Identification of consistent QTL and candidate genes associated with seed traits in common bean by combining GWAS and RNA-Seq.

KEY MESSAGE: Association analysis, colocation study with previously reported QTL, and differential expression analyses allowed the identification of the consistent QTLs and main candidate genes controlling seed traits. Common beans show wide seed variations in shape, size, water uptake, and coat proportion. This study aimed to identify consistent genomic regions and candidate genes involved in the genetic control of seed traits by combining association and differential expression analyses. In total, 298 lines from the Spanish Diversity Panel were genotyped with 4,658 SNP and phenotyped for seven seed traits in three seasons. Thirty-eight significant SNP-trait associations were detected, which were grouped into 23 QTL genomic regions with 1,605 predicted genes. The positions of the five QTL regions associated with seed weight were consistent with previously reported QTL. HCPC analysis using the SNP that tagged these five QTL regions revealed three main clusters with significantly different seed weights. This analysis also separated groups that corresponded well with the two gene pools described: Andean and Mesoamerican. Expression analysis was performed on the seeds of the cultivar 'Xana' in three seed development stages, and 1,992 differentially expressed genes (DEGs) were detected, mainly when comparing the early and late seed development stages (1,934 DEGs). Overall, 91 DEGs related to cell growth, signaling pathways, and transcriptomic factors underlying these 23 QTL were identified. Twenty-two DEGs were located in the five QTL regions associated with seed weight, suggesting that they are the main set of candidate genes controlling this character. The results confirmed that seed weight is the sum of the effects of a complex network of loci, and contributed to the understanding of seed phenotype control.

Genome-wide association study for the extractable phenolic profile and coat color of common bean seeds (Phaseolus vulgaris L.).

BACKGROUND: A large variation in seed coat colors and seed phenolic metabolites is present in common bean (Phaseolus vulgaris L.). The study of the relationships between seed coat color phenotype and the phenolic profile is an important step in the elucidation of the gene network involved in the phenylpropanoid biosynthetic pathway. However, this relationship is still poorly understood in this species. RESULTS: A genome-wide association study (GWAS) was used to investigate the genomic regions associated with the synthesis of 10 flavonoids (5 anthocyanins and 5 flavonols) and with 10 seed coat color traits using a set of 308 common bean lines of the Spanish Diversity Panel (SDP) which have been genotyped with 11,763 SNP markers.. A total of 31 significant SNP-trait associations (QTNs) were identified, grouped in 20 chromosome regions: 6 for phenolic metabolites on chromosomes Pv01, Pv02, Pv04, Pv08, and Pv09, 13 for seed coat color on chromosomes Pv01, Pv02, Pv06, Pv07, and Pv10, and 1 including both types of traits located on chromosome Pv08. In all, 58 candidate genes underlying these regions have been proposed, 31 of them previously described in the phenylpropanoid pathway in common bean, and 27 of them newly proposed in this work based on the association study and their homology with Arabidopsis anthocyanin genes. CONCLUSIONS: Chromosome Pv08 was identified as the main chromosome involved in the phenylpropanoid pathway and in consequence in the common bean seed pigmentation, with three independent chromosome regions identified, Phe/C_Pv08(2.7) (expanding from 2.71 to 4.04 Mbp), C_Pv08(5.8) (5.89-6.59 Mbp), and Phe_Pv08(62.5) (62.58 to 63.28 Mbp). Candidate genes previously proposed by other authors for the color genes V and P were validated in this GWAS. Candidate genes have been tentatively proposed from this study for color genes B and Rk on Pv02, Asp on Pv07, and complex C on Pv08. These results help to clarify the complex network of genes involved in the genetic control of phenolic compounds and seed color in common bean and provide the opportunity for future validation studies.

Genetic control of pod morphological traits and pod edibility in a common bean RIL population.

