Beating tissue factor at its own game: Design and properties of a soluble tissue factor-independent coagulation factor VIIa.

Two decades of research have uncovered the mechanism by which the complex of tissue factor (TF) and the plasma serine protease factor VIIa (FVIIa) mediates the initiation of blood coagulation. Membrane-anchored TF directly interacts with substrates and induces allosteric effects in the protease domain of FVIIa. These properties are also recapitulated by the soluble ectodomain of TF (sTF). At least two interdependent allosteric activation pathways originate at the FVIIa:sTF interface are proposed to enhance FVIIa activity upon sTF binding. Here, we sought to engineer an sTF-independent FVIIa variant by stabilizing both proposed pathways, with one pathway terminating at segment 215-217 in the activation domain and the other pathway terminating at the N terminus insertion site. To stabilize segment 215-217, we replaced the flexible 170 loop of FVIIa with the more rigid 170 loop from trypsin and combined it with an L163V substitution (FVIIa-VYT). The FVIIa-VYT variant exhibited 60-fold higher amidolytic activity than FVIIa, and displayed similar FX activation and antithrombin inhibition kinetics to the FVIIa.sTF complex. The sTF-independent activity of FVIIa-VYT was partly mediated by an increase in the N terminus insertion and, as shown by X-ray crystallography, partly by Tyr-172 inserting into a cavity in the activation domain stabilizing the S1 substrate-binding pocket. The combination with L163V likely drove additional changes in a delicate hydrogen-bonding network that further stabilized S1-S3 sites. In summary, we report the first FVIIa variant that is catalytically independent of sTF and provide evidence supporting the existence of two TF-mediated allosteric activation pathways.

Norovirus and rotavirus in children hospitalised with diarrhoea after rotavirus vaccine introduction in Burkina Faso.

Several studies report norovirus as the new leading cause of severe gastroenteritis in children after the global introduction of rotavirus vaccines. Burkina Faso introduced general rotavirus vaccination with the oral pentavalent vaccine RotaTeq in November 2013 and quickly reached a vaccine coverage of >90%. This study describes detection rates, clinical profiles and the molecular epidemiology of norovirus and rotavirus infections in 146 children aged <5 years with severe acute gastroenteritis in Ouagadougou, consecutively enrolled from a hospital between January 2015 and December 2015. Virus detection was performed with an antigen test or real-time polymerase chain reaction (PCR) and genotyping was performed by nucleotide sequencing or multiplex PCR. Rotavirus was found in 14% and norovirus in 20% of faecal samples. Norovirus infection was significantly more associated with severe dehydration compared to rotavirus (P < 0.001). Among genotyped norovirus samples 48% (12/25) belonged to GII.4 which caused significantly more diarrhoeal episodes than non-GII.4 genotypes (P = 0.01). The most common rotavirus genotypes were G2P[4] (30%), G12P[6] (25%) and G12P[8] (20%). Fifty percent of the rotavirus positive children were infected with fully or partly heterotypic strains. In conclusion, this study found a higher proportion of norovirus causing more severe symptoms in children with diarrhoea in Burkina Faso after the introduction of rotavirus vaccination.

Detection of Campylobacter spp. in water by dead-end ultrafiltration and application at farm level.

AIMS: The purposes were to evaluate the detection of low levels of Campylobacter in water by dead-end ultrafiltration (DEUF) to determine the sensitivity and suitability for use under field condition. METHODS AND RESULTS: The DEUF technique followed by detection according to ISO 10272 was tested on artificially and naturally contaminated water. Campylobacter were detected in all samples spiked with more than 10 CFU 60 l-1 and in four of nine samples with a concentration below 10 CFU 60 l-1 water. Naturally contaminated water from five different broiler producers was analysed. Campylobacter were detected in four of 12 samples from ponds near the houses and in three of 24 samples from water pipes inside the broiler houses, but not in tap water sampled at the entrance of the broiler houses. CONCLUSIONS: The results indicate that DEUF is useful for detection of low numbers of Campylobacter in large volumes of water. SIGNIFICANCE AND IMPACT OF THE STUDY: Contaminated water is an important source for transmission of Campylobacter to broilers and humans. The concentration of Campylobacter is usually low with a high level of background microbiota. This study shows the advantages of DEUF both in the laboratory and under field conditions.

