Cell Wall Composition and Structure Define the Developmental Fate of Embryogenic Microspores in Brassica napus

[EN] Microspore cultures generate a heterogeneous population of embryogenic structures that can be grouped into highly embryogenic structures [exine-enclosed (EE) and loose bicellular structures (LBS)] and barely embryogenic structures [compact callus (CC) and loose callus (LC) structures]. Little is known about the factors behind these different responses. In this study we performed a comparative analysis of the composition and architecture of the cell walls of each structure by confocal and quantitative electron microscopy. Each structure presented specific cell wall characteristics that defined their developmental fate. EE and LBS structures, which are responsible for most of the viable embryos, showed a specific profile with thin walls rich in arabinogalactan proteins (AGPs), highly and low methyl-esterified pectin and callose, and a callose-rich subintinal layer not necessarily thick, but with a remarkably high callose concentration. The different profiles of EE and LBS walls support the development as suspensorless and suspensor-bearing embryos, respectively. Conversely, less viable embryogenic structures (LC) presented the thickest walls and the lowest values for almost all of the studied cell wall components. These cell wall properties would be the less favorable for cell proliferation and embryo progression. High levels of highly methyl-esterified pectin are necessary for wall flexibility and growth of highly embryogenic structures. AGPs seem to play a role in cell wall stiffness, possibly due to their putative role as calcium capacitors, explaining the positive relationship between embryogenic potential and calcium levels.This work was supported by grant PID2020-115763RBI00 to JS-S from Spanish MICINN and by a Juan de la Cierva -Incorporacion Fellowship and a Marie Sklodowska-Curie Individual Fellowship (656579) to PC-M. RM holds a CDEIGENT (2018/023) fellowship from Generalitat Valenciana.Camacho-Fernández, C.; Seguí-Simarro, JM.; Mir Moreno, R.; Boutilier, K.; Corral-Martínez, P. (2021). Cell Wall Composition and Structure Define the Developmental Fate of Embryogenic Microspores in Brassica napus. Frontiers in Plant Science. 12:1-16. https://doi.org/10.3389/fpls.2021.7371391161

Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis

[EN] Doubled haploid (DH) technology allows for the production of pure lines, useful for plant breeding, through a one-generation procedure that reduces considerably the time and resources needed to produce them. Despite the advantages of microspore culture to obtain DHs, this technique is still insufficiently developed in eggplant, where DHs are produced from microsporederived calli through organogenesis. At present, very little is known on the best in vitro conditions to promote this process. This is why in this work we addressed the optimization of the process of regeneration of eggplant DH plants from microspore-derived calli. We evaluated the effect of different media compositions in the induction of organogenesis, in the promotion of shoot growth and elongation, and in root growth. According to our results, we propose the repeated subculture of the calli in MS medium with 0.2 mg/l IAA and 4 mg/l zeatin to produce shoots, and then the repeated subculture of the excised shoots in basal MS medium to promote their conversion into entire plantlets. This procedure yielded 7.6 plants per 100 cultured calli, which represents a *49 increase with respect to previous reports. We also evaluated by flow cytometry and SSR molecular markers the effect of these in vitro culture conditions in the rate of DH plant production, finding that*70 % of the regenerated plants were true DHs. These results substantially improve the efficiencies of DH recovery published to date in eggplant, and may be useful to those working in the field of eggplant doubled haploidy and breeding.We acknowledge Dr. Rosa Peiro for her statistical advice, and the staff of the COMAV greenhouses for their valuable help. This work was supported by the AGL2014-55177-R grant from Spanish MINECO to JMSS.Rivas Sendra, A.; Corral Martínez, P.; Camacho Fernández, C.; Seguí-Simarro, JM. (2015). Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis. Plant Cell, Tissue and Organ Culture. 122(3):759-765. https://doi.org/10.1007/s11240-015-0791-6S7597651223Asif M, Eudes F, Randhawa H, Amundsen E, Spaner D (2014) Phytosulfokine alpha enhances microspore embryogenesis in both triticale and wheat. Plant Cell Tissue Organ Cult 116:125–130Borgato L, Conicella C, Pisani F, Furini A (2007) Production and characterization of arboreous and fertile Solanum melongena plus Solanum marginatum somatic hybrid plants. Planta 226:961–969Castillo AM, Nielsen NH, Jensen A, Vallés MP (2014) Effects of n-butanol on barley microspore embryogenesis. Plant Cell Tissue Organ Cult 117:411–418Corral-Martínez P, Seguí-Simarro JM (2012) Efficient production of callus-derived doubled haploids through isolated microspore culture in eggplant (Solanum melongena L.). Euphytica 187:47–61Corral-Martínez P, Seguí-Simarro JM (2014) Refining the method for eggplant microspore culture: effect of abscisic acid, epibrassinolide, polyethylene glycol, naphthaleneacetic acid, 6-benzylaminopurine and arabinogalactan proteins. Euphytica 195:369–382Dhooghe E, Van Laere K, Eeckhaut T, Leus L, Van Huylenbroeck J (2011) Mitotic chromosome doubling of plant tissues in vitro. Plant Cell Tissue Organ Cult 104:359–373Dumas de Vaulx R, Chambonnet D (1982) Culture in vitro d’anthères d’aubergine (Solanum melongena L.): stimulation de la production de plantes au moyen de traitements à 35°C associés à de faibles teneurs en substances de croissance. Agronomie 2:983–988Dunwell JM (2010) Haploids in flowering plants: origins and exploitation. 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Effects of growth conditions of donor plants and in vitro culture environment in the viability and the embryogenic response of microspores of different eggplant genotypes

