A high-density and high-confinement tokamak plasma regime for fusion energy.

The tokamak approach, utilizing a toroidal magnetic field configuration to confine a hot plasma, is one of the most promising designs for developing reactors that can exploit nuclear fusion to generate electrical energy1,2. To reach the goal of an economical reactor, most tokamak reactor designs3-10 simultaneously require reaching a plasma line-averaged density above an empirical limit-the so-called Greenwald density11-and attaining an energy confinement quality better than the standard high-confinement mode12,13. However, such an operating regime has never been verified in experiments. In addition, a long-standing challenge in the high-confinement mode has been the compatibility between a high-performance core and avoiding large, transient edge perturbations that can cause very high heat loads on the plasma-facing-components in tokamaks. Here we report the demonstration of stable tokamak plasmas with a line-averaged density approximately 20% above the Greenwald density and an energy confinement quality of approximately 50% better than the standard high-confinement mode, which was realized by taking advantage of the enhanced suppression of turbulent transport granted by high density-gradients in the high-poloidal-beta scenario14,15. Furthermore, our experimental results show an integration of very low edge transient perturbations with the high normalized density and confinement core. The operating regime we report supports some critical requirements in many fusion reactor designs all over the world and opens a potential avenue to an operating point for producing economically attractive fusion energy.

Next-generation sequencing can reveal in vitro-generated PCR crossover products: some artifactual sequences correspond to HLA alleles in the IMGT/HLA database.

The high-resolution human leukocyte antigen (HLA) genotyping assay that we developed using 454 sequencing and Conexio software uses generic polymerase chain reaction (PCR) primers for DRB exon 2. Occasionally, we observed low abundance DRB amplicon sequences that resulted from in vitro PCR 'crossing over' between DRB1 and DRB3/4/5. These hybrid sequences, revealed by the clonal sequencing property of the 454 system, were generally observed at a read depth of 5%-10% of the true alleles. They usually contained at least one mismatch with the IMGT/HLA database, and consequently, were easily recognizable and did not cause a problem for HLA genotyping. Sometimes, however, these artifactual sequences matched a rare allele and the automatic genotype assignment was incorrect. These observations raised two issues: (1) could PCR conditions be modified to reduce such artifacts? and (2) could some of the rare alleles listed in the IMGT/HLA database be artifacts rather than true alleles? Because PCR crossing over occurs during late cycles of PCR, we compared DRB genotypes resulting from 28 and (our standard) 35 cycles of PCR. For all 21 cell line DNAs amplified for 35 cycles, crossover products were detected. In 33% of the cases, these hybrid sequences corresponded to named alleles. With amplification for only 28 cycles, these artifactual sequences were not detectable. To investigate whether some rare alleles in the IMGT/HLA database might be due to PCR artifacts, we analyzed four samples obtained from the investigators who submitted the sequences. In three cases, the sequences were generated from true alleles. In one case, our 454 sequencing revealed an error in the previously submitted sequence.

High throughput HLA genotyping using 454 sequencing and the Fluidigm Access Array™ System for simplified amplicon library preparation.

The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome; distinguishing the thousands of HLA alleles is challenging. Next generation sequencing of exonic amplicons with the 454 genome sequence (GS) FLX System and Conexio Assign ATF 454 software provides high resolution, high throughput HLA genotyping for eight class I and class II loci. HLA typing of potential donors for unrelated bone marrow donor registries typically uses a subset of these loci at high sample throughput and low cost per sample. The Fluidigm Access Array System enables the incorporation of 48 different multiplex identifiers (MIDs) corresponding to 48 genomic DNA samples with up to 48 different primer pairs in a microfluidic device generating 2304 parallel polymerase chain reactions (PCRs). Minimal volumes of reagents are used. During genomic PCR, in this 4-primer system, the outer set of primers containing the MID and the 454 adaptor sequences are incorporated into an amplicon generated by the inner HLA target-specific primers each containing a common sequence tag at the 5' end of the forward and reverse primers. Pools of the resulting amplicons are used for emulsion PCR and clonal sequencing on the 454 Life Sciences GS FLX System, followed by genotyping with Conexio software. We have genotyped 192 samples with 100% concordance to known genotypes using 8 primer pairs (covering exons 2 and 3 of HLA-A, B and C, and exon 2 of DRB1, 3/4/5 and DQB1) and 96 MIDs in a single GS FLX run. An average of 166 reads per amplicon was obtained. We have also genotyped 96 samples at high resolution (14 primer pairs covering exons 2, 3, and 4 of the class I loci and exons 2 of DRB1, 3/4/5, DQA1, DQB1, DPB1, and exon 3 of DQB1), recovering an average of 173 sequence reads per amplicon.

