High Inter- and Intra- Diversity of Amino Acid Content and Protein Digestibility Disclosed in Five Cool Season Legume Species with a Growing Market Demand.

Legumes have been sought as alternative protein sources to ensure food security and environmental sustainability. Characterizing their protein content and quality, including in underutilized grain legumes, e.g., grass pea, gives value to the legumes' underexplored variability. To fill the gap of knowledge in legumes' protein quality, for the first time, five extensive collections of cool season grain legumes were cropped under the same environmental conditions and further analyzed. Multivariate analysis showed the existent intra- and inter-species variability. The legume species with the highest protein content, grass pea, Lathyrus sativus (LS), was not the one with the overall highest individual amino acids content and in vitro protein digestibility. With these last characteristics lentil, Lens culinaris (LC), was highlighted. The highest average values of arginine (Arg), glutamic acid (Glu), and threonine (Thr) were found in LS and Vicia faba (VF). Cicer arietinum (CA) stood out as the species with the highest values of Thr and methionine (Met). Regarding the in vitro protein digestibility (IVPD), LC, followed by Pisum sativum (PS) and LS, were the legume species with the highest values. Ultimately, this study bought to the fore legume species that are not commonly used in western diets but have high adaptability to the European agricultural systems.

Organ donation after circulatory death as compared with organ donation after brain death in Switzerland - an observational study.

AIMS OF THE STUDY: Organ donation after circulatory death (DCD) was reintroduced in Switzerland in 2011 and accounts for a third of deceased organ donors today. Controversy persists if DCD transplants are of similar quality to transplants following donation after brain death (DBD), mainly due to warm ischaemia time DCD organs are exposed to. We compared DCD with DBD in Switzerland. METHODS: Data on deceased adults who were referred to and approved for organ donation from 1 September 2011 to 31 December 2019 were retrospectively analysed (217 DCD, 840 DBD donors). We compared DCD and DBD donor/organ characteristics, transplant rates of lungs, liver, kidneys, and pancreas, and early liver and kidney graft function in the recipient. The effect of DCD/DBD on transplant rates (organ transplanted or not) and 72-hour recipient graft function (moderate/good vs delayed graft function / organ loss) was analysed using multivariable logistic regression. Among utilised DCD donors, we analysed the effect of functional warm ischaemia time (FWIT) and donor age on 72-hour post-transplant liver and kidney graft function, also using multivariable logistic regression. RESULTS: DCD donors were more often male (64.5% vs 56.8% p = 0.039), presented with heart disease (36.4% vs 25.5%, p <0.001), were resuscitated before hospital admission (41.9% vs 30.7%, p = 0.006), and died from anoxia (41.9% vs 23.9%). Kidney function before transplantation was comparable, lung, liver and pancreas function were poorer in DCD than DBD. Eighty-one and 91% of approved DCD and DBD donors were utilised (p <0.001). Median FWIT in DCD was 29 minutes (interquartile range 25-35). DCD transplant rates ranged from 4% (pancreas) to 73% (left kidney) and were all lower compared with DBD. Seventy-two-hour liver graft function was comparable between DCD and DBD (94.2% vs 96.6% moderate/good, p = 0.199). DCD kidney transplants showed increased risk of delayed graft function or early organ loss (odds ratios 8.32 and 5.05; 95% confidence intervals CI 5.28-13.28 and 3.22-7.95; both p <0.001, for left and right kidney transplants, respectively). No negative effect of prolonged FWIT or higher donor age was detected. CONCLUSION: Despite less favourable donor/organ characteristics compared with donation after brain death, donation after circulatory death donors are increasingly referred and today provide an important source for scarce transplants in Switzerland. We identified a higher risk for delayed graft function or early organ loss for DCD kidney transplants, but not for DCD liver transplants. When carefully selected and allowed for other risk factors in organ allocation, prolonged functional warm ischaemia time or higher age in donation after circulatory death does not seem to be associated with impaired graft function early after transplantation.

Grass pea natural variation reveals oligogenic resistance to Fusarium oxysporum f. sp. pisi.