KEY MESSAGE: QTL mapping, association analysis, and colocation study with previously reported QTL revealed three main regions controlling pod morphological traits and two loci for edible pod characteristics on the common bean chromosomes Pv01 and Pv06. Bean pod phenotype is a complex characteristic defined by the combination of different traits that determine the potential use of a genotype as a snap bean. In this study, the TUM RIL population derived from a cross between 'TU' (dry) and 'Musica' (snap) was used to investigate the genetic control of pod phenotype. The character was dissected into pod morphological traits (PMTs) and edible pod characteristics (EPC). The results revealed 35 QTL for PMTs located on seven chromosomes, suggesting a strong QTL colocation on chromosomes Pv01 and Pv06. Some QTL were colocated with previously reported QTL, leading to the mapping of 15 consensus regions associated with bean PMTs. Analysis of EPC of cooked beans revealed that two major loci with epistatic effect, located on chromosomes Pv01 and Pv06, are involved in the genetic control of this trait. An association study using a subset of the Spanish Diversity Panel (snap vs. non-snap) detected 23 genomic regions, with three regions being mapped at a position similar to those of two loci identified in the TUM population. The results demonstrated the relevant roles of Pv01 and Pv06 in the modulation of bean pod phenotype. Gene ontology enrichment analysis revealed a significant overrepresentation of genes regulating the phenylpropanoid metabolic process and auxin response in regions associated with PMTs and EPC, respectively. Both biological functions converged in the lignin biosynthetic pathway, suggesting the key role of the pathway in the genetic control of bean pod phenotype.

PM2.5 chemical composition and health risks by inhalation near a chemical complex.

Particulate matter (PM2.5) samples were collected in the vicinity of an industrial chemical pole and analysed for organic and elemental carbon (OC and EC), 47 trace elements and around 150 organic constituents. On average, OC and EC accounted for 25.2% and 11.4% of the PM2.5 mass, respectively. Organic compounds comprised polycyclic aromatic hydrocarbons (PAHs), alkylated PAHs, anhydrosugars, phenolics, aromatic ketones, glycerol derivatives, aliphatic alcohols, sterols, and carboxyl groups, including aromatic, carboxylic and dicarboxylic acids. Enrichment factors > 100 were obtained for Pb, Cd, Zn, Cu, Sn, B, Se, Bi, Sb and Mo, showing the contribution of industrial emissions and nearby major roads. Principal component analysis revealed that vehicle, industrial and biomass burning emissions accounted for 66%, 11% and 9%, respectively, of the total PM2.5-bound PAHs. Some of the detected organic constituents are likely associated with plasticiser ingredients and thermal stabilisers used in the manufacture of PVC and other plastics in the industrial complex. Photooxidation products of both anthropogenic (e.g., toluene) and biogenic (e.g., isoprene and pinenes) precursors were also observed. It was estimated that biomass burning accounted for 13.8% of the PM2.5 concentrations and that secondary OC represented 37.6% of the total OC. The lifetime cancer risk from inhalation exposure to PM2.5-bound PAHs was found to be negligible, but it exceeded the threshold of 10-6 for metal(loi)s, mainly due to Cr and As.

Kynurenine inhibits melanogenesis in human melanocyte-keratinocyte co-cultures and in a reconstructed 3D skin model.

Kynurenine (KYN), the most abundant metabolite of tryptophan, is classically associated with immune tolerance and tumor immune escape. In the last years, KYN is in the spotlight in other biological processes. Here, we showed that KYN inhibited tyrosinase expression and melanin content in primary human melanocyte and keratinocyte co-cultures. Furthermore, KYN decreased melanosome content in a 3D human skin reconstruction model. In these experiments, we used tyrosine + NH4 Cl to induce pigmentation. We compared the inhibitory effect of KYN on melanogenesis with the already known inhibitory effect promoted by IFN-γ. Since increased KYN production depends on the IFN-γ-inducible enzyme indoleamine-2,3-dioxygenase (IDO), we propose that part of the effect of IFN-γ on melanogenesis involves KYN production. From that, we tested if, during melanogenesis, changes in tryptophan metabolism would occur. For this purpose, we measured tryptophan, KYN and downstream products along with pigmentation. There were no significant changes in Trp metabolism, except for the high consumption of kynurenic acid. Our data identify the skin as a potential target for the action of KYN relevant for skin physiology and pigmentation. The results are discussed concerning the high production of KYN in skin inflammatory disorders and cancer.

