Quantitative trait loci for resistance to infection in sheep using a live Salmonella Abortusovis vaccine.

Quantitative trait loci (QTL) mapping for susceptibility to a Salmonella Abortusovis vaccinal strain was performed using an experimental design involving 30 Romane sheep sire families (1216 progenies). Nine QTL corresponding to bacterial load, weight variations and antibody response criteria were mapped on eight chromosomes, including the major histocompatibility complex area on chromosome 20. Surprisingly, none was found to be significant in the SLC11A1 region (formerly NRAMP1) that has been shown to influence Salmonella susceptibility in other species.

Measurements of multiple heat flux components at the divertor target by using surface eroding thermocouples (invited).

The Surface Eroding Thermocouple (SETC) is a robust diagnostic utilized in DIII-D to provide fast, edge-localized modes (ELMs) resolved heat flux measurements, in particular in geometric regions that are too shadowed for traditional infrared thermography. In order to further investigate the power dissipation in the divertor region, a combination of flush-mounted and recessed SETCs was developed to assess the effect on surface heating from non-charged particles at the divertor target. Utilizing the Divertor Materials Evaluation System sample exposure platform, the first demonstration of the feasibility of using this new method to distinguish between the heat flux from charged particles and that from neutrals and radiative heating was achieved. This paper details the process of using the combination of flush SETCs and recessed SETCs to measure the multiple heat flux components at the divertor target and further discusses how to determine two important ratios, α (ratio of heat flux from charged particles deposit on recessed SETC to that deposit on flush SETC) and ß (ratio of heat flux from non-charged particles deposit on recessed SETC to that deposit on flush SETC), in the estimation of the heat flux from non-charged particle sources. Using a time dependent ratio α, it was found that ∼50% of the total incident heat flux is attributable to the non-charged particles in the fully detached open divertor in DIII-D. Finally, the new application of similar SETC diagnostics in the Small Angle Slot divertor with a V-like configuration and partial tungsten coated surface (SAS-VW) is also introduced.

Performance demonstration of vacuum microwave components critical for the operation of the ITER low-field side reflectometer.

Final design studies in preparation for manufacturing have been performed for functional components of the vacuum portion of the ITER Low-Field Side Reflectometer (LFSR). These components consist of an antenna array, electron cyclotron heating (ECH) protection mirrors, phase calibration mirrors, and vacuum windows. Evaluation of these components was conducted at the LFSR test facility and DIII-D. The antenna array consists of six corrugated-waveguide antennas for simultaneous profile, fluctuation, and Doppler measurements. A diffraction grating, incorporated into the plasma-facing miter bend, provides protection of sensitive components from stray ECH at 170 GHz. For in situ phase calibration of the LFSR profile reflectometer, an embossed mirror is incorporated into the adjacent miter bend. Measurements of the radiated beam profile indicate that these components have a small, acceptable effect on mode conversion and beam quality. Baseline transmission characteristics of the dual-disk vacuum window are obtained and are used to guide ongoing developments. Preliminary simulations indicate that a surface-relief structure on the window surfaces can greatly improve transmission. The workability of real-time phase measurements was demonstrated on the DIII-D profile reflectometer. The new automated real-time analysis agrees well with the standard post-processing routine.

Diagnosis of fast ions produced by negative-ion neutral-beam injection with fast-ion deuterium-alpha spectroscopy.

Negative-ion neutral-beam injection (NNBI) is an important source of heating and current drive for next-step fusion devices where the injected energy can range from hundreds of keV to 1 MeV. Few diagnostics are suitable for phase-space resolved measurements of fast ions with energy in excess of 100 keV. A study to assess the feasibility of fast-ion deuterium-alpha (FIDA) spectroscopy to diagnose high-energy ions produced by NNBI is presented. Case studies with the Large Helical Device (LHD) and JT-60SA illustrate possible solutions for the measurement. The distribution function of fast ions produced by NNBI is calculated for both devices, and the FIDA spectrum is predicted by synthetic diagnostic simulation. Results with 180 keV NNBI in LHD show that, with a judicious choice of viewing geometry, the FIDA intensity is comparable to that obtained with the existing FIDA system. The measurement is more challenging with the 500 keV NNBI in JT-60SA. Simulations predict the FIDA intensity to be about 1% of the background bremsstrahlung, which is small compared to existing FIDA implementations with positive neutral-beam injection where signal levels are an order of magnitude larger. The sampling time required to extract the small FIDA signal is determined using a probabilistic approach. Results indicate that long averaging periods, from ones to tens of seconds, are needed to resolve the FIDA signal in JT-60SA. These long averaging times are suitable in long-pulse (∼100 s), steady-state devices like JT-60SA where an important measurement objective is the spatial profile of the slowing-down distribution of fast ions.

