Phage tail fibre assembly proteins employ a modular structure to drive the correct folding of diverse fibres.

Phage tail fibres are elongated protein assemblies capable of specific recognition of bacterial surfaces during the first step of viral infection1-4. The folding of these complex trimeric structures often requires a phage-encoded tail fibre assembly (Tfa) protein5-7. Despite the wide occurrence of Tfa proteins, their functional mechanism has not been elucidated. Here, we investigate the tail fibre and Tfa of Escherichia coli phage Mu. We demonstrate that Tfa forms a stable complex with the tail fibre, and present a 2.1 Å resolution X-ray crystal structure of this complex. We find that Tfa proteins are comprised of two domains: a non-conserved N-terminal domain that binds to the C-terminal region of the fibre and a conserved C-terminal domain that probably mediates fibre oligomerization and assembly. Tfa forms rapidly exchanging multimers on its own, but not a stable trimer, implying that Tfa does not specify the trimeric state of the fibre. We propose that the key conserved role of Tfa is to ensure that fibre assembly and multimerization initiates at the C terminus, ensuring that the intertwined and repetitive structural elements of fibres come together in the correct sequence. The universal importance of correctly aligning the C termini of phage fibres is highlighted by our work.

Subtle changes in host cell density cause a serious error in monitoring of the intracellular growth of Chlamydia trachomatis in a low-oxygen environment: Proposal for a standardized culture method.

We monitored Chlamydia trachomatis growth in HeLa cells cultured with either DMEM or RPMI medium containing 10% FCS under 2% or 21% O2 conditions for 2 days. Bacterial numbers, host cell numbers, and fibrosis-related gene expression in the host cells were estimated by an inclusion forming unit assay, a cell counting assay, and a PCR array, respectively. In contrast to RPMI, bacterial growth under low oxygen conditions in DMEM rapidly decreased with increasing host cell density. The addition of supplements (glucose, glutamine, vitamin B12, D-biotin, non-essential amino acids, glutathione) to the media had no effect. The growth of host cells in DMEM under low oxygen conditions rapidly decreased, although the cells remained healthy morphologically. Furthermore, the downregulation of 17 genes was observed under low oxygen in DMEM. Whereas no effect on bacterial growth was observed when culturing in RPMI medium at low oxygen, and the downregulation of three genes (CTGF, SERPINE1, JUN) was observed following bacterial infection compared with the uninfected control cells. Thus, our findings indicate the need for carefully selected culture conditions when performing experiments with C. trachomatis under low-oxygen environments, and RPMI (rather than DMEM) is recommended when a low host cell density is to be used, proposing the major modification of cell culturing method of C. trachomatis in a low-oxygen environment.

Impact of bacterial traces belonging to the Enterobacteriaceae on the prevalence of Chlamydia trachomatis in women visiting a community hospital in Japan.

We explored the bacteria present in the vaginal microbiota facilitating the prevalence of Chlamydia trachomatis in women visiting a community hospital in Sapporo, Japan, by amplicon sequencing. A total of 273 cervical swab samples were collected, and bacterial vaginosis was evaluated in all specimens by assessment of the Nugent score. In 16 of the samples, bacterial 16S rDNA could not be detected and they were therefore omitted from subsequent experiments (n = 257). A significant negative correlation was observed between the Nugent scores and the amount of Lactobacillus 16S rDNA. Among the 257 samples, chlamydial plasmid was detected in 20 samples and was used for amplicon sequencing. No significant association between the Nugent score and the prevalence of C. trachomatis was detected. Based on the results of chlamydial plasmid detection and the Nugent score, chlamydia-negative samples (n = 27) were randomly selected. Finally, the number of operational taxonomic units (OTUs) obtained from amplicon sequencing was compared between chlamydia-positive (n = 20) and -negative samples (n = 27), revealing that a significant difference was only detected for the OTU numbers of Enterobacteriaceae between the C. trachomatis-positive and -negative groups. However, almost all of the samples utilized for amplicon sequencing failed to grow on MacConkey agar plates and produce indole. Taken together, we concluded that traces of bacteria, not live bacteria, belonging to the Enterobacteriaceae indicated the flow of bacteria through the anogenital route along with gut indole, and the resulting impact on the prevalence of C. trachomatis in the cervicogenital tract of women in Japan.

