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

Additional file 3: Figure S2. of Interferon-stimulated genes—essential antiviral effectors implicated in resistance to Theiler’s virus-induced demyelinating disease

ISG15, PKR, and OAS1 protein expression in the spinal cord of mock- and TMEV-infected C57BL/6 at 14 and 98 dpi. ISG15 (A–B), PKR (C–D), and OAS1 (E–F) protein expression in spinal cord gray (A, C, E) and white matter (B, D, F). Shown is the percentage of immunopositive area using Box-and-Whisker plots and significant differences between groups based on Mann-Whitney U tests (*P ≤ 0.05; **P ≤ 0.01). (TIF 3543 kb

Additional file 1: Table S1. of Interferon-stimulated genes—essential antiviral effectors implicated in resistance to Theiler’s virus-induced demyelinating disease

Transcriptional changes of the type I and II IFN signaling pathway in TMEV- compared to mock-infected SJL/J mice. Shown are fold changes at 14, 42, 98, and 196 dpi including P values based on Mann-Whitney U tests. Bold type indicates statistically significant up-regulation (P < 0.05). (DOC 233 kb

Additional file 2: Figure S1. of Interferon-stimulated genes—essential antiviral effectors implicated in resistance to Theiler’s virus-induced demyelinating disease

IFN-α, IFN-β, IRF7, ISG15, and PKR mRNA levels in the spinal cord of mock- and TMEV-infected SJL/J and C57BL/6 mice at 14 and 98 dpi. Shown are IFN-α (A), IFN-β (B), IRF7 (C), ISG15 (D), and PKR (E) transcript numbers using Box-and-Whisker plots and significant differences between groups based on Mann-Whitney U tests (*P ≤ 0.05; **P ≤ 0.01). (TIF 10550 kb

Analysis of microarray data of manually selected glial cell marker genes.

†<p>Kruskal−Wallis-test and independent pairwise <i>post-hoc</i> Mann-Whitney U-tests; group 1 = control dogs, group 2 = acute CDV leukoencephalitis, group 3 = subacute CDV leukoencephalitis, group 4 = chronic CDV leukoencephalitis.</p><p>CNP = 2′,3′-cyclic nucleotide 3′ phosphodiesterase; CSPG4 = chondroitin sulfate proteoglycan 4; GFAP = glial fibrillary acidic protein; MAG = myelin associated glycoprotein; MBP = myelin basic protein; MOBP = myelin-associated oligodendrocyte basic protein; MOG = myelin oligodendrocyte glycoprotein; MPZ = myelin protein zero; NGFR = nerve growth factor receptor; PDGFRA = platelet-derived growth factor receptor, alpha polypeptide; PMP22 = peripheral myelin protein 22; PLP1 = proteolipid protein 1.</p

Transcriptional Changes in Canine Distemper Virus-Induced Demyelinating Leukoencephalitis Favor a Biphasic Mode of Demyelination

<div><p><i>Canine distemper virus (CDV)</i>-induced demyelinating leukoencephalitis in dogs (<i>Canis familiaris</i>) is suggested to represent a naturally occurring translational model for subacute sclerosing panencephalitis and multiple sclerosis in humans. The aim of this study was a hypothesis-free microarray analysis of the transcriptional changes within cerebellar specimens of five cases of acute, six cases of subacute demyelinating, and three cases of chronic demyelinating and inflammatory CDV leukoencephalitis as compared to twelve non-infected control dogs. Frozen cerebellar specimens were used for analysis of histopathological changes including demyelination, transcriptional changes employing microarrays, and presence of CDV nucleoprotein RNA and protein using microarrays, RT-qPCR and immunohistochemistry. Microarray analysis revealed 780 differentially expressed probe sets. The dominating change was an up-regulation of genes related to the innate and the humoral immune response, and less distinct the cytotoxic T-cell-mediated immune response in all subtypes of CDV leukoencephalitis as compared to controls. Multiple myelin genes including <i>myelin basic protein</i> and <i>proteolipid protein</i> displayed a selective down-regulation in subacute CDV leukoencephalitis, suggestive of an oligodendrocyte dystrophy. In contrast, a marked up-regulation of multiple <i>immunoglobulin-like expressed sequence tags</i> and the <i>delta polypeptide of the CD3 antigen</i> was observed in chronic CDV leukoencephalitis, in agreement with the hypothesis of an immune-mediated demyelination in the late inflammatory phase of the disease. Analysis of pathways intimately linked to demyelination as determined by morphometry employing correlation-based Gene Set Enrichment Analysis highlighted the pathomechanistic importance of up-regulated genes comprised by the gene ontology terms “viral replication” and “humoral immune response” as well as down-regulated genes functionally related to “metabolite and energy generation”.</p></div

Pathohistological changes characteristic for the different subtypes of CDV leukoencephalitis.

