The simultaneous quantification of phytosterols and tocopherols in liposomal formulations using validated atmospheric pressure chemical ionization- liquid chromatography -tandem mass spectrometry.

A novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously quantify phytosterols (brassicasterol, campesterol, stigmasterol and ß-sitosterol) and tocopherols (alpha, beta, gamma and delta) entrapped in the lipid bilayer of a liposomal formulation. Apart from liposomes (a pharmaceutical product), the developed method was able to quantify target analytes in agricultural products, thus showing wide applications. Atmospheric pressure chemical ionization (APCI) was employed due to the enhanced ionization of phytosterols and tocopherols in comparison to electrospray ionization. Unlike published work, the chromatographic conditions were modified to simplify the analytical approach. For the first time, a simple isocratic elution (acetonitrile:methanol 99:1 v/v) was utilized for the separation of four phytosterols and four tocopherols in a single run. A substantially better baseline separation of phytosterols were obtained in comparison to reported methods by using poroshell C18 column. The method has a total run time of 7 min, which is the shortest run time among all reported quantitative methods for the simultaneous determination of four phytosterols and four tocopherols. Calibration curves for all phytosterols were linear in the range of 0.05-10 µg/mL. In the case of tocopherols, alpha tocopherol showed linear response in the range of 0.25-10 µg/mL. However, gamma and delta tocopherols exhibited quadratic relationship in the same concentration range (0.25-10 µg/mL). Validation parameters met the International Conference on Harmonization (ICH) guidelines in terms of selectivity, accuracy, precision, repeatability, sensitivity, matrix effects, dilution integrity and stability. The method was, for the first time, successfully applied for the quantifying phytosterols and tocopherols entrapped inside liposomes. An interesting chromatographic phenomenon was observed during sample analysis. Alpha tocopherol (entrapped in the liposomal lipid bilayer) was found to elute at two retention times, 2.53 min and 3.60 min. Such dual separation was not observed in calibration standards and quality controls. It was concluded that the chiral recognition ability of liposomes made up of phosphatidylcholine separated the enantiomers of alpha tocopherol, giving rise to two peaks at two different retention time. To sum, the reported novel LC-MS/MS method addresses three major analytical shortcomings, namely i)longer run time, ii)complex gradient elution and iii)poor baseline separation of phytosterols and tocopherols.

The Sclerotinia sclerotiorum Slt2 mitogen-activated protein kinase ortholog, SMK3, is required for infection initiation but not lesion expansion.

Mitogen-activated protein kinases (MAPKs) play a central role in transferring signals and regulating gene expression in response to extracellular stimuli. An ortholog of the Saccharomyces cerevisiae cell wall integrity MAPK was identified in the phytopathogenic fungus Sclerotinia sclerotiorum. Disruption of the S. sclerotiorum Smk3 gene severely reduced virulence on intact host plant leaves but not on leaves stripped of cuticle wax. This was attributed to alterations in hyphal apical dominance leading to the inability to aggregate and form infection cushions. The mutation also caused loss of the ability to produce sclerotia, increased aerial hyphae formation, and altered hyphal hydrophobicity and cell wall integrity. Mutants had slower radial expansion rates on solid media but more tolerance to elevated temperatures. Loss of the SMK3 cell wall integrity MAPK appears to have impaired the ability of S. sclerotiorum to sense its surrounding environment, leading to misregulation of a variety of functions. Many of the phenotypes were similar to those observed in S. sclerotiorum adenylate cyclase and SMK1 MAPK mutants, suggesting that these signaling pathways co-regulate aspects of fungal growth, physiology, and pathogenicity.

Brassica napus polygalacturonase inhibitor proteins inhibit Sclerotinia sclerotiorum polygalacturonase enzymatic and necrotizing activities and delay symptoms in transgenic plants.

Sclerotinia sclerotiorum releases a battery of polygalacturonases (PGs) during infection, which the host plant may cope with through production of polygalacturonase inhibitor proteins (PGIPs). To study the interaction between S. sclerotiorum PGs and Brassica napus PGIPs, 5 S. sclerotiorum PGs and 4 B. napus PGIPs were expressed in Pichia pastoris. SsPG3, SsPG6, and BnPGIP1 were successfully produced in the yeast system, and BnPGIP1 inhibited SsPG6 enzymatic activity in vitro. SsPG3 and SsPG6 both induced light-dependent necrosis when infiltrated into leaves, which was reduced in an Arabidopsis thaliana line expressing BnPGIP2 and to a lesser extent in a line expressing BnPGIP1. The line expressing BnPGIP2 also exhibited a delay in the onset of symptoms upon S. sclerotiorum inoculation, but no long-term effect on S. sclerotiorum disease progression was observed. The P. pastoris system was found to be suitable for expressing high levels of some S. sclerotiorum PGs, but PGIP interaction studies were best performed in planta. Arabidopsis thaliana forms necrotic lesions upon infiltration of PGs, is susceptible to S. sclerotiorum, and is easily transformed, and thus, is well-suited for the qualitative study of PG-PGIP interactions.

Isolation of fungal homokaryotic lines from heterokaryotic transformants by sonic disruption of mycelia.

Fungal hyphae--and in some cases, spores--are multi-nucleate. During genetic transformation of these spores or mycelia, only one nucleus generally receives the transferred T-DNA generating heterokaryotic colonies. Characterization of genetic changes, such as the effects of gene disruption in the transformants, requires purified homokaryotic lines. Hyphal tip transfer has conventionally been used to isolate homokaryons. We developed an alternative method for purification of fungal homokaryons from transformed heterokaryotic lines of Sclerotinia sclerotiorum. Ultrasound pulses were used to generate bi-septate, unicellular hyphal fragments that regenerate under selection to produce homokaryotic lines that can be easily identified using colony PCR. This technique facilitates the purification of transformed lines, which allows for routine genome manipulation, and should be adaptable for other filamentous fungi.