GWAS of pod morphological and color characters in common bean.

BACKGROUND: Common bean (Phaseolus vulgaris L.) is an important legume species which can be consumed as immature pods and dry seeds after re-hydration and cooking. Many genes and QTL, and epistatic interactions among them, condition pod morphological traits. However, not all them have been mapped or validated nor candidate genes proposed. We sought to investigate the genomic regions conditioning pod morphological and color characters through GWAS. RESULTS: Single and multi-locus genome wide association analysis was used to investigate pod traits for a set of 301 bean lines of the Spanish Diversity Panel (SDP). The SDP was genotyped with 32,812 SNPs obtained from Genotyping by Sequencing. The panel was grown in two seasons and phenotypic data were recorded for 17 fresh pods traits grouped in four pod characters: pod length, pod cross-section, pod color, and number of seeds per pod. In all, 23 QTL for pod length, 6 for cross-section, 18 for pod color, 6 for number of seeds per pod and 9 associated to two or more pod characters were detected. Most QTL were located in the telomeric region of chromosomes Pv01, Pv02, Pv04, Pv08, Pv09 and Pv10. Eighteen detected QTL co-localized with 28 previously reported QTL. Twenty-one potential candidate genes involving developmental processes were detected underlying 11 QTL for pod morphological characters, four of them homologous to A. thaliana genes FIS2, SPL10, TTG2 and AML4 affecting silique size. Eight potential candidate genes involved in pigment synthesis, were found underlying five QTL for pod color. CONCLUSIONS: GWAS for pod morphological and color characters in the bean Spanish Diversity Panel revealed 62 QTL, 18 co-localized with previously reported QTL, and 16 QTL were underlain by 25 candidate genes. Overall 44 new QTL identified and 18 existing QTL contribute to a better understanding of the complex inheritance of pod size and color traits in common bean and open the opportunity for future validation works.

Pod indehiscence in common bean is associated with the fine regulation of PvMYB26.

In legumes, pod shattering occurs when mature pods dehisce along the sutures, and detachment of the valves promotes seed dispersal. In Phaseolus vulgaris (L)., the major locus qPD5.1-Pv for pod indehiscence was identified recently. We developed a BC4/F4 introgression line population and narrowed the major locus down to a 22.5 kb region. Here, gene expression and a parallel histological analysis of dehiscent and indehiscent pods identified an AtMYB26 orthologue as the best candidate for loss of pod shattering, on a genomic region ~11 kb downstream of the highest associated peak. Based on mapping and expression data, we propose early and fine up-regulation of PvMYB26 in dehiscent pods. Detailed histological analysis establishes that pod indehiscence is associated with the lack of a functional abscission layer in the ventral sheath, and that the key anatomical modifications associated with pod shattering in common bean occur early during pod development. We finally propose that loss of pod shattering in legumes resulted from histological convergent evolution and that it is the result of selection at orthologous loci.

Dissecting the genetic control of seed coat color in a RIL population of common bean (Phaseolus vulgaris L.).