<p>(<b>A</b>) The cerebella of the non-infected control dogs (group 1) displayed no histological alterations. (<b>B</b>) The cerebella of dogs affected by acute CDV leukoencephalitis (group 2) showed focal astro- and microgliosis (arrow) and occasionally few vacuolated myelin sheaths. (<b>C</b>) The cerebella of dogs affected by subacute CDV leukoencephalitis with demyelination but without inflammation (group 3) exhibited focally demyelinated white matter (asterix) combined with astro- and microgliosis (arrow). (<b>D</b>) The cerebella of dogs affected by chronic CDV leukoencephalitis with demyelination and with inflammation (group 4) displayed focally demyelinated white matter (asterix), combined with astro- and microgliosis (arrow) as well as perivascular inflammatory infiltrates (arrowhead). Luxol fast blue-cresyl violet. Scale bars = 200 µm.</p

Transcriptional differences between controls (group 1) and subtypes of CDV leukoencephalitis (group 2–4).

†<p>Multigroup test followed by independent pairwise <i>post-hoc</i> comparisons employing LIMMA with a false discovery rate (Benjamini and Hochberg) filter of q≤0.05, and a fold change filter of ≥2.0 or ≤ −2.0.</p>‡<p>Gene as defined by the DAVID knowledgebase. Multiple probe sets may match to a single gene, and probe sets may be unassigned (expressed sequence tags).</p>§<p>Biological modules associated with the DEG as detected by the DAVID functional annotation clustering algorithm using orthologous mouse genes.</p><p>DEGs = differentially expressed genes; DEPs = differentially expressed probe sets; ES = enrichment score; group 1 = control dogs, group 2 = acute CDV leukoencephalitis, group 3 = subacute CDV leukoencephalitis, group 4 = chronic CDV leukoencephalitis.</p

Gene ontology terms correlated to demyelination as revealed by Gene Set Enrichment Analysis.

<p>CDKN2C = cyclin-dependent kinase inhibitor 2C; AFAP1L2 = actin filament associated protein 1-like 2; ALDH5A1 = aldehyde dehydrogenase 5 family, member A1; BLNK = B-cell linker; BTG3 = BTG family, member 3; CALCOCO2 = calcium binding and coiled-coil domain 2; CCL2 = chemokine (C-C motif) ligand 2; CCND2 = cyclin D2; CD81 = CD81 molecule; CDK2AP1 = CDK2-associated protein 1; CDKN1A = cyclin-dependent kinase inhibitor 1A; COX17 = cytochrome c oxidase, subunit XVII assembly protein homolog; DDO = D-aspartate oxidase; DNAJC1 = DnaJ (Hsp40) homolog, subfamily C, member 1; GSK3B = glycogen synthase kinase 3 beta; HBXIP = hepatitis B virus x interacting protein; IRAK3 = interleukin-1 receptor-associated kinase 3; KIF23 = kinesin family member 23; LY86 = lymphocyte antigen 86; NEK6 = NIMA (never in mitosis gene a)-related kinase 6; PPOX = protoporphyrinogen oxidase; PSMB10 = proteasome (prosome, macropain) subunit, beta type, 10; SMC4 = structural maintenance of chromosomes 4; TIMP1 = tissue inhibitor of metalloproteinase 1; TLR1 = toll-like receptor 1; TLR3 = toll-like receptor 3; TOP2A = topoisomerase (DNA) II alpha 170 kDa; TPX2 = TPX2, microtubule-associated, homolog.</p

Experimental design and signalement of dogs with CDV leukoencephalitis and controls.

<p>Group ID: 1 = control dogs, 2 = acute CDV leukoencephalitis, 3 = subacute CDV leukoencephalitis, 4 = chronic CDV leukoencephalitis; f = female; ID = identifier; m = male; n.d. = not determined.</p