Digoxin extracted from the foxglove plant is a widely prescribed natural product for treating heart failure. It is listed as an essential medicine by the World Health Organization. However, how the foxglove plant synthesizes digoxin is mostly unknown, especially the cytochrome P450 sterol side chain cleaving enzyme (P450scc), which catalyzes the first and rate-limiting step. Here we identify the long-speculated foxglove P450scc through differential transcriptomic analysis. This enzyme converts cholesterol and campesterol to pregnenolone, suggesting that digoxin biosynthesis starts from both sterols, unlike previously reported. Phylogenetic analysis indicates that this enzyme arises from a duplicated cytochrome P450 CYP87A gene and is distinct from the well-characterized mammalian P450scc. Protein structural analysis reveals two amino acids in the active site critical for the foxglove P450scc’s sterol cleavage ability. Identifying the foxglove P450scc is a crucial step toward completely elucidating digoxin biosynthesis and expanding the therapeutic applications of digoxin analogs in future work.

Digoxin from the foxglove plant is a commonly prescribed plant natural product for treating heart failure and atrial fibrillation. Despite its medicinal prominence, how foxglove synthesizes digoxin is largely unknown, especially the cytochrome P450 sterol side chain cleaving enzyme (P450scc), which catalyzes the first and rate-limiting step in this pathway. Here we report the identification of the foxglove P450scc, the gatekeeping enzyme that channels sterols to digoxin. This enzyme converts both cholesterol and campesterol to pregnenolone, thus explaining how pregnenolone is synthesized in plants. Phylogenetic analysis indicates that this enzyme arose from a duplicatedCYP87Agene and does not share clear homology with the mammalian P450scc. Identifying this long-speculated plant P450sccenzyme suggests that the digoxin biosynthetic pathway starts from both cholesterol and phytosterols, instead of just cholesterol as previously thought. The identification of this gatekeeping enzyme is a key step towards complete elucidation of digoxin biosynthesis and expanding the therapeutic applications of digoxin analogs in future work.

Cardenolides are steroidal metabolites in Digitalis lanata with potent cardioactive effects on animals. In plants, cardenolides are likely involved in various stress responses. However, the molecular mechanism of cardenolide increase during stresses is mostly unknown. Additionally, cardenolides are proposed to arise from cholesterol, but indirect results show that phytosterols may also be substrates for cardenolide biosynthesis. Here we show that cardenolides increased after methyl jasmonate (MJ), sorbitol, potassium chloride (KCl), and salicylic acid analog (BTH: 2,1,3-benzothiadiazole) treatments. However, the expression of three known genes for cardenolide biosynthesis did not correlate well with these increases. Specifically, the expression of progesterone-5β-reductases (P5βR and P5βR2) did not correlate with cardenolide increase. The expression of 3β-hydroxysteroid dehydrogenase (3βHSD) correlated with changes in cardenolide levels only during the BTH treatment. Mining the D. lanata transcriptome identified genes involved in cholesterol and phytosterol biosynthesis: C24 sterol sidechain reductase 1 (SSR1) and C4 sterol methyl oxidase 1 and 3 (SMO1 and SMO3). Surprisingly, the expression of all three genes correlated well with the cardenolide increase after the BTH treatment. Phylogenetic analysis showed that SSR1 is likely involved in both cholesterol and phytosterol biosynthesis. In addition, SMO1 is likely specific to phytosterol biosynthesis, and SMO3 is specific to cholesterol biosynthesis. These results suggest that stress-induced increase of cardenolides in foxglove may correlate with cholesterol and phytosterol biosynthesis. In summary, this work shows that cardenolides are important for stress responses in D. lanata and reveals a potential link between phytosterol and cardenolide biosynthesis.

Salicylic acid (SA) is a phytohormone essential for plant defense and development. Exogenously applied SA protects crops from abiotic and biotic stresses and improves yield. However, SA at higher concentrations induces cell death. Here, we test if a naturally occurring SA conjugate, salicylate glucoside (SAG), can be used as a nontoxic plant protectant. We show that Arabidopsis leaves treated with 5 mM SAG neither decreased chlorophyll content nor accumulated reactive oxygen species (ROS), contrary to SA-treated samples. Furthermore, externally applied SAG did not induce the expression of pathogenesis-related genes. When infected by Pseudomonas syringae, SAG-treated Arabidopsis showed a 97% ± 1.1% reduction of bacterial counts compared to the control. In conclusion, surface-applied SAG has the potential to be used as a nontoxic crop protectant against various stresses. Recent high-titer production of SAG in engineered Escherichia coli makes this compound accessible for agricultural applications.

AimTo evaluate and compare the antibacterial efficacy and horizontal depth of penetration of various irrigants into the dentinal tubules when used alone and when combined with lasers.Materials and methodsAn experimental study was done on 42 single-rooted teeth. Access cavity preparation was done and the canals were enlarged up to a ProTaper file size F2 of length 25 mm. They were inoculated with 0.1 mL of Enterococcus faecalis and the samples were randomly assigned into six different groups of seven teeth each. The following irrigation systems were used individually and in combinations—normal saline, sodium hypochlorite, chlorhexidine gluconate, diode laser and erbium, chromium: ytrrium scandium gallium garnet laser (Er,Cr:YSGG laser). The colony-forming units (CFU) of bacteria before and after disinfection and the penetration depth of different groups were determined. Statistical analysis was done by an ANOVA test.ResultsThe highest number of CFU of bacteria was shown by the group where saline was used and it also showed the least penetration depth compared to that of the Er,Cr:YSGG laser group.ConclusionEr,Cr:YSGG laser when used along with sodium hypochlorite and chlorhexidine gluconate showed the highest reduction in the CFU of bacteria and the greatest penetration depth when observed under a stereomicroscope.Clinical significanceLaser-assisted irrigation regimes have a high antibacterial efficacy and more penetration depth into the dentinal tubules.How to cite this articleSubramani SM, Anjana G, et al. Evaluation of Antimicrobial Efficacy and Penetration Depth of Various Irrigants into the Dentinal Tubules with and without Lasers: A Stereomicroscopic Study. Int J Clin Pediatr Dent 2019;12(4):273–279.