A Traceable DNA-Replicon Derived Vector to Speed Up Gene Editing in Potato: Interrupting Genes Related to Undesirable Postharvest Tuber Traits as an Example.

In potato (Solanum tuberosum L.), protoplast techniques are limited to a few genotypes; thus, the use of regular regeneration procedures of multicellular explants causes us to face complexities associated to CRISPR/Cas9 gene editing efficiency and final identification of individuals. Geminivirus-based replicons contained in T-DNAs could provide an improvement to these procedures considering their cargo capability. We built a Bean yellow dwarf virus-derived replicon vector, pGEF-U, that expresses all the editing reagents under a multi-guide RNA condition, and the Green Fluorescent Protein (GFP) marker gene. Agrobacterium-mediated gene transfer experiments were carried out on 'Yagana-INIA', a relevant local variety with no previous regeneration protocol. Assays showed that pGEF-U had GFP transient expression for up to 10 days post-infiltration when leaf explants were used. A dedicated potato genome analysis tool allowed for the design of guide RNA pairs to induce double cuts of genes associated to enzymatic browning (StPPO1 and 2) and to cold-induced sweetening (StvacINV1 and StBAM1). Monitoring GFP at 7 days post-infiltration, explants led to vector validation as well as to selection for regeneration (34.3% of starting explants). Plant sets were evaluated for the targeted deletion, showing individuals edited for StPPO1 and StBAM1 genes (1 and 4 lines, respectively), although with a transgenic condition. While no targeted deletion was seen in StvacINV1 and StPPO2 plant sets, stable GFP-expressing calli were chosen for analysis; we observed different repair alternatives, ranging from the expected loss of large gene fragments to those showing punctual insertions/deletions at both cut sites or incomplete repairs along the target region. Results validate pGEF-U for gene editing coupled to regular regeneration protocols, and both targeted deletion and single site editings encourage further characterization of the set of plants already generated.

The norpurpureine alkaloid from Annona purpurea inhibits human platelet activation in vitro.

BACKGROUND: The leaves of Annona purpurea have yielded several alkaloids with anti-aggregation activities against rabbit platelets. This is promising in the search for agents that might act against platelets and reduce the incidence of cardiovascular diseases. Since significant differences in platelet function have been reported between human and animal platelets, a study focusing on the effect of A. purpurea extracts against human platelet activation is necessary. METHODS: The compounds in an A. purpurea ethanolic extract underwent bio-guided fractionation and were used for in vitro human platelet aggregation assays to isolate the compounds with anti-platelet activity. The bioactive compounds were identified by spectroscopic analysis. Additional platelet studies were performed to characterize their action as inhibitors of human platelet activation. RESULTS: The benzylisoquinoline alkaloid norpurpureine was identified as the major anti-platelet compound. The IC50 for norpurpureine was 80 µM against platelets when stimulated with adenosine 5'-diphosphate (ADP), collagen and thrombin. It was pharmacologically effective from 20 to 220 µM. Norpurpureine (220 µM) exhibited its in vitro effectiveness in samples from 30 healthy human donors who did not take any drugs during the 2 weeks prior to the collection. Norpurpureine also gradually inhibited granule secretion and adhesion of activated platelets to immobilized fibrinogen. At the intra-platelet level, norpurpureine prevented agonist-stimulated calcium mobilization and cAMP reduction. Structure-activity relationship analysis indicates that the lack of a methyl group at the nitrogen seems to be key in the ability of the compound to interact with its molecular target. CONCLUSION: Norpurpureine displays a promising in vitro pharmacological profile as an inhibitor of human platelet activation. Its molecular target could be a common effector between Ca2+ and cAMP signaling, such as the PLC-PKC-Ca2+ pathway and PDEs. This needs further evaluation at the protein isoform level.

Chemical constituents from Licania cruegeriana and their cardiovascular and antiplatelet effects.

Three new lupane-type triterpenoids: 6ß,30-dihydroxybetulinic acid glucopyranosyl ester (4), 6ß,30-dihydroxybetulinic acid (5) and 6ß-hydroxybetulinic acid (6), were isolated from Licania cruegeriana Urb. along with six known compounds. Their structures were elucidated on the basis of spectroscopic methods, including IR, ESIMS, 1D- and 2D-NMR experiments, as well as by comparison of their spectral data with those of related compounds. All compounds were evaluated in vivo for their effects on the mean arterial blood pressure (MABP) and heart rate (HR) of spontaneously hypertensive rats (SHR) and also in vitro for their capacity to inhibit the human platelet aggregation. None of the isolated flavonoids 1-3 showed cardiovascular effects on SHR and among the isolated triterpenoids 4-9 only 5 and 6 produced a significant reduction in MABP (60.1% and 17.2%, respectively) and an elevation in HR (11.0% and 41.2%, respectively). Compounds 3, 4, 5 and 6 were able to inhibit human platelet aggregation induced by ADP, collagen and arachidonic acid with different selectivity profiles.