STRUCTURAL AND FUNCTIONAL STUDIES OF AN ATYPICAL OMPR/PHOB TRANSCRIPTIONAL REGULATOR, CHXR, FROM CHLAMYDIA TRACHOMATIS

Chlamydia infections have an immense impact on public health and are associated with diverse disease manifestations including atherosclerosis, blindness, and sterility. The chlamydial developmental cycle is intrinsically linked with the ability of the organism to cause disease. The mechanisms that regulate the developmental cycle are poorly understood; however, transcription appears to play a governing role. An OmpR/PhoB subfamily response regulator termed ChxR exhibits expression patterns that indicate an important role during the developmental cycle. Previously, ChxR was demonstrated to interact with its own promoter and facilitate the transcriptional activation of the chxR gene. To begin to understand the functional role of ChxR, I identified the DNA sequence recognized by ChxR to identify its gene targets. Primarily using gel mobility shift assays, I determined that ChxR interacts with, and has differential affinity for six binding sites in the chxR promoter region. Using the DNA sequences from these binding sites, I elucidated the ChxR cis-acting recognition sequence. Additionally, I was interested in elucidating the ChxR mechanism of transcriptional activation. Usually as a result of phosphorylation, OmpR/PhoB response regulators form homodimers through a receiver domain as an integral step in transcriptional activation. Dimer formation facilitates an interaction of the effector domain interaction with DNA and transcriptional machinery to regulate transcription. ChxR is an atypical OmpR/PhoB response regulator because it is active in the absence of phosphorylation. We hypothesized that the intra- and intermolecular interactions involved in forming a transcriptionally competent ChxR protein are distinct from the canonical phosphorylation (activation) paradigm in the OmpR/PhoB response regulator subfamily. Using biochemical techniques, I demonstrated that ChxR forms homodimers through the receiver domain and the effector domain interacts with DNA similar to phosphorylation-induced and transcriptionally active OmpR/PhoB response regulators. Additionally, the structures of the two domains were solved to direct functional studies to identify the residues important in homodimer formation, interaction with DNA, and interaction with RNA polymerase machinery. Both structures had unique features that are not found in other OmpR/PhoB subfamily members. The combination of these results suggests that ChxR is a member of the OmpR/PhoB subfamily, although many of the characteristics of the subfamily are not shared in ChxR

Extending long-range phasing and haplotype library imputation methods to impute genotypes on sex chromosomes

AlphaImpute is a flexible and accurate genotype imputation tool that was originally designed for the imputation of genotypes on autosomal chromosomes. In some species, sex chromosomes comprise a large portion of the genome. For example, chromosome Z represents approximately 8% of the chicken genome and therefore is likely to be important in determining genetic variation in a population. When breeding programs make selection decisions based on genomic information, chromosomes that are not represented on the genotyping platform will not be subject to selection. Therefore imputation algorithms should be able to impute genotypes for all chromosomes. The objective of this research was to extend AlphaImpute so that it could impute genotypes on sex chromosomes. The accuracy of imputation was assessed using different genotyping strategies in a real commercial chicken population. The correlation between true and imputed genotypes was high in all the scenarios and was 0.96 for the most favourable scenario. Overall, the accuracy of imputation of the sex chromosome was slightly lower than that of autosomes for all scenarios considered

Simulated Data for Genomic Selection and Genome-Wide Association Studies Using a Combination of Coalescent and Gene Drop Methods

An approach is described for simulating data sequence, genotype, and phenotype data to study genomic selection and genome-wide association studies (GWAS). The simulation method, implemented in a software package called AlphaDrop, can be used to simulate genomic data and phenotypes with flexibility in terms of the historical population structure, recent pedigree structure, distribution of quantitative trait loci effects, and with sequence and single nucleotide polymorphism-phased alleles and genotypes. Ten replicates of a representative scenario used to study genomic selection in livestock were generated and have been made publically available. The simulated data sets were structured to encompass a spectrum of additive quantitative trait loci effect distributions, relationship structures, and single nucleotide polymorphism chip densities

