Copper and zinc ligands of pisum sativum and expression of psMT(_A)

The gene, PsMT(_A) is highly expressed in the roots of the garden pea, Pisum sativum. The predicted primary structures of homologues of the PsMT(_A) gene from a range of plant species were compared. Common features are amino and carboxyl terminal domains of approximately 20 residues which are rich in cysteine residues many of which are arranged in the cysteine-Xaa-cysteine (Xaa is not a cysteine) motifs characteristic of metallothionein. The greatest degree of sequence conservation between these predicted gene products occurs within the cysteine rich domains. Two principal (most highly represented in the available sample) categories of sequence were identified on the basis of the arrangement of the cysteine residues within the amino termina domain. A secondary structure motif (β-strand) was predicted (using a computer algorithm) to occur in a conserved position in the central domain linking the two cysteine rich domains of these predicted proteins. This feature is not apparent in the structure of metallothioneins from other species. A recombinant GST-PsMT(_A) fusion protein was isolated from crude lysates of Escherichia coli. containing the plasmid pGEX3X with PsMT(_A) coding sequence. When isolated from Escherichia coli cells grown in zinc supplemented media it was demonstrated that zinc was associated with the PsMT(_A) moiety of the fusion protein. In aqueous solution the PsMT(_A) moiety of the fusion protein formed discreet degradation products. The fusion protein was insoluble at concentrations greater than 20 mg ml(^-1) which rendered it unsuitable for a structural study of the putative metal binding site(s) by (^113)Cd NMR. An antibody to the GST- PsMT(_A) fusion protein was characterised and the epitope was found to lie within the GST moiety. Comparison of the arrangement of cysteines in the amino terminal domains with the different domains of mammalian metallothionein suggested that the two principal categories of predicted plant metallothionein- like gene products may have different affinities for zinc. The predicted products of the metallothionein-like gene highly expressed in the leaves of Arabidopsis thali ana (AtMT-t2) and the PsMT(_A) gene are representative of the two principal categories identified by sequence analysis. The AtMT-t2 coding sequence was amplified from an Arabidopsis thaliana leaf cDNA library and cloned into the pGEX3X plasmid to allow expression of the protein as a fusion to GST in Escherichia coli. Zinc was associated with the AtMT-t2 moiety of the fusion protein. The pH of 50 % displacement for the GST-AtMT-t2 fusion protein with respect to zinc was 0.25 pH units lower than that for the GST-PsMT(_A) fusion protein indicating that putative metallothionein-like protein from Arabidopsis thaliana may have a higher affinity for zinc. It is feasible that this difference allows AtMT-t2 to compete with endogenous ligands of zinc more effectively than PsMT(_A). Expression of the AtMT-t2 gene in a zinc metallothionein deficient strain of Synechococcus (in collaboration with Dr. J.S. Turner) partially restored zinc tolerance to the transformed cells. The similarity of the cysteine rich domains of the predicted metallothionein-like proteins with metallothionein and the demonstration of metal binding in vitro suggested that these genes may be metal regulated. The effect of variations in the exogenous concentration of the trace metals copper zinc and iron on the expression of the PsMT(_A) gene in the roots of Pisum sativum seedlings was investigated by northern analysis. Induction of PsMT(_A) expression was seen with increasing iron concentrations up to 2.0 µM iron chelate. At this concentration of iron chelate, and above, expression of PsMT(_A), decreased. At concentrations of copper above 100 nM induction of PsMT(_A) expression was seen. Below 100 nM copper PsMT(_A) expression increased with decreasing copper concentrations. This response has not been reported for metallothioneins from other species and may be significant for the role of PsMT(_A) in root tissue. In the presence of 2.0 µM iron zinc concentrations above 5.0 µM induced expression of PsMT(_A). The response to changes in exogenous metal concentrations was rapid. Complete repression of PsMT(_A) transcription occurred within 1 h of transfer to media supplemented with 2.0 µM iron. To date no translational products of plant metallothionein-like genes (excluding the E(_c) protein from Tritticum aestivum) have been identified in plant tissue. Two copper and one zinc complex were identified following ion exchange chromatography of soluble extracts from roots of Pisum sativum. The quantity' of the zinc complex eluted from the matrix was reduced by the addition of iron chelate to the growth media. There was no consistent change in the quantity of copper complex recovered in response to iron. Two copper and one zinc component of low molecular weight were identified following gel filtration chromatography of the above complexes. Following polyacrylamide gel electrophoresis of extracts from roots of Pisum sativum, labelled in vivo with (^35)S cysteine, a band was identified with the characteristics predicted for the product of the PsMT(_A) gene. On two dimensional polyacrylamide gels a doublet of spots was identified (migrating to a low pH as predicted for PsMT(_A)) in an extract from roots of seedlings grown in media not supplemented with iron which were not observed on gels of extracts from seedlings grown in media supplemented with iron. The identity of these polypeptides was not established by sequence analysis

