Paternity analysis using microsatellite markers to identify pollen donors in an olive grove

Olive (Olea europaea L.) is a wind-pollinated, allogamous species that is generally not considered to be self-compatible. In addition, cross-incompatibilities exist between cultivars that can result in low fruit set if compatible pollinisers are not planted nearby. In this study, microsatellite markers were used to identify 17 genotypes that were potential pollen donors in a commercial olive orchard. DNA typing with the same primers was also applied to 800 olive embryos collected from five cultivars in the grove over 2 years of study. Pollen donors for the cultivars Barnea, Corregiola, Kalamata, Koroneiki, and Mission were estimated by paternity analysis, based on the parental contribution of alleles in the genotypes of the embryos. The exclusion probability for the marker set was 0.998 and paternity was assigned on the basis of the ‘most likely method’. Different pollen donors were identified for each of the maternal cultivars indicating that cross-compatibilities and incompatibilities varied between the genotypes studied. Cross-pollination was the principal method of fertilization, as selfing was only observed in two of the embryos studied and both of these were from the cultivar Mission. This is the first report where these techniques have been applied to survey the pollination patterns in an olive grove. The results indicate that careful planning in orchard design is required for efficient pollination between olive cultivars.     

The role of RIPK1 mediated cell death in acute on chronic liver failure

Acute-on-chronic liver failure (ACLF) is characterized predominantly by non-apoptotic forms of hepatocyte cell death. Necroptosis is a form of programmed lytic cell death in which receptor interacting protein kinase (RIPK) 1, RIPK3 and phosphorylated mixed lineage kinase domain-like (pMLKL) are key components. This study was performed to determine the role of RIPK1 mediated cell death in ACLF. RIPK3 plasma levels and hepatic expression of RIPK1, RIPK3, and pMLKL were measured in healthy volunteers, stable patients with cirrhosis, and in hospitalized cirrhotic patients with acutely decompensated cirrhosis, with and without ACLF (AD). The role of necroptosis in ACLF was studied in two animal models of ACLF using inhibitors of RIPK1, necrostatin-1 (NEC-1) and SML2100 (RIPA56). Plasma RIPK3 levels predicted the risk of 28- and 90-day mortality (AUROC, 0.653 (95%CI 0.530–0.776), 0.696 (95%CI 0.593–0.799)] and also the progression of patients from no ACLF to ACLF [0.744 (95%CI 0.593–0.895)] and the results were validated in a 2^nd patient cohort. This pattern was replicated in a rodent model of ACLF that was induced by administration of lipopolysaccharide (LPS) to bile-duct ligated rats and carbon tetrachloride-induced fibrosis mice administered galactosamine (CCL₄/GalN). Suppression of caspase-8 activity in ACLF rodent model was observed suggesting a switch from caspase-dependent cell death to necroptosis. NEC-1 treatment prior to administration of LPS significantly reduced the severity of ACLF manifested by reduced liver, kidney, and brain injury mirrored by reduced hepatic and renal cell death. Similar hepato-protective effects were observed with RIPA56 in a murine model of ACLF induced by CCL₄/GalN. These data demonstrate for the first time the importance of RIPK1 mediated cell death in human and rodent ACLF. Inhibition of RIPK1 is a potential novel therapeutic approach to prevent progression of susceptible patients from no ACLF to ACLF.Subject terms: Hepatitis, Liver diseases     

High Capacity and High‐Rate NASICON‐Na3.75V1.25Mn0.75(PO4)3 Cathode for Na‐Ion Batteries via Modulating Electronic and Crystal Structures

Sodium superionic conductor (NASICON) cathodes are attractive for Na‐ion battery applications as they exhibit both high structural stability and high sodium ion mobility. Herein, a comprehensive study is presented on the structural and electrochemical properties of the NASICON‐Na_3+yV_2?yMn_y(PO₄)₃ (0 ≤ y ≤ 1) series. A phase miscibility gap is observed at y = 0.5, defining two solid solution domains with low and high Mn contents. Although, members of each of these domains Na_3.25V_1.75Mn_0.25(PO₄)₃ and Na_3.75V_1.25Mn_0.75(PO₄)₃ reversibly exchange sodium ions with high structural integrity, the activity of the Mn³⁺/Mn²⁺ redox couple is found to be absent and present in the former and latter candidate, respectively. Galvanostatic cycling and rate studies reveal higher capacity and rate capability for the Na_3.75V_1.25Mn_0.75(PO₄)₃ cathode (100 and 89 mA h g^?1 at 1C and 5C rate, respectively) in the Na_3+yV_2?yMn_y(PO₄)₃ series. Such a remarkable performance is attributed to optimum bottleneck size (≈5 Ų) and modulated V‐ and Mn‐redox centers as deduced from Rietveld analysis and DFT calculations, respectively. This study shows how important it is to manipulate electronic and crystal structures to achieve high‐performance NASICON cathodes.     

Characteristics of the turnover of uncoupling protein 3 by the ubiquitin proteasome system in isolated mitochondria

Uncoupling protein 3 (UCP3) is implicated in mild uncoupling and the regulation of mitochondrial ROS production. We previously showed that UCP3 turns over rapidly in C2C12 myoblasts, with a half-life of 0.5-4h, and that turnover can be reconstituted in vitro. We show here that rapid degradation of UCP3 in vitro in isolated brown adipose tissue mitochondria required the 26S proteasome, ubiquitin, ATP, succinate to generate a high membrane potential, and a pH of 7.4 or less. Ubiquitin containing lysine-48 was both necessary and sufficient to support UCP3 degradation, implying a requirement for polyubiquitylation at this residue. The 20S proteasome did not support degradation. UCP3 degradation was prevented by simultaneously blocking matrix ATP generation and import, showing that ATP in the mitochondrial matrix was required. Degradation did not appear to require a transmembrane pH gradient, but was very sensitive to membrane potential: degradation was halved when membrane potential decreased 10-20mV from its resting value, and was not significant below about 120mV. We propose that matrix ATP and a high membrane potential are needed for UCP3 to be polyubiquitylated through lysine-48 of ubiquitin and exported to the cytosolic 26S proteasome, where it is de-ubiquitylated and degraded.