Genetic analysis of global faba bean diversity, agronomic traits and selection signatures.

KEY MESSAGE: We identified marker-trait associations for key faba bean agronomic traits and genomic signatures of selection within a global germplasm collection. Faba bean (Vicia faba L.) is a high-protein grain legume crop with great potential for sustainable protein production. However, little is known about the genetics underlying trait diversity. In this study, we used 21,345 high-quality SNP markers to genetically characterize 2678 faba bean genotypes. We performed genome-wide association studies of key agronomic traits using a seven-parent-MAGIC population and detected 238 significant marker-trait associations linked to 12 traits of agronomic importance. Sixty-five of these were stable across multiple environments. Using a non-redundant diversity panel of 685 accessions from 52 countries, we identified three subpopulations differentiated by geographical origin and 33 genomic regions subjected to strong diversifying selection between subpopulations. We found that SNP markers associated with the differentiation of northern and southern accessions explained a significant proportion of agronomic trait variance in the seven-parent-MAGIC population, suggesting that some of these traits were targets of selection during breeding. Our findings point to genomic regions associated with important agronomic traits and selection, facilitating faba bean genomics-based breeding.

QTL dissection and mining of candidate genes for Ascochyta fabae and Orobanche crenata resistance in faba bean (Vicia faba L.).

BACKGROUND: Ascochyta blight caused by Ascochyta fabae Speg. and broomrape (Orobanche crenata) are among the economically most significant pathogens of faba bean. Several QTLs conferring resistance against the two pathogens have been identified and validated in different genetic backgrounds. The aim of this study was to saturate the most stable QTLs for ascochyta and broomrape resistance in two Recombinant Inbred Line (RIL) populations, 29H x Vf136 and Vf6 x Vf136, to identify candidate genes conferring resistance against these two pathogens. RESULTS: We exploited the synteny between faba bean and the model species Medicago truncatula by selecting a set of 219 genes encoding putative WRKY transcription factors and defense related proteins falling within the target QTL intervals, for genotyping and marker saturation in the two RIL populations. Seventy and 50 of the candidate genes could be mapped in 29H x Vf136 and Vf6 x Vf136, respectively. Besides the strong reduction of the QTL intervals, the mapping process allowed replacing previous dominant and pedigree-specific RAPD flanking markers with robust and transferrable SNP markers, revealing promising candidates for resistance against the two pathogens. CONCLUSIONS: Although further efforts in association mapping and expression studies will be required to corroborate the candidate genes for resistance, the fine-mapping approach proposed here increases the genetic resolution of relevant QTL regions and paves the way for an efficient deployment of useful alleles for faba bean ascochyta and broomrape resistance through marker-assisted breeding.

Bond Strength of Composite Resin Restoration Repair: Influence of Silane and Adhesive Systems.

AIM: The aim of this study is to evaluate the effect of silane (Si) application and different adhesive systems on the bond strength of composite resin repair. MATERIALS AND METHODS: One hundred composite truncated cone-shaped specimens were prepared and submitted to 5,000 thermal cycles to simulate existing restorations. Their top surfaces were airborne particle abraded with aluminum oxide, etched with phosphoric acid, and divided into two groups (n = 50) with or without Si application. Each group was divided into five subgroups (n = 10) according to the adhesive system applied: Solobond Plus Primer and Adhesive (SPA)-two-bottle, Solobond Plus adhesive (SA), Admira Bond (A)-one bottle, Futurabond DC (FDC)-self-etch, and Futurabond M (FM)-self-etch. New composite resin was applied over the bonded area. A control group was prepared to evaluate the cohesive strength of the composite resin. Specimens were submitted to tensile stress. Data were analyzed with two-way analysis of variance (ANOVA), and the Tukey and Dunnett tests. RESULTS: Si application reduced the bond strength of all adhesives (p = 0.001). Groups SA and SPA showed higher bond strengths in relation to other groups (p = 0.01). Groups FDC + Si, FM, FM + Si, and A + Si showed smaller mean bond strength values than that of the control group (p < 0.05). CONCLUSION: Previous Si application reduced bond strength values. The two-bottle adhesive showed better results than one-bottle or self-etching systems for composite resin repairs. CLINICAL SIGNIFICANCE: The kind of adhesive system applied for composite resin repairs has a great influence on bond strength values. The use of Si in this situation is not recommended.

