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Design randomizations and spatial corrections have increased understanding of genotypic, spatial, and residual effects in field experiments, but precisely measuring spatial heterogeneity in the field remains a challenge. To this end, our study evaluated approaches to improve spatial modeling using high-throughput phenotypes (HTP) via unoccupied aerial vehicle (UAV) imagery. The normalized difference vegetation index was measured by a multispectral MicaSense camera and processed using ImageBreed. Contrasting to baseline agronomic trait spatial correction and a baseline multitrait model, a two-stage approach was proposed. Using longitudinal normalized difference vegetation index data, plot level permanent environment effects estimated spatial patterns in the field throughout the growing season. Normalized difference vegetation index permanent environment were separated from additive genetic effects using 2D spline, separable autoregressive models, or random regression models. The Permanent environment were leveraged within agronomic trait genomic best linear unbiased prediction either modeling an empirical covariance for random effects, or by modeling fixed effects as an average of permanent environment across time or split among three growth phases. Modeling approaches were tested using simulation data and Genomes-to-Fields hybrid maize (Zea maysL.) field experiments in 2015, 2017, 2019, and 2020 for grain yield, grain moisture, and ear height. The two-stage approach improved heritability, model fit, and genotypic effect estimation compared to baseline models. Electrical conductance and elevation from a 2019 soil survey significantly improved model fit, while 2D spline permanent environment were most strongly correlated with the soil parameters. Simulation of field effects demonstrated improved specificity for random regression models. In summary, the use of longitudinal normalized difference vegetation index measurements increased experimental accuracy and understanding of field spatio-temporal heterogeneity.Precisely measuring spatial heterogeneity in field experiments is a persistent challenge in crop research. Here, Morales et al. evaluate approaches to improve spatial modeling using high-throughput phenotypes via unoccupied aerial vehicle imagery. The authors test these approaches with simulation data and data from four Genomes-to-Fields hybrid maize field experiments, finding a two-stage approach improves heritability, model fit, and genotypic effect estimation compared to baseline models.

Variation in crop performance is directly affected by the environment in which the plant grows. Analyses and estimation of genotype × environment interactions (G×E) have the potential to provide information about the characteristics of genotypes, identify elite genotypes and suitable environmental conditions, establish breeding objectives, and make recommendations for crop management practices. For the past half century, a variety of statistical models have been used for estimating G×E in plant breeding field experiments to facilitate the allocation of superior genotypes to the target population of environments. The most commonly used models are described in this review starting with linear regression and ANOVA models. We then describe modifications in the form of multiplicative models, models that can accommodate external variables, and mixed effect models. Quantification of differential effects of segments of a genome across environments is shown by exploiting marker × environment (M×E) interactions. We close with a brief overview of some nonparametric concepts that aim to understand genotypic stability.

Pairwise distance data for maize (Zea maysL.) inbred lines generated using sets of single nucleotide polymorphisms (SNPs) selected from a 50k Infinium array were compared with pairwise distances generated using a set of 163 simple sequence repeat (SSR) loci previously identified to help determine essentially derived variety (EDV) status (UPOV, 1991). Final comparisons were made using 26,874 SNPs after discarding SNPs with insufficient data quality or vulnerability to ascertainment bias. Inbred lines developed in the United States or in western Europe that had been previously published to establish SSR‐based thresholds provided the means to determine equivalent SNP‐based protocols. Use of 3072 SNPs selected to provide even genomic coverage according to genetic and physical maps provided robust, precise, high discrimination among inbred lines with consistent zonal classification with up to 20% missing data. Comparisons of intercepts and slopes for SSR and SNP inbred pairwise distance data translated the 82% SSR green‐orange similarity threshold to 91% using SNPs and the 90% SSR orange‐red threshold to 95% using SNPs. Information required to conduct analyses using these 3072 SNPS is presented.

