Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the identification of two functional promoters of the Hsa-mir-200b cluster, methylation of which is associated with metastasis or hormone receptor status in advanced breast cancer

MicroRNAs (miRNAs) are small non-coding RNAs of 20 nt in length that are capable of modulating gene expression post-transcriptionally. Although miRNAs have been implicated in cancer, including breast cancer, the regulation of miRNA transcription and the role of defects in this process in cancer is not well understood. In this study we have mapped the promoters of 93 breast cancer-associated miRNAs, and then looked for associations between DNA methylation of 15 of these promoters and miRNA expression in breast cancer cells. The miRNA promoters with clearest association between DNA methylation and expression included a previously described and a novel promoter of the Hsa-mir-200b cluster. The novel promoter of the Hsa-mir-200b cluster, denoted P2, is located 2 kb upstream of the 5′ stemloop and maps within a CpG island. P2 has comparable promoter activity to the previously reported promoter (P1), and is able to drive the expression of miR-200b in its endogenous genomic context. DNA methylation of both P1 and P2 was inversely associated with miR-200b expression in eight out of nine breast cancer cell lines, and in vitro methylation of both promoters repressed their activity in reporter assays. In clinical samples, P1 and P2 were differentially methylated with methylation inversely associated with miR-200b expression. P1 was hypermethylated in metastatic lymph nodes compared with matched primary breast tumours whereas P2 hypermethylation was associated with loss of either oestrogen receptor or progesterone receptor. Hypomethylation of P2 was associated with gain of HER2 and androgen receptor expression. These data suggest an association between miR-200b regulation and breast cancer subtype and a potential use of DNA methylation of miRNA promoters as a component of a suite of breast cancer biomarkers.

Genetic determinants of bone density and fracture risk: State of the art and future directions

Context: Osteoporosis is a common, highly heritable condition that causes substantial morbidity and mortality, the etiopathogenesis of which is poorly understood. Genetic studies are making increasingly rapid progress in identifying the genes involved. Evidence Acquisition and Synthesis: In this review, we will summarize the current understanding of the genetics of osteoporosis based on publications from PubMed from the year 1987 onward. Conclusions: Most genes involved in osteoporosis identified to date encode components of known pathways involved in bone synthesis or resorption, but as the field progresses, new pathways are being identified. Only a small proportion of the total genetic variation involved in osteoporosis has been identified, and new approaches will be required to identify most of the remaining genes.

Genetic, functional, and histopathological evaluation of two C-terminal BRCA1 missense variants

Background: The vast majority of BRCA1 missense sequence variants remain uncharacterised for their possible effect on protein expression and function, and therefore are unclassified in terms of their pathogenicity. BRCA1 plays diverse cellular roles and it is unlikely that any single functional assay will accurately reflect the total cellular implications of missense mutations in this gene. Objective: To elucidate the effect of two BRCA1 variants, 5236G>C (G1706A) and 5242C>A (A1708E) on BRCA1 function, and to survey the relative usefulness of several assays to direct the characterisation of other unclassified variants in BRCA genes. Methods and Results: Data from a range of bioinformatic, genetic, and histopathological analyses, and in vitro functional assays indicated that the 1708E variant was associated with the disruption of different cellular functions of BRCA1. In transient transfection experiments in T47D and 293T cells, the 1708E product was mislocalised to the cytoplasm and induced centrosome amplification in 293T cells. The 1708E variant also failed to transactivate transcription of reporter constructs in mammalian transcriptional transactivation assays. In contrast, the 1706A variant displayed a phenotype comparable to wildtype BRCA1 in these assays. Consistent with functional data, tumours from 1708E carriers showed typical BRCA1 pathology, while tumour material from 1706A carriers displayed few histopathological features associated with BRCA1 related tumours. Conclusions: A comprehensive range of genetic, bioinformatic, and functional analyses have been combined for the characterisation of BRCA1 unclassified sequence variants. Consistent with the functional analyses, the combined odds of causality calculated for the 1706A variant after multifactorial likelihood analysis (1:142) indicates a definitive classification of this variant as "benign". In contrast, functional assays of the 1708E variant indicate that it is pathogenic, possibly through subcellular mislocalisation. However, the combined odds of 262:1 in favour of causality of this variant does not meet the minimal ratio of 1000:1 for classification as pathogenic, and A1708E remains formally designated as unclassified. Our findings highlight the importance of comprehensive genetic information, together with detailed functional analysis for the definitive categorisation of unclassified sequence variants. This combination of analyses may have direct application to the characterisation of other unclassified variants in BRCA1 and BRCA2.