The effect of genomic information on optimal contribution selection in livestock breeding programs

BACKGROUND: Long-term benefits in animal breeding programs require that increases in genetic merit be balanced with the need to maintain diversity (lost due to inbreeding). This can be achieved by using optimal contribution selection. The availability of high-density DNA marker information enables the incorporation of genomic data into optimal contribution selection but this raises the question about how this information affects the balance between genetic merit and diversity. METHODS: The effect of using genomic information in optimal contribution selection was examined based on simulated and real data on dairy bulls. We compared the genetic merit of selected animals at various levels of co-ancestry restrictions when using estimated breeding values based on parent average, genomic or progeny test information. Furthermore, we estimated the proportion of variation in estimated breeding values that is due to within-family differences. RESULTS: Optimal selection on genomic estimated breeding values increased genetic gain. Genetic merit was further increased using genomic rather than pedigree-based measures of co-ancestry under an inbreeding restriction policy. Using genomic instead of pedigree relationships to restrict inbreeding had a significant effect only when the population consisted of many large full-sib families; with a half-sib family structure, no difference was observed. In real data from dairy bulls, optimal contribution selection based on genomic estimated breeding values allowed for additional improvements in genetic merit at low to moderate inbreeding levels. Genomic estimated breeding values were more accurate and showed more within-family variation than parent average breeding values; for genomic estimated breeding values, 30 to 40% of the variation was due to within-family differences. Finally, there was no difference between constraining inbreeding via pedigree or genomic relationships in the real data. CONCLUSIONS: The use of genomic estimated breeding values increased genetic gain in optimal contribution selection. Genomic estimated breeding values were more accurate and showed more within-family variation, which led to higher genetic gains for the same restriction on inbreeding. Using genomic relationships to restrict inbreeding provided no additional gain, except in the case of very large full-sib families

Nimbus-7 ERB Solar Analysis Tape (ESAT) user's guide

Seven years and five months of Nimbus-7 Earth Radiation Budget (ERB) solar data are available on a single ERB Solar Analysis Tape (ESAT). The period covered is November 16, 1978 through March 31, 1986. The Nimbus-7 satellite performs approximately 14 orbits per day and the ERB solar telescope observes the sun once per orbit as the satellite crosses the southern terminator. The solar data were carefully calibrated and screened. Orbital and daily mean values are given for the total solar irradiance plus other spectral intervals (10 solar channels in all). In addition, selected solar activity indicators are included on the ESAT. The ESAT User's Guide is an update of the previous ESAT User's Guide (NASA TM 86143) and includes more detailed information on the solar data calibration, screening procedures, updated solar data plots, and applications to solar variability. Details of the tape format, including source code to access ESAT, are included

A Common Dataset for Genomic Analysis of Livestock Populations

Although common datasets are an important resource for the scientific community and can be used to address important questions, genomic datasets of a meaningful size have not generally been available in livestock species. We describe a pig dataset that PIC (a Genus company) has made available for comparing genomic prediction methods. We also describe genomic evaluation of the data using methods that PIC considers best practice for predicting and validating genomic breeding values, and we discuss the impact of data structure on accuracy. The dataset contains 3534 individuals with high-density genotypes, phenotypes, and estimated breeding values for five traits. Genomic breeding values were calculated using BayesB, with phenotypes and de-regressed breeding values, and using a single-step genomic BLUP approach that combines information from genotyped and un-genotyped animals. The genomic breeding value accuracy increased with increased trait heritability and with increased relationship between training and validation. In nearly all cases, BayesB using de-regressed breeding values outperformed the other approaches, but the single-step evaluation performed only slightly worse. This dataset was useful for comparing methods for genomic prediction using real data. Our results indicate that validation approaches accounting for relatedness between populations can correct for potential overestimation of genomic breeding value accuracies, with implications for genotyping strategies to carry out genomic selection programs