A Faraday-effect polarimeter for fast magnetic dynamics measurement on DIII-D.

A Faraday-effect-based radial-interferometer-polarimeter diagnostic has been developed to explore fast magnetic dynamics in high-performance DIII-D plasmas. The instrument measures radial magnetic field perturbations using three chords positioned near the magnetic axis. Newly developed solid-state sources operating at 650 GHz provide phase noise down to 0.01°/ k H z and tunable bandwidth up to 10 MHz. Various systematic errors which can contaminate the polarimetric measurement have been investigated in detail. Distortion of circular polarization due to non-ideal optical components is calibrated using a rotating quarter wave plate technique. The impact of perpendicular magnetic field, i.e., the Cotton-Mouton effect, is evaluated. The error due to non-collinearity of probe beams is minimized to less than 0.5° for electron density up to 7 × 1019 m-3 by alignment optimization. Optical feedback, due to multiple reflections induced by the double-pass configuration, is identified and reduced. Coherent and broadband high-frequency magnetic fluctuations for DIII-D H-mode plasmas are observed.

Thomson scattering measurements on DIII-D using in-vessel laser mirrors and lenses to diagnose a new divertor location.

Translatable in-vessel mirrors have enabled the DIII-D Thomson scattering system to diagnose the divertor plasma in high triangularity shaped plasmas. Previous divertor Thomson scattering measurements in DIII-D were restricted to spatial locations along a Nd:YAG laser beam that was directed through a vertical port. This only allowed measurements to be made in low triangularity shaped plasmas. The new mirrors re-route the laser underneath floor tiles to a position of smaller major radius as necessary for high triangularity plasmas. New in-vessel collection optics transmit scattered light from regions inaccessible to external lenses. Damage to mirrors and high stray light levels are challenges that were overcome to successfully make these measurements. Through the careful use of baffles and light shields, stray light leakage into polychromator detector channels was reduced to negligible levels, allowing temperature measurements below 1 eV. The system is described and the initial results presented.

The surface eroding thermocouple for fast heat flux measurement in DIII-D.

A novel type of surface eroding thermocouple (SETC) has been tested and demonstrated in the small angle slot (SAS) divertor of DIII-D for fast local heat flux measurements. The thermojunction of the SETC is formed between two thin (10 µm) ribbons, which are filed over to create microfiber junctions. These thermocouples are able to be exposed directly to the plasma at surface temperatures exceeding 2000 °C and are capable of sub-10 ms time resolution. Before installation in SAS, the SETCs were exposed in the lower DIII-D divertor during L-mode and H-mode discharges, from which results are presented. In preliminary tests, SETCs proved to be a qualified diagnostic to accurately measure both the intra-edge localized mode (ELM) and inter-ELM heat flux during H-mode shots with high frequency ELMs (hundreds of Hz) and to resolve heat flux profiles during strike point sweeps. The heat fluxes measured by using SETCs are consistent with the heat fluxes measured by using IR cameras and Langmuir probes. These new diagnostic capabilities will complement the existing IR camera measurements and will be of particularly significant value to measure surface heat flux in the SAS divertor or other regions where the IR camera lacks line of sight.

Tests of a full-scale ITER toroidal interferometer and polarimeter (TIP) prototype on the DIII-D tokamak (invited).

