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Project acronym 100 Archaic Genomes
Project Genome sequences from extinct hominins
Researcher (PI) Svante PÄÄBO
Summary Neandertals and Denisovans, an Asian group distantly related to Neandertals, are the closest evolutionary relatives of present-day humans. They are thus of direct relevance for understanding the origin of modern humans and how modern humans differ from their closest relatives. We will generate genome-wide data from a large number of Neandertal and Denisovan individuals from across their geographical and temporal range as well as from other extinct hominin groups which we may discover. This will be possible by automating highly sensitive approaches to ancient DNA extraction and DNA libraries construction that we have developed so that they can be applied to many specimens from many sites in order to identify those that contain retrievable DNA. Whenever possible we will sequence whole genomes and in other cases use DNA capture methods to generate high-quality data from representative parts of the genome. This will allow us to study the population history of Neandertals and Denisovans, elucidate how many times and where these extinct hominins contributed genes to present-day people, and the extent to which modern humans and archaic groups contributed genetically to Neandertals and Denisovans. By retrieving DNA from specimens that go back to the Middle Pleistocene we will furthermore shed light on the early history and origins of Neandertals and Denisovans.
Neandertals and Denisovans, an Asian group distantly related to Neandertals, are the closest evolutionary relatives of present-day humans. They are thus of direct relevance for understanding the origin of modern humans and how modern humans differ from their closest relatives. We will generate genome-wide data from a large number of Neandertal and Denisovan individuals from across their geographical and temporal range as well as from other extinct hominin groups which we may discover. This will be possible by automating highly sensitive approaches to ancient DNA extraction and DNA libraries construction that we have developed so that they can be applied to many specimens from many sites in order to identify those that contain retrievable DNA. Whenever possible we will sequence whole genomes and in other cases use DNA capture methods to generate high-quality data from representative parts of the genome. This will allow us to study the population history of Neandertals and Denisovans, elucidate how many times and where these extinct hominins contributed genes to present-day people, and the extent to which modern humans and archaic groups contributed genetically to Neandertals and Denisovans. By retrieving DNA from specimens that go back to the Middle Pleistocene we will furthermore shed light on the early history and origins of Neandertals and Denisovans.
Project acronym 2-HIT
Project Genetic interaction networks: From C. elegans to human disease
Researcher (PI) Ben Lehner
Host Institution (HI) FUNDACIO CENTRE DE REGULACIO GENOMICA
Summary Most hereditary diseases in humans are genetically complex, resulting from combinations of mutations in multiple genes. However synthetic interactions between genes are very difficult to identify in population studies because of a lack of statistical power and we fundamentally do not understand how mutations interact to produce phenotypes. C. elegans is a unique animal in which genetic interactions can be rapidly identified in vivo using RNA interference, and we recently used this system to construct the first genetic interaction network for any animal, focused on signal transduction genes. The first objective of this proposal is to extend this work and map a comprehensive genetic interaction network for this model metazoan. This project will provide the first insights into the global properties of animal genetic interaction networks, and a comprehensive view of the functional relationships between genes in an animal. The second objective of the proposal is to use C. elegans to develop and validate experimentally integrated gene networks that connect genes to phenotypes and predict genetic interactions on a genome-wide scale. The methods that we develop and validate in C. elegans will then be applied to predict phenotypes and interactions for human genes. The final objective is to dissect the molecular mechanisms underlying genetic interactions, and to understand how these interactions evolve. The combined aim of these three objectives is to generate a framework for understanding and predicting how mutations interact to produce phenotypes, including in human disease.
Most hereditary diseases in humans are genetically complex, resulting from combinations of mutations in multiple genes. However synthetic interactions between genes are very difficult to identify in population studies because of a lack of statistical power and we fundamentally do not understand how mutations interact to produce phenotypes. C. elegans is a unique animal in which genetic interactions can be rapidly identified in vivo using RNA interference, and we recently used this system to construct the first genetic interaction network for any animal, focused on signal transduction genes. The first objective of this proposal is to extend this work and map a comprehensive genetic interaction network for this model metazoan. This project will provide the first insights into the global properties of animal genetic interaction networks, and a comprehensive view of the functional relationships between genes in an animal. The second objective of the proposal is to use C. elegans to develop and validate experimentally integrated gene networks that connect genes to phenotypes and predict genetic interactions on a genome-wide scale. The methods that we develop and validate in C. elegans will then be applied to predict phenotypes and interactions for human genes. The final objective is to dissect the molecular mechanisms underlying genetic interactions, and to understand how these interactions evolve. The combined aim of these three objectives is to generate a framework for understanding and predicting how mutations interact to produce phenotypes, including in human disease.
Project acronym 20SComplexity
Project An integrative approach to uncover the multilevel regulation of 20S proteasome degradation
Researcher (PI) Michal Sharon
Host Institution (HI) WEIZMANN INSTITUTE OF SCIENCE
Summary For many years, the ubiquitin-26S proteasome degradation pathway was considered the primary route for proteasomal degradation. However, it is now becoming clear that proteins can also be targeted for degradation by a ubiquitin-independent mechanism mediated by the core 20S proteasome itself. Although initially believed to be limited to rare exceptions, degradation by the 20S proteasome is now understood to have a wide range of substrates, many of which are key regulatory proteins. Despite its importance, little is known about the mechanisms that control 20S proteasomal degradation, unlike the extensive knowledge acquired over the years concerning degradation by the 26S proteasome. Our overall aim is to reveal the multiple regulatory levels that coordinate the 20S proteasome degradation route. To achieve this goal we will carry out a comprehensive research program characterizing three distinct levels of 20S proteasome regulation: Intra-molecular regulation- Revealing the intrinsic molecular switch that activates the latent 20S proteasome. Inter-molecular regulation- Identifying novel proteins that bind the 20S proteasome to regulate its activity and characterizing their mechanism of function. Cellular regulatory networks- Unraveling the cellular cues and multiple pathways that influence 20S proteasome activity using a novel systematic and unbiased screening approach. Our experimental strategy involves the combination of biochemical approaches with native mass spectrometry, cross-linking and fluorescence measurements, complemented by cell biology analyses and high-throughput screening. Such a multidisciplinary approach, integrating in vitro and in vivo findings, will likely provide the much needed knowledge on the 20S proteasome degradation route. When completed, we anticipate that this work will be part of a new paradigm – no longer perceiving the 20S proteasome mediated degradation as a simple and passive event but rather a tightly regulated and coordinated process.
For many years, the ubiquitin-26S proteasome degradation pathway was considered the primary route for proteasomal degradation. However, it is now becoming clear that proteins can also be targeted for degradation by a ubiquitin-independent mechanism mediated by the core 20S proteasome itself. Although initially believed to be limited to rare exceptions, degradation by the 20S proteasome is now understood to have a wide range of substrates, many of which are key regulatory proteins. Despite its importance, little is known about the mechanisms that control 20S proteasomal degradation, unlike the extensive knowledge acquired over the years concerning degradation by the 26S proteasome. Our overall aim is to reveal the multiple regulatory levels that coordinate the 20S proteasome degradation route. To achieve this goal we will carry out a comprehensive research program characterizing three distinct levels of 20S proteasome regulation: Intra-molecular regulation- Revealing the intrinsic molecular switch that activates the latent 20S proteasome. Inter-molecular regulation- Identifying novel proteins that bind the 20S proteasome to regulate its activity and characterizing their mechanism of function. Cellular regulatory networks- Unraveling the cellular cues and multiple pathways that influence 20S proteasome activity using a novel systematic and unbiased screening approach. Our experimental strategy involves the combination of biochemical approaches with native mass spectrometry, cross-linking and fluorescence measurements, complemented by cell biology analyses and high-throughput screening. Such a multidisciplinary approach, integrating in vitro and in vivo findings, will likely provide the much needed knowledge on the 20S proteasome degradation route. When completed, we anticipate that this work will be part of a new paradigm – no longer perceiving the 20S proteasome mediated degradation as a simple and passive event but rather a tightly regulated and coordinated process.
Project acronym 2SEXES_1GENOME
Project Sex-specific genetic effects on fitness and human disease
Researcher (PI) Edward Hugh Morrow
Host Institution (HI) THE UNIVERSITY OF SUSSEX
Summary Darwin's theory of natural selection rests on the principle that fitness variation in natural populations has a heritable component, on which selection acts, thereby leading to evolutionary change. A fundamental and so far unresolved question for the field of evolutionary biology is to identify the genetic loci responsible for this fitness variation, thereby coming closer to an understanding of how variation is maintained in the face of continual selection. One important complicating factor in the search for fitness related genes however is the existence of separate sexes – theoretical expectations and empirical data both suggest that sexually antagonistic genes are common. The phrase "two sexes, one genome" nicely sums up the problem; selection may favour alleles in one sex, even if they have detrimental effects on the fitness of the opposite sex, since it is their net effect across both sexes that determine the likelihood that alleles persist in a population. This theoretical framework raises an interesting, and so far entirely unexplored issue: that in one sex the functional performance of some alleles is predicted to be compromised and this effect may account for some common human diseases and conditions which show genotype-sex interactions. I propose to explore the genetic basis of sex-specific fitness in a model organism in both laboratory and natural conditions and to test whether those genes identified as having sexually antagonistic effects can help explain the incidence of human diseases that display sexual dimorphism in prevalence, age of onset or severity. This multidisciplinary project directly addresses some fundamental unresolved questions in evolutionary biology: the genetic basis and maintenance of fitness variation; the evolution of sexual dimorphism; and aims to provide novel insights into the genetic basis of some common human diseases.
Darwin's theory of natural selection rests on the principle that fitness variation in natural populations has a heritable component, on which selection acts, thereby leading to evolutionary change. A fundamental and so far unresolved question for the field of evolutionary biology is to identify the genetic loci responsible for this fitness variation, thereby coming closer to an understanding of how variation is maintained in the face of continual selection. One important complicating factor in the search for fitness related genes however is the existence of separate sexes – theoretical expectations and empirical data both suggest that sexually antagonistic genes are common. The phrase "two sexes, one genome" nicely sums up the problem; selection may favour alleles in one sex, even if they have detrimental effects on the fitness of the opposite sex, since it is their net effect across both sexes that determine the likelihood that alleles persist in a population. This theoretical framework raises an interesting, and so far entirely unexplored issue: that in one sex the functional performance of some alleles is predicted to be compromised and this effect may account for some common human diseases and conditions which show genotype-sex interactions. I propose to explore the genetic basis of sex-specific fitness in a model organism in both laboratory and natural conditions and to test whether those genes identified as having sexually antagonistic effects can help explain the incidence of human diseases that display sexual dimorphism in prevalence, age of onset or severity. This multidisciplinary project directly addresses some fundamental unresolved questions in evolutionary biology: the genetic basis and maintenance of fitness variation; the evolution of sexual dimorphism; and aims to provide novel insights into the genetic basis of some common human diseases.
