Whole-cell living biosensors--are they ready for environmental application?

Since the development of the first whole-cell living biosensor or bioreporter about 15 years ago, construction and testing of new genetically modified microorganisms for environmental sensing and reporting has proceeded at an ever increasing rate. One and a half decades appear as a reasonable time span for a new technology to reach the maturity needed for application and commercial success. It seems, however, that the research into cellular biosensors is still mostly in a proof-of-principle or demonstration phase and not close to extensive or commercial use outside of academia. In this review, we consider the motivations for bioreporter developments and discuss the suitability of extant bioreporters for the proposed applications to stimulate complementary research and to help researchers to develop realistic objectives. This includes the identification of some popular misconceptions about the qualities and shortcomings of bioreporters.

FTO genotype is associated with phenotypic variability of body mass index.

There is evidence across several species for genetic control of phenotypic variation of complex traits, such that the variance among phenotypes is genotype dependent. Understanding genetic control of variability is important in evolutionary biology, agricultural selection programmes and human medicine, yet for complex traits, no individual genetic variants associated with variance, as opposed to the mean, have been identified. Here we perform a meta-analysis of genome-wide association studies of phenotypic variation using ∼170,000 samples on height and body mass index (BMI) in human populations. We report evidence that the single nucleotide polymorphism (SNP) rs7202116 at the FTO gene locus, which is known to be associated with obesity (as measured by mean BMI for each rs7202116 genotype), is also associated with phenotypic variability. We show that the results are not due to scale effects or other artefacts, and find no other experiment-wise significant evidence for effects on variability, either at loci other than FTO for BMI or at any locus for height. The difference in variance for BMI among individuals with opposite homozygous genotypes at the FTO locus is approximately 7%, corresponding to a difference of ∼0.5 kilograms in the standard deviation of weight. Our results indicate that genetic variants can be discovered that are associated with variability, and that between-person variability in obesity can partly be explained by the genotype at the FTO locus. The results are consistent with reported FTO by environment interactions for BMI, possibly mediated by DNA methylation. Our BMI results for other SNPs and our height results for all SNPs suggest that most genetic variants, including those that influence mean height or mean BMI, are not associated with phenotypic variance, or that their effects on variability are too small to detect even with samples sizes greater than 100,000.

Full-waveform inversion of cross-hole ground-penetrating radar data to characterize a gravel aquifer close to the Thur River, Switzerland

Cross-hole radar tomography is a useful tool for mapping shallow subsurface electrical properties viz. dielectric permittivity and electrical conductivity. Common practice is to invert cross-hole radar data with ray-based tomographic algorithms using first arrival traveltimes and first cycle amplitudes. However, the resolution of conventional standard ray-based inversion schemes for cross-hole ground-penetrating radar (GPR) is limited because only a fraction of the information contained in the radar data is used. The resolution can be improved significantly by using a full-waveform inversion that considers the entire waveform, or significant parts thereof. A recently developed 2D time-domain vectorial full-waveform crosshole radar inversion code has been modified in the present study by allowing optimized acquisition setups that reduce the acquisition time and computational costs significantly. This is achieved by minimizing the number of transmitter points and maximizing the number of receiver positions. The improved algorithm was employed to invert cross-hole GPR data acquired within a gravel aquifer (4-10 m depth) in the Thur valley, Switzerland. The simulated traces of the final model obtained by the full-waveform inversion fit the observed traces very well in the lower part of the section and reasonably well in the upper part of the section. Compared to the ray-based inversion, the results from the full-waveform inversion show significantly higher resolution images. At either side, 2.5 m distance away from the cross-hole plane, borehole logs were acquired. There is a good correspondence between the conductivity tomograms and the natural gamma logs at the boundary of the gravel layer and the underlying lacustrine clay deposits. Using existing petrophysical models, the inversion results and neutron-neutron logs are converted to porosity. Without any additional calibration, the values obtained for the converted neutron-neutron logs and permittivity results are very close and similar vertical variations can be observed. The full-waveform inversion provides in both cases additional information about the subsurface. Due to the presence of the water table and associated refracted/reflected waves, the upper traces are not well fitted and the upper 2 m in the permittivity and conductivity tomograms are not reliably reconstructed because the unsaturated zone is not incorporated into the inversion domain.

Genome-wide association study identifies loci influencing concentrations of liver enzymes in plasma.

