Domain within the helicase subunit Mcm4 integrates multiple kinase signals to control DNA replication initiation and fork progression

Eukaryotic DNA synthesis initiates from multiple replication origins and progresses through bidirectional replication forks to ensure efficient duplication of the genome. Temporal control of initiation from origins and regulation of replication fork functions are important aspects for maintaining genome stability. Multiple kinase-signaling pathways are involved in these processes. The Dbf4-dependent Cdc7 kinase (DDK), cyclin-dependent kinase (CDK), and Mec1, the yeast Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related checkpoint regulator, all target the structurally disordered N-terminal serine/threonine-rich domain (NSD) of mini-chromosome maintenance subunit 4 (Mcm4), a subunit of the mini-chromosome maintenance (MCM) replicative helicase complex. Using whole-genome replication profile analysis and single-molecule DNA fiber analysis, we show that under replication stress the temporal pattern of origin activation and DNA replication fork progression are altered in cells with mutations within two separate segments of the Mcm4 NSD. The proximal segment of the NSD residing next to the DDK-docking domain mediates repression of late-origin firing by checkpoint signals because in its absence late origins become active despite an elevated DNA damage-checkpoint response. In contrast, the distal segment of the NSD at the N terminus plays no role in the temporal pattern of origin firing but has a strong influence on replication fork progression and on checkpoint signaling. Both fork progression and checkpoint response are regulated by the phosphorylation of the canonical CDK sites at the distal NSD. Together, our data suggest that the eukaryotic MCM helicase contains an intrinsic regulatory domain that integrates multiple signals to coordinate origin activation and replication fork progression under stress conditions.Eukaryotic DNA replication initiates from multiple replication origins within each chromosome to duplicate the large genome efficiently. To ensure DNA synthesis occurs once and only once across the genome, cells adopt a two-step process to activate replication origins during two separate stages of the cell-division cycle. The first step is licensing of replication origins, which occurs only when cyclin-dependent kinase (CDK) activity is low. In Saccharomyces cerevisiae, origins of DNA replication are licensed in G1 by the formation of a prereplicative complex (pre-RC). The process begins with the origin recognition complex binding to replication origins and recruiting the licensing factor Cdc6, which facilitates loading of the Cdt1-bound minichromosome maintenance (MCM) complex composed of Mcm2–Mcm7 (Mcm2–7). The hexameric Mcm2–7 is the core of the replicative helicase that unwinds DNA during replication. Within the pre-RC Mcm2–7 is loaded as an inactive double hexamer. The next step, activation of licensed origins (origin firing), occurs throughout the S phase and requires the continuous presence of two kinases, the S phase CDKs and the Dbf4-dependent Cdc7 kinase (DDK). CDK phosphorylates Sld2 and Sld3 to allow their binding to Dpb11 (1, 2), facilitating recruitment of Cdc45 and GINS (composed of protein subunits Sld5, Psf1, Psf2 and Psf3; Go, Ichi, Nii, and San stand for five, one, two, and three in Japanese, respectively) to Mcm2–7 to create an active helicase. DDK phosphorylates Mcm2–7 and blocks an intrinsic initiation inhibitory activity residing in the N terminus of the Mcm4 subunit (3). The concerted action of these S-phase kinases transforms the inactive Mcm2–7 double hexamer into the active helicase complex composed of Cdc45, Mcm2-7, and GINS (the CMG complex) (4–6). Upon initiation, DNA polymerases and other components of the replication machinery are recruited to form replisomes and establish replication forks, where DNA synthesis ensues.Kinase-signaling pathways target various components of the replication machinery. Both CDK and DDK target replication proteins in addition to their essential targets described above. Furthermore, Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related (ATM/ATR) signaling targets components of the CMG helicase complex under replication stress (7–10). In the yeast S. cerevisiae, DNA damage activates the checkpoint kinase Rad53, which phosphorylates both Sld3 and Dbf4 to inhibit late origin firing (11, 12). The yeast ATM/ATR homolog Mec1 also targets Mcm4 (13). The stress-activated protein kinase Hog1 targets an auxiliary replisome component Mrc1 to regulate both origin firing and fork progression (14). Although we now have a better understanding of the essential functions of protein kinases in controlling the initiation of replication, we do not completely understand how the separate kinase signaling pathways are coordinated to regulate both initiation and replication fork progression.The structurally disordered N-terminal serine/threonine-rich domain (NSD) of Mcm4 is a target of multiple kinases, including DDK, CDK, and Mec1 (3, 13, 15, 16). Within this region we have identified two functionally distinct domains that exert different functions and are regulated by different kinase systems even though they overlap extensively in primary amino acid sequences. The segment of the Mcm4 NSD proximal to the DDK-docking domain (DDD) (15), and hence termed “proximal NSD,” blocks initiation until it is phosphorylated by DDK. In contrast, the distal segment of the NSD at the N terminus, away from the DDD, is targeted by additional kinases and contributes positively to promote S-phase progression. In this study we present a comprehensive analysis of the pattern of origin activation, replication fork progression, and the checkpoint response in cells under replication stress caused by the inhibition of ribonucleotide reductase (RNR). We show that the distal and proximal NSD segments contribute differently to origin activation and DNA replication fork progression. Furthermore, they exert opposing effects on checkpoint signaling under replication stress. All these effects are regulated by phosphorylation. We suggest that the Mcm4 NSD, a regulatory domain intrinsic to the replicative helicase, mediates the control of multiple aspects of DNA replication. Our data reveal a sophisticated mechanism to fine-tune S-phase progression in response to changing environments.     

