A divertor scraper observation system for the Wendelstein 7-X stellarator.

Two graphite divertor elements called scrapers have been installed on the Wendelstein 7-X stellarator in the throat of the magnetic island divertor. To diagnose one, we have designed, built, calibrated, and installed a new infrared/visible imaging endoscope system to enable detailed observations of the plasma interactions and heat loads at one of the scrapers and the nearby divertor surfaces. The new system uses a shuttered pinhole-protected pair of 90° off-axis 228 mm focal length aluminum parabolic mirrors, and two flat turning metal mirrors, to send light to a sapphire vacuum window 1.6 meters away, beyond which we have co-located telephoto lens-based infrared and visible cameras. The back-to-back off-axis parabolas serve to cancel out most aberrations, enabling the use of off-the-shelf commercial optics outside of the vessel. For the infrared, we use a 3-5 µm 1-megapixel FLIR SC8303HD camera and for the visible, a 5-megapixel CMOS PCO 5.5 edge camera. A short 1-m quartz pickoff fiber is used to send 200-1100 nm light to a compact spectrometer, also located in the same iron shield box as the cameras. The camera field of view covers the 700 mm length of the scraper, and includes locations monitored by thermocouples and Langmuir probes embedded in some of the scraper tiles. Predicted and actual optical test performances of the overall system are compared.

Fatty acid kinase A is an important determinant of biofilm formation in Staphylococcus aureus USA300.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA)-USA300 is notorious for its ability to cause community- and healthcare-acquired infections, which are even more difficult to treat when associated with a biofilm phenotype. We aimed to characterize the genetic determinants of biofilm formation in a USA300 skin abscess isolate (UAS391) that formed prolific biofilms. METHODS: USA300 S. aureus strains, TCH1516 and FPR3757, were found to be closely related based on whole genome mapping (Argus™ Optical Mapping System, Opgen Inc, Gaithersburg, USA) to UAS391 (96.3-99.1 % similarity, P=0.0151), however differed markedly in biofilm formation (P=0.0001) on a dynamic assay (BioFlux 200, Fluxion Biosciences, USA). Comparison of whole genome sequences of these strains identified differences in a total of 18 genes. Corresponding Tn (bursa aurealis-bearing) knockout mutants in these target genes were obtained from a publicly available mutant library of the same clonal lineage (USA300-JE2) and were characterized phenotypically for biofilm formation. Tn mutants showing significant differences in biofilm formation were utilized for transduction into a plasmid-cured erythromycin-sensitive derivative of UAS391 and for complementation experiments. All strains were tested on the dynamic assay, and 17h-biofilms were stained (SYTO9, Life Technologies) and fluorescence intensity quantified by microscopy (Zeiss, ImageJ). Gene expression levels in Tn and transduced mutants were studied by quantitative reverse transcriptase PCR (StepOnePlusTM, Applied Biosystems®). RESULTS: Comparison of the sequenced genomes of TCH1516, FPR3757 and UAS391 yielded a limited number of variant genes (n=18) that were hypothesized to account for the observed difference in biofilm-forming capacity. Screening of Tn mutants disrupted in these target genes identified one mutant (NE229) bearing a transposon insertion in SAUSA300_1119 (fakA), which exhibited increased biofilm formation similar to UAS391 (P=0.9320). Transduction experiments confirmed that fakA::Tn corresponded to 1.9- to 4.6-fold increase in biofilm formation depending on the USA300 strain background (P≤0.0007), while complementation of the TCH1516 wild-type fakA allele in UAS391 resulted in a 4.3-fold reduction in biofilm formation (P<0.0001). CONCLUSIONS: This sequential approach, consisting of strain typing, genome comparison and functional genomics, identified fakA, a recently described fatty acid kinase in S. aureus that is essential for phospholipid synthesis and also impacts the transcription of numerous virulence factors, as a negative regulator of biofilm formation in S. aureus USA300.

Complete Genome Sequences of Two Prolific Biofilm-Forming Staphylococcus aureus Isolates Belonging to USA300 and EMRSA-15 Clonal Lineages.