Supplemental oxygen therapy does not affect the systemic inflammatory response to acute myocardial infarction.

BACKGROUND: Oxygen therapy has been used routinely in normoxemic patients with suspected acute myocardial infarction (AMI) despite limited evidence supporting a beneficial effect. AMI is associated with a systemic inflammation. Here, we hypothesized that the inflammatory response to AMI is potentiated by oxygen therapy. METHODS: The DETermination of the role of Oxygen in suspected Acute Myocardial Infarction (DETO2X-AMI) multicentre trial randomized patients with suspected AMI to receive oxygen at 6 L min-1 for 6-12 h or ambient air. For this prespecified subgroup analysis, we recruited patients with confirmed AMI from two sites for evaluation of inflammatory biomarkers at randomization and 5-7 h later. Ninety-two inflammatory biomarkers were analysed using proximity extension assay technology, to evaluate the effect of oxygen on the systemic inflammatory response to AMI. RESULTS: Plasma from 144 AMI patients was analysed whereof 76 (53%) were randomized to oxygen and 68 (47%) to air. Eight biomarkers showed a significant increase, whereas 13 were decreased 5-7 h after randomization. The inflammatory response did not differ between the two treatment groups neither did plasma troponin T levels. After adjustment for increase in troponin T over time, age and sex, the release of inflammation-related biomarkers was still similar in the groups. CONCLUSIONS: In a randomized controlled setting of normoxemic patients with AMI, the use of supplemental oxygen did not have any significant impact on the early release of systemic inflammatory markers.

The challenges and possibilities of public access defibrillation.

Out-of-hospital cardiac arrest (OHCA) is a major health problem that affects approximately four hundred and thousand patients annually in the United States alone. It is a major challenge for the emergency medical system as decreased survival rates are directly proportional to the time delay from collapse to defibrillation. Historically, defibrillation has only been performed by physicians and in-hospital. With the development of automated external defibrillators (AEDs), rapid defibrillation by nonmedical professionals and subsequently by trained or untrained lay bystanders has become possible. Much hope has been put to the concept of Public Access Defibrillation with a massive dissemination of public available AEDs throughout most Western countries. Accordingly, current guidelines recommend that AEDs should be deployed in places with a high likelihood of OHCA. Despite these efforts, AED use is in most settings anecdotal with little effect on overall OHCA survival. The major reasons for low use of public AEDs are that most OHCAs take place outside high incidence sites of cardiac arrest and that most OHCAs take place in residential settings, currently defined as not suitable for Public Access Defibrillation. However, the use of new technology for identification and recruitment of lay bystanders and nearby AEDs to the scene of the cardiac arrest as well as new methods for strategic AED placement redefines and challenges the current concept and definitions of Public Access Defibrillation. Existing evidence of Public Access Defibrillation and knowledge gaps and future directions to improve outcomes for OHCA are discussed. In addition, a new definition of the different levels of Public Access Defibrillation is offered as well as new strategies for increasing AED use in the society.

Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity.

The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215-217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215-217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity.

Defibrillation before EMS arrival in western Sweden.