Minimally invasive plate osteosynthesis in humerus nonunion after intramedullary nailing.

INTRODUCTION: The purpose of this study was to evaluate the results achieved after the use of lateral minimally invasive plate osteosynthesis (MIPO) in oligotrophic humerus nonunions resulting from failed intramedullary nailing (IM). METHOD: We evaluated nine patients with humerus nonunion after failed locked intramedullary nailing, all treated using 3.5-mm locked compression plates (LCP) placed through lateral minimally invasive approaches, between 2010 and 2016. Patient's age averaged 39.7 years. All nonunions were diaphyseal and oligotrophic. All nonunions had previous surgical treatment with static locked nails (seven antegrade and two retrograde). The IM nails were all well inserted in the humerus (none of them protruded or had rotator cuff lesions associated). Pre-operative Disabilities of the Arm, Shoulder and Hand (DASH) score averaged 25.5 points. Pre-operative Constant's score averaged 80.2. Pre-operative visual analog scale of pain averaged 2.4 points. RESULTS: Follow-up averaged 17.7 months. Time between initial surgery and revision procedure averaged 11.7 months. Union was achieved in all cases, after an average of 4.8 months. DASH score at last follow-up averaged 5.1 points, and final Constant's score averaged 93.7 points. The analog scale of pain averaged 0.7 points. Time from definitive surgery to work return averaged 3.9 months. Long 3.5-mm LCPs were used (plate length averaged 16.9 screw holes). In two cases, a third 4-cm incision at the nonunion site was performed and cancellous autologous iliac crest bone graft was associated. CONCLUSION: In our series of nine patients, we achieved union and good objective and subjective results, with high patient satisfaction, using a lateral MIPO technique and placing long 3.5-mm LCPs in selected oligotrophic humerus nonunions after failed IM nailing.

Effects of Different Surface Treatments on Composite Repairs.

PURPOSE: To evaluate the influence of different surface treatments on roughness and bond strength of composite repairs. MATERIALS AND METHODS: 120 truncated conical specimens were prepared with composite Grandio SO (VOCO) and submitted to 5000 thermal cycles. Specimens were divided into 12 groups (n = 10) regarding the surface treatments: negative control (NC), without treatment; medium-grit diamond bur (MGD); coarse-grit diamond bur (CGD); conventional carbide bur (ConC); crosscut carbide bur (CutC); chemical vapor deposition diamond bur (CVD); sandblasting with aluminum oxide (AlO); Er:YAG laser 200 mJ/10 Hz (Er200); Er:YAG laser 60 mJ/10 Hz (Er50); Nd:YAG laser 120 mJ/15 Hz (Nd120); Nd:YAG laser 60 mJ/ 15Hz (Nd60); air abrasion with 110-µm silica modified aluminum oxide (Rocatec Plus-3M) (SIL). After the surface treatments, the surface roughness (Ra) was measured using a profilometer, and then the adhesive system Admira Bond (VOCO) was applied. Another truncated conical restoration was built up with the same composite over the bonded area of each specimen. In order to evaluate the cohesive strength, double-cone specimens were made and considered as a control group (CoheC). The specimens were submitted to tensile bond strength testing and the obtained data (MPa) were evaluated by one-way ANOVA, Tukey's and correlation tests. RESULTS: ANOVA showed significant differences among experimental groups for roughness and adhesive strength (p < 0.00). The roughness values (Ra) were: NC (0.21 ± 0.19)(c); ConC (0.30 ± 0.08)(c); CutC (0.50 ± 0.22)(cd); CVD (0.74 ± 0.14)(bd); MGD (0.89 ± 0.39)(ab); Er50 (0.89 ± 0.14)(ab); AlO (0.90 ± 0.07)(ab); Nd60 (0.94 ± 0.33ab; SIL (0.98 ± 0.07)(ab); Nd120 (1.10 ± 0.19)(a); CGD (1.10 ± 0.32)(a); Er200 (1.12 ± 0.21)(a). The results of the tensile bond strength test in MPa were: CGD (11.58 ± 3.03)(a); MGD (12.66 ± 3.82)(ab); NC (13.51 ± 3.95(ab); Nd120 (14.11 ± 5.95)(ab); ConC (14.73 ± 6.12)(ab); Er200 (15.51 ± 1.45)(abc); CVD (15.61 ± 5.00(abc); Er50 (16.44 ± 2.75) (abc); CutC (16.79 ± 2.98)(abc); Nd60 (17.72 ± 2.45)(abcd); AlO (18.33 ± 3.19)(bcd); SIL (21.13 ± 4.48(cd); CoheC (23.50 ± 5.81)(d). The groups followed by the same letters were not statistically significantly different (Tukey's test). No correlation was found between bond strength and roughness (r = 0.007). CONCLUSION: Air abrasion with silica coating (Rocatec) was the only method which resulted in significantly higher bond strength in relation to the negative control group. The increase in laser energy produced a rougher surface, but reduced the bond strength.