Methicillin-resistant Staphylococcus aureus (MRSA) causes serious infections that are even more difficult to treat when associated with a biofilm phenotype that facilitates evasion of the host immune system and antibiotics. As a first step toward understanding the mechanisms underlying biofilm formation, we sequenced the genomes of two prolific biofilm-forming strains belonging to the two most important globally disseminated clonal lineages, USA300 and EMRSA-15.

Complete genome sequence of the novel Escherichia coli phage phAPEC8.

Bacteriophage phAPEC8 is an Escherichia coli-infecting myovirus, isolated on an avian pathogenic Escherichia coli (APEC) strain. APEC strains cause colibacillosis in poultry, resulting in high mortality levels and important economic losses. Genomic analysis of the 147,737-bp double-stranded DNA phAPEC8 genome revealed that 53% of the 269 encoded proteins are unique to this phage. Its closest relatives include the Salmonella phage PVP-SE1 and the coliphage rv5, with 19% and 18% similar proteins, respectively. As such, phAPEC8 represents a novel, phylogenetically distinct clade within the Myoviridae, with molecular properties suitable for phage therapy applications.

Surgical treatment of the isolated left ostial stenosis.

AIM: Conventional grafting for left main coronary artery (LMCA) stenosis provides a retrograde perfusion to extensive myocardial area, leads prospectively to totally occlusion of the LMCA and to competitive flow of the non-occluded coronaries thus consuming the grafts. Surgical reconstruction of the LMCA with patch-angioplasty is an alternative method that eliminates these drawbacks. METHODS: Between February 1997 and August 2009, 45 patients with isolated LMCA stenosis were referred for surgical ostial reconstruction. In 32 patients (71%) surgical angioplasties have been performed. All patients were followed-up clinically and with transesophageal echocardiography (TEE) and coronary angiography when required. RESULTS: In 13 patients (29%) a LMCA stenosis could not be confirmed. There were no early mortality or perioperative myocardial infarctions. The postoperative course was uneventful in all patients. In 30 patients, TEE demonstrated a wide open main stem flow pattern one to six months after reconstruction of the left main coronary artery with one patch mild aneurysmal dilated. CONCLUSION: The results after surgical reconstruction with patch-angioplasty are good and comparable with those after CABG. The endarterectomy and reconstruction should be avoided in the case of distal stenosis of LMCA and excessive calcification. Almost one third of the study group had no really LMCA stenosis. These patients have benefited of the plan to perform a reconstruction: antegrade flow pattern remained sustained and the arterial grafts have been spared.

Equal intensity double plasmon resonance of bimetallic quasi-nanocomposites based on sandwich geometry.

We report a strategy to achieve a material showing equal intensity double plasmon resonance (EIDPR) based on sandwich geometry. We studied the interaction between localized plasmon resonances associated with different metal clusters (Au/Ag) on Teflon AF (TAF) in sandwich geometry. Engineering the EIDPR was done by tailoring the amount of Au/Ag and changing the TAF thickness. The samples were investigated by transmission electron microscopy (TEM) and UV-visible spectroscopy. Interestingly, and in agreement with the dipole-surface interaction, the critical barrier thickness for an optimum EIDPR was observed at 3.3 nm. The results clearly show a plasmon sequence effect and visualize the role of plasmon decay.

Plasmonic properties of Ag nanoclusters in various polymer matrices.

Nanocomposite films containing Ag nanoparticles embedded in a polymer matrix of Teflon AF, poly(methyl methacrylate) (PMMA) and Nylon 6 were prepared by vapour phase co-deposition in high vacuum. A large variation of the particle plasmon resonance frequency in the visible region was obtained by increasing the Ag volume fraction from 4-80%. The metal volume fraction was measured by energy dispersive x-ray spectrometry (EDX) and the film thickness was measured by surface profilometry. The position, width and strength of the plasmon resonance depend strongly on the metal filling factor, cluster size and interparticle distance. The microstructure of the nanocomposites (shape, size, size distribution and interparticle separation of metal clusters) was determined by transmission electron microscopy. The effect of the surrounding dielectric medium on the optical properties of nanocomposites was investigated by comparing the Teflon AF/Ag, PMMA/Ag and Nylon/Ag composites.