Prognostic CpG Methylation Biomarkers Identified by Methylation Array in Esophageal Squamous Cell Carcinoma Patients

Background: Esophageal squamous cell carcinoma (ESCC) is an aggressive cancer with poor prognosis. We aimed to identify a panel of CpG methylation biomarkers for prognosis prediction of ESCC patients.Methods: Illumina''s GoldenGate methylation array, supervised principal components, Kaplan-Meier survival analyses and Cox regression model were conducted on dissected tumor tissues from a training cohort of 40 ESCC patients to identify potential CpG methylation biomarkers. Pyrosequencing quantitative methylation assay were performed to validate prognostic CpG methylation biomarkers in 61 ESCC patients. The correlation between DNA methylation and RNA expression of a validated marker, SOX17, was examined in a validation cohort of 61 ESCC patients.Results: We identified a panel of nine CpG methylation probes located at promoter or exon1 region of eight genes including DDIT3, FES, FLT3, NTRK3, SEPT5, SEPT9, SOX1, and SOX17, for prognosis prediction in ESCC patients. Risk score calculated using the eight-gene panel statistically predicted poor outcome for patients with high risk score. These eight-gene also showed a significantly higher methylation level in tumor tissues than their corresponding normal samples in all patients analyzed. In addition, we also detected an inverse correlation between CpG hypermethylation and the mRNA expression level of SOX17 gene in ESCC patients, indicating that DNA hypermethylation was responsible for decreased expression of SOX17.Conclusions: This study established a proof-of-concept CpG methylation biomarker panel for ESCC prognosis that can be further validated by multiple cohort studies. Functional characterization of the eight prognostic methylation genes in our biomarker panel could help to dissect the mechanism of ESCC tumorigenesis.     

Comparison of recurrence rates after anterior colporrhaphy for cystocele using three different surgical techniques

AIMS: To compare the recurrence rates after anterior colporrhaphy for cystocele using three different surgical techniques: standard plicating, purse-string, and rolling anterior colporrhaphy. METHODS: A retrospective chart review of women undergoing anterior colporrhaphy for cystocele with or without concomitant operations between 1988 and 2000 was conducted. Women were assessed by vaginal examination pre- and postoperatively at 6 weeks and over 2 years. Grades of cystocele were assessed by Baden-Walker halfway grading system. chi(2) tests and ANOVAs were performed for categorical and continuous variables, respectively. Cox regressions were used to calculate recurrent rates. RESULTS: Among the studied 363 women, 296, 33, and 34 underwent standard plicating, purse-string and rolling techniques, respectively. There was no significant difference in age, parity, menopausal status, or mean length of follow-up among the three groups. The purse-string group revealed a significantly higher risk of recurrence than the standard plicating group (hazard ratio: 2.67; 95% CI: 1.05-6.08). The severity of cystocele was another key role of determining the prognosis for recurrences (grades 3 & 4 relative to grade 1, hazard ratio: 3.35; 95% CI: 1.34-8.38). CONCLUSION: Our study showed that standard plicating and rolling technique provided similar anatomic cure rates. However, the purse-string technique was less effective in restoring vaginal anatomy than standard plicating anterior colporrhaphy.     

