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We have developed a genotyping assay that produces fully phased, unambiguous HLA-E genotyping using Pacific Biosciences' single molecule real-time DNA sequencing. In total 212 cell lines were genotyped, including the panel of 107 established at the 10th International Histocompatibility Workshop. Our results matched the previously known HLA-E genotype in 94 (44.3%) cell lines, in all cases either improving or equalling previous genotyping resolution. Three (1.4%) cells had discrepant HLA-E genotyping data and 115 (54.2%) had no previous HLA-E data. The HLA-E genotypes for four (1.9%) cell lines resulted in a change of zygosity by identifying two distinct haplotypes. We discovered eight novel HLA-E alleles, extended the known reference sequence of seven and confirmed the existence of a further 10.

As the primary genetic determinant of immune recognition of self and non-self, the hyperpolymorphic HLA genes play key roles in disease association and transplantation. The large, variably sized HLA class II genes have historically been less well characterized than the shorter HLA class I genes. Here, we have used Pacific Biosciences Single Molecule Real-Time (SMRT®) DNA sequencing to perform four-field resolution HLA typing of HLA-DRB1/3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 from a panel of 181 B-lymphoblastoid cell lines from the International HLA and Immunogenetics Workshops. By interrogating all exons, introns, and the untranslated regions of these important reference cells, we have improved their HLA typing resolution on the IPD-IMGT/HLA database. We observed widespread non-coding polymorphism, with over twice as many unique genomic sequences identified compared with coding sequences (CDS). We submitted 263 unique sequences to the IPD-IMGT/HLA Database, often from multiple cell lines, including 114 confirmations of existing alleles, of which 30 were also extensions to full-length genomic sequences where only CDS was available previously. A total of 149 novel alleles were identified, largely differing from their closest reference allele sequences by a single nucleotide polymorphism (SNP). However, some highly divergent alleles were deemed to be recombinants, only detectable by full-length sequencing with long, phased reads. The fourth-field variation we observed allowed fine mapping of linkage disequilibrium patterns and haplotypes to particular ancestries. This study has highlighted the under-appreciated non-coding diversity in HLA class II genes, with potential implications for population genetic and clinical studies.

Two novel HLA class II alleles were characterized, HLA-DRB1*03:01:01:05 and HLA-DPB1*04:02:01:21.

Unlike traditional forage grasses, turfgrasses, which were developed to be tolerant of foot traffic and close mowing, may be suitable as alternative ground cover in areas of high hoof traffic such as dry lots. The objective of this study was to evaluate the potential of eight cool-season turfgrasses as alternative ground cover in heavy use areas. Cultivars were established via seeding in four replicated plots. To simulate horse traffic at a trot, a Baldree traffic simulator was driven over a section of the plot, either 0 (CON), 1 (LOW), or 2 (HIGH) times per week for 6 weeks followed by 4 weeks of rest in the spring, summer, and fall over a 2-year period. Variables assessed include compaction, biomass available for grazing, vegetative cover (persistence), and nutrient composition. Soil compaction increased with the application of traffic treatments (P.0001). Traffic treatment reduced persistence following traffic by 19%-36% across all trials in both years (P = .0003). For most trials, biomass available for grazing was reduced after traffic treatment by 19% and 43% (P = .02). Overall, tall fescue cultivars were most traffic tolerant followed by hard fescue. Cultivars considered "low" in nonstructural carbohydrate (15%) included creeping bentgrass in Year 1 and hard fescue and chewings fescue in Year 2. In Year 2, the Ca:P ratio declined beyond what is recommended for horses. Tall fescue, hard fescue, and creeping bentgrass cultivars are recommended for further evaluation as suitable ground cover in areas of heavy use on equine operations.

The new allele HLA-DRB1*16:02:10 showed one synonymous nucleotide difference with HLA-DRB1*16:02:01:01 in codon 58.