TROY is a promising prognostic biomarker in patients with colorectal cancer.

Tumor necrosis factor receptor superfamily member 19 (TROY) is involved in the Wnt/ß-catenin signaling pathway and interacts with leucine-rich repeat containing G-protein-coupled receptor 5 (LGR5), which is a well-known biomarker of cancer stem cells and a prognostic marker of colorectal cancer (CRC). Because there have been no studies to evaluate the prognostic significance of TROY, we performed the present study to determine whether TROY can be a prognostic biomarker in CRC patients. We evaluated TROY expression levels in 100 CRC tissues by quantitative real-time PCR and investigated the association of TROY expression levels with clinicopathologic features. Cancer stage and TROY expression level were found to be independent prognostic factors of disease-free survival. Moreover, TROY overexpression was the sole independent prognostic factor of disease-free survival in patients with stage II and III CRC. These results suggest that analysis of TROY might help predict clinical outcome in patients with CRC. To support our findings, confirmatory studies using independent data sets are needed.

Fusobacterium nucleatum as a prognostic marker of colorectal cancer in a Japanese population.

BACKGROUND: Accumulating evidence shows an overabundance of Fusobacterium nucleatum in colorectal tumor tissues. However, the correlation between the absolute copy number of F. nucleatum in colorectal cancer tissues and colorectal cancer progression is unclear from previous reports. Therefore, we performed a study to compare the abundance of F. nucleatum in colorectal tissues with clinicopathologic and molecular features of colorectal cancer. METHODS: We collected 100 colorectal cancer tissues and 72 matched normal-appearing mucosal tissues. Absolute copy numbers of F. nucleatum were measured by droplet digital PCR. RESULTS: The detection rates of F. nucleatum were 63.9% (46/72) in normal-appearing mucosal tissues and 75.0% (75/100) in CRC tissue samples. The median copy number of F. nucleatum was 0.4/ng DNA in the normal-appearing colorectal mucosa in patients with colorectal cancer and 1.9/ng DNA in the colorectal cancer tissues (P = 0.0031). F. nucleatum copy numbers in stage IV colorectal cancer tissues were significantly higher than those in the normal-appearing mucosa in patients with colorectal cancer (P = 0.0016). The abundance of F. nucleatum in colorectal cancer tissues correlated with tumor size and KRAS mutation and was significantly associated with shorter overall survival times; this trend was notable in the patients with stage IV colorectal cancer. Focusing on normal-appearing mucosa in the patients with colorectal cancer, the F. nucleatum copy number was significantly higher in the patients with stage IV rather than stages I-III. CONCLUSION: These results suggest that determining F. nucleatum levels may help predict clinical outcomes in colorectal cancer patients. Further confirmatory studies using independent datasets are required to confirm our findings.

Walker occupancy has an impact on changing airborne bacterial communities in an underground pedestrian space, as small-dust particles increased with raising both temperature and humidity.

