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The final output of WP8 is a web-based Decision Support System Pathfinder for assessment of the sustainability of legume agri-food value chains. The conceptual structure of the system, the decision models that it is comprised of, as well as the technical implementation of the system are described in Deliverables 8.1 to 8.4. This deliverable serves as a user manual with instructions about the use of the PathFinder. It is also included in the web-based Decision Support System PathFinder to guide the users through the functionalities of the system.

This report should be assimilated in parallel with the ‘Activity Plan – Year IV’ for the final year of the TRUE project, and it should also be noted that this report supersedes some of the information given in Deliverable 9.9 (Project Review, Impact and Legacy Plan – Report I) due to changes in the delivery plan, and delays associated with the impacts of COVID-19. Also, as the TRUE project draws to completion on Sept. 30th 2021, and effort progresses into a phase which is not specifically funded, it is important that effort is focused upon those key activities which has the potential to establish improved legume-based futures for all those who will become involved.

This Deliverable presents our final policy evaluation report giving, ‘An overview and classification of European and international policies to support legume-based systems’. This key output was developed to deliver four policy briefs - to help build positive, proactive policies to support the realisation of more sustainable legume-based systems.

Graphical abstract, The monosaccharide l-Rhamnose is an important component of bacterial cell walls. The first step in the l-rhamnose biosynthetic pathway is catalysed by glucose-1-phosphate thymidylyltransferase (RmlA), which condenses glucose-1-phosphate (Glu-1-P) with deoxythymidine triphosphate (dTTP) to yield dTDP-d-glucose. In addition to the active site where catalysis of this reaction occurs, RmlA has an allosteric site that is important for its function. Building on previous reports, SAR studies have explored further the allosteric site, leading to the identification of very potent P. aeruginosa RmlA inhibitors. Modification at the C6-NH2 of the inhibitor’s pyrimidinedione core structure was tolerated. X-ray crystallographic analysis of the complexes of P. aeruginosa RmlA with the novel analogues revealed that C6-aminoalkyl substituents can be used to position a modifiable amine just outside the allosteric pocket. This opens up the possibility of linking a siderophore to this class of inhibitor with the goal of enhancing bacterial cell wall permeability.

This Deliverable describes the Project Legacy Plan (version I) of the TRUE-Project. It formally details how the various products developed from TRUE-Projectactivities will be manged and coordinated to ensure project impact legacy beyond the funded project term. To improve this aim the bulk of this document present a highly detailed series of vignettes that capturea wide range of perspectives from the exercise of, 'considering legumes in context'. Going forward, the TRUE-Project’s implementation plans will be developed based on these insights. Also, delivered collectively, they are expected to encourage transition from current conventional food- and feed-networks, to those which are legume-based and more sustainable.

Though a widely used metal-catalyzed cross-coupling process, the Mizoroki–Heck (MH) reaction can be a capricious transformation. This is particularly true for oxidation-prone alkene substrates containing ligating heteroatoms, as in the case of N-alkyl tetrahydropyridines, whose MH reactions have been underexplored due to the many side reactions that hamper the process. Since the products of tetrahydropyridine Heck reactions are direct precursors to potent pharmacophores, and therefore of commercial value, this is a significant drawback. We report here the results of our study designed to deliver an optimized, scalable MH procedure for N-alkyltetrahydropyridines and its exemplification in a gram-scale synthesis of the drug substance preclamol.

From analysing trade and infrastructures, a map has beenproduced detailing legume food supply chains. Rooted in project case studies and the Legume Innovation Network (LIN) workshops, the analysis will cover both conventional and organic production. Production, location, processing, actors, infrastructure, products, prices and markets have been analysed, including the institutional framework of the quality chains. The results have been compiled into thisreport, whichdetails the current and futureEU market-pull for legumes used as food, and it includesa map of infrastructure for processing and trade.

This document outlines the work carried out to complete Deliverable D4.3 ‘Review of historic datasets covering multiple stresses in tomato’. A systematic methodology has been thoroughly designed andapplied to identify, collate and synthesise the available evidence on techniques and management approaches that aim that optimise the productivity of tomatoes under conditions of water and/ornutrient (nitrogen and phosphorous) deficit. A systematic map was considered the most appropriate approach to meet the objective of the review, which includes the identification of the current state ofa range of interventions and extent to which each has been studied. The insight will help direct the foci of researchers, growers and policy makers.