Effect of Matrix Metalloproteinase-inhibiting Solutions and Aging Methods on Dentin Bond Strength.

PURPOSE: This study examined the effects of matrix metalloproteinase-inhibiting solutions and aging methods on the bond strength between resin composite and human dentin. MATERIALS AND METHODS: Crown segments of 105 human non-carious molars were bonded using simulated pulpal pressure at 20 cm water pressure. The teeth were randomly split into 5 groups according to the solution applied: CG (control, no solution), CHX (0.2% chlorhexidine), EPE (10% ethanolic propolis extract), APE (aqueous propolis extract), and E (70% ethanol). Each solution was left on the acid-etched dentin for 1 min. Adper Single Bond 2 and resin composite (Filtek Z350 XT) were applied to all specimens. The 5 groups were subdivided according to the aging method: SI (sectioned immediately); S (storage in artificial saliva for 6 months); and T (thermomechanical aging with 240,000 mechanical cycles and 1000 thermal cycles). Specimens were sectioned into sticks and subjected to microtensile testing. Bond strength data were analyzed by two-factor ANOVA followed by a post-hoc Tukey's test (α=0.05). RESULTS: For the factor "solution", there was no significant difference among the groups (p=0.32). For the factor "aging method", significant differences were found (p<0.001), with the following mean bond strengths (MPa): SI: 31.1; S: 24.4; T: 26.8. CONCLUSIONS: The use of matrix metalloproteinase-inhibiting solutions on dentin as an adjunct to the application of an etch-and-rinse adhesive does not prevent the loss of bond strength after aging. Nevertheless, these solutions have no adverse effect on adhesion to tooth structure.

Health literacy, health information seeking behaviors and internet use among patients attending a private and public clinic in the same geographic area.

Despite the growing body of health information available online, patients with limited health literacy may lack either internet access or skills necessary to utilize this information. Nonetheless, patients at all health literacy levels may prefer other primary sources to obtain health information. We conducted a cross-sectional study to measure health literacy of patients attending two clinics in Dallas, TX and determine associations between health literacy, health information access and internet usage before and after controlling for confounders. Patients from both clinics (county N = 265; private N = 233) completed a brief survey which included sociodemographics, internet patterns, confidence in filling out medical forms and a self-administered Newest Vital Sign to measure health literacy. In the county clinic, most patients (61.5 %) were Hispanic, had low income (<$19,000/year), limited education (<11th grade) and a high likelihood or possibility of limited health literacy (68.5 %). In the private clinic, participants were mostly black (40.4 %) or white (38.6 %), had higher incomes (≥$46,000), higher education (technical college or college) and adequate health literacy (75.1 %). The primary source of obtaining health information in both clinics was their health care professional (50.6 % county; 40.1 % private). In multivariate analyses to determine differences by health literacy level, there were no statistically significant differences between patients with limited and adequate health literacy and their primary information source. Regardless of health literacy, patients rely on their health care providers to obtain health information. These results showcase the importance of providers' effective communication with patients to make shared decisions about their health regardless of other factors.