AbstractAntibiotic growth promoters (AGPs) have been used as feed additives to improve average daily weight gain and feed efficiency in food animals for more than five decades. However, use of AGPs is associated with the emergence of antibiotic-resistant human pathogens of animal origin, posing a significant threat to food safety and public health. Thus, development of novel alternatives to AGPs is important to mitigate antimicrobial resistance in foodborne pathogens. To achieve this goal, the mode of action of AGPs should be elucidated. In this study, the response of the chicken gut microbiota to AGPs was examined using two culture-independent approaches: phospholipid fatty acid (PLFA) biomarker analysis and 16S rDNA clone library sequencing. PLFA analysis showed that AGP tylosin treatment changed composition of the microbiota in various intestinal sites; however, total viable bacterial biomass in intestine was not affected by tylosin treatment. PLFA analysis also revealed an abundant viable fungal population in chicken microbiota. Eight 16S rDNA libraries (96 clones per library) were constructed using ileal samples from chickens that received either antibiotic-free or medicated feed. The 16S rDNA clone analysis of the growth-relevant samples showed the AGP treatment influenced the diversity of ileum microbiota in the chickens primarily in the Firmicutes division. In particular, Lactobacillusspp. populations in the ileum of AGP-treated chickens were significantly lower than those from chickens receiving antibiotic-free feed. Together, this study revealed novel features of the intestinal microbiota in response to AGP treatment and suggested approach to develop potential alternatives to AGPs for mitigation of antimicrobial resistance in foodborne pathogens.

Stromal cell derived factor-1 (SDF-1 or CXCL12) and its receptor CXCR4 are involved in the directional homing to the bone marrow niches and in peripheral mobilization of normal and transformed hematopoietic stem and myeloid progenitor cells. Elevated CXCR4 expression confers poor prognosis, whereas inhibition of CXCR4 signaling overcomes stroma-mediated chemoresistance in acute myeloid leukemia (AML). Here, we demonstrate that treatment with the pan-histone deacetylase inhibitor panobinostat (PS) depleted the mRNA and protein levels of CXCR4 in the cultured and primary AML cells. PS-induced acetylation of the heat shock protein (hsp) 90 reduced the chaperone association between CXCR4 and hsp90, directing CXCR4 to degradation by the 20S proteasome. PS treatment also depleted G protein–coupled receptor kinase 3, as well as attenuated the phosphorylation of AKT and ERK1/2 in AML cells, which was not affected by cotreatment with CXCL12. Compared with each agent alone, cotreatment with PS and CXCR4 antagonist AMD3100 or FC-131 synergistically induced apoptosis of cultured and primary AML cells. PS and FC-131 exerted more lethal effects on primary AML versus normal CD34+bone marrow progenitor cells. These findings support the rationale to test the in vivo efficacy of PS in enhancing the lethal effects of CXCR4 antagonists against AML cells.

Stromal cell derived factor-1 (SDF-1 or CXCL12) and its receptor CXCR4 are involved in the directional homing to the bone marrow niches and in peripheral mobilization of normal and transformed hematopoietic stem and myeloid progenitor cells. Elevated CXCR4 expression confers poor prognosis, whereas inhibition of CXCR4 signaling overcomes stroma-mediated chemoresistance in acute myeloid leukemia (AML). Here, we demonstrate that treatment with the pan-histone deacetylase inhibitor panobinostat (PS) depleted the mRNA and protein levels of CXCR4 in the cultured and primary AML cells. PS-induced acetylation of the heat shock protein (hsp) 90 reduced the chaperone association between CXCR4 and hsp90, directing CXCR4 to degradation by the 20S proteasome. PS treatment also depleted G protein–coupled receptor kinase 3, as well as attenuated the phosphorylation of AKT and ERK1/2 in AML cells, which was not affected by cotreatment with CXCL12. Compared with each agent alone, cotreatment with PS and CXCR4 antagonist AMD3100 or FC-131 synergistically induced apoptosis of cultured and primary AML cells. PS and FC-131 exerted more lethal effects on primary AML versus normal CD34+ bone marrow progenitor cells. These findings support the rationale to test the in vivo efficacy of PS in enhancing the lethal effects of CXCR4 antagonists against AML cells.

Human embryonic stem cells (hESC) have the potential to produce all of the cells in the body. They are able to self-renew indefinitely, potentially making them a source for large-scale production of therapeutic cell lines. Here, we developed a monolayer differentiation culture that induces hESC (WA09 and BG01) to form epithelial sheets with mesodermal gene expression patterns (BMP4, RUNX1,and GATA4). These E-cadherin+CD90lowcells then undergo apparent epithelial–mesenchymal transition for the derivation of mesenchymal progenitor cells (hESC-derived mesenchymal cells [hES-MC]) that by flow cytometry are negative for hematopoietic (CD34, CD45, and CD133) and endothelial (CD31 and CD146) markers, but positive for markers associated with mesenchymal stem cells (CD73, CD90, CD105, and CD166). To determine their functionality, we tested their capacity to produce the three lineages associated with mesenchymal stem cells and found they could form osteogenic and chondrogenic, but not adipogenic lineages. The derived hES-MC were able to remodel and contract collagen I lattice constructs to an equivalent degree as keloid fibroblasts and were induced to express α-smooth muscle actin when exposed to transforming growth factor (TGF)-β1, but not platelet derived growth factor-B (PDGF-B). These data suggest that the derived hES-MC are multipotent cells with potential uses in tissue engineering and regenerative medicine and for providing a highly reproducible cell source for adult-like progenitor cells.