Background There is an urgent need to ensure that food production is maintained in response to either a reduction in use or lack of availability of natural resources. To this end, several strategies have been investigated to determine which agronomic approaches may improve crop yields under conditions of reduced water and/or nutrients provision, with special attention upon nitrogen (N) and phosphorus (P). New technologies and practices have been developed for key commercial crops, such as tomatoes. However, few of these are widely adopted in the field and evidence of their value in this production setting is limited. Methods This protocol sets out a systematic map methodology that aims to provide a coherent synthesis of the available evidence among the literature on the techniques and management approaches that may ensure the productivity of field-grown tomatoes under conditions of water-, N- and/or P-deficits, either as single or combined stresses. To conduct the literature search, a search strategy was produced to define the scope of the systematic map and to allow reproducibility of the approach. A list of published and unpublished sources of literature were selected and a preliminary trial identified best-fit-for-purpose search-terms and -strings. A literature screening process was set with consistency checks amongst reviewers at the title, abstract and full text screening stages. A series of eligibility criteria were defined to ensure objectivity and consistency in the selection of studies that are best suited to address the research question of the systematic map. In addition, a coding strategy was designed to set the means for meta-data extraction out from the literature for review. A drafted structured questionnaire will serve as the base for collating the meta-data to produce a database where variables will be queried for the evidence synthesis. This work is expected to inform stakeholders, researchers and policy makers regarding the extent and nature of the existing evidence base, and so serve as a basis by-which specific approaches may be highlighted as potential focal-areas in future.

The common bean (Phaseolous vulgaris L.) is a grain legume functionally characterized by its capacity for symbiotic of biological nitrogen fixation. As such, it does not demand the application of synthetic nitrogenous fertilizer and can offer environmental benefits as part of holistic cropping systems. While common bean commodities are highly nutritious, commercial cultivation of this crop is declining in already-industrialized countries. However, recent interest of consumers towards diets that benefit environmental and personal health has rekindled commercial interest in legumes, including the common bean. The aim of this protocol is to identify agronomic practices that are capable of increasing the yield and quality of the common bean for use as food. To address this research question, published literature will be screened for inclusion on the basis of defined eligibility criteria to ensure data sources are selected in an objective and consistent manner. Consistency checks will be carried out for the title, abstract and full texts of the literature collated. The output is expected to be a summary of the knowledge available to maximize the productivity and quality of the common bean as food. This anticipated synthesis will be of utility for a wide range of value-chain stakeholders from farmers and consumers, to research scientists and policy makers.

The authors gratefully acknowledge financial support from the European Commission (SuBiCat Initial Training Network, Call FP7-PEOPLE-2013-ITN, grant no. 607044). Conversion of lignin into well-defined aromatic chemicals is a highly attractive goal, but is often hampered by recondensation of the formed fragments, especially in acidolysis. Here, we describe new strategies that markedly suppress such undesired pathways to result in diverse aromatic compounds previously not systematically targeted from lignin. Model studies established that a catalytic amount of triflic acid is very effective in cleaving the β-O-4 linkage, most abundant in lignin. An aldehyde product was identified as the main cause of side reactions under cleavage conditions. Capturing this unstable compound by reaction with diols and by in situ catalytic hydrogenation or decarbonylation lead to three distinct groups of aromatic compounds in high yields acetals, ethanol and ethyl aromatics, and methyl aromatics. Notably, the same product groups were obtained when these approaches were successfully extended to lignin. In addition, the formation of higher molecular weight side products was markedly suppressed, indicating that the aldehyde intermediates play a significant role in these processes. The described strategy has the potential to be generally applicable for the production of interesting aromatic compounds from lignin. Postprint

Functionalized phenolic monomers have been generated and isolated from an organosolv lignin through a two-step depolymerization process. Chemoselective catalytic oxidation of β-O-4 linkages promoted by the DDQ/tBuONO/O2 system was achieved in model compounds, including polymeric models and in real lignin. The oxidized β-O-4 linkages were then cleaved on reaction with zinc. Compared to many existing methods, this protocol, which can be achieved in one pot, is highly selective, giving rise to a simple mixture of products that can be readily purified to give pure compounds. The functionality present in these products makes them potentially valuable building blocks.

This work was funded by the EP/J018139/1, EP/K00445X/1 grants (NJW and PCJK), an EPSRC Doctoral Prize Fellowship (CSL), and the European Union (Marie Curie ITN ‘SuBiCat’ PITN-GA-2013-607044, CWL, NJW, PCJK, PJD, KB, JdeV). The development of fundamentally new approaches for lignin depolymerization is challenged by the complexity of this aromatic biopolymer. While overly simplified model compounds often lack relevance to the chemistry of lignin, the direct use of lignin streams poses significant analytical challenges to methodology development. Ideally, new methods should be tested on model compounds that are complex enough to mirror the structural diversity in lignin but still of sufficiently low molecular weight to enable facile analysis. In this contribution, we present a new class of advanced (β-O-4)-(β-5) dilinkage models that are highly realistic representations of a lignin fragment. Together with selected β-O-4, β-5, and β–β structures, these compounds provide a detailed understanding of the reactivity of various types of lignin linkages in acid catalysis in conjunction with stabilization of reactive intermediates using ethylene glycol. The use of these new models has allowed for identification of novel reaction pathways and intermediates and led to the characterization of new dimeric products in subsequent lignin depolymerization studies. The excellent correlation between model and lignin experiments highlights the relevance of this new class of model compounds for broader use in catalysis studies. Only by understanding the reactivity of the linkages in lignin at this level of detail can fully optimized lignin depolymerization strategies be developed. Postprint