Concentrations of liver enzymes in plasma are widely used as indicators of liver disease. We carried out a genome-wide association study in 61,089 individuals, identifying 42 loci associated with concentrations of liver enzymes in plasma, of which 32 are new associations (P = 10(-8) to P = 10(-190)). We used functional genomic approaches including metabonomic profiling and gene expression analyses to identify probable candidate genes at these regions. We identified 69 candidate genes, including genes involved in biliary transport (ATP8B1 and ABCB11), glucose, carbohydrate and lipid metabolism (FADS1, FADS2, GCKR, JMJD1C, HNF1A, MLXIPL, PNPLA3, PPP1R3B, SLC2A2 and TRIB1), glycoprotein biosynthesis and cell surface glycobiology (ABO, ASGR1, FUT2, GPLD1 and ST3GAL4), inflammation and immunity (CD276, CDH6, GCKR, HNF1A, HPR, ITGA1, RORA and STAT4) and glutathione metabolism (GSTT1, GSTT2 and GGT), as well as several genes of uncertain or unknown function (including ABHD12, EFHD1, EFNA1, EPHA2, MICAL3 and ZNF827). Our results provide new insight into genetic mechanisms and pathways influencing markers of liver function.

Self reported physical activity, public health, and perceived environment: results from a comparative European study

The focus of physical activity promotion is moving from methods for increasing health enhancing physical activity on the individual level to higher level strategies including environmental and policy approaches. Scientific inquiry, traditionally related to individual-based strategies, requires adaptation and refinement when environmental and policy changes become more relevant. The objective of this study is to investigate the significance for behaviour and health of community-based environments that encourage physical activity. DESIGN AND SETTING The article presents data and results from a cross sectional comparative survey of the general population in six European countries (Belgium, Finland, Germany (East and West), Netherlands, Spain, Switzerland). Specifically, the relation between perceived community-based opportunities for physical activity, self reported physical activity, and self rated health status is investigated. PARTICIPANTS Representative samples of general populations (adults 18 years or older). Overall response rate: 53.5%. Sample sizes realised: Belgium: n=389; Finland: n=400; Germany (East): n = 913; Germany (West): n=489; Netherlands: n=366; Spain: n=380; Switzerland: n=406. MAIN RESULTS Analyses show that best opportunities are reported by people who are lightly to moderately physically active. People's self rated health is moderately, but significantly associated with both perceived opportunities, and physical activity itself. These predictors interact in that especially for women, the health impact of physical activity is more pronounced in case of good opportunities. CONCLUSIONS The paper shows the potential of opportunities within residential and community environments with regard to physical activity, both for behaviour and health. Opportunities may enable the population, especially women, to develop an active lifestyle, and thus improve their health. Future studies with objective indicators for physical activity related environments should test the findings that are based on perceptions.

Current features of infective endocarditis in elderly patients: results of the International Collaboration on Endocarditis Prospective Cohort Study.

BACKGROUND: Elderly patients are emerging as a population at high risk for infective endocarditis (IE). However, adequately sized prospective studies on the features of IE in elderly patients are lacking. METHODS: In this multinational, prospective, observational cohort study within the International Collaboration on Endocarditis, 2759 consecutive patients were enrolled from June 15, 2000, to December 1, 2005; 1056 patients with IE 65 years or older were compared with 1703 patients younger than 65 years. Risk factors, predisposing conditions, origin, clinical features, course, and outcome of IE were comprehensively analyzed. RESULTS: Elderly patients reported more frequently a hospitalization or an invasive procedure before IE onset. Diabetes mellitus and genitourinary and gastrointestinal cancer were the major predisposing conditions. Blood culture yield was higher among elderly patients with IE. The leading causative organism was Staphylococcus aureus, with a higher rate of methicillin resistance. Streptococcus bovis and enterococci were also significantly more prevalent. The clinical presentation of elderly patients with IE was remarkable for lower rates of embolism, immune-mediated phenomena, or septic complications. At both echocardiography and surgery, fewer vegetations and more abscesses were found, and the gain in the diagnostic yield of transesophageal echocardiography was significantly larger. Significantly fewer elderly patients underwent cardiac surgery (38.9% vs 53.5%; P < .001). Elderly patients with IE showed a higher rate of in-hospital death (24.9% vs 12.8%; P < .001), and age older than 65 years was an independent predictor of mortality. CONCLUSIONS: In this large prospective study, increasing age emerges as a major determinant of the clinical characteristics of IE. Lower rates of surgical treatment and high mortality are the most prominent features of elderly patients with IE. Efforts should be made to prevent health care-associated acquisition and improve outcomes in this major subgroup of patients with IE.

Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.

Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the entire HSC pool turns over every few weeks, suggesting that HSCs regularly enter and exit cell cycle. Here, we combine flow cytometry with label-retaining assays (BrdU and histone H2B-GFP) to identify a population of dormant mouse HSCs (d-HSCs) within the lin(-)Sca1+cKit+CD150+CD48(-)CD34(-) population. Computational modeling suggests that d-HSCs divide about every 145 days, or five times per lifetime. d-HSCs harbor the vast majority of multilineage long-term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis, activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress.

