Periodic Reporting for period 2 - VISGEN (Transcribing the processes of life: Visual Genetics) Reporting period: 2019-03-01 to 2022-02-28 Summary of the context and overall objectives of the project The genetic basis of most brain diseases is still unknown. Many dozens or even hundreds of mutations may contribute to schizophrenia, depression or autism. Often we do not even know when and where these mutations act in the brain. The current ignorance in this area limits not only the diagnosis of neuro-psychiatric disorders, but also the therapeutic possibilities. It is therefore one of the dreams of neuroscience to see in real time what genetic changes are happening inside nerve cells, how genes are activated across the brain during development and behaviour and to compare the healthy to the diseased state. This is challenging as it requires two technical breakthroughs: first, labeling the building blocks of genetic material (while not hindering normal cell function) and, second, making the labeled molecules visible in the living brain. Visualisation of transcription (the first step of gene expression, in which a particular segment of DNA is copied into RNA) in living systems has never been witnessed directly. The overall objective of the VISGEN project is to develop a technology to be able to see which genes are active in a living neuron. This multidisciplinary and international project will herald a new era where this idea becomes a regular research tool and translates to a clinical and diagnostic technology in the future. The team will use a unique biotagging platform to develop the technology that is required to interrogate transcription. The effort requires the amalgamation of knowledge from neuroscientists, synthetic chemists, engineers, physicists, analytical chemists, behavioural scientists, laser technology and image processing experts. The consortium combines expertise from fourteen organisations from the academic and business sectors from six countries to build the multidisciplinary team and share the knowledge that addresses and will overcome the task of realising real-time and spatially resolved genetic studies. Real-time visual genetics will transform our understanding of the state-of-the-art and herald transformative changes in the field of neuroscience, and in general life science. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The first part of the project covers the following areas: 1) project management; 2) chemical synthesis of tagged molecules; 3) building a microscope capable of visualizing the tagged molecules; 4) coupling an SRS (Stimulated Raman Spectroscope) laser to the microscope; 5) develop a genetic tool to visualize gene activity in live cells.For this international project we use internet-based project management tool that is widely used in the industrial field (University of Birmingham, University of Pecs). It helps us to assigned subtasks, set deadlines and responsible person and track the progress. Importantly, the progress is visible to every partner providing a transparent platform where the building blocks of the project can be viewed by all. We also set up a website (visgeneu.wordpress.com) where the public can view the development of the project. The chemistry team (University of Birmingham, UK) was successfully developed a method that attaches an untrasmall tag to the building blocks of RNAs (nucleotide). This tagged molecules were used to build up specific RNA segments that binds to certain genes. The team was able to synthetize enough molecules for the use of biological experiments. The commercial partner (Femtonics Ltd, Hungary) that is specialized in building microscopes has delivered 2 microscopes to two partners already. They have also written the necessary customized software that enables the laser physics team to couple the laser apparatus in order to visualize the tagged nucleotides in live cells.The laser physics team’s job (Wigner research Institute, Hungary) was to link the SRS laser unit to the custom-built microscope. Using various optical elements the laser was successfully coupled to the microscope and the laser signal was modulated with the required speed to achieve SRS scanning capabilities. The genetics team (University of Birmingham) designed a unique method to visualize gene activity in live cells. First they used commercially available fluorescent tags and a traditional confocal microscopes to tune the method. Zebrafish embryos were used as model organism to visualize gene activity in numerous cells in parallel. The team with the help of researchers from University of Pecs and Wigner Research Institute tracked the fluorescent signal both spatially and temporally. The team in Shanghai, China at Fudan University prepared the mesoporus magnetic nanomarticles to introduce the tagged nucleotides to mammalian neurons. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The method, termed Visual Genetics, which is based on the physical phenomenon of Raman scattering, is a major development not only in brain research, but also in other medical applications including early stage cancer detection and toxicity studies. We already have projects to investigate gene activity in human cells (induced pluripotent stem cells, IPSCs) from control subjects and Alzheimer patients to investigate genetic differences in Alzheimer disease. In addition we also set up a project with neurosurgeons at University of Pecs to investigate gene activity in human brain tissue dissected from both epileptic patients and brain tumor sufferers. These investigations will provide essential information about the cause of targeted brain disorders (Alzheimer, epilepsy, and brain tumor) that have an associated social and economic burden that costs society billions of euros every year.