HIV-1 maturation inhibitors are a novel class of antiretroviral compounds that consist of two structurally distinct chemical classes: betulinic acid derivatives and the pyridone-based compound PF-46396. It is currently believed that both classes act by similar modes of action to generate aberrant noninfectious particles via inhibition of CA-SP1 cleavage during Gag proteolytic processing. In this study, we utilized a series of novel analogues with decreasing similarity to PF-46396 to determine the chemical groups within PF-46396 that contribute to antiviral activity, Gag binding, and the relationship between these essential properties. A spectrum of antiviral activity (active, intermediate, and inactive) was observed across the analogue series with respect to CA-SP1 cleavage and HIV-1 (NL4-3) replication kinetics in Jurkat T cells. We demonstrate that selected inactive analogues are incorporated into wild-type (WT) immature particles and that one inactive analogue is capable of interfering with PF-46396 inhibition of CA-SP1 cleavage. Mutations that confer PF-46396 resistance can impose a defective phenotype on HIV-1 that can be rescued in a compound-dependent manner. Some inactive analogues retained the capacity to rescue PF-46396-dependent mutants (SP1-A3V, SP1-A3T, and CA-P157S), implying that they can also interact with mutant Gag. The structure-activity relationships observed in this study demonstrate that (i) the tert-butyl group is essential for antiviral activity but is not an absolute requirement for Gag binding, (ii) the trifluoromethyl group is optimal but not essential for antiviral activity, and (iii) the 2-aminoindan group is important for antiviral activity and Gag binding but is not essential, as its replacement is tolerated. IMPORTANCE Combinations of antiretroviral drugs successfully treat HIV/AIDS patients; however, drug resistance problems make the development of new mechanistic drug classes an ongoing priority. HIV-1 maturation inhibitors are novel as they target the Gag protein, specifically by inhibiting CA-SP1 proteolytic cleavage. The lack of high-resolution structural information of the CA-SP1 target in Gag has hindered our understanding of the inhibitor-binding pocket and maturation inhibitor mode of action. Therefore, we utilized analogues of the maturation inhibitor PF-46396 as chemical tools to determine the chemical components of PF-46396 that contribute to antiviral activity and Gag binding and the relationship between these essential properties. This is the first study to report structure-activity relationships of the maturation inhibitor PF-46396. PF-46396 is chemically distinct from betulinic acid-derived maturation inhibitors; therefore, our data provide a foundation of knowledge that will aid our understanding of how structurally distinct maturation inhibitors act by similar modes of action.

This work was supported in part by National Institutes of Health Grants AI-110027 and AI-096836 (to S. N. J. M.) and 1DP5OD017892 (to S. L.). The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca2+. Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca2+ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca2+ signaling in the model apicomplexan Toxoplasma gondii. In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca2+. We define the pool of Ca2+ regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca2+ signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca2+. The enhancers identified are capable of releasing intracellular Ca2+ stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii. The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum. Inhibition of Ca2+-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca2+ stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca2+, underscoring the importance of these pathways and the therapeutic potential of their inhibition. Publisher PDF

The tenovins are small molecule inhibitors of the NAD(+)-dependent family of protein deacetylases known as the sirtuins. There remains considerable interest in inhibitors of this enzyme family due to possible applications in both cancer and neurodegenerative disease therapy. Through the synthesis of novel tenovin analogues, further insights into the structural requirements for activity against the sirtuins in vitro are provided. In addition, the activity of one of the analogues in cells led to an improved understanding of the function of SirT1 in cells.