KEY MESSAGE: Three genes associated with the seed coat color in a TU/Musica RIL population were located on a genetic map, and two candidate genes proposed to control black seed coat in the TU genotype were characterized. Seed coat color is an important characteristic of common bean (Phaseolus vulgaris L.) associated with the marketability of dry bean cultivars, quality and nutritional characteristics of seed, as well as response to pathogens. In this study, the genetic control of seed coat color in a recombinant inbred line population (175 lines) obtained from the cross 'TU' × 'Musica' was investigated. Phenotypic segregation fitted 1:1 for white vs. nonwhite, and 3:1 for brown versus black, indicating the involvement of three independent genes, one controlling white color and two (with epistatic interaction) controlling black color. Using a genetic map built with 842 SNPs, the gene responsible for the white seed coat was mapped on the linkage group Pv07, in the position previously described for the P gene. For the black seed coat phenotype, two genes were mapped to the beginning of chromosomes Pv06 and Pv08, in the positions estimated for the V gene and the complex C locus, respectively, by classical studies. The involvement of these two genomic regions was verified through two crosses between three selected RILs exhibiting complementary and dominant inheritance, in which the TU alleles for both genes resulted in a black phenotype. Two genes involved in the anthocyanin biosynthesis pathway were proposed as candidate genes: Phvul.006G018800 encoding a flavonoid 3'5'hydroxylase and Phvul.008G038400 encoding MYB113 transcription factor. These findings add knowledge to the complex network of genes controlling seed coat color in common bean as well as providing genetic markers to be used in future genetic analysis or plant breeding.

Indoleamine 2,3-dioxygenase in melanoma progression and BRAF inhibitor resistance.

Indoleamine 2,3-dioxygenase (IDO) is associated with the progression of many types of tumors, including melanoma. However, there is limited information about IDO modulation on tumor cell itself and the effect of BRAF inhibitor (BRAFi) treatment and resistance. Herein, IDO expression was analyzed in different stages of melanoma development and progression linked to BRAFi resistance. IDO expression was increased in primary and metastatic melanomas from patients' biopsies, especially in the immune cells infiltrate. Using a bioinformatics approach, we also identified an increase in the IDO mRNA in the vertical growth and metastatic phases of melanoma. Using in silico analyses, we found that IDO mRNA was increased in BRAFi resistance. In an in vitro model, IDO expression and activity induced by interferon-gamma (IFNγ) in sensitive melanoma cells was decreased by BRAFi treatment. However, cells that became resistant to BRAFi presented random IDO expression levels. Also, we identified that treatment with the IDO inhibitor, 1-methyltryptophan (1-MT), was able to reduce clonogenicity for parental and BRAFi-resistant cells. In conclusion, our results support the hypothesis that the decreased IDO expression in tumor cells is one of the many additional outcomes contributing to the therapeutic effects of BRAFi. Still, the IDO production changeability by the BRAFi-resistant cells reiterates the complexity of the response arising from resistance, making it not possible, at this stage, to associate IDO expression in tumor cells with resistance. On the other hand, the maintenance of 1-MT off-target effect endorses its use as an adjuvant treatment of melanoma that has become BRAFi-resistant.

Gene coding for an elongation factor is involved in resistance against powdery mildew in common bean.

KEY MESSAGE: Genetic control of the resistance response against powdery mildew in common bean was studied combining genetic, genomic and transcriptomic analyses. A candidate resistance gene in cultivar Porrillo Sintetico was proposed. The species causing the fungal disease powdery mildew (PM) in the local common bean crop was identified as Erysiphe polygoni through the molecular analysis of the internal transcribed spacer region. A genetic analysis of the resistance in cultivar Porrillo Sintetico was conducted using different F2:3 populations, and a dominant gene conferring total resistance against a local PM isolate was physically located between 84,188 and 218,664 bp of chromosome Pv04. An in silico analysis of this region, based on the common bean reference sequence, revealed four genes candidate to be involved in the resistance reaction. Relative expression levels of these genes after PM infection showed a significant over-expression of the candidate gene Phvul.004G001500 in the resistant genotype Porrillo Sintetico. This gene was re-sequenced in the parental genotypes X2776 and Porrillo Sintetico to explain their different phenotypic responses against PM. Several substitutions where identified in exon regions, all of them synonymous, so differences in the produced amino acid sequence were not expected. However, a total of 37 mutations were identified in non-coding regions of the gene sequence, suggesting that intron variation could be responsible for the different gene expression levels after PM infection. No evidence of other regulatory mechanisms, such as alternative splicing or methylation, was identified. Candidate resistance gene Phvul.004G001500 codes for an elongation factor that is not a typical gene related to recognition of specific pathogens in plants, suggesting its involvement in the resistance through plant immune system.