Report on initial experience with local anaesthesia for carotid artery endarterectomy.

PURPOSE OF THE STUDY: This prospective study reports our preliminary results with local anaesthesia (LA) for carotid endarterectomy (CEA). MATERIAL AND METHODS: Twenty CEA in nineteen patients were performed using a three-stage local infiltration technique. CEA were performed through a short Duplex-assisted skin incision (median length: 55 mm) using a retro-jugular approach and polyurethane patch closure (median length: 35 mm). RESULTS: There were 13 men and 6 women with a mean age of 71.2 years. The indications of CEA were asymptomatic lesions in 11 cases, stroke in 7 cases and transient ischaemic attack in 2 cases. The median degree of internal carotid artery stenosis was 90%. One patient (5%) required an intraluminal shunt. There were no peri-operative deaths, stroke or conversion to general anaesthesia (GA). The median length of stay was 3 days. CONCLUSIONS: LA is a good alternative to GA. It can be used after a feasibility study and a short teaching procedure. In our centre, it is a safe and effective procedure associated with low morbidity, high acceptance by patients and a short hospital stay.

Meta-analysis identifies multiple loci associated with kidney function-related traits in east Asian populations.

Chronic kidney disease (CKD), impairment of kidney function, is a serious public health problem, and the assessment of genetic factors influencing kidney function has substantial clinical relevance. Here, we report a meta-analysis of genome-wide association studies for kidney function-related traits, including 71,149 east Asian individuals from 18 studies in 11 population-, hospital- or family-based cohorts, conducted as part of the Asian Genetic Epidemiology Network (AGEN). Our meta-analysis identified 17 loci newly associated with kidney function-related traits, including the concentrations of blood urea nitrogen, uric acid and serum creatinine and estimated glomerular filtration rate based on serum creatinine levels (eGFRcrea) (P < 5.0 × 10(-8)). We further examined these loci with in silico replication in individuals of European ancestry from the KidneyGen, CKDGen and GUGC consortia, including a combined total of ∼110,347 individuals. We identify pleiotropic associations among these loci with kidney function-related traits and risk of CKD. These findings provide new insights into the genetics of kidney function.

Therapeutic drug monitoring and pharmacogenetic tests as tools in pharmacovigilance.

Therapeutic drug monitoring (TDM) and pharmacogenetic tests play a major role in minimising adverse drug reactions and enhancing optimal therapeutic response. The response to medication varies greatly between individuals, according to genetic constitution, age, sex, co-morbidities, environmental factors including diet and lifestyle (e.g. smoking and alcohol intake), and drug-related factors such as pharmacokinetic or pharmacodynamic drug-drug interactions. Most adverse drug reactions are type A reactions, i.e. plasma-level dependent, and represent one of the major causes of hospitalisation, in some cases leading to death. However, they may be avoidable to some extent if pharmacokinetic and pharmacogenetic factors are taken into consideration. This article provides a review of the literature and describes how to apply and interpret TDM and certain pharmacogenetic tests and is illustrated by case reports. An algorithm on the use of TDM and pharmacogenetic tests to help characterise adverse drug reactions is also presented. Although, in the scientific community, differences in drug response are increasingly recognised, there is an urgent need to translate this knowledge into clinical recommendations. Databases on drug-drug interactions and the impact of pharmacogenetic polymorphisms and adverse drug reaction information systems will be helpful to guide clinicians in individualised treatment choices.

Internal arsenite bioassay calibration using multiple reporter cell lines

Bioassays with bioreporter bacteria are usually calibrated with analyte solutions of known concentrations that are analysed along with the samples of interest. This is done as bioreporter output (the intensity of light, fluorescence or colour) does not only depend on the target concentration, but also on the incubation time and physiological activity of the cells in the assay. Comparing the bioreporter output with standardized colour tables in the field seems rather difficult and error-prone. A new approach to control assay variations and improve application ease could be an internal calibration based on the use of multiple bioreporter cell lines with drastically different reporter protein outputs at a given analyte concentration. To test this concept, different Escherichia coli-based bioreporter strains expressing either cytochrome c peroxidase (CCP, or CCP mutants) or β-galactosidase upon induction with arsenite were constructed. The reporter strains differed either in the catalytic activity of the reporter protein (for CCP) or in the rates of reporter protein synthesis (for β-galactosidase), which, indeed, resulted in output signals with different intensities at the same arsenite concentration. Hence, it was possible to use combinations of these cell lines to define arsenite concentration ranges at which none, one or more cell lines gave qualitative (yes/no) visible signals that were relatively independent of incubation time or bioreporter activity. The discriminated concentration ranges would fit very well with the current permissive (e.g. World Health Organization) levels of arsenite in drinking water (10 µg l−1).

