Artificial nanovesicles for dsRNA delivery in spray-induced gene silencing for crop protection

Spray-induced gene silencing (SIGS) is an innovative and eco-friendly technology where topical application of pathogen gene-targeting RNAs to plant material can enable disease control. SIGS applications remain limited because of the instability of RNA, which can be rapidly degraded when exposed to various environmental conditions. Inspired by the natural mechanism of cross-kingdom RNAi through extracellular vesicle trafficking, we describe herein the use of artificial nanovesicles (AVs) for RNA encapsulation and control against the fungal pathogen, Botrytis cinerea. AVs were synthesized using three different cationic lipid formulations, DOTAP + PEG, DOTAP and DODMA, and examined for their ability to protect and deliver double stranded RNA (dsRNA). All three formulations enabled dsRNA delivery and uptake by B. cinerea. Further, encapsulating dsRNA in AVs provided strong protection from nuclease degradation and from removal by leaf washing. This improved stability led to prolonged RNAi-mediated protection against B. cinerea both on pre- and post-harvest plant material using AVs. Specifically, the AVs extended the protection duration conferred by dsRNA to 10 days on tomato and grape fruits and to 21 days on grape leaves. The results of this work demonstrate how AVs can be used as a new nanocarrier to overcome RNA instability in SIGS for crop protection.     

Epimeletic behavior in a free-ranging female Risso’s dolphin (Grampus griseus)

acta ethologica - Here, we describe the epimeletic behavior of an adult Risso’s dolphin towards a deceased newborn calf of the same species across several days with photographs, acoustic...     

The differential effect of N6-benzyl-adenine and N6-(Δ2-isopentenyl)-adenine on in vitro propagation of Paeonia suffruticosa Andr. is correlated with different hormone contents

Summary N⁶-benzyl-adenine (BA) enhanced phyllogenesis and axillary bud development of Paeonia suffruticosa during in vitro culture allowing good propagation while N⁶-(²isopentenyl)adenine (iP) did not. During the first five days of culture, the mitotic activity of BA-treated explants was higher than in the iP-treated ones. High BA levels were detected in the BA-treated explants, and this was correlated with the absence of or the low indole-3-acetic acid (IAA) content. The low iP levels measured in iP-treated explants were correlated with high endogenous IAA content; the new cytokinin / auxin ratio could explain the lack of axillary buds and the development of only one leaf. Abscisic acid (ABA) was detected neither in the controls nor in the cytokinin-treated explants during the first week. However, intensive restoration of ABA accumulation was observed in controls from the third week onwards. Both BA and iP-treated explants accumulated less ABA than the controls but this hormone appeared later in the BA-treated explants than in the iP-treated ones.Abbreviations ABA abscisic acid - BA N⁶-benzyl-adenine - BHT butyl-hydroxy-toluene - ELISA enzyme linked immunosorbent assay - FM fresh mass - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - iP N⁶-(²-isopentenyl)adenine - MI mitotic index - 9RBA 9-ß-D-ribofuranosyl-BA - 9RiP 9-ß-Dribofuranosyl-iP - 9RZ 9-ß-D-ribofuranosyl-zeatin - Z zeatin     

Agrobacterium-mediated transformation of Asparagus officinalis L. long-term embryogenic callus and regeneration of transgenic plants

Summary Twenty-three independent kanamycin resistant lines were obtained after cocultivation of longterm embryogenic cultures of three Asparagus officinalis L. genotypes with an Agrobacterium tumefaciens strain harboring ß-glucuronidase and neomycin phosphotransferase II genes. All the lines showed ß-glucuronidase activity by histological staining. DNA analysis by Southern blots of the kanamycin resistant embryogenic lines and of a plant regenerated from one of them confirmed the integration of the T-DNA.Abbreviations GUS ß-glucuronidase - X-Gluc 5-bromo-4-chloro-3indolyl ß-D-glucuronic acid - NPT II neomycin phosphotransferase II     

Dual mechanism of laminin modulation of ecto-5′-nucleotidase activity

The myoblast cell surface activity of ecto-5′-nucleotidase was stimulated by a laminin substrate, whereas fibronectin and gelatin did not increase the AMPase activity of ecto-5′-nucleotidase. This increase was related to a higher expression of ecto-5′-nucleotidase on the surface of cells seeded on a laminin substrate, but without the mobilization of an intracellular pool of enzyme. Furthermore, laminin and its fragments E′₁ and E₈ modified the AMPase activity of the ecto-5′-nucleotidase purified from chicken striated muscle and reconstituted in liposomes. Over the range of concentrations used, intact laminin and its fragment E₈, consisting of the distal half of the long arm, stimulated the AMPase activity of ecto-5′-nucleotidase. By contrast, the large fragment derived from the short arms, designated E′₁, inhibited the AMPase activity. Furthermore, the monoclonal anti-ecto-5′-nucleotidase antibody, CG37, abolished the stimulatory effect of fragment E₈ on the AMPase activity of ecto-5′-nucleotidase but did not reverse the inhibitory effect of fragment E′₁. In conclusion, laminin stimulates the AMPase activity of ecto-5′-nucleotidase by two mechanisms: inducing the expression of ecto-5′-nucleotidase to the cell surface and direct modulation of the enzymatic activity.     