Warm season wear-tolerant turfgrasses, such as those used on golf courses and athletic fields, may be valuable forages on equine operations because of their potential to remain viable during heavy hoof traffic. Crabgrass may also be suitable as it thrives in conditions where other grasses have limited success. The objective of this study was to assess the relative traffic tolerance and nutritional composition of five warm-season (WS) turfgrass cultivars of bermudagrass and zoysiagrass and one WS forage-type crabgrass. All cultivars were established by seed in replicated monoculture plots. Simulated hoof traffic treatments consisted of either none, one, or two passes of a Baldree Traffic Simulator. Traffic was applied weekly for 6 weeks in the summer of 2016 and 2017, with each treatment period followed by a 4-week rest period. Plots were assessed for compaction, biomass, and persistence before and after treatment and rest periods. Nutritional composition was assessed throughout the growing seasons. Soil compaction increased as treatment level increased for all cultivars (P.0001). There was no effect of treatment on cultivar persistence. Biomass available for grazing was increased in year 1 by the application of LOW traffic treatment (P = .0193). Both bermudagrass and zoysiagrass cultivars showed promise for use in areas of heavy traffic on equine operations, however, zoysiagrass cultivars were more suitable as they were highest ranking in relative traffic tolerance, moderate in yield, and low nonstructural carbohydrates (12% NSC). Future on-farm studies evaluating bermudagrass and zoysiagrass to determine ideal stocking rate, management methods, and persistence under grazing are warranted.

Novel allele HLA-B*56:67 potentially formed by recombination between B*56:01:01:03 and B*40:01:01.

The novel HLA-DQA1*03:03:05 allele, first described in a potential bone marrow donor from Brazil.

The hyperpolymorphic HLA genes play important roles in disease and transplantation and act as genetic markers of migration and evolution. A panel of 107 B-lymphoblastoid cell lines (B-LCLs) was established in 1987 at the 10th International Histocompatibility Workshop as a resource for the immunogenetics community. These B-LCLs are well characterised and represent diverse ethnicities and HLA haplotypes. Here we have applied Pacific Biosciences' Single Molecule Real-Time (SMRT) DNA sequencing to HLA type 126 B-LCL, including the 107 International HLA and Immunogenetics Workshop (IHIW) cells, to ultra-high resolution. Amplicon sequencing of full-length HLA class I genes (HLA-A, -B and -C) and partial length HLA class II genes (HLA-DRB1, -DQB1 and -DPB1) was performed. We typed a total of 931 HLA alleles, 895 (96%) of which were consistent with the typing in the IPD-IMGT/HLA Database (Release 3.27.0, January 20, 2017), with 595 (64%) typed at a higher resolution. Discrepant types, including novel alleles (n = 10) and changes in zygosity (n = 13), as well as previously unreported types (n = 34) were observed. In addition, patterns of linkage disequilibrium were distinguished by four-field resolution typing of HLA-B and HLA-C. By improving and standardising the HLA typing of these B-LCLs, we have ensured their continued usefulness as a resource for the immunogenetics community in the age of next generation DNA sequencing.

A novel variant of HLA-C*03:04:01:47 was identified using Pacific Biosciences SMRT sequencing platform. This article is protected by copyright. All rights reserved.

The influence of wheat (modern wheat, both bread and pasta, their wild ancestors and synthetic hybrids) on the microbiota of their roots and surrounding soil is characterized. We isolated lines of bread wheat by hybridizing diploid (Aegilops tauschii) with tetraploid Triticum durum and crossed it with a modern cultivar of Triticum aestivum. The newly created, synthetic hybrid wheat, which recapitulate the breeding history of wheat through artificial selection, is found to support a microbiome enriched in beneficial Glomeromycetes fungi, but also in, potentially detrimental, Nematoda. We hypothesize that during wheat domestication this plant-microbe interaction diminished, suggesting an evolutionary tradeoff; sacrificing advantageous nutrient acquisition through fungal interactions to minimize interaction with pathogenic fungi. Increased plant selection for Glomeromycetes and Nematoda is correlated with the D genome derived from A. tauschii. Despite differences in their soil microbiota communities, overall wheat plants consistently show a low ratio of eukaryotes to prokaryotes. We propose that this is a mechanism for protection against soil-borne fungal disease and appears to be deeply rooted in the wheat genome. We suggest that the influence of plants on the composition of their associated microbiota is an integral factor, hitherto overlooked, but intrinsic to selection during wheat domestication.

