No Origin, No Problem for Yeast DNA Replication

Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins (origins). In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unknown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that these deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism

Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) is a cell-cycle checkpoint effector and inducer of senescence, regulated by p53. Yet, evidence suggests that p21 could also be oncogenic, through a mechanism that has so far remained obscure. We report that a subset of atypical cancerous cells strongly expressing p21 showed proliferation features. This occurred predominantly in p53-mutant human cancers, suggesting p53-independent upregulation of p21 selectively in more aggressive tumour cells. Multifaceted phenotypic and genomic analyses of p21-inducible, p53-null, cancerous and near-normal cellular models showed that after an initial senescence-like phase, a subpopulation of p21-expressing proliferating cells emerged, featuring increased genomic instability, aggressiveness and chemoresistance. Mechanistically, sustained p21 accumulation inhibited mainly the CRL4–CDT2 ubiquitin ligase, leading to deregulated origin licensing and replication stress. Collectively, our data reveal the tumour-promoting ability of p21 through deregulation of DNA replication licensing machinery—an unorthodox role to be considered in cancer treatment, since p21 responds to various stimuli including some chemotherapy drugs

Single Molecule Analysis of Replicated DNA Reveals the Usage of Multiple KSHV Genome Regions for Latent Replication

Kaposi's sarcoma associated herpesvirus (KSHV), an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA). Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors. Our previous studies identified and characterized another latent origin, which supported the replication of plasmids ex-vivo without LANA expression in trans. Therefore identification of an additional origin site prompted us to analyze the entire KSHV genome for replication initiation sites using single molecule analysis of replicated DNA (SMARD). Our results showed that replication of DNA can initiate throughout the KSHV genome and the usage of these regions is not conserved in two different KSHV strains investigated. SMARD also showed that the utilization of multiple replication initiation sites occurs across large regions of the genome rather than a specified sequence. The replication origin of the terminal repeats showed only a slight preference for their usage indicating that LANA dependent origin at the terminal repeats (TR) plays only a limited role in genome duplication. Furthermore, we performed chromatin immunoprecipitation for ORC2 and MCM3, which are part of the pre-replication initiation complex to determine the genomic sites where these proteins accumulate, to provide further characterization of potential replication initiation sites on the KSHV genome. The ChIP data confirmed accumulation of these pre-RC proteins at multiple genomic sites in a cell cycle dependent manner. Our data also show that both the frequency and the sites of replication initiation vary within the two KSHV genomes studied here, suggesting that initiation of replication is likely to be affected by the genomic context rather than the DNA sequences

How to do an evaluation: pitfalls and traps

The recent literature is replete with papers evaluating computational tools (often those operating on 3D structures) for their performance in a certain set of tasks. Most commonly these papers compare a number of docking tools for their performance in cognate re-docking (pose prediction) and/or virtual screening. Related papers have been published on ligand-based tools: pose prediction by conformer generators and virtual screening using a variety of ligand-based approaches. The reliability of these comparisons is critically affected by a number of factors usually ignored by the authors, including bias in the datasets used in virtual screening, the metrics used to assess performance in virtual screening and pose prediction and errors in crystal structures used

Suicide-related behaviors in older patients with new anti-epileptic drug use: data from the VA hospital system

<p>Abstract</p> <p>Background</p> <p>The U.S. Food and Drug Administration (FDA) recently linked antiepileptic drug (AED) exposure to suicide-related behaviors based on meta-analysis of randomized clinical trials. We examined the relationship between suicide-related behaviors and different AEDs in older veterans receiving new AED monotherapy from the Veterans Health Administration (VA), controlling for potential confounders.</p> <p>Methods</p> <p>VA and Medicare databases were used to identify veterans 66 years and older, who received a) care from the VA between 1999 and 2004, and b) an incident AED (monotherapy) prescription. Previously validated ICD-9-CM codes were used to identify suicidal ideation or behavior (suicide-related behaviors cases), epilepsy, and other conditions previously associated with suicide-related behaviors. Each case was matched to controls based on prior history of suicide-related behaviors, year of AED prescription, and epilepsy status.</p> <p>Results</p> <p>The strongest predictor of suicide-related behaviors (N = 64; Controls N = 768) based on conditional logistic regression analysis was affective disorder (depression, anxiety, or post-traumatic stress disorder (PTSD); Odds Ratio 4.42, 95% CI 2.30 to 8.49) diagnosed before AED treatment. Increased suicide-related behaviors were not associated with individual AEDs, including the most commonly prescribed AED in the US - phenytoin.</p> <p>Conclusion</p> <p>Our extensive diagnostic and treatment data demonstrated that the strongest predictor of suicide-related behaviors for older patients newly treated with AED monotherapy was a previous diagnosis of affective disorder. Additional, research using a larger sample is needed to clearly determine the risk of suicide-related behaviors among less commonly used AEDs.</p

