In vitro platforms are key tools to answer fundamental questions about biology and diseases. One of the strategies to produce a complex cell culture system is micropatterning, enabling control over cell and tissue architecture in vitro to explore and dissect the relationship between cell and tissue architecture and resulting function and behaviour configurations in various fields such as disease modelling, immunology, or neurobiology. The two main techniques used for micropatterning, micro contract printing and deep UV micropatterning, require complex, multistep, manual operations, lengthy incubation time, and are limited in application and reproducibility therefore not suitable for high throughput assays and limited in their applications and development. As part of its Biomaterial and Microfluidics service, the Making Lab wishes to take advantage of the recent advances in molecular plasma for patterning to propose a cutting-edge fully automated equipment, whose rapid implementation, simplicity of operation and versatility will immediately benefit research at the Crick Cold atmospheric Molecular plasma is an interesting technology that use the plasma as a vector to graft various chemistry (antibodies, peptides, proteins, epoxy, acrylic, etc) directly onto any substrate in a single-step, solvent-free, scalable atmospheric process at room temperature. Moreover, this process can be adapted to any type of support used in biology including s challenging ones used for low volumes or high throughput experiments. Combining micropatterning masks (i.e. mask with negative features letting the plasma and molecules imprint them on the support's surface), a cold atmospheric plasma with coaxial biomolecule deposition, and a computer numerical control (CNC) manufacturing system, it allows programming and automation of surface treatment. In practice, a stream of inert gas is used to create a plasma projected through a pattern on the support's surface while a solution containing the molecule in aerosol form is coaxially distributed to bind to the plasma on the surface. The coating sequence is programmed on the machine's software and can be adapted to any type of surface and dish fitting into an A4 surface. A custom adapter placed on top of the plasma allows the use of multiple heads to allow the coating of the entire surface, parts only or patterns. The proposed development of a custom writing plasma head for the instrument and the associated protocols to apply it to biological research represents a unique opportunity to significantly increase the impact that this instrument could have on the development of new project Ultimately this equipment allows to address a wide range of areas such as cell growth, cell migration, organdies, microfabrication of microstructures substrate and microfluidics. This technology will immediately benefit laboratories working in the areas of bioengineering, developmental biology, cell biology, infection and immunity, gene regulation, neuroscience, nanofabrication, biosensing. For example, it will help better understand the dynamic interactions between human macrophages and Mycobacterium tuberculosis (Mtb) during early infection state, or describe the dynamic behind the vascular topology, contribute to finding ways to model the nervous system more accurately and see how conditions like motor neurone disease (MND) damage it and to automatically assay arrays of 3D cell cultures to enable their use in drug discovery.

Plasmodium vivax Malaria can cause severe illness in people. The parasite is thought to stick to tissues and organs resulting in disease. P. vivax is a chronic infection resulting in low levels of parasites in blood for a long time. One reason for this is that the parasite avoids the host?s antibody response. The malaria parasite lives for much of its life in red blood cells (RBC), and should be invisible to host antibodies. However, the parasite needs to communicate with the environment outside the RBC, and therefore produces molecules present on the RBC surface for this. This means that the parasite is no longer invisible and can be eliminated by antibodies. To avoid this, the proteins at the RBC surface constantly change by switching on and off genes that code for different variants, so that the parasite stays one step ahead of the antibody response. We will use a mouse model of malaria to investigate how the expression of these proteins is regulated, whether they are recognized by antibodies, and whether they are responsible for adhesion in organs. It might possible to use this knowledge to prevent adhesion and thus disease, and to develop vaccines.

The global spread of H5N1 highly pathogenic avian influenza viruses and their ability to infect not only birds but humans emphasises that human and animal health are unavoidably linked. At present avian influenza remains an animal disease problem under urgent need for control but control in birds will also reduce the potential for a human influenza pandemic. Our knowledge of the behaviour of avian influenza viruses in domestic fowl and wild birds is limited. This proposal poses some fundamental questions that address how the easily the virus can infect chickens, turkeys and ducks; how much, and for how long, virus is shed following infection in each species; and how avian influenza virus infection is controlled by the immune response of birds. Fundamental studies of this type will be critical to the design and implementation of control measures in the short term and the long term.

Lymphocytes are central for the regulation of an immune response. CD4+ T lymphocytes can differentiate to functionally distinct subsets and classified based on their cytokine secretion profiles as well as other properties. Proper regulation and balance between various lymphocyte subsets is central for the appropriate function of the immune system and required e.g. for host defence against pathogens or cancer as well as protection from autoimmune diseases. Recently, a new effector CD4+ T cell subset Th17 was discovered and named according to the main cytokine they secrete, IL17. Besides being involved in host defence to extracellular pathogens, Th17 cells play a central role in the pathogenesis of inflammatory diseases in human and experimental mouse models. The recent discovery by the Stockinger group links the aryl hydrocarbon receptor, a transcription factor widely studied in the toxicology field, and the Th17 system, which provides new hypotheses on the potential mechanisms underlying the well-established connection between the environmental factors and autoimmune diseases. The cellular response to an external stimulus is mediated through a series of overlapping networks that include the signalling network, the epigenetic and transcription factor control mechanisms at genome level, and the gene regulatory. In this project we will study these control mechanisms during the differentiation of Th17 cells by combining genome-wide experimental and computational approaches. We expect that our project results in the identification of the key regulatory components of each of the networks involved, as well as provide insights into the nature of molecular interactions between them. Such regulatory hubs, or the pathways that they participate in, provide targets for rational chemotherapy either for the immune-mediated diseases described above, or for enhancing protective immunity against infectious agents. Our multidisciplinary consortium provides cutting-edge complementary expertise to achieve the goals.

The Crick achieves operational and research efficiencies and economies-of-scale through centralised facilities and functions, known as Science Technology Platforms, that provide all researchers at the Crick, irrespective of affiliation, with access to cutting-edge equipment for research and laboratory enabling functions such as the High Throughput Screening facility (HTS). The Crick Covid Surveillance unit (CCSU) is an offshoot of the HTS facility and was established to run a pipeline of activity delivering a high throughput live-virus antibody neutralisation assay to measure the ability of patient serum to prevent cell infection by SARS-CoV-2 across thousands of samples simultaneously. This unique neutralisation assay platform (developed in the HTS facility) is essential to many ongoing research studies, for example those assessing the likely impact of Variants of Concern on current vaccinated populations and in clinically vulnerable cohorts. The pipeline is able to generate neutralization data against new VOCs (e.g. Delta and Omicron) within 2-3 weeks upon receipt of a novel virus sample and can process thousands of samples per week at GCP level. Data from this pipeline has measured neutralising antibody titres against Delta and Omicron variants in participants in the Crick-Legacy study (recipients of both the Pfizer and Oxford-AstraZeneca vaccines). These data are shared with UK government scientific advisory boards, contributing to the extension of UK pandemic restrictions to allow more people to receive a second vaccine dose in summer 2021 and the successful booster campaign. Publications reporting these findings have been cited nearly 600 times. This pipeline also enabled the comparison of antibody titres in healthy adults with those of both cancer patients (Crick-CAPTURE study) and kidney dialysis patients (Crick-NAOMI study), providing important data on prioritisation of vulnerable patient groups. In addition, data on the in vitro neutralising efficacy of the synthetic monoclonal antibody, Sotrovimab was shared with NHSE and the Chief Medical Officer and supported the ongoing use of this drug for patients infected with Omicron BA.1 and BA.2. There are no other similar pipelines capable of this scale of live virus assay in the UK (possibly internationally) and with near real time reporting, the data produced remains a vital tool in the UK and international pandemic preparedness and response. Currently, the neutralisation pipeline is made robust by using multiple HTS liquid handling devices to ensure continuity of delivery. However, the primary imaging (and measurements derived from them) is restricted to one microscope, the Opera Phenix. This reliance on one imaging device is a weak point for this pipeline. This proposal is to deploy the Celigo scanning cytometer in this assay and thereby remove the dependence on one machine for a pipeline that needs to remain operational. Moreover, acquisition on the Celigo is 4-5 times faster meaning that pipeline throughput and capacity can be significantly increased. The Covid neutralisation pipeline accounted for 50% of HTS usage over 2020/21. Having a high-end microscope occupied with one type of assay necessarily restricts access by other users (currently measured in weeks of delay for access to the Opera Phenix). By moving the primary imaging platform for the CCSU activity to the Celigo, we would greatly increase the availability of high content microscopy to other Crick researchers and thereby improve service delivery for more than 100 Crick researchers who used the HTS facility over 20/21. The availability of a Celigo imager will also enable improvements to existing activities requiring analysis of cell populations (at scale) in the areas of radiation biology, stem cell biology and general cell viability. In addition, there will be new opportunities for users to explore automation of previously manual assays e.g. Influenza plaque assays