Serum amyloid A1 is upregulated in human glioblastoma.

Serum amyloid A1 (SAA1) is a sensitive acute phase reactant primarily produced by the liver in response to acute inflammation. We have recently shown that SAA affects proliferation, migration, and invasion of glioblastoma cell lines, which suggest its participation in the malignant process. Consistently, levels of SAA have been used as a non-invasive biomarker for the prognosis of many cancers. In this study, we aimed to investigate SAA serum levels and expression of SAA genes in human astrocytomas tissues. Serum and tissue samples were obtained from patients with astrocytoma grades I to III and glioblastoma (GBM or grade IV). Levels of circulating SAA were significantly higher in the serum of patients with AGII-IV when compared to non-neoplastic samples derived from non-neoplastic patients (NN) (p > 0.0001). Quantitative real time PCR (qRT-PCR) of 148 astrocytomas samples (grades I-IV) showed that SAA1 mRNA was significantly higher in GBM when compared to AGI-III and NN samples (p < 0.0001). Immunohistochemistry analysis revealed cytoplasmic positivity for SAA in GBM. There was no correlation of SAA1 with clinical end-point of overall survival among GBM patients. However, it was found a positive correlation between SAA1 and genes involved in tumor progression, such as: HIF1A (r = 0.50; p < 0.00001), CD163 (r = 0.52; p < 0.00001), CXCR4 (r = 0.42; p < 0.00001) and CXCR7 (r = 0.33; p = 0.002). In conclusions, we show that astrocytoma patients have increased levels of serum SAA and SAA1 is expressed and secreted in GBM, and its co-expression with tumor-related genes supports its involvement in GBM angiogenesis and progression.

Identification of Clusters that Condition Resistance to Anthracnose in the Common Bean Differential Cultivars AB136 and MDRK.

The correct identification of the anthracnose resistance systems present in the common bean cultivars AB136 and MDRK is important because both are included in the set of 12 differential cultivars proposed for use in classifying the races of the anthracnose causal agent, Colletrotrichum lindemuthianum. In this work, the responses against seven C. lindemuthianum races were analyzed in a recombinant inbred line population derived from the cross AB136 × MDRK. A genetic linkage map of 100 molecular markers distributed across the 11 bean chromosomes was developed in this population to locate the gene or genes conferring resistance against each race, based on linkage analyses and χ2 tests of independence. The identified anthracnose resistance genes were organized in clusters. Two clusters were found in AB136: one located on linkage group Pv07, which corresponds to the anthracnose resistance cluster Co-5, and the other located at the end of linkage group Pv11, which corresponds to the Co-2 cluster. The presence of resistance genes at the Co-5 cluster in AB136 was validated through an allelism test conducted in the F2 population TU × AB136. The presence of resistance genes at the Co-2 cluster in AB136 was validated through genetic dissection using the F2:3 population ABM3 × MDRK, in which it was directly mapped to a genomic position between 46.01 and 47.77 Mb of chromosome Pv11. In MDRK, two independent clusters were identified: one located on linkage group Pv01, corresponding to the Co-1 cluster, and the second located on LG Pv04, corresponding to the Co-3 cluster. This report enhances the understanding of the race-specific Phaseolus vulgaris-C. lindemuthianum interactions and will be useful in breeding programs.

Serum amyloid A links endotoxaemia to weight gain and insulin resistance in mice.