A full-scale ITER toroidal interferometer and polarimeter (TIP) prototype, including an active feedback alignment system, has been installed and tested on the DIII-D tokamak. In the TIP prototype, a two-color interferometry measurement of line-integrated density is carried out at 10.59 µm and 5.22 µm using a CO2 and quantum cascade laser, respectively, while a separate polarimetry measurement of the plasma-induced Faraday effect is made at 10.59 µm. The TIP prototype is equipped with a piezo tip/tilt stage active feedback alignment system that minimizes noise in the measurement and keeps the diagnostic aligned throughout DIII-D discharges. The measured phase resolution for the polarimeter and interferometer is 0.05° (100 Hz bandwidth) and 1.9° (1 kHz bandwidth), respectively. The corresponding line-integrated density resolution for the vibration-compensated interferometer is δnL = 1.5 × 1018 m-2, and the magnetic field-weighted line-integrated density from the polarimeter is δnBL = 1.5 × 1019 Tm-2. Both interferometer and polarimeter measurements during DIII-D discharges compare well with the expectations based on calculations using Thomson scattering measured density profiles and magnetic equilibrium reconstructions. Additionally, larger bandwidth interferometer measurements show that the diagnostic is a sensitive monitor of core density fluctuations with demonstrated measurements of Alfvén eigenmodes and tearing modes.

A heterodyne dispersion interferometer for wide bandwidth density measurements on DIII-D.

In order to improve both the density and particularly the temporal resolution beyond previous dispersion interferometers (DIs), a heterodyne technique based on an acousto-optic (AO) cell has been added to the DI. A 40 MHz drive frequency for the AO cell allows density fluctuation measurements into the MHz range. A CO2 laser-based heterodyne DI (HDI) installed on DIII-D has demonstrated that the HDI is capable of tracking the density evolution throughout DIII-D discharges, including disruption events and other rapid transient phenomena. The data also show good agreement with independent density measurements obtained with the existing DIII-D two-color interferometer. The HDI line-integrated density resolution sampled over a 1 s interval is ∼9 × 1017 m-2. Density fluctuations induced by MHD instabilities are also successfully measured by the HDI.

Faraday-effect polarimeter diagnostic for internal magnetic field fluctuation measurements in DIII-D.

Motivated by the need to measure fast equilibrium temporal dynamics, non-axisymmetric structures, and core magnetic fluctuations (coherent and broadband), a three-chord Faraday-effect polarimeter-interferometer system with fast time response and high phase resolution has recently been installed on the DIII-D tokamak. A novel detection scheme utilizing two probe beams and two detectors for each chord results in reduced phase noise and increased time response [δb ∼ 1G with up to 3 MHz bandwidth]. First measurement results were obtained during the recent DIII-D experimental campaign. Simultaneous Faraday and density measurements have been successfully demonstrated and high-frequency, up to 100 kHz, Faraday-effect perturbations have been observed. Preliminary comparisons with EFIT are used to validate diagnostic performance. Principle of the diagnostic and first experimental results is presented.

Upgraded divertor Thomson scattering system on DIII-D.

A design to extend the unique divertor Thomson scattering system on DIII-D to allow measurements of electron temperature and density in high triangularity plasmas is presented. Access to this region is selectable on a shot-by-shot basis by redirecting the laser beam of the existing divertor Thomson system inboard - beneath the lower floor using a moveable, high-damage threshold, in-vacuum mirror - and then redirecting again vertically. The currently measured divertor region remains available with this mirror retracted. Scattered light is collected from viewchords near the divertor floor using in-vacuum, high temperature optical elements and relayed through the port window, before being coupled into optical fiber bundles. At higher elevations from the floor, measurements are made by dynamically re-focusing the existing divertor system collection optics. Nd:YAG laser timing, analysis of the scattered light spectrum via polychromators, data acquisition, and calibration are all handled by existing systems or methods of the current multi-pulse Thomson scattering system. Existing filtered polychromators with 7 spectral channels are employed to provide maximum measurement breadth (Te in the range of 0.5 eV-2 keV, ne in the range of 5 × 1018-1 × 1021 m3) for both low Te in detachment and high Te measurement up beyond the separatrix.

Bench testing of a heterodyne CO2 laser dispersion interferometer for high temporal resolution plasma density measurements.

A heterodyne detection scheme is combined with a 10.59 µm CO2 laser dispersion interferometer for the first time to allow large bandwidth measurements in the 10-100 MHz range. The approach employed utilizes a 40 MHz acousto-optic cell operating on the frequency doubled CO2 beam which is obtained using a high 2nd harmonic conversion efficiency orientation patterned gallium arsenide crystal. The measured standard deviation of the line integrated electron density equivalent phase resolution obtained with digital phase demodulation technique, is 4 × 1017 m-2. Air flow was found to significantly affect the baseline of the phase signal, which an optical table cover was able to reduce considerably. The heterodyne dispersion interferometer (DI) approach is found to be robustly insensitive to motion, with measured phase shifts below baseline drifts even in the presence of several centimeters of retroreflector induced path length variations. Plasma induced dispersion was simulated with a wedged ZnSe plate and the measured DI phase shifts are consistent with expectations.

Stage-specific transcription of the homebox gene Bnhd1 in young tissues and flowers of Brassica napus.

The Bnhd1 cDNA, only distantly related to published homebox gene sequences, was isolated from Brassica napus by the polymerase chain reaction. The Bnhd1 transcript was detected in all organs of young seedlings, but only in the youngest leaves and flowers of mature plants. A 4- and 7-fold increase in transcription levels was observed after wounding of young roots and leaves, respectively.

Potent inhibition of steroid sulfatase activity by 3-O-sulfamate 17alpha-benzyl(or 4'-tert-butylbenzyl)estra-1,3,5(10)-trienes: combination of two substituents at positions C3 and c17alpha of estradiol.

Steroid sulfates are precursors of hormones that stimulate androgen- and estrogen-dependent cancers. Thus, steroid sulfatase, the enzyme that catalyzes conversion of DHEAS and E1S to the corresponding unconjugated steroids DHEA and E1, appears to be one of the key enzymes regulating the level of active androgenic and estrogenic steroids. Since 17alpha-substituted benzylestradiols and 3-O-sulfamate estrone (EMATE) represent two families of steroid sulfatase inhibitors that probably act through different mechanisms, we synthesized compounds 3-O-sulfamate 17alpha-benzylestradiol (4) and 3-O-sulfamate 17alpha-(tert-butylbenzyl)estradiol (5) that contain two kinds of substituents on the same molecule. In our enzymatic assay using a homogenate of human embryonal (293) cells transfected with steroid sulfatase, compounds 4 and 5 were found to be more potent inhibitors than already known steroid sulfatase inhibitors that have only a C17alpha-substituent or only a C3-sulfamate group (EMATE). The IC50 values of 4 and 5 were, respectively, 0.39 and 0.15 nM for the transformation of E1S to E1 and 4.1 and 1.4 nM for the transformation of DHEAS to DHEA. Compound 5 inhibited the steroid sulfatase activity in intact transfected (293) cell culture assays by inactivating the enzyme activity. Compound 5 also inactivates the steroid sulfatase activity at lower concentration than EMATE in microsomes of transfected (293) cells. In this assay, an excess of natural substrate E1S protects enzyme against inactivation by 5 or EMATE. Furthermore, the unsulfamoylated analogue of 5, compound 3, did not inactivate the steroid sulfatase.

Structure-activity relationships of 17alpha-derivatives of estradiol as inhibitors of steroid sulfatase.