One-pot near-ambient temperature syntheses of aryl(difluoroenol) derivatives from trifluoroethanol

Difluoroalkenylzinc reagents prepared from 1-(2’-methoxy-ethoxymethoxy)-2,2,2-trifluoroethane and 1-(N,N-diethylcarbamoyloxy)-2,2,2-trifluoroethane at ice bath temperatures, underwent Negishi coupling with a range of aryl halides in a convenient one pot procedure. While significant differences between the enol acetal and carbamate reagents were revealed, the Negishi protocol compared very favourably with alternative coupling procedures in terms of overall yields from trifluoroethanol

Effect of silver content on the structure and antibacterial activity of silver-doped phosphate-based glasses

Staphylococcus aureus can cause a range of diseases, such as osteomyelitis, as well as colonize implanted medical devices. In most instances the organism forms biofilms that not only are resistant to the body's defense mechanisms but also display decreased susceptibilities to antibiotics. In the present study, we have examined the effect of increasing silver contents in phosphate-based glasses to prevent the formation of S. aureus biofilms. Silver was found to be an effective bactericidal agent against S. aureus biofilms, and the rate of silver ion release (0.42 to 1.22 µg·mm–2·h–1) from phosphate-based glass was found to account for the variation in its bactericidal effect. Analysis of biofilms by confocal microscopy indicated that they consisted of an upper layer of viable bacteria together with a layer (20 µm) of nonviable cells on the glass surface. Our results showed that regardless of the silver contents in these glasses (10, 15, or 20 mol%) the silver exists in its +1 oxidation state, which is known to be a highly effective bactericidal agent compared to that of silver in other oxidation states (+2 or +3). Analysis of the glasses by 31P nuclear magnetic resonance imaging and high-energy X-ray diffraction showed that it is the structural rearrangement of the phosphate network that is responsible for the variation in silver ion release and the associated bactericidal effectiveness. Thus, an understanding of the glass structure is important in interpreting the in vitro data and also has important clinical implications for the potential use of the phosphate-based glasses in orthopedic applications to deliver silver ions to combat S. aureus biofilm infections

Descriptive epidemiology of domain-specific sitting in working adults: the Stormont Study

Background Given links between sedentary behaviour and unfavourable health outcomes, there is a need to understand the influence of socio-demographic factors on sedentary behaviour to inform effective interventions. This study examined domain-specific sitting times reported across socio-demographic groups of office workers. Methods The analyses are cross-sectional and based on a survey conducted within the Stormont Study, which is tracking employees in the Northern Ireland Civil Service. Participants self-reported their daily sitting times across multiple domains (work, TV, travel, PC use and leisure) on workdays and non-workdays, along with their physical activity and socio-demographic variables (sex, age, marital status, BMI, educational attainment and work pattern). Total and domain-specific sitting on workdays and non-workdays were compared across socio-demographic groups using multivariate analyses of covariance. Results Completed responses were obtained from 4436 participants. For the whole sample, total daily sitting times were higher on workdays in comparison to non-workdays (625 ± 168 versus 469 ± 210 min/day, P < 0.001). On workdays and non-workdays, higher sitting times were reported by individuals aged 18–29 years, obese individuals, full-time workers and single/divorced/widowed individuals (P < 0.001). Conclusions Interventions are needed to combat the high levels of sedentary behaviour observed in office workers, particularly among the highlighted demographic groups. Interventions should target workplace and leisure-time sitting

