Eliminating exposure to aqueous solvents is necessary for the early detection and ultrastructural elemental analysis of sites of calcium and phosphorus enrichment in mineralizing UMR106-01 osteoblastic cultures

The mechanism underlying the mineralization of bone is well studied and yet it remains controversial. Inherent difficulties of imaging mineralized tissues and the aqueous solubility of calcium and phosphate, the 2 ions which combine to form bone mineral crystals, limit current analyses of labile diffusible, amorphous, and crystalline intermediates by electron microscopy. To improve the retention of calcium and phosphorus, we developed a pseudo nonaqueous processing approach and used it to characterize biomineralization foci, extracellular sites of hydroxyapatite deposition in osteoblastic cell cultures. Since mineralization of UMR106-01 osteoblasts is temporally synchronized and begins 78 h after plating, we used these cultures to evaluate the effectiveness of our method when applied to cells just prior to the formation of the first mineral crystals. Our approach combines for the first time 3 well-established methods with a fourth one, i.e. dry ultrathin sectioning. Dry ultrathin sectioning with an oscillating diamond knife was used to produce electron spectroscopic images of mineralized biomineralization foci which were high-pressure frozen and freeze substituted. For comparison, cultures were also treated with conventional processing and wet sectioning. The results show that only the use of pseudo nonaqueous processing was able to detect extracellular sites of early calcium and phosphorus enrichment at 76 h, several hours prior to detection of mineral crystals within biomineralization foci.

Ethics and social networking sites : a disclosive analysis of Facebook

Purpose – This paper aims to provide insights into the moral values embodied by a popular social networking site (SNS), Facebook. Design/methodology/approach – This study is based upon qualitative fieldwork, involving participant observation, conducted over a two-year period. The authors adopt the position that technology as well as humans has a moral character in order to disclose ethical concerns that are not transparent to users of the site. Findings – Much research on the ethics of information systems has focused on the way that people deploy particular technologies, and the consequences arising, with a view to making policy recommendations and ethical interventions. By focusing on technology as a moral actor with reach across and beyond the internet, the authors reveal the complex and diffuse nature of ethical responsibility and the consequent implications for governance of SNS. Research limitations/implications – The authors situate their research in a body of work known as disclosive ethics, and argue for an ongoing process of evaluating SNS to reveal their moral importance. Along with that of other authors in the genre, this work is largely descriptive, but the paper engages with prior research by Brey and Introna to highlight the scope for theory development. Practical implications – Governance measures that require the developers of social networking sites to revise their designs fail to address the diffuse nature of ethical responsibility in this case. Such technologies need to be opened up to scrutiny on a regular basis to increase public awareness of the issues and thereby disclose concerns to a wider audience. The authors suggest that there is value in studying the development and use of these technologies in their infancy, or if established, in the experiences of novice users. Furthermore, flash points in technological trajectories can prove useful sites of investigation. Originality/value – Existing research on social networking sites either fails to address ethical concerns head on or adopts a tool view of the technologies so that the focus is on the ethical behaviour of users. The authors focus upon the agency, and hence the moral character, of technology to show both the possibilities for, and limitations of, ethical interventions in such cases.

Networked masculinities and social networking sites : a call for the analysis of men and contemporary digital media

It is of course recognised that technology can be gendered and implicated in gender relations. However, it continues to be the case that men’s experiences with technology are underexplored and the situation is even more problematic where digital media is concerned. Over the past 30 years we have witnessed a dramatic rise in the pervasiveness of digital media across many parts of the world and as associated with wide ranging aspects of our lives. This rise has been fuelled over the last decade by the emergence of Web 2.0 and particularly Social Networking Sites (SNS). Given this context, I believe it is necessary for us to undertake more work to understand men’s engagements with digital media, the implications this might have for masculinities and the analysis of gender relations more generally. To begin to unpack this area, I engage theorizations of the properties of digital media networks and integrate this with the masculinity studies field. Using this framework, I suggest we need to consider the rise in what I call networked masculinities – those masculinities (co)produced and reproduced with digitally networked publics. Through this analysis I discuss themes related to digital mediators, relationships, play and leisure, work and commerce, and ethics. I conclude that as masculinities can be, and are being, complicated and given agency by advancing notions and practices of connectivity, mobility, classification and convergence, those engaged with masculinity studies and digital media have much to contribute.

Three-dimensional analysis of budding sites and released virus suggests a revised model for HIV-1 morphogenesis

Current models of HIV-1 morphogenesis hold that newly synthesized viral Gag polyproteins traffic to and assemble at the cell membrane into spherical protein shells. The resulting late-budding structure is thought to be released by the cellular ESCRT machinery severing the membrane tether connecting it to the producer cell. Using electron tomography and scanning transmission electron microscopy, we find that virions have a morphology and composition distinct from late-budding sites. Gag is arranged as a continuous but incomplete sphere in the released virion. In contrast, late-budding sites lacking functional ESCRT exhibited a nearly closed Gag sphere. The results lead us to propose that budding is initiated by Gag assembly, but is completed in an ESCRT-dependent manner before the Gag sphere is complete. This suggests that ESCRT functions early in HIV-1 release-akin to its role in vesicle formation-and is not restricted to severing the thin membrane tether.