Transcriptome Analysis of Stigmas of Vicia faba L. Flowers.

Pollination in angiosperms depends on complex communication between pollen grains and stigmas, classified as wet or dry, depending on the presence or absence of secretions at the stigma surface, respectively. In species with wet stigma, the cuticle is disrupted and the presence of exudates is indicative of their receptivity. Most stigma studies are focused on a few species and families, many of them with self-incompatibility systems. However, there is scarce knowledge about the stigma composition in Fabaceae, the third angiosperm family, whose stigmas have been classified as semidry. Here we report the first transcriptome profiling and DEGs of Vicia faba L. styles and stigmas from autofertile (flowers able to self-fertilize in the absence of manipulation, whose exudate is released spontaneously) and autosterile (flowers that need to be manipulated to break the cuticle and release the exudates to be receptive) inbred lines. From the 76,269 contigs obtained from the de novo assembly, only 45.1% of the sequences were annotated with at least one GO term. A total of 115,920, 75,489, and 70,801 annotations were assigned to Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) categories, respectively, and 5918 differentially expressed genes (DEGs) were identified between the autofertile and the autosterile lines. Among the most enriched metabolic pathways in the DEGs subset were those related with amino acid biosynthesis, terpenoid metabolism, or signal transduction. Some DEGs have been related with previous QTLs identified for autofertility traits, and their putative functions are discussed. The results derived from this work provide an important transcriptomic reference for style-stigma processes to aid our understanding of the molecular mechanisms involved in faba bean fertilization.

Genome-wide association study for yield-related traits in faba bean (Vicia faba L.).

Yield is the most complex trait to improve crop production, and identifying the genetic determinants for high yield is a major issue in breeding new varieties. In faba bean (Vicia faba L.), quantitative trait loci (QTLs) have previously been detected in studies of biparental mapping populations, but the genes controlling the main trait components remain largely unknown. In this study, we investigated for the first time the genetic control of six faba bean yield-related traits: shattering (SH), pods per plant (PP), seeds per pod (SP), seeds per plant (SPL), 100-seed weight (HSW), and plot yield (PY), using a genome-wide association study (GWAS) on a worldwide collection of 352 homozygous faba bean accessions with the aim of identifying markers associated with them. Phenotyping was carried out in field trials at three locations (Spain, United Kingdom, and Serbia) over 2 years. The faba bean panel was genotyped with the Affymetrix faba bean SNP-chip yielding 22,867 SNP markers. The GWAS analysis identified 112 marker-trait associations (MTAs) in 97 candidate genes, distributed over the six faba bean chromosomes. Eight MTAs were detected in at least two environments, and five were associated with multiple traits. The next step will be to validate these candidates in different genetic backgrounds to provide resources for marker-assisted breeding of faba bean yield.

A reference consensus genetic map for molecular markers and economically important traits in faba bean (Vicia faba L.).

BACKGROUND: Faba bean (Vicia faba L.) is among the earliest domesticated crops from the Near East. Today this legume is a key protein feed and food worldwide and continues to serve an important role in culinary traditions throughout Middle East, Mediterranean region, China and Ethiopia. Adapted to a wide range of soil types, the main faba bean breeding objectives are to improve yield, resistance to biotic and abiotic stresses, seed quality and other agronomic traits. Genomic approaches aimed at enhancing faba bean breeding programs require high-quality genetic linkage maps to facilitate quantitative trait locus analysis and gene tagging for use in a marker-assisted selection. The objective of this study was to construct a reference consensus map in faba bean by joining the information from the most relevant maps reported so far in this crop. RESULTS: A combination of two approaches, increasing the number of anchor loci in diverse mapping populations and joining the corresponding genetic maps, was used to develop a reference consensus map in faba bean. The map was constructed from three main recombinant inbreed populations derived from four parental lines, incorporates 729 markers and is based on 69 common loci. It spans 4,602 cM with a range from 323 to 1041 loci in six main linkage groups or chromosomes, and an average marker density of one locus every 6 cM. Locus order is generally well maintained between the consensus map and the individual maps. CONCLUSION: We have constructed a reliable and fairly dense consensus genetic linkage map that will serve as a basis for genomic approaches in faba bean research and breeding. The core map contains a larger number of markers than any previous individual map, covers existing gaps and achieves a wider coverage of the large faba bean genome as a whole. This tool can be used as a reference resource for studies in different genetic backgrounds, and provides a framework for transferring genetic information when using different marker technologies. Combined with syntenic approaches, the consensus map will increase marker density in selected genomic regions and will be useful for future faba bean molecular breeding applications.

