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The number of people with type 2 diabetes (T2DM) and obesity (the major risk factor for the development of T2DM) has reached epidemic proportions worldwide. 80% of people with T2DM will die from the complications of cardiovascular atherosclerosis (furring of the arteries) resulting in an increased risk of death equivalent to 15 years of aging. We recently demonstrated that despite the use of contemporary secondary prevention therapies patients with T2DM, sustaining an acute myocardial infarction (AMI) or ?heart attack? have not benefited from the improvement in mortality seen in similar patients without T2DM. A central feature of T2DM is resistance to the actions of insulin the hormone that is released to reduce blood sugar this process is known as insulin resistance. Insulin resistance has been demonstrated in many tissues of patients with T2DM. In addition to lowering glucose, insulin is thought to stimulate the release of a substance from the blood vessel wall known as nitric oxide (NO). NO protects the artery against atherosclerosis. It has recently emerged that patients with T2DM have reduced NO. This may therefore contribute to the accelerated atherosclerosis seen in patients with T2DM. We have performed studies that demonstrate a novel mechanism by which insulin resistance may reduce NO actions. We have shown that an enzyme in the blood vessel wall (NADPH oxidase) that produces a damaging gas that mops up NO becomes overactive in insulin resistance. We plan to perform studies examining the effect of reducing the activity of NADPH oxidase in different models of insulin resistance. The results of this study may guide us towards new treatments to prevent AMI in patients with T2DM.

Chikungunya virus (CHIKV) is a mosquito-transmitted positive-stranded RNA virus that causes incapacitating chronic joint pain in humans. Having re-emerged as an epidemic in 2004 around the Indian Ocean the virus has been imported into many countries, with >1,135,000 cases reported in the Americas and Europe by 2015. There remains no vaccine or specific antiviral therapy, with development hampered by lack of insight into its biology. Given the scale and expanding geographic distribution of these outbreaks, more research on this virus is urgently needed. In many positive-strand RNA viruses essential mechanisms for regulating of early replication events involve stem-loops within the virus genome. Such structures can act as RNA-replication elements (RRE) and through interaction with host and viral proteins regulate a range of essential processes such as virus translation and genome replication. In preliminary studies we used a combination of structural and reverse genetic analysis to investigate RNA stem-loop elements conserved in protein coding and non-coding regions of the CHIKV genome. In preliminary studies leading to this application we demonstrate that these stem-loops function as RREs during the early stages of CHIKV replication. Silent mutations within each element inhibited CHIKV replication in human cell culture while enhancing it in mosquito cells. In both instances wild-type levels of replication were restored following further mutagenesis to reinstate stem-loop structure. This and further evidence from our preliminary studies lead us to propose that the CHIKV RREs function during early virus replication events, regulating initial translation and genome replication via host specific interaction with trans-activating proteins. I seek support to define host-specific genetic and structural determinants of RRE function, precise roles during early virus replication and specific protein trans-activator interactions. Comparison of results between human and mosquito cell systems, using sub-genomic replicons and infectious virus, will enhance detailed dissection of mechanisms involved. OBJECTIVE-1: Through analysis of engineered mutants across a range of complimentary systems, we will determine precise functional domains within each RRE during early stages of CHIKV replication and generate information on functional local or long-range RNA-RNA/RNA-viral protein interactions. RRE function during CHIKV translation and genome replication will be defined in human and mosquito cell culture. OBJECTIVE-2: Comparative RNA structural analysis will be undertaken using a variety of established and innovative SHAPE methods, producing complimentary quantitative information on RRE structure. Direct insight into the relationship between RNA conformation and function will be provided by comparative analysis of mutants and wild-type under different physiologically relevant conditions and within the replication complex of infected cells. OBJECTIVE-3: Proteomic analysis will be undertaken using a range of in vitro and intracellular techniques, including SILAC mass spectroscopy and PAR-CLIP. Dissecting differences between RRE-proteome interactions in human vs. mosquito cells and native vs. mutated elements will pinpoint interactions relevant to the function of individual RNA structures and stem-loop regions. Through these complimentary in vitro and physiological approaches we will define structural and trans-activating interactions essential to RRE function during CHIKV replication. Roles in translation and genome replication will be defined and through comparison of contrasting host-specific and mutant phenotypes we will dissect functional mechanisms. We expect these results to have direct application in identification of novel therapeutic targets. Furthermore, mechanistic understanding of RRE-mutant attenuation will have direct application in studies towards a genetically stable attenuated vaccine.

E. coli RNase E is a major regulator of gene expression and homologues are found in many bacteria and plant plastids. In a series of landmark papers published this year, we have shown that contrary to current models RNase E can survey multiple sites directly without being constrained by 5'-end tethering. We have also outlined the underlying mechanism. This work, which has important ramifications, was funded by the BBSRC and featured on their website. Objectives: In addition to the above, we have made a finding that may produce a seismic shift in our understanding of gene regulation. We have found that RNase E reaches beyond the RNA it cleaves and binds to virtually any it encounters. The broad objective of this project is to establish whether the binding of 5' untranslated regions (UTRs) by RNase E represents a major route by which translation is regulated in E. coli. The specific aims are to (1) confirm that RNase E binds to many 5' UTRs with high affinity, (2) establish that binding of 30S ribosomal subunits can be outcompeted by RNase E, and (3) determine the overall influence of RNase E binding on translation. Novelty: The hypothesis is novel. Never before has it been proposed that RNase E, or indeed any other ribonuclease, can regulate translation by outcompeting ribosomes for binding to 5' UTRs. Moreover, the inactivation of mRNAs before the first cleavage could prevent the futile and perhaps deleterious translation of partially degraded transcripts. Our most recent direct entry paper was published as a "Breakthrough" article and was given the front cover. Timeliness: The proposed project not only comes on the back of a series of strong publications plus recent findings that were unexpected and unprecedented, it fits with major initiatives in synthetic biology and the development of new antibiotics.

This research will identify important similarities and differences in the way animals as diverse as arthropods (i.e. fruit flies, beetles and spiders) and vertebrates (i.e. fish, mice and humans) develop their body during embryogenesis. This work is crucial for understanding how the genetic and developmental mechanisms forming animal body plans changed and diverged over evolutionary time to produce the diverse types of animal body plans we see in nature today. It is also important for understanding how the common ancestor of all animals developed its body plan, deep in evolutionary history, over 550 million years ago. Humans, and other vertebrates, possess a segmented body plan: internal parts of our body are divided into separated and repeated structures, for example our vertebrae and ribs. Vertebrate embryos grow during their development, with head structures formed first and the trunk grown in an anterior to posterior sequence. During this process, the cell populations that will later give rise to ribs/vertebrae are determined early, and one-by-one, in an anterior to posterior sequence. This process is controlled by a network of genes that are repeatedly turned on and off to define each group of cells (i.e. each future rib/vertebra). This complex oscillating network of genes is called the vertebrate segmentation clock. Arthropods, including fruit flies, beetles and spiders, also have a visibly segmented body plan. The abdominal body segments of the red flour beetle Tribolium castaneum form sequentially, one-by-one, in an anterior to posterior progression during beetle development, in a process similar to that described above for vertebrates. I have recently shown that this developmental process is also controlled by a network of genes that oscillate to sequentially pattern abdominal segments - the arthropod segmentation clock. Interestingly, the fruit fly Drosophila melanogaster has evolved to speed up its development, such that all body segments form simultaneously in the egg in a process that is already well understood at the genetic level by biologists. The first objective of this work programme is to determine whether the gene network underlying the arthropod segmentation clock is organized in a similar way to the vertebrate segmentation clock. If striking similarities are found, it could suggest that the segmentation clock was a feature of the common ancestor of arthropods and vertebrates. Alternatively, if there are striking differences, it could suggest that disparate animal groups have evolved similar genetic mechanisms independently and in parallel. Either way, this research promises to reveal important information about our evolutionary history. The second objective of this work programme is to determine how a sequential segmentation mechanism involving a segmentation clock was modified in evolution to produce the mechanism controlling the simultaneous formation of segments in Drosophila. Identifying the changes in gene regulation that facilitated this transition could reveal general principles by which developmental mechanisms evolved to produce the wide variety of animal body plans we see in nature today. The third objective of this work programme is to develop new genetic techniques that will advance Tribolium castaneum as a cheap, amenable, ethically acceptable, invertebrate model with which to study segmentation clocks. The general public will benefit from this research via an increase in our understanding of human and animal evolution, through the development of a powerful invertebrate genetic model for studying the genetic principles underlying segmentation clocks thus reducing the current dependence on less ethically acceptable vertebrate models, and through better value for money from future research on segmentation clocks via the use of Tribolium, a cheaper and potentially more amenable alternative to vertebrate models.