AIMS/HYPOTHESIS: Pre-adipocytes and adipocytes are responsive to the acute phase protein serum amyloid A (SAA). The combined effects triggered by SAA encompass an increase in pre-adipocyte proliferation, an induction of TNF-α and IL-6 release and a decrease in glucose uptake in mature adipocytes, strongly supporting a role for SAA in obesity and related comorbidities. This study addressed whether SAA depletion modulates weight gain and insulin resistance induced by a high-fat diet (HFD). METHODS: Male Swiss Webster mice were fed an HFD for 10 weeks under an SAA-targeted antisense oligonucleotide (ASOSAA) treatment in order to evaluate the role of SAA in weight gain. RESULTS: With ASOSAA treatment, mice receiving an HFD did not differ in energy intake when compared with their controls, but were prevented from gaining weight and developing insulin resistance. The phenotype was characterised by a lack of adipose tissue expansion, with low accumulation of epididymal, retroperitoneal and subcutaneous fat content and decreased inflammatory markers, such as SAA3 and toll-like receptor (TLR)-4 expression, as well as macrophage infiltration into the adipose tissue. Furthermore, a metabolic status similar to chow-fed mice counterparts could be observed, with equivalent levels of leptin, adiponectin, IGF-I, SAA, fasting glucose and insulin, and remarkable improvement in glucose and insulin tolerance test profiles. Surprisingly, the expected HFD-induced metabolic endotoxaemia was also prevented by the ASOSAA treatment. CONCLUSIONS/INTERPRETATION: This study provides further evidence of the role of SAA in weight gain and insulin resistance. Moreover, we also suggest that beyond its proliferative and inflammatory effects, SAA is part of the lipopolysaccharide signalling pathway that links inflammation to obesity and insulin resistance.

Melanocytes are more responsive to IFN-γ and produce higher amounts of kynurenine than melanoma cells.

A key link between amino acid catabolism and immune regulation in cancer is the augmented tryptophan (Trp) catabolism through the kynurenine pathway (KP), a metabolic route induced by interferon-γ (IFN-γ) and related to poor prognosis in melanomas. Besides its role in cancer, IFN-γ plays a key role in the control of pigmentation homeostasis. Here we measured KP metabolites in human melanoma lines and skin melanocytes and fibroblasts in response to IFN-γ. In general, IFN-γ affected KP in skin cells more than in melanoma cells, supporting IFN-γ roles in skin physiology and that of stromal cells in modulating the tumor microenvironment.

Myeloperoxidase in human peripheral blood lymphocytes: Production and subcellular localization.

Myeloperoxidase (MPO) is an important enzyme in the front-line protection against microorganisms. In peripheral blood, it is accepted that MPO is only produced by myeloid-lineage cells. Thus, MPO presence is unexpected in lymphocytes. We showed recently that B1-lymphocytes from mice have MPO. Here, we showed that subsets of human peripheral B, CD4(+) and CD8(+) T lymphocytes express MPO. The content of MPO in lymphocytes was very low compared to neutrophils/monocytes with a preferential distribution in the nucleus and perinuclear region. Also, we performed a MPO mRNA expression analysis from human blood cells derived from microarray raw data publicly available, showing that MPO is modulated in infectious disease. MPO was increased in CD4(+) T lymphocytes from HIV chronic infection and in CD8(+) T lymphocytes from HCV-positive patients. Our study points out MPO as a multifunctional protein due to its subcellular localization and expression modulation in lymphocytes indicating alternative unknown functions for MPO in lymphocytes.

Genetic analysis of the response to eleven Colletotrichum lindemuthianum races in a RIL population of common bean (Phaseolus vulgaris L.).