Characterization of HbpR binding by site-directed mutagenesis of its DNA-binding site and by deletion of the effector domain.

In the presence of 2-hydroxybiphenyl, the enhancer binding protein, HbpR, activates the sigma54-dependent P(hbpC) promoter and controls the initial steps of 2-hydroxybiphenyl degradation in Pseudomonas azelaica. In the activation process, an oligomeric HbpR complex of unknown subunit composition binds to an operator region containing two imperfect palindromic sequences. Here, the HbpR-DNA binding interactions were investigated by site-directed mutagenesis of the operator region and by DNA-binding assays using purified HbpR. Mutations that disrupted the twofold symmetry in the palindromes did not affect the binding affinity of HbpR, but various mutations along a 60 bp region, and also outside the direct palindromic sequences, decreased the binding affinity. Footprints of HbpR on mutant operator fragments showed that a partial loss of binding contacts occurs, suggesting that the binding of one HbpR 'protomer' in the oligomeric complex is impaired whilst leaving the other contacts intact. An HbpR variant, devoid of its N-terminal sensing A-domain, was unable to activate transcription from the hbpC promoter while maintaining protection of the operator DNA in footprints. Wild-type HbpR was unable to activate transcription from the hbpC promoter when delta A-HbpR was expressed in the same cell, suggesting the formation of (repressing) hetero-oligomers. This model implies that HbpR can self-associate on its operator DNA without effector recognition or ATP binding. Furthermore, our findings suggest that the N-terminal sensing domain of HbpR is needed to activate the central ATPase domain rather than to repress a constitutively active C domain, as is the case for the related regulatory protein XylR.

A new green fluorescent protein-based bacterial biosensor for analysing phenanthrene fluxes.

The polycyclic aromatic hydrocarbon (PAH)-degrading strain Burkholderia sp. RP007 served as host strain for the design of a bacterial biosensor for the detection of phenanthrene. RP007 was transformed with a reporter plasmid containing a transcriptional fusion between the phnS putative promoter/operator region and the gene encoding the enhanced green fluorescent protein (GFP). The resulting bacterial biosensor--Burkholderia sp. strain RP037--produced significant amounts of GFP after batch incubation in the presence of phenanthrene crystals. Co-incubation with acetate did not disturb the phenanthrene-specific response but resulted in a homogenously responding population of cells. Active metabolism was required for induction with phenanthrene. The magnitude of GFP induction was influenced by physical parameters affecting the phenanthrene flux to the cells, such as the contact surface area between solid phenanthrene and the aqueous phase, addition of surfactant, and slow phenanthrene release from Model Polymer Release System beads or from a water-immiscible oil. These results strongly suggest that the bacterial biosensor can sense different phenanthrene fluxes while maintaining phenanthrene metabolism, thus acting as a genuine sensor for phenanthrene bioavailability. A relationship between GFP production and phenanthrene mass transfer is proposed.

Genome-wide analysis of Sphingomonas wittichii RW1 behaviour during inoculation and growth in contaminated sand.

The efficacy of inoculation of single pure bacterial cultures into complex microbiomes, for example, in order to achieve increased pollutant degradation rates in contaminated material (that is, bioaugmentation), has been frustrated by insufficient knowledge on the behaviour of the inoculated bacteria under the specific abiotic and biotic boundary conditions. Here we present a comprehensive analysis of genome-wide gene expression of the bacterium Sphingomonas wittichii RW1 in contaminated non-sterile sand, compared with regular suspended batch growth in liquid culture. RW1 is a well-known bacterium capable of mineralizing dibenzodioxins and dibenzofurans. We tested the reactions of the cells both during the immediate transition phase from liquid culture to sand with or without dibenzofuran, as well as during growth and stationary phase in sand. Cells during transition show stationary phase characteristics, evidence for stress and for nutrient scavenging, and adjust their primary metabolism if they were not precultured on the same contaminant as found in the soil. Cells growing and surviving in sand degrade dibenzofuran but display a very different transcriptome signature as in liquid or in liquid culture exposed to chemicals inducing drought stress, and we obtain evidence for numerous 'soil-specific' expressed genes. Studies focusing on inoculation efficacy should test behaviour under conditions as closely as possible mimicking the intended microbiome conditions.

Classification of current anticancer immunotherapies.

During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into "passive" and "active" based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches.