This proposal addresses a major need to increase understanding and improve treatment of a group of severe developmental diseases caused by activating mutations in the PIK3CA gene. These are collectively called the PIK3CA-related Overgrowth Spectrum (PROS), and they begin before birth. The PIK3CA gene controls cell and tissue growth, and when it is genetically activated, the rules that ensure orderly, co-ordinated tissue growth are broken. Unhealthy increased and disorganised growth ensues. Mutations are found in only some cells and tissues, a situation called genetic mosaicism, and this produces patchy, asymmetric excess growth. This often severely affects blood vessel formation to produce major risks of blood clots, bleeding and infections that can be life threatening, while other functions (e.g. breathing, walking) are also commonly affected by increased tissue bulk. PROS is incredibly variable despite the common underlying mechanism. Many affected patients wait years for specific diagnosis, if it is ever made, as PROS does not "belong" to any one medical or surgical speciality. When PIK3CA mutations were found to cause these diverse diseases, new opportunities to treat them emerged. This is because the same PIK3CA mutations causing PROS are common in cancers. Although PROS causes cancer very rarely indeed, PIK3CA-targeting drugs developed for cancer have raised great hopes in PROS, with one drug recently licenced in the USA based on an uncontrolled registry study. However while this has some benefits, these are partial, and side effects are frequent. Thus major need exists to improve medical treatment. In the rush to test cancer drugs in PROS, major questions about how PIK3CA mutations cause the disease have not yet been unanswered, although these may hold the key to developing new, smarter treatment. The scientific and clinical complexity of PROS means that ambitious multimodal translational research is required to advance understanding. In this project a team of two clinician scientists and three fundamental scientists will combine expertise in clinical medicine, genetics, stem cell biology, animal disease modelling and sophisticated data analysis. The proposal has been informed by working with a PROS patient advocacy group (clovessyndrome.org) that has funded key preliminary work. The project will use cutting edge single cell analysis of affected human tissue from PROS patients, combining this with the power of disease modelling in human pluripotent stem cells and zebrafish. Findings from human tissue and the model systems will be combined and used to identify new mechanisms driving PROS that might be targeted. Time windows when the existing clinical drug has its greatest effect will be identified, and by studying why sensitivity is greatest in these windows, we will devise and test new ways to increase the sensitivity of mutation bearing but not healthy cells. We envisage two potential new strategies: (1) to "trick" affected cells to enter a state more vulnerable to current inhibitors; and (2) to identify messages from affected cells that trigger healthy cells around them to grow too much. If these messages can be disrupted then this may produce a new line of attack to combat excess growth in PROS. Outcomes of the study will include 1) identification of new strategies worth testing as PROS treatments; 2) fundamental insights into the way tissue growth is regulated that are relevant also for cancer, and 3) key lessons about the best ways to study other "mosaic" genetic diseases.

Our detailed understanding of the history of life relies on the fossil record, and most especially on the very rare and scientifically important fossil deposits that preserve not simply the hard parts of animals but their entire bodies, soft parts and all. Geological deposits that show such exceptional preservation are known as fossil Lagerstätten, and these tell us much more about ancient biotas and communities than the normal shelly fossil record; they provide unique windows on past life. One such deposit is the Herefordshire Lagerstätte from the Welsh Borderland, which contains spectacular fossils of small marine invertebrates that lived about 425 million years ago, during the Silurian Period. These animals were preserved when they were engulfed in ash from a volcanic eruption. The animals themselves soon rotted away, but their shapes were faithfully recorded, initially by the ash itself, and then by crystals of calcite that grew within the resulting voids. These crystalline shapes are now found within hard nodules in the ash layer, and not only do the fossils preserve entire animals in fine detail, but almost uniquely they are fully three-dimensional rather than squashed flat. The Herefordshire fossils cannot be extracted whole from the rock by mechanical preparation or acid digestion techniques. Nor can images of them be obtained by use of more recent technologies such as X-ray computed tomography (CT scanning) or magnetic resonance imagery (MRI). Instead, they are reconstructed and studied, by a team of scientists from Oxford, Leicester, London (imperial College) and Yale universities, using a novel approach. This involves computer technology and, paradoxically, the destruction of the fossils themselves. Specimens are ground away 30 microns at a time, and a photograph is taken of each freshly exposed surface. Tens to hundreds of these photographs are then used to create a high-fidelity 'virtual fossil' in the round, which can be rotated or even dissected on a computer screen. The fossils themselves are in the range of a few millimetres to about five centimetres, but scaled-up physical models can also be made of them through rapid-prototyping technologies. The Herefordshire animals date from a period of time for which we have little knowledge of soft-bodied faunas. Sediments deposited during the Cambrian Period (about 542-488 million years ago) are relatively rich in fossil Lagerstätten, and those deposited during the Devonian (about 416-359 million years ago) reasonably so, but there are very few such horizons represented in sediments laid down in the intervening time, during the Ordovician and Silurian periods. Thus the Herefordshire fossils are of great importance in helping to fill in this gap in the history of life. Various of the Herefordshire animals, such as the worm-like mollusc Acaenoplax and the tiny, Limulus-like arthropod Offacolus, have already been researched by the team. These and other fossils from this Lagerstätte have proved to be representatives of previously unknown evolutionary lineages, and helped to resolve controversies about the relationships both of extinct animals and of those alive today. Some animals from the Herefordshire Lagerstätte remain to be investigated. They include several arthropods, a mollusc, two echinoderms, a polychaete worm, a brachiopod (lamp-shell) and a sponge, and it is these that will provide the focus of a new research programme. Individually, they are anticipated to be as exciting and as scientifically significant for each of their respective major animal groups as those Herefordshire fossils that have already been studied. Together, their study will allow, for the first time, syntheses of the composition, community structure and ecology of the Herefordshire fauna, and comparison of it with other exceptionally preserved faunas. This will give us an unrivalled view of life on the seabed 425 million years ago, during the Silurian period.

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Since the discovery of fire and the development of agriculture, humans have been releasing carbon dioxide (CO2) to the Earth's atmosphere. We have known about the effect that burning of fossil fuels and deforestation has on the amount of CO2 in the atmosphere as well as its influence on global temperatures for many years now. However, the CO2 we put in the atmosphere does not all just stay there / because CO2 reacts with water, about a third of current fossil fuel emissions is removed by the ocean. This effect would be really helpful for us in preventing more extreme global warming from taking place, except ... in past few years scientists have realized that because CO2 dissolved in seawater creates a weak acid, we are causing the pH of the ocean to steadily decrease in a process known as 'ocean acidification'. There are currently about 380 molecules of CO2 in the atmosphere for every million of all gases combined ('parts per million' or ppm). Atmospheric CO2 is predicted to steadily increase in the coming decades, reaching 450-550 ppm by the year 2050 / a concentration that our Planet has not experienced in at least the past 3 million years. As atmospheric CO2 increases, so does the rate at which it will dissolve in seawater, forcing the pH of the surface ocean lower and lower. It is likely that ocean pH will reach values seen only rarely since the time of the Dinosaurs. Most organisms alive in the ocean today have never experienced such a large change in all their evolutionary history. Is this important? From laboratory experiments it seems that ocean acidification will affect marine organisms, particularly those that make shells and skeletons out of calcium carbonate, because calcium carbonate minerals become less stable as waters become more acidic and will eventually dissolve. If we fail to control CO2 emissions to keep ocean pH change within the limits calcifying organisms can cope with in the future, we may see dissolution of their shells, slower growth, failure to reproduce, dwarfism, or reduced activity, with impacts further the ecosystem. Unrestricted industrial activities may even push these organisms over an ecological precipice and cause extinctions. So what is going to happen in the future? In the geological past, organisms normally had thousands to millions of years to adapt and evolve in response to global environmental change. Although the global environmental change we are causing now is many hundreds of times faster, it would still take laboratory experiments conducted over decades to tell us whether marine organisms will be able to adapt to ocean acidification. By the time we know the answer, it may be too late! Luckily, there is an alternative path; one that lies hidden in rocks. The geological record, stored in the mud at the bottom of the ocean is packed with millions of microfossils that record how much change organisms can tolerate and how much is too much. We will take samples of ancient sediments that have been drilled from the ocean floor, analyse these samples using a range of state-of-the-art techniques involving detailed laboratory analyses, and apply complex computer models to help make complete sense of the numbers. This will tell us how the pH of the ocean changed in the past. By linking this information with observations of ecosystem changes and species extinctions will provide vital clues to what changes in marine ecosystems we might expect in the future if we do not make much greater efforts to curtail our greenhouse gas emissions now.