The steroid sulfatase or steryl sulfatase is a microsomal enzyme widely distributed in human tissues that catalyzes the hydrolysis of sulfated 3-hydroxy steroids to the corresponding free active 3-hydroxy steroids. Since androgens and estrogens may be synthesized inside the cancerous cells starting from dehydroepiandrosterone sulfate (DHEAS) and estrone sulfate (E(1)S) available in blood circulation, the use of therapeutic agents that inhibit steroid sulfatase activity may be a rewarding approach to the treatment of androgeno-sensitive and estrogeno-sensitive diseases. In the present study, we report the chemical synthesis and biological evaluation of a new family of steroid sulfatase inhibitors. The inhibitors were designed by adding an alkyl, a phenyl, a benzyl, or a benzyl substituted at position 17alpha of estradiol (E(2)), a C18-steroid, and enzymatic assays were performed using the steroid sulfatase of homogenized JEG-3 cells or transfected in HEK-293 cells. We observed that a hydrophobic substituent induces powerful inhibition of steroid sulfatase while a hydrophilic one was weak. Although a hydrophobic group at the 17alpha-position increased the inhibitory activity, the steric factors contribute to the opposite effect. As exemplified by 17alpha-decyl-E(2) and 17alpha-dodecyl-E(2), a long flexible side chain prevents adequate fitting into the enzyme catalytic site, thus decreasing capacity to inhibit the steroid sulfatase activity. In the alkyl series, the best compromise between hydrophobicity and steric hindrance was obtained with the octyl group (IC(50) = 440 nM), but judicious branching of side chain could improve this further. Benzyl substituted derivatives of estradiol were better inhibitors than alkyl analogues. Among the series of 17alpha-(benzyl substituted)-E(2) derivatives studied, the 3'-bromobenzyl, 4'-tert-butylbenzyl, 4'-butylbenzyl, and 4'-benzyloxybenzyl groups provided the most potent inhibition of steroid sulfatase transformation of E(1)S into E(1) (IC(50) = 24, 28, 25, and 22 nM, respectively). As an example, the tert-butylbenzyl group increases the ability of the E(2) nucleus to inhibit the steroid sulfatase by 3000-fold, and it also inhibits similarly the steroid sulfatase transformations of both natural substrates, E(1)S and DHEAS. Interestingly, the newly reported family of steroid sulfatase inhibitors acts by a reversible mechanism of action that is different from the irreversible mechanism of the known inhibitor estrone sulfamate (EMATE).

Cytotoxic effect of multinucleation in HeLa cell cultures associated with the presence of Vir plasmid in Escherichia coli strains.

Escherichia coli strain S5 possesses a virulent plasmid termed Vir, which codes for a lethal toxin and a surface antigen. This strain and two trans-conjugant strains, which have received the Vir plasmid from S5, produced a specific and thermolabile cytopathic effect of multinucleation in an HeLa cell cultures assay, whereas isogenic Vir- strains did not. Moreover sonicates of two epidemiologically unrelated Vir+ strains, exerted the same type of cytotoxicity. This effect, together with lethality for chicken, was specifically neutralized by a rabbit antiserum prepared against Vir+ sonicates. The Vir cytopathic effect appeared morphologically distinct from the one caused by the cytotoxic necrotizing factor of E. coli, which was partially related immunologically. We therefore propose to call this type of toxin 'Vir cytotoxin'.

17Alpha-alkan (or alkyn) amide derivatives of estradiol as inhibitors of steroid-sulfatase activity.

To develop inhibitors of steroid sulfatase without residual estrogenic activity, we have designed a series of estradiol (E2) derivatives bearing an alkan (or alkyn) amide side chain at position 17alpha. A hydrophobic alkyl group was selected from our previous study where 17alpha-octyl-E2 was found to inhibit strongly the steroid-sulfatase activity. Furthermore, it is known that an alkylamide side chain blocks the estrogen-receptor activation. Starting from ethynylestradiol, the chemical synthesis of target compounds was short and efficient with overall yields of 22-42% (3 or 4 steps). Among these compounds, N-octyl,N-methyl-3-(3',17'beta-dihydroxy-1',3',5'(10')-estratrien- 17'alpha-yl)-propanamide (15) was the most potent inhibitor, with an IC50 value of 0.08 microM for the transformation of estrone sulfate (E1S) to estrone (E1) by homogenated JEG-3 cells. N-butyl, N-hexyl, and N,N-dioctyl propanamide derivatives of E2 (IC50 values of 6.4, 2.8, and >20 microM, respectively) were less potent inhibitors than N-octyl analog 15. Furthermore, the unsaturated propynamide analog of 15 gave lower inhibition (four times) than the saturated compound. Compound 15 is also about 100-fold more effective in interacting with the enzyme than substrate E1S itself. The ability of target compounds to bind the estrogen receptor, to stimulate the proliferation of estrogen-sensitive ZR-75-1 cells, or to inhibit the E2-stimulation of ZR-75-1 cells was also evaluated. Although a mixed estrogenic/anti-estrogenic activity was obtained for tested compounds at 1 microM, no estrogenic activity was observed at 0.03 microM for 15. In conclusion, a promising inhibitor of steroid-sulfatase activity was obtained by introducing a hydrophobic octyl group in a 17alpha-propanamide side chain of E2, but further structure-activity relationships (SAR) studies are necessary to minimize the residual estrogenic activity.