Effect of decellularization on the load-bearing characteristics of articular cartilage matrix

The application of decellularized extracellular matrices to aid tissue regeneration in reconstructive surgery and regenerative medicine has been promising. Several decellularization protocols for removing cellular materials from natural tissues such as heart valves are currently in use. This paper evaluates the feasibility of potential extension of this methodology relative to the desirable properties of load bearing joint tissues such as stiffness, porosity and ability to recover adequately after deformation to facilitate physiological function. Two decellularization protocols, namely: Trypsin and Triton X-100 were evaluated against their effects on bovine articular cartilage, using biomechanical, biochemical and microstructural techniques. These analyses revealed that decellularization with trypsin resulted in severe loss of mechanical stiffness including deleterious collapse of the collagen architecture which in turn significantly compromised the porosity of the construct. In contrast, triton X-100 detergent treatment yielded samples that retain mechanical stiffness relative to that of the normal intact cartilage sample, but the resulting construct contained ruminant cellular constituents. We conclude that both of these common decellularization protocols are inadequate for producing constructs that can serve as effective replacement and scaffolds to regenerate articular joint tissue.

Nonlinear Enzyme Kinetics Can Lead to High Metabolic Flux Control Coefficients: Implications for the Evolution of Dominance

In a classic study, Kacser & Burns (1981, Genetics 97, 639-666) demonstrated that given certain plausible assumptions, the flux in a metabolic pathway was more or less indifferent to the activity of any of the enzymes in the pathway taken singly. It was inferred from this that the observed dominance of most wild-type alleles with respect to loss-of-function mutations did not require an adaptive, meaning selectionist, explanation. Cornish-Bowden (1987, J. theor. Biol. 125, 333-338) showed that the Kacser-Burns inference was not valid when substrate concentrations were large relative to the relevant Michaelis constants. We find that in a randomly constructed functional pathway, even when substrate levels are small, one can expect high values of control coefficients for metabolic flux in the presence of significant nonlinearities as exemplified by enzymes with Hill coefficients ranging from two to six, or by the existence of oscillatory loops. Under these conditions the flux can be quite sensitive to changes in enzyme activity as might be caused by inactivating one of the two alleles in a diploid. Therefore, the phenomenon of dominance cannot be a trivial ''default'' consequence of physiology but must be intimately linked to the manner in which metabolic networks have been moulded by natural selection.

Surface chemical studies of Thiobacillus ferrooxidans with reference to copper tolerance

A strain of Thiobacillus ferrooxidans was adapted to grow at higher concentrations of copper by single step culturing in the presence of 20 g/L (0.314 mol/L) cupric ions added to 9K medium. Exposure to copper results in change in the surface chemistry of the microorganism. The isoelectric point of the adapted strain (pI=4.7) was observed to be at a higher pH than that of the wild unadapted strain(pI=2.0). Compared to the wild strain, the copper adapted strain was found to be more hydrophobic and showed enhanced attachment efficiency to the pyrite mineral. The copper adsorption ability of the adapted strain was also found to be higher than that of the wild strain. Fourier transform infrared spectroscopy of adapted cells suggested that a proteinaceous new cell surface component is synthesized by the adapted strain. Treatment of adapted cells with proteinase-K, resulted in complete loss of tolerance to copper, reduction in copper adsorption and hydrophobicity of the adapted cells. These observations strongly suggest a role played by cell surface modifications of Thiobacillus ferrooxidans in imparting the copper tolerance to the cells and bioleaching of sulphide minerals.