Immune concept of human papillomaviruses and related antigens in local cancer milieu of human cervical neoplasia

It is presently the right time for clarifying human papillomavirus (HPV)-associated cellular immunity and clinical implications before global HPV vaccination programs begin. Infection with oncogenic HPV associates with the progression of cervical neoplasia. Both cellular and humoral immune responses are essential for the clearance of HPV-associated cervical lesions. There is increasing evidence that the immune system plays a pivotal role in determining the outcome of HPV infection. Viruses and associated neoplastic cells are proposed to have evolved mechanisms to avoid immune attack. T-cell-mediated immune responses against oncogenic HPV are believed to play a central role in cervical carcinogenesis. The presence of HPV-specific cytotoxic T lymphocytes (CTL) in a majority of human cervical cancer patients provides an approach for further study of their functional role in modulating this malignancy. Tumor-infiltrating lymphocytes (TIL) develop as manifestations of the recognition and defense against malignant cells by the host immune system. Cancer cells may overcome immune surveillance, either by downregulating the proliferation of HPV-specific CTL, or altering the effector compositions of immune cells against HPV infections. TIL in the tumor microenvironment can be functionally inhibited and lose the ability of clonal proliferation as a result of depressed expression of IL-2Ralpha. The upregulation of inhibitory signaling relates to the modulation of the virus- and/or tumor-specific immune responses. Alteration of host genetic susceptibility may also lead to abnormal immune response as a general genomic instability resulting from virus persistence. Induction of HPV-specific immune responses is anticipated as an intimate point for the treatment of cervical neoplasia.     

Usefulness of Upstroke Time per Cardiac Cycle for Cardiovascular and All-Cause Mortality Prediction in Patients with Normal Ankle-Brachial Index

Aim: Abnormal ankle-brachial index (ABI) is regarded as peripheral artery disease and can be used to predict cardiovascular (CV) outcomes. However, the usefulness of ABI for the prediction of CV outcome in patients with normal ABI is limited. Upstroke time per cardiac cycle (UTCC) is recently reported to be associated with mortality in patients with acute myocardial infarction and the elderly. Therefore, we aimed to evaluate UTCC, left ventricular ejection fraction (LVEF), brachial-ankle pulse wave velocity (baPWV), and ABI for the prediction of mortality in patients with normal ABI. Methods: Patients arranged for echocardiographic examinations were enrolled, and 1076 patients with normal ABI were included. ABI, baPWV, and UTCC were measured by an ABI-form device. Results: The median follow-up to mortality was 95 months. There were 88 CV and 244 all-cause deaths. After multivariate analysis, UTCC was associated with increased CV and all-cause mortality ( P ≤ 0.004). Age, diabetes, heart failure, left ventricular hypertrophy, baPWV, and LVEF were also independent predictors of CV and all-cause mortality, but ABI was not. Furthermore, UTCC had a better additive predictive value than ABI, baPWV, and LVEF for CV mortality ( P ≤ 0.012). It also had a better additive predictive value than ABI and LVEF for all-cause mortality ( P ≤ 0.013). Conclusions: UTCC is an independent predictor for CV and all-cause mortality in patients with normal ABI. It also has a better additive predictive value of CV and all-cause mortality than ABI and LVEF. Therefore, UTCC is a simple, novel, and useful parameter for identifying high-risk patients with normal ABI.     

Successful retrieval of dislodged paclitaxel-eluting stent with a nitinol loop snare: a case report

Coronary stent dislodgment or embolization before deployment is a rare but challenging complication in interventional cardiology. Intracoronary embolization of the dislodged stent is associated with a high risk of coronary occlusion, due to thrombus formation and subsequent myocardial infarction. Furthermore, systemic embolization may cause severe cerebrovascular events. Nonsurgical retrieval strategies for this complication have been suggested, but bailout cardiac surgery may be indicated if percutaneous retrieval attempts fail. To our knowledge, this is the first case report of intracoronary drug-eluting stent dislodgment, and successful retrieval was accomplished by a loop snare technique. With the increasing trend of using drug-eluting stents in percutaneous coronary intervention, the likelihood of stent dislodgment or embolization may increase. It should be kept in mind, especially by coronary interventionists, how to manage this complication.     