BACKGROUND: Bystanders play a vital role in public access defibrillation (PAD) in out-of-hospital cardiac arrest (OHCA). Dual dispatch of first responders (FR) alongside emergency medical services (EMS) can reduce time to first defibrillation. The aim of this study was to describe the use of automated external defibrillators (AEDs) in OHCAs before EMS arrival. METHODS: All OHCA cases with a shockable rhythm in which an AED was used prior to the arrival of EMS between 2008 and 2015 in western Sweden were eligible for inclusion. Data from the Swedish Register for Cardiopulmonary Resuscitation (SRCR) were used for analysis, on-site bystander and FR defibrillation were compared with EMS defibrillation in the final analysis. RESULTS: Of the reported 6675 cases, 24% suffered ventricular fibrillation (VF), 162 patients (15%) of all VF cases were defibrillated before EMS arrival, 46% with a public AED on site. The proportion of cases defibrillated before EMS arrival increased from 5% in 2008 to 20% in 2015 (p<0.001). During this period, 30-day survival increased in patients with VF from 22% to 28% (p=0.04) and was highest when an AED was used on site (68%), with a median delay of 6.5min from collapse to defibrillation. Adjusted odds ratio for on-site defibrillation versus dispatched defibrillation for 30-day survival was 2.45 (95% CI: 1.02-5.95). CONCLUSIONS: The use of AEDs before the arrival of EMS increased over time. This was associated with an increased 30-day survival among patients with VF. Thirty-day survival was highest when an AED was used on site before EMS arrival.

Lymphatic Drainage in the Breast Before and Up to Five Years After a Reduction Mammaplasty.

The aim of this study was to investigate lymph circulation before and after breast reduction mammaplasty in different parts of the breast and with two different carriers of the radiopharmaceutical. Nine patients with breast hypertrophy planned for bilateral breast reduction mammaplasty were prospectively included in the study. The breast operation procedure was decided on intraoperatively. The regional lymph circulation in the breast was measured preoperatively by Technetium (99mTc) clearance in 4 different locations in each breast 1, 2 and 3 hours after injection. The procedure was repeated at one month and in six of the nine women also five years postoperatively with injection sites chosen to correspond to the preoperative location of that breast pedicle. Two different types of carriers of the radiopharmaceutical were tested, dextran in the right and nanocoll in the left breast. Dextran had a much more rapid clearance than nanocoll. There was no significant regional difference in lymph drainage up to five years after the mammaplasty, independent of dextran or nanocoll as being the carrier of the radiopharmaceutical.

Dental anxiety, concomitant factors and change in prevalence over 50 years.

OBJECTIVE: To analyse the prevalence of Dental Anxiety (DA) in the general adult population of Sweden, to study concomitant factors of DA and also to compare the prevalence of DA in 1962 with that in 2013. METHOD: The national study for 2013 included 3,500 individuals, randomly selected from the Swedish population. The data sampling was performed as a telephone survey including 38 questions and this report is a selection of those questions with the focus on DA. The national study from 1962 was a face-to-face survey of 1,331 individuals randomly selected from the Swedish population. Both surveys were conducted by the same company. RESULTS: In 2013, severe DA was reported in 4.7%, moderate DA in 4.5%, low DA in 9.8% and no DA in 80.9% of the subjects. Most (72.9%) of the subjects who reported severe DA attended dental care regularly. Important predictive factors of DA were age, gender, education, and self-rated poor oral and general health. The analysis showed a decrease in the prevalence of DA between 1962 and 2013, specifically a change towards more individuals reporting no dental anxiety (38.5% vs. 80.9%) but also smaller proportions of individuals having low and high DA (46.4% vs 9.8% and 15.1% vs 9.2%, respectively). CONCLUSIONS: In this national representative sample of Swedish adults the prevalence of severe DA was 4.7%. The main finding revealed a significant decrease of the prevalence of DA over 50 years.

Patients with lymphatic malformations who receive the immunostimulant OK-432 experience excellent long-term outcomes.

AIM: Sclerotherapy is the primary treatment for lymphatic malformations. The aim of this study was to evaluate the long-term outcome in patients with lymphatic malformations treated with the immunostimulant OK-432 as a sclerosant. METHODS: Between 1998 and 2013, we enrolled 131 of 138 eligible patients treated with OK-432 for lymphatic malformations in a retrospective study. The malformations were categorised according to the International Society for the Study of Vascular Anomalies. The outcome was assessed with a clinical examination and a questionnaire. RESULTS: The lymphatic malformations were localised to the head/neck (60%), the trunk (20%) and the extremities (6%) or involved with more than one region (14%). Patients with microcystic (10%), macrocystic (21%) and mixed lymphatic malformations (69%) underwent a median number of three, two and two injection treatments, respectively. The median age at the first injection was 3.4 years. Good or excellent clinical outcomes were seen in 70% of the patients. The number of injections, previous treatment and lesion localisation, but not time to follow-up and cyst size, predicted the clinical outcome. CONCLUSION: OK-432 treatment resulted in a successful outcome in 70% of patients with lymphatic malformations. The long-term outcome was comparable to the short-term outcome.