Project acronym 3CBIOTECH
Project Cold Carbon Catabolism of Microbial Communities underprinning a Sustainable Bioenergy and Biorefinery Economy
Researcher (PI) Gavin James Collins
Host Institution (HI) NATIONAL UNIVERSITY OF IRELAND GALWAY
Summary The applicant will collaborate with Irish, European and U.S.-based colleagues to develop a sustainable biorefinery and bioenergy industry in Ireland and Europe. The focus of this ERC Starting Grant will be the application of classical microbiological, physiological and real-time polymerase chain reaction (PCR)-based assays, to qualitatively and quantitatively characterize microbial communities underpinning novel and innovative, low-temperature, anaerobic waste (and other biomass) conversion technologies, including municipal wastewater treatment and, demonstration- and full-scale biorefinery applications. Anaerobic digestion (AD) is a naturally-occurring process, which is widely applied for the conversion of waste to methane-containing biogas. Low-temperature (<20 degrees C) AD has been applied by the applicant as a cost-effective alternative to mesophilic (c. 35C) AD for the treatment of several waste categories. However, the microbiology of low-temperature AD is poorly understood. The applicant will work with microbial consortia isolated from anaerobic bioreactors, which have been operated for long-term experiments (>3.5 years), and include organic acid-oxidizing, hydrogen-producing syntrophic microbes and hydrogen-consuming methanogens. A major focus of the project will be the ecophysiology of psychrotolerant and psychrophilic methanogens already identified and cultivated by the applicant. The project will also investigate the role(s) of poorly-understood Crenarchaeota populations and homoacetogenic bacteria, in complex consortia. The host organization is a leading player in the microbiology of waste-to-energy applications. The applicant will train a team of scientists in all aspects of the microbiology and bioengineering of biomass conversion systems.
The applicant will collaborate with Irish, European and U.S.-based colleagues to develop a sustainable biorefinery and bioenergy industry in Ireland and Europe. The focus of this ERC Starting Grant will be the application of classical microbiological, physiological and real-time polymerase chain reaction (PCR)-based assays, to qualitatively and quantitatively characterize microbial communities underpinning novel and innovative, low-temperature, anaerobic waste (and other biomass) conversion technologies, including municipal wastewater treatment and, demonstration- and full-scale biorefinery applications. Anaerobic digestion (AD) is a naturally-occurring process, which is widely applied for the conversion of waste to methane-containing biogas. Low-temperature (<20 degrees C) AD has been applied by the applicant as a cost-effective alternative to mesophilic (c. 35C) AD for the treatment of several waste categories. However, the microbiology of low-temperature AD is poorly understood. The applicant will work with microbial consortia isolated from anaerobic bioreactors, which have been operated for long-term experiments (>3.5 years), and include organic acid-oxidizing, hydrogen-producing syntrophic microbes and hydrogen-consuming methanogens. A major focus of the project will be the ecophysiology of psychrotolerant and psychrophilic methanogens already identified and cultivated by the applicant. The project will also investigate the role(s) of poorly-understood Crenarchaeota populations and homoacetogenic bacteria, in complex consortia. The host organization is a leading player in the microbiology of waste-to-energy applications. The applicant will train a team of scientists in all aspects of the microbiology and bioengineering of biomass conversion systems.
Project acronym 3D-OA-HISTO
Project Development of 3D Histopathological Grading of Osteoarthritis
Researcher (PI) Simo Jaakko Saarakkala
Host Institution (HI) OULUN YLIOPISTO
Summary "Background: Osteoarthritis (OA) is a common musculoskeletal disease occurring worldwide. Despite extensive research, etiology of OA is still poorly understood. Histopathological grading (HPG) of 2D tissue sections is the gold standard reference method for determination of OA stage. However, traditional 2D-HPG is destructive and based only on subjective visual evaluation. These limitations induce bias to clinical in vitro OA diagnostics and basic research that both rely strongly on HPG. Objectives: 1) To establish and validate the very first 3D-HPG of OA based on cutting-edge nano/micro-CT (Computed Tomography) technologies in vitro; 2) To use the established method to clarify the beginning phases of OA; and 3) To validate 3D-HPG of OA for in vivo use. Methods: Several hundreds of human osteochondral samples from patients undergoing total knee arthroplasty will be collected. The samples will be imaged in vitro with nano/micro-CT and clinical high-end extremity CT devices using specific contrast-agents to quantify tissue constituents and structure in 3D in large volume. From this information, a novel 3D-HPG is developed with statistical classification algorithms. Finally, the developed novel 3D-HPG of OA will be applied clinically in vivo. Significance: This is the very first study to establish 3D-HPG of OA pathology in vitro and in vivo. Furthermore, the developed technique hugely improves the understanding of the beginning phases of OA. Ultimately, the study will contribute for improving OA patients' quality of life by slowing the disease progression, and for providing powerful tools to develop new OA therapies."
"Background: Osteoarthritis (OA) is a common musculoskeletal disease occurring worldwide. Despite extensive research, etiology of OA is still poorly understood. Histopathological grading (HPG) of 2D tissue sections is the gold standard reference method for determination of OA stage. However, traditional 2D-HPG is destructive and based only on subjective visual evaluation. These limitations induce bias to clinical in vitro OA diagnostics and basic research that both rely strongly on HPG. Objectives: 1) To establish and validate the very first 3D-HPG of OA based on cutting-edge nano/micro-CT (Computed Tomography) technologies in vitro; 2) To use the established method to clarify the beginning phases of OA; and 3) To validate 3D-HPG of OA for in vivo use. Methods: Several hundreds of human osteochondral samples from patients undergoing total knee arthroplasty will be collected. The samples will be imaged in vitro with nano/micro-CT and clinical high-end extremity CT devices using specific contrast-agents to quantify tissue constituents and structure in 3D in large volume. From this information, a novel 3D-HPG is developed with statistical classification algorithms. Finally, the developed novel 3D-HPG of OA will be applied clinically in vivo. Significance: This is the very first study to establish 3D-HPG of OA pathology in vitro and in vivo. Furthermore, the developed technique hugely improves the understanding of the beginning phases of OA. Ultimately, the study will contribute for improving OA patients' quality of life by slowing the disease progression, and for providing powerful tools to develop new OA therapies."
Project acronym 3D-REPAIR
Project Spatial organization of DNA repair within the nucleus
Researcher (PI) Evanthia Soutoglou
Host Institution (HI) CENTRE EUROPEEN DE RECHERCHE EN BIOLOGIE ET MEDECINE
Summary Faithful repair of double stranded DNA breaks (DSBs) is essential, as they are at the origin of genome instability, chromosomal translocations and cancer. Cells repair DSBs through different pathways, which can be faithful or mutagenic, and the balance between them at a given locus must be tightly regulated to preserve genome integrity. Although, much is known about DSB repair factors, how the choice between pathways is controlled within the nuclear environment is not understood. We have shown that nuclear architecture and non-random genome organization determine the frequency of chromosomal translocations and that pathway choice is dictated by the spatial organization of DNA in the nucleus. Nevertheless, what determines which pathway is activated in response to DSBs at specific genomic locations is not understood. Furthermore, the impact of 3D-genome folding on the kinetics and efficiency of DSB repair is completely unknown. Here we aim to understand how nuclear compartmentalization, chromatin structure and genome organization impact on the efficiency of detection, signaling and repair of DSBs. We will unravel what determines the DNA repair specificity within distinct nuclear compartments using protein tethering, promiscuous biotinylation and quantitative proteomics. We will determine how DNA repair is orchestrated at different heterochromatin structures using a CRISPR/Cas9-based system that allows, for the first time robust induction of DSBs at specific heterochromatin compartments. Finally, we will investigate the role of 3D-genome folding in the kinetics of DNA repair and pathway choice using single nucleotide resolution DSB-mapping coupled to 3D-topological maps. This proposal has significant implications for understanding the mechanisms controlling DNA repair within the nuclear environment and will reveal the regions of the genome that are susceptible to genomic instability and help us understand why certain mutations and translocations are recurrent in cancer
Faithful repair of double stranded DNA breaks (DSBs) is essential, as they are at the origin of genome instability, chromosomal translocations and cancer. Cells repair DSBs through different pathways, which can be faithful or mutagenic, and the balance between them at a given locus must be tightly regulated to preserve genome integrity. Although, much is known about DSB repair factors, how the choice between pathways is controlled within the nuclear environment is not understood. We have shown that nuclear architecture and non-random genome organization determine the frequency of chromosomal translocations and that pathway choice is dictated by the spatial organization of DNA in the nucleus. Nevertheless, what determines which pathway is activated in response to DSBs at specific genomic locations is not understood. Furthermore, the impact of 3D-genome folding on the kinetics and efficiency of DSB repair is completely unknown. Here we aim to understand how nuclear compartmentalization, chromatin structure and genome organization impact on the efficiency of detection, signaling and repair of DSBs. We will unravel what determines the DNA repair specificity within distinct nuclear compartments using protein tethering, promiscuous biotinylation and quantitative proteomics. We will determine how DNA repair is orchestrated at different heterochromatin structures using a CRISPR/Cas9-based system that allows, for the first time robust induction of DSBs at specific heterochromatin compartments. Finally, we will investigate the role of 3D-genome folding in the kinetics of DNA repair and pathway choice using single nucleotide resolution DSB-mapping coupled to 3D-topological maps. This proposal has significant implications for understanding the mechanisms controlling DNA repair within the nuclear environment and will reveal the regions of the genome that are susceptible to genomic instability and help us understand why certain mutations and translocations are recurrent in cancer
Project acronym 3D_Tryps
Project The role of three-dimensional genome architecture in antigenic variation
Researcher (PI) Tim Nicolai SIEGEL
Summary Antigenic variation is a widely employed strategy to evade the host immune response. It has similar functional requirements even in evolutionarily divergent pathogens. These include the mutually exclusive expression of antigens and the periodic, nonrandom switching in the expression of different antigens during the course of an infection. Despite decades of research the mechanisms of antigenic variation are not fully understood in any organism. The recent development of high-throughput sequencing-based assays to probe the 3D genome architecture (Hi-C) has revealed the importance of the spatial organization of DNA inside the nucleus. 3D genome architecture plays a critical role in the regulation of mutually exclusive gene expression and the frequency of translocation between different genomic loci in many eukaryotes. Thus, genome architecture may also be a key regulator of antigenic variation, yet the causal links between genome architecture and the expression of antigens have not been studied systematically. In addition, the development of CRISPR-Cas9-based approaches to perform nucleotide-specific genome editing has opened unprecedented opportunities to study the influence of DNA sequence elements on the spatial organization of DNA and how this impacts antigen expression. I have adapted both Hi-C and CRISPR-Cas9 technology to the protozoan parasite Trypanosoma brucei, one of the most important model organisms to study antigenic variation. These techniques will enable me to bridge the field of antigenic variation research with that of genome architecture. I will perform the first systematic analysis of the role of genome architecture in the mutually exclusive and hierarchical expression of antigens in any pathogen. The experiments outlined in this proposal will provide new insight, facilitating a new view of antigenic variation and may eventually help medical intervention in T. brucei and in other pathogens relying on antigenic variation for their survival.