Although human occupancy is a source of airborne bacteria, the role of walkers on bacterial communities in built environments is poorly understood. Therefore, we visualized the impact of walker occupancy combined with other factors (temperature, humidity, atmospheric pressure, dust particles) on airborne bacterial features in the Sapporo underground pedestrian space in Sapporo, Japan. Air samples (n = 18; 4,800L/each sample) were collected at 8:00 h to 20:00 h on 3 days (regular sampling) and at early morning / late night (5:50 h to 7:50 h / 22:15 h to 24:45 h) on a day (baseline sampling), and the number of CFUs (colony forming units) OTUs (operational taxonomic units) and other factors were determined. The results revealed that temperature, humidity, and atmospheric pressure changed with weather. The number of walkers increased greatly in the morning and evening on each regular sampling day, although total walker numbers did not differ significantly among regular sampling days. A slight increase in small dust particles (0.3-0.5µm) was observed on the days with higher temperature regardless of regular or baseline sampling. At the period on regular sampling, CFU levels varied irregularly among days, and the OTUs of 22-phylum types were observed, with the majority being from Firmicutes or Proteobacteria (γ-), including Staphylococcus sp. derived from human individuals. The data obtained from regular samplings reveled that although no direct interaction of walker occupancy and airborne CFU and OTU features was observed upon Pearson's correlation analysis, cluster analysis indicated an obvious lineage consisting of walker occupancy, CFU numbers, OTU types, small dust particles, and seasonal factors (including temperature and humidity). Meanwhile, at the period on baseline sampling both walker and CFU numbers were similarly minimal. Taken together, the results revealed a positive correlation of walker occupancy with airborne bacteria that increased with increases in temperature and humidity in the presence of airborne small particles. Moreover, the results indicated that small dust particles at high temperature and humidity may be a crucial factor responsible for stabilizing the bacteria released from walkers in built environments. The findings presented herein advance our knowledge and understanding of the relationship between humans and bacterial communities in built environments, and will help improve public health in urban communities.

The Role of Epigenetic Regulation in Epstein-Barr Virus-Associated Gastric Cancer.

The Epstein-Barr virus (EBV) is detected in about 10% of gastric carcinoma cases throughout the world. In EBV-associated gastric carcinoma (EBVaGC), all tumor cells harbor the clonal EBV genome. The expression of latent EBV genes is strictly regulated through the methylation of EBV DNA. The methylation of viral DNA regulates the type of EBV latency, and methylation of the tumor suppressor genes is a key abnormality in EBVaGC. The methylation frequencies of several tumor suppressor genes and cell adhesion molecules are significantly higher in EBVaGC than in control cases. EBV-derived microRNAs repress translation from viral and host mRNAs. EBV regulates the expression of non-coding RNA in gastric carcinoma. With regard to the clinical application of demethylating agents against EBVaGC, we investigated the effects of decitabine against the EBVaGC cell lines. Decitabine inhibited the cell growth of EBVaGC cells. The promoter regions of p73 and Runt-related transcription factor 3(RUNX3) were demethylated, and their expression was upregulated by the treatment. We review the role of epigenetic regulation in the development and maintenance of EBVaGC and discuss the therapeutic application of DNA demethylating agents for EBVaGC.

Decitabine inhibits tumor cell proliferation and up-regulates e-cadherin expression in Epstein-Barr virus-associated gastric cancer.

The present study investigated the effect of a DNA demethylating agent, decitabine, against Epstein-Barr virus-associated gastric cancer (EBVaGC). Decitabine inhibited cell growth and induced G2/M arrest and apoptosis in EBVaGC cell lines. The expression of E-cadherin was up-regulated and cell motility was significantly inhibited in the cells treated with decitabine. The promoter regions of p73 and RUNX3 were demethylated, and their expression was up-regulated by decitabine. They enhanced the transcription of p21, which induced G2/M arrest and apoptosis through down-regulation of c-Myc. Decitabine also induced the expression of BZLF1 in SNU719. Induction of EBV lytic infection was an alternative way to cause apoptosis of the host cells. This study is the first report to reveal the effectiveness of a demethylating agent in inhibiting tumor cell proliferation and up-regulation of E-cadherin in EBVaGC. J. Med. Virol. 89:508-517, 2017. © 2016 Wiley Periodicals, Inc.

Highly sensitive stool DNA testing of Fusobacterium nucleatum as a marker for detection of colorectal tumours in a Japanese population.

