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This study examined the effects of sesamin inclusion in vegetable oil-based diets fed to Atlantic salmon ( Salmo salar L.). The diets used differed in n-6/n-3 fatty acid (FA) ratio (0.5 and 1) and sesamin content (high 5.8 g/kg, low 1.16 g/kg and no sesamin). The oils used in the feeds were a mixture of rapeseed, linseed and palm oil. Fish were fed for 4 months. Fatty acids and expression of hepatic genes involved in transcription, lipid uptake, desaturation, elongation and β-oxidation were measured. No major effects on the percentage of DHA in white muscle, liver triacylglycerol and phospholipid fraction were detected. Genes involved in β-oxidation, elongation and desaturation were affected by sesamin addition. Limited effects were seen on any of the transcription factors tested and no effect was seen on the expression of peroxisome proliferator-activated receptors (PPAR). Expression of both SREBP-1 and SREBP-2 increased with sesamin addition. It was concluded that supplementation of fish feed with a high level of sesamin had a negative effect on the growth rate and live weight and did not alter the proportions of DHA in tissues even though gene expression was affected. Thus, more studies are needed to formulate a diet that would increase the percentage of DHA in fish without negative effects on fish growth.

BackgroundUnderstanding of up and down regulation of genes, post-transcriptional changes as well as variations in protein translation are insufficient to fully comprehend onset of disease and disease progression as well as response to treatment.The cell surface proteome is spatially dynamic and changes with the state of the cell, which in turn determines its activity in health and disease. Protein spatial architecture enables cell-cell communication, mobility, structure, and immunological activities. Molecular Pixelation enables the study of these fundamental aspects of immune cell biology at an unprecedented scale enabling data driven research into immunology, drug development and future diagnostics.MethodsPixelgen Technologies has developed the Molecular Pixelation workflow1 for single cell analysis of immune cells which generates location data on spatial cell surface proteins. Delivering 76 immune-cell-specific proteins with spatial resolution in a multiplex assay panel.The Molecular Pixelation protocol (figure 1) is initiated with antibody-oligo conjugates binding to proteins on the surface of PFA-fixed cells. This is followed by two rounds of pixelation. Pixelation A involves the addition of DNA-pixels A, where each A pixel binds to many antibody-oligo conjugates in proximity, generating small connected protein neighbourhoods followed by a round of Pixelation B, connecting the protein neighbourhoods into a protein map. This protein map is amplified and converted into a Illumina compatible next-generation sequencing (NGS) library.The NGS FASTQ files are imported to Pixelgen’s software Pixelator, undergoing several steps of QC and analysis. The read sequences are built to produce a graph, ultimately generating a network of protein connections. Each graph is a reconstruction of the surface of a cell.ResultsOne of the major mechanisms of action for rituximab is antibody-dependent cellular cytotoxicity (ADCC), which is mediated by natural killer (NK) cells e.g. Rituximab clusters CD20 on B-cell cancers, recognized by NK-cells activating ADCC. This is clearly shown by the Molecular Pixelation Polarity scores which were significantly elevated in Rituximab stimulated samples compared to controls. Orthogonal comparison with fluorescent microscopy for Rituximab was performed on fixed cells to validate the spatial distribution of the target proteins upon stimulation (figure 2).ConclusionsThis work presents the applicability of Molecular Pixelation for single-cell analyses as a multiplexed, 3D spatial proteomics method, without any dedicated instrumentation. As illustrated, high protein multiplexing with spatial dimensions can provide insights into essential processes in immune therapies of cancer.AcknowledgementsThe work was performed by the following employees and consultants of Pixelgen Technologies: Filip Karlsson, Tomasz Kallas, Divya Thiagarajan, Max Karlsson, Maud Schweitzer, Jose Fernandez Navarro, Louise Leijonancker, Sylvain Geny, Erik Pettersson, Jan Rhomberg-Kauert, Marcela Gonzalez Granillo, Jessica Bunz, Johan Dahlberg, Michele Simonetti, Prajakta Sathe, Petter Brodin, Alvaro Martinez Barrio and Simon FredrikssonReferenceKarlsson F, Kallas T, Thiagarajan D, Karlsson M, Schweitzer M, Fernandez Navarro J, Leijonancker L, Geny S, Pettersson E, Rhomberg-Kauert J, Gonzalez Granillo M, Bunz J, Dahlberg J, Simonetti M, Sathe P, Brodin P, Martinez Barrio A, Fredriksson S. Molecular Pixelation: Single cell spatial proteomics by sequencing bioRxiv 2023.06.05.543770; doi: https://doi.org/10.1101/2023.06.05.543770Abstract 174 Figure 1Abstract 174 Figure 2