[EN] Notwithstanding the importance of eggplant in global horticulture, doubled haploid production in this species is still far from being efficient. Although acknowledged to have a role in the efficiency of androgenesis induction, factors such as the growth conditions of donor plant or the in vitro culture environment have not been deeply explored or not explored at all in eggplant, which leaves room for further improvement. In this work, we investigated the effects of different in vivo and in vitro parameters on the androgenic performance of different eggplant genotypes, including two hybrids and a DH line. The in vivo parameters included the exposure of donor plants to different temperature and light conditions and to increased levels of boron. The in vitro parameters included the use of different concentrations of NLN medium components, sucrose and growth regulators, and the suspension of microspores at different densities. Our results showed that whereas greenhouse temperature variations or boron application did not to have a positive influence, greenhouse lighting influenced their viability, thereby conditioning the embryogenic response. Changes in different sucrose, salts and hormone levels had different effects in the genotypes studied, which correlated with their genetic constitution. Finally, we determined the best microspore density, different from that previously proposed. Our work shed light on the role of different factors involved in eggplant microspore cultures, some of them not yet studied, contributing to make microspore culture a more efficient tool in eggplant breeding.This work was supported by Grant AGL2017-88135-R to JMSS from Spanish MICINN, respectively, jointly funded by FEDER. ARS and CCF were supported by predoctoral fellowships from the FPI Programs of Universitat Politecnica de Valencia and Generalitat Valenciana, respectively.Rivas-Sendra, A.; Corral Martínez, P.; Camacho-Fernández, C.; Porcel, R.; Seguí-Simarro, JM. (2020). Effects of growth conditions of donor plants and in vitro culture environment in the viability and the embryogenic response of microspores of different eggplant genotypes. Euphytica. 216(11):1-15. https://doi.org/10.1007/s10681-020-02709-4S11521611Abdollahi MR, Corral-Martinez P, Mousavi A, Salmanian AH, Moieni A, Seguí-Simarro JM (2009) An efficient method for transformation of pre-androgenic, isolated Brassica napus microspores involving microprojectile bombardment and Agrobacterium-mediated transformation. Acta Physiol Plant 31:1313–1317Aulinger IE (2002) Combination of in vitro androgenesis and biolistic transformation: an approach for breeding transgenic maize (Zea mays L.) lines. 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Embryogenic competence of microspores is associated to their ability to form a callosic, osmoprotective subintinal layer

[EN] Microspore embryogenesis is an experimental morphogenic pathway with important applications in basic research and applied plant breeding, but its genetic, cellular, and molecular bases are poorly understood. We applied a multi-disciplinary approach using confocal and electron microscopy, detection of Ca2+, callose, and cellulose, treatments with caffeine, digitonin, and endosidin7, morphometry, qPCR, osmometry, and viability assays in order to study the dynamics of cell wall formation during embryogenesis induction in a high-response rapeseed (Brassica napus) line and two recalcitrant rapeseed and eggplant (Solanum melongena) lines. Formation of a callose-rich subintinal layer (SL) was common to microspore embryogenesis in the different genotypes. However, this process was directly related to embryogenic response, being greater in high-response genotypes. A link could be established between Ca2+ influx, abnormal callose/cellulose deposition, and the genotype-specific embryogenic competence. Callose deposition in inner walls and SLs are independent processes, regulated by different callose synthases. Viability and control of internal osmolality are also related to SL formation. In summary, we identified one of the causes of recalcitrance to embryogenesis induction: a reduced or absent protective SL. In responding genotypes, SLs are markers for changes in cell fate and serve as osmoprotective barriers to increase viability in imbalanced in vitro environments. Genotype-specific differences relate to different responses against abiotic (heat/osmotic) stresses.Thanks are due to the Electron Microscopy Service of Universitat Politecnica de Valencia, Marisol Gascon (IBMCP Microscopy Service), Dr Kim Boutilier (WUR, Wageningen) for hosting ARS at her lab, and Dr Samantha Vernhettes (INRA Versailles) for kindly providing us with S4B. 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Evolution of the microstructure, chemical composition and magnetic behaviour during the synthesis of alkanethiol-capped gold nanoparticles