Genome-wide association analysis for drought tolerance and associated traits in faba bean (Vicia faba L.).

Faba bean (Vicia faba L.) is an important high protein legume adapted to diverse climatic conditions with multiple benefits for the overall sustainability of the cropping systems. Plant-based protein demand is being expanded and faba bean is a good candidate to cover this need. However, the crop is very sensitive to abiotic stresses, especially drought, which severely affects faba bean yield and development worldwide. Therefore, identifying genes associated with drought stress tolerance is a major challenge in faba bean breeding. Although the faba bean response to drought stress has been widely studied, the molecular approaches to improve drought tolerance in this crop are still limited. Here we built on recent genomic advances such as the development of the first high-density SNP genotyping array, to conduct a genome-wide association study (GWAS) using thousands of genetic polymorphisms throughout the entire faba bean genome. A worldwide collection of 100 faba bean accessions was grown under control and drought conditions and 10 morphological, phenological and physiological traits were evaluated to identify single nucleotide polymorphism (SNP) markers associated with drought tolerance. We identified 29 SNP markers significantly correlated with these traits under drought stress conditions. The flanking sequences were blasted to the Medicago truncatula reference genomes in order to annotate potential candidate genes underlying the causal variants. Three of the SNPs for chlorophyll content after the stress, correspond to uncharacterized proteins indicating the presence of novel genes associated with drought tolerance in faba bean. The significance of stress-inducible signal transducers provides valuable information on the possible mechanisms underlying the faba bean response to drought stress, thus providing a foundation for future marker-assisted breeding in the crop.

Variability in drought stress response in a panel of 100 faba bean genotypes.

Faba bean is an important protein crop for food and feed worldwide and provides a range of advantages in crop rotations. Its limited use in modern agriculture is mainly due to the high fluctuations in yield. A well known limiting factor in most legumes, and particularly in faba bean, is the high sensitivity to water shortage, which is further aggravated by climate change. The present study was undertaken to exploit the genetic variation in drought stress response in a faba bean collection of 100 accessions with diverse origins and to assess selection criteria for identifying drought tolerant genotypes. Physiological, phenological and yield related traits evaluated under drought or water-sufficient conditions responded significantly to the end-terminated drought stress. Comparison of yield relations showed the advantage of using a stress tolerance index (STI) to identify genotypes combining high yield potential with high stress yield. With regard to physiological traits, SPAD (chlorophyll content) values were significantly related to yield as well as to STI, while the other traits also contributed to different extents to variation in yield formation. Among the yield related traits, seeds per plant proved to be the most important trait followed by pods per plant. Interestingly, the eight genotypes with the best STI performance use different strategies to cope with drought stress.

A Clear Cell Sarcoma Case: A Diagnostic and Treatment Challenge, with a Promising Response to Trabectedin.

Introduction: Clear cell sarcoma (CCS) is a rare and aggressive soft tissue sarcoma. CCS is characterized by the translocation t(12;22) (q13;q12), involving the fusion of EWSR1 and ATF1 genes, and less frequently the fusion gene EWSR1-CREB1. Usually, CCSs are considered poorly responsive to conventional chemotherapy. However, trabectedin has shown activity against translocation-related sarcomas. Furthermore, preclinical results suggest that trabectedin is a promising antitumor agent for CCS, potentially inducing melanocytic differentiation. Case Presentation: We report the case of a challenging anatomopathological diagnosis in a patient with an aggressive metastatic CCS. Following the diagnosis of CCS, the patient experienced a clinical and radiological tumor response to trabectedin after four lines of treatment. Conclusion: This is a novel report of CCS treated with trabectedin that resulted in a partial response and suggests the need for further research on trabectedin as a therapeutic option for CCS.

