Quantification and confocal imaging of protein specific molecularly imprinted polymers

We have employed FITC-albumin as the protein template molecule in an aqueous phase molecular imprinted polymer (HydroMIP) strategy. For the first time, the use of a fluorescently labelled template is reported, with subsequent characterisation of the smart material to show that the HydroMIP possess a significant molecular memory in comparison to that of the nonimprinted control polymer (HydroNIP). The imaging of the FITC-albumin imprinted HydroMIP using confocal microscopy is described, with the in situ removal of imprinted protein displayed in terms of observed changes in the fluorescence of the imprinted polymer, both before and after template elution (using a 10% SDS/10% AcOH (w/v) solution). We also report the imaging of a bovine haemoglobin (BHb) imprinted HydroMIP using two-photon confocal microscopy, and describe the effects of template elution upon protein autofluorescence. The findings further contribute to the understanding of aqueous phase molecular imprinting protocols, and document the use of fluorescence as a useful tool in template labelling/detection and novel imaging strategies

Making sense of the early-2000s warming slowdown

It has been claimed that the early-2000s global warming slowdown or hiatus, characterized by a reduced rate of global surface warming, has been overstated, lacks sound scientific basis, or is unsupported by observations. The evidence presented here contradicts these claims

Cloning, expression and characterization of l-asparaginase from Withania somnifera L. for large scale production

l-Asparaginase (E.C. 3.5.1.1) is used as a therapeutic agent in the treatment of acute childhood lymphoblastic leukemia. It is found in a variety of organisms such as microbes, plants and mammals. In plants, l-asparaginase enzymes are required to catalyze the release of ammonia from asparagine, which is the main nitrogen-relocation molecule in these organisms. An Indian medicinal plant, Withania somnifera was reported as a novel source of l-asparaginase. l-Asparaginase from W. somnifera was cloned and overexpressed in E. coli. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (Km, kcat) for a number of substrates were determined. The kinetic parameters of selected substrates were determined at various pH and the pH- and temperature-dependence profiles were analyzed. WA gene successfully cloned into E. coli BL21 (DE3) showed high asparaginase activity with a specific activity of 17.3 IU/mg protein

An empirical model for probabilistic decadal prediction: global attribution and regional hindcasts

Empirical models, designed to predict surface variables over seasons to decades ahead, provide useful benchmarks for comparison against the performance of dynamical forecast systems; they may also be employable as predictive tools for use by climate services in their own right. A new global empirical decadal prediction system is presented, based on a multiple linear regression approach designed to produce probabilistic output for comparison against dynamical models. A global attribution is performed initially to identify the important forcing and predictor components of the model . Ensemble hindcasts of surface air temperature anomaly fields are then generated, based on the forcings and predictors identified as important, under a series of different prediction ‘modes’ and their performance is evaluated. The modes include a real-time setting, a scenario in which future volcanic forcings are prescribed during the hindcasts, and an approach which exploits knowledge of the forced trend. A two-tier prediction system, which uses knowledge of future sea surface temperatures in the Pacific and Atlantic Oceans, is also tested, but within a perfect knowledge framework. Each mode is designed to identify sources of predictability and uncertainty, as well as investigate different approaches to the design of decadal prediction systems for operational use. It is found that the empirical model shows skill above that of persistence hindcasts for annual means at lead times of up to 10 years ahead in all of the prediction modes investigated. It is suggested that hindcasts which exploit full knowledge of the forced trend due to increasing greenhouse gases throughout the hindcast period can provide more robust estimates of model bias for the calibration of the empirical model in an operational setting. The two-tier system shows potential for improved real-time prediction, given the assumption that skilful predictions of large-scale modes of variability are available. The empirical model framework has been designed with enough flexibility to facilitate further developments, including the prediction of other surface variables and the ability to incorporate additional predictors within the model that are shown to contribute significantly to variability at the local scale. It is also semi-operational in the sense that forecasts have been produced for the coming decade and can be updated when additional data becomes available

Irreducible uncertainty in near-term climate projections

Model simulations of the next few decades are widely used in assessments of climate change impacts and as guidance for adaptation. Their non-linear nature reveals a level of irreducible uncertainty which it is important to understand and quantify, especially for projections of near-term regional climate. Here we use large idealised initial condition ensembles of the FAMOUS global climate model with a 1 %/year compound increase in CO2 levels to quantify the range of future temperatures in model-based projections. These simulations explore the role of both atmospheric and oceanic initial conditions and are the largest such ensembles to date. Short-term simulated trends in global temperature are diverse, and cooling periods are more likely to be followed by larger warming rates. The spatial pattern of near-term temperature change varies considerably, but the proportion of the surface showing a warming is more consistent. In addition, ensemble spread in inter-annual temperature declines as the climate warms, especially in the North Atlantic. Over Europe, atmospheric initial condition uncertainty can, for certain ocean initial conditions, lead to 20 year trends in winter and summer in which every location can exhibit either strong cooling or rapid warming. However, the details of the distribution are highly sensitive to the ocean initial condition chosen and particularly the state of the Atlantic meridional overturning circulation. On longer timescales, the warming signal becomes more clear and consistent amongst different initial condition ensembles. An ensemble using a range of different oceanic initial conditions produces a larger spread in temperature trends than ensembles using a single ocean initial condition for all lead times. This highlights the potential benefits from initialising climate predictions from ocean states informed by observations. These results suggest that climate projections need to be performed with many more ensemble members than at present, using a range of ocean initial conditions, if the uncertainty in near-term regional climate is to be adequately quantified