16(th) IHIW : review of HLA typing by NGS.

Human leucocyte antigen (HLA) genes play an important role in the success of organ transplantation and are associated with autoimmune and infectious diseases. Current DNA-based genotyping methods, including Sanger sequence-based typing (SSBT), have identified a high degree of polymorphism. This level of polymorphism makes high-resolution HLA genotyping challenging, resulting in ambiguous typing results due to an inability to resolve phase and/or defining polymorphisms lying outside the region amplified. Next-generation sequencing (NGS) may resolve the issue through the combination of clonal amplification, which provides phase information, and the ability to sequence larger regions of genes, including introns, without the additional effort or cost associated with current methods. The NGS HLA sequencing project of the 16IHIW aimed to discuss the different approaches to (i) template preparation including short- and long-range PCR amplicons, exome capture and whole genome; (ii) sequencing platforms, including GS 454 FLX, Ion Torrent PGM, Illumina MiSeq/HiSeq and Pacific Biosciences SMRT; (iii) data analysis, specifically allele-calling software. The pilot studies presented at the workshop demonstrated that although individual sequencers have very different performance characteristics, all produced sequence data suitable for the resolution of HLA genotyping ambiguities. The developments presented at this workshop clearly highlight the potential benefits of NGS in the HLA laboratory.

16(th) IHIW: immunogenomic data-management methods. report from the immunogenomic data analysis working group (IDAWG).

SUMMARY: The goal of the immunogenomic data analysis working group (IDAWG) is to facilitate the consistent analysis of HLA and KIR data, and the sharing of those data among the immunogenomic and larger genomic communities. However, the data management approaches currently applied by immunogenomic researchers are not widely discussed or reported in the literature, and the effect of different approaches on data analyses is not known. With ASHI's support, the IDAWG developed a 45 question survey on HLA and KIR data generation, data management and data analysis practices. Survey questions detailed the loci genotyped, typing systems used, nomenclature versions reported, computer operating systems and software used to manage and transmit data, the approaches applied to resolve HLA ambiguity and the methods used for basic population-level analyses. Respondents were invited to demonstrate their HLA ambiguity resolution approaches in simulated data sets. By May 2012, 156 respondents from 35 nations had completed the survey. These survey respondents represent a broad sampling of the Immunogenomic community; 52% were European, 30% North American, 10% Asian, 4% South American and 4% from the Pacific. The project will continue in conjunction with the 17th Workshop, with the aim of developing community data sharing standards, ambiguity resolution documentation formats, single-task data Management tools and novel data analysis methods and applications. While additional project details and plans for the 17th IHIW will be forthcoming, we welcome the input and participation in these projects from the histocompatibility and immunogenetics community.

A multi-site study using high-resolution HLA genotyping by next generation sequencing.

The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and CONEXIO ATF software allows reliable identification of HLA genotypes at high resolution.

Spectrally resolved polarization angle across the motional Stark effect spectrum.

A new diagnostic technique has been developed that couples a spectrometer and an image-intensified camera into the traditional motional Stark effect (MSE) system on DIII-D. The image-intensified camera syncs with the photo-elastic modulators to spectrally resolve the Stokes parameters across the Stark multiplet. Polarization dependent phase shift, likely from a plasma facing mirror, leads to the spectropolarimeter measuring a variation in the polarization angle across the MSE spectrum of ∼8°.

Evidence for fast-ion transport by microturbulence.

Cross-field diffusion of energetic ions by microturbulence is measured during neutral-beam injection into the DIII-D tokamak. Fast-ion D(alpha), neutron, and motional Stark effect measurements diagnose the fast-ion distribution function. As expected for transport by plasma turbulence, anomalies relative to the classical prediction are greatest in high temperature plasmas, at low fast-ion energy, and at larger minor radius. Theoretical estimates of fast-ion diffusion are comparable to experimental levels.

Immunolocalization of Kex2 protease identifies a putative late Golgi compartment in the yeast Saccharomyces cerevisiae.

The Kex2 protein of the yeast Saccharomyces cerevisiae is a membrane-bound, Ca2(+)-dependent serine protease that cleaves the precursors of the mating pheromone alpha-factor and the M1 killer toxin at pairs of basic residues during their transport through the secretory pathway. To begin to characterize the intracellular locus of Kex2-dependent proteolytic processing, we have examined the subcellular distribution of Kex2 protein in yeast by indirect immunofluorescence. Kex2 protein is located at multiple, discrete sites within wild-type yeast cells (average, 3.0 +/- 1.7/mother cell). Qualitatively similar fluorescence patterns are observed at elevated levels of expression, but no signal is found in cells lacking the KEX2 gene. Structures containing Kex2 protein are not concentrated at a perinuclear location, but are distributed throughout the cytoplasm at all phases of the cell cycle. Kex2-containing structures appear in the bud at an early, premitotic stage. Analysis of conditional secretory (sec) mutants demonstrates that Kex2 protein ordinarily progresses from the ER to the Golgi but is not incorporated into secretory vesicles, consistent with the proposed localization of Kex2 protein to the yeast Golgi complex.

Secretory vesicles externalize the major plasma membrane ATPase in yeast.

Yeast cell surface growth is accomplished by constitutive secretion and plasma membrane assembly, culminating in the fusion of vesicles with the bud membrane. Coordination of secretion and membrane assembly has been investigated by examining the biogenesis of plasma membrane ATPase (PM ATPase) in secretion-defective (sec) strains of Saccharomyces cerevisiae. PM ATPase is synthesized as a approximately 106-kD polypeptide that is not detectably modified by asparagine-linked glycosylation or proteolysis during transit to the plasma membrane. Export of the PM ATPase requires the secretory pathway. In sec1, a mutant defective in the last step of secretion, large amounts of Golgi-derived vesicles are accumulated. Biochemical characterization of this organelle has demonstrated that PM ATPase and the secretory enzyme, acid phosphatase, are transported in a single vesicle species.

Four-dimensional calibration turntable of the motional Stark effect diagnostic on EAST.

The motional Stark effect (MSE) diagnostic is applied to measure the safety factor q and current density profile of a tokamak device, which are important parameters in realizing the high-performance and long-pulse steady state of a tokamak. A single-channel MSE diagnostic based on dual photoelastic modulators, whose sightline meets with the neutral beam injection at a major radius of R = 2.12 m, has been built for the D window of the Experimental Advanced Superconducting Tokamak (EAST). According to the requirements of MSE diagnostic polarimetric calibration, a high-precision four-dimensional calibration turntable, driven by four stepping motors and controlled by software running on the computer, was designed for EAST. The turntable allows us to rapidly calibrate the MSE diagnostic in a series of positions and angles during EAST maintenance. The turntable can move in four dimensions of translation, yaw, pitch, and roll of the polarizer and can create linearly polarized light at any given angle with accuracy of ∼0.05° for the MSE system offline calibration. The experimental results of the MSE diagnostic calibration in the laboratory show that the turntable has the advantages of high positioning accuracy, flexible spatial movement, and convenient control and fully meets the calibration requirements of an MSE diagnosis system.

Asparagine-linked glycosylation in Saccharomyces cerevisiae: genetic analysis of an early step.

