A p53-independent role for the MDM2 antagonist Nutlin-3 in DNA damage response initiation.

BACKGROUND: The mammalian DNA-damage response (DDR) has evolved to protect genome stability and maximize cell survival following DNA-damage. One of the key regulators of the DDR is p53, itself tightly regulated by MDM2. Following double-strand DNA breaks (DSBs), mediators including ATM are recruited to the site of DNA-damage. Subsequent phosphorylation of p53 by ATM and ATM-induced CHK2 results in p53 stabilization, ultimately intensifying transcription of p53-responsive genes involved in DNA repair, cell-cycle checkpoint control and apoptosis. METHODS: In the current study, we investigated the stabilization and activation of p53 and associated DDR proteins in response to treatment of human colorectal cancer cells (HCT116p53+/+) with the MDM2 antagonist, Nutlin-3. RESULTS: Using immunoblotting, Nutlin-3 was observed to stabilize p53, and activate p53 target proteins. Unexpectedly, Nutlin-3 also mediated phosphorylation of p53 at key DNA-damage-specific serine residues (Ser15, 20 and 37). Furthermore, Nutlin-3 induced activation of CHK2 and ATM - proteins required for DNA-damage-dependent phosphorylation and activation of p53, and the phosphorylation of BRCA1 and H2AX - proteins known to be activated specifically in response to DNA damage. Indeed, using immunofluorescent labeling, Nutlin-3 was seen to induce formation of γH2AX foci, an early hallmark of the DDR. Moreover, Nutlin-3 induced phosphorylation of key DDR proteins, initiated cell cycle arrest and led to formation of γH2AX foci in cells lacking p53, whilst γH2AX foci were also noted in MDM2-deficient cells. CONCLUSION: To our knowledge, this is the first solid evidence showing a secondary role for Nutlin-3 as a DDR triggering agent, independent of p53 status, and unrelated to its role as an MDM2 antagonist

Macrocyclic colibactin induces DNA double-strand breaks via copper-mediated oxidative cleavage.

Colibactin is an assumed human gut bacterial genotoxin, whose biosynthesis is linked to the clb genomic island that has a widespread distribution in pathogenic and commensal human enterobacteria. Colibactin-producing gut microbes promote colon tumour formation and enhance the progression of colorectal cancer via cellular senescence and death induced by DNA double-strand breaks (DSBs); however, the chemical basis that contributes to the pathogenesis at the molecular level has not been fully characterized. Here, we report the discovery of colibactin-645, a macrocyclic colibactin metabolite that recapitulates the previously assumed genotoxicity and cytotoxicity. Colibactin-645 shows strong DNA DSB activity in vitro and in human cell cultures via a unique copper-mediated oxidative mechanism. We also delineate a complete biosynthetic model for colibactin-645, which highlights a unique fate of the aminomalonate-building monomer in forming the C-terminal 5-hydroxy-4-oxazolecarboxylic acid moiety through the activities of both the polyketide synthase ClbO and the amidase ClbL. This work thus provides a molecular basis for colibactin's DNA DSB activity and facilitates further mechanistic study of colibactin-related colorectal cancer incidence and prevention

Disparities and risks of sexually transmissible infections among men who have sex with men in China: a meta-analysis and data synthesis.

BACKGROUND: Sexually transmitted infections (STIs), including Hepatitis B and C virus, are emerging public health risks in China, especially among men who have sex with men (MSM). This study aims to assess the magnitude and risks of STIs among Chinese MSM. METHODS: Chinese and English peer-reviewed articles were searched in five electronic databases from January 2000 to February 2013. Pooled prevalence estimates for each STI infection were calculated using meta-analysis. Infection risks of STIs in MSM, HIV-positive MSM and male sex workers (MSW) were obtained. This review followed the PRISMA guidelines and was registered in PROSPERO. RESULTS: Eighty-eight articles (11 in English and 77 in Chinese) investigating 35,203 MSM in 28 provinces were included in this review. The prevalence levels of STIs among MSM were 6.3% (95% CI: 3.5-11.0%) for chlamydia, 1.5% (0.7-2.9%) for genital wart, 1.9% (1.3-2.7%) for gonorrhoea, 8.9% (7.8-10.2%) for hepatitis B (HBV), 1.2% (1.0-1.6%) for hepatitis C (HCV), 66.3% (57.4-74.1%) for human papillomavirus (HPV), 10.6% (6.2-17.6%) for herpes simplex virus (HSV-2) and 4.3% (3.2-5.8%) for Ureaplasma urealyticum. HIV-positive MSM have consistently higher odds of all these infections than the broader MSM population. As a subgroup of MSM, MSW were 2.5 (1.4-4.7), 5.7 (2.7-12.3), and 2.2 (1.4-3.7) times more likely to be infected with chlamydia, gonorrhoea and HCV than the broader MSM population, respectively. CONCLUSION: Prevalence levels of STIs among MSW were significantly higher than the broader MSM population. Co-infection of HIV and STIs were prevalent among Chinese MSM. Integration of HIV and STIs healthcare and surveillance systems is essential in providing effective HIV/STIs preventive measures and treatments. TRIAL REGISTRATION: PROSPERO NO: CRD42013003721

The importance of interacting climate modes on Australia’s contribution to global carbon cycle extremes