How to do an evaluation: pitfalls and traps

The recent literature is replete with papers evaluating computational tools (often those operating on 3D structures) for their performance in a certain set of tasks. Most commonly these papers compare a number of docking tools for their performance in cognate re-docking (pose prediction) and/or virtual screening. Related papers have been published on ligand-based tools: pose prediction by conformer generators and virtual screening using a variety of ligand-based approaches. The reliability of these comparisons is critically affected by a number of factors usually ignored by the authors, including bias in the datasets used in virtual screening, the metrics used to assess performance in virtual screening and pose prediction and errors in crystal structures used

Histone acetylation controls the inactive X chromosome replication dynamics

In mammals, dosage compensation between male and female cells is achieved by inactivating one female X chromosome (Xi). Late replication of Xi was proposed to be involved in the maintenance of its silenced state. Here, we show a highly synchronous replication of the Xi within 1 to 2 h during early-mid S-phase by following DNA replication in living mammalian cells with green fluorescent protein-tagged replication proteins. The Xi was replicated before or concomitant with perinuclear or perinucleolar facultative heterochromatin and before constitutive heterochromatin. Ectopic expression of the X-inactive-specific transcript (Xist) gene from an autosome imposed the same synchronous replication pattern. We used mutations and chemical inhibition affecting different epigenetic marks as well as inducible Xist expression and we demonstrate that histone hypoacetylation has a key role in controlling Xi replication. The epigenetically controlled, highly coordinated replication of the Xi is reminiscent of embryonic genome replication in flies and frogs before genome activation and might be a common feature of transcriptionally silent chromatin

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

<p>Abstract</p> <p>Background</p> <p>Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of <it>KRAS </it>are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells.</p> <p>Methods</p> <p>DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for <it>KRAS </it>amplification by quantitative PCR, and investigated <it>KRAS </it>amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of <it>KRAS </it>knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively.</p> <p>Results</p> <p>DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the <it>KRAS </it>gene locus. Amplification of the <it>KRAS </it>locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). <it>KRAS </it>mutations were identified in two of the three cell lines in which <it>KRAS </it>was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased <it>KRAS </it>copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type <it>KRAS</it>, but not in cells with amplified mutant <it>KRAS</it>. Knock-down of <it>KRAS </it>in gastric cancer cells that carried amplified wild-type <it>KRAS </it>resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity.</p> <p>Conclusion</p> <p>Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified <it>KRAS </it>as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of <it>KRAS </it>amplification and overexpression.</p

Will emergency and surgical patients participate in and complete alcohol interventions? A systematic review

<p>Abstract</p> <p>Background</p> <p>In the everyday surgical life, staff may experience that patients with Alcohol Use Disorders (AUDs) seem reluctant to participate in alcohol intervention programs. The objective was therefore to assess acceptance of screening and intervention as well as adherence to the intervention program among emergency department (ED) and surgical patients with AUDs.</p> <p>Methods</p> <p>A systematic literature search was followed by extraction of acceptance and adherence rates in ED and surgical patients. Numbers needed to screen (NNS) were calculated. Subgroup analyses were carried out based on different study characteristics.</p> <p>Results</p> <p>The literature search revealed 33 relevant studies. Of these, 31 were randomized trials, 28 were conducted in EDs and 31 evaluated the effect of brief alcohol intervention. Follow-up was mainly conducted after six and/or twelve months.</p> <p>Four in five ED patients accepted alcohol screening and two in three accepted participation in intervention. In surgical patients, two in three accepted screening and the intervention acceptance rate was almost 100%. The adherence rate was above 60% for up to twelve months in both ED and surgical patients. The NNS to identify one eligible AUD patient and to get one eligible patient to accept participation in alcohol intervention varied from a few up to 70 patients.</p> <p>The rates did not differ between randomized and non-randomized trials, brief and intensive interventions or validated and self-reported alcohol consumption. Adherence rates were not affected by patients' group allocation and type of follow-up.</p> <p>Conclusions</p> <p>Most emergency and surgical patients with AUD accept participation in alcohol screening and interventions and complete the intervention program.</p