A large-scale analysis of genetic variants within putative miRNA binding sites in prostate cancer

Prostate cancer is the second most common malignancy among men worldwide. Genome-wide association studies have identified 100 risk variants for prostate cancer, which can explain approximately 33% of the familial risk of the disease. We hypothesized that a comprehensive analysis of genetic variations found within the 3' untranslated region of genes predicted to affect miRNA binding (miRSNP) can identify additional prostate cancer risk variants. We investigated the association between 2,169 miRSNPs and prostate cancer risk in a large-scale analysis of 22,301 cases and 22,320 controls of European ancestry from 23 participating studies. Twenty-two miRSNPs were associated (P<2.3×10(-5)) with risk of prostate cancer, 10 of which were within 7 genes previously not mapped by GWAS studies. Further, using miRNA mimics and reporter gene assays, we showed that miR-3162-5p has specific affinity for the KLK3 rs1058205 miRSNP T-allele, whereas miR-370 has greater affinity for the VAMP8 rs1010 miRSNP A-allele, validating their functional role. SIGNIFICANCE Findings from this large association study suggest that a focus on miRSNPs, including functional evaluation, can identify candidate risk loci below currently accepted statistical levels of genome-wide significance. Studies of miRNAs and their interactions with SNPs could provide further insights into the mechanisms of prostate cancer risk.

Additional binding sites in lysozyme. X-ray analysis of lysozyme complexes with bromophenol red and bromophenol blue

The binding sites in hen egg-white lysozyme for neutral bromophenol red (BPR) and ionized bromophenol blue (BPB) have been characterized at 2 Å resolution. In either case, the dye-bound enzyme is active against the polysaccharide, but not against the cell wall. Both binding sites are outside, but close to, the hexasaccharide binding cleft in the enzyme. The binding site of BPR made up of Arg5, Lys33, Phe34, Asn37, Phe38, Ala122, Trp123 and possibly Arg125, is dose to subsite F while that of BPB made up of Tyr20, Arg21, Asn93, Lys96, Lys97 and Ser100, is close to subsites A and B. The binding sites of the neutral dye and the ionized dye are thus spatially far apart. The peptide component of the bacterial cell wall probably interacts with these cells during enzyme action. Such interactions are perhaps necessary for appropriately positioning the enzyme molecule on the bacterial cell wall.

Toxicant distributions and impact models in environmental risk analysis of waste sites

Myrkyllisten aineiden jakaumat ja vaikutusmallit jätealueiden ympäristöriskien analyysissä.

Polypurine-polypyrimidine sequences adopt unwound structure in pBR322 form V DNA as probed by single-hit analysis of HpaII sites.

Structure at the polypurine-polypyrimidine sequences flanking the HpaII sites (CCGG) in pBR322 form V DNA was probed employing single-hit analysis using HpaII restriction endonuclease. Reduced cleavage efficiency of HpaII sites flanked by polypurine-polypyrimidine sequences suggested that under high torsional stress these sequences adopt unwound structures rendering these sites insensitive to restriction enzyme cleavage. In addition to polypurine-polypyrimidine sequences. HpaII sites flanked by alternating purine-pyrimidine sequence, a potential motif of left handed Z-DNA, were also found to be resistant to HpaII cleavage. Results obtained from various studies implicating structure sensitivity of restriction endonucleases and methylases were compiled and a direct correlation was observed between the occurrence of altered sites in a domain and its G/C content in pBR322 form V DNA.

Synthesis and crystal structure of copper (II) uracil ternary polymeric complex with 1,10-phenanthroline along with the Hirshfeld surface analysis of the metal binding sites for the uracil ligand

The study of models for ``metal-enzyme-substrate'' interaction has been a proactive area of research owing to its biological and pharmacological importance. In this regard the ternary copper uracil complex with 1,10-phenanthroline represents metal-enzyme-substrate system for DNA binding enzymes. The synthesis of the complex, followed by slow evaporation of the reaction mixture forms two concomitant solvatomorph crystals viz., {Cu(phen)(mu-ura)(H2O)](n)center dot H2O (1a)} and {Cu(phen)(mu-ura)(H2O)](n)center dot CH3OH (1b)}. Both complexes are structurally characterized, while elemental analysis, IR and EPR spectra were recorded for 1b (major product). In both complexes, uracil coordinates uniquely via N1 and N3 nitrogen atom acting as a bidentate bridging ligand forming a 1-D polymer. The two solvatomorphs were quantitatively analyzed for the differences with the aid of Hirshfeld surface analysis. (C) 2014 Elsevier B.V. All rights reserved.

