Human origin recognition complex binds to the region of the latent origin of DNA replication of Epstein-Barr virus

Epstein-Barr virus (EBV) replicates in its latent phase once per cell cycle in proliferating B cells. The latent origin of DNA replication, oriP, supports replication and stable maintenance of the EBV genome. OriP comprises two essential elements: the dyad symmetry (DS) and the family of repeats (FR), both containing clusters of binding sites for the transactivator EBNA1. The DS element appears to be the functional replicator. It is not yet understood how oriP-dependent replication is integrated into the cell cycle and how EBNA1 acts at the molecular level. Using chromatin immunoprecipitation experiments, we show that the human origin recognition complex (hsORC) binds at or near the DS element. The association of hsORC with oriP depends on the DS element. Deletion of this element not only abolishes hsORC binding but also reduces replication initiation at oriP to background level. Co-immunoprecipitation experiments indicate that EBNA1 is associated with hsORC in vivo. These results indicate that oriP might use the same cellular initiation factors that regulate chromosomal replication, and that EBNA1 may be involved in recruiting hsORC to oriP.     

Separate SCF(CDC4) recognition elements target Cdc6 for proteolysis in S phase and mitosis

The Cdc6 DNA replication initiation factor is targeted for ubiquitin-mediated proteolysis by the E3 ubiquitin ligase SCF(CDC4) from the end of G1phase until mitosis in the budding yeast Saccharomyces cerevisiae. Here we describe a dominant-negative CDC6 mutant that, when overexpressed, arrests the cell cycle by inhibiting cyclin-dependent kinases (CDKs) and, thus, prevents passage through mitosis. This mutant protein inhibits CDKs more efficiently than wild-type Cdc6, in part because it is completely refractory to SCF(CDC4)-mediated proteolysis late in the cell cycle and consequently accumulates to high levels. The mutation responsible for this phenotype destroys a putative CDK phosphorylation site near the middle of the Cdc6 primary amino acid sequence. We show that this site lies within a novel Cdc4-interacting domain distinct from a Cdc4-interacting site identified previously near the N-terminus of the protein. We show that both sites can target Cdc6 for proteolysis in late G1/early S phase whilst only the newly identified site can target Cdc6 for proteolysis during mitosis.     

Treatment and outcome of patients with metastatic NSCLC: a retrospective institution analysis of 493 patients

Background

Most patients with metastatic non-small cell lung cancer (NSCLC) will face treatment with systemic therapy. Current clinical studies are demonstrating improvements in chemotherapy and overall survival. However, it remains unclear whether these results are translated into clinical practice.

Methods

We reviewed all stage IV NSCLC patients without second malignancies that were diagnosed from 2004 to 2006 at our institution. 493 consecutive patients were included into this retrospective analysis and were followed-up until end of 2011.

Results

352 patients (71.4%) received systemic therapy for up to 7 lines. For most patients, adjustments of dosages or applications had to be made at some point of the treatment, but the total applied dose remained generally close to the intended dose. The best disease control (BDC) rate decreased with increasing therapy lines from 59.7% to about 35%. Patients with palliative local therapy but no systemic treatment demonstrated inferior survival (median 2.9 versus 8.7 months, p < 0.001). The median interval between last treatment and death was 50 days and 15 days for chemotherapy and anti-EGFR therapy, respectively. BDC to the previous therapy lines was predictive for improved BDC to third- but not second-line therapy. Performing multivariate analysis, BDC to previous therapy, never-/ former-smoking status, and age > 70 years were associated with improved survival performing third-line therapy.

Conclusions

Stage IV NSCLC patients may receive substantial systemic therapy resulting in response and median survival rates that are comparable to data from clinical studies. However, preselection factors are increasingly important to improve therapy outcome and life quality.     