Reporting and evaluating genetic association studies

Genetic association studies have become an important part of our scientific landscape. This commentary discusses some basic scientific issues which should be considered when reporting and evaluating such studies including SNP Discovery, Genotyping and Haplotype Analysis; Population Size, Matching of Cases and Controls, and Population Stratification; Phenotype Definition and Multiple Related Phenotypes; Multiple Testing; Replication; Genome-wide Association Studies (GWAS); and the Role of Functional Studies. All of these elements are important in evaluating such studies and should be carefully considered when these studies are conceived and carried out

Plasmin activity promotes amyloid deposition in a transgenic model of human transthyretin amyloidosis

Cardiac ATTR amyloidosis, a serious but much under-diagnosed form of cardiomyopathy, is caused by deposition of amyloid fibrils derived from the plasma protein transthyretin (TTR), but its pathogenesis is poorly understood and informative in vivo models have proved elusive. Here we report the generation of a mouse model of cardiac ATTR amyloidosis with transgenic expression of human TTRS52P. The model is characterised by substantial ATTR amyloid deposits in the heart and tongue. The amyloid fibrils contain both full-length human TTR protomers and the residue 49-127 cleavage fragment which are present in ATTR amyloidosis patients. Urokinase-type plasminogen activator (uPA) and plasmin are abundant within the cardiac and lingual amyloid deposits, which contain marked serine protease activity; knockout of α2-antiplasmin, the physiological inhibitor of plasmin, enhances amyloid formation. Together, these findings indicate that cardiac ATTR amyloid deposition involves local uPA-mediated generation of plasmin and cleavage of TTR, consistent with the previously described mechano-enzymatic hypothesis for cardiac ATTR amyloid formation. This experimental model of ATTR cardiomyopathy has potential to allow further investigations of the factors that influence human ATTR amyloid deposition and the development of new treatments

A falls prevention programme to improve quality of life, physical function and falls efficacy in older people receiving home help services: study protocol for a randomised controlled trial

BACKGROUND: Falls and fall-related injuries in older adults are associated with great burdens, both for the individuals, the health care system and the society. Previous research has shown evidence for the efficiency of exercise as falls prevention. An understudied group are older adults receiving home help services, and the effect of a falls prevention programme on health-related quality of life is unclear. The primary aim of this randomised controlled trial is to examine the effect of a falls prevention programme on quality of life, physical function and falls efficacy in older adults receiving home help services. A secondary aim is to explore the mediating factors between falls prevention and health-related quality of life. METHODS: The study is a single-blinded randomised controlled trial. Participants are older adults, aged 67 or older, receiving home help services, who are able to walk with or without walking aids, who have experienced at least one fall during the last 12 months and who have a Mini Mental State Examination of 23 or above. The intervention group receives a programme, based on the Otago Exercise Programme, lasting 12 weeks including home visits and motivational telephone calls. The control group receives usual care. The primary outcome is health-related quality of life (SF-36). Secondary outcomes are leg strength, balance, walking speed, walking habits, activities of daily living, nutritional status and falls efficacy. All measurements are performed at baseline, following intervention at 3 months and at 6 months' follow-up. Sample size, based on the primary outcome, is set to 150 participants randomised into the two arms, including an estimated 15-20% drop out. Participants are recruited from six municipalities in Norway. DISCUSSION: This trial will generate new knowledge on the effects of an exercise falls prevention programme among older fallers receiving home help services. This knowledge will be useful for clinicians, for health managers in the primary health care service and for policy makers

Common ADRB2 Haplotypes Derived from 26 Polymorphic Sites Direct β2-Adrenergic Receptor Expression and Regulation Phenotypes

The beta2-adrenergic receptor (beta2AR) is expressed on numerous cell-types including airway smooth muscle cells and cardiomyocytes. Drugs (agonists or antagonists) acting at these receptors for treatment of asthma, chronic obstructive pulmonary disease, and heart failure show substantial interindividual variability in response. The ADRB2 gene is polymorphic in noncoding and coding regions, but virtually all ADRB2 association studies have utilized the two common nonsynonymous coding SNPs, often reaching discrepant conclusions.We constructed the 8 common ADRB2 haplotypes derived from 26 polymorphisms in the promoter, 5'UTR, coding, and 3'UTR of the intronless ADRB2 gene. These were cloned into an expression construct lacking a vector-based promoter, so that beta2AR expression was driven by its promoter, and steady state expression could be modified by polymorphisms throughout ADRB2 within a haplotype. "Whole-gene" transfections were performed with COS-7 cells and revealed 4 haplotypes with increased cell surface beta2AR protein expression compared to the others. Agonist-promoted downregulation of beta2AR protein expression was also haplotype-dependent, and was found to be increased for 2 haplotypes. A phylogenetic tree of the haplotypes was derived and annotated by cellular phenotypes, revealing a pattern potentially driven by expression.Thus for obstructive lung disease, the initial bronchodilator response from intermittent administration of beta-agonist may be influenced by certain beta2AR haplotypes (expression phenotypes), while other haplotypes may influence tachyphylaxis during the response to chronic therapy (downregulation phenotypes). An ideal clinical outcome of high expression and less downregulation was found for two haplotypes. Haplotypes may also affect heart failure antagonist therapy, where beta2AR increase inotropy and are anti-apoptotic. The haplotype-specific expression and regulation phenotypes found in this transfection-based system suggest that the density of genetic information in the form of these haplotypes, or haplotype-clusters with similar phenotypes can potentially provide greater discrimination of phenotype in human disease and pharmacogenomic association studies