The Cheshire Meres an analysis of data and a prioritisation of sites

This is a report on the Analysis of Data and a Prioritisation of Sites at the Cheshire Meres by the Institute of Freshwater Ecology. The report addresses data collected by the Agency for 24 basin sites in Cheshire. At least two samples were collected from each site, though not simultaneously. Sites were visited in May/June and in November. The determinands are standard and they included: water, temperature, conductivity, pH, DO, fractional white light penetration, TSS, chlorophyll, TP, ortho-phosphate, nitrate-, nitrite-, ammonium and silicate. Though concentrations were often higher than for other lakes in the region, rather exceeding criteria for classification as eutrophic lakes, the results confirmed that the series of lakes is, naturally, highly eutrophic and nothing in the present data differs so far from expectation that is persuasive that the ecosystems are reacting adversely to environmental stress. The data set is review and summarised, site-by-site, in an appendix. The grounds for prioritisation are discussed. Whether or not this preferred prioritised option is adopted, the Agency is recommended to review the way it carries out monitoring. The determinands and the sampling frequency need to be geared to the information that is required.

Distance between home and work: A multilevel analysis of individual workers, neighbourhoods, and employment sites in Northern Ireland

Abstract. We explore the distances between home and work for employees at twenty-eight different employment sites across Northern Ireland. Substantively, this is important for better understanding the geography of labour catchments. Methodologically, with data on the distances between place of residence (566 wards) and place of work for some 15 000 workers, and the use of multilevel modelling (MLM), the analysis adds to the evidence derived from other census-based and survey-based studies. Descriptive analysis is supplemented with MLM that simultaneously explores individual, neighbourhood, and site variations in travel-to-work patterns using hierarchical and cross-classified model specifications, including individual and ecological predictor variables (and their cross-level interactions). In doing so we apportion variability to different levels and spatial contexts, and also outline the factors that shape spatial mobility. We find, as expected, that factors such as gender and occupation influence the distance between home and work, and also confirm the importance of neighbourhood characteristics (such as population density observed in ecological analyses at ward level) in shaping individual outcomes, with major differences found between urban and rural locations. Beyond this, the analysis of variability also points to the relative significance of residential location, with less individual variability in travel-to-work distance between workers within wards than within employment sites. We conclude by suggesting that, whilst some general ‘rules’ about the factors that shape labour catchments are possible (eg workers in rural areas and in higher occupations travel further than others), the complex variability between places highlighted by the MLM analysis illustrates the salience of place-specific uniqueness.

Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation

The p63 transcription factor (TP63) is critical in development, growth and differentiation of stratifying epithelia. This is highlighted by the severity of congenital abnormalities caused by TP63 mutations in humans, the dramatic phenotypes in knockout mice and de-regulation of TP63 expression in neoplasia altering the tumour suppressive roles of the TP53 family. In order to define the normal role played by TP63 and provide the basis for better understanding how this network is perturbed in disease, we used chromatin immunoprecipitation combined with massively parallel sequencing (ChIP-seq) to identify >7500 high-confidence TP63-binding regions across the entire genome, in primary human neonatal foreskin keratinocytes (HFKs). Using integrative strategies, we demonstrate that only a subset of these sites are bound by TP53 in response to DNA damage. We identify a role for TP63 in transcriptional regulation of multiple genes genetically linked to cleft palate and identify AP-2alpha (TFAP2A) as a co-regulator of a subset of these genes. We further demonstrate that AP-2gamma (TFAP2C) can bind a subset of these regions and that acute depletion of either TFAP2A or TFAP2C alone is sufficient to reduce terminal differentiation of organotypic epidermal skin equivalents, indicating overlapping physiological functions with TP63.

REMA: A computer-based mapping tool for analysis of restriction sites in multiple DNA sequences

REMA is an interactive web-based program which predicts endonuclease cut sites in DNA sequences. It analyses Multiple sequences simultaneously and predicts the number and size of fragments as well as provides restriction maps. The users can select single or paired combinations of all commercially available enzymes. Additionally, REMA permits prediction of multiple sequence terminal fragment sizes and suggests suitable restriction enzymes for maximally discriminatory results. REMA is an easy to use, web based program which will have a wide application in molecular biology research. Availability: REMA is written in Perl and is freely available for non-commercial use. Detailed information on installation can be obtained from Jan Szubert (jan.szubert@gmail.com) and the web based application is accessible on the internet at the URL http://www.macaulay.ac.uk/rema. Contact: b.singh@macaulay.ac.uk. (C) 2007 Elsevier B.V. All rights reserved.