Expression and localization of trefoil factor family genes in rat submandibular glands

The trefoil factor (TFF) family, which comprises TFF1, TFF2 and TFF3, plays an essential role in epithelial regeneration within the gastrointestinal tract. All three TFFs are present in human saliva; TFF3 is the predominant trefoil peptide. Little is known about the expression and tissue distribution of TFFs in rats, which are commonly used as a model system for human studies. We investigated the localization of the TFF genes that encode secretory peptides in rat submandibular glands (SMG). All three TFFs were expressed in rat SMG, although their location varied. Substantial amounts of TFF1 were detected only in the cytoplasm of epithelial cells in the SMG granular convoluted tubules (GCT), while TFF2 and TFF3 were widely distributed in the cytoplasm of epithelial cells of intercalated ducts (ID), striated ducts (SD) and interlobular ducts (ILD). The three TFFs also were detected especially in the lumens of the SD and ILD. Semi-quantitative RT-PCR and in situ hybridization experiments confirmed TFF1, TFF2 and TFF3 mRNA expressions in the SMG. Greater expression of TFF peptides and mRNA was observed in male rats than in females. The broad expression of TFFs in rat SMG cells and lumens suggests that TFFs function in this organ by their secretion into the duct lumens. We also found differences in TFF expression profiles between rat and human SMG; therefore, caution should be exercised when using rats as a model for human TFF studies.     

tigaR: integrative significance analysis of temporal differential gene expression induced by genomic abnormalities

Background

To determine which changes in the host cell genome are crucial for cervical carcinogenesis, a longitudinal in vitro model system of HPV-transformed keratinocytes was profiled in a genome-wide manner. Four cell lines affected with either HPV16 or HPV18 were assayed at 8 sequential time points for gene expression (mRNA) and gene copy number (DNA) using high-resolution microarrays. Available methods for temporal differential expression analysis are not designed for integrative genomic studies.

Results

Here, we present a method that allows for the identification of differential gene expression associated with DNA copy number changes over time. The temporal variation in gene expression is described by a generalized linear mixed model employing low-rank thin-plate splines. Model parameters are estimated with an empirical Bayes procedure, which exploits integrated nested Laplace approximation for fast computation. Iteratively, posteriors of hyperparameters and model parameters are estimated. The empirical Bayes procedure shrinks multiple dispersion-related parameters. Shrinkage leads to more stable estimates of the model parameters, better control of false positives and improvement of reproducibility. In addition, to make estimates of the DNA copy number more stable, model parameters are also estimated in a multivariate way using triplets of features, imposing a spatial prior for the copy number effect.

Conclusion

With the proposed method for analysis of time-course multilevel molecular data, more profound insight may be gained through the identification of temporal differential expression induced by DNA copy number abnormalities. In particular, in the analysis of an integrative oncogenomics study with a time-course set-up our method finds genes previously reported to be involved in cervical carcinogenesis. Furthermore, the proposed method yields improvements in sensitivity, specificity and reproducibility compared to existing methods. Finally, the proposed method is able to handle count (RNAseq) data from time course experiments as is shown on a real data set.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-327) contains supplementary material, which is available to authorized users.     

Is the MCM2-7 complex the eukaryotic DNA replication fork helicase?

The MCM2-7 complex is essential for both the initiation and elongation phases of eukaryotic chromosome replication. There is some evidence that MCM2-7 proteins may act as a DNA helicase; at the same time, a variety of other DNA helicases have also been implicated in the replication of eukaryotic chromosomes.     

Do leucocytes reflect condition in nestling burrowing parrots Cyanoliseus patagonus in the wild?