Cost-effectiveness of adult circumcision in a resource-rich setting for HIV prevention among men who have sex with men

Background.&nbsp; We examined the effects and cost‐effectiveness of 4 strategies of circumcision in a resource‐rich setting (Australia) in a population of men who have sex with men (MSM).Method.&nbsp; We created a dynamic mathematical transmission model and performed an economic analysis to estimate the costs, outcomes, and cost‐effectiveness of different strategies, compared with those of the status quo. Strategies included circumcision of all MSM at age 18 years, circumcision of all MSM aged 35&ndash;44 years, circumcision of all insertive MSM aged 18 years, and circumcision of all MSM aged 18 years . All costs are reported in US dollars, with a cost‐effectiveness threshold of 42,000 per quality‐adjusted life‐year.Results.  We find that 2%–5% of human immunodeficiency virus (HIV) infections would be averted per year, with initial costs ranging from 3.6 million to $95.1 million, depending on the strategy. The number of circumcisions needed to prevent 1 HIV infection would range from 118 through 338. Circumcision of predominately insertive MSM would save$21.7 million over 25 years with a $62.2 million investment. Strategies to circumcise 100% of all MSM and to circumcise MSM aged 35&ndash;44 years would be cost‐effective; the latter would require a smaller investment. The least cost‐effective approach is circumcision of young MSM close to their sexual debut. Results are very sensitive to assumptions about the cost of circumcision, the efficacy of circumcision, sexual preferences, and behavioral disinhibition.Conclusions.&nbsp; Circumcision of adult MSM may be cost‐effective in this resource‐rich setting. However, the intervention costs are high relative to the costs spent on other HIV prevention programs.<br /

The role of pre-existing structures during rifting, continental breakup and transform system development, offshore West Greenland

Continental breakup between Greenland and North America produced the small oceanic basins of the Labrador Sea and Baffin Bay, which are connected via the Davis Strait, a region mostly comprised of continental crust. This study contributes to the debate regarding the role of pre-existing structures on rift development in this region using seismic reflection data from the Davis Strait data to produce a series of seismic surfaces, isochrons and a new offshore fault map from which three normal fault sets were identified as (i) NE-SW, (ii) NNW-SSE and (iii) NW-SE. These results were then integrated with plate reconstructions and onshore structural data allowing us to build a two-stage conceptual model for the offshore fault evolution in which basin formation was primarily controlled by rejuvenation of various types of pre-existing structures. During the first phase of rifting between at least Chron 27 (ca. 62 Ma; Palaeocene), but potentially earlier, and Chron 24 (ca. 54 Ma; Eocene) faulting was primarily controlled by pre-existing structures with oblique normal reactivation of both the NE-SW and NW-SE structural sets in addition to possible normal reactivation of the NNW-SSE structural set. In the second rifting stage between Chron 24 (ca. 54 Ma; Eocene) and Chron 13 (ca. 35 Ma; Oligocene), the sinistral Ungava transform fault system developed due to the lateral offset between the Labrador Sea and Baffin Bay. This lateral offset was established in the first rift stage possibly due to the presence of the Nagssugtoqidian and Torngat terranes being less susceptible to rift propagation. Without the influence of pre-existing structures the manifestation of deformation cannot be easily explained during either of the rifting phases. Although basement control diminished into the post-rift, the syn-rift basins from both rift stages continued to influence the location of sedimentation possibly due to differential compaction effects. Variable lithospheric strength through the rifting cycle may provide an explanation for the observed diminishing role of basement structures through time

Fine-scale climate change: modelling spatial variation in biologically meaningful rates of warming

