Validating Functional Mechanisms For Non-Coding Genetic Variants Associated With Complex Traits

Genome-wide association studies (GWAS) have identified a large number of genetic variants associated with disease as well as normal phenotypic variation for complex traits. However challenges remain in determining the functional relevance of human DNA sequence variants. Even after fine mapping, most variants are located in non-coding regions making it difficult to infer mechanisms linking individual genetic variants with the disease trait. In addition, we do not know under which environmental conditions the sequence variants have a functional impact, and whether they become one of many factors involved in complex phenotypes at the organismal level. Chapter 1 describes computational methods to predict causal GWAS variants, validation of a subset for ASE using a traditional reporter assay, and development of a method to identify ASE in high throughput assays. These methods improved positive detection of enhancer activity and ASE, and this analysis pipeline will continue to be useful as more researchers begin using high throughput assays to identify allelic effects. Chapter 2 improves upon chapter 1 with the development of a new modification of STARR-seq in order to streamline the assay and improve power to detect ASE through the addition of an UMI. Additionally, by integrating BiT-STARR-seq with a high throughput allele-specific EMSA, we are able to identify the mechanism behind many ASE variants. Studying GxE in human studies is extremely difficult, so our approach of using an in-vitro method and modeling molecular phenotypes is a useful alternative. Chapter 3 describes the investigation of GxE with complex traits. Using GEMMA, we were able to identify environments that were enriched for complex traits. With ATAC-seq data we were able to identify differentially accessible regions, TF footprints, and differential TF footprints. Integrating this data with BiT-STARR-seq, we were able to identify enrichment for these differential chromatin accessibility regions with ASE. Overall, these chapters show the integration of computational predictions with experimental validation in order to identify allelic effects. This design is a useful approach to validate the molecular mechanism for specific transcription factors, and link these to the context of human health

Optimized utilization of Salix-Perspectives for the genetic improvement toward sustainable biofuel value chains

Bioenergy will be one of the most important renewable energy sources in the conversion from fossil fuels to bio-based products. Short rotation coppice Salix could be a key player in this conversion since Salix has rapid growth, positive energy balance, easy to manage cultivation system with vegetative propagation of plant material and multiple harvests from the same plantation. The aim of the present paper is to provide an overview of the main challenges and key issues in willow genetic improvement toward sustainable biofuel value chains. Primarily based on results from the research project "Optimized Utilization of Salix" (OPTUS), the influence of Salix wood quality on the potential for biofuel use is discussed, followed by issues related to the conversion of Salix biomass into liquid and gaseous transportation fuels. Thereafter, the studies address genotypic influence on soil carbon sequestration in Salix plantations, as well as on soil carbon dynamics and climate change impacts. Finally, the opportunities for plant breeding are discussed using willow as a resource for sustainable biofuel production. Substantial phenotypic and genotypic variation was reported for different wood quality traits important in biological (i.e., enzymatic and anaerobic) and thermochemical conversion processes, which is a prerequisite for plant breeding. Furthermore, different Salix genotypes can affect soil carbon sequestration variably, and life cycle assessment illustrates that these differences can result in different climate mitigation potential depending on genotype. Thus, the potential of Salix plantations for sustainable biomass production and its conversion into biofuels is shown. Large genetic variation in various wood and biomass traits, important for different conversion processes and carbon sequestration, provides opportunities to enhance the sustainability of the production system via plant breeding. This includes new breeding targets in addition to traditional targets for high yield to improve biomass quality and carbon sequestration potential

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

Comparison of the activities of two allelic variants of the human wildtype p53 protein

The human wildtype p53 tumor suppressor gene contains a polymorphism at amino acid residue 72 which results in either an arginine (p53 Arg-72) or proline (p53 Pro-72) at this codon. In the present study I have examined this polymorphism at the molecular level to determine whether differences exist in the biochemical functions of these two p53 variants. No differences were observed in their sequence-specific DNA binding abilities, nor in their ability to be targeted by HPV-18 E6 oncoprotein for degradation by ubiquitination in vitro. However, differences were observed in the ability of these two variants to function as transcriptional activators: p53 Pro-72 was more transcriptionally active than p53 Arg-72. I propose that the polymorphism at codon 72 may affect the structure of the N-terminal transactivation domain of the p53 protein, which would then have an effect on the ability of these variants to interact with transcription factors in order to initiate transcription of target genes and function as a tumor suppressor

Inclusive diets within planetary boundaries

Our food production system is unsustainable and threatening planetary boundaries. Yet, a quarter of the global population still lacks access to safe and nutritious food, while suboptimal diets account for 11 million adult deaths per year. This Voices asks: what critical barriers must be overcome to enable sustainable, healthy, accessible, and equitable diets for all