Aortic valve replacement with the Cryolife-O'Brien stentless aortic bioprosthesis: five-year experience.

AIM: Improved hemodynamics with stentless bioprosthesis compared to stented valves have been well documented. It has been suggested that a simplified implant model, the Cryolife-O'Brien, offers less satisfactory outcomes compared with standard stentless models. This study was conducted to prospectively evaluate the midterm results after aortic valve replacement with the Cryolife-O'Brien stentless bioprosthesis. METHODS: In 1996, the prospective clinical trial using different stentless valves was initiated in our center. From September 1996 through August 2001, 132 consecutive patients with a mean age of 72.5 years underwent aortic valve replacement with the Cryolife-O'Brien porcine stentless bioprosthesis by the same surgeon. The predominant aortic valve lesion was stenosis in 110 cases and insufficiency in 22 cases. Patients have been followed-up from 2 to 60 months, mean 28 months. Echocardiography was performed by the same echocardiographer preoperatively, intraoperatively, postoperatively at discharge, 2 to 6 months later and annually thereafter. RESULTS: Sixty-five percent of patients received a valve 25 mm in diameter or larger, 42% had concomitant coronary bypass grafting. The 30-day operative mortality rate was 6.8 %. Nine late deaths, none related to the valve, have occurred. Severe aortic insufficiency caused by oversizing led to early reoperation in 3 patients. The peak and mean systolic gradients decreased significantly during the first 12 months after implantation (p<0.001) and the effective valve areas increased significantly during this time interval (p<0.001). Eleven patients have aortic insufficiency, trivial in 7 and mild in 4. The actuarial survival at 5 years was 86+/-3%. The rate for freedom from endocarditis was 100% and for freedom from thromboembolic events 92%. CONCLUSIONS: The Cryolife-OBrien stentless bioprosthesis has superior hemodynamics and a low rate of valve-related complications thus representing a very good alternative to conventional stented bioprosthesis. The midterm results are encouraging but further follow-up is needed to determine the valve's durability.

Comparison of three different types of stentless valves: full root or subcoronary.

BACKGROUND: It is believed that, compared with stented valves, stentless bioprostheses at the aortic position offer a larger orifice area. METHODS: During the past 45 months, we have implanted 211 various types of aortic prostheses in our clinic. In the subcoronary position, we have used the Medtronic Freestyle, Toronto SPV, and Cryolife O'Brien prostheses, and as an aortic root replacement, the Medtronic Freestyle. There were no special indications for selection of each prosthesis except in 8 patients suffering from a disease of the ascending aorta in addition or in a redo procedure because of endocarditis or valve degeneration in which we implanted the full root Freestyle prosthesis. All patients had echocardiographic examinations postoperatively and after 1 year. RESULTS: Although the implantations took significantly longer time initially, recently the complication rate has shown itself to be no greater than in comparable patients with stented prostheses. The hemodynamic results are very good with the exception of the Freestyle prosthesis implanted in the subcoronary position. The gradients of the remaining three prosthesis after 1 year are between 5 mm Hg and 10 mm Hg, and the effective valve orifice is between 2 and 3 cm2 depending on valve size. CONCLUSIONS: The use of stentless tissue valves offers better hemodynamic results than that of stented valves with essentially no increased operative risk.

[Coronary bypass surgery in May-Hegglin anomaly]. / Koronare Bypassoperation bei May-Hegglin-Anomalie.