Background Accumulating evidence shows an over-abundance of Fusobacterium nucleatum in colorectal tumour tissues. Although stool DNA testing of Fusobacterium nucleatum might be a potential marker for the detection of colorectal tumours, the difficulty in detecting Fusobacterium nucleatum in stool by conventional methods prevented further explorations. Therefore, we developed a droplet digital polymerase chain reaction (PCR) assay for detecting Fusobacterium nucleatum in stool and investigated its clinical utility in the management of colorectal tumours in a Japanese population. Methods Feces were collected from 60 healthy subjects (control group) and from 11 patients with colorectal non-advanced adenomas (non-advanced adenoma group), 19 patients with colorectal advanced adenoma/carcinoma in situ (advanced adenoma/carcinoma in situ (CIS) group) and 158 patients with colorectal cancer of stages I to IV (colorectal cancer group). Absolute copy numbers of Fusobacterium nucleatum were measured by droplet digital PCR. Results The median copy number of Fusobacterium nucleatum was 17.5 in the control group, 311 in the non-advanced adenoma group, 122 in the advanced adenoma/CIS group, and 317 in the colorectal cancer group. In comparison with that in the control group, the Fusobacterium nucleatum level was significantly higher in the non-advanced adenoma group, the advanced adenoma/CIS group and the colorectal cancer group. Conclusions This study illustrates the potential of stool DNA testing of Fusobacterium nucleatum by droplet digital PCR to detect individuals with colorectal tumours in a Japanese population.

Surgical intervention strategies for pediatric congenital cystic lesions of the lungs: A 20-year single-institution experience.

BACKGROUND: The aim of this study was to assess surgical intervention strategies for congenital cystic lesions of the lungs (CCL), focusing on the safety of lung resection. MATERIALS AND METHODS: The clinical features of 27 children (CCAM, n=16; bronchial atresia, n=4; bronchogenic cyst, n=3; pulmonary sequestration, n=3; lobar emphysema, n=1) who were treated at our institution between 1995 and 2014 were analyzed. RESULTS: Of the 27 patients, 14 were asymptomatic, and 13 were symptomatic. The youngest symptomatic patient presented with pneumonia at 9months of age. The mean age at surgery was 4months in the asymptomatic group and 4.1years in the symptomatic group. The mean operating time was 167minutes in the asymptomatic group and 275minutes in the symptomatic group (P<0.001). The mean amount of intraoperative bleeding was 15g in the asymptomatic group and 83.4g in the symptomatic group (P<0.05). All of the prenatally diagnosed patients underwent surgery within six months of birth. Three patients had remnant cystic lesions, all of which involved cystic lesions located over the lobulation anomalies of the lung. CONCLUSIONS: To minimize surgical invasiveness, surgery for CCL should be performed during the asymptomatic period or within six months after birth.

Dendritic cells transfected with heat-shock protein 70 messenger RNA for patients with hepatitis C virus-related hepatocellular carcinoma: a phase 1 dose escalation clinical trial.

BACKGROUND: We previously reported overexpression of heat-shock protein (HSP) 70 in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) using proteomic profiling and immunohistochemical staining (IHS). This suggested that HSP70 could be a molecular target for treatment of HCC. METHODS: Twelve patients with HCV-related HCC were enrolled in a phase 1 clinical trial. Dendritic cells (DCs) transfected with HSP70 mRNA (HSP70-DCs) induced by electroporation were injected intradermally. Patients were treated three times every 3 weeks. The number of HSP70-DCs injected was 1 × 10(7) as the lowest dose, then 2 × 10(7) as the medium dose, and then 3 × 10(7) as the highest dose. Immunological analyses were performed. FINDINGS: No adverse effects of grade III/IV, except one grade III liver abscess at the 3 × 10(7) dose, were observed. Thus, we added three more patients to confirm whether 3 × 10(7) is an appropriate dose. Eventually, we chose 3 × 10(7) as the recommended dose of DCs. Complete response (CR) without any recurrence occurred in two patients, stable disease in five, and progression of disease in five. The two patients with CR have had no recurrence for 44 and 33 months, respectively. IHS in one patient who underwent partial hepatectomy showed infiltration of CD8+ T cells and granzyme B in tumors, indicating that the dominant immune effector cells were cytotoxic T lymphocytes with tumor-killing activity. INTERPRETATION: This study demonstrated that HSP70-DCs therapy is both safe and feasible in patients with HCV-related HCC. Further clinical trials should be considered.