Antigenic variation is a widely employed strategy to evade the host immune response. It has similar functional requirements even in evolutionarily divergent pathogens. These include the mutually exclusive expression of antigens and the periodic, nonrandom switching in the expression of different antigens during the course of an infection. Despite decades of research the mechanisms of antigenic variation are not fully understood in any organism. The recent development of high-throughput sequencing-based assays to probe the 3D genome architecture (Hi-C) has revealed the importance of the spatial organization of DNA inside the nucleus. 3D genome architecture plays a critical role in the regulation of mutually exclusive gene expression and the frequency of translocation between different genomic loci in many eukaryotes. Thus, genome architecture may also be a key regulator of antigenic variation, yet the causal links between genome architecture and the expression of antigens have not been studied systematically. In addition, the development of CRISPR-Cas9-based approaches to perform nucleotide-specific genome editing has opened unprecedented opportunities to study the influence of DNA sequence elements on the spatial organization of DNA and how this impacts antigen expression. I have adapted both Hi-C and CRISPR-Cas9 technology to the protozoan parasite Trypanosoma brucei, one of the most important model organisms to study antigenic variation. These techniques will enable me to bridge the field of antigenic variation research with that of genome architecture. I will perform the first systematic analysis of the role of genome architecture in the mutually exclusive and hierarchical expression of antigens in any pathogen. The experiments outlined in this proposal will provide new insight, facilitating a new view of antigenic variation and may eventually help medical intervention in T. brucei and in other pathogens relying on antigenic variation for their survival.
Project acronym 3DBIOLUNG
Project Bioengineering lung tissue using extracellular matrix based 3D bioprinting
Researcher (PI) Darcy WAGNER
Summary Chronic lung diseases are increasing in prevalence with over 65 million patients worldwide. Lung transplantation remains the only potential option at end-stage disease. Around 4000 patients receive lung transplants annually with more awaiting transplantation, including 1000 patients in Europe. New options to increase available tissue for lung transplantation are desperately needed. An exciting new research area focuses on generating lung tissue ex vivo using bioengineering approaches. Scaffolds can be generated from synthetic or biologically-derived (acellular) materials, seeded with cells and grown in a bioreactor prior to transplantation. Ideally, scaffolds would be seeded with cells derived from the transplant recipient, thus obviating the need for long-term immunosuppression. However, functional regeneration has yet to be achieved. New advances in 3D printing and 3D bioprinting (when cells are printed) indicate that this once thought of science-fiction concept might finally be mature enough for complex tissues, including lung. 3D bioprinting addresses a number of concerns identified in previous approaches, such as a) patient heterogeneity in acellular human scaffolds, b) anatomical differences in xenogeneic sources, c) lack of biological cues on synthetic materials and d) difficulty in manufacturing the complex lung architecture. 3D bioprinting could be a reproducible, scalable, and controllable approach for generating functional lung tissue. The aim of this proposal is to use custom 3D bioprinters to generate constructs mimicking lung tissue using an innovative approach combining primary cells, the engineering reproducibility of synthetic materials, and the biologically conductive properties of acellular lung (hybrid). We will 3D bioprint hybrid murine and human lung tissue models and test gas exchange, angiogenesis and in vivo immune responses. This proposal will be a critical first step in demonstrating feasibility of 3D bioprinting lung tissue.
Chronic lung diseases are increasing in prevalence with over 65 million patients worldwide. Lung transplantation remains the only potential option at end-stage disease. Around 4000 patients receive lung transplants annually with more awaiting transplantation, including 1000 patients in Europe. New options to increase available tissue for lung transplantation are desperately needed. An exciting new research area focuses on generating lung tissue ex vivo using bioengineering approaches. Scaffolds can be generated from synthetic or biologically-derived (acellular) materials, seeded with cells and grown in a bioreactor prior to transplantation. Ideally, scaffolds would be seeded with cells derived from the transplant recipient, thus obviating the need for long-term immunosuppression. However, functional regeneration has yet to be achieved. New advances in 3D printing and 3D bioprinting (when cells are printed) indicate that this once thought of science-fiction concept might finally be mature enough for complex tissues, including lung. 3D bioprinting addresses a number of concerns identified in previous approaches, such as a) patient heterogeneity in acellular human scaffolds, b) anatomical differences in xenogeneic sources, c) lack of biological cues on synthetic materials and d) difficulty in manufacturing the complex lung architecture. 3D bioprinting could be a reproducible, scalable, and controllable approach for generating functional lung tissue. The aim of this proposal is to use custom 3D bioprinters to generate constructs mimicking lung tissue using an innovative approach combining primary cells, the engineering reproducibility of synthetic materials, and the biologically conductive properties of acellular lung (hybrid). We will 3D bioprint hybrid murine and human lung tissue models and test gas exchange, angiogenesis and in vivo immune responses. This proposal will be a critical first step in demonstrating feasibility of 3D bioprinting lung tissue.
Project acronym 3DBrainStrom
Project Brain metastases: Deciphering tumor-stroma interactions in three dimensions for the rational design of nanomedicines
Researcher (PI) Ronit Satchi Fainaro
Summary Brain metastases represent a major therapeutic challenge. Despite significant breakthroughs in targeted therapies, survival rates of patients with brain metastases remain poor. Nowadays, discovery, development and evaluation of new therapies are performed on human cancer cells grown in 2D on rigid plastic plates followed by in vivo testing in immunodeficient mice. These experimental settings are lacking and constitute a fundamental hurdle for the translation of preclinical discoveries into clinical practice. We propose to establish 3D-printed models of brain metastases (Aim 1), which include brain extracellular matrix, stroma and serum containing immune cells flowing in functional tumor vessels. Our unique models better capture the clinical physio-mechanical tissue properties, signaling pathways, hemodynamics and drug responsiveness. Using our 3D-printed models, we aim to develop two new fronts for identifying novel clinically-relevant molecular drivers (Aim 2) followed by the development of precision nanomedicines (Aim 3). We will exploit our vast experience in anticancer nanomedicines to design three therapeutic approaches that target various cellular compartments involved in brain metastases: 1) Prevention of brain metastatic colonization using targeted nano-vaccines, which elicit antitumor immune response; 2) Intervention of tumor-brain stroma cells crosstalk when brain micrometastases establish; 3) Regression of macrometastatic disease by selectively targeting tumor cells. These approaches will materialize using our libraries of polymeric nanocarriers that selectively accumulate in tumors. This project will result in a paradigm shift by generating new preclinical cancer models that will bridge the translational gap in cancer therapeutics. The insights and tumor-stroma-targeted nanomedicines developed here will pave the way for prediction of patient outcome, revolutionizing our perception of tumor modelling and consequently the way we prevent and treat cancer.
Brain metastases represent a major therapeutic challenge. Despite significant breakthroughs in targeted therapies, survival rates of patients with brain metastases remain poor. Nowadays, discovery, development and evaluation of new therapies are performed on human cancer cells grown in 2D on rigid plastic plates followed by in vivo testing in immunodeficient mice. These experimental settings are lacking and constitute a fundamental hurdle for the translation of preclinical discoveries into clinical practice. We propose to establish 3D-printed models of brain metastases (Aim 1), which include brain extracellular matrix, stroma and serum containing immune cells flowing in functional tumor vessels. Our unique models better capture the clinical physio-mechanical tissue properties, signaling pathways, hemodynamics and drug responsiveness. Using our 3D-printed models, we aim to develop two new fronts for identifying novel clinically-relevant molecular drivers (Aim 2) followed by the development of precision nanomedicines (Aim 3). We will exploit our vast experience in anticancer nanomedicines to design three therapeutic approaches that target various cellular compartments involved in brain metastases: 1) Prevention of brain metastatic colonization using targeted nano-vaccines, which elicit antitumor immune response; 2) Intervention of tumor-brain stroma cells crosstalk when brain micrometastases establish; 3) Regression of macrometastatic disease by selectively targeting tumor cells. These approaches will materialize using our libraries of polymeric nanocarriers that selectively accumulate in tumors. This project will result in a paradigm shift by generating new preclinical cancer models that will bridge the translational gap in cancer therapeutics. The insights and tumor-stroma-targeted nanomedicines developed here will pave the way for prediction of patient outcome, revolutionizing our perception of tumor modelling and consequently the way we prevent and treat cancer.
Project acronym 3DCellPhase-
Project In situ Structural Analysis of Molecular Crowding and Phase Separation
Researcher (PI) Julia MAHAMID
Host Institution (HI) EUROPEAN MOLECULAR BIOLOGY LABORATORY
Summary This proposal brings together two fields in biology, namely the emerging field of phase-separated assemblies in cell biology and state-of-the-art cellular cryo-electron tomography, to advance our understanding on a fundamental, yet illusive, question: the molecular organization of the cytoplasm. Eukaryotes organize their biochemical reactions into functionally distinct compartments. Intriguingly, many, if not most, cellular compartments are not membrane enclosed. Rather, they assemble dynamically by phase separation, typically triggered upon a specific event. Despite significant progress on reconstituting such liquid-like assemblies in vitro, we lack information as to whether these compartments in vivo are indeed amorphous liquids, or whether they exhibit structural features such as gels or fibers. My recent work on sample preparation of cells for cryo-electron tomography, including cryo-focused ion beam thinning, guided by 3D correlative fluorescence microscopy, shows that we can now prepare site-specific 'electron-transparent windows' in suitable eukaryotic systems, which allow direct examination of structural features of cellular compartments in their cellular context. Here, we will use these techniques to elucidate the structural principles and cytoplasmic environment driving the dynamic assembly of two phase-separated compartments: Stress granules, which are RNA bodies that form rapidly in the cytoplasm upon cellular stress, and centrosomes, which are sites of microtubule nucleation. We will combine these studies with a quantitative description of the crowded nature of cytoplasm and of its local variations, to provide a direct readout of the impact of excluded volume on molecular assembly in living cells. Taken together, these studies will provide fundamental insights into the structural basis by which cells form biochemical compartments.
This proposal brings together two fields in biology, namely the emerging field of phase-separated assemblies in cell biology and state-of-the-art cellular cryo-electron tomography, to advance our understanding on a fundamental, yet illusive, question: the molecular organization of the cytoplasm. Eukaryotes organize their biochemical reactions into functionally distinct compartments. Intriguingly, many, if not most, cellular compartments are not membrane enclosed. Rather, they assemble dynamically by phase separation, typically triggered upon a specific event. Despite significant progress on reconstituting such liquid-like assemblies in vitro, we lack information as to whether these compartments in vivo are indeed amorphous liquids, or whether they exhibit structural features such as gels or fibers. My recent work on sample preparation of cells for cryo-electron tomography, including cryo-focused ion beam thinning, guided by 3D correlative fluorescence microscopy, shows that we can now prepare site-specific 'electron-transparent windows' in suitable eukaryotic systems, which allow direct examination of structural features of cellular compartments in their cellular context. Here, we will use these techniques to elucidate the structural principles and cytoplasmic environment driving the dynamic assembly of two phase-separated compartments: Stress granules, which are RNA bodies that form rapidly in the cytoplasm upon cellular stress, and centrosomes, which are sites of microtubule nucleation. We will combine these studies with a quantitative description of the crowded nature of cytoplasm and of its local variations, to provide a direct readout of the impact of excluded volume on molecular assembly in living cells. Taken together, these studies will provide fundamental insights into the structural basis by which cells form biochemical compartments.