HLA matching at an allelic-level resolution for volunteer unrelated donor (VUD) hematopoietic cell transplantation (HCT) results in improved survival and fewer post-transplant complications. Limitations in typing technologies used for the hyperpolymorphic HLA genes have meant that variations outside of the antigen recognition domain (ARD) have not been previously characterized in HCT. Our aim was to explore the extent of diversity outside of the ARD and determine the impact of this diversity on transplant outcome. Eight hundred ninety-one VUD-HCT donors and their recipients transplanted for a hematologic malignancy in the United Kingdom were retrospectively HLA typed at an ultra-high resolution (UHR) for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 using next-generation sequencing technology. Matching was determined at full gene level for HLA class I and at a coding DNA sequence level for HLA class II genes. The HLA matching status changed in 29.1% of pairs after UHR HLA typing. The 12/12 UHR HLA matched patients had significantly improved 5-year overall survival when compared with those believed to be 12/12 HLA matches based on their original HLA typing but were found to be mismatched after UHR HLA typing (54.8% versus 30.1%, P = .022). Survival was also significantly better in 12/12 UHR HLA-matched patients when compared with those with any degree of mismatch at this level of resolution (55.1% versus 40.1%, P = .005). This study shows that better HLA matching, found when typing is done at UHR that includes exons outside of the ARD, introns, and untranslated regions, can significantly improve outcomes for recipients of a VUD-HCT for a hematologic malignancy and should be prospectively performed at donor selection.

HLA-DPB1*18:01:01:04 differs from HLA-DPB1*18:01:01:01 by four substitutions within intron 1.

Pacific Bioscience's SMRT DNA sequencing was used to identify two novel intronic variants of HLA-A*32:01:01:01.

Pacific Bioscience's SMRT sequencing was used to confirm and extend HLA-DPB1*64:01N and 701:01.

The novel allele, HLA-C*07:01:01:30 differs by one nucleotide substitution with HLA-C*07:01:01:01 at gDNA 627.

Extended molecular characterization of HLA genes in the IHWG reference B-lymphoblastoid cell lines (B-LCLs) was one of the major goals for the 17(th) International HLA and Immunogenetics Workshop (IHIW). Although reference B-LCLs have been examined extensively in previous workshops complete high-resolution typing was not completed for all the classical class I and class II HLA genes. To address this, we conducted a single-blind study where select panels of B-LCL genomic DNA samples were distributed to multiple laboratories for HLA genotyping by next-generation sequencing methods. Identical cell panels comprised of 24 and 346 samples were distributed and typed by at least four laboratories in order to derive accurate consensus HLA genotypes. Overall concordance rates calculated at both 2- and 4-field allele-level resolutions ranged from 90.4% to 100%. Concordance for the class I genes ranged from 91.7 to 100%, whereas concordance for class II genes was variable; the lowest observed at HLA-DRB3 (84.2%). At the maximum allele-resolution 78 B-LCLs were defined as homozygous for all 11 loci. We identified 11 novel exon polymorphisms in the entire cell panel. A comparison of the B-LCLs NGS HLA genotypes with the HLA genotypes catalogued in the IPD-IMGT/HLA Database Cell Repository, revealed an overall allele match at 68.4%. Typing discrepancies between the two datasets were mostly due to the lower-resolution historical typing methods resulting in incomplete HLA genotypes for some samples listed in the IPD-IMGT/HLA Database Cell Repository. Our approach of multiple-laboratory NGS HLA typing of the B-LCLs has provided accurate genotyping data. The data generated by the tremendous collaborative efforts of the 17(th) IHIW participants is useful for updating the current cell and sequence databases and will be a valuable resource for future studies.