Long-fragment DNA as a potential marker for stool-based detection of colorectal cancer.

Neoplastic cells that are exfoliated from the colorectal epithelium exhibit dysfunctional apoptotic mechanisms, and thus it is possible to identify high-molecular-weight DNA fragments (long DNA) in feces. In the present study, the sensitivity and specificity of fecal-based long DNA assays were evaluated for the detection of colorectal cancer (CRC). Feces were collected from 54 healthy volunteers and 130 patients with CRC prior to surgical treatment. The presence of long DNA of the adenomatosis polyposis coli, Kirsten rat sarcoma viral oncogene homolog (KRAS), B-raf proto-oncogene, serine/threonine kinase and p53 genes was assessed by polymerase chain reaction followed by electrophoresis. The identification of long DNA in feces was found to exhibit a sensitivity of 56.2% and specificity of 96.3% for CRC detection. In addition, long DNA was identified in the feces of 58/90 (64.4%) patients with distal CRC and 15/40 (37.5%) patients with proximal CRC. This study indicates the potential of the fecal long DNA assay as a non-invasive and easily performed method for the detection of individuals with CRC.

Epstein-barr virus in gastric carcinoma.

The Epstein-Barr virus (EBV) is detected in about 10% of gastric carcinoma cases throughout the world. In EBV-associated gastric carcinoma, all tumor cells harbor the clonal EBV genome. Gastric carcinoma associated with EBV has distinct clinicopathological features, occurs predominately in men and in younger-aged individuals, and presents a generally diffuse histological type. Most cases of EBV-associated gastric carcinoma exhibit a histology rich in lymphocyte infiltration. The immunological reactiveness in the host may represent a relatively preferable prognosis in EBV-positive cases. This fact highlights the important role of EBV in the development of EBV-associated gastric carcinoma. We have clearly proved direct infection of human gastric epithelialcells by EBV. The infection was achieved by using a recombinant EBV. Promotion of growth by EBV infection was observed in the cells. Considerable data suggest that EBV may directly contribute to the development of EBV-associated GC. This tumor-promoting effect seems to involve multiple mechanisms, because EBV affects several host proteins and pathways that normally promote apoptosis and regulate cell proliferation.

Identification of genes specifically methylated in Epstein-Barr virus-associated gastric carcinomas.

We studied the comprehensive DNA methylation status in the naturally derived gastric adenocarcinoma cell line SNU-719, which was infected with the Epstein-Barr virus (EBV) by methylated CpG island recovery on chip assay. To identify genes specifically methylated in EBV-associated gastric carcinomas (EBVaGC), we focused on seven genes, TP73, BLU, FSD1, BCL7A, MARK1, SCRN1, and NKX3.1, based on the results of methylated CpG island recovery on chip assay. We confirmed DNA methylation of the genes by methylation-specific PCR and bisulfite sequencing in SNU-719. The expression of the genes, except for BCL7A, was upregulated by a combination of 5-Aza-2'-deoxycytidine and trichostatin A treatment in SNU-719. After the treatment, unmethylated DNA became detectable in all seven genes by methylation-specific PCR. We verified DNA methylation of the genes in 75 primary gastric cancer tissues from 25 patients with EBVaGC and 50 EBV-negative patients who were controls. The methylation frequencies of TP73, BLU, FSD1, BCL7A, MARK1, SCRN1, and NKX3.1 were significantly higher in EBVaGC than in EBV-negative gastric carcinoma. We identified seven genes with promoter regions that were specifically methylated in EBVaGC. Inactivation of these genes may suppress their function as tumor suppressor genes or tumor-associated antigens and help to develop and maintain EBVaGC.

Possible involvement of Wnt11 in colorectal cancer progression.