A framing analysis on Nepalese media portrayal of disaster events using news frames and PPRR cycle

This study seeks to understand the prevailing status of Nepalese media portrayal of natural disasters and develop a disaster management framework to improve the effectiveness and efficiency of news production through the continuum of prevention, preparedness, response and recovery (PPRR) phases of disaster management. The study is currently under progress. It is being undertaken in three phases. In phase-1, a qualitative content analysis is conducted. The news contents are categorized in frames as proposed in the 'Framing theory' and pre-defined frames. However, researcher has looked at the theories of the Press, linking to social responsibility theory as it is regarded as the major obligation of the media towards the society. Thereafter, the contents are categorized as per PPRR cycle. In Phase-2, based on the findings of content analysis, 12 in-depth interviews with journalists, disaster managers and community leaders are conducted. In phase-3, based on the findings of content analysis and in-depth interviews, a framework for effective media management of disaster are developed using thematic analysis. As the study is currently under progress hence, findings from the pilot study are elucidated. The response phase of disasters is most commonly reported in Nepal. There is relatively low coverage of preparedness and prevention. Furthermore, the responsibility frame in the news is most prevalent following human interest. Economic consequences and conflict frames are also used while reporting and vulnerability assessment has been used as an additional frame. The outcomes of this study are multifaceted: At the micro-level people will be benefited as it will enable a reduction in the loss of human lives and property through effective dissemination of information in news and other mode of media. They will be ‘well prepared for', 'able to prevent', 'respond to' and 'recover from' any natural disasters. At the meso level the media industry will be benefited and have their own 'disaster management model of news production' as an effective disaster reporting tool which will improve in media's editorial judgment and priority. At the macro-level it will assist government and other agencies to develop appropriate policies and strategies for better management of natural disasters.

Thermal shock characteristics of plasma sprayed mullite coatings

Commercially available mullite (3Al(2)O(3). 2SiO(2)) powders containing oxides of calcium and iron as impurities, have been made suitable for plasma spraying by using an organic binder. Stainless steel substrates covered with Ni-22Cr-10Al-1.0Y bond coat were spray coated with mullite, The 425 mu m thick coatings were subjected to thermal shock cycling under burner rig conditions between 1000 and 1200 degrees C and less than 200 degrees C with holding times of 1, 5, and 30 min. While the coatings withstood as high as 1000 shock cycles without failure between 1000 and 200 degrees C, spallation occurred early at 120 cycles when shocked from 1200 degrees C, The coatings appeared to go through a process of self erosion at high temperatures resulting in loss of material. Also observed were changes attributable to melting of the silicate grains, which smooth down the surface. Oxidation of the bond coat did not appear to influence the failure, These observations were supported by detailed scanning electron microscopy and quantitative chemical composition analysis, differential thermal analysis, and surface roughness measurements.

Determination of the effect of JO146, a CtHtrA inhibitor, on the chlamydial developmental cycle and persistence

The role of Chlamydia trachomatis HtrA (CtHtrA) for the growth and pathogenesis of this organism is presented. Inhibition of CtHtrA led to loss of infectious progeny in Chlamydia, particularly during heat stress or recovery from penicillin persistence. Isolation of CtHtrA inhibitor resistant mutants identified positive selection for mutants in fatty acid related pathways. Importantly, HtrA inhibition was effective against clinical isolates of C. trachomatis. Thus the findings combined indicate that CtHtrA is an important chlamydial growth factor that has the potential to be targeted for the development of anti-chlamydial drugs in the future.

UVB irradiation alters the activities and kinetic properties of the enzymes of energy metabolism in rat lens during aging

Ultraviolet (UV) radiation is one of the major risk factors of cataract (loss of eye-lens transparency). The influence of UVB radiation (300 nm, 100 mu W cm(-2)) on the activity and apparent kinetic constants (K-m and V-max) of rat lens hexokinase (HK;EC2.7.1.1), phosphofructokinase (PFK;EC2.7.1.11), isocitrate dehydrogenase (ICDH;EC1.1.1.41) and malate dehydrogenase (MDH;EC1.1.1.37) of energy metabolism has been investigated by irradiating the lens homogenate of three-and 12-month-old rats. In the three-month-old group specific activities of HK and PFK are reduced by 56 and 43 %, respectively, and there is no change in ICDH and MDH activities after a 24 h exposure. On the other hand, in the 12-month-old group the decreases are 72, 71, 24 and 16 % for HK, PFK. ICDH and MDH, respectively. UVB irradiation increases the apparent K-m of HK and PFK (in both age groups), whereas the K-m of ICDH and MDH is not altered. While the decrease in V-max of these enzymes due to UVB exposure is only marginal in three-month-old rats, it is more pronounced (significant) in 12-month-old rats. A similar decrease in enzyme activities of HK and PFK is also observe upon UVB exposure of the intact rat lens. The photoinduced changes in energy metabolism may in turn have a bearing on lens transparency, particularly at an older age.