Sesamin (S) is a known lipid modulator and has been shown to increase the conversion of α-linolenic acid (ALA) to docosahexaenoic acid in rainbow trout ( Oncorhynchus mykiss ) fed vegetable oil mixtures including linseed oil. In this study, we evaluated the effects of S supplementation in linseed oil-based diets, content of α- and γ-tocopherols, fatty acid (FA) composition, as well as the gene expression of lipid-related genes. Fish with an average weight of 36.5 g were fed different combinations of commercial linseed oil (LO), purified linseed oil triacylglycerols (TAG) with polar fraction removed and a mixed linseed-sunflower oil (6:4 v/v) (MO). S was added at 0.58 g 100 −1 g feed and fed to the fish for a period of 58 days. Expression of PPARα was downregulated in white muscle of fish fed S containing diets ( P  < 0.05). The expression of PPARβ1A was not affected by S supplementation except where TAG oil was used. The expression of PPARβ1A declined significantly in TAG + S fed group ( P  < 0.05), which indicates that some minor compounds in linseed oil might suppress the effect of S on the expression of PPARβ1A. The expression of PPARγ(long) declined in LO + S and MO + S fed group ( P  < 0.05). The β-oxidation-related genes CPT1 and ACO were upregulated by vegetable oils compared to fish oil. S decreased percentage of ALA in white muscle of fish fed LO + S ( P  < 0.05). The increased desaturation index and the decreased ALA levels suggest that S may increase the biosynthesis of highly unsaturated FA in rainbow trout.

Metabolic transformations of two substrates for human cytochrome P450 (CYP450) 2C9, tolbutamide and diclofenac, were investigated in hepatic microsomes from Atlantic salmon (Salmo salar L.). Tolbutamide hydroxylation followed Michaelis–Menten kinetics. Mean apparent Michaelis–Menten constant (Kₘ) and maximum reaction velocity (Vₘₐₓ) values for 4-hydroxytolbutamide (TBOH) formation were 0.09 ± 0.031 mM and 49.5 ± 6.03 pmol/min/mg, respectively. Addition of sulfaphenazole, an inhibitor for mammalian CYP2C9, in a range from 1 to 200 μM decreased formation of TBOH in a concentration-dependent manner, but not to 50%. Neither fluconazole, an inhibitor of human CYP2C9, nor ketoconazole, inhibitor of CYP1A and CYP3A in fish, affected TBOH formation. In contrast ellipticine, an inhibitor of CYP1A in fish inhibited TBOH formation with the IC₅₀ value of 12.1 μM. The rate of TBOH formation was competitively inhibited by 100 μM of sesamin in the incubations, but the degree of inhibition did not increase with increased sesamin concentration. Ethoxyresorufin hydroxylase (EROD) activity was inhibited by tolbutamide in a non-competitive manner (inhibition constant Kᵢ = 218 μM). Our data suggest that tolbutamide is metabolized by salmon microsomes with formation of TBOH. CYP1A might be involved in this reaction as suggested by decreased TBOH formation in the presence of ellipticine and decreased EROD activity in the presence of tolbutamide. Incubation of diclofenac with the microsomes yielded no metabolite formation, suggesting that salmon does not possess diclofenac-metabolizing activity.