5-Hydroxymethylcytosine is a predominantly stable DNA modification.

5-Hydroxymethylcytosine (hmC) is an oxidation product of 5-methylcytosine which is present in the deoxyribonucleic acid (DNA) of most mammalian cells. Reduction of hmC levels in DNA is a hallmark of cancers. Elucidating the dynamics of this oxidation reaction and the lifetime of hmC in DNA is fundamental to understanding hmC function. Using stable isotope labelling of cytosine derivatives in the DNA of mammalian cells and ultrasensitive tandem liquid-chromatography mass spectrometry, we show that the majority of hmC is a stable modification, as opposed to a transient intermediate. In contrast with DNA methylation, which occurs immediately during replication, hmC forms slowly during the first 30 hours following DNA synthesis. Isotopic labelling of DNA in mouse tissues confirmed the stability of hmC in vivo and demonstrated a relationship between global levels of hmC and cell proliferation. These insights have important implications for understanding the states of chemically modified DNA bases in health and disease.We would like to acknowledge the CRUK CI Flow Cytometry and Histopathology/ISH core facilities for their contributions, David Oxley, Clive d’Santos and Donna Michelle-Smith for their support with mass spectrometry, Xiangang Zou for his help with mES cells and David Tannahill for critical reading of the manuscript. This work was funded by Cancer Research UK (all authors) and the Wellcome Trust Senior Investigator Award (S.B.).This is the accepted manuscript. The final version is available from Nature Chemistry at http://www.nature.com/nchem/journal/vaop/ncurrent/full/nchem.2064.html

Caveolin-1 protects B6129 mice against Helicobacter pylori gastritis.

Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25+ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies ("humming bird") compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells

A novel route for catalytic activation of peroxymonosulfate by oxygen vacancies improved bismuth-doped titania for the removal of recalcitrant organic contaminant

In this work, bismuth-doped titania (BixTiO2) with improved oxygen vacancies was synthesized by sol-gel protocol as a novel peroxymonosulfate (PMS, HSO5−) activator. HSO5− and adsorbed oxygen molecules could efficiently be transformed into their respective radicals through defect ionization to attain charge balance after their trapping on oxygen vacancies of the catalyst. XRD study of BixTiO2 with 5 wt% Bi (5BiT) revealed anatase, crystalline nature, and successful doping of Bi into TiO2 crystal lattice. The particle size obtained from BET data and SEM observations was in good agreement. PL spectra showed the formation rates of •OH by 3BiT, 7BiT, 5BiTC, and 5BiT as 0.720, 1.200, 1.489, and 2.153 μmol/h, respectively. 5BiT catalyst with high surface area (216.87 m2 g−1) and high porosity (29.81%) was observed the excellent HSO5− activator. The catalytic performance of 0BiT, 3BiT, 5BiT, and 7BiT when coupled with 2 mM HSO5− for recalcitrant flumequine (FLU) removal under dark was 10, 27, 55, and 37%, respectively. Only 5.4% decrease in catalytic efficiency was observed at the end of seventh cyclic run. Radical scavenging studies indicate that SO4•− is the dominant species that caused 62.0% degradation. Moreover, strong interaction between Bi and TiO2 through Bi-O-Ti bonds prevents Bi leaching (0.081 mg L−1) as shown by AAS. The kinetics, degradation pathways, ecotoxicity, and catalytic mechanism for recalcitrant FLU were also elucidated. Cost-efficient, environment-friendly, and high mineralization recommends this design strategy; BixTiO2/HSO5− system is a promising advanced oxidation process for the aquatic environment remediation

Defending the genome from the enemy within:mechanisms of retrotransposon suppression in the mouse germline

The viability of any species requires that the genome is kept stable as it is transmitted from generation to generation by the germ cells. One of the challenges to transgenerational genome stability is the potential mutagenic activity of transposable genetic elements, particularly retrotransposons. There are many different types of retrotransposon in mammalian genomes, and these target different points in germline development to amplify and integrate into new genomic locations. Germ cells, and their pluripotent developmental precursors, have evolved a variety of genome defence mechanisms that suppress retrotransposon activity and maintain genome stability across the generations. Here, we review recent advances in understanding how retrotransposon activity is suppressed in the mammalian germline, how genes involved in germline genome defence mechanisms are regulated, and the consequences of mutating these genome defence genes for the developing germline

Synergistic activity of polarised osteoblasts inside condensations cause their differentiation