Waste not want not: Applying an old adage to the modern issue of pharmaceutical waste

Our predecessors taught us, ‘waste not, want not’ – if we did not waste anything we would always have enough. Unfortunately, we did not heed their sage advice. Over the last three centuries, human kind’s wastefulness, or lack of respect for the finite resources of this planet, has contributed to climate change and negatively impacted on ‘ecosystem services’ with a significant, irreversible loss of biodiversity...

Stability of Dimeric Interface in Banana Lectin: Insight from Molecular Dynamics Simulations

Banana lectin (Banlec) is a homodimeric non-glycosylated protein. It exhibits the b-prism I structure. High-temperature molecular dynamics simulations have been utilized to monitor and understand early stages of thermally induced unfolding of Banlec. The present study elucidates the behavior of the dimeric protein at four different temperatures and compares the structural and conformational changes to that of the minimized crystal structure. The process of unfolding was monitored by following the radius of gyration, the rms deviation of each residue, change in relative solvent accessibility and the pattern of inter- and intra-subunit interactions. The overall study demonstrates that the Banlec dimer is a highly stable structure, and the stability is mostly contributed by interfacial interactions. It maintains its overall conformation during high-temperature (400–500 K) simulations, with only the unstructured loop regions acquiring greater momentum under such condition. Nevertheless, at still higher temperatures (600 K) the tertiary structure is gradually lost which later extends to loss of secondary structural elements. The pattern of hydrogen bonding within the subunit and at the interface across different stages has been analyzed and has provided rationale for its intrinsic high stability.

Urban growth and development of contemporary neighbourhood public space in Kathmandu Valley, Nepal

Rapid and unplanned growth of Kathmandu Valley towns over the past decades has resulted in the haphazard development of new neighbourhoods with significant consequences on their public space. This paper examines the development of public space in the valley’s new neighbourhoods in the context of the current urban growth. A case study approach of three new neighbourhoods was developed to examine the provision of public space with data collected from site observations, interviews with neighbourhood residents and other secondary sources. The cases studies consist of both planned and unplanned new neighbourhoods. Findings reveal a severe loss of public space in the unplanned new neighbourhoods. In planned new neighbourhoods, the provision of public space remains poor in terms of physical features, and thus, does not support community activities and needs. Several factors, which are an outcome of the lack of proper urban growth initiatives and control measures, such as an overall drawback in the formation of new neighbourhoods, the poor capacity of local community-based organisations and the encroachment of public land are responsible for the present development of neighbourhood public space. The problems with ongoing management of public spaces are a significant issue in both unplanned and planned new neighbourhoods.

Effects of mass of charge on loop heat pipe operational characteristics

Experimental results on a loop heat pipe, using R134a as the working fluid, indicates that the liquid inventory in the compensation chamber can significantly influence the operating characteristics. The large liquid inventory in the compensation chamber, under terrestrial conditions, can result in loss of thermal coupling between the compensation chamber and the evaporator core. This causes the operating temperature to increase monotonically. This phenomenon, which has been experimentally observed, is reported in this paper. A theoretical model to predict the steady-state performance of a loop heat pipe with a weak thermal link between the compensation chamber and the core, as observed in the experiment, is also presented. The predicted and the experimentally determined temperatures correlate well.

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme a reductase by cytosolic proteins - real or artifact

The inhibitory effect of FeSO4-dependent cytosolic protein on microsomal HMGCoA reductase is on the enzyme activity and not an artifact of loss of the product, mevalonate, through phosphorylation, unlike that of ATP.Mg effect.