Human GII.4 norovirus VLP induces membrane invaginations on giant unilamellar vesicles containing secretor gene dependent α1,2-fucosylated glycosphingolipids.

Norovirus is a non-enveloped virus causing acute gastroenteritis. For human norovirus, no simple cell culture system is available and consequently knowledge on cellular entry of the virus is limited. The virus binds to ABH histo-blood group glycans on glycoproteins and glycosphingolipids. Non-secretors, characterized by the lack of ABH histo-blood group glycans in the gastrointestinal tract, are resistant to most norovirus infections, suggesting that these glycans may be part of the viral receptor. Recent studies have shown that polyomavirus enters the cell via membrane invaginations induced by the multivalent binding of the virus to receptor glycosphingolipids. In this study, we have investigated whether norovirus has the ability to induce membrane invaginations on giant unilamellar vesicles (GUVs) containing purified glycosphingolipids. First, we characterized the glycosphingolipid binding pattern of VLPs from the Dijon strain (genogroup II.4), using thin-layer chromatography. The VLP recognized the ABH active glycosphingolipids H type 1, Lewis b, B type 1, A type 1 and A Lewis b, but not lactotetraosylceramide or Lewis a, typically found in non-secretors. The binding pattern to glycosphingolipids incorporated into GUVs was in full agreement with the thin-layer chromatography experiments. Upon binding to the vesicles, the VLPs formed highly mobile clusters on the surface of the GUVs. VLP containing tubular invaginations were seen on the GUVs containing glycosphingolipids recognized by the VLP. In conclusion, this study suggests that human norovirus has the ability to induce membrane curvature by binding to and clustering glycosphingolipids, which may reflect the first step in cellular entry of the virus.

Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model.

Hemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (K(D) = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.

Out-of-hospital cardiac arrest: 10 years of progress in research and treatment.

Cardiac disease is the most common cause of mortality in Western countries, with most deaths due to out-of-hospital cardiac arrest (OHCA). In Sweden, 5000-10 000 OHCAs occur annually. During the last decade, the time from cardiac arrest to start of cardiopulmonary resuscitation (CPR) and defibrillation has increased, whereas survival has remained unchanged or even increased. Resuscitation of OHCA patients is based on the 'chain-of-survival' concept, including early (i) access, (ii) CPR, (iii) defibrillation, (iv) advanced cardiac life support and (v) post-resuscitation care. Regarding early access, agonal breathing, telephone-guided CPR and the use of 'track and trigger systems' to detect deterioration in patients' condition prior to an arrest are all important. The use of compression-only CPR by bystanders as an alternative to standard CPR in OHCA has been debated. Based on recent findings, guidelines recommend telephone-guided chest compression-only CPR for untrained rescuers, but trained personnel are still advised to give standard CPR with both compressions and ventilation, and the method of choice for this large group remains unclear and demands for a randomized study. Data have shown the benefit of public access defibrillation for dispatched rescuers (e.g. police and fire fighters) but data are not as strong for the use of automated defibrillators (AEDs) by trained or untrained rescuers. Postresuscitation, use of therapeutic hypothermia, the importance of specific prognostic survival factors in the intensive care unit and the widespread use of percutaneous coronary intervention have all been considered. Despite progress in research and improved treatment regimens, most patients do not survive OHCA. Particular areas of interest for improving survival include (i) identification of high-risk patients prior to their arrest (e.g. early warning symptoms and genes); (ii) increased use of bystander CPR training (e.g. in schools) and simplified CPR techniques; (iii) better identification of high-incidence sites and better recruitment of AEDs (via mobile phone solutions?); (iv) improved understanding of the use of therapeutic hypothermia; (v) determining which patients should undergo immediate coronary angiography on hospital admission; and (vi) clarifying the importance of extracorporeal membrane oxygenation during CPR.

