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.

Impact de la prophylaxie hormonale sur les fractures du fémur proximal des femmes postménopausiques: une étude de simulation. [Impact of hormonal prevention on fractures of the proximal femur in postmenopausal women: a simulation study].

A simulation model of the effects of hormone replacement therapy (HRT) on hip fractures and their consequences is based on a population of 100,000 post-menopausal women. This cohort is confronted with literature derived probabilities of cancers (endometrium or breast, which are contra-indications to HRT), hip fracture, disability requiring nursing home or home care, and death. Administration of HRT for life prevents 55,5% of hip fractures, 22,6% of years with home care and 4,4% of years in nursing homes. If HRT is administered for 15 years, these results are 15,5%, 10% and 2,2%, respectively. A slight gain in life expectancy is observed for both durations of HRT. The net financial loss in the simulated population is 222 million Swiss Francs (cost/benefit ratio 1.25) for lifelong administration of HRT, and 153 million Swiss Francs (cost/benefit ratio 1.42) if HRT is administered during 15 years.

The clc element of Pseudomonas sp. strain B13, a genomic island with various catabolic properties.

Pseudomonas sp. strain B13 is a bacterium known to degrade chloroaromatic compounds. The properties to use 3- and 4-chlorocatechol are determined by a self-transferable DNA element, the clc element, which normally resides at two locations in the cell's chromosome. Here we report the complete nucleotide sequence of the clc element, demonstrating the unique catabolic properties while showing its relatedness to genomic islands and integrative and conjugative elements rather than to other known catabolic plasmids. As far as catabolic functions, the clc element harbored, in addition to the genes for chlorocatechol degradation, a complete functional operon for 2-aminophenol degradation and genes for a putative aromatic compound transport protein and for a multicomponent aromatic ring dioxygenase similar to anthranilate hydroxylase. The genes for catabolic functions were inducible under various conditions, suggesting a network of catabolic pathway induction. For about half of the open reading frames (ORFs) on the clc element, no clear functional prediction could be given, although some indications were found for functions that were similar to plasmid conjugation. The region in which these ORFs were situated displayed a high overall conservation of nucleotide sequence and gene order to genomic regions in other recently completed bacterial genomes or to other genomic islands. Most notably, except for two discrete regions, the clc element was almost 100% identical over the whole length to a chromosomal region in Burkholderia xenovorans LB400. This indicates the dynamic evolution of this type of element and the continued transition between elements with a more pathogenic character and those with catabolic properties.

Acute toxicity of curative radiotherapy for intermediate- and high-risk localised prostate cancer in the EORTC trial 22991.

INTRODUCTION: This trial randomly assessed short-term adjuvant hormonal therapy added to radiotherapy (RT) for intermediate- and high-risk (UICC 1997 cT2a or cT1b-c with high PSA or Gleason score) localised prostate cancer. We report acute toxicity (CTCAE v2) assessed weekly during RT in relation to radiation parameters. PATIENTS AND METHODS: Centres selected the RT dose (70, 74 or 78Gy) and RT technique. Statistical significance is at 0.05. RESULTS: Of 791 patients, 652 received 3D-CRT (70Gy: 195, 74Gy: 376, 78Gy: 81) and 139 received IMRT (74Gy: 28, 78Gy: 111). During RT, grade 3 gastrointestinal (GI) and genitourinary (GU) toxicities were reported by 7 (0.8%) and 50 (6.3%) patients, respectively. No grade 4 was reported. The risk of grade 2 GI toxicity increased significantly with increasing D50%-rectum (p=0.004) and that of grade 2 GU toxicity correlated only to Dmax-bladder (p=0.051). 3D-RT technique, increasing total dose and V95% >400 cc increased D50% and Dmax. One month after RT, only 14 patients (1.8%) reported grade 3 toxicity. AST did not seem to influence the risk of GU or GI acute toxicity. CONCLUSION: RT up to 78Gy was well tolerated. Dmax-bladder and D50%-rectum influenced the risk of grade 2 GU toxicity and GI toxicity, respectively. Both were lower with IMRT but remained high for an irradiated RT volume>400 cc for 3D-RT and for a dose of 78Gy. Hormonal treatment did not influence acute toxicity.

Inflammation-induced changes in expression and glycosylation of genetic variants of alpha 1-acid glycoprotein. Studies with human sera, primary cultures of human hepatocytes and transgenic mice.

The relative occurrence of genetic variants of human alpha 1-acid glycoprotein (AGP) in relation to changes in glycosylation was studied in sera of patients with burn injury, media of cytokine-treated primary cultures of human hepatocytes and Hep 3B cells, and sera of transgenic mice expressing the human AGP-A gene. It is concluded (i) that the glycosylation of AGP was not dependent on its genetic expression and (ii) that both the variants determined by the AGP-A gene as well as by the AGP-B/B' genes are increased after inflammation or treatment with interleukins 1 and 6.