The global carbon cycle is highly sensitive to climate-driven fluctuations of precipitation, especially in the Southern Hemisphere. This was clearly manifested by a 20% increase of the global terrestrial C sink in 2011 during the strongest sustained La Niña since 1917. However, inconsistencies exist between El Niño/La Niña (ENSO) cycles and precipitation in the historical record; for example, significant ENSO-precipitation correlations were present in only 31% of the last 100 years, and often absent in wet years. To resolve these inconsistencies, we used an advanced temporal scaling method for identifying interactions amongst three key climate modes (El Niño, the Indian Ocean dipole, and the southern annular mode). When these climate modes synchronised (1999-2012), drought and extreme precipitation were observed across Australia. The interaction amongst these climate modes, more than the effect of any single mode, was associated with large fluctuations in precipitation and productivity. The long-term exposure of vegetation to this arid environment has favoured a resilient flora capable of large fluctuations in photosynthetic productivity and explains why Australia was a major contributor not only to the 2011 global C sink anomaly but also to global reductions in photosynthetic C uptake during the previous decade of drought

STK295900, a Dual Inhibitor of Topoisomerase 1 and 2, Induces G<inf>2</inf> Arrest in the Absence of DNA Damage

STK295900, a small synthetic molecule belonging to a class of symmetric bibenzimidazoles, exhibits antiproliferative activity against various human cancer cell lines from different origins. Examining the effect of STK295900 in HeLa cells indicates that it induces G2 phase arrest without invoking DNA damage. Further analysis shows that STK295900 inhibits DNA relaxation that is mediated by topoisomerase 1 (Top 1) and topoisomerase 2 (Top 2) in vitro. In addition, STK295900 also exhibits protective effect against DNA damage induced by camptothecin. However, STK295900 does not affect etoposide-induced DNA damage. Moreover, STK295900 preferentially exerts cytotoxic effect on cancer cell lines while camptothecin, etoposide, and Hoechst 33342 affected both cancer and normal cells. Therefore, STK295900 has a potential to be developed as an anticancer chemotherapeutic agent. © 2013 Kim et al

Membrane Surface Nanostructures and Adhesion Property of T Lymphocytes Exploited by AFM

The activation of T lymphocytes plays a very important role in T-cell-mediated immune response. Though there are many related literatures, the changes of membrane surface nanostructures and adhesion property of T lymphocytes at different activation stages have not been reported yet. However, these investigations will help us further understand the biophysical and immunologic function of T lymphocytes in the context of activation. In the present study, the membrane architectures of peripheral blood T lymphocytes were obtained by AFM, and adhesion force of the cell membrane were measured by acquiring force–distance curves. The results indicated that the cell volume increased with the increases of activation time, whereas membrane surface adhesion force decreased, even though the local stiffness for resting and activated cells is similar. The results provided complementary and important data to further understand the variation of biophysical properties of T lymphocytes in the context of in vitro activation

DNA Suspension Arrays: Silencing Discrete Artifacts for High-Sensitivity Applications

Detection of low frequency single nucleotide polymorphisms (SNPs) has important implications in early screening for tumorgenesis, genetic disorders and pathogen drug resistance. Nucleic acid arrays are a powerful tool for genome-scale SNP analysis, but detection of low-frequency SNPs in a mixed population on an array is problematic. We demonstrate a model assay for HIV-1 drug resistance mutations, wherein ligase discrimination products are collected on a suspension array. In developing this system, we discovered that signal from multiple polymorphisms was obscured by two discrete hybridization artifacts. Specifically: 1) tethering of unligated probes on the template DNA elicited false signal and 2) unpredictable probe secondary structures impaired probe capture and suppressed legitimate signal from the array. Two sets of oligonucleotides were used to disrupt these structures; one to displace unligated reporter labels from the bead-bound species and another to occupy sequences which interfered with array hybridization. This artifact silencing system resulted in a mean 21-fold increased sensitivity for 29 minority variants of 17 codons in our model assay for mutations most commonly associated with HIV-1 drug resistance. Furthermore, since the artifacts we characterized are not unique to our system, their specific inhibition might improve the quality of data from solid-state microarrays as well as from the growing number of multiple analyte suspension arrays relying on sequence-specific nucleic acid target capture

Differential attraction and repulsion of Staphylococcus aureus and Pseudomonas aeruginosa on molecularly smooth titanium films

Magnetron sputtering techniques were used to prepare molecularly smooth titanium thin films possessing an average roughness between 0.18 nm and 0.52 nm over 5 μm × 5 μm AFM scanning areas. Films with an average roughness of 0.52 nm or lower were found to restrict the extent of P. aeruginosa cell attachment, with less than 0.5% of all available cells being retained on the surface. The attachment of S. aureus cells was also limited on films with an average surface roughness of 0.52 nm, however they exhibited a remarkable propensity for attachment on the nano-smoother 0.18 nm average surface roughness films, with the attachment density being almost twice as great as that observed on the nano-rougher film. The difference in attachment behaviour can be attributed to the difference in morphology of the rod-shaped P. aeruginosa compared to the spherical S. aureus cells

The impact of point mutations in the human androgen receptor : classification of mutations on the basis of transcriptional activity

Peer reviewedPublisher PD