Double tension band osteosynthesis in transverse supracondylar distal humerus fractures and nonunions.

PURPOSE: The purpose of this study was to evaluate the objective and subjective results achieved after double tension band fixation in transverse extra-articular intracapsular supracondylar distal humerus fractures and nonunions in adults. METHODS: Nine patients presenting six fractures and three nonunions of the distal humerus, treated with double tension band wiring between 1998 and 2011, were retrospectively evaluated. Two fractures were type A2 and four type A3, and the nonunions were oligotrophic; all nine lesions had a supracondylar intracapsular transverse orientation, that passed through the olecranon fossa, in a direction parallel to the joint line, and they compromised both columns of the distal humerus. Patient's age averaged 70 years (range, 56-82). RESULTS: Follow-up averaged 24.6 months (range, 12-53). All fractures and nonunions united; there were no infections, elbow stiffness or heterotopic bone formations. DASH score at final follow-up averaged 14.2 points (range, 4-22). The analog scale of pain averaged 1.1 points (range, 0-3). Elbow range of motion averaged 100° (range, 100-120°). Flexion averaged 123° (range, 115-130°) and elbow extension loss averaged 15.5° (range, 10-25°). CONCLUSIONS: The results achieved with double tension band fixation in transverse extra-articular intracapsular supracondylar distal humerus fractures and nonunions are comparable to the results that can be expected when using other available fixation methods; this technique is faster, less demanding and cheaper, and surgeons should have it in mind when leading with these particular types of distal humerus fractures and nonunions.

Identification of Leukemia-Associated Immunophenotypes by Databaseguided Flow Cytometry Provides a Highly Sensitive and Reproducible Strategy for the Study of Measurable Residual Disease in Acute Myeloblastic Leukemia.

Background: Multiparametric Flow Cytometry (MFC) is an essential tool to study the involved cell lineages, the aberrant differentiation/maturation patterns and the expression of aberrant antigens in acute myeloid leukemia (AML). The characterization of leukemia-associated immunophenotypes (LAIPs) at the moment of diagnosis is critical to establish reproducible strategies for the study of measurable residual disease using MFC (MFC-MRD). Methods: In this study, we identify and characterize LAIPs by comparing the leukemic populations of 145 AML patients, using the EuroFlow AML/ MDS MFC panel, with six databases of normal myeloid progenitors (MPCs). Principal component analysis was used to identify and characterize the LAIPs, which were then used to generate individual profiles for MFC-MRD monitoring. Furthermore, we investigated the relationship between the expression patterns of LAIPs and the different subtypes of AML. The MFC-MRD study was performed by identifying residual AML populations that matched with the LAIPs at diagnosis. To further validate this approach, the presence of MRD was also assessed by qPCR (qPCR-MRD). Finally, we studied the association between MFC-MRD and progression-free survival (PFS). Results: The strategy used in this study allowed us to describe more than 300 different LAIPs and facilitated the association of specific phenotypes with certain subtypes of AML. The MFC-MRD monitoring based on LAIPs with good/strong specificity was applicable to virtually all patients and showed a good correlation with qPCR-MRD and PFS. Conclusions: The described methodology provides an objective method to identify and characterize LAIPs. Furthermore, it provides a theoretical basis to develop highly sensitive MFC-MRD strategies.

Antioxidant and Chemopreventive Activity of Protein Hydrolysates from Raw and Germinated Flour of Legumes with Commercial Interest in Colorectal Cancer.