Condensation of pre-osteogenic, or pre-chondrogenic, cells is the first of a series of processes that initiate skeletal development. We present a validated, novel, three-dimensional agent-based model of in vitro intramembranous osteogenic condensation. The model, informed by system heterogeneity and relying on an interaction-reliant strategy, is shown to be sensitive to 'rules' capturing condensation growth and can be employed to track activity of individual cells to observe their macroscopic impact. It, therefore, makes available previously inaccessible data, offering new insights and providing a new context for exploring the emergence, as well as normal and abnormal development, of osteogenic structures. Of the several stages of condensation we investigate osteoblast 'burial' within the osteoid they deposit. The mechanisms underlying entrapment - required for osteoblasts to differentiate into osteocytes - remain a matter of conjecture with several hypotheses claiming to capture this important transition. Computational examination of this transition indicates that osteoblasts neither turn off nor slow down their matrix secreting genes - a widely held view; nor do they secrete matrix randomly. The model further reveals that osteoblasts display polarised behaviour to deposit osteoid. This is both an important addition to our understanding of condensation and an important validation of the model's utility

Medulloblastoma and ependymoma cells display levels of 5-carboxylcytosine and elevated TET1 expression

Background Alteration of DNA methylation (5-methylcytosine, 5mC) patterns represents one of the causes of tumorigenesis and cancer progression. Tet proteins can oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine and 5-carboxylcytosine (5caC). Although the roles of these oxidised forms of 5mC (oxi-mCs) in cancer pathogenesis are still largely unknown, there are indications that they may be involved in the mechanisms of malignant transformation. Thus, reduction of 5hmC content represents an epigenetic hallmark of human tumours and, according to our recent report; 5caC is enriched in a proportion of breast cancers and gliomas. Nevertheless, the distribution of oxi-mCs in paediatric brain tumours has not been assessed. Findings Here we analyse the global levels and spatial distribution of 5hmC and 5caC in 4 brain tumour cell lines derived from paediatric sonic hedgehog (SHH) pathway activated medulloblastomas (Daoy and UW228-3) and ependymomas (BXD-1425EPN and DKFZEP1NS). We show that, unlike HeLa cells, the paediatric tumour cell lines possess both 5hmC and 5caC at immunochemically detectable levels, and demonstrate that both modifications display high degrees of spatial overlap in the nuclei of medulloblastomas and ependymomas. Moreover, although 5hmC levels are comparable in the 4 brain tumour cell lines, 5caC staining intensities differ dramatically between them with highest levels of this mark in a subpopulation of DKFZ-EP1NS cells. Remarkably, the 5caC enrichment does not correlate with 5hmC levels and is not associated with alterations in Thymine DNA Glycosylase (TDG) expression in SHH medulloblastoma and ependymoma cell lines, but corresponds to elevated levels of TET1 transcript in UW228-3 and DKFZ-EP1NS cells. Conclusions We demonstrate that both 5caC enrichment and elevated TET1 expression are observed in SHH medulloblastomas and ependymomas. Our results suggest that increased Tet-dependent 5mC oxidation may represent one of the epigenetic signatures of cancers with neural stem cell origin and, thus, may contribute to development of novel approaches for diagnosis and therapy of the brain tumours

A multidisciplinary systematic review of the use of diagrams as a means of collecting data from research subjects: application, benefits and recommendations

BACKGROUND: In research, diagrams are most commonly used in the analysis of data and visual presentation of results. However there has been a substantial growth in the use of diagrams in earlier stages of the research process to collect data. Despite this growth, guidance on this technique is often isolated within disciplines. METHODS: A multidisciplinary systematic review was performed, which included 13 traditional healthcare and non-health-focused indexes, non-indexed searches and contacting experts in the field. English-language articles that used diagrams as a data collection tool and reflected on the process were included in the review, with no restriction on publication date. RESULTS: The search identified 2690 documents, of which 80 were included in the final analysis. The choice to use diagrams for data collection is often determined by requirements of the research topic, such as the need to understand research subjects' knowledge or cognitive structure, to overcome cultural and linguistic differences, or to understand highly complex subject matter. How diagrams were used for data collection varied by the degrees of instruction for, and freedom in, diagram creation, the number of diagrams created or edited and the use of diagrams in conjunction with other data collection methods. Depending on how data collection is structured, a variety of options for qualitative and quantitative analysis are available to the researcher. The review identified a number of benefits to using diagrams in data collection, including the ease with which the method can be adapted to complement other data collection methods and its ability to focus discussion. However it is clear that the benefits and challenges of diagramming depend on the nature of its application and the type of diagrams used. DISCUSSION/CONCLUSION: The results of this multidisciplinary systematic review examine the application of diagrams in data collection and the methods for analyzing the unique datasets elicited. Three recommendations are presented. Firstly, the diagrammatic approach should be chosen based on the type of data needed. Secondly, appropriate instructions will depend on the approach chosen. And thirdly, the final results should present examples of original or recreated diagrams. This review also highlighted the need for a standardized terminology of the method and a supporting theoretical framework