HISTORY: Twelve years prior to hospitalization because of a severe bleeding, a 70 year old patient was diagnosed with a May-Hegglin anomaly, which is a rare autosomal dominant inherited form of thrombocytopenia. INVESTIGATIONS: The blood smear contained signs of the May-Hegglin anomaly: Döhle's inclusion bodies and giant platelets. Platelet counts were around 30 G/l. Coronary angiography revealed a highly severe left main stenosis. TREATMENT AND COURSE: Even using maximum drug therapy, angina pectoris could not be stabilized. Therefore emergency coronary artery bypass grafting had to be performed. Before skin incision 3 micrograms/kg DDAVP (Desmopressin) were administered and after extracorporeal circulation 8 units of platelets were transfused. In addition, perioperative coagulation management was performed according to usual standards. There were no bleeding complications. The patient could leave the clinic after 11 days in stable condition. CONCLUSION: Patients showing May-Hegglin anomaly, even with serious thrombocytopenia, can be operated using extracorporal circulation without a high risk of bleeding.

Early clinical experience with the On-X prosthetic heart valve.

BACKGROUND AND AIM OF THE STUDY: The study was designed to investigate the clinical performance of the On-X prosthetic heart valve in a multicenter experience. METHODS: Between September 1996 and September 1999, 301 patients (56% males) underwent isolated On-X valve replacement (184 isolated aortic (AVR), 117 isolated mitral (MVR)) at 11 European centers under a standardized protocol. Average age at implant was 60.2 years. Office or hospital follow up was required by the protocol; average follow up on all patients was 11 months. RESULTS: Thirty-day mortality in the study was 2.2% for AVR and 6.0% for MVR, with valve-related mortality of 0.5% for AVR. There were eight late deaths (0.7%/pt-yr AVR and 2.3%/pt-yr MVR). Two of these deaths were sudden, and thus possibly valve-related (one AVR, one MVR). Early total valve-related morbidity was 3.5% for AVR and 2.6% for MVR. In total, 13 thromboembolic events occurred; one early event in AVR resulted in death (0.5%), and one transient early event occurred in MVR (0.9%). There were 11 late events (seven AVR (1.7%/pt-yr) and four MVR (1.8%/pt-yr)), for a two-year freedom from thromboembolism of 96.6% after AVR and 97.1% after MVR. Three late bleeding events occurred, all after AVR (0.7%/pt-yr and 98.9% free at two years). Major paravalvular leaks requiring reoperation occurred on two occasions early (one AVR (0.5%), one MVR (0.8%)) and once late in MVR (0.5%/pt-yr). Late minor, untreated paravalvular leaks occurred in three AVR patients (0.7%/pt-yr) and in one MVR patient (0.5%/pt-yr). Prosthetic endocarditis occurred four times (two AVR (0.5%/pt-yr), two MVR (0.9%/pt-yr)), all within the first 12 months of surgery. Actuarial freedom from all valve-related events at two years was 88.7% for AVR and 88.1% for MVR. NYHA class was improved in 75.8% of AVR patients and 70.6% of MVR patients at two years after surgery. CONCLUSION: These early results indicate that the On-X valve provides satisfactory clinical outcome in the immediate postoperative period, and that the valve is both safe and effective.

Analysis of calcium imaging signals from the honeybee brain by nonlinear models.

Recent Ca(2+)-imaging studies on the antennal lobe of the honeybee (Apis mellifera) have shown that olfactory stimuli evoke complex spatiotemporal changes of the intracellular Ca(2+) concentration, in which stimulus-dependent subsets of glomeruli are highlighted. In this work we use nonlinear models for the quantitative identification of the spatial and temporal properties of the Ca(2+)-dependent fluorescence signal. This technique describes time series of the Ca(2+) signal as a superposition of biophysically motivated model functions for photobleaching and Ca(2+) dynamics and provides optimal estimates of their amplitudes (signal strengths) and time constants together with error measures. Using this method, we can reliably identify two different stimulus-dependent signal components. Their delays and rise times, delta(c1) = (0.4 +/- 0.3) s, tau(c1) = (3.8 +/- 1.2) s for the fast component and delta(c2) = (2.4 +/- 0.6) s, tau(c2) = (10.3 +/- 3.2) s for the slow component, are constant over space and across different odors and animals. In chronological experiments, the amplitude of the fast (slow) component often decreases (increases) with time. The pattern of the Ca(2+) dynamics in space and time can be reliably described as a superposition of only two spatiotemporally separable patterns based on the fast and slow components. However, the distributions of both components over space turn out to differ from each other, and more work has to be done in order to specify their relationship with neuronal activity.