Grass pea (Lathyrus sativus L.) is an annual legume species, phylogenetically close to pea (Pisum sativum L.), that may be infected by Fusarium oxysporum f. sp. pisi (Fop), the causal agent of fusarium wilt in peas with vast worldwide yield losses. A range of responses varying from high resistance to susceptibility to this pathogen has been reported in grass pea germplasm. Nevertheless, the genetic basis of that diversity of responses is still unknown, hampering its breeding exploitation. To identify genomic regions controlling grass pea resistance to fusarium wilt, a genome-wide association study approach was applied on a grass pea worldwide collection of accessions inoculated with Fop race 2. Disease responses were scored in this collection that was also subjected to high-throughput based single nucleotide polymorphisms (SNP) screening through genotyping-by-sequencing. A total of 5,651 high-quality SNPs were considered for association mapping analysis, performed using mixed linear models accounting for population structure. Because of the absence of a fully assembled grass pea reference genome, SNP markers' genomic positions were retrieved from the pea's reference genome v1a. In total, 17 genomic regions were associated with three fusarium wilt response traits in grass pea, anticipating an oligogenic control. Seven of these regions were located on pea chromosomes 1, 6, and 7. The candidate genes underlying these regions were putatively involved in secondary and amino acid metabolism, RNA (regulation of transcription), transport, and development. This study revealed important fusarium wilt resistance favorable grass pea SNP alleles, allowing the development of molecular tools for precision disease resistance breeding.

Towards a Trait-Based Approach to Potentiate Yield under Drought in Legume-Rich Annual Forage Mixtures.

Mediterranean annual forage mixtures are facing the impact of climate change, especially higher frequencies of winter-time drought. Increased mixture plasticity to climate variability is needed to mitigate this impact. However, little information exists regarding the specificities and complementarities of each forage species component to potentiate mixture resilience under drought. In this study, we identified traits with breeding potential under water scarcity through a detailed characterization of leaf and root-related parameters of 10 legume and grass species components of Mediterranean annual forage mixtures, complemented by their photosynthetic response evaluation under well-watered and water deficit conditions. This integrated approach also allowed us to identify the most resilient species to water deficit. In particular, we found that the highest canopy height and root to shoot ratio of grass components complemented well the highest aerial and root biomass and superior photosynthetic performance of the legume components. Trifolium squarrosum and Triticosecale showed the most adequate combination of traits and the best photosynthetic performance under water deficit within each species family. Although some of these traits are not commonly used in annual forage selection, they may in part explain the potential higher resilience of the grass-legume mixture under water deficit and should be considered in forage breeding.

Accessing Ancestral Origin and Diversity Evolution by Net Divergence of an Ongoing Domestication Mediterranean Olive Tree Variety.

Olea europaea 'Galega vulgar' variety is a blend of West and Central Mediterranean germplasm with cultivated-wild admixture characteristics. 'Galega vulgar' is known for its high rusticity and superior-quality olive oil, being the main Portuguese variety with high impact for bioeconomy. Nevertheless, it has been replaced by higher-yielding and more adapted to intensive production foreign varieties. To clarify the potential ancestral origin, genetic diversity evolution, and existing genetic relationships within the national heritage of 'Galega vulgar', 595 trees, belonging to ancient and centenary age groups and prospected among ten traditional production regions, were characterized using 14 SSR markers after variety validation by endocarp measurements. Ninety-five distinguishable genets were identified, revealing the presence of a reasonable amount of intra-genetic and morphological variability. A minimum spanning tree, depicting the complete genealogy of all identified genets, represented the 'Galega vulgar' intra-varietal diversity, with 94% of the trees showing only a two-allele difference from the most frequent genet (C001). Strong correlations between the number of differentiating alleles from C001, the clonal size, and their net divergence suggested an ancestral monoclonal origin of the 'Galega vulgar', with the most frequent genet identified as the most likely origin of all the genets and phenotypic diversification occurring through somatic mutations. Genetic erosion was detected through the loss of some allele combinations across time. This work highlights the need to recover the lost diversity in this traditional olive variety by including ancient private genets (associated with potential adaptation traits) in future breeding programs and investing in the protection of these valuable resources in situ by safeguarding the defined region of origin and dispersion of 'Galega vulgar'. Furthermore, this approach proved useful on a highly diverse olive variety and thus applicable to other diverse varieties due either to their intermediate nature between different gene pools or to the presence of a mixture of cultivated and wild traits (as is the case of 'Galega vulgar').

Traditional Foods From Maize (Zea mays L.) in Europe.

Maize (Zea mays L.) is one of the major crops of the world for feed, food, and industrial uses. It was originated in Central America and introduced into Europe and other continents after Columbus trips at the end of the 15th century. Due to the large adaptability of maize, farmers have originated a wide variability of genetic resources with wide diversity of adaptation, characteristics, and uses. Nowadays, in Europe, maize is mainly used for feed, but several food specialties were originated during these five centuries of maize history and became traditional food specialties. This review summarizes the state of the art of traditional foodstuffs made with maize in Southern, South-Western and South-Eastern Europe, from an historic evolution to the last research activities that focus on improving sustainability, quality and safety of food production.

Volatilome-Genome-Wide Association Study on Wholemeal Maize Flour.