Trophic ecology of three stingrays (Myliobatoidei: Dasyatidae) off the Brazilian north-eastern coast: Habitat use and resource partitioning

Understanding the ecological role of species with overlapping distributions is central to inform ecosystem management. Here we describe the diet, trophic level and habitat use of three sympatric stingrays, Hypanus guttatus, H. marianae and H. berthalutzae, through combined stomach content and stable isotope (δ¹³C and δ¹⁵N) analyses. Our integrated approach revealed that H. guttatus is a mesopredator that feeds on a diverse diet of benthic and epibenthic marine and estuarine organisms, principally bivalve molluscs, Alpheus shrimp and teleost fishes. Isotopic data supported movement of this species between marine and estuarine environments. H. berthalutzae is also a marine generalist feeder, but feeds primarily on teleost fishes and cephalopods, and consequently occupies a higher trophic level. In contrast, H. marianae is a mesopredator specialized on shrimps and polychaetas occurring only in the marine environment and occupying a low niche breadth. While niche overlap occurred, the three stingrays utilized the same prey resources at different rates and occupied distinct trophic niches, potentially limiting competition for resources and promoting coexistence. These combined data demonstrate that these three mesopredators perform different ecological roles in the ecosystems they occupy, limiting functional redundancy.     

Construction and characterization of a YAC library with a lowfrequency of chimeric clones from flow-sorted human chromosome 9

Human chromosome 9 DNA, flow-sorted from somaticcell hybrid PK-87-9, has been used to construct two complete digest YAC libraries. The combined representation of chromosome 9 in these libraries, estimated by hybridization of chromosome 9-specific sequences to YAC colony grids, is 95%. The frequency of chimeric clones, analyzed by fluorescence in situ hybridization of chromosome 9 YACs to human metaphase chromosomes, was estimated to be 4%. These libraries provide a resource for physical mapping and for moving from genetic markers to disease loci on chromosome 9.     

Human pluripotent stem cell-derived extracellular vesicles: From now to the future

Extracellular vesicles (EVs) are nanometric particles that enclose cell-derived bioactive molecules in a lipid bilayer and serve as intercellular communication tools. Accordingly, in various biological contexts, EVs are reported to engage in immune modulation, senescence, and cell proliferation and differentiation. Therefore, EVs could be key elements for potential off-the-shelf cell-free therapy. Little has been studied regarding EVs derived from human pluripotent stem cells (hPSC-EVs), even though hPSCs offer good opportunities for induction of tissue regeneration and unlimited proliferative ability. In this review article, we provide an overview of studies using hPSC-EVs, focusing on identifying the conditions in which the cells are cultivated for the isolation of EVs, how they are characterized, and applications already demonstrated. The topics reported in this article highlight the incipient status of the studies in the field and the significance of hPSC-EVs’ prospective applications as PSC-derived cell-free therapy products.     

Bioguided Fractionation and Isolation of an Antiplasmodial Saponin from the Roots of Nauclea xanthoxylon (A.Chev.) Aubrév. (Rubiaceae)

The root extract of Nauclea xanthoxylon (A.Chev.) Aubrév. displayed significant 50 % inhibition concentration (IC₅₀s) of 0.57 and 1.26 μg/mL against chloroquine resistant and sensitive Plasmodium falciparum (Pf) Dd2 and 3D7 strains, respectively. Bio-guided fractionation led to an ethyl acetate fraction with IC₅₀s of 2.68 and 1.85 μg/mL and subsequently, to the new quinovic acid saponin named xanthoxyloside ( 1 ) with IC₅₀s of 0.33 and 1.30 μM, respectively against the tested strains. Further compounds obtained from ethyl acetate and hexane fractions were the known clethric acid ( 2 ), ursolic acid ( 3 ), quafrinoic acid ( 4 ), quinovic acid ( 5 ), quinovic acid 3-O-β-D-fucopyranoside ( 6 ), oleanolic acid ( 7 ), oleanolic acid 3-acetate ( 8 ), friedelin ( 9 ), β-sitosterol ( 10a ), stigmasterol ( 10b ) and stigmasterol 3-O-β-D-glucopyranoside ( 11 ). Their structures were characterised with the aid of comprehensive spectroscopic methods (1 and 2D NMR, Mass). Bio-assays were performed using nucleic acid gel stain (SYBR green I)-based fluorescence assay with chloroquine as reference. Extracts and compounds exhibited good selectivity indices (SIs) of >10. Significant antiplasmodial activities measured for the crude extract, the ethyl acetate fraction and xanthoxyloside ( 1 ) from that fraction can justify the use of the root of N. xanthoxylon in ethnomedicine to treat malaria.