Twenty-minute pad test: comparison of infusion of 250 ml of water with strong-desire amount in the bladder in women with stress urinary incontinence

OBJECTIVE: The objective was to compare the sensitivity of the 20-min pad test by infusion of 250 ml of water with the strong-desire amount in the bladder in women with stress urinary incontinence (SUI). STUDY DESIGN: Eighty-three women with SUI were enrolled between November 2005 and January 2006. The 20-min pad test by infusion of 250 ml of water was performed before urodynamic study (UDS). The strong-desire amount pad test was done after UDS. The results were analyzed by Pearson's chi(2) and Wilcoxon's signed-rank tests. RESULTS: The sensitivity by infusion of the strong-desire amount was better than infusion of 250 ml of water in the 20-min pad test (P<0.001). In the quantitative study, the two pad tests had fair agreement and the pad weight results of the infusion of the strong-desire amount were statistically higher than the infusion of 250 ml of water (P=0.0004). CONCLUSIONS: The infusion of the strong-desire amount had better sensitivity measured by the 20-min pad test in women with SUI compared with infusion of 250 ml of water in the bladder.     

Laparoscopic-assisted vaginal hysterectomy with in situ morcellation for large uteri

STUDY OBJECTIVE: To estimate whether laparoscopic in situ morcellation (LISM) can facilitate laparoscopic-assisted vaginal hysterectomy (LAVH) for large uteri. DESIGN: Prospective study (Canadian Task Force classification II-1). SETTING: University-affiliated hospital. PATIENTS: In all, 147 women with myoma or adenomyosis weighing more than 500 g from January 2004 through December 2007 were enrolled. The patients were divided into 4 subgroups: patients with uteri weighing 500 to 749 g who had traditional LAVH without LISM (group 1A, n = 69) or with LISM (group 1B, n = 16); and patients with uteri weighing 750 g or more who were treated by traditional LAVH without LISM (group 2A, n = 38) or with LISM (group 2B, n = 24). INTERVENTIONS: Laparoscopic-assisted vaginal hysterectomy with or without LISM. MEASUREMENT AND MAIN RESULTS: No significant differences existed in age, body mass index, preoperative diagnoses, complications, or duration of hospital stay among groups. The mean uterine weights were 608 +/- 75, 597 +/- 66, 989 +/- 179, and 935 +/- 226 g for groups 1A, 1B, 2A, and 2B, respectively. The operative time (120 +/- 16 vs 157 +/- 36 minutes, p <.001; 140 +/- 19 vs 224 +/- 57 minutes, p <.001) were significantly shorter in patients with LISM than without in both groups 1 and 2. The estimated blood loss was highest in group 2A. Six (16%) patients lost more than 500 mL of blood and 3 (8%) of them needed blood transfusions. Conversion to laparotomy occurred in 1 (2.6%) of 38 patients in group 2A. No repeated surgery or surgical mortality occurred. CONCLUSION: Laparoscopic-assisted vaginal hysterectomy with LISM was an efficient and safe procedure for removal of large uteri during LAVH.     

Oxygenated cembranoids from a Formosan soft coral Sinularia gibberosa

Chemical investigation of a Formosan soft coral, Sinularia gibberosa, has led to the isolation of eight oxygenated cembranoids, 1- 8, including seven new compounds, gibberosenes A-G ( 2- 8). None of these compounds were found to be cytotoxic toward a limited panel of cancer cell lines. Compound 1 significantly inhibited the accumulation of the pro-inflammatory COX-2 protein of the LPS-stimulated RAW264.7 macrophage cells.     

Anti-inflammatory cembranoids from the soft corals Sinularia querciformis and Sinularia granosa

Four new cembranoids, querciformolides A-D (1-4), along with two known cembranoids, 7 and 8, have been isolated from the soft coral Sinularia querciformis. Furthermore, chemical investigation of Sinularia granosa has afforded three new cembranoids, querciformolide B (2) and granosolides A (5) and B (6). The structures of the new metabolites were elucidated on the basis of extensive spectroscopic methods, and that of 2 was further confirmed by X-ray diffraction analysis. The absolute configurations of 1 and 2 were determined by a modified Mosher's method. Among these metabolites, 2-6 are rarely found cembranoids possessing a tetrahydrofuran moiety with a 4,7-ether linkage; in addition, 1 is the first epsilon-lactone cembrane found that possesses a tetrahydropyran moiety with a 4,8-ether linkage. None of these compounds were found to be cytotoxic toward a limited panel of cancer cell lines. However, compounds 3, 7, and 8 significantly inhibited the accumulation of the pro-inflammatory iNOS and COX-2 proteins in LPS-stimulated RAW264.7 macrophage cells.