Plant–microbe interactions in the rhizosphere have important roles in biogeochemical cycling, and maintenance of plant health and productivity, yet remain poorly understood. Using RNA-based metatranscriptomics, the global active microbiomes were analysed in soil and rhizospheres of wheat, oat, pea and an oat mutant (sad1) deficient in production of anti-fungal avenacins. Rhizosphere microbiomes differed from bulk soil and between plant species. Pea (a legume) had a much stronger effect on the rhizosphere than wheat and oat (cereals), resulting in a dramatically different rhizosphere community. The relative abundance of eukaryotes in the oat and pea rhizospheres was more than fivefold higher than in the wheat rhizosphere or bulk soil. Nematodes and bacterivorous protozoa were enriched in all rhizospheres, whereas the pea rhizosphere was highly enriched for fungi. Metabolic capabilities for rhizosphere colonisation were selected, including cellulose degradation (cereals), H2 oxidation (pea) and methylotrophy (all plants). Avenacins had little effect on the prokaryotic community of oat, but the eukaryotic community was strongly altered in the sad1 mutant, suggesting that avenacins have a broader role than protecting from fungal pathogens. Profiling microbial communities with metatranscriptomics allows comparison of relative abundance, from multiple samples, across all domains of life, without polymerase chain reaction bias. This revealed profound differences in the rhizosphere microbiome, particularly at the kingdom level between plants.

Plant genomes contribute to the structure and function of the plant microbiome, a key determinant of plant health and productivity. High-throughput technologies are revealing interactions between these complex communities and their hosts in unprecedented detail.

The rhizosphere is a nutrient-rich area surrounding roots with increased microbial number and activity compared with bulk soil. Plant–microbe interactions in the rhizosphere are an essential determinant of plant health and productivity, as well as biogeochemical cycling. Traditionally, genomics has focussed on the small proportion of cultivable microbes, but recent advances in culture-independent molecular tools, high-throughput deoxyribonucleic acid (DNA) sequencing, and bioinformatics are revolutionising our understanding of plant–microbe interactions. Identification of total DNA (metagenomics), ribonucleic acid (RNA) (metatranscriptomics) and expressed proteins (metaproteomics) and metabolites (metabolomics) are now applicable to complex microbial communities such as those in the rhizosphere. Combining these genomic technologies will enable us to analyse the rhizosphere at the systems level. Key Concepts: Soil microbial diversity is vast and plant interfaces are a focus of microbial activity. Plant–microbial interactions are an essential determinant of plant health and productivity. Microbes in the rhizosphere play a key role in biogeochemical cycling. Genomic technologies enable the investigation of the genetic and functional diversity of noncultivable microbes. Next generation sequencing technologies are revolutionising the study of microbial community analysis. Keywords: Genomics; rhizosphere; soil; plant–microbe interaction; microbial community; roots; metagenomics; metatranscriptomics; metaproteomics; metabolomics

A human B-lymphoblastoid cell line, designated MCL-5, constitutively expressing human cytochrome P-450 CYP1A1 and also expressing five transfected human cDNAs encoding drug-metabolizing enzymes, has been developed. cDNAs encoding CYP1A2, CYP2A6, and microsomal epoxide hydrolase (mEH) were introduced by using a vector conferring hygromycin B resistance, and cDNAs encoding CYP2E1 and CYP3A4 were introduced by using a vector conferring resistance to 1-histidinol. MCL-5 cells stably expressed all five cDNAs and the native CYP1A1 as determined by measurement of form-specific enzyme activity levels. The mutagenicity of seven model procarcinogens to MCL-5 cells was examined at the hypoxanthine guanine phosphoribosyltransferase (hprt) and thymidine kinase (tk) loci. Exposure to benzo[a]pyrene (BP), 3-methylcholanthrene (3MC), N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), aflatoxin B1, (AFB1), 2-(acetylamino)fluorene (AAF), or benzidine (BZD) induced a statistically significant increase in mutant frequency. Linear interpolation of the concentration of procarcinogen necessary to produce a doubling of the mutant fraction at the hprt locus in MCL-5 cells and the parent AHH-1 cell line revealed that, for each of the chemicals examined, except BZD, MCL-5 cells were significantly more sensitive than the parent AHH-1 cells. The increase in sensitivity to mutagenicity ranged from 3-fold for AAF to greater than 40,000-fold for NDMA. MCL-5 cells have great potential as a screening system for the analysis of human procarcinogen/promutagen activation.