The weanling pig's requirement for total dietary aromatic amino acids (TAAA) and the portion of this requirement that can be efficiently provided by tyrosine were studied. An amino acid-fortified corn diet that was first-limiting in TAAA was used in all feeding trials. Chick and microbiological growth assays established that the corn contained .31% bioavailable phenylalanine and .48% bioavailable tyrosine. The optimum crude protein level for the basal corn diet (adjusted by varying the level of L-glutamic acid) was found to be 12% (i.e., 6.72% supplemental glutamic acid), since pigs gained faster and more efficiently at this protein level than at 8, 16 or 20% crude protein.The TAAA study consisted of adding varied levels of L-phenylalanine and L-tyrosine to the diet containing 12% crude protein, .24% L-tyrosine and .16% L-phenylalanine. Gain and gain/ feed were assessed in young pigs (10 to 20 kg) during a 16-day feeding trial. Optimal gain and gain/feed occurred at .56% TAAA. Furthermore, there was no depression in gain or gain/feed when tyrosine was increased from 43% to 49% of the TAAA in the diet containing .56% TAAA. This indicates that the weanling pig's requirement for TAAA is .56% of the diet at 12% protein and that 49% of this requirement can be supplied as tyrosine.

Two least squares methods of estimating nutrient requirements from growth data were compared. One method involved fitting a broken line by the method of least squares. The requirement was taken as the abscissa of the breakpoint in the curve. The other method involved fitting an appropriate exponential function to the growth data and estimating the requirement as the abscissa of the point on the fitted curve whose ordinate was 95% of the upper asymptote. For the nine sets of data studied, the broken line provided adequate fits for only six. The nonlinear models provided adequate fits for all the data studied. When both the broken line and the chosen nonlinear model provided adequate fits, the estimated requirements were nearly the same. However, the consistently good fits obtained with the nonlinear models suggest that this approach may generally be more useful.

Two experiments with purified crystalline amino acid diets were conducted to determine the effect of dietary potassium level on chick breast muscle free amino acid concentrations. In both experiments, concentrations of muscle free glutamine, lysine and arginine increased as dietary potassium increased from 0.12 to 0.18% and decreased as dietary potassium exceeded 0.18%. As dietary potassium increased, muscle potassium concentration increased linearly with an equivalent linear decrease in muscle sodium concentration. Peak concentrations of muscle free glutamine, lysine and arginine occurred in muscle with a K+:Na+ratio of approximately 1.4. Concentrations of the three amino acids were less in muscle with K+:Na+ratios either less than or greater than 1.4. Breast muscle concentrations of free histidine decreased slightly, while muscle free carnosine increased substantially when dietary potassium exceeded 0.18%.

Studies were conducted to determine the nutritional value of lysinoalanine for rats and chicks and the nutritional value of lanthionine for chicks. For each experiment, purified crystalline amino acid diets and crystalline lysinoalanine and lanthionine were employed. Results indicated that the lysine moiety of lysinoalanine is completely unavailable to the rat and only partially available to the chick. Of 10 rats fed lysinoalanine, seven developed nephrocytomegaly. The lanthionine studies indicated that the cysteine moiety of lanthionine is 32% available when fed as the racemic mixture and 52% available when fed as the L-DL-isomeric mixture. Thus, it appears that lysinoalanine formation in alkali-treated proteins results in a corresponding decrease in nutritionally available lysine. However, the lanthionine formed under similar conditions results in loss of only one-half of the corresponding level of nutritionally available cysteine.