Low bone mass in microscopic colitis

<p>Abstract</p> <p>Background</p> <p>Microscopic colitis presents with similar symptoms to classic inflammatory bowel diseases. Osteoporosis is a common complication of Crohn's disease but there are no data concerning bone metabolism in microscopic colitis.</p> <p>Aims</p> <p>The aim of the present study was to evaluate bone density and metabolism in patients with microscopic colitis.</p> <p>Methods</p> <p>Fourteen patients microscopic colitis were included in the study, and 28 healthy persons and 28 age and gender matched Crohn's disease patients were enrolled as controls. Bone mineral density was measured using dual x-ray absorptiometry at the lumbar spine, femoral neck and the radius. Serum bone formation and bone resorption markers (osteocalcin and beta-crosslaps, respectively) were measured using immunoassays.</p> <p>Results</p> <p>Low bone mass was measured in 57.14% patients with microscopic colitis. Bone mineral density at the femoral neck in patients suffering from microscopic colitis and Crohn's disease was lower than in healthy controls (0.852 ± 0.165 and 0.807 ± 0.136 vs. 1.056 ± 0.126 g/cm²; p < 0.01). Bone mineral density at the non-dominant radius was decreased in microscopic colitis patients (0.565 ± 0.093 vs. 0.667 ± 0.072 g/cm²; p < 0.05) but unaffected in Crohn's disease patients (0.672 ± 0.056 g/cm²). Mean beta-crosslaps concentration was higher in microscopic colitis and Crohn's disease patients than controls (417.714 ± 250.37 and 466.071 ± 249.96 vs. 264.75 ± 138.65 pg/ml; p < 0.05). A negative correlation between beta-crosslaps concentration and the femoral and radius t-scores was evident in microscopic colitis patients.</p> <p>Conclusions</p> <p>Low bone mass is frequent in microscopic colitis, and alterations to bone metabolism are similar to those present in Crohn's disease. Therefore, microscopic colitis-associated osteopenia could be a significant problem in such patients.</p

Chemokine gene expression in lung CD8 T cells correlates with protective immunity in mice immunized intra-nasally with Adenovirus-85A

<p>Abstract</p> <p>Background</p> <p>Immunization of BALB/c mice with a recombinant adenovirus expressing <it>Mycobacterium tuberculosis </it>(<it>M. tuberculosis</it>) antigen 85A (Ad85A) protects against aerosol challenge with <it>M. tuberculosis </it>only when it is administered intra-nasally (i.n.). Immunization with Ad85A induces a lung-resident population of activated CD8 T cells that is antigen dependent, highly activated and mediates protection by early inhibition of <it>M. tuberculosis </it>growth. In order to determine why the i.n. route is so effective compared to parenteral immunization, we used microarray analysis to compare gene expression profiles of pulmonary and splenic CD8 T cells after i.n. or intra-dermal (i.d.) immunization.</p> <p>Method</p> <p>Total RNA from CD8 T cells was isolated from lungs or spleens of mice immunized with Ad85A by the i.n. or i.d. route. The gene profiles generated from each condition were compared. Statistically significant (p ≤ 0.05) differentially expressed genes were analyzed to determine if they mapped to particular molecular functions, biological processes or pathways using Gene Ontology and Panther DB mapping tools.</p> <p>Results</p> <p>CD8 T cells from lungs of i.n. immunized mice expressed a large number of chemokines chemotactic for resting and activated T cells as well as activation and survival genes. Lung lymphocytes from i.n. immunized mice also express the chemokine receptor gene <it>Cxcr6</it>, which is thought to aid long-term retention of antigen-responding T cells in the lungs. Expression of CXCR6 on CD8 T cells was confirmed by flow cytometry.</p> <p>Conclusions</p> <p>Our microarray analysis represents the first <it>ex vivo </it>study comparing gene expression profiles of CD8 T cells isolated from distinct sites after immunization with an adenoviral vector by different routes. It confirms earlier phenotypic data indicating that lung i.n. cells are more activated than lung i.d. CD8 T cells. The sustained expression of chemokines and activation genes enables CD8 T cells to remain in the lungs for extended periods after i.n. immunization. This may account for the early inhibition of <it>M. tuberculosis </it>growth observed in Ad85A i.n. immunized mice and explain the effectiveness of i.n. compared to parenteral immunization with this viral vector.</p