BACKGROUND: Bean anthracnose is caused by the fungus Colletotrichum lindemuthianum (Sacc. & Magnus) Lams.- Scrib. Resistance to C. lindemuthianum in common bean (Phaseolus vulgaris L.) generally follows a qualitative mode of inheritance. The pathogen shows extensive pathogenic variation and up to 20 anthracnose resistance loci (named Co-), conferring resistance to specific races, have been described. Anthracnose resistance has generally been investigated by analyzing a limited number of isolates or races in segregating populations. In this work, we analyzed the response against eleven C. lindemuthianum races in a recombinant inbred line (RIL) common bean population derived from the cross Xana × Cornell 49242 in which a saturated linkage map was previously developed. RESULTS: A systematic genetic analysis was carried out to dissect the complex resistance segregations observed, which included contingency analyses, subpopulations and genetic mapping. Twenty two resistance genes were identified, some with a complementary mode of action. The Cornell 49242 genotype carries a complex cluster of resistance genes at the end of linkage group (LG) Pv11 corresponding to the previously described anthracnose resistance cluster Co-2. In this position, specific resistance genes to races 3, 6, 7, 19, 38, 39, 65, 357, 449 and 453 were identified, with one of them showing a complementary mode of action. In addition, Cornell 49242 had an independent gene on LG Pv09 showing a complementary mode of action for resistance to race 453. Resistance genes in genotype Xana were located on three regions involving LGs Pv01, Pv02 and Pv04. All resistance genes identified in Xana showed a complementary mode of action, except for two controlling resistance to races 65 and 73 located on LG Pv01, in the position of the previously described anthracnose resistance cluster Co-1. CONCLUSIONS: Results shown herein reveal a complex and specific interaction between bean and fungus genotypes leading to anthracnose resistance. Organization of specific resistance genes in clusters including resistance genes with different modes of action (dominant and complementary genes) was also confirmed. Finally, new locations for anthracnose resistance genes were identified in LG Pv09.

The term basal plate of the human placenta as a source of functional extravillous trophoblast cells.

BACKGROUND: Extravillous trophoblast (EVT) cells are of pivotal importance in human embryo implantation and homeostasis of the maternal fetal interface. Invasion of the endometrium by EVT contributes to placental anchorage, spiral artery remodeling, immunological defense, tolerogenic responses, and several collaborative cross talks involved in establishing and maintaining a successful pregnancy. We report here an improved protocol for the isolation of fully differentiated EVT cells from the basal plate of the human term placenta. METHODS: The basal plate was carefully dissected from the villous tissue and the amniochorion membrane prior to enzymatic digestion. Term basal EVT cells were isolated using a 30 and 60% Percoll gradient. A panel of markers and characteristics of the isolated cells were used to confirm the specificity and efficiency of the method so that their potential as an investigative tool for placental research could be ascertained. RESULTS: Isolated cells were immunoreactive for cytokeratin-7 (CK-7), placental growth factor, placental alkaline phosphatase, human leukocyte antigen G1 (HLA-G1), and α1 and α5 integrins, similarly to the EVT markers from first trimester placental villi. Around 95% of the isolated cells labeled positively for CK-7 and 82% for HLA-G1. No significant change in viability was observed during 48 h of EVT culture as indicated by propidium iodide incorporation and trypan blue test exclusion. Genes for metalloproteinases MMP-2 and MMP9 (positive regulators of trophoblast invasiveness) were expressed up to 48 h of culturing, as also the gelatinolytic activity of the isolated cells. Transforming growth factor (TGF)-beta, which inhibits proliferation, migration, and invasiveness of first-trimester EVT cells, also reduced invasion of isolated term EVT cells in transwell assays, whereas epidermal growth factor was a positive modulator. CONCLUSIONS: Term basal plate may be a viable source of functional EVT cells that is an alternative to villous explant-derived EVT cells and cell lines. Isolated term EVT cells may be particularly useful in investigation of the role of trophoblast cells in pathological gestations, in which the precise regulation and interactive ability of extravillous trophoblast has been impaired.

Screening for resistance to four fungal diseases and associated genomic regions in a snap bean diversity panel.