Project acronym 3DEpi
Project Transgenerational epigenetic inheritance of chromatin states : the role of Polycomb and 3D chromosome architecture
Researcher (PI) Giacomo CAVALLI
Summary Epigenetic inheritance entails transmission of phenotypic traits not encoded in the DNA sequence and, in the most extreme case, Transgenerational Epigenetic Inheritance (TEI) involves transmission of memory through multiple generations. Very little is known on the mechanisms governing TEI and this is the subject of the present proposal. By transiently enhancing long-range chromatin interactions, we recently established isogenic Drosophila epilines that carry stable alternative epialleles, defined by differential levels of the Polycomb-dependent H3K27me3 mark. Furthermore, we extended our paradigm to natural phenotypes. These are ideal systems to study the role of Polycomb group (PcG) proteins and other components in regulating nuclear organization and epigenetic inheritance of chromatin states. The present project conjugates genetics, epigenomics, imaging and molecular biology to reach three critical aims. Aim 1: Analysis of the molecular mechanisms regulating Polycomb-mediated TEI. We will identify the DNA, protein and RNA components that trigger and maintain transgenerational chromatin inheritance as well as their mechanisms of action. Aim 2: Role of 3D genome organization in the regulation of TEI. We will analyze the developmental dynamics of TEI-inducing long-range chromatin interactions, identify chromatin components mediating 3D chromatin contacts and characterize their function in the TEI process. Aim 3: Identification of a broader role of TEI during development. TEI might reflect a normal role of PcG components in the transmission of parental chromatin onto the next embryonic generation. We will explore this possibility by establishing other TEI paradigms and by relating TEI to the normal PcG function in these systems and in normal development. This research program will unravel the biological significance and the molecular underpinnings of TEI and lead the way towards establishing this area of research into a consolidated scientific discipline.
Epigenetic inheritance entails transmission of phenotypic traits not encoded in the DNA sequence and, in the most extreme case, Transgenerational Epigenetic Inheritance (TEI) involves transmission of memory through multiple generations. Very little is known on the mechanisms governing TEI and this is the subject of the present proposal. By transiently enhancing long-range chromatin interactions, we recently established isogenic Drosophila epilines that carry stable alternative epialleles, defined by differential levels of the Polycomb-dependent H3K27me3 mark. Furthermore, we extended our paradigm to natural phenotypes. These are ideal systems to study the role of Polycomb group (PcG) proteins and other components in regulating nuclear organization and epigenetic inheritance of chromatin states. The present project conjugates genetics, epigenomics, imaging and molecular biology to reach three critical aims. Aim 1: Analysis of the molecular mechanisms regulating Polycomb-mediated TEI. We will identify the DNA, protein and RNA components that trigger and maintain transgenerational chromatin inheritance as well as their mechanisms of action. Aim 2: Role of 3D genome organization in the regulation of TEI. We will analyze the developmental dynamics of TEI-inducing long-range chromatin interactions, identify chromatin components mediating 3D chromatin contacts and characterize their function in the TEI process. Aim 3: Identification of a broader role of TEI during development. TEI might reflect a normal role of PcG components in the transmission of parental chromatin onto the next embryonic generation. We will explore this possibility by establishing other TEI paradigms and by relating TEI to the normal PcG function in these systems and in normal development. This research program will unravel the biological significance and the molecular underpinnings of TEI and lead the way towards establishing this area of research into a consolidated scientific discipline.
Project acronym 3DMOSHBOND
Project Three-Dimensional Mapping Of a Single Hydrogen Bond
Researcher (PI) Adam Marc SWEETMAN
Host Institution (HI) UNIVERSITY OF LEEDS
Summary All properties of matter are ultimately governed by the forces between single atoms, but our knowledge of interatomic, and intermolecular, potentials is often derived indirectly. In 3DMOSHBOND, I outline a program of work designed to create a paradigm shift in the direct measurement of complex interatomic potentials via a fundamental reimagining of how atomic resolution imaging, and force measurement, techniques are applied. To provide a clear proof of principle demonstration of the power of this concept, I propose to map the strength, shape and extent of single hydrogen bonding (H-bonding) interactions in 3D with sub-Angstrom precision. H-bonding is a key component governing intermolecular interactions, particularly for biologically important molecules. Despite its critical importance, H-bonding is relatively poorly understood, and the IUPAC definition of the H-bond was changed as recently as 2011- highlighting the relevance of a new means to engage with these fundamental interactions. Hitherto unprecedented resolution and accuracy will be achieved via a creation of a novel layer of vertically oriented H-bonding molecules, functionalisation of the tip of a scanning probe microscope with a single complementary H-bonding molecule, and by complete characterisation of the position of all atoms in the junction. This will place two H-bonding groups "end on" and map the extent, and magnitude, of the H-bond with sub-Angstrom precision for a variety of systems. This investigation of the H-bond will present us with an unparalleled level of information regarding its properties. Experimental results will be compared with ab initio density functional theory (DFT) simulations, to investigate the extent to which state-of-the-art simulations are able to reproduce the behaviour of the H-bonding interaction. The project will create a new generalised probe for the study of single atomic and molecular interactions.
All properties of matter are ultimately governed by the forces between single atoms, but our knowledge of interatomic, and intermolecular, potentials is often derived indirectly. In 3DMOSHBOND, I outline a program of work designed to create a paradigm shift in the direct measurement of complex interatomic potentials via a fundamental reimagining of how atomic resolution imaging, and force measurement, techniques are applied. To provide a clear proof of principle demonstration of the power of this concept, I propose to map the strength, shape and extent of single hydrogen bonding (H-bonding) interactions in 3D with sub-Angstrom precision. H-bonding is a key component governing intermolecular interactions, particularly for biologically important molecules. Despite its critical importance, H-bonding is relatively poorly understood, and the IUPAC definition of the H-bond was changed as recently as 2011- highlighting the relevance of a new means to engage with these fundamental interactions. Hitherto unprecedented resolution and accuracy will be achieved via a creation of a novel layer of vertically oriented H-bonding molecules, functionalisation of the tip of a scanning probe microscope with a single complementary H-bonding molecule, and by complete characterisation of the position of all atoms in the junction. This will place two H-bonding groups "end on" and map the extent, and magnitude, of the H-bond with sub-Angstrom precision for a variety of systems. This investigation of the H-bond will present us with an unparalleled level of information regarding its properties. Experimental results will be compared with ab initio density functional theory (DFT) simulations, to investigate the extent to which state-of-the-art simulations are able to reproduce the behaviour of the H-bonding interaction. The project will create a new generalised probe for the study of single atomic and molecular interactions.
Project acronym 3DPROTEINPUZZLES
Project Shape-directed protein assembly design
Researcher (PI) Lars Ingemar ANDRÉ
Summary Large protein complexes carry out some of the most complex functions in biology. Such structures are often assembled spontaneously from individual components through the process of self-assembly. If self-assembled protein complexes could be engineered from first principle it would enable a wide range of applications in biomedicine, nanotechnology and materials science. Recently, approaches to rationally design proteins to self-assembly into predefined structures have emerged. The highlight of this work is the design of protein cages that may be engineered into protein containers. However, current approaches for self-assembly design does not result in the assemblies with the required structural complexity to encode many of the sophisticated functions found in nature. To move forward, we have to learn how to engineer protein subunits with more than one designed interface that can assemble into tightly interacting complexes. In this proposal we propose a new protein design paradigm, shape directed protein design, in order to address shortcomings of the current methodology. The proposed method combines geometric shape matching and computational protein design. Using this approach we will de novo design assemblies with a wide variety of structural states, including protein complexes with cyclic and dihedral symmetry as well as icosahedral protein capsids built from novel protein building blocks. To enable these two design challenges we also develop a high-throughput assay to measure assembly stability in vivo that builds on a three-color fluorescent assay. This method will not only facilitate the screening of orders of magnitude more design constructs, but also enable the application of directed evolution to experimentally improve stable and assembly properties of designed containers as well as other designed assemblies.
Large protein complexes carry out some of the most complex functions in biology. Such structures are often assembled spontaneously from individual components through the process of self-assembly. If self-assembled protein complexes could be engineered from first principle it would enable a wide range of applications in biomedicine, nanotechnology and materials science. Recently, approaches to rationally design proteins to self-assembly into predefined structures have emerged. The highlight of this work is the design of protein cages that may be engineered into protein containers. However, current approaches for self-assembly design does not result in the assemblies with the required structural complexity to encode many of the sophisticated functions found in nature. To move forward, we have to learn how to engineer protein subunits with more than one designed interface that can assemble into tightly interacting complexes. In this proposal we propose a new protein design paradigm, shape directed protein design, in order to address shortcomings of the current methodology. The proposed method combines geometric shape matching and computational protein design. Using this approach we will de novo design assemblies with a wide variety of structural states, including protein complexes with cyclic and dihedral symmetry as well as icosahedral protein capsids built from novel protein building blocks. To enable these two design challenges we also develop a high-throughput assay to measure assembly stability in vivo that builds on a three-color fluorescent assay. This method will not only facilitate the screening of orders of magnitude more design constructs, but also enable the application of directed evolution to experimentally improve stable and assembly properties of designed containers as well as other designed assemblies.
Project acronym 3Ps
Project 3Ps Plastic-Antibodies, Plasmonics and Photovoltaic-Cells: on-site screening of cancer biomarkers made possible
Researcher (PI) Maria Goreti Ferreira Sales
Host Institution (HI) INSTITUTO SUPERIOR DE ENGENHARIA DO PORTO
Summary This project presents a new concept for the detection, diagnosis and monitoring of cancer biomarker patterns in point-of-care. The device under development will make use of the selectivity of the plastic antibodies as sensing materials and the interference they will play on the normal operation of a photovoltaic cell. Plastic antibodies will be designed by surface imprinting procedures. Self-assembled monolayer and molecular imprinting techniques will be merged in this process because they allow the self-assembly of nanostructured materials on a "bottom-up" nanofabrication approach. A dye-sensitized solar cell will be used as photovoltaic cell. It includes a liquid interface in the cell circuit, which allows the introduction of the sample (also in liquid phase) without disturbing the normal cell operation. Furthermore, it works well with rather low cost materials and requires mild and easy processing conditions. The cell will be equipped with plasmonic structures to enhance light absorption and cell efficiency. The device under development will be easily operated by any clinician or patient. It will require ambient light and a regular multimeter. Eye detection will be also tried out.
This project presents a new concept for the detection, diagnosis and monitoring of cancer biomarker patterns in point-of-care. The device under development will make use of the selectivity of the plastic antibodies as sensing materials and the interference they will play on the normal operation of a photovoltaic cell. Plastic antibodies will be designed by surface imprinting procedures. Self-assembled monolayer and molecular imprinting techniques will be merged in this process because they allow the self-assembly of nanostructured materials on a "bottom-up" nanofabrication approach. A dye-sensitized solar cell will be used as photovoltaic cell. It includes a liquid interface in the cell circuit, which allows the introduction of the sample (also in liquid phase) without disturbing the normal cell operation. Furthermore, it works well with rather low cost materials and requires mild and easy processing conditions. The cell will be equipped with plasmonic structures to enhance light absorption and cell efficiency. The device under development will be easily operated by any clinician or patient. It will require ambient light and a regular multimeter. Eye detection will be also tried out.