Effect of white-matter lesions on the risk of periprocedural stroke after carotid artery stenting versus endarterectomy in the International Carotid Stenting Study (ICSS): a prespecified analysis of data from a randomised trial.

BACKGROUND: Findings from randomised trials have shown a higher early risk of stroke after carotid artery stenting than after carotid endarterectomy. We assessed whether white-matter lesions affect the perioperative risk of stroke in patients treated with carotid artery stenting versus carotid endarterectomy. METHODS: Patients with symptomatic carotid artery stenosis included in the International Carotid Stenting Study (ICSS) were randomly allocated to receive carotid artery stenting or carotid endarterectomy. Copies of baseline brain imaging were analysed by two investigators, who were masked to treatment, for the severity of white-matter lesions using the age-related white-matter changes (ARWMC) score. Randomisation was done with a computer-generated sequence (1:1). Patients were divided into two groups using the median ARWMC. We analysed the risk of stroke within 30 days of revascularisation using a per-protocol analysis. ICSS is registered with controlled-trials.com, number ISRCTN 25337470. FINDINGS: 1036 patients (536 randomly allocated to carotid artery stenting, 500 to carotid endarterectomy) had baseline imaging available. Median ARWMC score was 7, and patients were dichotomised into those with a score of 7 or more and those with a score of less than 7. In patients treated with carotid artery stenting, those with an ARWMC score of 7 or more had an increased risk of stroke compared with those with a score of less than 7 (HR for any stroke 2·76, 95% CI 1·17-6·51; p=0·021; HR for non-disabling stroke 3·00, 1·10-8·36; p=0·031), but we did not see a similar association in patients treated with carotid endarterectomy (HR for any stroke 1·18, 0·40-3·55; p=0·76; HR for disabling or fatal stroke 1·41, 0·38-5·26; p=0·607). Carotid artery stenting was associated with a higher risk of stroke compared with carotid endarterectomy in patients with an ARWMC score of 7 or more (HR for any stroke 2·98, 1·29-6·93; p=0·011; HR for non-disabling stroke 6·34, 1·45-27·71; p=0·014), but there was no risk difference in patients with an ARWMC score of less than 7. INTERPRETATION: The presence of white-matter lesions on brain imaging should be taken into account when selecting patients for carotid revascularisation. Carotid artery stenting should be avoided in patients with more extensive white-matter lesions, but might be an acceptable alternative to carotid endarterectomy in patients with less extensive lesions. FUNDING: Medical Research Council, the Stroke Association, Sanofi-Synthélabo, the European Union Research Framework Programme 5.

In the eye of the listener: pupil dilation elucidates discourse processing.

The current study investigated cognitive resource allocation in discourse processing by means of pupil dilation and behavioral measures. Short question-answer dialogs were presented to listeners. Either the context question queried a new information focus in the successive answer, or else the context query was corrected in the answer sentence (correction information). The information foci contained in the answer sentences were either adequately highlighted by prosodic means or not. Participants had to judge the adequacy of the focus prosody with respect to the preceding context question. Prosodic judgment accuracy was higher in the conditions bearing adequate focus prosody than in the conditions with inadequate focus prosody. Latency to peak pupil dilation was longer when new information foci were perceived compared to correction foci. Moreover, for the peak dilation, an interaction of focus type and prosody was found. Post hoc statistical tests revealed that prosodically adequate correction focus positions were processed with smaller peak dilation in comparison to all other dialog conditions. Thus, pupil dilation and results of a principal component analysis suggest an interaction of focus type and focus prosody in discourse processing.

Expression and function of macrophage migration inhibitory factor (MIF) in melioidosis.

BACKGROUND: Macrophage migration inhibitory factor (MIF) has emerged as a pivotal mediator of innate immunity and has been shown to be an important effector molecule in severe sepsis. Melioidosis, caused by Burkholderia pseudomallei, is an important cause of community-acquired sepsis in Southeast-Asia. We aimed to characterize the expression and function of MIF in melioidosis. METHODOLOGY AND PRINCIPAL FINDINGS: MIF expression was determined in leukocytes and plasma from 34 melioidosis patients and 32 controls, and in mice infected with B. pseudomallei. MIF function was investigated in experimental murine melioidosis using anti-MIF antibodies and recombinant MIF. Patients demonstrated markedly increased MIF mRNA leukocyte and MIF plasma concentrations. Elevated MIF concentrations were associated with mortality. Mice inoculated intranasally with B. pseudomallei displayed a robust increase in pulmonary and systemic MIF expression. Anti-MIF treated mice showed lower bacterial loads in their lungs upon infection with a low inoculum. Conversely, mice treated with recombinant MIF displayed a modestly impaired clearance of B. pseudomallei. MIF exerted no direct effects on bacterial outgrowth or phagocytosis of B. pseudomallei. CONCLUSIONS: MIF concentrations are markedly elevated during clinical melioidosis and correlate with patients' outcomes. In experimental melioidosis MIF impaired antibacterial defense.