Asparagine-linked glycosylation is a form of covalent modification that distinguishes proteins that are either membrane bound or are in cellular compartments topologically outside of the cell from those proteins that remain soluble in the cytoplasm. This type of glycosylation occurs stepwise, with core oligosaccharide added in the endoplasmic reticulum and subsequent modifications occurring in the golgi. We used tunicamycin, an inhibitor of one of the earliest steps in the synthesis of N-linked oligosaccharide, to select for mutants that are resistant to this antibiotic. Genetic, biochemical, and physiological experiments led to the following conclusions. The synthesis of N-linked oligosaccharide is an essential function in cells. In contrast to mammalian cells, yeast cells do not transport tunicamycin by a glucosamine transport function. We identified a gene, ALG7, that is probably the structural gene for UDP-N-acetylglucosamine-1-P transferase, the enzyme inhibited by tunicamycin. Dominant mutations in this gene result in increased activity of the transferase and loss of the ability of the cell to sporulate. In addition, we identified another gene, TUN1, in which recessive mutations result in resistance to tunicamycin. The ALG7 and TUN1 genes both map on chromosome VII.

Asymmetries in the motional Stark effect emission on the DIII-D tokamak.

Spectrometer measurements and filter upgrades to a motional Stark effect polarimeter measuring the outer half-radius of the DIII-D tokamak helped to identify asymmetries in the polarization angle of Stark-split emission. The measured polarization angle of the π components differs and is not orthogonal to the σ component. These differences persist over a range of densities and with low levels of background light. It is suggested that the difference in the polarization angle between components is from a change in the ellipticity of the emitted light across the Stark components coupled with imperfect polarization preservation from an in-vessel mirror.

Very high resolution single pass HLA genotyping using amplicon sequencing on the 454 next generation DNA sequencers: Comparison with Sanger sequencing.

Compared to Sanger sequencing, next-generation sequencing offers advantages for high resolution HLA genotyping including increased throughput, lower cost, and reduced genotype ambiguity. Here we describe an enhancement of the Roche 454 GS GType HLA genotyping assay to provide very high resolution (VHR) typing, by the addition of 8 primer pairs to the original 14, to genotype 11 HLA loci. These additional amplicons help resolve common and well-documented alleles and exclude commonly found null alleles in genotype ambiguity strings. Simplification of workflow to reduce the initial preparation effort using early pooling of amplicons or the Fluidigm Access Array™ is also described. Performance of the VHR assay was evaluated on 28 well characterized cell lines using Conexio Assign MPS software which uses genomic, rather than cDNA, reference sequence. Concordance was 98.4%; 1.6% had no genotype assignment. Of concordant calls, 53% were unambiguous. To further assess the assay, 59 clinical samples were genotyped and results compared to unambiguous allele assignments obtained by prior sequence-based typing supplemented with SSO and/or SSP. Concordance was 98.7% with 58.2% as unambiguous calls; 1.3% could not be assigned. Our results show that the amplicon-based VHR assay is robust and can replace current Sanger methodology. Together with software enhancements, it has the potential to provide even higher resolution HLA typing.

Interactions of zinc and selenium on the binding of cadmium to rat tissue proteins.

The administration of cadmium to rats by either oral or injection routes causes zinc to accumulate in the low molecular weight (MW) protein metallothionein (MT) of liver and kidney, but not in a low MW protein in the testis. Preliminary evidence indicates that the low MW cadmium-binding protein in testes is not MT. Feeding high levels of zinc to rats results in its accumulation with tissue MT, and the zinc is very labile. In contrast, the zinc that accumulates in MT as the result of cadmium exposure is not very labile. In the reverse situation, zinc does not cause cadmium to accumulate in MT. The turnover of MT is shorter when saturated with zinc than when cadmium is the predominant metal bound to it. Even though selenium will counteract testicular damage due to cadmium, it causes cadmium to accumulate in this organ at higher levels than in animals exposed to cadmium without selenium. Injection of selenate, and selenide--but not selenomethionine or selenocystine--diverts the binding of injected cadmium from low MW proteins to high MW ones in the testes. Selenium injections had only minor influences on the binding of zinc to testicular proteins. In contrast, very little diversion occurred in the binding of either cadmium or zinc in tissues of rats fed high levels of selenium. The data suggest that it is the selenide form of selenium that causes the diversion of cadmium binding in tissues, thus providing protection of testes against cadmium exposure.

The Swan-Ganz catheter and management of patients undergoing pelvic exenteration.