Project acronym 3S-BTMUC
Project Soft, Slimy, Sliding Interfaces: Biotribological Properties of Mucins and Mucus gels
Researcher (PI) Seunghwan Lee
Summary Mucins are a family of high-molecular-weight glycoproteins and a major macromolecular constituent in slimy mucus gels that are covering the surface of internal biological tissues. A primary role of mucus gels in biological systems is known to be the protection and lubrication of underlying epithelial cell surfaces. This is intuitively well appreciated by both science community and the public, and yet detailed lubrication properties of mucins and mucus gels have remained largely unexplored to date. Detailed and systematic understanding of the lubrication mechanism of mucus gels is significant from many angles; firstly, lubricity of mucus gels is closely related with fundamental functions of various human organs, such as eye blinking, mastication in oral cavity, swallowing through esophagus, digestion in stomach, breathing through air way and respiratory organs, and thus often indicates the health state of those organs. Furthermore, for the application of various tissue-contacting devices or personal care products, e.g. catheters, endoscopes, and contact lenses, mucus gel layer is the first counter surface that comes into the mechanical and tribological contacts with them. Finally, remarkable lubricating performance by mucins and mucus gels in biological systems may provide many useful and possibly innovative hints in utilizing water as base lubricant for man-made engineering systems. This project thus proposes to carry out a 5 year research program focusing on exploring the lubricity of mucins and mucus gels by combining a broad range of experimental approaches in biology and tribology.
Mucins are a family of high-molecular-weight glycoproteins and a major macromolecular constituent in slimy mucus gels that are covering the surface of internal biological tissues. A primary role of mucus gels in biological systems is known to be the protection and lubrication of underlying epithelial cell surfaces. This is intuitively well appreciated by both science community and the public, and yet detailed lubrication properties of mucins and mucus gels have remained largely unexplored to date. Detailed and systematic understanding of the lubrication mechanism of mucus gels is significant from many angles; firstly, lubricity of mucus gels is closely related with fundamental functions of various human organs, such as eye blinking, mastication in oral cavity, swallowing through esophagus, digestion in stomach, breathing through air way and respiratory organs, and thus often indicates the health state of those organs. Furthermore, for the application of various tissue-contacting devices or personal care products, e.g. catheters, endoscopes, and contact lenses, mucus gel layer is the first counter surface that comes into the mechanical and tribological contacts with them. Finally, remarkable lubricating performance by mucins and mucus gels in biological systems may provide many useful and possibly innovative hints in utilizing water as base lubricant for man-made engineering systems. This project thus proposes to carry out a 5 year research program focusing on exploring the lubricity of mucins and mucus gels by combining a broad range of experimental approaches in biology and tribology.
Project acronym 3SPIN
Project Three Dimensional Spintronics
Researcher (PI) Russell Paul Cowburn
Summary Spintronics, in which both the spin and the charge of the electron are used, is one of the most exciting new disciplines to emerge from nanoscience. The 3SPIN project seeks to open a new research front within spintronics: namely 3-dimensional spintronics, in which magnetic nanostructures are formed into a 3-dimensional interacting network of unrivalled density and hence technological benefit. 3SPIN will explore early-stage science that could underpin 3-dimensional metallic spintronics. The thesis of the project is: that by careful control of the constituent nanostructure properties, a 3-dimensional medium can be created in which a large number of topological solitons can exist. Although hardly studied at all to date, these solitons should be stable at room temperature, extremely compact and easy to manipulate and propagate. This makes them potentially ideal candidates to form the basis of a new spintronics in which the soliton is the basic transport vector instead of electrical current. ¬3.5M of funding is requested to form a new team of 5 researchers who, over a period of 60 months, will perform computer simulations and experimental studies of solitons in 3-dimensional networks of magnetic nanostructures and develop a laboratory demonstrator 3-dimensional memory device using solitons to represent and store data. A high performance electron beam lithography system (cost 1M¬) will be purchased to allow state-of-the-art magnetic nanostructures to be fabricated with perfect control over their magnetic properties, thus allowing the ideal conditions for solitons to be created and controllably manipulated. Outputs from the project will be a complete understanding of the properties of these new objects and a road map charting the next steps for research in the field.
Spintronics, in which both the spin and the charge of the electron are used, is one of the most exciting new disciplines to emerge from nanoscience. The 3SPIN project seeks to open a new research front within spintronics: namely 3-dimensional spintronics, in which magnetic nanostructures are formed into a 3-dimensional interacting network of unrivalled density and hence technological benefit. 3SPIN will explore early-stage science that could underpin 3-dimensional metallic spintronics. The thesis of the project is: that by careful control of the constituent nanostructure properties, a 3-dimensional medium can be created in which a large number of topological solitons can exist. Although hardly studied at all to date, these solitons should be stable at room temperature, extremely compact and easy to manipulate and propagate. This makes them potentially ideal candidates to form the basis of a new spintronics in which the soliton is the basic transport vector instead of electrical current. ¬3.5M of funding is requested to form a new team of 5 researchers who, over a period of 60 months, will perform computer simulations and experimental studies of solitons in 3-dimensional networks of magnetic nanostructures and develop a laboratory demonstrator 3-dimensional memory device using solitons to represent and store data. A high performance electron beam lithography system (cost 1M¬) will be purchased to allow state-of-the-art magnetic nanostructures to be fabricated with perfect control over their magnetic properties, thus allowing the ideal conditions for solitons to be created and controllably manipulated. Outputs from the project will be a complete understanding of the properties of these new objects and a road map charting the next steps for research in the field.
Project acronym 4-TOPS
Project Four experiments in Topological Superconductivity.
Researcher (PI) Laurens Molenkamp
Summary Topological materials have developed rapidly in recent years, with my previous ERC-AG project 3-TOP playing a major role in this development. While so far no bulk topological superconductor has been unambiguously demonstrated, their properties can be studied in a very flexible manner by inducing superconductivity through the proximity effect into the surface or edge states of a topological insulator. In 4-TOPS we will explore the possibilities of this approach in full, and conduct a thorough study of induced superconductivity in both two and three dimensional HgTe based topological insulators. The 4 avenues we will follow are: -SQUID based devices to investigate full phase dependent spectroscopy of the gapless Andreev bound state by studying their Josephson radiation and current-phase relationships. -Experiments aimed at providing unambiguous proof of localized Majorana states in TI junctions by studying tunnelling transport into such states. -Attempts to induce superconductivity in Quantum Hall states with the aim of creating a chiral topological superconductor. These chiral superconductors host Majorana fermions at their edges, which, at least in the case of a single QH edge mode, follow non-Abelian statistics and are therefore promising for explorations in topological quantum computing. -Studies of induced superconductivity in Weyl semimetals, a completely unexplored state of matter. Taken together, these four sets of experiments will greatly enhance our understanding of topological superconductivity, which is not only a subject of great academic interest as it constitutes the study of new phases of matter, but also has potential application in the field of quantum information processing.
Topological materials have developed rapidly in recent years, with my previous ERC-AG project 3-TOP playing a major role in this development. While so far no bulk topological superconductor has been unambiguously demonstrated, their properties can be studied in a very flexible manner by inducing superconductivity through the proximity effect into the surface or edge states of a topological insulator. In 4-TOPS we will explore the possibilities of this approach in full, and conduct a thorough study of induced superconductivity in both two and three dimensional HgTe based topological insulators. The 4 avenues we will follow are: -SQUID based devices to investigate full phase dependent spectroscopy of the gapless Andreev bound state by studying their Josephson radiation and current-phase relationships. -Experiments aimed at providing unambiguous proof of localized Majorana states in TI junctions by studying tunnelling transport into such states. -Attempts to induce superconductivity in Quantum Hall states with the aim of creating a chiral topological superconductor. These chiral superconductors host Majorana fermions at their edges, which, at least in the case of a single QH edge mode, follow non-Abelian statistics and are therefore promising for explorations in topological quantum computing. -Studies of induced superconductivity in Weyl semimetals, a completely unexplored state of matter. Taken together, these four sets of experiments will greatly enhance our understanding of topological superconductivity, which is not only a subject of great academic interest as it constitutes the study of new phases of matter, but also has potential application in the field of quantum information processing.
Project acronym 4D-GenEx
Project Spatio-temporal Organization and Expression of the Genome
Researcher (PI) Antoine COULON
Summary This project investigates the two-way relationship between spatio-temporal genome organization and coordinated gene regulation, through an approach at the interface between physics, computer science and biology. In the nucleus, preferred positions are observed from chromosomes to single genes, in relation to normal and pathological cellular states. Evidence indicates a complex spatio-temporal coupling between co-regulated genes: e.g. certain genes cluster spatially when responding to similar factors and transcriptional noise patterns suggest domain-wide mechanisms. Yet, no individual experiment allows probing transcriptional coordination in 4 dimensions (FISH, live locus tracking, Hi-C...). Interpreting such data also critically requires theory (stochastic processes, statistical physics…). A lack of appropriate experimental/analytical approaches is impairing our understanding of the 4D genome. Our proposal combines cutting-edge single-molecule imaging, signal-theory data analysis and physical modeling to study how genes coordinate in space and time in a single nucleus. Our objectives are to understand (a) competition/recycling of shared resources between genes within subnuclear compartments, (b) how enhancers communicate with genes domain-wide, and (c) the role of local conformational dynamics and supercoiling in gene co-regulation. Our organizing hypothesis is that, by acting on their microenvironment, genes shape their co-expression with other genes. Building upon my expertise, we will use dual-color MS2/PP7 RNA labeling to visualize for the first time transcription and motion of pairs of hormone-responsive genes in real time. With our innovative signal analysis tools, we will extract spatio-temporal signatures of underlying processes, which we will investigate with stochastic modeling and validate through experimental perturbations. We expect to uncover how the functional organization of the linear genome relates to its physical properties and dynamics in 4D.
This project investigates the two-way relationship between spatio-temporal genome organization and coordinated gene regulation, through an approach at the interface between physics, computer science and biology. In the nucleus, preferred positions are observed from chromosomes to single genes, in relation to normal and pathological cellular states. Evidence indicates a complex spatio-temporal coupling between co-regulated genes: e.g. certain genes cluster spatially when responding to similar factors and transcriptional noise patterns suggest domain-wide mechanisms. Yet, no individual experiment allows probing transcriptional coordination in 4 dimensions (FISH, live locus tracking, Hi-C...). Interpreting such data also critically requires theory (stochastic processes, statistical physics…). A lack of appropriate experimental/analytical approaches is impairing our understanding of the 4D genome. Our proposal combines cutting-edge single-molecule imaging, signal-theory data analysis and physical modeling to study how genes coordinate in space and time in a single nucleus. Our objectives are to understand (a) competition/recycling of shared resources between genes within subnuclear compartments, (b) how enhancers communicate with genes domain-wide, and (c) the role of local conformational dynamics and supercoiling in gene co-regulation. Our organizing hypothesis is that, by acting on their microenvironment, genes shape their co-expression with other genes. Building upon my expertise, we will use dual-color MS2/PP7 RNA labeling to visualize for the first time transcription and motion of pairs of hormone-responsive genes in real time. With our innovative signal analysis tools, we will extract spatio-temporal signatures of underlying processes, which we will investigate with stochastic modeling and validate through experimental perturbations. We expect to uncover how the functional organization of the linear genome relates to its physical properties and dynamics in 4D.