In the present paper, we show an exhaustive microstructural characterization of thiol-capped gold nanoparticles (NPs) with two different average particle sizes. These samples are compared with the polymer-like Au(I) phase formed as a precursor during the synthesis of the thiol-capped gold NPs. The set of analysed samples shows different microstructures at the nanoscale with different proportions of Au atoms bonded either to S or to Au atoms. It has been experimentally shown that the presence of a ferromagnetic-like behaviour is associated to the formation of NPs with simultaneous presence of Au–Au and Au–S bonds. In order to explain such magnetic behaviour a possible model is proposed based on the spin–orbit coupling so that localized charges and/or spins (Au–S bonds) can trap conduction electrons (Au–Au bonds) in orbits.XAS facilities at BM29 in ESRF and the technical support from G.L. Ciatto are acknowledged. Financial support from the Spanish MEC (NAN2004-09125-C07) and “Junta de Andalucía” is also acknowledged. E. Guerrero thanks the Spanish MEC for financial support.Peer reviewe

Magnetic and microstructural analysis of palladium nanoparticles with different capping systems

Palladium nanoparticles capped with different protective systems in a size range between 1.2 and 2.4 nm have been obtained by varying the preparation chemical method. Magnetization curves for all the samples show hysteresis loops, evidencing a ferromagnetic or a permanent magnetism in the nanoparticles. The microstructure of the nanoparticles has been analyzed by x-ray absorption and transmission electron microscopy. The nature of the magnetic behavior found for all these Pd nanoparticles (NPs) is different depending on their sizes and structural features and is explained on the basis of two different suggested mechanisms. The particles protected by means of a surfactant (tetralkylammonium salts), present a ferromagnetic order related to the factors increasing the density of states just below the Fermi level. Whereas, when the nanoparticles are stabilized by covalent bonds with protective species (thiol derivatized alkane chains or surface oxidized Pd NPs), the increase of the 4d density of holes, localized by the bonded atoms (S or O), is giving rise to the observed ferromagneticlike behavio

Analysis of endogenous peptides released from osteoarthritic articular cartilage unravels novel pathogenic markers

[Abstract] Osteoarthritis (OA) is a pathology characterized by the loss of articular cartilage. In this study, we performed a peptidomic strategy to identify endogenous peptides (neopeptides) that are released from human osteoarthritic tissue, which may serve as disease markers. With this aim, secretomes of osteoarthritic and healthy articular cartilages obtained from knee and hip were analyzed by shotgun peptidomics. This discovery step led to the identification of 1175 different peptides, corresponding to 101 proteins, as products of the physiological or pathological turnover of cartilage extracellular matrix. Then, a targeted multiple reaction monitoring-mass spectrometry method was developed to quantify the panel of best marker candidates on a larger set of samples (n = 62). Statistical analyses were performed to evaluate the significance of the observed differences and the ability of the neopeptides to classify the tissue. Eight of them were differentially abundant in the media from wounded zones of OA cartilage compared with the healthy tissue (p < 0.05). Three neopeptides belonging to Clusterin and one from Cartilage Oligomeric Matrix Protein showed a disease-dependent decrease specifically in hip OA, whereas two from Prolargin (PRELP) and one from Cartilage Intermediate Layer Protein 1 were significantly increased in samples from knee OA. The release of one peptide from PRELP showed the best metrics for tissue classification (AUC = 0.834). The present study reveals specific neopeptides that are differentially released from knee or hip human osteoarthritic cartilage compared with healthy tissue. This evidences the intervention of characteristic pathogenic pathways in OA and provides a novel panel of peptidic candidates for biomarker development.Instituto de Salud Carlos III; PI14/01707Instituto de Salud Carlos III; PI16/02124Instituto de Salud Carlos III; PI17/00404Instituto de Salud Carlos III; CIBER-CB06/01/0040Instituto de Salud Carlos III; DTS17/00200Instituto de Salud Carlos III; RETIC-RIER-RD16/0012/0002Instituto de Salud Carlos III; PT17/0019/001

Risk factors, clinical features, and outcomes of listeriosis in solid-organ transplant recipients: a matched case-control study