Impact of mycelia on the accessibility of fluorene to PAH-degrading bacteria.

Mycelia have been recently shown to actively transport polycyclic aromatic hydrocarbons (PAH) in water-unsaturated soil over the range of centimeters, thereby efficiently mobilizing hydrophobic PAH beyond their purely diffusive transport in air and water. However, the question if mycelia-based PAH transport has an effect on PAH biodegradation was so far unsolved. To address this, we developed a laboratory model microcosm mimicking air-water interfaces in soil. Chemical analyses demonstrated transport of the PAH fluorene (FLU) by the mycelial oomycete Pythium ultimum that was grown along the air-water interfaces. Furthermore, degradation of mycelia-transported FLU by the bacterium Burkholderia sartisoli RP037-mChe was indicated. Since this organism expresses eGFP in response to a FLU flux to the cell, it was also as a bacterial reporter of FLU bioavailability in the vicinity of mycelia. Confocal laser scanning microscopy (CLSM) and image analyses revealed a significant increase of eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU. Hence, we could show that physically separated FLU becomes bioavailable to bacteria after transport by mycelia. Experiments with silicon coated glass fibers capturing mycelia-transported FLU guided us to propose a three-step mechanism of passive uptake, active transport and diffusion-driven release. These experiments were also used to evaluate the contributions of these individual steps to the overall mycelial FLU transport rate.

Genomic islands: tools of bacterial horizontal gene transfer and evolution.

Abstract Bacterial genomes evolve through mutations, rearrangements or horizontal gene transfer. Besides the core genes encoding essential metabolic functions, bacterial genomes also harbour a number of accessory genes acquired by horizontal gene transfer that might be beneficial under certain environmental conditions. The horizontal gene transfer contributes to the diversification and adaptation of microorganisms, thus having an impact on the genome plasticity. A significant part of the horizontal gene transfer is or has been facilitated by genomic islands (GEIs). GEIs are discrete DNA segments, some of which are mobile and others which are not, or are no longer mobile, which differ among closely related strains. A number of GEIs are capable of integration into the chromosome of the host, excision, and transfer to a new host by transformation, conjugation or transduction. GEIs play a crucial role in the evolution of a broad spectrum of bacteria as they are involved in the dissemination of variable genes, including antibiotic resistance and virulence genes leading to generation of hospital 'superbugs', as well as catabolic genes leading to formation of new metabolic pathways. Depending on the composition of gene modules, the same type of GEIs can promote survival of pathogenic as well as environmental bacteria.

The role of embolic protection devices during carotid stenting prior to cardiac surgery in asymptomatic patients: Empty filters?

Objectives: The purpose of this study was to analyze the debris captured in the distal protection filters used during carotid artery stenting (CAS). Background: CAS is an option available to high-risk patients requiring revascularization. Filters are suggested for optimal stroke prevention during CAS. Methods: From May 2005 to June 2007, filters from 59 asymptomatic patients who underwent CAS were collected and sent to a specialized laboratory for light-microscope and histological analysis. Peri- and postprocedural outcomes were assessed during 1-year follow-up. Results: On the basis of biomedical imaging of the filter debris, the captured material could not be identified as embolized particles from the carotid plaque. On histological analysis the debris consisted mainly of red blood cell aggregates and/ or platelets, occasionally accompanied by granulocytes. We found no consistent histological evidence of embolized particles originating from atherosclerotic plaques. Post-procedure, three neurological events were reported: two (3.4%) transient ischemic attacks (TIA) and one (1.7%) ipsilateral minor stroke. Conclusion: The filters used during CAS in asymptomatic patients planned for cardiac surgery often remained empty. These findings may be explained by assuming that asymptomatic patients feature a different atherosclerotic plaque composition or stabilization through antiplatelet medication. Larger, randomized trials are clearly warranted, especially in the asymptomatic population. © 2012 Wiley Periodicals, Inc.

The clc element and related genomic islands in Proteobacteria

Genomic islands are large DNA segments, present in most bacterial genome, that are acquired via horizontal gene transfer and contribute to the rapid bacterial evolution and adaptation of the host cell. Here we focus on the clc element (or ICEclc), a 103‑kb genomic island first discovered in Pseudomonas knackmussii B13, as a model of this diverse group of mobile genetic elements. ICEclc is normally integrated in the host bacterial chromosome but can excise and transfer to a new host by conjugation. In this chapter we review the basic features of ICEclc, the mechanisms of its life‑style as well as evolutionary relationships with other known and unknown elements in a variety of Proteobacteria.