Human Immunodeficiency Virus type 1 (HIV-1), the causative agent of the AIDS pandemic, encodes two accessory proteins (Vpr and Vpu) that potently suppress proinflammatory signalling via the NFkB family of transcription factors during the early and late phases of viral replication in CD4+ T cells and macrophages. This appears, at least in part, to be a mechanism to inhibit the induction of innate immune to viral replication at vulnerable stages of the lifecycle. Despite this, the integrated HIV-1 provirus is an NFkB-regulated gene which requires NFkB to be activated to initiate viral gene expression. Our recent publications on Vpu and unpublished work on Vpr from clinical isolates shows that the potency of NFkB suppression by these proteins has been underestimated. Both exert their functions through markedly different mechanisms that are incompletely understood at the molecular and cellular level. We have shown that Vpu promotes the degradation of the bTrCP1 adaptor of the SCF ubiquitin ligase, leading to a potent block of both classical and alternative NFkB pathways. But how this specificity is achieved is unclear. By contrast Vpr appears to block nuclear transit of inflammatory transcription factors by association with the nuclear pore and karyopherins. However, the requirement for its cognate ubiquitin ligase Cullin4DCAF1 implies Vpr targets an unknown factor to exert this activity. Since Vpr is a constituent of the incoming viral particle and Vpu is expressed 'late', these data imply that HIV-1 dynamically regulates NFkB across its replication cycle to balance innate immune evasion and viral production. A key barrier to curing individuals of HIV-1 is the presence of a pool of infected CD4+T cells harbouring transcriptionally silent integrated proviruses - the so-called latent reservoir. While individuals living with HIV-1 are take combined antiretroviral therapy (cART) this latent reservoir is of little consequence. However, upon therapy withdrawal re-activation of some of these reservoir cells leads to rapid re-emergence of the virus. Therefore, aside from a handful of cases where HIV+ blood cancer patients were transplanted with bone marrow stem cells genetically resistant to infection, people living with HIV cannot be cured and must take cART for the rest of their lives. Thus, purging or silencing of this reservoir is viewed as the key goal of HIV-1 cure research, and ultimately this is dependent on the level of NFkB activation in individual latently infected cells. However, a key gap in knowledge is what viral and cellular factors promote the establishment of latent proviruses in the first place and whether these activities limit the success and sustainability of latency-reversing agents (LRAs) currently being developed to facilitate reservoir reduction - so called 'shock and kill' strategies. It is our hypothesis that dynamic manipulation of NFkB in HIV-1-infected primary cells by Vpr and Vpu tips the balance to promote the establishment of a latent provirus. Furthermore, their potency at inhibiting NFkB may limit the sustained gene expression required for sufficient reservoir purging by LRAs/cure strategies activating these pathways. We therefore seek MRC programmatic support for interlinked basic and translational studies to understand: 1. The consequences of dynamic regulation of NFkB for both viral and host gene expression across the viral replication cycle 2. The molecular mechanisms underlying the suppression of NFkB by HIV-1 accessory proteins 3. How accessory protein function affects the establishment, maintenance, and sustainable reversion of HIV-1 latency.