Legumes are a highly nutritious source of plant protein, fiber, minerals and vitamins. However, they also contain several bioactive compounds with significant potential benefits for human health. The objectives of this study were to evaluate the antioxidant, antitumor and chemopreventive activity of functional extracts from legumes using raw and germinated flours of six legume species of commercial interest. The methodology carried out consisted on the development of protein hydrolysates, assessment of their antioxidant capacity and in vitro tests on T84, HCT15 and SW480 colorectal cancer (CRC) cell lines. Our results showed a high antitumor activity of protein hydrolysate from M. sativa. Likewise, when combined with 5-Fluorouracile (5-Fu), there was a synergistic effect using extract concentrations from 50 to 175 µg/mL and 5-Fu concentrations from 1.5 to 5 µM. Similarly, the induction effect on detoxifying enzymes by the extracts of M. sativa, germinated V. faba Baraca × LVzt1 and V. narbonensis, which produced a higher induction rate than the positive control sulforaphane (10 µM), should be highlighted. Therefore, incorporating these enzymes into the diet could provide nutritional effects, as well as play an effective role in cancer chemoprevention and therapy.

The bHLH transcription factor VfTT8 underlies zt2, the locus determining zero tannin content in faba bean (Vicia faba L.).

Faba bean (Vicia faba L.) is an important protein-rich fodder crop, which is widely cultivated in temperate areas. However, antinutritional compounds such as condensed tannins, limit the use of this protein source in monogastric feed formulations. Previous studies demonstrated that two recessive and complementary genes, zt1 and zt2, control absence of tannin and white flower colour in faba bean. An ortholog of the Medicago WD40 transcription factor TTG1 was reported to encode the zt1 phenotype, but the responsible gene for zt2 is still unknown. Here we used a candidate gene approach combined with linkage mapping, comparative genomics and gene expression to fine map the zt2 genomic region and to identify the regulatory gene controlling both traits. Seventy-two genes, including 23 MYB and bHLH regulatory genes predicted to be associated with anthocyanin expression together with WRKY proteins, were screened and genotyped in three mapping populations. The linkage groups constructed identified the regulatory gene, TRANSPARENT TESTA8 (TT8), encoding a basic helix-loop-helix (bHLH) transcription factor, as the candidate for zt2. This finding was supported by qPCR analysis and further validated in different genetic backgrounds. Accordingly, VfTT8 was downregulated in white flowered types while showing high levels of expression in wild genotypes. Our results provide new insights on the regulatory mechanisms of tannin biosynthesis in faba bean and will facilitate the development of an ultimate zt2 diagnostic marker for the fast generation of new value-added cultivars free of tannins and with improved nutritional value.

Outcomes of COVID-19 in Patients With Lung Cancer Treated in a Tertiary Hospital in Madrid.

Background: Cancer patients represent a vulnerable population for COVID-19 illness. We aimed to analyze outcomes of lung cancer patients affected by COVID-19 in a tertiary hospital of a high-incidence region during the pandemic. Methods: We annotated 23 lung cancer patients consecutively diagnosed with COVID-19 at our institution (HGUGM; Madrid, Spain) between March 4th, 2020 and May 12th, 2020. Only patients with a confirmatory SARS-CoV-2 RT-PCR were included in the study. Results: All patients had at least 1 COVID-19 related symptom; cough (48%), shortness of breath (48%), fever (39%), and low-grade fever (30%) were the most common. Time from symptoms onset to first positive SARS-CoV-2 PCR was 5.5 days (range 1-17), with 13% of cases needed from a 2nd PCR to confirm diagnosis. There was a high variability on thoracic imaging findings, with multilobar pneumonia as the most commonly found pattern (74%). Main lab test abnormalities were low lymphocytes count (87%), high neutrophil to lymphocyte ratio -NLR- (78%), and elevated inflammatory markers: fibrinogen (91%), c-reactive protein -CRP- (87%), and D-dimer (70%). In our series, hospitalization rate was 74%, 39% of patients developed acute respiratory distress syndrome (ARDS), and the case-fatality rate was 35% (8/23). 87% of patients received anti-viral treatment (87% hydroxychloroquine, 74% lopinavir/ritonavir, 13% azithromycin), 43% corticosteroids, 26% interferon-ß, 4% tocilizumab, and 82% of hospitalized patients received anticoagulation. High-oxygen requirements were needed in 39% of patients, but only 1 pt was admitted for invasive MV and was discharged 42 days after admission. Multiple variables related to tumor status, clinical baseline conditions, and inflammation markers were associated with mortality but did not remain statistically significant in a multivariate model. In patients with lung cancer receiving systemic therapy (n = 242) incidence and mortality from COVID-19 were 4.5, and 2.1%, respectively, with no differences found by type of treatment. Conclusions: Lung cancer patients represent a vulnerable population for COVID-19, according to the high rate of hospitalization, onset of ARDS, and high mortality rate. Although larger series are needed, no differences in mortality were found by type of cancer treatment. Measures to minimize the risk of SARS-CoV-2 infection remain key to protect lung cancer patients.