Our previous report revealed that the expression of Frizzled-7 (FZD7) in colorectal cancer (CRC) and its possible role in CRC progression. In this study we measured the expression levels of candidate FZD7 ligands, Wnt3 and Wnt11 in colon cancer cell lines (n = 7) and primary CRC tissues (n = 133) by quantitative RT-PCR. We also examined the functional effects of Wnt11 with the use of Wnt11 transfectants of colon cancer HCT-116 cells. Wnt11 transfectants showed the increased proliferation and migration/invasion activities compared to mock cells. Western blot analysis of transfectants revealed that phosphorylation of JNK and c-jun was increased after Wnt11 transfection. Wnt11 mRNA expression was significantly higher in the stage I, II, III, or IV tumor tissues than in non-tumor tissues (overall P < 0.003), while there was no significant difference in Wnt3 mRNA expression between tumor and non-tumor tissues. In addition, Wnt11 mRNA expression was significantly higher in patients with recurrence or death after surgery than in those with no recurrence (disease free) after surgery (P = 0.018). We also compared the expression levels of Wnt11 mRNA with those of FZD7 mRNA in the same CRC samples. Wnt11 mRNA expression was significantly higher in patients with higher FZD7 mRNA levels than in those with lower FZD7 mRNA levels (P = 0.0005). The expression levels of Wnt11 mRNA were correlated with those of FZD7 mRNA (P < 0.0001). These data suggest that Wnt11 may play an important role in CRC progression.

Role of DNA methylation in the development of Epstein-Barr virus-associated gastric carcinoma.

The frequencies of DNA methylation of certain tumor-related genes are higher in Epstein-Barr virus (EBV)-associated gastric carcinomas than in EBV-negative gastric carcinomas. EBV-associated gastric carcinomas have distinct clinicopathological features; however, there are no case-control studies comparing methylation frequency between EBV-associated gastric carcinomas and controls that have been adjusted according to the clinicopathological features of EBV-associated gastric carcinomas. This study evaluated 25 EBV-associated gastric carcinomas that were positive for EBV-encoded small RNA 1 (EBER-1) by in situ hybridization and 50 EBV-negative gastric carcinomas that were matched with the EBV-associated gastric carcinomas by age, sex, histology, depth of tumor invasion, and stage. Methylation status of 16 loci associated with tumor-related genes was analyzed by methylation-specific polymerase chain reaction (PCR) to identify genes in which DNA methylation specifically occurred in EBV-associated gastric carcinomas. Methylation frequencies of 12 of the 16 genes were higher in EBV-associated gastric carcinomas than in EBV-negative controls, and the frequency of methylation of 6 specific loci (MINT2, MINT31, p14, p16, p73, and RUNX3) was significantly higher in EBV-associated gastric carcinomas than in EBV-negative controls. There were no significant differences in the methylation frequencies of the other genes. The mean methylation index in EBV-associated gastric carcinomas was significantly higher than that in EBV-negative controls. DNA methylation of tumor suppressor genes that regulate the cell cycle and apoptosis specifically occurred in EBV-associated gastric carcinomas. Aberrant DNA methylation might lead to the development and progression of EBV-associated gastric carcinoma.

The importance of evaluation of DNA amplificability in KRAS mutation testing with dideoxy sequencing using formalin-fixed and paraffin-embedded colorectal cancer tissues.

OBJECTIVE: We evaluated DNA amplificability to achieve a 100% success rate in KRAS mutation testing with dideoxy sequencing using formalin-fixed and paraffin-embedded colorectal cancer tissue samples obtained from a recent clinical trial. METHODS: We evaluated the effects of deparaffinization, formalin fixation or storage time, and amplicon size on the amplificability of DNAs extracted from 19 formalin-fixed and paraffin-embedded colorectal cancer tissue samples. We subjected to KRAS mutation analysis 112 samples from metastatic colorectal cancer patients in 31 hospitals enrolled in a Phase II trial of a second-line FOLFIRI (5-fluorouracil+ leucovorin + irinotecan) + cetuximab regimen. RESULTS: Deparaffinization, formalin fixation and storage times did not appear to affect the recovery and amplificability of DNAs. However, amplicon size had a remarkable effect on the amplificability of DNAs. The smaller fragments with a size of ≤278 bp (96-278 bp) were successfully amplified with polymerase chain reaction in all samples tested, whereas the larger fragments with a size of ≥298 bp (298-565 bp) were not amplified. All samples from our clinical trial were successfully analyzed using three sets of primers with the amplicon sizes of 201, 221 and 240 bp, and KRAS mutations in exons 2 and 3 were detected in 49 of the 112 cases (43.8%). CONCLUSIONS: These data suggest that the evaluation of DNA amplificability and amplicon size is important for the success of mutation detection tests such as the KRAS test with dideoxy sequencing using formalin-fixed and paraffin-embedded tissue samples in the clinical setting.