Odor and aroma, resulting from the perception of volatiles by the olfactory receptors, are important in consumer food acceptance. To develop more efficient molecular breeding tools to improve the odor/aroma on maize (Zea mays L.), a staple food crop, increasing the knowledge on the genetic basis of maize volatilome is needed. In this work, we conducted a genome-wide association study on a unique germplasm collection to identify genomic regions controlling maize wholemeal flour's volatilome. We identified 64 regions on the maize genome and candidate genes controlling the levels of 15 volatiles, mainly aldehydes. As an example, the Zm00001d033623 gene was within a region associated with 2-octenal (E) and 2-nonenal (E), two byproducts of linoleic acid oxidation. This gene codes for linoleate 9S-lipoxygenase, an enzyme responsible for oxidizing linoleic acid. This knowledge can now support the development of molecular tools to increase the selection efficacy/efficiency of these volatiles within maize breeding programs.

Alleles to Enhance Antioxidant Content in Maize-A Genome-Wide Association Approach.

The interest in antioxidant compound breeding in maize (Zea mays L.), a major food crop, has increased in recent years. However, breeding of antioxidant compounds in maize can be hampered, given the complex genetic nature of these compounds. In this work, we followed a genome-wide association approach, using a unique germplasm collection (containing Portuguese germplasm), to study the genetic basis of several antioxidants in maize. Sixty-seven genomic regions associated with seven antioxidant compounds and two color-related traits were identified. Several significant associations were located within or near genes involved in the carotenoid (Zm00001d036345) and tocopherol biosynthetic pathways (Zm00001d017746). Some indications of a negative selection against α-tocopherol levels were detected in the Portuguese maize germplasm. The strongest single nucleotide polymorphism (SNP)-trait associations and the SNP alleles with larger effect sizes were pinpointed and set as priority for future validation studies; these associations detected now constitute a benchmark for developing molecular selection tools for antioxidant compound selection in maize.

Genome-wide association study for kernel composition and flour pasting behavior in wholemeal maize flour.

BACKGROUND: Maize is a crop in high demand for food purposes and consumers worldwide are increasingly concerned with food quality. However, breeding for improved quality is a complex task and therefore developing tools to select for better quality products is of great importance. Kernel composition, flour pasting behavior, and flour particle size have been previously identified as crucial for maize-based food quality. In this work we carried out a genome-wide association study to identify genomic regions controlling compositional and pasting properties of maize wholemeal flour. RESULTS: A collection of 132 diverse inbred lines, with a considerable representation of the food used Portuguese unique germplasm, was trialed during two seasons, and harvested samples characterized for main compositional traits, flour pasting parameters and mean particle size. The collection was genotyped with the MaizeSNP50 array. SNP-trait associations were tested using a mixed linear model accounting for genetic relatedness. Fifty-seven genomic regions were identified, associated with the 11 different quality-related traits evaluated. Regions controlling multiple traits were detected and potential candidate genes identified. As an example, for two viscosity parameters that reflect the capacity of the starch to absorb water and swell, the strongest common associated region was located near the dull endosperm 1 gene that encodes a starch synthase and is determinant on the starch endosperm structure in maize. CONCLUSIONS: This study allowed for identifying relevant regions on the maize genome affecting maize kernel composition and flour pasting behavior, candidate genes for the majority of the quality-associated genomic regions, or the most promising target regions to develop molecular tools to increase efficacy and efficiency of quality traits selection (such as "breadability") within maize breeding programs.

First genetic linkage map of Lathyrus cicera based on RNA sequencing-derived markers: Key tool for genetic mapping of disease resistance.

The Lathyrus cicera transcriptome was analysed in response to rust (Uromyces pisi) infection to develop novel molecular breeding tools with potential for genetic mapping of resistance in this robust orphan legume species. One RNA-seq library each was generated from control and rust-inoculated leaves from two L. cicera genotypes with contrasting quantitative resistance, de novo assembled into contigs and sequence polymorphisms were identified. In toto, 19,224 SNPs differentiate the susceptible from the partially resistant genotype's transcriptome. In addition, we developed and tested 341 expressed E-SSR markers from the contigs, of which 60.7% varied between the two L. cicera genotypes. A first L. cicera linkage map was created using part of the developed markers in a RIL population from the cross of the two genotypes. This map contains 307 markers, covered 724.2 cM and is organised in 7 major and 2 minor linkage groups, with an average mapping interval of 2.4 cM. The genic markers also enabled us to compare their position in L. cicera map with the physical position of the same markers mapped on Medicago truncatula genome, highlighting a high macrosyntenic conservation between both species. This study provides a large new set of genic polymorphic molecular markers with potential for mapping rust resistances. It represents the first step towards genomics-assisted precision breeding in L. cicera.

Long-term on-farm participatory maize breeding by stratified mass selection retains molecular diversity while improving agronomic performance.