Improvements in logistics could increase survival after out-of-hospital cardiac arrest in Sweden.

OBJECTIVES: In a review based on estimations and assumptions, to report the estimated number of survivors after out-of-hospital cardiac arrest (OHCA) in whom cardiopulmonary resuscitation (CPR) was started and to speculate about possible future improvements in Sweden. DESIGN: An observational study. SETTING: All ambulance organisations in Sweden. SUBJECTS: Patients included in the Swedish Cardiac Arrest Registry who suffered an OHCA between January 1, 2008 and December 31, 2010. Approximately 80% of OHCA cases in Sweden in which CPR was started are included. INTERVENTIONS: None RESULTS: In 11 005 patients, the 1-month survival rate was 9.4%. There are approximately 5000 OHCA cases annually in which CPR is started and 30-day survival is achieved in up to 500 patients yearly (6 per 100 000 inhabitants). Based on findings on survival in relation to the time to calling for the Emergency Medical Service (EMS) and the start of CPR and defibrillation, it was estimated that, if the delay from collapse to (i) calling EMS, (ii) the start of CPR, and (iii) the time to defibrillation were reduced to <2 min, <2 min, and <8 min, respectively, 300-400 additional lives could be saved. CONCLUSION: Based on findings relating to the delay to calling for the EMS and the start of CPR and defibrillation, we speculate that 300-400 additional OHCA patients yearly (4 per 100 000 inhabitants) could be saved in Sweden.

Computational design of N-linked glycans for high throughput epitope profiling.

Efficient identification of epitopes is crucial for drug discovery and design as it enables the selection of optimal epitopes, expansion of lead antibody diversity, and verification of binding interface. Although high-resolution low throughput methods like x-ray crystallography can determine epitopes or protein-protein interactions accurately, they are time-consuming and can only be applied to a limited number of complexes. To overcome these limitations, we have developed a rapid computational method that incorporates N-linked glycans to mask epitopes or protein interaction surfaces, thereby providing a mapping of these regions. Using human coagulation factor IXa (fIXa) as a model system, we computationally screened 158 positions and expressed 98 variants to test experimentally for epitope mapping. We were able to delineate epitopes rapidly and reliably through the insertion of N-linked glycans that efficiently disrupted binding in a site-selective manner. To validate the efficacy of our method, we conducted ELISA experiments and high-throughput yeast surface display assays. Furthermore, x-ray crystallography was employed to verify the results, thereby recapitulating through the method of N-linked glycans a coarse-grained mapping of the epitope.

Translating clinical activity and gene expression signatures of etanercept and ciclosporin to the psoriasis xenograft SCID mouse model.

BACKGROUND: The psoriasis xenograft severe combined immunodeficiency (SCID) mouse model is used in drug discovery to obtain preclinical proof-of-principle of new antipsoriatic drug candidates. Validation of this model by antipsoriatic therapeutic agents in clinical use is important to understand its utility as well as its limitations. The effects of the clinically efficacious antitumour necrosis factor-α biologics have not yet been demonstrated in the psoriasis xenograft SCID mouse model. OBJECTIVES: To investigate the effect of etanercept and to explore the time-dependent changes induced by ciclosporin on psoriatic biomarkers at the gene expression level in the psoriasis xenograft SCID mouse model. METHODS: Xenografted SCID mice were treated either with etanercept and vehicle for 2 weeks or with ciclosporin and vehicle for 2 and 4 weeks, respectively. Treatment-induced changes in the psoriatic grafts were assessed by gene expression analysis and compared with published clinical microarray data. The grafts were further evaluated by histology and immunohistochemistry. RESULTS: Etanercept induced normalization of gene expression, which correlated with a significant reduction in epidermal thickness as well as a decrease in the number of proliferative cells. Anti-inflammatory activity induced by ciclosporin preceded the reduction in epidermal hyperplasia. Comparison of the etanercept- and ciclosporin-induced gene expression signatures with clinical microarray data showed significant correlations. CONCLUSIONS: Efficacy of etanercept and ciclosporin could be translated to the psoriasis xenograft SCID mouse model.