Structural basis of carbohydrate recognition by lectin II from Ulex europaeus, a protein with a promiscuous carbohydrate-binding site.

Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins. The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework.

Examination of hemolytic potential with the On-X(R) prosthetic heart valve.

BACKGROUND AND AIM OF THE STUDY: Mechanical valves are known to produce chronic, subclinical hemolysis in most patients. Generally, haptoglobin is reduced to below normal in most patients, while lactate dehydrogenase (LDH) is increased to as much as 200% above the upper normal, sometimes resulting in anemia. The study was designed to investigate the clinical hemolysis of the On-X(R) prosthetic heart valve in a multicenter experience with a standard protocol and a single clinical laboratory. METHODS: Between September 1996 and August 1998, 248 patients underwent isolated valve replacement at 10 European centers. Blood samples were collected from these preoperatively and at 3-6 months and one year postoperatively. All samples were analyzed at a central laboratory, thus assuring poolability of the data. In total, 151 patients were tested at 3-6 months, and 62 at one year. Blood parameters measured were LDH, haptoglobin, hematocrit, total hemoglobin, red cell count and reticulocyte count. Paired analysis was used to compare preoperative baseline values with 3-6-month and one-year values. Data were analyzed with regard to both valve position and size. RESULTS: At 3-6 months and one year after surgery, average values for hematocrit, hemoglobin, red cell count and reticulocyte count were all near the center of the normal range, regardless of valve position or size. Statistically significant increases in red cell count and decreases in reticulocyte count occurred after both aortic valve replacement (AVR) and mitral valve replacement (MVR). These changes were of no clinical importance, but indicate that anemia has not occurred in these patients. At 3-6 months, haptoglobin was reduced to below normal in 86% of both AVR and MVR patients; this also occurred after one year and was statistically significant. Postoperatively, the mean LDH value in AVR was 228 U/l (91% of upper normal, 250 U/l) at 3-6 months, and 246 U/l (98% of upper normal) at one year. In MVR, these LDH values were 271 U/l (108% of upper normal) and 265 U/l (106% of upper normal). CONCLUSIONS: These results indicate that the On-X valve causes lower levels of chronic hemolysis in the immediate postoperative period and up to one year after surgery, especially when compared with reports of LDH elevations up to 200% of upper normal. Hemolytic anemia has not occurred in this patient population.

The trehalose operon of Pseudomonas fluorescens ATCC 17400.

The trehalose operon of Pseudomonas fluorescens ATCC 17400 consists of treP, treA and treR. The gene treP codes for a putative enzyme II subunit of the phosphotransferase system that catalyzes the phosphorylation of trehalose together with its translocation across the cell membrane and treA encodes a putative phosphotrehalase, which hydrolyzes the incoming trehalose-6-phosphate into glucose and glucose-6-phosphate. Both genes are negatively regulated by TreR, a repressor of the FadR-GntR family of transcription regulators. The operon that is induced by trehalose present in the medium shows a high similarity both in the function of genes and in the regulation with the trehalose operon of Bacillus subtilis.

Identification and molecular characterization of a novel Salmonella enteritidis pathogenicity islet encoding an ABC transporter.

Using a newly constructed minitransposon with a phoA reporter gene in a Salmonella enteritidis phoN mutant, we have identified an iron- and pH-inducible lipoprotein gene sfbA, which is a component of a novel ABC-type transporter system required for virulence. This gene is located on a 4 kb Salmonella-specific chromosomal segment, which constitutes a new pathogenicity islet. This islet encodes an outer membrane protein, OmpX, and contains the operon designated sfbABC (Salmonella ferric binding) encoding a putative periplasmic iron-binding lipoprotein SfbA, a nucleotide-binding ATPase SfbB and a cytoplasmic permease SfbC, as predicted by their characteristic signature sequences. Inactivation of the sfbA gene resulted in a mutant that is avirulent and induces protective immunity in BALB/c mice. The wild-type phenotype could be restored by in vivo complementation with the sfbABC operon. This novel transporter might be involved in iron uptake in Salmonella.