Expression, purification, crystallization and preliminary X-ray analysis of the DNA-binding domain of a Chlamydia trachomatis OmpR/PhoB-subfamily response regulator homolog, ChxR

This is the published version. Copyright 2009 by the International Union of Crystallography.Two-component signal transduction systems in bacteria are a primary mechan­ism for responding to environmental stimuli and adjusting gene expression accordingly. Generally in these systems a sensor kinase phosphorylates a response regulator that regulates transcription. Response regulators contain two domains: a receiver domain and an effector domain. The receiver domain is typically phosphorylated and as a result facilitates the DNA-binding and transcriptional activity of the effector domain. The OmpR/PhoB subfamily is the largest of the response-regulator subfamilies and is primarily defined by the winged helix-turn-helix DNA-binding motif within the effector domain. The overall structure of effector domains is highly conserved and contains three defined elements that are critical for transcriptional regulation: a DNA major-groove binding helix, a DNA minor-groove binding wing and a transcriptional activation loop. These functional elements are often diverse in sequence and conformation and reflect the functional differences observed between individual subfamily members. ChxR from Chlamydia trachomatis is an atypical OmpR/PhoB response regulator homolog that has transcriptional activity in the absence of phos­phorylation. To facilitate the precise identification of the functional elements of the ChxR effector domain, this protein was cloned, expressed, purified and crystallized. Crystals were obtained from two separate mother liquors, producing two morphologically distinct crystals. The space group of both crystals was P43212 (or its enantiomorph P41212) with isomorphous unit-cell parameters; the crystals diffracted to 2.2-2.5 Å resolution

Formulation development of a recombinant protein based non-replicating rotavirus (NRRV) vaccine candidate: Antigen-adjuvant-preservative interactions

Rotavirus is the leading cause of acute diarrhea and gastroenteritis among infants and young children worldwide. Over 215,000 children under five years of age die from rotavirus infection each year, mostly in developing world1. Currently two live attenuated oral rotavirus vaccines are available globally (Rotarix® and RotaTeq®) to reduce the burden of this disease with an efficacy of \u3e90% in developed countries2. Vaccine efficacy is lower, however, in developing countries due to a variety of factors3. To this end, a non-replicating rotavirus (NRRV) vaccine candidate, containing three recombinant protein antigens (P2-VP8-P[4], P2-VP8-P[6] and P2-VP8-P[8]), is being developed by PATH and its partners as a trivalent vaccine for use in the developing world4. This trivalent rotavirus vaccine candidate includes the three antigens from the most prevalent serotypes associated with \u3e90% of rotavirus gastroenteritis worldwide. In the present study, the following formulation development issues were examined: (1) establish stability-indicating physicochemical assays for a NRRV protein antigen (P[8]) bound to an aluminum hydroxide adjuvant (Alhydrogel®), which include primary and higher-order structures, chemical and conformational stability of the protein on Alhydrogel, and the ability to desorb the antigen from Alhydrogel; (2) examine the adsorptive capacity and coefficients of Alhydrogel® for the P[8] antigen in several candidate drug product formulations; (3) investigate the effects of binding to Alhydrogel® and the addition of two antimicrobial preservatives (2-phenoxyethanol or thimerosal) on the structural integrity and conformational stability of P[8], the latter of which were found to be potent destabilizers of the antigen; and (4) monitor the real-time and accelerated storage stability over 3 months of P[8] bound to Alhydrogel® in several candidate formulations with and without thimerosal at different temperatures. In the absence of preservative, the P[8] protein antigen was overall stable with only a small amount of Asn deamidation observed in samples stored under real-time (4˚C) or accelerated (25˚C) temperatures. Similarly, little (if any) changes were observed in the real-time stability of the antigen on Alhydrogel® in the presence of thimerosal. Under accelerated storage temperatures (25 or 37˚C) however, the preservative caused an increase in inter-molecular disulfide bonding, decrease of apparent enthalpy as measured by DSC, and a decrease in in-vitro antigenicity. Similar stability studies are currently ongoing with the P[4] and P[6] protein antigens. Acknowledgements: Funding provided by the Bill & Melinda Gates Foundation References: 1. Tate et al 2016. Clinical Infectious Diseases 62:S96-S105 2. Tissera et al. 2017. Human Vaccines & Immunotherapeutics 13(4):921-927 3. Glass et al. 2014. Journal of Infection 68: S9-S18. 4. Groome et al. 2017. Lancet Infectious Diseases17(8): 843-853