The research reported here was funded by BBSRC and a consortium of industry partners comprising the IBTI Club. The NMR component of this work (FT, NJW) was funded by the EPSRC grants EP/J018139/1 and EP/K00445X/1 and through the International Training Network SuBiCat. Four lignin preparations with different contents of alkyl aryl ether bonds were depolymerised using an alumina supported platinum catalyst. The results showed that the proportion of β-O-4 linkages is the crucial factor for both the yield and the nature of the monomeric products. Highly condensed lignin generated mainly non-alkylated phenolic products while uncondensed lignin generated mainly phenolic products retaining the 3-carbon side-chain. These phenolic products with the 3-carbon chain still attached were considerably less abundant than the maximum potential yield calculated from selective cleavage of alkyl aryl ether bonds by thioacidolysis, demonstrating that scope for improved yield remains. Although the catalytic conversion yield rose with increasing content of labile ether linkages in the lignin structure, optimisation of the catalytic depolymerisation was increasingly required to minimize side reactions. Gel permeation chromatography showed that the products converged towards the same molecular weight distribution regardless of the starting material. The full potential of the highly uncondensed lignin was reached only after the minimisation of condensation reactions during the catalytic conversion. Publisher PDF

This work was supported by US Public Health Service grant AI054961 The yeast three-hybrid (Y3H) approach shows considerable promise for the unbiased identification of novel small molecule-protein interactions. In recent years, it has been successfully used to link a number of bioactive molecules to novel protein binding partners. However despite its potential importance as a protein target identification method, the Y3H technique has not yet been widely adopted, in part due to the challenges associated with the synthesis of the complex chemical inducers of dimerisation (CIDs). The development of a modular approach using potentially "off the shelf" synthetic components was achieved and allowed the synthesis of a family of four triazole-containing CIDs, MTX-Cmpd2.2-2.5. These CIDs were then compared using the Y3H approach with three of them giving a strong positive interaction with a known target of compound 2, TgCDPK1. These results showed that the modular nature of our synthetic strategy may help to overcome the challenges currently encountered with CID synthesis and should contribute to the Y3H approach reaching its full potential as an unbiased target identification strategy. Publisher PDF Publisher PDF

This work was supported by National Institutes of Health (NIH) grant AI054961 to GEW, and Royal Society University Research grant 516002.K5682/KK to NJW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Differentiation of the protozoan parasite Toxoplasma gondii into its latent bradyzoite stage is a key event in the parasite’s life cycle. Compound 2 is an imidazopyridine that was previously shown to inhibit the parasite lytic cycle, in part through inhibition of parasite cGMP-dependent protein kinase. We show here that Compound 2 can also enhance parasite differentiation, and we use yeast three-hybrid analysis to identify TgBRADIN/GRA24 as a parasite protein that interacts directly or indirectly with the compound. Disruption of the TgBRADIN/GRA24 gene leads to enhanced differentiation of the parasite, and the TgBRADIN/GRA24 knockout parasites show decreased susceptibility to the differentiation-enhancing effects of Compound 2. This study represents the first use of yeast three-hybrid analysis to study small-molecule mechanism of action in any pathogenic microorganism, and it identifies a previously unrecognized inhibitor of differentiation in T. gondii. A better understanding of the proteins and mechanisms regulating T. gondii differentiation will enable new approaches to preventing the establishment of chronic infection in this important human pathogen. Publisher PDF

This work was supported in part by NIH grant 1DP5OD017892 to S.L Toxoplasma gondii is a parasite of humans and animals, and a model for other apicomplexans including Plasmodium spp., the causative agents of malaria. Despite many advances, manipulating the T. gondii genome remains labor intensive, and is often restricted to lab-adapted strains or lines carrying mutations that enable selection. Here, we use the RNA-guided Cas9 nuclease to efficiently generate knockouts without selection, and to introduce point mutations and epitope tags into the T. gondii genome. These methods will streamline the functional analysis of parasite genes and enable high-throughput engineering of their genomes. Publisher PDF

This recipe book aims to present the positive properties of pulses and to highlight their valuable contribution to an improved food system. By modifying our daily diets to increase our consumption of pulses, we can help to bring about long-term changes for more environmentally friendly agriculture. Key to this transition is the consumers and the daily decisions they make whilst cooking or eating. The book showcases examples of recipes featuring pulses and encourages cookers to be creative with them. It also demonstrates the local legume traditions that have prevailed in Europe for many centuries and the cultural roots that are connected to them. Finally, the nutritional values and ecological footprint data of each recipe underpin the benefits of legumes for both health and the environment. This book is available digitally to reach a wide audience. It will also be distributed through the Slow Food networks and Slow Food Germany will distribute printed copies at various events and workshops to further promote the benefits of legumes.

This is the final report on this deliverable. It presents a methodological description of the sector model, CAPRI, and the scenarios used to examine legume production under three different pathways which were assumed to be the conditions which would lead to increase legume production in the EU27+UK. Three alternative legume production scenarios have been identified and described including changes in legume support payment (policy), dietary change (consumer demand) and soya import ban (trade). The scenario results were compared with a baseline scenario, analysed and presented in this report.