Project acronym 4D-PET
Project Innovative PET scanner for dynamic imaging
Researcher (PI) José María BENLLOCH BAVIERA
Host Institution (HI) AGENCIA ESTATAL CONSEJO SUPERIOR DEINVESTIGACIONES CIENTIFICAS
Summary The main objective of 4D-PET is to develop an innovative whole-body PET scanner based in a new detector concept that stores 3D position and time of every single gamma interaction with unprecedented resolution. The combination of scanner geometrical design and high timing resolution will enable developing a full sequence of all gamma-ray interactions inside the scanner, including Compton interactions, like in a 3D movie. 4D-PET fully exploits Time Of Flight (TOF) information to obtain a better image quality and to increase scanner sensitivity, through the inclusion in the image formation of all Compton events occurring inside the detector, which are always rejected in state-of-the-art PET scanners. The new PET design will radically improve state-of-the-art PET performance features, overcoming limitations of current PET technology and opening up new diagnostic venues and very valuable physiological information
The main objective of 4D-PET is to develop an innovative whole-body PET scanner based in a new detector concept that stores 3D position and time of every single gamma interaction with unprecedented resolution. The combination of scanner geometrical design and high timing resolution will enable developing a full sequence of all gamma-ray interactions inside the scanner, including Compton interactions, like in a 3D movie. 4D-PET fully exploits Time Of Flight (TOF) information to obtain a better image quality and to increase scanner sensitivity, through the inclusion in the image formation of all Compton events occurring inside the detector, which are always rejected in state-of-the-art PET scanners. The new PET design will radically improve state-of-the-art PET performance features, overcoming limitations of current PET technology and opening up new diagnostic venues and very valuable physiological information
Project acronym 5D Heart Patch
Project A Functional, Mature In vivo Human Ventricular Muscle Patch for Cardiomyopathy
Researcher (PI) Kenneth Randall Chien
Summary Developing new therapeutic strategies for heart regeneration is a major goal for cardiac biology and medicine. While cardiomyocytes can be generated from human pluripotent stem (hPSC) cells in vitro, it has proven difficult to use these cells to generate a large scale, mature human heart ventricular muscle graft on the injured heart in vivo. The central objective of this proposal is to optimize the generation of a large-scale pure, fully functional human ventricular muscle patch in vivo through the self-assembly of purified human ventricular progenitors and the localized expression of defined paracrine factors that drive their expansion, differentiation, vascularization, matrix formation, and maturation. Recently, we have found that purified hPSC-derived ventricular progenitors (HVPs) can self-assemble in vivo on the epicardial surface into a 3D vascularized, and functional ventricular patch with its own extracellular matrix via a cell autonomous pathway. A two-step protocol and FACS purification of HVP receptors can generate billions of pure HVPs- The current proposal will lead to the identification of defined paracrine pathways to enhance the survival, grafting/implantation, expansion, differentiation, matrix formation, vascularization and maturation of the graft in vivo. We will captalize on our unique HVP system and our novel modRNA technology to deliver therapeutic strategies by using the in vivo human ventricular muscle to model in vivo arrhythmogenic cardiomyopathy, and optimize the ability of the graft to compensate for the massive loss of functional muscle during ischemic cardiomyopathy and post-myocardial infarction. The studies will lead to new in vivo chimeric models of human cardiac disease and an experimental paradigm to optimize organ-on-organ cardiac tissue engineers of an in vivo, functional mature ventricular patch for cardiomyopathy
Developing new therapeutic strategies for heart regeneration is a major goal for cardiac biology and medicine. While cardiomyocytes can be generated from human pluripotent stem (hPSC) cells in vitro, it has proven difficult to use these cells to generate a large scale, mature human heart ventricular muscle graft on the injured heart in vivo. The central objective of this proposal is to optimize the generation of a large-scale pure, fully functional human ventricular muscle patch in vivo through the self-assembly of purified human ventricular progenitors and the localized expression of defined paracrine factors that drive their expansion, differentiation, vascularization, matrix formation, and maturation. Recently, we have found that purified hPSC-derived ventricular progenitors (HVPs) can self-assemble in vivo on the epicardial surface into a 3D vascularized, and functional ventricular patch with its own extracellular matrix via a cell autonomous pathway. A two-step protocol and FACS purification of HVP receptors can generate billions of pure HVPs- The current proposal will lead to the identification of defined paracrine pathways to enhance the survival, grafting/implantation, expansion, differentiation, matrix formation, vascularization and maturation of the graft in vivo. We will captalize on our unique HVP system and our novel modRNA technology to deliver therapeutic strategies by using the in vivo human ventricular muscle to model in vivo arrhythmogenic cardiomyopathy, and optimize the ability of the graft to compensate for the massive loss of functional muscle during ischemic cardiomyopathy and post-myocardial infarction. The studies will lead to new in vivo chimeric models of human cardiac disease and an experimental paradigm to optimize organ-on-organ cardiac tissue engineers of an in vivo, functional mature ventricular patch for cardiomyopathy
Project acronym 5HT-OPTOGENETICS
Project Optogenetic Analysis of Serotonin Function in the Mammalian Brain
Researcher (PI) Zachary Mainen
Summary Serotonin (5-HT) is implicated in a wide spectrum of brain functions and disorders. However, its functions remain controversial and enigmatic. We suggest that past work on the 5-HT system have been significantly hampered by technical limitations in the selectivity and temporal resolution of the conventional pharmacological and electrophysiological methods that have been applied. We therefore propose to apply novel optogenetic methods that will allow us to overcome these limitations and thereby gain new insight into the biological functions of this important molecule. In preliminary studies, we have demonstrated that we can deliver exogenous proteins specifically to 5-HT neurons using viral vectors. Our objectives are to (1) record, (2) stimulate and (3) silence the activity of 5-HT neurons with high molecular selectivity and temporal precision by using genetically-encoded sensors, activators and inhibitors of neural function. These tools will allow us to monitor and control the 5-HT system in real-time in freely-behaving animals and thereby to establish causal links between information processing in 5-HT neurons and specific behaviors. In combination with quantitative behavioral assays, we will use this approach to define the role of 5-HT in sensory, motor and cognitive functions. The significance of the work is three-fold. First, we will establish a new arsenal of tools for probing the physiological and behavioral functions of 5-HT neurons. Second, we will make definitive tests of major hypotheses of 5-HT function. Third, we will have possible therapeutic applications. In this way, the proposed work has the potential for a major impact in research on the role of 5-HT in brain function and dysfunction.
Serotonin (5-HT) is implicated in a wide spectrum of brain functions and disorders. However, its functions remain controversial and enigmatic. We suggest that past work on the 5-HT system have been significantly hampered by technical limitations in the selectivity and temporal resolution of the conventional pharmacological and electrophysiological methods that have been applied. We therefore propose to apply novel optogenetic methods that will allow us to overcome these limitations and thereby gain new insight into the biological functions of this important molecule. In preliminary studies, we have demonstrated that we can deliver exogenous proteins specifically to 5-HT neurons using viral vectors. Our objectives are to (1) record, (2) stimulate and (3) silence the activity of 5-HT neurons with high molecular selectivity and temporal precision by using genetically-encoded sensors, activators and inhibitors of neural function. These tools will allow us to monitor and control the 5-HT system in real-time in freely-behaving animals and thereby to establish causal links between information processing in 5-HT neurons and specific behaviors. In combination with quantitative behavioral assays, we will use this approach to define the role of 5-HT in sensory, motor and cognitive functions. The significance of the work is three-fold. First, we will establish a new arsenal of tools for probing the physiological and behavioral functions of 5-HT neurons. Second, we will make definitive tests of major hypotheses of 5-HT function. Third, we will have possible therapeutic applications. In this way, the proposed work has the potential for a major impact in research on the role of 5-HT in brain function and dysfunction.
Project acronym 5HTCircuits
Project Modulation of cortical circuits and predictive neural coding by serotonin
Summary Serotonin (5-HT) is a central neuromodulator and a major target of therapeutic psychoactive drugs, but relatively little is known about how it modulates information processing in neural circuits. The theory of predictive coding postulates that the brain combines raw bottom-up sensory information with top-down information from internal models to make perceptual inferences about the world. We hypothesize, based on preliminary data and prior literature, that a role of 5-HT in this process is to report prediction errors and promote the suppression and weakening of erroneous internal models. We propose that it does this by inhibiting top-down relative to bottom-up cortical information flow. To test this hypothesis, we propose a set of experiments in mice performing olfactory perceptual tasks. Our specific aims are: (1) We will test whether 5-HT neurons encode sensory prediction errors. (2) We will test their causal role in using predictive cues to guide perceptual decisions. (3) We will characterize how 5-HT influences the encoding of sensory information by neuronal populations in the olfactory cortex and identify the underlying circuitry. (4) Finally, we will map the effects of 5-HT across the whole brain and use this information to target further causal manipulations to specific 5-HT projections. We accomplish these aims using state-of-the-art optogenetic, electrophysiological and imaging techniques (including 9.4T small-animal functional magnetic resonance imaging) as well as psychophysical tasks amenable to quantitative analysis and computational theory. Together, these experiments will tackle multiple facets of an important general computational question, bringing to bear an array of cutting-edge technologies to address with unprecedented mechanistic detail how 5-HT impacts neural coding and perceptual decision-making.
Serotonin (5-HT) is a central neuromodulator and a major target of therapeutic psychoactive drugs, but relatively little is known about how it modulates information processing in neural circuits. The theory of predictive coding postulates that the brain combines raw bottom-up sensory information with top-down information from internal models to make perceptual inferences about the world. We hypothesize, based on preliminary data and prior literature, that a role of 5-HT in this process is to report prediction errors and promote the suppression and weakening of erroneous internal models. We propose that it does this by inhibiting top-down relative to bottom-up cortical information flow. To test this hypothesis, we propose a set of experiments in mice performing olfactory perceptual tasks. Our specific aims are: (1) We will test whether 5-HT neurons encode sensory prediction errors. (2) We will test their causal role in using predictive cues to guide perceptual decisions. (3) We will characterize how 5-HT influences the encoding of sensory information by neuronal populations in the olfactory cortex and identify the underlying circuitry. (4) Finally, we will map the effects of 5-HT across the whole brain and use this information to target further causal manipulations to specific 5-HT projections. We accomplish these aims using state-of-the-art optogenetic, electrophysiological and imaging techniques (including 9.4T small-animal functional magnetic resonance imaging) as well as psychophysical tasks amenable to quantitative analysis and computational theory. Together, these experiments will tackle multiple facets of an important general computational question, bringing to bear an array of cutting-edge technologies to address with unprecedented mechanistic detail how 5-HT impacts neural coding and perceptual decision-making.