Three assays with crystalline amino acid diets were conducted to delineate the histidine requirement of chicks of various ages, and to compare weight gain, plasma free histidine and breast muscle carnosine as parameters of response. Data from assays 1 and 2 indicated a histidine requirement for maximal weight gain of close to 0.32% of the diet for all age periods between hatching and 29 days posthatching. Graded increments of L-histidine between 0 and 0.57% were fed to chicks between 8 and 20 days posthatching in assay 3. Least squares analysis indicated a linear response in weight gain up to 0.285 ± 0.002% histidine, with no additional response above this point. Muscle carnosine concentration remained constant at all dietary levels of histidine below 0.268 ± 0.041%; between this level and 0.368 ± 0.028%, however, carnosine concentration increased linearly from 750 µg/g to 3,500 µg/g wet tissue and then plateaued. Plasma free histidine concentration remained low and unchanged until dietary histidine reached 0.410 ± 0.011% at which point it began to increase markedly. Administration of low levels of L-carnosine to histidine-deprived chicks increased growth rate but had little effect on either plasma histidine or muscle carnosine. These results suggest that a) muscle carnosine is labile and readily metabolized to histidine and β-alanine, b) protein retention has priority over muscle carnosine accumulation when deficient levels of histidine are fed, and c) accumulation of free histidine in plasma does not occur until the histidine need for bothoptimal weight gain and muscle carnosine synthesis has been satisfied.

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Van Laar: Signal Genetics: Employment, Equity Ownership. Ali:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc: Employment. Zangari:Norvartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Research Funding; Millennium: Research Funding. van Rhee:Millenium: Speakers Bureau; Sanofi: Speakers Bureau; Celgene: Speakers Bureau; Janssen: Speakers Bureau. Morgan:Celgene Corp: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Myeloma UK: Membership on an entity's Board of Directors or advisory committees; International Myeloma Foundation: Membership on an entity's Board of Directors or advisory committees; The Binding Site: Membership on an entity's Board of Directors or advisory committees; MMRF: Membership on an entity's Board of Directors or advisory committees.

Background:Diagnostic and therapeutic advances have significantly improved the outcomes for multiple myeloma (MM) patients. However, pts who are refractory to or relapse after therapy with immune modulatory drugs and proteasome inhibitors remain a therapeutic challenge. Comprehensive genomic profiling via clinical next generation sequencing (NGS)-based assays studies of MM cases have revealed multiple targetable mutations that were previously unexploited in MM.

Human Embryonic stem cells (hESC) offers scientist a new tool to better understand human germ cell specification and signaling pathways associated with germ cell development. Two markers often associated with germ cell differentiation, vasa and SSEA-1 are not expressed in undifferentiated human embryonic stem cells. Therefore monitoring the expression of these markers during differentiation may provide information on germ cell formation. Also, basic fibroblast growth factor (β-FGF) has been shown to increase the proportion of mouse embryonic stem cells that differentiate toward the germ cell lineage. The objectives of this study were to determine the effect of time in culture and β-FGF on expression of SSEA-1 and Vasa markers in differentiating populations of hESCs. The experiment was a 2 by 3 factorial with time points at day 3, 10 and 30 with and without β-FGF. H9 hESCs were maintained on mitomycin C inactivated mouse embryonic feeder (MEF) layers in DMEM/F12, 20% Knockout Serum Replacer, 2 mM L-glutamine, 0.1 mM MEM non-essential amino acids, 50 U/ml penicillin, 50 μg/ml streptomycin (all from Gibco/Invitrogen), 0.1 mM βME (Sigma), and 4 ng/ml βFGF (Sigma). Undifferentiated hESC were plated as clumps of manually disassociated colonies onto 4 well chamber slides that were previously plated with inactivated MEFs. The cells were cultured in the presence or absence βFGF for the appropriate amount of time (3, 10 and 30 days). At the end of these three time points, cells were fixed and immunocytochemical analysis was performed for SSEA-1 and Vasa epitopes. We found that there was a subpopulation of differentiating cells that expressed the germ cell markers SSEA-1 and Vasa. There was a trending increase in SSEA-1 expression from day 3 to day 10 and a statistically significant decrease in SSEA-1 expression on day 30, but there was no effect of β-FGF during this time. There was no affect of time and β-FGF on vasa expression however we observed a clear trend toward increased vasa expression after prolonged culture in the presence of β- FGF. Importantly, vasa expression was localized in the nucleolus. Cellular localization of vasa expression has not been previously determined in any species and this study provides new insight into the possible role for vasa expression in the differentiation human germ cells. (poster)