The existence of fine‐grain climate heterogeneity has prompted suggestions that species may be able to survive future climate change in pockets of suitable microclimate, termed ‘microrefugia’. However, evidence for microrefugia is hindered by lack of understanding of how rates of warming vary across a landscape. Here, we present a model that is applied to provide fine‐grained, multidecadal estimates of temperature change based on the underlying physical processes that influence microclimate. Weather station and remotely derived environmental data were used to construct physical variables that capture the effects of terrain, sea surface temperatures, altitude and surface albedo on local temperatures, which were then calibrated statistically to derive gridded estimates of temperature. We apply the model to the Lizard Peninsula, United Kingdom, to provide accurate (mean error = 1.21 °C; RMS error = 1.63 °C) hourly estimates of temperature at a resolution of 100 m for the period 1977–2014. We show that rates of warming vary across a landscape primarily due to long‐term trends in weather conditions. Total warming varied from 0.87 to 1.16 °C, with the slowest rates of warming evident on north‐east‐facing slopes. This variation contributed to substantial spatial heterogeneity in trends in bioclimatic variables: for example, the change in the length of the frost‐free season varied from +11 to −54 days and the increase in annual growing degree‐days from 51 to 267 °C days. Spatial variation in warming was caused primarily by a decrease in daytime cloud cover with a resulting increase in received solar radiation, and secondarily by a decrease in the strength of westerly winds, which has amplified the effects on temperature of solar radiation on west‐facing slopes. We emphasize the importance of multidecadal trends in weather conditions in determining spatial variation in rates of warming, suggesting that locations experiencing least warming may not remain consistent under future climate change

Examining Criminogenic Risk Levels Among People with Mental Illness Incarcerated in US Jails and Prisons

This study examines criminogenic risk levels of individuals with serious mental illness (SMI) involved in the justice system compared to justice-involved individuals without mental illness. The sample (N = 436) consisted of ninety-three individuals with SMI incarcerated in a county jail in a mid-size Midwest city, 217 individuals with SMI incarcerated in a state prison in the US Northeast, and 126 individuals without mental illness incarcerated in a state prison in the US Southwest. Results indicated that people with SMI incarcerated in jail and prison had higher overall criminal risk levels than prison inmates without mental illness. Results further demonstrated that, on average, higher percentages of persons with SMI had high/very high criminogenic risk scores. Finally, we noted that persons with SMI scored higher on most of the eight criminogenic risk domains measured by the Level of Service Inventory. These findings are possibly the most compelling to date in the growing body of literature demonstrating that justice-involved people with SMI have elevated criminogenic risk comparable to or greater than their non-mentally ill peers involved in the justice system. Consequently, treatment programs and interventions for justice-involved individuals with SMI need to explicitly target criminogenic needs into treatment efforts

A comparison of collision cross section values obtained via travelling wave ion mobility-mass spectrometry and ultra high performance liquid chromatography-ion mobility-mass spectrometry : application to the characterisation of metabolites in rat urine

A comprehensive Collision Cross Section (CCS) library was obtained via Travelling Wave Ion Guide mobility measurements through direct infusion (DI). The library consists of CCS and Mass Spectral (MS) data in negative and positive ElectroSpray Ionisation (ESI) mode for 463 and 479 endogenous metabolites, respectively. For both ionisation modes combined, TWCCSN2 data were obtained for 542 non-redundant metabolites. These data were acquired on two different ion mobility enabled orthogonal acceleration QToF MS systems in two different laboratories, with the majority of the resulting TWCCSN2 values (from detected compounds) found to be within 1% of one another. Validation of these results against two independent, external TWCCSN2 data sources and predicted TWCCSN2 values indicated to be within 1-2% of these other values. The same metabolites were then analysed using a rapid reversed-phase ultra (high) performance liquid chromatographic (U(H)PLC) separation combined with IM and MS (IM-MS) thus providing retention time (tr), m/z and TWCCSN2 values (with the latter compared with the DI-IM-MS data). Analytes for which TWCCSN2 values were obtained by U(H)PLC-IM-MS showed good agreement with the results obtained from DI-IM-MS. The repeatability of the TWCCSN2 values obtained for these metabolites on the different ion mobility QToF systems, using either DI or LC, encouraged the further evaluation of the U(H)PLC-IM-MS approach via the analysis of samples of rat urine, from control and methotrexate-treated animals, in order to assess the potential of the approach for metabolite identification and profiling in metabolic phenotyping studies. Based on the database derived from the standards 63 metabolites were identified in rat urine, using positive ESI, based on the combination of tr, TWCCSN2 and MS data.</p