Project acronym A-DIET
Project Metabolomics based biomarkers of dietary intake- new tools for nutrition research
Researcher (PI) Lorraine Brennan
Host Institution (HI) UNIVERSITY COLLEGE DUBLIN, NATIONAL UNIVERSITY OF IRELAND, DUBLIN
Summary In todays advanced technological world, we can track the exact movement of individuals, analyse their genetic makeup and predict predisposition to certain diseases. However, we are unable to accurately assess an individual's dietary intake. This is without a doubt one of the main stumbling blocks in assessing the link between diet and disease/health. The present proposal (A-DIET) will address this issue with the overarching objective to develop novel strategies for assessment of dietary intake. Using approaches to (1) identify biomarkers of specific foods (2) classify people into dietary patterns (nutritypes) and (3) develop a tool for integration of dietary and biomarker data, A-DIET has the potential to dramatically enhance our ability to accurately assess dietary intake. The ultimate output from A-DIET will be a dietary assessment tool which can be used to obtain an accurate assessment of dietary intake by combining dietary and biomarker data which in turn will allow investigations into relationships between diet, health and disease. New biomarkers of specific foods will be identified and validated using intervention studies and metabolomic analyses. Methods will be developed to classify individuals into dietary patterns based on biomarker/metabolomic profiles thus demonstrating the novel concept of nutritypes. Strategies for integration of dietary and biomarker data will be developed and translated into a tool that will be made available to the wider scientific community. Advances made in A-DIET will enable nutrition epidemiologist's to properly examine the relationship between diet and disease and develop clear public health messages with regard to diet and health. Additionally results from A-DIET will allow researchers to accurately assess people's diet and implement health promotion strategies and enable dieticians in a clinical environment to assess compliance to therapeutic diets such as adherence to a high fibre diet or a gluten free diet.
In todays advanced technological world, we can track the exact movement of individuals, analyse their genetic makeup and predict predisposition to certain diseases. However, we are unable to accurately assess an individual's dietary intake. This is without a doubt one of the main stumbling blocks in assessing the link between diet and disease/health. The present proposal (A-DIET) will address this issue with the overarching objective to develop novel strategies for assessment of dietary intake. Using approaches to (1) identify biomarkers of specific foods (2) classify people into dietary patterns (nutritypes) and (3) develop a tool for integration of dietary and biomarker data, A-DIET has the potential to dramatically enhance our ability to accurately assess dietary intake. The ultimate output from A-DIET will be a dietary assessment tool which can be used to obtain an accurate assessment of dietary intake by combining dietary and biomarker data which in turn will allow investigations into relationships between diet, health and disease. New biomarkers of specific foods will be identified and validated using intervention studies and metabolomic analyses. Methods will be developed to classify individuals into dietary patterns based on biomarker/metabolomic profiles thus demonstrating the novel concept of nutritypes. Strategies for integration of dietary and biomarker data will be developed and translated into a tool that will be made available to the wider scientific community. Advances made in A-DIET will enable nutrition epidemiologist's to properly examine the relationship between diet and disease and develop clear public health messages with regard to diet and health. Additionally results from A-DIET will allow researchers to accurately assess people's diet and implement health promotion strategies and enable dieticians in a clinical environment to assess compliance to therapeutic diets such as adherence to a high fibre diet or a gluten free diet.
Project acronym A-FRO
Project Actively Frozen - contextual modulation of freezing and its neuronal basis
Researcher (PI) Marta de Aragão Pacheco Moita
Summary When faced with a threat, an animal must decide whether to freeze, reducing its chances of being noticed, or to flee to the safety of a refuge. Animals from fish to primates choose between these two alternatives when confronted by an attacking predator, a choice that largely depends on the context in which the threat occurs. Recent work has made strides identifying the pre-motor circuits, and their inputs, which control freezing behavior in rodents, but how contextual information is integrated to guide this choice is still far from understood. We recently found that fruit flies in response to visual looming stimuli, simulating a large object on collision course, make rapid freeze/flee choices that depend on the social and spatial environment, and the fly's internal state. Further, identification of looming detector neurons was recently reported and we identified the descending command neurons, DNp09, responsible for freezing in the fly. Knowing the sensory input and descending output for looming-evoked freezing, two environmental factors that modulate its expression, and using a genetically tractable system affording the use of large sample sizes, places us in an unique position to understand how a information about a threat is integrated with cues from the environment to guide the choice of whether to freeze (our goal). To assess how social information impinges on the circuit for freezing, we will examine the sensory inputs and neuromodulators that mediate this process, mapping their connections to DNp09 neurons (Aim 1). We ask whether learning is required for the spatial modulation of freezing, which cues flies are using to discriminate different places and which brain circuits mediate this process (Aim 2). Finally, we will study how activity of DNp09 neurons drives freezing (Aim 3). This project will provide a comprehensive understanding of the mechanism of freezing and its modulation by the environment, from single neurons to behaviour.
When faced with a threat, an animal must decide whether to freeze, reducing its chances of being noticed, or to flee to the safety of a refuge. Animals from fish to primates choose between these two alternatives when confronted by an attacking predator, a choice that largely depends on the context in which the threat occurs. Recent work has made strides identifying the pre-motor circuits, and their inputs, which control freezing behavior in rodents, but how contextual information is integrated to guide this choice is still far from understood. We recently found that fruit flies in response to visual looming stimuli, simulating a large object on collision course, make rapid freeze/flee choices that depend on the social and spatial environment, and the fly's internal state. Further, identification of looming detector neurons was recently reported and we identified the descending command neurons, DNp09, responsible for freezing in the fly. Knowing the sensory input and descending output for looming-evoked freezing, two environmental factors that modulate its expression, and using a genetically tractable system affording the use of large sample sizes, places us in an unique position to understand how a information about a threat is integrated with cues from the environment to guide the choice of whether to freeze (our goal). To assess how social information impinges on the circuit for freezing, we will examine the sensory inputs and neuromodulators that mediate this process, mapping their connections to DNp09 neurons (Aim 1). We ask whether learning is required for the spatial modulation of freezing, which cues flies are using to discriminate different places and which brain circuits mediate this process (Aim 2). Finally, we will study how activity of DNp09 neurons drives freezing (Aim 3). This project will provide a comprehensive understanding of the mechanism of freezing and its modulation by the environment, from single neurons to behaviour.
Project acronym AAMDDR
Project DNA damage response and genome stability: The role of ATM, ATR and the Mre11 complex
Researcher (PI) Vincenzo Costanzo
Host Institution (HI) CANCER RESEARCH UK LBG
Summary Chromosomal DNA is continuously subjected to exogenous and endogenous damaging insults. In the presence of DNA damage cells activate a multi-faceted checkpoint response that delays cell cycle progression and promotes DNA repair. Failures in this response lead to genomic instability, the main feature of cancer cells. Several cancer-prone human syndromes including the Ataxia teleangiectasia (A-T), the A-T Like Disorder (ATLD) and the Seckel Syndrome reflect defects in the specific genes of the DNA damage response such as ATM, MRE11 and ATR. DNA damage response pathways are poorly understood at biochemical level in vertebrate organisms. We have established a cell-free system based on Xenopus laevis egg extract to study molecular events underlying DNA damage response. This is the first in vitro system that recapitulates different aspects of the DNA damage response in vertebrates. Using this system we propose to study the biochemistry of the ATM, ATR and the Mre11 complex dependent DNA damage response. In particular we will: 1) Dissect the signal transduction pathway that senses DNA damage and promotes cell cycle arrest and DNA damage repair; 2) Analyze at molecular level the role of ATM, ATR, Mre11 in chromosomal DNA replication and mitosis during normal and stressful conditions; 3) Identify substrates of the ATM and ATR dependent DNA damage response using an innovative screening procedure.
Chromosomal DNA is continuously subjected to exogenous and endogenous damaging insults. In the presence of DNA damage cells activate a multi-faceted checkpoint response that delays cell cycle progression and promotes DNA repair. Failures in this response lead to genomic instability, the main feature of cancer cells. Several cancer-prone human syndromes including the Ataxia teleangiectasia (A-T), the A-T Like Disorder (ATLD) and the Seckel Syndrome reflect defects in the specific genes of the DNA damage response such as ATM, MRE11 and ATR. DNA damage response pathways are poorly understood at biochemical level in vertebrate organisms. We have established a cell-free system based on Xenopus laevis egg extract to study molecular events underlying DNA damage response. This is the first in vitro system that recapitulates different aspects of the DNA damage response in vertebrates. Using this system we propose to study the biochemistry of the ATM, ATR and the Mre11 complex dependent DNA damage response. In particular we will: 1) Dissect the signal transduction pathway that senses DNA damage and promotes cell cycle arrest and DNA damage repair; 2) Analyze at molecular level the role of ATM, ATR, Mre11 in chromosomal DNA replication and mitosis during normal and stressful conditions; 3) Identify substrates of the ATM and ATR dependent DNA damage response using an innovative screening procedure.
Project acronym ABDESIGN
Project Computational design of novel protein function in antibodies
Researcher (PI) Sarel-Jacob Fleishman
Summary We propose to elucidate the structural design principles of naturally occurring antibody complementarity-determining regions (CDRs) and to computationally design novel antibody functions. Antibodies represent the most versatile known system for molecular recognition. Research has yielded many insights into antibody design principles and promising biotechnological and pharmaceutical applications. Still, our understanding of how CDRs encode specific loop conformations lags far behind our understanding of structure-function relationships in non-immunological scaffolds. Thus, design of antibodies from first principles has not been demonstrated. We propose a computational-experimental strategy to address this challenge. We will: (a) characterize the design principles and sequence elements that rigidify antibody CDRs. Natural antibody loops will be subjected to computational modeling, crystallography, and a combined in vitro evolution and deep-sequencing approach to isolate sequence features that rigidify loop backbones; (b) develop a novel computational-design strategy, which uses the >1000 solved structures of antibodies deposited in structure databases to realistically model CDRs and design them to recognize proteins that have not been co-crystallized with antibodies. For example, we will design novel antibodies targeting insulin, for which clinically useful diagnostics are needed. By accessing much larger sequence/structure spaces than are available to natural immune-system repertoires and experimental methods, computational antibody design could produce higher-specificity and higher-affinity binders, even to challenging targets; and (c) develop new strategies to program conformational change in CDRs, generating, e.g., the first allosteric antibodies. These will allow targeting, in principle, of any molecule, potentially revolutionizing how antibodies are generated for research and medicine, providing new insights on the design principles of protein functional sites.
We propose to elucidate the structural design principles of naturally occurring antibody complementarity-determining regions (CDRs) and to computationally design novel antibody functions. Antibodies represent the most versatile known system for molecular recognition. Research has yielded many insights into antibody design principles and promising biotechnological and pharmaceutical applications. Still, our understanding of how CDRs encode specific loop conformations lags far behind our understanding of structure-function relationships in non-immunological scaffolds. Thus, design of antibodies from first principles has not been demonstrated. We propose a computational-experimental strategy to address this challenge. We will: (a) characterize the design principles and sequence elements that rigidify antibody CDRs. Natural antibody loops will be subjected to computational modeling, crystallography, and a combined in vitro evolution and deep-sequencing approach to isolate sequence features that rigidify loop backbones; (b) develop a novel computational-design strategy, which uses the >1000 solved structures of antibodies deposited in structure databases to realistically model CDRs and design them to recognize proteins that have not been co-crystallized with antibodies. For example, we will design novel antibodies targeting insulin, for which clinically useful diagnostics are needed. By accessing much larger sequence/structure spaces than are available to natural immune-system repertoires and experimental methods, computational antibody design could produce higher-specificity and higher-affinity binders, even to challenging targets; and (c) develop new strategies to program conformational change in CDRs, generating, e.g., the first allosteric antibodies. These will allow targeting, in principle, of any molecule, potentially revolutionizing how antibodies are generated for research and medicine, providing new insights on the design principles of protein functional sites.
