The impact of upstream and downstream processing on the quality of oil bodies of partially de-hulled sunflower seeds

Few publications on oil bodies or oleosomes seem concerned about their quality (chemical and physical) ex-vivo. This work attempts to identify the main factors (processing and pre-processing) that affect the quality/integrity of sunflower seed oil bodies recovered through a wet-milling process. The physical state of seeds during wet milling had a significant impact on the quality of the oil body suspension. Pre-soaking for 6 hours before wet milling and multiple washing with alkaline buffer (0.1M sodium bicarbonate) was performed to isolate high quality oil body suspensions. It was evident from different physical measurements such as particle size, ζ-potential and light microscopy that pre-soaking had a positive influence on the quality of oil body suspensions with no significant signs of aggregation or coalescence. It was also observed that the resultant washed oil body suspensions were highly surface charged (-28.4 ± 1.2 mV) indicating very stable suspension phase behavior. Washing oil bodies not only removes non-integral, extraneous proteins (derived from the seed matrix) but enriches the lipid content including Tocopherol (α-tocopherol: 491.6 mg/kg of washed oil bodies compared with 252.6 mg/kg crude oil bodies). Changes in the composition of oil bodies after washing have been observed before, but this research also monitored the size of oil bodies after washing, and our results indicate that certain factors can shift the distribution of droplet size. It is believed that any change in average size of droplets indicate the presence of disrupted oil bodies whose surface chemistry has changed enough to compromise their integrity on washing. The retention of droplet size on washing may, therefore, be diagnostic for the recovery of intact oil bodies. An assessment of the integrity of oil bodies recovered from sunflower seeds after accelerated aging (5 months) was carried out. Free fatty acid was more pronounced in oil rather than oil bodies, this could be due to the elimination of some of the free acid bound to oil body during washing. Although some minor variation was observed during seed aging, however, the oil bodies remained stable in the final suspension. The results indicate that oil body membrane was extremely robust under extreme conditions and the integrity of oil bodies was preserved. In addition, oil bodies obtained in this study were resistant to oxidation due to the presence of naturally occurring antioxidants (including vitamin E) associated with them.. The results indicate that the physical barrier of surface membrane protein (oelosin) protect oil bodies against pro-oxidants

Persistent CDV infection of DH82 cells causes a significant differential regulation of genes associated with epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET), as determined by Wilcoxon-Mann-Whitney test p ≤ 0.05 and fold change cut-off ≤ -2 or ≥ 2.

<p>Persistent CDV infection of DH82 cells causes a significant differential regulation of genes associated with epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET), as determined by Wilcoxon-Mann-Whitney test p ≤ 0.05 and fold change cut-off ≤ -2 or ≥ 2.</p

Confocal laser microscopy of non-infected and persistently CDV-Ond infected DH82 cells.

<p>(a) Confocal laser microscopy of non-infected and persistently CDV-Ond infected DH82 cells demonstrates significant differences in the intracellular cortactin distribution (significance is highlighted by asterisks). Depicted are differences in cortical cortactin distribution between non-infected and persistently CDV-Ond infected DH82 cells at the same time point (Figs 5 b-i). Immunofluorescence of non-infected (Figs 5b,d,f,h) and persistently CDV-Ond infected (Figs 5c,e,g,i) DH82 cells, 6h (Figs 5b,c), 1d (Figs 5d,e), 3d (Figs 5f,g) and 5d (Figs 5h,i) after seeding (red: cortactin; blue: nuclear staining with bisbenzimide) highlights the differences in the cortactin distribution pattern and the tendency of both cell types to exhibit a diffuse cytoplasmic cortactin distribution at later time points.</p

Persistent Morbillivirus Infection Leads to Altered Cortactin Distribution in Histiocytic Sarcoma Cells with Decreased Cellular Migration Capacity