Cytotoxins in non-enterotoxigenic strains of Escherichia coli isolated from feces of diarrheic calves.

We have examined the cytotoxic responses produced in HeLa and Vero cell cultures by sonicates from 15 non-enterotoxigenic (STa-, LT-) strains of E. coli, highly lethal for mice parenterally LD50 less than 3 X 10(7) CFU), which had been isolated from feces of diarrheic calves. Three types of cytotoxic responses were observed. Type 1 (five strains) consisted of enlargement, rounding and polynucleation of HeLa cells, an effect previously reported with cytotoxic necrotizing factor (CNF) in E. coli from infant and piglet enteritis. Type 2 toxicity (three strains and the control Vir strain S5) was also characterized by enlargement and polynucleation of HeLa cells, but in contrast to Type 2 effect, cells were elongated. Sonicates from the latter strains were lethal for chickens, producing the lesions previously described with Vir strains. Type 3 toxicity (two strains and the control VT strain H19), produced an extensive destruction of both Vero and HeLa cell cultures. Cytotoxic effects were completely abolished upon heating for 1 h at 60 degrees C for Type 1 and 2 extracts and at 80 degrees C for Type 3 extracts. Seroneutralization assays showed that cytotoxins of the same type were closely related antigenically. In addition, a slight cross-neutralization was observed between Type 1 (CNF) and Type 2 (Vir) toxins.

Detection of Escherichia coli strains producing cytotoxic necrotizing factor type two (CNF2) by enzyme-linked immunosorbent assay.

Sheep and rabbit antisera were produced against lysates of E. coli strain 711 (pVir). This K-12 strain carries the Vir plasmid which codes for Cytotoxic Necrotizing Factor type 2 (CNF2). Immunoglobulin G (IgG) fractions of both immune sera were subsequently purified by a two-step precipitation method. To increase the specificity for CNF2, the sheep IgG preparation was extensively adsorbed against both a sonicated extract of isogenic K-12 strain 711 and intact phenol-treated cells of vaccine strain 711 (pVir). An enzyme-linked immunosorbent assay (ELISA) was developed to detect clinical isolates of E. coli producing CNF2, using the final preparations of rabbit and sheep IgG in a double sandwich technique. The results obtained with this CNF2-ELISA were compared to those obtained with the conventional HeLa cell cytotoxicity assay. The testing of 133 E. coli strains (49 CNF2 positive strains and 84 negative strains) resulted in no false-negative and no false-positive. Therefore, the CNF2-ELISA offers a good alternative to the HeLa cell culture assay for the detection of CNF2-producing strains where facilities for and experience with cell cultures is lacking.

Cytokine gene expression in lymph node and spleen of sheep in response to Salmonella infection by two serotypes displaying different host specificity.

In order to investigate the determinism of the host specificity and to better understand the host resistance mechanisms, infections of sheep were performed with either S. abortusovis, serotype specific for ovine species, or with S. dublin, serotype adapted to cattle and accidentally transmissible to human. Following a subcutaneous challenge, S. dublin disseminated more rapidly towards lymphoid tissues than S. abortusovis. However, S. abortusovis tended to persist in spleen more efficiently than S. dublin. Using a quantitative RT-PCR method, the expression level of ovine cytokines genes was measured in the draining lymph node and in the spleen, in the course of infection. Inflammatory cytokine response was characterised by an early and strong increase of IL-1beta and TNFalpha mRNA in both lymphoid organs following S. dublin infection, while S. abortusovis challenge only induced IL-1beta mRNA increase in the spleen at day 3 post-inoculation. Likewise, S. dublin infection provoked a marked increase of IL-12 mRNA and a slight up-regulation of IFNgamma gene transcription in the local lymphoid site, in contrast to S. abortusovis infection. Elsewhere, both serotypes induced a strong and early IL-10 mRNA production and had no effect on IL-4 gene expression. Finally, taken together, these data suggest that the intensity of inflammatory and anti-infectious cytokine responses, but not the type 2 cytokine response, is serotype-dependent. They also suggest that the host-specific serotype, by limiting the host cytokine-mediated defence, could favour its persistence within lymphoid organs.