Expression, purification and crystallization of a novel metagenome-derived salicylaldehyde dehydrogenase from Alpine soil.

Salicylaldehyde dehydrogenase (SALD) catalyses the last reaction in the upper pathway of naphthalene degradation: the oxidation of salicylaldehyde to salicylate. This enzyme has been isolated and studied from a few organisms that belong to the betaproteobacteria and gammaproteobacteria, predominantly Pseudomonas putida. Furthermore, there is only one crystal structure of this enzyme, which was obtained from P. putida G7. Here, crystallographic studies and analysis of the crystal structure of an Alpine soil metagenome-derived SALD (SALDAP) from an alphaproteobacterium are presented. The SALDAP gene was discovered using gene-targeted sequence assembly and it was cloned into a pLATE51 vector. The recombinant protein was overexpressed in Escherichia coli BL21 (DE3) cells and the soluble protein was purified to homogeneity. The protein crystallized at 20°C and diffraction data from the crystals were collected at a resolution of 1.9 Å. The crystal belonged to the orthorhombic space group C2221, with unit-cell parameters a = 116.8, b = 121.7, c = 318.0 Å. Analysis of the crystal structure revealed its conformation to be similar to the organization of the aldehyde dehydrogenase superfamily with three domains: the catalytic, NAD+-binding and bridging domains. The crystal structure of NahF from P. putida G7 was found to be the best structural homologue of SALDAP, even though the enzymes share only 48% amino-acid identity. Interestingly, a carboxylic acid (protocatechuic acid) was found to be a putative ligand of the enzyme and differential scanning fluorimetry was employed to confirm ligand binding. These findings open up the possibility of studying the mechanism(s) of product inhibition and biocatalysis of carboxylic acids using this enzyme and other related aldehyde dehydrogenases.

Silencing of STE20-type kinase STK25 in human aortic endothelial and smooth muscle cells is atheroprotective.

Recent studies highlight the importance of lipotoxic damage in aortic cells as the major pathogenetic contributor to atherosclerotic disease. Since the STE20-type kinase STK25 has been shown to exacerbate ectopic lipid storage and associated cell injury in several metabolic organs, we here investigate its role in the main cell types of vasculature. We depleted STK25 by small interfering RNA in human aortic endothelial and smooth muscle cells exposed to oleic acid and oxidized LDL. In both cell types, the silencing of STK25 reduces lipid accumulation and suppresses activation of inflammatory and fibrotic pathways as well as lowering oxidative and endoplasmic reticulum stress. Notably, in smooth muscle cells, STK25 inactivation hinders the shift from a contractile to a synthetic phenotype. Together, we provide several lines of evidence that antagonizing STK25 signaling in human aortic endothelial and smooth muscle cells is atheroprotective, highlighting this kinase as a new potential therapeutic target for atherosclerotic disease.

Crystal structure of DNA polymerase I from Thermus phage G20c.

This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SßαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SßαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SßαR motif, was first determined to 2.19 Šresolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Šresolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.

Interaction of single viruslike particles with vesicles containing glycosphingolipids.

Glycosphingolipids are involved in the first steps of virus-cell interaction, where they mediate specific recognition of the host cell membrane. We have employed total-internal-reflection fluorescence microscopy to explore the interaction kinetics between individual unlabeled noroviruslike particles, which are attached to a glycosphingolipid-containing lipid bilayer, and fluorescent vesicles containing different types and concentrations of glycosphingolipids. Under association equilibrium, the vesicle-binding rate is found to be kinetically limited, yielding information on the corresponding activation energy. The dissociation kinetics are logarithmic over a wide range of time. The latter is explained by the vesicle-size-related distribution of the dissociation activation energy. The biological, pharmaceutical, and diagnostic relevance of the study is briefly discussed.