<div><p>Histiocytic sarcomas represent rare but fatal neoplasms in humans. Based on the absence of a commercially available human histiocytic sarcoma cell line the frequently affected dog displays a suitable translational model. Canine distemper virus, closely related to measles virus, is a highly promising candidate for oncolytic virotherapy. Therapeutic failures in patients are mostly associated with tumour invasion and metastasis often induced by misdirected cytoskeletal protein activities. Thus, the impact of persistent canine distemper virus infection on the cytoskeletal protein cortactin, which is frequently overexpressed in human cancers with poor prognosis, was investigated <i>in vitro</i> in a canine histiocytic sarcoma cell line (DH82). Though phagocytic activity, proliferation and apoptotic rate were unaltered, a significantly reduced migration activity compared to controls (6 hours and 1 day after seeding) accompanied by a decreased number of cortactin mRNA transcripts (1 day) was detected. Furthermore, persistently canine distemper virus infected DH82 cells showed a predominant diffuse intracytoplasmic cortactin distribution at 6 hours and 1 day compared to controls with a prominent membranous expression pattern (p ≤ 0.05). Summarized, persistent canine distemper virus infection induces reduced tumour cell migration associated with an altered intracellular cortactin distribution, indicating cytoskeletal changes as one of the major pathways of virus-associated inhibition of tumour spread.</p></div

Immunofluorescence of non-infected DH82 cells and persistently CDV-Ond infected DH82 cells 1 day post seeding using anti-CDV nucleoprotein and anti-cleaved caspase 3 antibodies.

<p>(a) CDV-immunoreactivity is lacking in non-infected DH82 cells using an anti-CDV nucleoprotein antibody (D110; mouse monoclonal; 1:100; kind gift from Prof. Dr. A. Zurbriggen, University of Bern, Switzerland). (b) Immunofluorescence of persistently CDV-Ond infected DH82 cells using an anti-CDV nucleoprotein antibody (D110; mouse monoclonal; 1:100; kind gift from Prof. Dr. A. Zurbriggen, University of Bern, Switzerland) reveals a median percentage of 94.15% positive (bright red, secondary antibody: Cy™ 3-conjugated goat-anti-mouse IgG (H + L) antibody; 1:100; Jackson ImmunoResearch Laboratories, Hamburg, Germany) cells at 1d post seeding (minimum 92.99%; maximum 98.36%). Bisbenzimide was used for nuclear counterstaining (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). (c, d) Immunofluorescence of non-infected and persistently CDV-Ond infected DH82 cells 1 day post seeding using an anti-cleaved caspase 3 antibody (Asp175; rabbit polyclonal; 1:900; Cat# 9661, RRID:AB_2341188; Cell Signaling Technology, Inc., Danvers, USA). Single immunopositive cells (bright red, secondary antibody: Cy™ 3-conjugated goat-anti-rabbit IgG (H + L) antibody; 1:100; Jackson ImmunoResearch Laboratories, Hamburg, Germany) are obvious in both non-infected (c) and CDV-infected cells (d) without significant differences between both conditions.</p

RT-qPCR of non-infected and persistently CDV-Ond infected DH82 cells.

<p>RT-qPCR confirms the significant difference in the relative cortactin gene expression in non-infected and persistently CDV-Ond infected DH82 cells as demonstrated by microarray analysis. The relative cortactin gene expression is calculated by normalisation against the housekeeping gene GAPDH. The relative percentage of target-specific gene expression was calculated as follows: X/Y × 100 = normalised target specific gene expression, where X = target-specific gene expression level and Y = housekeeping gene (GAPDH) expression level. (significance (p ≤ 0.05) is highlighted by an asterisk).</p

Persistent CDV infection causes a significant difference (Wilcoxon-Mann-Whitney test p ≤ 0.05 and fold change ≤ -2 or ≥ 2) in the expression of genes associated with invadopodia formation.

<p>Persistent CDV infection causes a significant difference (Wilcoxon-Mann-Whitney test p ≤ 0.05 and fold change ≤ -2 or ≥ 2) in the expression of genes associated with invadopodia formation.</p

Overview of CDV induced changes on cell mechanisms including cell doubling, apoptosis and migration.

<p>(a) Persistent CDV-Ond infection of DH82 cells has no influence on cell proliferation as demonstrated by cells lacking a significant difference (p ≥ 0.05) in the cell doubling assay of non-infected and persistently infected DH82 cells. (b) Immunofluorescence of non-infected and persistently CDV infected DH82 cells reveals no significant difference (p ≥ 0.05) in the percentage of cleaved caspase 3 positive cells indicating a similar apoptotic rate following persistent CDV-Ond infection. Median, minimum and maximum percentages of immunopositive cells are presented. (c) Transwell migration assay of non-infected and persistently CDV-Ond infected DH82 cells reveals a significant difference (depicted by the asterisk, p ≤ 0.05) in the number of migrated cells 6h and 1d after seeding, thus indicating a reduced migratory activity of DH82 cells following persistent CDV-Ond infection. Median, minimum and maximum of counted cells are presented.</p