Sodium fluoride was found to induce gene-locus mutations at the thymidine kinase (tk) and hypoxanthine guanine phosphoribosyl transferase (hgprt) loci in human lymphoblastoid cells. A single, 28 hr exposure to up to 600 micrograms/ml sodium fluoride induced a concentration-dependent increase in mutant fraction at both gene loci and reduced cell survival to 12% relative to negative control cultures. When cells were exposed to sodium fluoride concentrations that were only minimally toxic using a 20 day treatment protocol, no detectable induction of mutation was ob-served at the hgprt locus, and induction of mutation was observed at the tk locus only for treatment with 65 micrograms/ml sodium fluoride; exposure to 50 and 35 micrograms/ml sodium fluoride did not induce detectable mutation. The assay protocol used was of sufficient statistical sensitivity to detect the level of mutation predicted based on a linear extrapolation of data obtained from a 28 hour exposure. The implications of these observations with regard to the extrapolability of mutagenicity data to low concentrations are discussed.

We report that, in a human cell line, human cytochrome P450IIA3 is capable of metabolizing aflatoxin B1, benzo[a]-pyrene, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) to cytotoxic and mutagenic species. Cytochrome P450IIA3-mediated activation of NDMA and NDEA was compared with human cytochrome P450IIE1-mediated activation in the same cell system. P450IIE1 was more effective at activating NDMA than P450IIA3, while P450IIA3 was more effective at activating NDEA than P450IIE1. Whole cells and microsomal fractions obtained from control cells and from cells expressing the P450IIA3 cDNA were characterized for expression of P450IIA3. Microsomal coumarin 7-hydroxylase activity was some 40 times greater in the transfected cells than in the control cells and was catalyzed by a protein that was immunochemically related to the rat liver cytochrome P450IIA gene family. Immunoblot analysis demonstrated that this protein was readily detectable in transfected cells but barely detectable in control cells. We also report the DNA and deduced amino acid sequence of the P450IIA3 cDNA isolate used in this study. Our isolate encodes a protein 489 amino acids that is five amino acids shorter at the N terminus but otherwise identical to a previously reported human P450IIA3 cDNA sequence.

In this book, one of America's leading assassination experts turns his attention to some of the major myths surrounding the sixteenth president. It is hardly surprising that an assassination schola...

The current Army drawdown and mandated budget cuts force examination of our paradigms in almost every area of defense. Only with a through look at the way we do business and why we do what we do will we achieve economies that allow us to maintain a trained and ready force in an era of shrinking resources. An obvious question that begs scrutiny is should we consolidate the Army War College (AWC) and the Command and General Staff College (CGSOC)? Do the obvious savings in faculty, library, school support and base operations that result from consolidation offset the advantages of the current geographical separation of the two schools? This study examines the historical evolution of senior officer education in the Army, the recommendations of the Panel on Military Education of the House Armed Services Committee, Navy and Air Force professional senior officer education, and recent Army studies in an attempt to determine if the unification of the AWC and the CGSOC under one command structure or if collocation of the schools would achieve cost savings without a degradation of the education officers' currently receive at those institutions. The author concludes that the Nation and Army are best served with an AWC that develops senior leaders prepared to operate at the strategic level in today's world and a CGSOC that develops leaders prepared to operate in a joint tactical and operational environment. These colleges are ideally located separately at Carlisle and Leavenworth. The current command structure is sound; the AWC as a Field Operating Agency of the DCSOPS and the CGSOC subordinate to TRADOC.