Characterization and diagnostic marker for TTG1 regulating tannin and anthocyanin biosynthesis in faba bean.

Condensed tannins, found in coloured-flowering varieties of faba bean (Vicia faba L) are, after vicine and convicine, one of the major anti-nutritional factors for monogastric animals. The development of tannin-free cultivars is a key goal in breeding to broaden the use of this legume in the animal feed industry. Two recessive genes, zt-1 and zt-2, control the zero-tannin content and promote white-flowered plants. Previous studies exploiting synteny with the model Medicago truncatula reported a mutation in TTG1, a gene encoding a WD40 transcription factor located in chromosome II, as the responsible for the zt-1 phenotypes. Here a comprehensive analysis of VfTTG1 (including phylogenetic relationships, gene structure and gene expression) has been conducted to confirm the identity of the gene and to reveal structural changes that may result in different functional alleles. The results confirmed the identity of the candidate and revealed the existence of two different alleles responsible for the phenotype: ttg1-a, probably due to a mutation in the promoter region, and ttg1-b caused by a deletion at the 5'end of VfTTG1. Based on the sequencing results, an allele-specific diagnostic marker was designed that differentiate zt-1 from wild and zt-2 genotypes and facilitates its deployment in faba bean breeding programs.

Species Deletions from Microbiome Consortia Reveal Key Metabolic Interactions between Gut Microbes.

The gut microbiome is a complex microbial community that plays a key role in human health. Diet is an important factor dictating gut microbiome composition. This is mediated by multiple microbe-microbe interactions that result in the fermentation of nondigestible carbohydrates and the production of short-chain fatty acids. Certain species play key metabolic roles in the microbiome, and their disappearance could result in dysbiosis. In this work, a synthetic consortium of 14 gut microbes was studied during the utilization of prebiotic inulin in batch bioreactors. Fermentations were repeated leaving one species out every time, in order to evaluate the impact of their elimination on the system. Substrate consumption, microbial composition, and metabolite production were determined. Single deletions never resulted in a complete loss of bacterial growth or inulin consumption, suggesting functional redundancy. Deletions of Bacteroides dorei and Lachnoclostridium clostridioforme resulted in lower biomass and higher residual inulin. The absence of B. dorei impacted the abundance of the other 10 species negatively. Lachnoclostridium symbiosum, a butyrate producer, appeared to be the most sensitive species to deletions, being stimulated by the presence of Escherichia coli, Bifidobacterium adolescentis, B. dorei, and Lactobacillus plantarum Conversely, bioreactors without these species did not show butyrate production. L. clostridioforme was observed to be essential for propionate production, and B. dorei for lactate production. Our analysis identified specific members that were essential for the function of the consortium. In conclusion, species deletions from microbial consortia could be a useful approach to identify relevant interactions between microorganisms and defining metabolic roles in the gut microbiome.IMPORTANCE Gut microbes associate, compete for, and specialize in specific metabolic tasks. These interactions are dictated by the cross-feeding of degradation or fermentation products. However, the individual contribution of microbes to the function of the gut microbiome is difficult to evaluate. It is essential to understand the complexity of microbial interactions and how the presence or absence of specific microorganisms affects the stability and functioning of the gut microbiome. The experimental approach of this study could be used for identifying keystone species, in addition to redundant functions and conditions that contribute to community stability. Redundancy is an important feature of the microbiome, and its reduction could be useful for the design of microbial consortia with desired metabolic properties enhancing the tasks of the keystone species.