A Swan-Ganz catheter has been used in 10 consecutive patients undergoing pelvic exenteration and has made the intraoperative and postoperative management of these patients a much easier task. Use of this catheter eliminates the guesswork involved in managing fluid and volume status by providing an accurate assessment of left ventricular end diastolic pressure. The complication rate is reported as 5% and consists mostly of ruptured balloons, infection, coiling of the catheter, and cardiac irritability. There have been no complications in the 10 patients in whom we have used the catheter. We believe that the use of the Swan-Ganz catheter in these difficult-to-manage patients is justified because of its low complication rate, easy use, and the accurate valuable information obtained.

Preventive health practices among female employees in an academic community.

This report presents data on the prevalence of seven preventive health practices among 202 female employees in an academic community. Favorable and unfavorable practices are compared according to selected demographic variables. Data were obtained from a self-administered questionnaire mailed to a random sample of female faculty and classified staff. The older women were more likely to eat breakfast every day but were also more likely to be overweight. Alcoholic beverages were consumed with greater frequency by nonmarried personnel. Women with higher educational levels were more likely to be drinkers but less likely to be current smokers. By occupation, classified staff had a significantly higher proportion of current smokers than did faculty. Differences in the seven health practices by living arrangement, employment status, and place of residence were minimal and nonsignificant.

Effects of prenatal testosterone propionate treatment on saccharin preference of adult rats exposed to ethanol in utero.

Prenatal exposure to alcohol feminizes saccharin consumption patterns in adult male rats. To study the involvement of testosterone in this effect, testosterone propionate (TP) was administered to pregnant dams in an attempt to reverse that feminized saccharin consumption pattern in the male offspring. Female offspring were also studied to determine the effect of TP on saccharin preference in normal males and females. During the last week of gestation, dams were administered a liquid diet containing 35% ethanol derived calories, an isocaloric liquid diet containing no ethanol, or Purina Lab Chow. Half of the dams in each group received twice daily injections of TP, the other half were injected with the oil vehicle. Saccharin consumption of adult fetal alcohol exposed (FAE) males from dams administered oil or TP was significantly greater than controls, indicating that the feminized pattern of saccharin consumption of FAE males cannot be overcome with TP administration during the prenatal period. In controls, prenatal TP exposure alone was found to increase adult saccharin consumption in both sexes. Prenatal administration of TP was also found to markedly depress body weight of offspring of dams receiving the liquid diets compared to offspring from dams receiving the same diets plus oil injections. Body weights of offspring from TP or oil injected dams receiving the chow fed diets during pregnancy did not differ.

Positive influence of age and education on food consumption and nutrient intakes of older women living alone.

OBJECTIVE: To examine the effect of age, education, and residence on food consumption and nutrient intakes of older women living alone. DESIGN: In-home interviews were conducted using the Health Habits and History Questionnaire developed by the National Cancer Institute. SUBJECTS: One hundred fifty-two free-living, healthy women who were between the ages of 65 and 94 years. STATISTICAL ANALYSIS: Analysis of variance was used to assess the effect of three independent variables on food consumption and nutrient intakes. RESULTS: The oldest participants did not report a significantly lower consumption of foods compared with younger participants. A significant (P > .05) interaction effect between age and education occurred for the mean weekly servings of vegetables. Weekly consumption of different fruits, vegetables, and meats was significantly (P > .05) higher for more highly educated respondents. Compared with rural residents, urban residents had higher consumption frequencies for all food categories, but only sweets showed a significant (P > .05) difference. Mean nutrient intakes were higher for the oldest age group compared with the younger age groups for all nutrients except sodium and dietary fiber. After controlling for education, only vitamin A and retinol showed a significant (P > .05) difference among the three age groups. More highly educated respondents had higher intakes of calcium, phosphorus, vitamin A, carotene, sodium, dietary fiber, and potassium than did respondents with less education. Comparisons between urban and rural respondents showed no significant effect of age or education for any of the nutrient intakes. CONCLUSIONS: Age and education had more influence on reported food consumption and nutrient intakes of older women living alone than did residence. Data from this study indicate that living alone in a rural area does not necessarily mean that an older woman is at risk for poor nutrition. An important target population for nutrition information may be younger retired women without postsecondary education regardless of residence.