Project acronym ABEL
Project "Alpha-helical Barrels: Exploring, Understanding and Exploiting a New Class of Protein Structure"
Summary "Recently through de novo peptide design, we have discovered and presented a new protein structure. This is an all-parallel, 6-helix bundle with a continuous central channel of 0.5 – 0.6 nm diameter. We posit that this is one of a broader class of protein structures that we call the alpha-helical barrels. Here, in three Work Packages, we propose to explore these structures and to develop protein functions within them. First, through a combination of computer-aided design, peptide synthesis and thorough biophysical characterization, we will examine the extents and limits of the alpha-helical-barrel structures. Whilst this is curiosity driven research, it also has practical consequences for the studies that will follow; that is, alpha-helical barrels made from increasing numbers of helices have channels or pores that increase in a predictable way. Second, we will use rational and empirical design approaches to engineer a range of functions within these cavities, including binding capabilities and enzyme-like activities. Finally, and taking the programme into another ambitious area, we will use the alpha-helical barrels to template other folds that are otherwise difficult to design and engineer, notably beta-barrels that insert into membranes to render ion-channel and sensor functions."
"Recently through de novo peptide design, we have discovered and presented a new protein structure. This is an all-parallel, 6-helix bundle with a continuous central channel of 0.5 – 0.6 nm diameter. We posit that this is one of a broader class of protein structures that we call the alpha-helical barrels. Here, in three Work Packages, we propose to explore these structures and to develop protein functions within them. First, through a combination of computer-aided design, peptide synthesis and thorough biophysical characterization, we will examine the extents and limits of the alpha-helical-barrel structures. Whilst this is curiosity driven research, it also has practical consequences for the studies that will follow; that is, alpha-helical barrels made from increasing numbers of helices have channels or pores that increase in a predictable way. Second, we will use rational and empirical design approaches to engineer a range of functions within these cavities, including binding capabilities and enzyme-like activities. Finally, and taking the programme into another ambitious area, we will use the alpha-helical barrels to template other folds that are otherwise difficult to design and engineer, notably beta-barrels that insert into membranes to render ion-channel and sensor functions."
Project acronym ABINITIODGA
Project Ab initio Dynamical Vertex Approximation
Researcher (PI) Karsten Held
Host Institution (HI) TECHNISCHE UNIVERSITAET WIEN
Summary Some of the most fascinating physical phenomena are experimentally observed in strongly correlated electron systems and, on the theoretical side, only poorly understood hitherto. The aim of the ERC project AbinitioDGA is the development, implementation and application of a new, 21th century method for the ab initio calculation of materials with such strong electronic correlations. AbinitioDGA includes strong electronic correlations on all time and length scales and hence is a big step beyond the state-of-the-art methods, such as the local density approximation, dynamical mean field theory, and the GW approach (Green function G times screened interaction W). It has the potential for an extraordinary high impact not only in the field of computational materials science but also for a better understanding of quantum critical heavy fermion systems, high-temperature superconductors, and transport through nano- and heterostructures. These four physical problems and related materials will be studied within the ERC project, besides the methodological development. On the technical side, AbinitioDGA realizes Hedin's idea to include vertex corrections beyond the GW approximation. All vertex corrections which can be traced back to a fully irreducible local vertex and the bare non-local Coulomb interaction are included. This way, AbinitioDGA does not only contain the GW physics of screened exchange and the strong local correlations of dynamical mean field theory but also non-local correlations beyond on all length scales. Through the latter, AbinitioDGA can prospectively describe phenomena such as quantum criticality, spin-fluctuation mediated superconductivity, and weak localization corrections to the conductivity. Nonetheless, the computational effort is still manageable even for realistic materials calculations, making the considerable effort to implement AbinitioDGA worthwhile.
Some of the most fascinating physical phenomena are experimentally observed in strongly correlated electron systems and, on the theoretical side, only poorly understood hitherto. The aim of the ERC project AbinitioDGA is the development, implementation and application of a new, 21th century method for the ab initio calculation of materials with such strong electronic correlations. AbinitioDGA includes strong electronic correlations on all time and length scales and hence is a big step beyond the state-of-the-art methods, such as the local density approximation, dynamical mean field theory, and the GW approach (Green function G times screened interaction W). It has the potential for an extraordinary high impact not only in the field of computational materials science but also for a better understanding of quantum critical heavy fermion systems, high-temperature superconductors, and transport through nano- and heterostructures. These four physical problems and related materials will be studied within the ERC project, besides the methodological development. On the technical side, AbinitioDGA realizes Hedin's idea to include vertex corrections beyond the GW approximation. All vertex corrections which can be traced back to a fully irreducible local vertex and the bare non-local Coulomb interaction are included. This way, AbinitioDGA does not only contain the GW physics of screened exchange and the strong local correlations of dynamical mean field theory but also non-local correlations beyond on all length scales. Through the latter, AbinitioDGA can prospectively describe phenomena such as quantum criticality, spin-fluctuation mediated superconductivity, and weak localization corrections to the conductivity. Nonetheless, the computational effort is still manageable even for realistic materials calculations, making the considerable effort to implement AbinitioDGA worthwhile.
Project acronym ABLASE
Project Advanced Bioderived and Biocompatible Lasers
Researcher (PI) Malte Christian Gather
Summary Naturally occurring optical phenomena attract great attention and transform our ability to study biological processes, with "the discovery and development of the green fluorescent protein (GFP)" (Nobel Prize in Chemistry 2008) being a particularly successful example. Although found only in very few species in nature, most organisms can be genetically programmed to produce the brightly fluorescent GFP molecules. Combined with modern fluorescence detection schemes, this has led to entirely new ways of monitoring biological processes. The applicant now demonstrated a biological laser – a completely novel, living source of coherent light based on a single biological cell bioengineered to produce GFP. Such a laser is intrinsically biocompatible, thus offering unique properties not shared by any existing laser. However, the physical processes involved in lasing from GFP remain poorly understood and so far biological lasers rely on bulky, impractical external resonators for optical feedback. Within this project, the applicant and his team will develop for the first time an understanding of stimulated emission in GFP and related proteins and create an unprecedented stand-alone single-cell biolaser based on intracellular optical feedback. These lasers will be deployed as microscopic and biocompatible imaging probes, thus opening in vivo microscopy to dense wavelength-multiplexing and enabling unmatched sensing of biomolecules and mechanical pressure. The evolutionarily evolved nano-structure of GFP will also enable novel ways of studying strong light-matter coupling and will bio-inspire advances of synthetic emitters. The proposed project is inter-disciplinary by its very nature, bridging photonics, genetic engineering and material science. The applicant's previous pioneering work and synergies with work on other lasers developed at the applicant's host institution provide an exclusive competitive edge. ERC support would transform this into a truly novel field of research.
Naturally occurring optical phenomena attract great attention and transform our ability to study biological processes, with "the discovery and development of the green fluorescent protein (GFP)" (Nobel Prize in Chemistry 2008) being a particularly successful example. Although found only in very few species in nature, most organisms can be genetically programmed to produce the brightly fluorescent GFP molecules. Combined with modern fluorescence detection schemes, this has led to entirely new ways of monitoring biological processes. The applicant now demonstrated a biological laser – a completely novel, living source of coherent light based on a single biological cell bioengineered to produce GFP. Such a laser is intrinsically biocompatible, thus offering unique properties not shared by any existing laser. However, the physical processes involved in lasing from GFP remain poorly understood and so far biological lasers rely on bulky, impractical external resonators for optical feedback. Within this project, the applicant and his team will develop for the first time an understanding of stimulated emission in GFP and related proteins and create an unprecedented stand-alone single-cell biolaser based on intracellular optical feedback. These lasers will be deployed as microscopic and biocompatible imaging probes, thus opening in vivo microscopy to dense wavelength-multiplexing and enabling unmatched sensing of biomolecules and mechanical pressure. The evolutionarily evolved nano-structure of GFP will also enable novel ways of studying strong light-matter coupling and will bio-inspire advances of synthetic emitters. The proposed project is inter-disciplinary by its very nature, bridging photonics, genetic engineering and material science. The applicant's previous pioneering work and synergies with work on other lasers developed at the applicant's host institution provide an exclusive competitive edge. ERC support would transform this into a truly novel field of research.
Project acronym ABSOLUTESPIN
Project Absolute Spin Dynamics in Quantum Materials
Researcher (PI) Christian Reinhard Ast
Summary One of the greatest challenges in exploiting the electron spin for information processing is that it is not a conserved quantity like the electron charge. In addition, spin lifetimes are rather short and correspondingly coherence is quickly lost. This challenge culminates in the coherent manipulation and detection of information from a single spin. Except in a few special systems, so far, single spins cannot be manipulated coherently on the atomic scale, while spin coherence times can only be measured on spin ensembles. A new concept is needed for coherence measurements on arbitrary single spins. Here, the principal investigator (PI) will combine a novel time- and spin-resolved low-temperature scanning tunneling microscope (STM) with the concept of pulsed electron paramagnetic resonance. With this unique and innovative setup, he will be able to address long-standing problems, such as relaxation and coherence times of arbitrary single spin systems on the atomic scale as well as individual spin interactions with the immediate surroundings. Spin readout will be realized through the detection of the absolute spin polarization in the tunneling current by a superconducting tip based on the Meservey-Tedrow-Fulde effect, which the PI has recently demonstrated for the first time in STM. For the coherent excitation, a specially designed pulsed GHz light source will be implemented. The goal is to better understand the spin dynamics and coherence times of single spin systems as well as the spin interactions involved in the decay mechanisms. This will have direct impact on the feasibility of quantum spin information processing with single spin systems on different decoupling surfaces and their scalability at the atomic level. A successful demonstration will enhance the detection limit of spins by several orders of magnitude and fill important missing links in the understanding of spin dynamics and quantum computing with single spins.
One of the greatest challenges in exploiting the electron spin for information processing is that it is not a conserved quantity like the electron charge. In addition, spin lifetimes are rather short and correspondingly coherence is quickly lost. This challenge culminates in the coherent manipulation and detection of information from a single spin. Except in a few special systems, so far, single spins cannot be manipulated coherently on the atomic scale, while spin coherence times can only be measured on spin ensembles. A new concept is needed for coherence measurements on arbitrary single spins. Here, the principal investigator (PI) will combine a novel time- and spin-resolved low-temperature scanning tunneling microscope (STM) with the concept of pulsed electron paramagnetic resonance. With this unique and innovative setup, he will be able to address long-standing problems, such as relaxation and coherence times of arbitrary single spin systems on the atomic scale as well as individual spin interactions with the immediate surroundings. Spin readout will be realized through the detection of the absolute spin polarization in the tunneling current by a superconducting tip based on the Meservey-Tedrow-Fulde effect, which the PI has recently demonstrated for the first time in STM. For the coherent excitation, a specially designed pulsed GHz light source will be implemented. The goal is to better understand the spin dynamics and coherence times of single spin systems as well as the spin interactions involved in the decay mechanisms. This will have direct impact on the feasibility of quantum spin information processing with single spin systems on different decoupling surfaces and their scalability at the atomic level. A successful demonstration will enhance the detection limit of spins by several orders of magnitude and fill important missing links in the understanding of spin dynamics and quantum computing with single spins.