BACKGROUND: Solid-organ transplant (SOT) recipients are classically considered to be at increased risk for listeriosis. However, risk factors for this infection have not been assessed. METHODS: We carried out a multicenter, matched case-control study (1:2 ratio) from January 1995 through December 2007. Control subjects were matched for center, transplant type, and timing. Conditional logistic regression was performed to identify independent risk factors. Clinical features and outcomes for all case patients were reviewed. RESULTS: Thirty patients (0.12%) with cases of listeriosis were identified among 25,997 SOT recipients at 15 Spanish transplant centers. In a comparison of case patients with 60 matched control subjects, the following independent risk factors for listeriosis were identified: diabetes mellitus (odds ratio [OR], 5.6; 95% confidence interval [CI], 1.6-19.6; ), P = .007 history of cytomegalovirus infection or disease within the preceding 6 months (OR, 35.9; 95% CI, 2.1-620; P = .014), receipt of high-dose prednisone within the preceding 6 months (OR, 6.2; 95% CI, 1.8-21.1; P = .003), and trimethoprim-sulfamethoxazole (TMP-SMZ) prophylaxis (OR, 0.07; 95% CI, 0.006-0.76; P = .029). Twenty-six patients (86.7%) had bacteremia, and 7 had shock at presentation. Other manifestations included meningoencephalitis (10 cases), spontaneous peritonitis (2), pleural empyema (1), brain abscesses (1), and liver abscesses (1). The 30-day mortality rate was 26.7% (8 of 30 patients died). CONCLUSIONS: Listeriosis in SOT recipients is uncommon but causes high mortality. Diabetes mellitus, cytomegalovirus infection or disease, and receipt of high-dose steroids are independent risk factors for this infection, whereas TMP-SMZ prophylaxis is a protective factor

Predictive modeling of therapeutic response to chondroitin sulfate/glucosamine hydrochloride in knee osteoarthritis

[Abstract] Background: In the present study, we explored potential protein biomarkers useful to predict the therapeutic response of knee osteoarthritis (KOA) patients treated with pharmaceutical grade Chondroitin sulfate/Glucosamine hydrochloride (CS+GH; Droglican, Bioiberica), in order to optimize therapeutic outcomes. Methods: A shotgun proteomic analysis by iTRAQ labelling and liquid chromatography–mass spectrometry (LC-MS/MS) was performed using sera from 40 patients enrolled in the Multicentre Osteoarthritis interVEntion trial with Sysadoa (MOVES). The panel of proteins potentially useful to predict KOA patient’s response was clinically validated in the whole MOVES cohort at baseline (n = 506) using commercially available enzyme-linked immunosorbent assays kits. Logistic regression models and receiver-operating-characteristics (ROC) curves were used to analyze the contribution of these proteins to our prediction models of symptomatic drug response in KOA. Results: In the discovery phase of the study, a panel of six putative predictive biomarkers of response to CS+GH (APOA2, APOA4, APOH, ITIH1, C4BPa and ORM2) were identified by shotgun proteomics. Data are available via ProteomeXchange with identifier PXD012444. In the verification phase, the panel was verified in a larger set of KOA patients (n = 262). Finally, ITIH1 and ORM2 were qualified by a blind test in the whole MOVES cohort at baseline. The combination of these biomarkers with clinical variables predict the patients’ response to CS+GH with a specificity of 79.5% and a sensitivity of 77.1%. Conclusions: Combining clinical and analytical parameters, we identified one biomarker that could accurately predict KOA patients’ response to CS+GH treatment. Its use would allow an increase in response rates and safety for the patients suffering KOA.Insituto de Salud Carlos III; PI14/01707Instituto de Salud Carlos III; PI16/02124Insituto de Salud Carlos III; PI17/00404Instituto de Salud Carlos III; DTS17/00200Instituto de Salud Carlos III; CIBER-CB06/01/0040Insituto de Salud Carlos III; RETIC-RIER-RD16/0012/000

Nephrin mutations cause childhood- and adult-onset focal segmental glomerulosclerosis

Mutations in the NPHS1 gene cause congenital nephrotic syndrome of the Finnish type presenting before the first 3 months of life. Recently, NPHS1 mutations have also been identified in childhood-onset steroid-resistant nephrotic syndrome and milder courses of disease, but their role in adults with focal segmental glomerulosclerosis remains unknown. Here we developed an in silico scoring matrix to evaluate the pathogenicity of amino-acid substitutions using the biophysical and biochemical difference between wild-type and mutant amino acid, the evolutionary conservation of the amino-acid residue in orthologs, and defined domains, with the addition of contextual information. Mutation analysis was performed in 97 patients from 89 unrelated families, of which 52 presented with steroid-resistant nephrotic syndrome after 18 years of age. Compound heterozygous or homozygous NPHS1 mutations were identified in five familial and seven sporadic cases, including one patient 27 years old at onset of the disease. Substitutions were classified as ‘severe’ or ‘mild’ using this in silico approach. Our results suggest an earlier onset of the disease in patients with two ‘severe’ mutations compared to patients with at least one ‘mild’ mutation. The finding of mutations in a patient with adult-onset focal segmental glomerulosclerosis indicates that NPHS1 analysis could be considered in patients with later onset of the disease