Anthracnose, white mold, powdery mildew, and root rot caused by Colletotrichum lindemuthianum, Scletorinia sclerotiorum, Erysiphe spp., and Pythium ultimum, respectively, are among the most frequent diseases that cause significant production losses worldwide in common bean (Phaseolus vulgaris L.). Reactions against these four fungal diseases were investigated under controlled conditions using a diversity panel of 311 bean lines for snap consumption (Snap bean Panel). The genomic regions involved in these resistance responses were identified based on a genome-wide association study conducted with 16,242 SNP markers. The highest number of resistant lines was observed against the three C. lindemuthianum isolates evaluated: 156 lines were resistant to CL124 isolate, 146 lines resistant to CL18, and 109 lines were resistant to C531 isolate. Two well-known anthracnose resistance clusters were identified, the Co-2 on chromosome Pv11 for isolates CL124 and CL18, and the Co-3 on chromosome Pv04 for isolates CL124 and C531. In addition, other lesser-known regions of anthracnose resistance were identified on chromosomes Pv02, Pv06, Pv08, and Pv10. For the white mold isolate tested, 24 resistant lines were identified and the resistance was localized to three different positions on chromosome Pv08. For the powdery mildew local isolate, only 12 resistant lines were identified, and along with the two previous resistance genes on chromosomes Pv04 and Pv11, a new region on chromosome Pv06 was also identified. For root rot caused by Pythium, 31 resistant lines were identified and two main regions were located on chromosomes Pv04 and Pv05. Relevant information for snap bean breeding programs was provided in this work. A total of 20 lines showed resistant or intermediate responses against four or five isolates, which can be suitable for sustainable farm production and could be used as resistance donors. Potential genes and genomic regions to be considered for targeted improvement were provided, including new or less characterized regions that should be validated in future works. Powdery mildew disease was identified as a potential risk for snap bean production and should be considered a main goal in breeding programs.

Indoor PM from residential coal combustion: Levels, chemical composition, and toxicity.

Indoor air quality is crucial for human health due to the significant time people spend at home, and it is mainly affected by internal sources such as solid fuel combustion for heating. This study investigated the indoor air quality and health implications associated with residential coal burning covering gaseous pollutants (CO, CO2 and total volatile organic compounds), particulate matter, and toxicity. The PM10 chemical composition was obtained by ICP-MS/OES (elements), ion chromatography (water-soluble ions) and thermal-optical analysis (organic and elemental carbon). During coal combustion, PM10 levels were higher (up to 8.8 times) than background levels and the indoor-to-outdoor ratios were, on average, greater than unity, confirming the existence of a significant indoor source. The chemical characterisation of PM10 revealed increased concentrations of organic carbon and elemental carbon during coal combustion as well as arsenic, cadmium and lead. Carcinogenic risks associated with exposure to arsenic exceeded safety thresholds. Indoor air quality fluctuated during the study, with varying toxicity levels assessed using the Aliivibrio fischeri bioluminescence inhibition assay. These findings underscore the importance of mitigating indoor air pollution associated with coal burning and highlight the potential health risks from long-term exposure. Effective interventions are needed to improve indoor air quality and reduce health risks in coal-burning households.

Intracellular localization of myeloperoxidase in murine peritoneal B-lymphocytes and macrophages.

Generation of hypochlorous acid (HOCl), an important microbicidal agent, is considered to be the main function of myeloperoxidase (MPO), an enzyme present in phagocytes. High amounts of MPO are present in neutrophil azurophilic granules, which are mobilized into the phagolysosome vacuole during phagocytosis. MPO is also present in monocytes and macrophages, although to a lesser degree than in neutrophils. In the present study, we investigated the distribution of MPO in murine peritoneal cells using flow cytometry, confocal microscopy (CM) and transmission electron microscopy (TEM). MPO was observed in macrophages, and surprisingly, we detected MPO in B lymphocytes, specifically in B1-a. MPO was present in cytoplasmic granules, vesicles, mitochondria and the nucleus of murine peritoneal cells. Together, these findings suggest that, in addition to its known microbicidal activity, MPO has a myriad of other unanticipated cellular functions.