. The processor can be taught a new training vector by viewing it only once. If the desired outputs are trained to be either +/-1, then the network can be configured to converge in one iteration.

Performance analysis of associative memories with nonlinearities in the correlation domain.

A matched filter-based architecture for associative memories (MFAMs) has been proposed by many researchers. The correlation from a leg of a matched filter bank, after being altered nonlinearly, weights its corresponding library vector. The weighted vectors are summed and clipped to give an estimate of the library vector closest to the input. We analyze the performance of such architectures for binary and/or bipolar inputs and libraries. Sufficient conditions are derived for the correlation nonlinearity so that the MFAM outputs the correct result. If, for example, N bipolar library vectors are stored, theicorrelation nonlinearity Z(x) = N(x/2) will always result in that library vector closest to the input in the Hamming sense.

Drinking styles and drinking problems.

Little research has focused on the range of drinking styles within a particular society. The intent of this study was to continue the development of an empirical typology of drinking behavior by replicating and extending the results of two previous projects. The earlier work had looked mainly at "normal" drinking, while this study placed more emphasis on types of problem drinking and on the relationships among various types of "normal" and "problem" drinking. The data were taken from a 1979 national probability sample of the adult population of the United States, with 1,169 cases classified as current drinkers. The results clearly showed that a variety of drinking styles exist, regardless of whether one is concerned with "normal" or "pathological" drinking. More importantly, certain types of "normal" drinking and types of comportment while drinking were powerful predictors of "problem" drinking.

Composite matched filter output partitioning.

A common pattern recognition problem is finding a library element closest, in some sense, to a given reception. In many scenarios, optimal detection requires N matched filters for N library elements. Since N can often be quite large, there is a need for suboptimal techniques that base their decisions on a reduced number of filters. The use of composite matched filters (CMFs) (also called synthetic discriminant functions or linear combination filters) is one technique to achieve this reduction. For two level CMF outputs, the reduction is from N to log(2)N matched filters. Previously, the coefficients of the CMF output were restricted to positive values-often 0 and 1. We refer to such filters as binary CMFs. An alternative approach is to use -1 and +1 for filter coefficients. This alternative filter will be called a bipolar CMF. This paper demonstrates how the extension from a binary to a bipolar CMF greatly improves the detection performance while still maintaining the reduced computational requirements of the binary CMF. Furthermore, the bipolar CMF is invariant to scale: multiplying the input by a positive constant gives the same processor output. This desirable behavior does not exist for the binary CMF.

Conventional and composite matched filters with error correction: a comparison.

A common pattern recognition problem is finding a library object which most closely matches a received image. For additive white Gaussian input noise, optimal detection performance is obtained using a matched filter for each of the N possible library objects. The use of composite matched filters (CMFs) (also called synthetic discriminant functions or linear combination filters) is one technique of reducing the number of filters required for the recognition problem. For two-level composite matched filter outputs, the reduction is from N to Q = log(2) (N) filters. The CMF's performance, however, can be suboptimum. Using CMFs with bipolar (+1,-1) outputs, this paper examines the detection performance improvement obtained by using error correcting codes. Use of varying levels of error correction is shown to allow trade-off between detection probability and the number of bank filters. Also, we show that in the case of inexact processing, the CMF can perform better than the conventional matched filter.

Synchronous vs asynchronous behavior of Hopfield's CAM neural net.

The performance of Hopfield's neural net operating in synchronous and asynchronous modes is contrasted. Two interconnect matrices are considered: (1) the original Hopfield interconnect matrix; (2) the original Hopfield interconnect matrix with self-neural feedback. Specific attention is focused on techniques to maximize convergence rates and avoid steady-state oscillation. We identify two oscillation modes. Vertical oscillation occurs when the net's energy changes during each iteration. A neural net operated asynchronously cannot oscillate vertically. Synchronous operation, on the other hand, can change a net's energy either positively or negatively and vertical oscillation can occur. Horizontal oscillation occurs when the net alternates between two or more states of the same energy. Certain horizontal oscillations can be avoided by adopting appropriate thresholding rules. We demonstrate, for example, that when (1) the states of neurons with an input sum of zero are assigned the complement of their previous state, (2) the net is operated asynchronously, and (3) nonzero neural autoconnects are allowed, the net will not oscillate either vertically or horizontally.

Controlled demonstration of metrazol kindling.

Convulsive responses elicited in rats by intraperitoneal injections of metrazol were intensified following a seriew of metrazol injections administered once every three days. However, neither handling alone nor placebo injections increased the susceptibility of control rats to metrazol-induced convulsive symptoms. Thus, although increases in the susceptibility to metrazol seizures following periodic placebo injections have been reported by others, such increases do not appear to be a critical factor in metrazol kindling.