Modern maize breeding programs gave rise to genetically uniform varieties that can affect maize's capacity to cope with increasing climate unpredictability. Maize populations, genetically more heterogeneous, can evolve and better adapt to a broader range of edaphic-climatic conditions. These populations usually suffer from low yields; it is therefore desirable to improve their agronomic performance while maintaining their valuable diversity levels. With this objective, a long-term participatory breeding/on-farm conservation program was established in Portugal. In this program, maize populations were subject to stratified mass selection. This work aimed to estimate the effect of on-farm stratified mass selection on the agronomic performance, quality, and molecular diversity of two historical maize populations. Multilocation field trials, comparing the initial populations with the derived selection cycles, showed that this selection methodology led to agronomic improvement for one of the populations. The molecular diversity analysis, using microsatellites, revealed that overall genetic diversity in both populations was maintained throughout selection. The comparison of quality parameters between the initial populations and the derived selection cycles was made using kernel from a common-garden experiment. This analysis showed that the majority of the quality traits evaluated progressed erratically over time. In conclusion, this breeding approach, through simple and low-cost methodologies, proved to be an alternative strategy for genetic resources' on-farm conservation.

Senescence-associated SIN3B promotes inflammation and pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is strikingly resistant to conventional therapeutic approaches. We previously demonstrated that the histone deacetylase-associated protein SIN3B is essential for oncogene-induced senescence in cultured cells. Here, using a mouse model of pancreatic cancer, we have demonstrated that SIN3B is required for activated KRAS-induced senescence in vivo. Surprisingly, impaired senescence as the result of genetic inactivation of Sin3B was associated with delayed PDAC progression and correlated with an impaired inflammatory response. In murine and human pancreatic cells and tissues, levels of SIN3B correlated with KRAS-induced production of IL-1α. Furthermore, evaluation of human pancreatic tissue and cancer cells revealed that Sin3B was decreased in control and PDAC samples, compared with samples from patients with pancreatic inflammation. These results indicate that senescence-associated inflammation positively correlates with PDAC progression and suggest that SIN3B has potential as a therapeutic target for inhibiting inflammation-driven tumorigenesis.

Phenocopy--a strategy to qualify chemical compounds during hit-to-lead and/or lead optimization.

A phenocopy is defined as an environmentally induced phenotype of one individual which is identical to the genotype-determined phenotype of another individual. The phenocopy phenomenon has been translated to the drug discovery process as phenotypes produced by the treatment of biological systems with new chemical entities (NCE) may resemble environmentally induced phenotypic modifications. Various new chemical entities exerting inhibition of the kinase activity of Transforming Growth Factor ß Receptor I (TGF-ßR1) were qualified by high-throughput RNA expression profiling. This chemical genomics approach resulted in a precise time-dependent insight to the TGF-ß biology and allowed furthermore a comprehensive analysis of each NCE's off-target effects. The evaluation of off-target effects by the phenocopy approach allows a more accurate and integrated view on optimized compounds, supplementing classical biological evaluation parameters such as potency and selectivity. It has therefore the potential to become a novel method for ranking compounds during various drug discovery phases.

Inhibition of pro-inflammatory cytokine production by the dual p38/JNK2 inhibitor BIRB796 correlates with the inhibition of p38 signaling.

The characterization of the potent p38 inhibitor BIRB796 as a dual inhibitor of p38/Jun N-terminal kinases (JNK) mitogen-activated protein kinases (EC 2.7.11.24) has complicated the interpretation of its reported anti-inflammatory activity. To better understand the contribution of JNK2 inhibition to the anti-inflammatory activities of BIRB796, we explored the relationship between the effects of BIRB796 and analogues on cytokine production and on cellular p38 and JNK signaling. We determined the binding affinity for BIRB796 and structural analogues to p38alpha and JNK2 and characterized compound 2 as a p38 inhibitor that binds to p38alpha with an affinity equivalent to BIRB796 but does not bind to any of the JNK isoforms. High-content imaging enabled us to show that the inhibition of p38 signaling by BIRB796 and analogues correlates with the ability of these compounds to inhibit the lipopolysaccharide (LPS)-induced TNF-alpha production in THP-1 monocytes. This finding was extended to cytokine release by disease-relevant human primary cells: to the production of TNF-alpha by peripheral blood mononuclear cells, and of IL-8 by neutrophils. Furthermore, BIRB796 and compound 2 inhibited the production of TNF-alpha in THP-1 monocytes and the IL-12/IL-18-induced production of interferon-gamma in human T-cells with similar potencies. In contrast, cellular JNK signaling in response to cytokines or stress stimuli was only weakly inhibited by BIRB796 and analogues and not affected by compound 2. In summary, our data suggest that p38 inhibition alone is sufficient to completely suppress cytokine production and that the added inhibition of JNK2 does not significantly contribute to the effects of BIRB796 on cytokine production.