Characterization of nonenterotoxigenic Escherichia coli strains producing F17 fimbriae isolated from diarrheic lambs and goat kids.

Forty-five ovine and caprine nonenterotoxigenic Escherichia coli strains producing F17-related fimbriae were characterized with respect to the fimbrial structural subunit and adhesin subtypes produced. In addition, several characteristics related to the virulence of strains producing F17 fimbriae were studied. Most of the strains (73%) possessed the f17cA structural subunit gene, whereas the f17aA and f17dA genes were detected only on three (6%) and two (4%) strains, respectively. The f17bA gene was not detected. All but one of these strains possessed the f17G genes of the adhesin subfamily II. The only strain having the f17G gene of subfamily I possessed the structural subunit gene f17dA. Sequencing of the f17A and f17G genes of four selected strains confirmed the association of f17cA and f17dA structural subunit genes with the f17G genes of the adhesin subfamily II. These results indicated that adhesins of the subfamily II are prominent among ovine and caprine isolates and that they are indistinctly associated with the F17 structural subunit subtypes on these field strains. CS31A- and CNF2-related genes were not detected. Most of the strains adhered in vitro to ovine intestinal brush borders (36 of 45) and agglutinated the erythrocytes of different species in the presence of D-mannose (39 of 45). F17-positive strains produced colicin V (57%) and were resistant to the bactericidal effect of serum (91%) in significantly higher percentages than F17-negative strains (34% produced colicin V, and 66% were serum resistant). Thus, most of the studied ovine and caprine strains showed phenotypic characteristics of septicemic strains.

Analysis of T-DNA-mediated translational beta-glucuronidase gene fusions.

Three random translational beta-glucuronidase (gus) gene fusions were previously obtained in Arabidopsis thaliana, using Agrobacterium-mediated transfer of a gus coding sequence without promoter and ATG initiation site. These were analysed by IPCR amplification of the sequence upstream of gus and nucleotide sequence analysis. In one instance, the gus sequence was fused, in inverse orientation, to the nos promoter sequence of a truncated tandem T-DNA copy and translated from a spurious ATG in this sequence. In the second transgenic line, the gus gene was fused to A. thaliana DNA, 27 bp downstream an ATG. In this line, a large deletion occurred at the target site of the T-DNA. In the third line, gus is fused in frame to a plant DNA sequence after the eighth codon of an open reading frame encoding a protein of 619 amino acids. This protein has significant homology with animal and plant (receptor) serine/threonine protein kinases. The twelve subdomains essential for kinase activity are conserved. The presence of a potential signal peptide and a membrane-spanning domain suggests that it may be a receptor kinase. These data confirm that plant genes can be tagged as functional translational gene fusions.

Trehalose induces antagonism towards Pythium debaryanum in Pseudomonas fluorescens ATCC 17400.

Pseudomonas fluorescens ATCC 17400 shows in vitro activity against Pythium debaryanum under conditions of iron limitation. A lacZ reporter gene introduced by transposon mutagenesis into the P. fluorescens ATCC 17400 trehalase gene (treA) was induced by a factor released by the phytopathogen Pythium debaryanum. The induction of the lacZ gene was lost upon treatment of the Pythium supernatant with commercial trehalase. A trehalose concentration as low as 1 microM could induce the expression of treA. The mutation did not affect the wild-type potential for fungus antagonism but drastically decreased the osmotolerance of the mutant in liquid culture and suppressed the ability of P. fluorescens ATCC 17400 to utilize trehalose as a carbon source. A subsequent transposon insertion in treP, one of the trehalose phosphotransferase genes upstream of treA, silenced the lacZ gene. This double mutant restricted fungal growth only under conditions of high osmolarity, which probably results in internal trehalose accumulation. These data confirm the role of the disaccharide trehalose in osmotolerance, and they indicate its additional role as an initiator of or a signal for fungal antagonism.