The different leucocyte types are an important part of the immune system. Thus, they have been used in ecological studies to assess immune function and physiological stress in wild birds. It is generally assumed that increased stress and decreased condition are associated with an increase in the ratio of heterophils to lymphocytes, the H/L ratio. We studied leucocyte profiles in relation to body condition in nestling Burrowing Parrots (Cyanoliseus patagonus) in North-eastern Patagonia, Argentina. As in other wild parrots, heterophils were the most numerous leucocyte type, suggesting strong investment into innate immunity. Leucocyte profiles did not change with the age, while nestlings in better body condition increased the number of heterophils. Because the number of lymphocytes was independent of body condition, as a result we observed a positive correlation between body condition and the H/L ratio. The total number of leucocytes relative to erythrocytes increased in nestlings in better body condition, indicating a larger overall investment into immune function in well-nourished nestlings. The observed heterophilic profiles of nestling Burrowing Parrots together with the positive relationship between H/L ratio and body condition may indicate a favoured investment in a robust innate immunity that reduces the risk of infection taking hold in these long-lived birds.     

Is Intracranial Atherosclerosis an Independent Risk Factor for Cerebral Atrophy? A Retrospective Evaluation

Background

Our purpose was to study the association between the intracranial atherosclerosis as measured by cavernous carotid artery calcification (ICAC) observed on head CT and atrophic changes of supra-tentorial brain demonstrated by MRI.

Methods

Institutional review board approval was obtained for this retrospective study incorporating 65 consecutive patients presenting acutely who had both head CT and MRI. Arterial calcifications of the intracranial cavernous carotids (ICAC) were assigned a number (1 to 4) in the bone window images from CT scans. These 4 groups were then combined into high (grades 3 and 4) and low calcium (grades 1 and 2) subgroups. Brain MRI was independently evaluated to identify cortical and central atrophy. Demographics and cardiovascular risk factors were evaluated in subjects with high and low ICAC. Relationship between CT demonstrated ICAC and brain atrophy patterns were evaluated both without and with adjustment for cerebral ischemic scores and cardiovascular risk factors.

Results

Forty-six of the 65 (71%) patients had high ICAC on head CT. Subjects with high ICAC were older, and had higher prevalence of hypertension, diabetes, coronary artery disease (CAD), atrial fibrillation and history of previous stroke (CVA) compared to those with low ICAC. Age demonstrated strong correlation with both supratentorial atrophy patterns. There was no correlation between ICAC and cortical atrophy. There was correlation however between central atrophy and ICAC. This persisted even after adjustment for age.

Conclusion

Age is the most important determinant of atrophic cerebral changes. However, high ICAC demonstrated age independent association with central atrophy.

     

The cyclin-dependent kinase Cdc28p regulates distinct modes of Cdc6p proteolysis during the budding yeast cell cycle

BACKGROUND: Cdc28p, the major cyclin-dependent kinase in budding yeast, prevents re-replication within each cell cycle by preventing the reassembly of Cdc6p-dependent pre-replicative complexes (pre-RCs) once origins have fired. Cdc6p is a rapidly degraded protein that must be synthesised in each cell cycle and is present only during the G1 phase. RESULTS: We found that, at different times in the cell cycle, there are distinct modes of Cdc6p proteolysis. Before Start, Cdc6p proteolysis did not require either the anaphase-promoting complex (APC/C) or the SCF complex, which mediate the major cell cycle regulated ubiquitination pathways, nor did it require Cdc28p activity or any of the potential Cdc28p phosphorylation sites in Cdc6p. In fact, the activation of B cyclin (Clb)-Cdc28p kinase inactivated this pathway of Cdc6p degradation later in the cell cycle. Activation of the G1 cyclins (Clns) caused Cdc6p degradation to become extremely rapid. This degradation required the SCF(CDC4) and Cdc28p consensus sites in Cdc6p, but did not require Clb5 and Clb6. Later in the cell cycle, SCF(CDC4)-dependent Cdc6p proteolysis remained active but became less rapid. CONCLUSIONS: Levels of Cdc6p are regulated in several ways by the Cdc28p cyclin-dependent kinase. The Cln-dependent elimination of Cdc6p, which does not require the S-phase-promoting cyclins Clb5 and Clb6, suggests that the ability to assemble pre-RCs is lost before, not concomitant with, origin firing.