Assessment of alternative genotyping strategies to maximize imputation accuracy at minimal cost

BACKGROUND: Commercial breeding programs seek to maximise the rate of genetic gain while minimizing the costs of attaining that gain. Genomic information offers great potential to increase rates of genetic gain but it is expensive to generate. Low-cost genotyping strategies combined with genotype imputation offer dramatically reduced costs. However, both the costs and accuracy of imputation of these strategies are highly sensitive to several factors. The objective of this paper was to explore the cost and imputation accuracy of several alternative genotyping strategies in pedigreed populations. METHODS: Pedigree and genotype data from a commercial pig population were used. Several alternative genotyping strategies were explored. The strategies differed in the density of genotypes used for the ancestors and the individuals to be imputed. Parents, grandparents, and other relatives that were not descendants, were genotyped at high-density, low-density, or extremely low-density, and associated costs and imputation accuracies were evaluated. RESULTS: Imputation accuracy and cost were influenced by the alternative genotyping strategies. Given the mating ratios and the numbers of offspring produced by males and females, an optimized low-cost genotyping strategy for a commercial pig population could involve genotyping male parents at high-density, female parents at low-density (e.g. 3000 SNP), and selection candidates at very low-density (384 SNP). CONCLUSIONS: Among the selection candidates, 95.5 % and 93.5 % of the genotype variation contained in the high-density SNP panels were recovered using a genotyping strategy that costs respectively, $24.74 and$20.58 per candidate

Exposure of Bifidobacterium longum subsp. infantis to Milk Oligosaccharides Increases Adhesion to Epithelial Cells and Induces a Substantial Transcriptional Response

Devon Kavanaugh is in receipt of a Teagasc Walsh Fellowship. The authors would also like to acknowledge the support of Science Foundation Ireland under Grant No. 08/SRC/B1393 and the Alimentary Glycoscience Research Cluster (AGRC).peer-reviewedIn this study, we tested the hypothesis that milk oligosaccharides may contribute not only to selective growth of bifidobacteria, but also to their specific adhesive ability. Human milk oligosaccharides (3′sialyllactose and 6′sialyllactose) and a commercial prebiotic (Beneo Orafti P95; oligofructose) were assayed for their ability to promote adhesion of Bifidobacterium longum subsp. infantis ATCC 15697 to HT-29 and Caco-2 human intestinal cells. Treatment with the commercial prebiotic or 3′sialyllactose did not enhance adhesion. However, treatment with 6′sialyllactose resulted in increased adhesion (4.7 fold), while treatment with a mixture of 3′- and 6′-sialyllactose substantially increased adhesion (9.8 fold) to HT-29 intestinal cells. Microarray analyses were subsequently employed to investigate the transcriptional response of B. longum subsp. infantis to the different oligosaccharide treatments. This data correlated strongly with the observed changes in adhesion to HT-29 cells. The combination of 3′- and 6′-sialyllactose resulted in the greatest response at the genetic level (both in diversity and magnitude) followed by 6′sialyllactose, and 3′sialyllactose alone. The microarray data was further validated by means of real-time PCR. The current findings suggest that the increased adherence phenotype of Bifidobacterium longum subsp. infantis resulting from exposure to milk oligosaccharides is multi-faceted, involving transcription factors, chaperone proteins, adhesion-related proteins, and a glycoside hydrolase. This study gives additional insight into the role of milk oligosaccharides within the human intestine and the molecular mechanisms underpinning host-microbe interactions.Science Foundation IrelandTeagasc Walsh Fellowship Programm