Response-time improved hydrothermal-method-grown ZnO scintillator for soft x-ray free-electron laser timing-observation.

For pump and probe experiments in x-ray free-electron laser (XFEL) facilities, accurate timing synchronization between short-wavelength femtosecond pulses from XFELs and short optical pulses from other light sources is required. For this purpose, the response time of a hydrothermal-method-grown ZnO is improved by over one order of magnitude via intentional iron ion doping. The fluorescence rise- and decay-time constants are measured to be less than 10 and 100 ps, respectively. Owing to its intense fluorescence even for single pulse XFEL excitation, the timing jitter of the soft x-ray pulse and timing electronics are evaluated to be less than 70 ps.

TWIST1 hypermethylation is observed frequently in colorectal tumors and its overexpression is associated with unfavorable outcomes in patients with colorectal cancer.

Although growing evidence demonstrates that TWIST1 is an interesting tumor biomarker, little is known about the clinical significance of TWIST1 expression and TWIST1 methylation in human primary colorectal cancer. In this study, we examined the association of TWIST1 expression and TWIST1 methylation with clinicopathologic features in human primary colorectal tumors. Primary colorectal cancer (CRC) specimens from 319 patients, corresponding normal colorectal nontumorous mucosa from 251 patients with cancer, and colorectal adenomas from 189 patients were used. Methylation and expression levels of TWIST1 were compared with clinicopathologic features. The TWIST1 methylation level was higher in colorectal adenoma and cancer than in normal colorectal mucosa. Elevated TWIST1 mRNA expression in normal colorectal mucosa in patients with CRC as well as in primary CRC specimens was associated with unfavorable outcomes. There was no correlation between TWIST1 methylation and TWIST1 expression. Our results suggest that TWIST1 methylation may be a useful biomarker for screening colorectal tumors. In addition, TWIST1 mRNA expression is a possible molecular marker for predicting the outcome in patients with CRC. Confirmatory studies using independent data sets are needed to confirm our findings.

Rac1-mediated Bcl-2 induction is critical in antigen-induced CD4 single-positive differentiation of a CD4+CD8+ immature thymocyte line.

Rac1, one of the Rho family small guanosine triphosphatases, has been shown to work as a "molecular switch" in various signal transduction pathways. To assess the function of Rac1 in the differentiation process of CD4 single-positive (CD4-SP) T cells from CD4CD8 double-positive (DP) cells, we used a DP cell line DPK, which can differentiate into CD4-SP cells upon TCR stimulation in vitro. DPK expressing dominant-negative (dn)Rac1 underwent massive apoptosis upon TCR stimulation and resulted in defective differentiation of CD4-SP cells. Conversely, overexpression of dnRac2 did not affect differentiation. TCR-dependent actin polymerization was inhibited, whereas early ERK activation was unaltered in dnRac1-expressing DPK. We found that TCR-dependent induction of Bcl-2 was suppressed greatly in dnRac1-expressing DPK, and this suppression was independent of actin rearrangement. Furthermore, introduction of exogenous Bcl-2 inhibited TCR-dependent induction of apoptosis and restored CD4-SP generation in dnRac1-expressing DPK without restoring TCR-induced actin polymerization. Collectively, these data indicate that Rac1 is critical in differentiation of CD4-SP from the DP cell line by preventing TCR-induced apoptosis via Bcl-2 up-regulation.